United States

Department

of Agriculture

Forest Service

Rocky Mountain

Research Station

Proceedings

RMRS-P-32

May 2004

IUFRO Working Party 2.02.15

International Conference

Medford, Oregon, USA

July 23-27, 2001

Breeding and Genetic

Resources of FiveNeedle Pines:

Growth, Adaptability, and

Pest Resistance
Abstract ___________________________________________________

Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel, Howard B. eds. 2004. Breeding and genetic

resources of five-needle pines: growth, adaptability, and pest resistance; 2001 July 23-27; Medford, OR, USA.

IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins, CO: U.S. Department of Agriculture, Forest

Service, Rocky Mountain Research Station. 259 p.

This volume presents 29 overview and research papers on the breeding, genetic variation, genecology, gene

conservation, and pest resistance of five-needle pines (Pinus L. subgenus Strobus Lemm.) from throughout the world.

Overview papers provide information on past and present research as well as future needs for research on white pines

from North America, Europe, and Asia. Research papers, more narrowly focused, cover various aspects of genetics.

Throughout the distribution of five-needle pines, but particularly in many of the nine North American species, the pathogen

Cronartium ribicola J.C. Fisch. continues to cause high levels of mortality and threatens ecosystems and plantations.

Studies on genetic resistance to C. ribicola are described in papers from different regions of the world. Use of P. strobus

as an exotic species in Europe and Russia and corresponding problems with white pine blister rust are discussed in several

papers. Other papers focus on examining and exploiting patterns of genetic variation of different species.

Key words: five-needle pines, white pines, Cronartium ribicola, genetic variation, conservation, restoration

Pinus L. Subgenus Strobus Lemm. Species

Classification of the species as used in these proceedings follows: Price, R.A., A. Liston, and S.H. Strauss.

1998. Phylogeny and systematics of Pinus. In Richardson, D.M. (ed.), Ecology and Biogeography of Pinus.

Cambridge University Press. p. 49-68.

Section Strobus, Subsection Strobi Loud.

P. armandii Franchet. Armand pine

P. ayacahuite Ehrenberg ex. Schlechtendahl. Mexican white pine

P. bhutanica Grierson, Long & Page. (no English common name)

P. chiapensis (Martinez) Andresen. (formerly P. strobus var. chiapensis) Mexican white pine

P. dabeshanensis (formerly syn. for P. armandii, now separate species)

P. dalatensis de Ferré (Vietnamese common names only)

P. fenzeliana Handel-Mazzetti (Vietnam; no English common name)

P. flexilis James. Limber pine

P. lambertiana Douglas. Sugar pine

P. monticola Douglas ex. D.Don. Western white pine

P. morrisonicola Hayata. Taiwan white pine

P. parviflora Siebold & Zuccarini. Japanese white pine

P. peuce Grisebach. Macedonian pine; Balkan pine

P. strobiformis Engelmann. Southwestern white pine

P. strobus Linnaeus. Eastern white pine

P. wallichiana A.B. Jackson (syn. P. griffithii McClelland). Blue pine; Himalayan

white pine

P. wangii Hu & Cheng. (no English common name)

Section Strobus, Subsection Cembrae Loud.

P. albicaulis Engelmann. Whitebark pine

P. cembra Linnaeus. Swiss stone pine; Arolla pine

P. koraiensis Siebold & Zuccarini. Korean pine

P. pumila von Regel. Japanese stone pine

P. sibirica du Tour. Siberian stone pine

Section Parrya Mayr, Subsection Balfourianae Engelm.

P. aristata Engelmann. Rocky Mountain bristlecone pine

P. balfouriana Greville & Balfour. Foxtail pine

P. longaeva D.K. Bailey. Great Basin bristlecone pine
Breeding and Genetic Resources of Five-Needle Pines:

Growth, Adaptability, and Pest Resistance

Proceedings of the

IUFRO Five-Needle Pines Working Party Conference

July 23-27, 2001

Medford, Oregon, USA

Organizing Committee

Richard A. Sniezko, USDA Forest Service, Chair

Scott E. Schlarbaum, University of Tennessee

Howard B. Kriebel, Ohio State University (retired)

Safiya Samman, USDA Forest Service

Harvey Koester, USDI Bureau of Land Management

Joseph Linn, USDA Forest Service

Sponsors

International Union of Forest Research Organizations (IUFRO)

U.S. Department of Agriculture, Forest Service

U.S. Department of the Interior, Bureau of Land Management

University of Tennessee

Washington Department of Natural Resources

Oregon Department of Forestry

U.S. Department of the Interior, Crater Lake National Park

Sierra Pacific Industries

Inland Empire Tree Improvement Cooperative (IETIC)

White Pine Working Group (IETIC)

Forest Renewal BC

IUFRO Working Party 2.02.15

Preface

An international conference on breeding and genetic resources of the five-needle pines took place

in southwestern Oregon, USA, July 23-27, 2001. The scope was worldwide, including 25 species of

subgenus Strobus found in North and Central America, Europe, and Asia. The conference was held

under the auspices of Working Unit 2.02.15 of the International Union of Forest Research Organizations (IRFRO), with the support of the USDA Forest Service and several other forestry organizations.

The goals of the conference were to review available knowledge from research on the genetics and

genetic resources of this diverse group of pines, and to report current research on genetic diversity and

natural hybridization and on the genetics of growth, adaptability, pest resistance, and other traits of

interest in applied tree genetics and gene resource conservation.

The five-needle pines are nearly all in three sections of Subgenus Strobus. Although there is no

universal agreement on the systematics of the subgenus, we have chosen to adhere to the recent

classification by Price and others (Richardson 1998), although new phylogenetic research results

based on isozyme and molecular analysis do not fully concur with this arrangement. We include two

sections, Section Strobus and Section Parrya, the latter including the foxtail and bristlecone pines,

two groups quite different from the rest of the species, of interest for in situ conservation of species

with an ecological role important in their habitat. Most genetic research to date has been conducted

within Subsections Strobi and Cembrae of Section Strobus. Until recently, the greatest attention was

given to the approximately 17 species of Subsection Strobi, which includes several species of great

importance as timber trees, many of which are capable of interspecific hybridization. More recently,

research in Subsection Cembrae, especially in Siberia, has been focused on genetic diversity and

natural hybridization. In addition to two important timber species, this subsection has some species

that, although slow-growing, have great importance in horticulture and watershed protection.

The conference had 53 participants from nine countries, including the USA, Canada, Germany,

Romania, Bulgaria, Russia, Pakistan, China, and South Korea. Papers were also contributed by

nonattending scientists in Japan, Austria, New Zealand, and Russia. Because of the worldwide

natural distribution of the five-needle pines, overview papers were invited covering the regions where

five-needle pines are of major importance from a forestry standpoint. The exception was the Mexico/

Central America region, from which we were unable to secure scientist participation. Research papers

addresssed genetics and genecology, blister rust resistance, breeding and propagation, genetic

diversity, and gene conservation. In addition to paper sessions, the conference, held in Medford,

Oregon, included excursions to seed orchards, research plantations, native stands, Crater Lake

National Park, and the Dorena Genetic Resource Center of the USDA Forest Service. Indigenous

species of five-needle pines included in the field trips were P. monticola, P. lambertiana and P.

albicaulis.

In Subsection Strobi, the white pine blister rust (Cronartium ribicola) is the major target of applied

research, with the goal of removing this obstacle to survival and growth of natural and artificial

stands, especially of sugar pine, western white pine, and eastern white pine. Resistance screening and

breeding are proving to be effective strategies for restoring susceptible western North American

species of five-needle pines. In eastern North America, rust resistance breeding has been more

difficult in eastern white pine, although screening and breeding continues in the USA and Canada.

In Europe, P. strobus would be a premier species for forestry were it not for the blister rust, which is

a serious problem throughout the region, including Russia. For this reason other species are employed

in forest planting.

The Balkan or Macedonian white pine has a dual role in the Balkans, critical for watershed

protection and to a lesser degree as a timber species. Provenance research has shown that geographic

variation in P. peuce can be exploited to some extent for optimum productivity, and population

research has facilitated gene conservation. The blue or Himalayan pine, P. wallichiana, is an

important timber tree, especially in India and Pakistan; its wide geographic and altitudinal range

requires much more research on population variation and gene diversity for effective conservation of

genetic resources. This will only be possible through regional cooperation with support from

international organizations.

Subsection Cembrae includes several cold-climate and high-elevation five-needle pines of Eurasia

and western North America. Research on the phylogenetics of the Siberian stone pines (P. sibirica and

P. pumila) and P. parvifloraindicates that the current partitioning of Subsections Strobi and Cembrae

needs revision. Research on within-species genetic diversity in Siberia shows that there are relatively

low interpopulation differences within the stone pines, and that natural hybridization occurs between

the species. Korean pine also has small genetic distances between populations, even over a wide area;

the main diversity occurs within populations. All of these species are nut pines, related to the North

iii
Acknowledgments

The impetus for this volume came from the IUFRO Five-Needle Pine Breeding and Genetic

Resources Working Party international conference that was held at the IUFRO XX World

Congress in Tampere, Finland, in 1995. Many people and organizations facilitated the

planning and undertaking of this conference and the subsequent compilation of this volume.

We wish to thank the USDA Forest Service (FS - the International Forestry, Research, and

Forest Health groups from the Washington Office as well as the Region 6 Genetics group all

provided vital contributions) and USDI Bureau of Land Management (BLM) which served

as local hosts for the meeting and field excursions. Thanks to Regional Geneticist Sheila

Martinson for opening remarks and support. Along with the editors, Harvey Koester and Joe

Linn helped coordinate local arrangements. Many people contributed to success of the

meeting including Andy Bower, Jeremy Kaufman, Jeremy Pinto, Bob Danchok, Sally Long,

Ryan Berdeen, Laura Berdeen, Jerry Berdeen and Clinton Armstrong from Dorena Genetic

Resource Center, Umpqua National Forest (FS); Tom Atzet and Don Goheen (FS); Liang

Hsin, Terry Tuttle, Gordon Lyford, Tammy Jebb, Larry Price, Bill Robinson, and Dennis

Pyle from BLM; Joel King and the staff (including Smokey Bear) at Prospect Ranger

District, Rogue River National Forest. We thank Mike Cloughesy, Cindy Wardles, and

Nathalie Gitt of the Forestry Outreach Education office at Oregon State University for

support in planning and logistics, and external reviewers S. Aitken, P. Berrang, J. Dunlap, J.

Hamlin, R. Hunt, R. Johnson, A. Kegley, B. Kinloch, J. King, S. Kolpak, S. Martinson, D. Oline,

and P. Zambino for their technical reviews of the papers. We specifically acknowledge

Konstantin Krutovskii (FS) for facilitating communication with Russian scientists and

assistance with Russian manuscripts.

The sponsors of the conference and this volume include IUFRO, USDA Forest Service, USDI

BLM, Crater Lake National Park, Oregon Department of Forestry, Washington Department

of Natural Resources, The University of Tennessee, Inland Empire Tree Improvement

Cooperative (IETIC), White Pine Working Group (IETIC), Sierra Pacific Industries, and

Forest Renewal BC (British Columbia); this array of sponsors allowed us to invite speakers

from throughout the world. The hospitality shown by both the conference hotel (Rogue

Regency Inn in Medford), and the town of Jacksonville was truly outstanding. The conference

banquet hosted by Dennis and Mary Ann Ramsden in the gardens of the McCully House Inn

in Jacksonville on a beautiful southern Oregon evening was truly superb.

We thank all participants of the conference and all authors of papers. A special thanks to

Angelia Kegley (Dorena Genetic Resource Center) who was invaluable with many phases from

conference planning to publication. We thank Louise Kingsbury (FS) and her staff at Rocky

Mountain Research Station Publishing Services for their patience and for preparing the final

publication and the RMRS for distribution of this volume.

American P. albicaulis. The interconnection between subsections is shown by the similarity of

ecological role between the high elevation species limber pine, the pines of Subsection Cembrae, and

the Rocky Mountain bristlecone pine (P. aristata), all of which occupy and stabilize habitats not likely

to be occupied by other, less tolerant tree species. They are for the most part, less susceptible to blister

rust, some much less, than is P. albicaulis.

These are but a few of the many findings of recent research brought out in the conference. The

extensive worldwide distribution of the five-needle pines, their varied and critical ecological roles in

the plant and animal diversity of the world’s forest ecosystems, and their aesthetic and economic

importance to human society are all indicators of the need for continued worldwide research on these

important forest trees. We look forward to continued cooperation and information exchange in the

future.

Howard Kriebel

Medford, New Jersey, USA

June 13, 2003

iv
Contents

Page

Preface ...............................................................................................................................................................iii

Acknowledgments ............................................................................................................................................. iv

Part I: Regional Overview Papers ................................................................................................................. 1

G. Daoust Genetics, Breeding, Improvement and Conservation of Pinus strobus

J. Beaulieu in Canada ............................................................................................................. 3

John N. King Five Needle Pines in British Columbia, Canada: Past, Present and Future ....... 12

Richard S. Hunt

Howard B. Kriebel Genetics and Breeding of Five-Needle Pines in the Eastern United States ....... 20

Geral McDonald Breeding Rust-Resistant Five-Needle Pines in the Western United States:

Paul Zambino Lessons from the Past and a Look to the Future ................................................ 28

Richard Sniezko

Ioan Blada Genetic Research and Development of Five-Needle Pines (Pinus

Flaviu Popescu subgenus Strobus) in Europe: An Overview ....................................................... 51

Alexander H. Alexandrov Genetic and Conservation Research on Pinus peuce in Bulgaria ...................... 61

Roumen Dobrev

Hristo Tsakov

Anatoly I. Iroshnikov Five-Needle Pines in Russia: Introduction and Breeding ................................... 64

Dmitri V. Politov

Huoran Wang Genetic Resources, Tree Improvement and Gene Conservation of

Jusheng Hong Five-Needle Pines in East Asia .......................................................................... 73

Shams R. Khan Genetic Variation in Blue Pine and Applications for Tree Improvement

in Pakistan, Europe and North America.............................................................. 79

Part II: Genetics, Genecology, and Breeding .............................................................................................. 83

Dmitri V. Politov Phylogenetics, Genogeography and Hybridization of Five-Needle Pines in

Konstantin Krutovsky Russia and Neighboring Countries ..................................................................... 85

Bruno Richard Stephan Studies of Genetic Variation with Five-Needle Pines in Germany ...................... 98

Jay H. Kitzmiller Adaptive Genetic Variation in Sugar Pine......................................................... 103

A.W. Schoettle Ecological Roles of Five-Needle Pines in Colorado: Potential

Consequences of Their Loss ............................................................................ 124

Raphael Thomas Klumpp Genetic Variation of Pinus cembra Along an Elevational

Marcus Stefsky Transect in Austria ............................................................................................ 136

J.T. Miller Five-Needle Pines in New Zealand: Plantings and Experience........................ 141

F.B. Knowles

R.D. Burdon

v
Kwan-Soo Woo Genetic and Environmentally Related Variation in Needle Morphology of

Lauren Fins Blister Rust Resistant and Nonresistant Pinus monticola ................................. 148

Geral I. McDonald

Andrew D. Bower Eight-Year Growth and Survival of a Western White Pine Evaluation

Richard A. Sniezko Plantation in the Southwestern Oregon Cascades ........................................... 154

Danilo D. Fernando Development of an In Vitro Technology for White Pine Blister

John N. Owens Rust Resistance................................................................................................ 163

Sergej N. Goroshkevich Natural Hybridization between Russian Stone Pine (Pinus siberica) and

Japanese Stone Pine (Pinus pumila)................................................................ 169

Wan-Yong Choi Estimation of Heritabilities and Clonal Contribution Based on the Flowering

Kyu-Suk Kang Assessment in Two Clone Banks of Pinus koraiensis Sieb et Zucc. ................ 172

Sang-Urk Han

Seong-Doo Hur

Part III: Genetic Diversity and Conservation ............................................................................................. 179

M.F. Mahalovich Whitebark Pine Genetic Restoration Program for the Intermountain

G. A. Dickerson West (United States)......................................................................................... 181

Sei-ichi Kanetani Diversity and Conservation of Genetic Resources of an Endangered

Takayuki Kawahara Five-Needle Pine Species, Pinus armandii Franch. var. amamiana

Ayako Kanazashi (Koidz.) Hatusima ............................................................................................. 188

Hiroshi Yoshimaru

Vladimir Potenko Genetic Diversity and Mating System of Korean Pine in Russia ...................... 192

Part IV: White Pine Blister Rust Resistance .............................................................................................. 201

R.A. Sniezko Variation in Cronartium ribicola Field Resistance Among 13

A.D. Bower Pinus monticola and 12 P. lambertiana Families: Early Results

A.J. Kegley from Happy Camp............................................................................................. 203

A.J. Kegley Variation in Blister Rust Resistance Among 226 Pinus monticola

R.A. Sniezko and 217 P. lambertiana Seedling Families in the Pacific Northwest................. 209

R.S. Hunt Confirmation of Dominant Gene Resistance (Cr2) in U.S. White

G.D. Jensen Pine Selections to White Pine Blister Rust Growing in British Columbia .......... 227

A.K. M. Ekramoddoullah

Ioan Blada Age Trends in Genetic Parameters of Blister Rust Resistance

Flaviu Popescu and Height Growth in a Pinus strobus x P. peuce F1 hybrid population........... 230

Richard A. Sniezko Field Resistance to Cronartium ribicola in Full-Sib Families of

Bohun B. Kinloch Jr. Pinus monticola in Oregon................................................................................ 243

Andrew D. Bower

Robert S. Danchok

Joseph M. Linn

Angelia J. Kegley

Kwan-Soo Woo Influence of Seedling Physiology on Expression of Blister Rust

Geral I. McDonald Resistance in Needles of Western White Pine ................................................. 250

Lauren Fins

Part V: Conference Attendees ................................................................................................................... 255

Conference Attendees ...................................................................................... 257

Page

vi
Part I: Regional

Overview Papers

Part III: Genetic Diversity

and Conservation

Part II: Genetics,

Genecology, and

Breeding

Part IV: White Pine Blister

Rust Resistance

Part V: Conference Attendees

USDA Forest Service Proceedings RMRS-P-32. 2004 1

Part I: Regional Overview Papers

P. balfouriana (foxtail pine), Pinus aristata (Rocky Mountain

bristlecone pine) and P. flexilis (limber pine)

P. aristata and P. flexilis photos courtesy of A. Schoettle

P. balfouriana photo courtesy of D. Burton
2 USDA Forest Service Proceedings RMRS-P-32. 2004
USDA Forest Service Proceedings RMRS-P-32. 2004 3

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

The authors are with the Natural Resources Canada, Canadian Forest

Service, Laurentian Forestry Centre, 1055 du P.E.P.S., P.O. Box 3800,

Sainte-Foy, QC, Canada, G1V 4C7. Tel.: (418) 648-5830, Fax: (418) 648-5849,

e-mail: gdaoust@cfl.forestry.ca

Abstract—The aim of this paper is to present an overview of the

research work carried out in eastern Canada over the last 50 years

to increase knowledge of the genetics of eastern white pine (Pinus

strobus L.), the most majestic conifer of eastern Canada. The intent

of the paper is also to describe the accomplishments achieved in

breeding and tree improvement by a number of private and public

sector organizations from different eastern Canadian provinces as

well as the activities that are currently underway. Ontario’s program, which has been cancelled but which comprised the production

of interspecific hybrids from rust-resistant species such as Himalayan white pine (P. wallichiana A.B.Jackson), is briefly described.

Results of recent studies of population structure of eastern and

western North American blister rust (Cronartium ribicola J.C.

Fisher) are reported. Estimated genetic gain for height 10 years

after plantation from a network of three provenance-progeny tests

established in Quebec is presented with the origin of the most

promising progenies. Other related subjects such as white pine

weevil, flower induction, somatic embryogenesis and seed orchard

production are discussed. Finally, work presently being carried out

for in situ and ex situ conservation of genetic resources of eastern

white pine in eastern Canada are summarized.

Key words: eastern white pine, Pinus strobus, genetics, interspecific hybrids, blister rust.

Introduction ____________________

Eastern white pine (Pinus strobus L.) is the most majestic

of all the conifer species growing in eastern Canada. It has

a very broad tolerance range and is found on a wide variety

of soils ranging from well-drained sands and rocky ridges

through sphagnum bogs (Farrar 1995). In Canada, eastern

white pine’s natural range is limited mainly to the southeast, and extends from eastern Manitoba all the way to

Newfoundland (fig. 1). This species is characteristic of

Canada’s Great Lakes and St. Lawrence Forest Region,

where fire plays a primary role in the establishment of

extensive stands of eastern white pine (Whitney 1986).

Genetics, Breeding, Improvement and

Conservation of Pinus strobus in Canada

G. Daoust

J. Beaulieu

Eastern white pine was overharvested for many decades,

owing to the huge size of the mature trees and their prized

wood qualities. By the end of the 19th century, the extensive

resources of this species had been irremediably decimated in

all of eastern Canada, from Ontario to Newfoundland. The

subsequent introduction of an exotic pathogen—white pine

blister rust (Cronartium ribicola J. C. Fisher)—caused major losses in areas that had been naturally and artificially

regenerated. Today, with the exception of some zones of

white pine in southeastern Ontario and southwestern Quebec, there are only scattered remnants of the beautiful

natural stands that once covered eastern Canada. In Quebec, for the year 1998, nearly 90 percent of the volume of

wood harvested out of the eastern white pine annual allowable cut of 730,000 m3

 came from the southwestern part of

the province (Bouillon 1998).

During the past century, the efforts devoted to reforestation of this species have varied considerably both spatially

and temporally. The virulence of pests like blister rust and

the white pine weevil (Pissodes strobi Peck) are largely to

blame for the failures and cutbacks that have occurred in

reforestation programs. At present, white pine makes up

just a little over 1 percent of the total number of conifer

seedlings planted in eastern Canada yearly. Scattered

distribution of the species at the landscape level, fire

Figure 1—Natural range of eastern white pine (Wendel

and Clay 1990).
4 USDA Forest Service Proceedings RMRS-P-32. 2004

Daoust and Beaulieu Genetics, Breeding, Improvement and Conservation of Pinus strobus in Canada

suppression, competition from other plants and blister rust

have affected natural regeneration and have dramatically

reduced the presence of the species in the landscape. In some

regions such as Anticosti Island, browsing from white tail

deer prevents development of advance regeneration. To put

harvested stands back into production, forest companies

currently rely mainly on silvicultural practices designed to

mimic natural disturbances that favour eastern white pine

natural regeneration.

Over the past 50 years, a number of private and public

sector organizations have made great efforts to learn more

about the genetics of this species, establishing breeding

programs and increasing knowledge of the most damaging

pests. This document provides an overview of the results

obtained to date and describes briefly the activities that are

currently underway in eastern Canada.

Population Genetics _____________

Population genetics studies help us to understand genetic

changes occurring within and among populations. Knowledge obtained can be used to devise breeding strategies

adapted to the species life history traits. During the last

decade, studies of eastern white pine population genetics

were carried out using isozymes as well as DNA markers.

Results of these studies showed that there was a high level

of genetic diversity in this species, and that about 95

percent of it is located within populations. Population

Figure 2—Location of some collections and some provenance-progeny tests in Ontario and Quebec.

differentiation was reported to be about 2 percent on average for populations sampled in Quebec (Beaulieu and Simon

1994, Isabel and others 1999) while it was about 6 percent for

populations located in Ontario and Newfoundland (Rajora

and others 1998). This low level of population differentiation

means that gene flow is extensive. It was also shown that

eastern white pine is a predominantly outcrossing species

with a very low selfing rate as reported in a study of its

mating system in two populations (Beaulieu and Simon

1995). Finally, while it was shown that the level of genetic

diversity in this species is relatively high, a loss of genetic

diversity was made clear in the St. Lawrence Lowlands and

it is believed to be caused by highgrading that was practiced

during the last century (Beaulieu and Simon 1994).

Provenance Testing _____________

Provenance testing of eastern white pine started as early

as the 1950s in Ontario, but these first trials were inconclusive and did not include a good representation of provenances (Zsuffa 1985). The first comprehensive information

on genetic variation in eastern white pine was derived from

a range-wide provenance test initiated by the U.S. Department of Agriculture in 1955. Two out of the 15 provenance

tests established were located in southern Ontario

(Ganaraska and Turkey Point; fig. 2) and included 12 out of

31 provenances tested in the range-wide study. However,

only three provenances of eastern Canada (Ontario, Quebec
USDA Forest Service Proceedings RMRS-P-32. 2004 5

Genetics, Breeding, Improvement and Conservation of Pinus strobus in Canada Daoust and Beaulieu

and Nova Scotia) were represented. Results observed in

these two tests located in southeastern Ontario were reported for 7-year (Fowler and Heimburger 1969) and 28-year

(Abubaker and Zsuffa 1991) morphological and growth traits.

Significant differences among provenances were found for

each of the 12 morphological and growth characters studied.

Variation in some characters was more significant than for

others. The fastest growing provenances (Pennsylvania,

Maine, New York in the U.S.A. and Nova Scotia in Canada)

had fewer forked trees, wider branch angles and finer

branch diameters and were all from the Atlantic coast. On a

broader perspective for provenance testing carried out in

North America, Wright and others (1979) demonstrated

that trees from the southern Appalachian mountains grew

the fastest in the eastern United States and eastern Canada.

The number of provenances originating from Quebec and

Ontario tested in the range-wide provenance trial was too

small to provide the basic information needed for the breeding

program initiated in the 1970s in Quebec (Corriveau and

Lamontagne 1977). Hence, a series of three provenanceprogeny tests was established in Quebec in 1988. Over 250

open-pollinated progenies belonging to 67 provenances coming from eastern Canada and the eastern United States were

tested. Four- and ten-year height data were analyzed by

Beaulieu and others (1996) and Li and others (1996). It was

shown that there is an extensive variation in eastern white

pine and that much of this variation is located within progenies and provenances. Significant differences among provenances and among progenies within provenances were disclosed. Furthermore, the estimates of heritability at the

family level were moderate (10-year) to high (4-year), suggesting that tree breeding would be successful. Culling the worst

progenies in the nursery could be effective without negatively

impacting the expected genetic gain. Nine out of the 10 best

provenances identified at 10 years of age were from outside

Quebec. Four of them were from the Atlantic coast and the five

others were from the Great Lakes region (Ontario in Canada

and Minnesota and Michigan in the U.S.A.).

Blister Rust ____________________

Introduced in the early 20th century into North America on

eastern white pine seedlings imported from Germany, white

pine blister rust is now considered to be the most prevalent

disease of eastern white pine in eastern Canada. In general,

disease incidence increases from west to east and is related

to total rainfall and the decrease in mean July and August

temperatures. Populations growing on coastal areas are

particularly affected by the disease. In Newfoundland, for

instance, all the trees in some young plantations, with some

as young as 6 years old, were killed by the rust (pers. comm.

J. Bérubé 2001). In other provinces, such as Quebec, areas

where eastern white pine is particularly susceptible to the

rust have been mapped (Lavallée 1986), and ecological

characteristics of plantation sites that reduce the risk of

infection have been identified (Lavallée 1991). Plantations

established in partial shade are generally less severely

affected than those established in open fields (Boulet 1998).

Recently, Et-touil and others (1999) studied the genetic

structure of nine populations of blister rust in eastern Canada.

They found that most of the total gene diversity (H T = 0.386)

was present within populations (Hw = 0.370), resulting in a

low level of genetic differentiation among populations (Fst =

0.062). No statistically significant genetic differences either

among provinces or among regions were revealed. The eastern Canadian provinces are considered to make up one large

white pine blister rust epidemiological unit. Furthermore,

gene flow between the populations is high and trees tested

in this unit could be infected by inoculum travelling hundreds of kilometres.

Comparing eastern Canadian blister rust populations

with western ones, Hamelin and others (2000) found that

the populations clustered into two distinct clades, one from

the east and one from the west. Furthermore, the genetic

differentiation was very high (Fst = 44 percent). Results of

this study suggest the presence of a barrier to gene flow

between blister rust populations from eastern and western

North America but that there may be zones in central North

America where the two populations can bridge. It will be

important to determine if the genetic differences between

eastern and western populations could also be translated

into differences in adaptation, virulence, or any other traits

having a high impact in terms of this host-pathogen

pathosystem.

White Pine Weevil _______________

The white pine weevil (Pissodes strobi Peck) is the native

insect that has the greatest impact on the quality of white

pine trees growing in plantations. Studies conducted on this

insect and its relationship with the species have focussed

primarily on management techniques (Stiell and Berry

1985) and on the environmental variables of plantation sites

(Lavallée 1992, Lavallée and others 1996) with the goal of

reducing tree susceptibility and the consequences of infestation. In eastern Canada, few genetic studies have been

carried out on family or individual resistance to the weevil

but it was demonstrated by Ledig and Smith (1981) that

there is genetic variation for weevil resistance in eastern

white pine. With regard to the susceptibility of different

provenances, Abubaker and Zsuffa (1991) showed that there

was a significant difference among the 12 North American

seed sources tested at two locations in Ontario. However, the

variance that could be attributed to the provenances accounted for only 11.5 percent of total variance. Although it is

possible to select specific phenotypes (narrow crown, slender

leader and resin flow) for their resistance to the weevil, this

phenotypical resistance appears to vary widely and even

disappear depending on the environment and the conditions

at the plantation site (Zsuffa 1985). In 1996, a farm-field test

comprising 14 open-pollinated families with some putatively resistant families was established at the Valcartier

Forest Station in Quebec and will make it possible in the

near future to study family variation for resistance to the

weevil (pers. comm. R. Lavallée 2001).

Breeding and Improvement _______

History and Present Status in Ontario

The first breeding program for eastern white pine in

eastern Canada was initiated in Ontario by C. Heimburger

in 1946. The main goal of this program, which was a major
6 USDA Forest Service Proceedings RMRS-P-32. 2004

Daoust and Beaulieu Genetics, Breeding, Improvement and Conservation of Pinus strobus in Canada

effort for that time, was to develop varieties resistant to

white pine blister rust. The program included selection in

natural stands and the propagation of white pines free of

symptoms of the disease, as well as the production of

interspecific hybrids from rust-resistant species such as

Himalayan white pine (P. wallichiana A.B.Jackson (syn.

P. griffithii McClelland). For the eastern white pine, any

significant resistance expressed after blister rust inoculation was particularly notable in the progenies coming from

healthy parents. Zufa (1971) found that the percentage of

diseased trees in progenies of both healthy and diseased

parents was similar and very high. No major genes for

resistance were found.

A great deal of effort was put into developing hybrids. At

the Maple Research Station (fig. 2) , 17 soft pine species were

tested and the breeding effort resulted in about 100 hybrids

(table 1). The program was successful, resulting in the

development of rust-resistant interspecific hybrids (Zsuffa

1981). Most notable were the eastern and Himalayan white

pine hybrids, because of their superior growth, high level of

resistance to blister rust and ability to transmit these

characteristics to future generations (Heimburger 1972,

Zsuffa 1976). However, selections of resistant material based

Table 1—List of the interspecific five-needle pine hybrids produced by the Ontario Ministry of Natural Resources and maintained in research

archives by the Ontario Forest Research Institute.

* Recognition of this name uncertain.

** Syn. P. griffithii McClelland

Source: Personal communication from B. Sinclair, Ontario Forest Research Institute, 2001.

armandii x albicaulis

ayacahuite x strobus

cembra x armandii

cembra x albicaulis

cembra x strobus

flexilis x wallichiana

koraiensis x albicaulis

koraiensis x lambertiana

lambertiana x koraiensis

monticola x ayacahuite

monticola x (wallichiana** x strobus (P. Schwerinii))

monticola x parviflora

(monticola x parviflora) x strobus

(monticola x parviflora) x (wallichiana x strobus)

(monticola x parviflora) x pentaphylla

monticola x pentaphylla

monticola x peuce

(monticola x peuce) x (wallichiana x strobus)

monticola x strobus

parviflora (glaucous*)

parviflora (glaucous*) x (strobus x parviflora)

parviflora glauca

parviflora glauca x strobus

parviflora x albicaulis

parviflora x wallichiana

parviflora x strobus

parviflora x (strobus x parviflora)

(parviflora x strobus) x strobus

(parviflora x strobus) x (strobus x parviflora)

pentaphylla x peuce

pentaphylla x (strobus x parviflora)

peuce x (flexilis x wallichiana)

peuce x (wallichiana x strobus )

peuce x flexilis

peuce x wallichiana

peuce x (monticola x parviflora)

peuce x parviflora

peuce x pentaphylla

peuce x (peuce x strobus)

peuce x strobus

(peuce x strobus (wind*) x (peuce x strobus)

(peuce x strobus (wind*) x strobus

peuce x (strobus x wallichiana)

(peuce x strobus) x (flexilis x wallichiana)

peuce x (strobus x peuce)

(peuce x strobus) x (peuce x strobus)

pumila x strobus

pumila x (wallichiana x strobus)

strobus x albicaulis

strobus x (flexilis x wallichiana)

(strobus x wallichiana) x (wallichiana x strobus)

(strobus x wallichiana) x (wallichiana x strobus) (P. Schwerinii)

strobus x (wallichiana x strobus)

strobus x wallichiana

strobus x monticola

strobus x parviflora

(strobus x parviflora) x peuce

(strobus x parviflora) x strobus

(strobus x parviflora) x (strobus x parviflora)

strobus x pentaphylla

strobus x peuce

(strobus x peuce) x wallichiana

(strobus x peuce) x peuce

(strobus x peuce ) x monticola

(strobus x peuce) x (peuce x strobus)

strobus x (peuce x strobus)

strobus x (strobus x parviflora)

wallichiana x albicaulis

wallichiana x ayacahuite (P. Holfordiana) x parviflora

wallichiana x (wallichiana x parviflora)

wallichiana x lambertiana

wallichiana x (wallichiana x strobus)

wallichiana x (wallichiana x strobus) (P. Schwerinii)

wallichiana x strobus

wallichiana x koraiensis

wallichiana x parviflora

(wallichiana x parviflora) x (wallichiana x parviflora)

wallichiana x pentaphylla

wallichiana x peuce

wallichiana x strobus (P. Schwerinii) x wallichiana

wallichiana x strobus (P. Schwerinii) x (wallichiana x strobus)

wallichiana x strobus (P. Schwerinii) x peuce

(wallichiana x strobus) x (wallichiana x parviflora)

(wallichiana x strobus) x (wallichiana x strobus)

(wallichiana x strobus) x (wallichiana x strobus) (P. Schwerinii)

(wallichiana x strobus) x pentaphylla

(wallichiana x strobus) x strobus

wallichiana x strobus (P. Schwerinii)
USDA Forest Service Proceedings RMRS-P-32. 2004 7

Genetics, Breeding, Improvement and Conservation of Pinus strobus in Canada Daoust and Beaulieu

on the results of this program were never made and included

in operational seed orchards (Cherry and others 2000). In a

study using eight P. wallichiana x P. strobus clones, Zsuffa

(1975) found moderate broad sense heritability values for

tree heights (0.62) and diameters (0.45) and moderately high

broad sense heritability values for branch lengths (0.76) and

branch angles (0.71).

Although significant efforts were also made to develop

varieties resistant to the white pine weevil, they were

mainly unsuccessful, particularly the hybrids of such promising species as Pinus peuce Griseb. and Pinus monticola

Doug. The P. monticola hybrids were poorly adapted to

climatic conditions in Ontario while the resistance of several

P. peucehybrids broke down (Zsuffa 1985). In the mid-1980s,

the financial resources allocated to the production and the

selection of interspecific hybrids and to the development of

blister rust resistant eastern white pine were reduced and

finally the program ended. The most promising material

(pure or hybrid species) produced in this program was

established on six different sites in Ontario and comprised,

at the beginning, more than 4500 trees. The Ontario Forest

Research Institute (OFRI) is conserving these research

archives. The status of these collections is however presently

unknown and there is a high probability that most of the

trees are dead due to several factors including lack of cold

hardiness (pers. comm. B. Sinclair 2001).

In the late 1970s, an intensive plus-tree selection program

was launched by the Ontario Ministry of Natural Resources

(OMNR) in all the major white pine regions to meet the

needs of an expanding reforestation program. By the late

1980s, the province had developed eight breeding populations and an extensive network of seed orchards, comprising

18 orchards covering over 130 hectares. However, few seeds

were collected from the selections and, therefore, no progeny

tests were carried out (Cherry and others 2000). Despite

warnings by Zsuffa (1985) that only continued efforts would

allow full benefits to be derived from all the work done to

date, significant budget cuts in the mid-1990s stripped

breeding programs to their bare bones. The white pine

breeding program and the development of seed orchards

were put on hold. Unfortunately, to all intents and purposes,

no improved seed has been obtained from the orchards for

reforestation. All the seeds being used in the current reforestation program were collected in natural stands during

logging operations (pers. comm. D. Joyce 2001).

In the mid-1990s, a genecology study of eastern white pine

that sampled the current Ontario natural range of eastern

white pine east of Lake Superior was initiated by the OMNR.

Genetic tests were set up and preliminary results were used

to establish a breeding zone for the ‘North Bay’ tree improvement program. Growth variation among populations was

significant and showed a clinal pattern along environment

gradients; southern populations generally grew faster than

the northern ones (pers. comm. P. Lu 2001). Now there is a

renewed interest in Ontario for research on eastern white

pine resistance to blister rust and progeny testing of the

genotypes present in the seed orchards (Cherry and others

2000). P. Lu has recently proposed a study of genetic resistance to blister rust (pers. comm. D. Joyce 2001). To meet

some forest management objectives, the forest company

Tembec Inc. has also recently reactivated a genetic improvement program for eastern white pine in the North Bay area.

Open-pollinated families collected on 265 clones present in

the regional seed orchard were recently sown. The company

plans to carry out progeny tests in 2002.

History and Present Status in Quebec

In the late 1970s, an eastern white pine breeding program

was initiated for Quebec by Corriveau and Lamontagne

(1977), under which genetically improved varieties would be

created by selecting and hybridizing superior genotypes for

growth, shape and resistance to white pine blister rust and

white pine weevil. Although a 1995 program review recommended this area of activity be transferred to the provincial

government, the program has remained headed by the

Canadian Forest Service (CFS). The ministère des Ressources

naturelles du Québec (MRNQ) has not been able to take it

over due to limited human and financial resources. Progress

and accomplishments of the breeding program were reported by Daoust and Beaulieu (1999).

From 1976 to 1986, over 150 selections were made to

create the first-generation breeding population in Quebec.

Selections were propagated by grafting and grown in a

breeding orchard at the Cap-Tourmente National Wildlife

Area east of Quebec City (fig. 2). Beginning in 1992, large

crops of seed and pollen cones allowed the production of fullsib families; in addition, a 6 x 6 diallel was created for a study

on genetic variation in the capacity of somatic embryogenesis initiation. Several experimental designs to evaluate

general and specific combining ability were developed in the

last few years and are now in production at the Valcartier

Forest Station or are in their first post-planting year. Seeds

produced in breeding orchards that are not needed for the

breeding program are collected by the MRNQ for its reforestation program. In the 1980s, plus-tree selections made in

natural stands by the MRNQ as well as selections formerly

made for the breeding program were used to establish a

network of six seed orchards for producing more than 3

million seedlings yearly. Significant seed production has

begun in two out of the six orchards. However, cones were

heavily damaged by a white pine cone beetle (Conophthorus

coniperda (Schwartz). Insect populations will have to be

monitored and controlled. Use of pheromones to control this

insect seems to be promising. A research project is in progress

at the Institut National de Recherche Scientifique (INRS) -

Institut Armand-Frappier (pers. comm. R. Trudel 2001).

Since the inception of the breeding program, seeds were

collected in more than 100 eastern white pine natural

populations in Quebec for ex situ conservation and

genecological studies. Seed lots from neighbouring provinces and states have also been obtained from a number of

collaborators. In 1986, the first phase of a genecology study

involving 300 progenies derived from 160 populations was

established on three different sites in Quebec (Fort-Coulonge,

Notre-Dame-du-Rosaire, Saint-Elzéar-de-Bonaventure; fig.

2). These tests were set up in cut strips under a partial

canopy of tolerant hardwoods, a plantation management

technique recommended to reduce risks of infestation by the

white pine weevil. Despite an initially high survival rate (+

80 percent after 6 years) and intensive stand tending, it

became clear that little valuable information about genetic

variation in juvenile growth could be obtained from these
8 USDA Forest Service Proceedings RMRS-P-32. 2004

Daoust and Beaulieu Genetics, Breeding, Improvement and Conservation of Pinus strobus in Canada

tests because blister rust was ravaging the plants, as was

significant browsing damage from hares. At Saint-Elzéarde-Bonaventure, the most eastern site, the survival rate was

only 46 percent 11 years after planting and half of the

survivors were rust infected; no progeny with more than 50

percent of unaffected seedlings was found. Only 14 percent

of the progenies tested showed a proportion between 30-43

percent of unaffected seedlings.

Fortunately for this first phase, growth and phenological

traits measured during production in the greenhouse (1

year) and nursery (3 years) made it possible to study the

genetic structure and patterns of variation of white pine

populations in Quebec (Li and others 1997). Data were also

used to delineate preliminary seed zones following a method

proposed by Campbell (1986), which estimates relative risks

in transferring seed sources. Principal component analysis

(PCA) was used to take into account all the traits at the same

time. Analysis of variance made it possible to show that

provenances were significantly different for each of these

traits as well as for the PCA scores. In examining the

patterns of variation, it was clear that even though southeastern provenances flushed later, they had superior growth

mainly because they set their buds later. Seed source transfer for eastern white pine in southwestern Quebec is now

controlled through estimates of relative risks obtained from

mathematical models that were developed.

In 1988, another series of three other genecological tests

was established (Grand-Mère, Notre-Dame-du-Laus, NotreDame-du Rosaire; fig. 2) under a partial canopy of mature

pioneer species. These tests included 250 progenies representing 67 provenances. Although intensive stand tending

was done from the beginning, the average survival rate for

each test 12 years after planting ranged from 53 percent to

69 percent. The main pest affecting tree survival in the tests

was blister rust. Genetic variation in juvenile growth was

analyzed using these tests and the main conclusions were

reported by Beaulieu and others (1996). Provenances and

progenies were shown to be phenotypically stable over the

three environments. Breeding values were estimated using

best linear predictions (BLP) and estimates of genetic gain

for height, 10 years after plantation, were obtained for the

best 50 progenies. Hence, a 14 percent (9-29 percent) genetic

gain is expected for height growth 12 years after planting.

For each progeny, three elite trees were chosen and propagated by grafting to create a breeding population. Out of the

50 progenies selected, 22 are from Quebec, 6 from Ontario,

10 from Vermont, 5 from Michigan and 7 from other U.S.

states.

During the 1990s, several other provenance-progeny tests

comprising a smaller number of progenies were established

to improve the distribution of experimental blocks in the

province. One of these is located in Béarn in the

Témiscamingue region and was carried out in co-operation

with Tembec Inc. These tests will supplement the information obtained to date and be used for estimating the number

of progenies required to accurately evaluate the value of a

provenance.

Up to now, little work has been done to select material

resistant to blister rust in eastern white pine. This is mainly

because breeders considered that the genetic variability in

blister rust resistance in eastern white pine is too low to

expect substantial gains through intra-specific selection and

breeding. So, most of the work has been directed toward

transferring blister rust resistance found in other species to

the eastern white pine. It is for this purpose that the most

interesting exotic material, identified in Ontario’s former

program, was included in the breeding program. Thus, 6, 12

and 22 genotypes of P. wallichiana, P. koraiensis and P.

peuce, respectively, were obtained from the collection gathered by the OFRI at the Maple Research Station and put

together with genotypes making up our eastern white pine

breeding population. Seeds obtained from these clones as

well as those from interspecific crosses were sown in 2000

and transplanted in the nursery at the Valcartier Forest

Station in 2001. Exotics and hybrids will eventually be

evaluated for growth, form and resistance.

History and Present Status in the Atlantic

Provinces

As mentioned earlier in this document, mortality caused

by blister rust infection in natural stands or in plantations

is more severe as we move east and closer to the maritime

climate. For example, in Newfoundland mortality can reach

30 percent in natural stands and even 100 percent in plantations (pers. comm. J. Bérubé 2001). So, the plus-tree

selection program carried out in the Atlantic provinces has

always considered blister rust as the main concern and all

the trees selected were free of symptoms of the disease at the

time of selection.

At present, there is no tree breeding program for eastern

white pine in New Brunswick that is headed by the government. However, J.D. Irving. Inc., a forest company, recently

initiated a breeding program by establishing a seed orchard

including plus-trees selected in New Brunswick and others

obtained via collaborators from neighbouring provinces such

as Quebec.

Nova Scotia has a breeding program underway for eastern

white pine. A clonal seed orchard, made up of 58 locally

selected genotypes, was set up in 1981. In 1998, it produced

over 26 kg of seed, which exceeds the requirements of the

province’s reforestation program for the species.

In Prince Edward Island, there is currently no genetic

improvement program underway and none is planned in the

short term since over 90 percent of forest lands are privately

owned. However, first-generation seed orchards were established at the end of the 1980s by the provincial government.

Cones have been collected on a regular basis since the mid1990s (2.7 kg of seeds in 1996).

Although Newfoundland has no active breeding program

for the species at this time, interest in eastern white pine is

growing. A seed orchard, made up of 200 clones from plustrees free of symptoms of blister rust selected in natural

populations on the island, was established between 1998

and 2000. The orchard will ensure a supply of high-quality

seed while allowing the ex situ conservation of genetic

diversity of the island (English and Linehan 2000).

Progress reports about eastern white pine breeding and

improvement are regularly produced and published by the

active members of the Canadian Tree Improvement Association in their biennial proceedings. A description of the

eastern white pine seed orchards in place in the eastern

Canadian provinces is presented in table 2.
USDA Forest Service Proceedings RMRS-P-32. 2004 9

Genetics, Breeding, Improvement and Conservation of Pinus strobus in Canada Daoust and Beaulieu

Flower Induction ________________

In the late 1980s, studies on flower induction in eastern

white pine were undertaken in Ontario and Quebec. In

Ontario, flower induction trials carried out on 3-5-year-old

potted grafts revealed that spraying GA4/7 at concentrations

of 250 and 500 mg /L were effective in promoting pollen- and

seed-cone production. Significant increases in pollen-cone

production were obtained when applications were made

during the period of rapid terminal shoot elongation whereas

applications made about one month after completion of the

terminal shoot elongation were the best for favouring seedcone production (Ho and Schnekenburger 1992). Similar

results were also obtained at the Valcartier Forest Station,

Quebec, between 1990 and 1992, where some trials including root pruning, spray application of GA4/7 and heat stress

were carried out on 1.5-m to 2-m high potted grafts. Despite

high clonal variation, some ramets produced over 200 seed

cones and over 500 pollen strobili (Daoust and Beaulieu

1999). For field-grown grafts, Ho and Eng (1995) found that

GA4/7 injection made during the period of shoot elongation

also promoted pollen- and seed-cone production.

Somatic Embryogenesis (SE) _____

Significant progress has been achieved in this research

field at the CFS since the first study was carried out in 1990.

Garin and others (1998) investigated the somatic embryogenic process using immature and mature zygotic embryos

of eastern white pine originating from 13 open-pollinated

seed families sampled in the breeding population maintained at the Cap-Tourmente National Wildlife Area. From

the immature zygotic embryos, embryogenic tissues were

obtained for 12 out of the 13 families with initiation rates

varying from 2.6 percent to 23 percent. Mature somatic

embryos were produced for 30 out of 52 cell lines and plants

were regenerated. Embryogenic tissues were also obtained

from mature zygotic embryos and embryogenic cell lines

developed for 5 out of the 13 families tested with a maximum

initiation rate of 2.7 percent. Plants were produced from four

cell lines. Those produced from immature as well as from

mature zygotic embryos are presently under evaluation at

the Valcartier Forest Station. After the success obtained in

SE, it was decided to make a complete 6 x 6 diallel with

maternal trees whose seed families had high, intermediate

and low initiation rates. This diallel will make it possible to

determine the genetic components and their effects on SE

initiation.

Recently, the optimal concentration of plant growth regulator to be put into the culture medium was found. It made

it possible to increase the SE initiation rate in eastern white

pine from approximately 20 percent to 53 percent

(Klimaszewska and others 2001). This study also demonstrated that a low level of plant growth regulator in the

medium used for initiation and proliferation consistently

produced a high number of somatic embryos. The different

concentrations of plant growth regulator tested allowed

somatic embryos to convert to plants at an overall frequency

of 76 percent. An estimated narrow-sense heritability for

somatic embryogenesis initiation (h2 ) of 0.25 was found and

indicates that selection of responsive families can be done.

According to the authors, with these improved protocols,

application of eastern white pine somatic embryogenesis in

commercial clonal forestry is feasible as an alternative to

traditional breeding for reforestation purposes.

Conservation ___________________

In eastern Canada, eastern white pine is not considered as

a local species at risk or as an endangered species at present.

The Ancient Forest Exploration & Research group from

Ontario estimated that less than 2 percent of our old-growth

white pine forests remain world-wide, making them an

endangered ecosystem type (Quinby 2000). After a report

prepared by the White Pine Working Group of Newfoundland, describing the deplorable situation of the species in the

province, the government issued a moratorium on domestic

and commercial cutting licences in 1998. The province’s

policy is underpinned by a reforestation program and precise guidelines on precommercial thinning operations. A

genetic diversity selection and conservation program was

launched on the island in 1999 and a 2-ha seed orchard was

established.

Presently on crown lands, where harvesting is carried out,

forest companies are required to regenerate the species

naturally or artifically and must also comply with the

sustainable management principles that are enforced in the

various provinces. In Ontario, for example, the Ministry of

Natural Resources put out a silviculture guide partly designed for white pine stands in 1998 (OMNR 1998) and

developed a conservation strategy for old-growth pine forest

ecosystems (OMNR 2001). However, it seems that in all the

Table 2—Description of the eastern white pine seed orchards in the eastern Canadian provinces.

No. of seed No. of Year of Breeding Area Seed

Province orchards clones establishment generation (ha) production

Ontario 18 2500 Late 80s First 130 No

Quebec 7 700 1981-91 First 33.3 Yes

1 140 1999 Second 1 No

New Brunswick 1 — 1998 First — No

Prince Edward Island 2 70 1988-90 First 2.8 Yes

1 — 1995-97 — — No

Nova Scotia 1 58 1981 First 2.1 Yes

Newfoundland 1 200 2002 First 2.0 No
10 USDA Forest Service Proceedings RMRS-P-32. 2004

Daoust and Beaulieu Genetics, Breeding, Improvement and Conservation of Pinus strobus in Canada

conservation programs in place in eastern Canada, not

enough resources are allocated to study the impact of blister

rust on the long-term survival of the species.

In situ conservation of the species is also being carried out

in existing parks and reserves in eastern Canada (Boyle

1992). This protection may be complete as is the case in

national parks or it may be partial, as in some provincial

parks. In the latter case, logging activities can take place but

they are subject to certain restrictions designed to ensure

natural regeneration of harvested species among other things.

In Ontario, following the recommendations of the Temagami

Comprehensive Planning Council, the government decided

in 1996 to protect 11 old-growth red and eastern white pine

stands ranging from 100 to 9,000 ha in the Temagami region

(Quinby 2000). In Quebec, the most representative ecosystems are protected by a network of ecological reserves. Other

stands, identified as exceptional forest ecosystems, will be

protected in the near future by a recently adopted law (pers.

comm. N. Villeneuve 2001).

In Mauricie National Park, Quebec, a master plan has

been drawn up for the ecological rehabilitation of white pine

(Quenneville and others 1998). Tests involving prescribed

burning were conducted but proved to be inconclusive since

they did not coincide with a good cone crop year. In eastern

Quebec, work is also in progress to establish a white pine

restoration program. In spring 2001, for instance, the Lower

St. Lawrence Model Forest collaborated on the establishment, within its territory, of two open-pollinated progeny

tests that will be used to estimate the breeding values of the

trees making up the first-generation breeding population

built up by the CFS. In Ontario, the OMNR has begun to

develop restoration goals for white pine ecosystems along

the northern edge of its range and a task force has been

mandated to develop principles, criteria and management

guidelines for ensuring the long-term persistence of oldgrowth forest in unprotected areas. The task force will use

white pine as a test case (pers. comm. D. Joyce 2001).

Ex situ conservation activities include all the experimental plots used for breeding programs, such as provenance

tests, progeny tests, breeding orchards and clonal archives.

Similarly, the regional seed orchards put in place by provincial governments and forestry companies are excellent tools

for conserving genetic resources. These orchards, which are

made up of locally selected genotypes, represent a broad

sample of the genetic diversity that is present in eastern

white pine in eastern Canada. The different provincial

reforestation programs, which are carried out using local

seed sources most of the time, also help to conserve genetic

diversity at the regional level.

Another valuable ex situ conservation approach is the

maintenance of seed banks such as the one that the CFS

maintains at its National Tree Seed Centre in Fredericton

(Simpson and Daigle 1998) and at the Laurentian Forestry

Centre in Quebec City. The seedlots in these collections

provide a very good sampling of the genetic diversity of

eastern white pine populations in northeastern North America.

Acknowledgments ______________

The authors thank the following persons who graciously

provided pertinent information allowing us to present an

accurate overview of the eastern white pine status in eastern

Canada: D. Joyce, B. Sinclair, P. Lu, from the Ontario

Ministry of Natural Resources; B. English, from the Newfoundland Department of Forest Resources and Agrifoods;

K. Tosh, from the New Brunswick Department of Natural

Resources and Energy; R. Lavallée, J. Bérubé, R. Hamelin

and D. Simpson, from Natural Resources Canada, Canadian

Forest Service; A. Deshaies, N. Villeneuve, from the ministère

des Ressources naturelles du Québec; and R. Trudel, from

the INRS, Institut Armand Frappier. They are also grateful

to Pamela Cheers, from Natural Resources Canada, Canadian Forest Service, for editing the paper.

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white pine (Pinus strobusL.): 28th- year results from two southern

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12 USDA Forest Service Proceedings RMRS-P-32. 2004

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

John N. King is Research Scientist, Research Branch, British Columbia

Ministry of Forests, P.O. Box 9519 Stn Prov Govt, Victoria, B.C. V8W 9C2,

Canada. Phone: 250 387-6476. Fax: 250 387-0046. Email: John.King@

gems7.gov.bc.ca. Richard S. Hunt is Research Pathologist, Pacific Forestry

Centre, Canadian Forest Service, 506 West Burnside Road, Victoria, B.C. V8Z

1M5, Canada. Email: R.Hunt@PFC.forestry.ca.

Five Needle Pines in British Columbia,

Canada: Past, Present and Future

John N. King

Richard S. Hunt

Abstract—In British Columbia (BC), Canada, we have been involved with white pine and blister rust since the rust’s discovery on

imported infected pines through the port of Vancouver in 1910. Just

after the rust’s introduction, the USDA Forest Service established

monitoring plots and species trials in BC, but these were abandoned

when the rust became well established in the USA. Resistance

research began again in 1946 with a collection of western white pine

(Pinus monticola Dougl. ex D.Don) seed that was sent to Ontario for

testing. In about 1950 grafted plus trees were inoculated in a disease

garden, but this work was also abandoned in 1960 when it was

demonstrated that seedlings from such selections could be susceptible. Parent tree selection and seedling inoculation of open-pollinated families of western white pine began again in earnest in 1987.

From this material we have the basis of a breeding and seed orchard

program based on partial resistance mechanisms. An F1 generation

is being produced for future research. Additionally, we are considering single gene resistance traits, such as MGR, which can be

pyramided onto the partial resistance of our breeding population.

Efforts, particularly for conservation interests, are also being started

for whitebark pine (P. albicaulis Engel.).

Key words: genetic resistance, western white pine, whitebark

pine, limber pine

Five Needle Pines in British

Columbia ______________________

Both western white pine (Pinus monticolaDougl. ex D.Don)

and whitebark pine (P. albicaulis Engel.) achieve the northern extent of their distributions in British Columbia (BC),

Canada, while limber pine (P. flexilis James) achieves the

limit of its distribution in the Canadian Rockies in both

Alberta and BC. The two alpine species, whitebark and

limber pine, provide valuable tree cover for wildlife in

exposed alpine country, food for birds and small mammals,

stabilizing elements for snow packs and soils in these steep

and fragile environments, and are an important feature of

the aesthetics of the high mountains. Western white pine,

besides providing many of these features, is also a fast

growing and highly valuable component of BC’s timber

industry. Although these species may suffer from the mountain pine beetle (Dendroctonus ponderosae Hopkins), and P.

albicaulis relies on Clark’s nutcracker (Nucifraga columbiana

Wilson) for regeneration, their most serious threat has been

the introduction of blister rust (Cronartium ribicola J.C.

Fischer) in the early part of the 1900s. So great was the

damage due to blister rust, that it was felt that these species

might be lost completely. The major research effort with the

white pines has therefore been an intensive search for

resistance to blister rust. To this end, the USDA Forest

Service established rust resistance programs in Idaho, Oregon, and California. The Canadian and BC Forest Services

also established rust screening programs in BC. The prognosis for western white pine is now considerably better –

although some of the other species such as whitebark pine

are still at a risk. We outline below the events and the

progress made to date and strategies we are hoping to

develop to protect this valuable natural resource.

Past—Introduction of Cronartium

ribicola ________________________

In 1911 British Columbia (BC) was experiencing a boom

with a population of 392,500, more than double the previous

10-year census. The population was largely farmers, loggers, and coal miners with little education. Quite likely Tom

Newman planned to take advantage of the boom when he

imported 1,000 exotic P. strobusL. seedlings from France for

resale in 1910. However, most of the recent immigrants were

from Britain, and in 1914 many of the men returned to

Europe to fight in World War I. Before the war, Newman and

others sold some imported pines, but sales crashed with the

outbreak of the war, and the remaining plants seem to have

been abandoned (Gussow 1923). Perhaps Newman never

returned from the European conflict; certainly many did not,

and the war had effectively put an end to the boom. Under

these circumstances it is amazing that blister rust was first

identified in BC only 3 years after the war, in the fall of 1921

(Gussow 1923). Although the potential value of the white

pines was understood, in those years in BC there was a

stronger commercial interest in currants (Ribes spp), the

alternate host of blister rust (Eastham 1922; 1922/3). However, the U.S. Forest Service was worried about the threat to

western white pines, particularly P. monticola, and they

dispatched field personnel to BC as early as 1922.
USDA Forest Service Proceedings RMRS-P-32. 2004 13

Five Needle Pines in British Columbia, Canada: Past, Present and Future King and Hunt

From the Portland, OR, office researchers came to

Vancouver, BC, then north via train to near present day

Whistler, BC. Here they established a species susceptibility

trial (Childs and Bedwell 1948) and various research plots

(Lachmund 1934; Childs and Kimmey 1938). Once it was

clear that the rust was well established in the United States,

no new plots were established in BC. About that time (1927)

the lone collaborating Canadian scientist was killed in a car

crash (Estey 1994), and Canadian rust research stopped

until 1946.

In 1946 the provincial chief forester had P. monticola seed

collected from the BC interior and sent to Heimburger in

Ontario for resistance testing. Both Heimburger and Riker

in Wisconsin were doing resistance testing in P. strobus. In

1948 the Canadian government hired Porter to do resistance

testing in BC. Porter followed Riker’s protocol of grafting

plus-trees and placing them in a ribes (disease) garden

(fig. 1). He obtained scions from survivors in the old U.S.

Forest Service plots near Whistler, trees in similar plots that

he had established, and a few trees recommended by the BC

Forest Service. He rated clones by percentage of ramets

cankered after 5 or 7 years in a disease garden. These were

also placed in three forest sites and subjected to natural

infection. The most promising P. strobus and P. monticola

from Heimburger and P. strobus from Riker were also placed

at these sites. When it was discovered that grafts from old

trees can produce susceptible offspring (Patton 1967), the

program ended, and Porter left to become a school teacher.

All the material from one field site was transferred to the

University of BC experimental forest, and the other sites

were abandoned. These sites were revisited in the 1980s,

and the cankering of the clones tended to follow Porter’s

(1960) original ranks (Hunt and Meagher 1989).

The success of Bingham’s resistance work in Idaho

(Bingham 1983) and the need for P. monticola for reforesting

laminated-root-rot (Phellinus weirii (Murr.) Gilb.) sites was

the catalyst for the BC Forest Service and Canadian Forest

Service (CFS) to sign a cooperative memorandum of understanding on blister rust resistance in 1983. Disease free

plus-trees were selected for both Interior and Coastal populations. Open-pollinated (OP) cones were collected from the

selected trees, and the resulting progeny seedlings were

exposed to blister rust in inoculation chambers. The first

successful inoculation occurred in 1987 and was repeated

annually to 1995. This material is now the basis for white

pine seed orchards and resistance breeding programs in BC.

Present ________________________

The resistance program continues in BC primarily through

the continuing cooperative relationship between the provincial government, the CFS, which provides pathology research and screening, and increasingly the Forest Industries, which provide technical support through their seed

orchard programs. The efforts of the past have allowed us, at

this stage, to assess the resistance found to date, not just in

the populations screened in BC, but also in other jurisdictions in Western North America. We can also assess the

transferability of seed sources of western white pine and are

looking at the most appropriate strategy of seed deployment

from our seed orchards in order to use the best available

resistance with well-adapted seed sources. Not all of these

questions can be answered at present, but current research

should answer them in the near future.

Figure 1—Porter’s screening for blister rust resistance by growing grafted Pinus monticola ramets from blister rust

resistant candidates in a disease (ribes) garden at Duncan BC in 1955.
14 USDA Forest Service Proceedings RMRS-P-32. 2004

King and Hunt Five Needle Pines in British Columbia, Canada: Past, Present and Future

Resistance Story to Date

Most of the resistance programs in Western North America

to date have concentrated on selections and screening of

open-pollinated families from surviving canker-free parent

trees. The strong selection pressure, first in the natural

stands and then in inoculation chambers, almost assures

that these are not mere “escapes” but that there is a genetic

basis to this resistance. However, as with the original Riker

method of screening grafts, it has been difficult to determine

the basis of resistance and how the resistance is inherited.

The exception is the case of the hypersensitive response

(HR), a major gene resistance (MGR) found in sugar pine (P.

lambertiana Dougl.) and some populations of P. monticola

(for example, Champion Mine) (Kinloch and others 1970,

Kinloch and others 2003). Although there are some complexities to MGR (Kinloch and Dupper 1998, Kinloch and

others 1999), it is relatively simple and easily understood

because it is a classical vertical resistance controlled by a

single dominant gene. More complex resistances, falling

under the headings of “partial resistance” and “tolerance”,

are more difficult to characterize, and we have a much poorer

understanding of their genetic basis.

In BC we have now made a series of nearly 600 selections

from the CFS screening program. This included a fairly

intensive parent tree selection from both the Interior and

Coastal BC (about 300 from each population) and rust

screening of the OP progeny for what may be considered two

“partial resistance” mechanisms: “slow-canker growth” (Hunt

1997) and “difficult-to-infect seedlings” (Hunt and others

1998). We have also selected a set of trees from established

plantation trials and from Texada Island where a stand was

characterized with “tolerance” and trees were selected for

their marked “bark reaction” response.

Although the first orchard selections were based on forward selection of the progeny from the screening trials,

lately, where it is feasible, we have switched to collecting

scion from the original selected parents. Selection of parent

material, rather than progeny, has allowed us to proceed

much faster with our breeding program, as seed cones can be

produced on ramets in as little as 2 years. Also the hypothesis presented that some of the resistance found in the Idaho

populations may be controlled by recessive genes (McDonald

and Hoff 1971; Hoff 1988) has encouraged us to use parents

rather than OP progeny and concentrate future screening on

a F1 population constructed from the best parents. Crossing

for this breeding program consists of crosses between parents of similar putative mechanisms, crosses with susceptible parents and selfs. Selfs, where they can be made, will

be particularly valuable if recessive genes are involved. The

construction of this F1 breeding population is now well under

way (fig. 2), making use of structured mating designs that

will help in future genetic interpretations.

Transferability and Adaptability of White

Pine Seed Sources

Seed transfer guidelines have been, and continue to be,

developed from three series of trials that test most of the

Figure 2—Pollination bags for breeding program crosses on top-pruned young grafts of

western white pine at CanFor Seed Orchard, Sechelt BC (photo courtesy R. Sniezko).
USDA Forest Service Proceedings RMRS-P-32. 2004 15

Five Needle Pines in British Columbia, Canada: Past, Present and Future King and Hunt

range of western white pine on 24 sites throughout BC

(fig. 3). The first series contrasted the R.T. Bingham

(Moscow, ID) arboretum seed source with a local BC

source. Trials were established within and north of the

species range. The second was established in nine root

disease sites and included 14 provenances covering the

range limits of the species (Hunt 1987). The third had 12

provenances with family structure on six sites. These trials

have been described in detail (Hunt 1987, Hunt 1994, Hunt

and Meagher 1989, Meagher and Hunt 1998, Meagher and

Hunt 1999), and these results are summarized below. We

also report results from recent assessments from two of the

family/provenance trials.

Results from these series show that western white pine

does not show a strong clinal response to growth or disease

resistance, but rather there are abrupt changes. Most striking are the southern populations from the Sierras, Klamath,

and Warner Mountains that grow poorly and are generally

highly susceptible to rust, even more so than the northern

populations (perhaps as a result of physiological opportunities that the rust can exploit) (Hunt 1994; Meagher and

Hunt 1998). Rehfeldt and others (1984) also showed this

absence of a strong geographic pattern of variation except in

these southern populations. Sources north of these (especially north of the Columbia River) and extending east as far

as Montana are less dramatic in their differences. Interior

sources, including Idaho, grow well at the coast (Bower 1987;

Meagher and Hunt 1998). Coastal sources tended to be

slightly inferior for growth (Meagher and Hunt 1998) and

less hardy (Thomas and Lester 1992) than interior sources

on the interior sites. Some of our more northerly populations

do well for juvenile vigour on our interior sites (Meagher and

Hunt 1998). The Idaho material was more winter-damaged

than BC sources in the trials north of the species range. The

resistance of the Moscow, ID, arboretum material held up

well in BC’s interior but was lower on coastal sites (Hunt and

Meagher 1989; Meagher and Hunt 1999). Thus, the Idaho

material is not recommended for the northern part of the

range nor at the coast but is recommended for the southern

Interior. At 5 years, the Champion Mine (MGR) source from

southern Oregon showed poor vigour as did the more northern Oregon sources (Mt. Hood and Willamette; fig. 3, sources

29 and 30). Based on this it was recommended that the

Columbia River should be a southern transfer boundary and

that sources from Oregon should not be used in BC (Meagher

and Hunt 1998).

Recent, 2001, Reassessment of Coastal

Family/Provenance Trials

In 2001 12-year assessments were made on the two coastal

family/provenance trial sites (Ladysmith and Sechelt, fig. 3,

locations close to sources 24 and 26) for growth and survival.

We present here some preliminary results that allow a

reflection on the above recommendations after rust has

greatly affected these two coastal sites. Details of the Provenance Plantations experiments are provided in Meagher

and Hunt (1998). But briefly this included 12 provenances

with four to five cone-parents per provenance with an additional five more bulked provenances. Figure 3 shows the

Figure 3—Distribution of western white pine (shaded area),

provenance origins (circled numbers) and plantations. Solid

circles indicate plantations using R.T. Bingham (Moscow) arboretum seed source contrasted to local sources. Solid squares

indicate plantations in the root-rot disease experiment sites with

provenances 1 through 14. Open squares indicate plantations

that have some or all of provenances 15 through 35. The

Ladysmith and Sechelt site are these latter open squares near

provenaces 26 and 24, respectively.
16 USDA Forest Service Proceedings RMRS-P-32. 2004

King and Hunt Five Needle Pines in British Columbia, Canada: Past, Present and Future

provenance collections site (numbers 15 through 31) and the

trial locations. Six plantations (3 Coastal and 3 Interior)

were established with 25 replicates per plantation. The

measurements reported here include height, rust and overall survival on two of the Coastal sites (fig. 3, trial sites close

to origin sources, 24 (Sechelt) and 26 (Ladysmith)). In terms

of survival, rust has not been the only mortality agent

although it is by far the most causative agent. Both sites

have been damaged by bough pickers (white pine is highly

desirable for Christmas decorations), but this was not deemed

to hinder our results and interpretations.

Anova models were run on each of these sites for the

following effects: replicates, geographic origin and families

within geographic origin. Means analysis – StudentNewman-Keuls (SNK) were also conducted on the geographic origin groupings (Steel and Torrie 1980). Geographic

origin groups included: Northern Interior BC (Valemont,

Raft River, Barriere and Mt. Revelstoke, fig. 3, sources 15,

16, 17, and 18); Southern Interior BC (Arrow and Trail,

fig. 3, sources 19 and 20); Idaho (bulk unselected collections

not F2, fig. 3, source 21); Vancouver Island (includes low

elevation Sunshine Coast) (fig. 3, sources 22, 24 and 26);

Lower Mainland High Elevation (Cascade) BC (Whistler

and Manning Park, fig. 3, sources 23 and 25); Washington

Olympic Peninsula (fig. 3, source 27); Northern Oregon

Cascade (Mt Hood and Willamette, fig. 3, sources 29 and 30);

Southern Washington Cascade (White River, fig. 3, source

28) and Dorena Oregon – “Champion Mine” (fig. 3, source

31). Also included were some selected seedlots. These include the Westar selections – Southern Interior BC but

selected as clean parent trees; the Dorena “Champion Mine”

MGR selections; and the Porter selections as described

above; and at the Ladysmith site, only exotics (mainly P.

strobus but also P. koraiensis Sieb. and Zucc.).

Results are presented for: the percentage canker-free

stems in 1995 assessment (CF95); the percentage cankerfree stems in 2001 (CF01); mean height and standard

errors (in cm) and finally - percent likely crop tree survivors

(CT) - those trees, both tall and healthy either canker-free or

just minor infections (table 1). Although survival in this last

measure reflects other factors such as vigour (height growth),

frost survival, and other factors, blister rust escape was by

far the major factor.

Ladysmith, although showing the results of blister rust

ahead of Sechelt, has now grown beyond the worst of the

infection, and 30 percent of the original healthy trees are

alive and likely to remain so (some as tall as 12m at age 12).

On this site three sources were significantly less infected

than the rest. One of the exotic species, P. koraiensis, showed

markedly less infections in the 1995 assessment (only 9

percent), but this species quickly fell behind for growth rate

and had faded from the planting by 2001. Although P.

strobus has continued to show good survival, it has also

shown signs of frost damage and poor overall vigour (table 1;

fig. 4). The Dorena seedlot did the best for rust survival (as

expected) but was lower ranked for growth (reflecting the

earlier assessment) (table 1; fig. 4). One of the more impressive lots was the Porter families, which were second only to

the Dorena source for both clean trees in 2001 (CF01) and

potential crop trees (CT). The Porter families were also the

tallest and were significantly different from the Dorena lot

for vigour (height growth) (table 1; fig. 4). This selected lot

screened for early survival, using Riker’s P. strobus protocols, appears to have been effective on a site such as

Ladysmith.

The Vancouver Island, Washington, and northern Oregon

sources performed quite similarly. The Idaho lots as a whole,

as in the earlier analysis, were poor on these coastal sites for

survival (mainly blister rust) but were good for growth;

these, however, were nonselected Idaho material. The Northern Interior BC source and the high elevation Lower Mainland BC were poor for both growth and survival. Although

geographic origins were significant in our model (P<0.0001)

so were families within origin. The Southern Interior BC

origin, which had the largest number of families, showed up

Table 1—Results showing : % clean trees 1995 – CF95, % clean trees 2001 – CF01, mean height and

standard error, and % crop trees (CT), which are defined as those trees that are alive and

healthy (either canker free, branch canker, or tolerant stem reaction) and greater than 3 m at

Ladysmith or greater than 2 m at Sechelt.

Trial Sites

Ladysmith Sechelt

Seedlots CF95 CF01 HT01 ± se CT CF95 CF01 HT01 ± se CT

Overall plantation 56 30 610 ± 4 27 65 20 461 ± 4 18

Northern Interior BC 46 15 576 ± 15 26 62 14 399 ± 12 12

Southern Interior BC 53 25 639 ± 24 36 73 20 467 ± 24 21

Westar S. Interior BC 52 27 627 ± 5 35 66 23 484 ± 7 21

Idaho 51 22 603 ± 12 29 56 9 460 ± 11 9

Coastal BC, High Elevation 44 10 551 ± 22 15 59 10 455 ± 17 8

Vancouver Island BC 46 20 614 ± 15 28 60 16 470 ± 14 17

Porter families BC 68 52 679 ± 13 55 78 25 510 ± 20 27

Olympic Peninsula WA 51 29 619 ± 31 37 70 22 452 ± 24 21

Southern Cascade WA 62 40 605 ± 25 42 72 33 426 ± 23 23

Northern Cascade OR 54 29 585 ± 20 35 64 17 457 ± 17 15

Dorena OR Champion Mine 74 65 573 ± 20 65 84 49 494 ± 17 40

Exotics (P strobus) 73 48 534 ± 15 46
USDA Forest Service Proceedings RMRS-P-32. 2004 17

Five Needle Pines in British Columbia, Canada: Past, Present and Future King and Hunt

well for growth but not survival; however; several families

showed consistently better survival over both sites.

Although the Porter families, screened for phenotypic

survival in ribes gardens (fig. 1), did well on such sites as

Ladysmith, on higher rust hazard sites such as Sechelt

heavy mortality continues (overall plantation infections

going from 35 percent to 80 percent in 5 years; table 1). The

phenotypic survival selection as conducted by Porter is

likely equivalent to the partial resistance screening of the

current programs. This indicates to us that on severe rust

sites, MGR, such as in the Dorena “Champion Mine” source,

will need to be combined with the partial resistances in order

to have any trees survive.

Future _________________________

The future prospect for western white pine against blister

rust is hopeful. Certainly compared to other exotic

pathosystems, such as chestnut blight (1904 introduction)

caused by Cryphonectria parasitic (Murrill) Barr. on American chestnut (Castenea dentata Marsh. Borkh.) or to Dutch

elm disease caused by Ophiostoma ulmi (Buism.) Nannf.

(1920’s introduction) and O. novo-ulmi Brasier (1940’s introduction) on elm species (Ulmus spp.), there does appear to be

a reasonable degree of native resistance. Confirmation of

this resistance from the inoculations to field trials is under

way through a series of excess stock trials (Hunt 2002).

Investigation of these trials together with continued measurements of the provenance trials will help us to establish

deployment guidelines for the orchard seed which will soon

be available.

Deployment Potential for Western White

Pine in BC

Orchards in the interior BC have a predominant element

of material from the Idaho program. The emphasis here will

be to incorporate our own selections and compare them to

Idaho material.

On the coast, three seed orchards will soon be producing

seed. Earlier use of seedling progeny for orchard establishment has now given way to the use of selected parents based

on results of the inoculation of their progeny. New material,

primarily selections from heavily infected trials, is also

being added. All of these selections fall under the general

categories of “partial resistance” or “tolerance”. In addition

to this, we have been encouraged to use “total resistance”

pollen based on the performance of “Champion Mine” and

“Champion Mine” pollinated seedlots in the “root disease”

trials (Hunt 1987, Hunt these proceedings) and shown here

(table 1; fig. 4). These seedlots (Dorena in table 1) have the

Cr2 gene which conditions a hypersensitive response (HR)

in western white pine. The strategy of pyramiding HR can

be implemented in seed orchards by either supplemental

mass pollination or mass control pollination. Both of these

methods have found practical use in BC (Webber 1995).

While Cr2 is a powerful form of resistance (the Dorena

seedlot, table 1), a pathotype of rust that overcomes it does

exist (vcr2), and Cr2 cannot be seen as an ultimate solution

(Kinloch and others 2003). Although there are few examples

of “total” or “vertical resistance” pathosystems being durable (Leach and others 2001), a completely durable resistance may not be required. Because most cankering occurs

close to the ground in BC (Hunt 1991), resistance may

therefore only be needed during the plantation’s early years.

How fast and far vcr2 will spread and its durability are the

more relevant questions. If vcr2 becomes widely distributed,

it would negate any further planting of single gene resistance solely based on Cr2. Investigations of Cr2 material in

the BC root disease trials have failed to show any virulent

pathotypes up to 15 years (Hunt and others these proceedings), and in a Bear Pass, OR, plantation some resistant Cr2

trees are still canker-free after more than 60 years (Sniezko,

pers. comm). However, the observation of vcr2, the virulent

strain, in a relatively small population (hence small selec0

10

20

30

40

50

60

70

80

Vancouver Island Southern Interior BC Idaho P. strobus Dorena OR Porter selections

Survival at Sechelt (%)

Survival at Ladysmith (%)

Height at Ladysmith (dm)

Figure 4—Height (dm) at

Ladysmith plantation and Survival (%) at Sechelt and

Ladysmith plantations for five

P. monticola sources and the

P. strobus source in 2001 at

age 12. (Survival refers to crop

tree survival as discussed in

the text.)
18 USDA Forest Service Proceedings RMRS-P-32. 2004

King and Hunt Five Needle Pines in British Columbia, Canada: Past, Present and Future

tion pressure) of P. monticola with Cr2 at the Happy Camp

field station in northern California (Sniezko and others

these proceedings) is most certainly disturbing. Some encouragement for using the strategy of pyramiding HR has

come from observations made in the long-term deployment

and monitoring of sugar pine with the Cr1 hypersensitive

response gene. As in western white pine, a pathotype of

blister rust virulent to Cr1 exists (Kinloch and Comstock

1980). Data indicate that the virulent strain of the rust

(vcr1) does not always arise quickly or spread rapidly (Kinloch

and Dupper 1998). This has encouraged us to develop a

deployment strategy that attempts to manage the Cr2/vcr2

pathosystem by integrating it into a silvicultural option that

would incorporate hazard assessment area to be planted and

distance from other plantations.

Future Research Directions

Further investigation of the Cr2 gene and its potential

durability is needed. This will include: careful investigation

of all plots in which it has been deployed in BC, follow up of

the material that the Dorena program has deployed, and

continued interaction with the Region 5 sugar pine program

which has provided a model for this deployment. Besides

Cr2, other “total resistant” genes may exist and be made

available. The Dorena Genetic Resource Center is investigating other potential dominant gene resistances in western

white pine (Sniezko pers. comm). Although P. monticola and

P. lambertiana do not naturally hybridize (Bingham 1972),

there are now in vitro fertilization methods (Fernando and

others 1997) which may permit such a cross, and thus add

Cr1 as a resistance gene in P. monticola. The multiplicity of

these “total resistance” genes should add to their durability

and strategies to use multiple “total resistance” genes need

developing.

The pyramiding of several race-specific resistances into a

single plant genotype theoretically has the ability to greatly

reduce the probability of a mutation to multiple virulence

(Wheeler and Diachun 1983). However, this assumes that

the mutations to virulence are independent of each other.

Empirical evidence from crop literature, however, points to

the fact that there is no clear association between the

number of resistance genes in cultivars and their durability

(Mundt 1990). Some single resistances have proven highly

durable while others have been highly ephemeral, and

combinations are not necessarily more durable unless specific resistances are included (Johnson 2000). It has been

hypothesized that the quality and durability of a plant

resistance gene is a function of the fitness penalty of virulence. Even where genes fail, they may be beneficial through

a residual effect because they may add a cost to the pathogen

of not having the avirulence (Leach and others 2001). The

advent of molecular genetics technology to investigate gene

function has offered some insights into the potential relationships between viurlence/avirulence and durability. Although avirulence can confer a high degree of fitness in some

cases (resulting in durability of HR), in others this does not

appear to be so, and these relationships can be complex

(Leach and others 2001). Bacterial blight resistance in rice

has shown such a positive functional relationship between

the avirulence gene in the pathogen and fitness through its

aggressiveness (rate a virulent isolate produces an amount

of disease) (Vera Cruz and others 2000). The study of gene

function and the protein – ligand relationships between

resistance, virulence/avirulence in HR pathosystems in the

white pines are being investigated by the CFS

(Ekramodddoullah and Tan 1998, Yu and others 2002) and

may lead to some insights and potential indicators of

durability.

HR total resistance is only one component of our resistant

breeding program. We will continue to rely on partial resistances and tolerances for the major part of our effort, and the

breeding program is directed to families and individuals

selected for this type of resistance. By using a structured

mating design and cloning of individuals we can begin to

construct pedigreed lines to more carefully observe the

partial resistances and be in a position to start to understand

some of the underlying genetics.

Another part of the investigation of resistance will be the

observation of blister rust as an endemic pathosystem with

Asian white pines. Some early work with Asian hybrids was

conducted by Heimberger in Ontario, and some of this

material may still be available (G. Daoust pers. comm).

Unlike the other two exotic pathosystems mentioned earlier

(chestnut blight and Dutch elm disease), we are not obliged

to use species hybrids and backcrossing to save our native

gene pool as there does appear to be ample resistance in our

native populations. However, the observation of the endemic

pathosystem in Asian species and their hybrids with North

American white pines should help us greatly in understanding resistance and identifying which resistances are likely to

be the most durable.

Biotechnology can help in our efforts. This will include invitro fertilization (Fernando and others 1997) to help in

hybrid crosses; embryogenesis to clone lines for the pedigreed breeding program (some successful lines have already

been produced); molecular biology to detect the protein

precursors of HR; and molecular genetic techniques to help

in understanding the genetic basis of resistance. A lot of

classical breeding and pathological research will need to be

continued to realize this effort.

Although a lot of effort has been spent on western white

pine to the point where we can start to see the results and

envision its return as an important species to our landscape

(Fins and others 2001), other species are still in danger.

Whitebark pine, P. albicaulus, is considered an endangered

species in BC and the U.S. Pacific Northwest (Krakowski

2001, Mahalovich, these proceedings). To this end we have

initiated a large-scale seed collection. This is both to preserve important gene pools that are under threat and to start

some initial screening in this species. The successes we have

had to date should encourage us to keep up the effort in

reestablishing these species and continue the co-operative

atmosphere of this effort throughout the regions where the

white pines grow.

References _____________________

Bingham, R.T. 1972. Taxonmy, crossability, and relative blister

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Misc. Publ. No. 1221, pp 271-280.

Bingham, R.T. 1983. Blister rust resistant western white pine for

the Inland Empire: USDA For. Serv. Gen. Tech. Rept. INT-146.
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Bower, R.C. 1987. Early comparison of an Idaho and a coastal source

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Childs, T.W. and J.W. Kimmey. 1938. Studies on probable damage

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Eastham, J.W. 1922. Report of provincial plant pathologist,

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Ekramodddoullah, A.K.M and Y. Tan. 1998. Differential accumulation of proteins in resistant and susceptible sugar pine inoculated

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Estey, R.H. 1994. Essays on the Early History of Plant Pathology

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Fernado, D.D., J.N. Owens, P. Von Aderkas, and T. Takaso. 1997. In

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McDonald, D. Miller, J. Schwandt, and A. Zack. 2001. Return of

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Gussow, H.T. 1923. Report of the dominion botanist for the year

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Hoff, R.J. 1988. Blister rust resistance in western white pine for

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Hunt, R.S. 1987. Is there a biological risk of western white pine

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removal of lower branches. For. Chron. 67:284-287.

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Hunt, R.S. 1997. Relative value of slow-canker growth and bark

reactions as resistance responses to white pine blister rust. Can.

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Hunt, R.S. 2002. Relationship between early family-selection traits

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Hunt, R.S. and M.D. Meagher.1989. Incidence of blister rust on

“resistant” white pine (Pinus monticola and P. strobus) in coastal

British Columbia plantations. Can. J. Plant Pathol. 11: 419-423.

Hunt, R.S., G.D. Jensen, A.K. Ekramoddoullah, and E.E. White.

1998. Western white pine improvement program for British

Columbia. Can. Tree Improv. Assoc. 26(1): 157-159.

Johnson, R. 2000. Classical plant breeding for durable resistance to

diseases. Jour. Plant Path. 82: 3-7.

Kinloch , B.B. Jr., G.K. Parks and C.W. Fowler. 1970. White pine

blister rust: Simply inherited resistance in sugar pine. Science

167:193-195.

Kinloch, B.B. Jr. and M. Comstock. 1980. Cotyledon test for major

gene resistance. Can. J. Bot 58:1912-1914.

Kinloch, B.B. and G.E. Dupper. 1998. Evidence of cytoplasmic

inheritance of virulence in Cronartium ribicola to major gene

resistance in sugar pine. Phytopathology 89:192-196.

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1999. A major gene for resistance to white pine blister rust in

western white pine from the western Cascade Range. Phytopathology 89: 861-867.

Kinloch, B.B., R.A. Sniezko, and G.E. Dupper. 2003. Origin and

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691-694.

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provenance and family variation in cold hardiness of Pinus

monticola. Can. J. For. Res. 22: 1917-1921.

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and J.E. Leach. 2000. Predicting durability of a disease resistance gene based on an assessment of the fitness loss and

epidemiological consequences of avirulence gene mutation. PNAS

97: 13500-13505.

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20 USDA Forest Service Proceedings RMRS-P-32. 2004

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

The author is with Ohio State University (Emeritus), 625 Medford Leas,

Medford, NJ 08055 USA. Tel.: 1-609-654-3625 or Fax: 1-609-654-3625 or Email: kriebel@medleas.com.

Abstract—Research and breeding of five-needle pines in the eastern USA has been concerned mainly with eastern white pine (Pinus

strobus L.), which has been found to be a highly variable species.

Principal attention has been given to the inheritance of growth

traits within and among stands and among provenances. Growth of

trees of Tennessee, North Carolina and Georgia provenances exceeds that of other origins in many areas of the US and even in other

countries. Research basic to breeding indicates that the genetic

barrier to species crossability in the soft pines is embryo failure,

whereas in hard pines it is pollen tube incompatibility. Patterns of

premature cone drop in relation to genetic affinity have also been

determined; cone retention is not closely related to capacity to

produce viable seed. The expression of inbreeding after selfing in

young plantations of P. strobus varies with the parents and the site,

but the species is more tolerant of inbreeding than most other

species of pines. Eastern white pine population studies show that

genetic variability may be maintained even in small isolated stands

and in the next generation after heavy thinning, although gene pool

deterioration may follow cutting if silviculture disregards gene pool

conservation. A genetic gain in volume of about 22 percent has been

obtained in P. strobus from age 13 family selections in first-generation progeny tests. Potentially useful species crosses are eastern

white pine x Himalayan pine and eastern white pine x western

white pine. The best hybrid families of the eastern white pine x

Himalayan cross have been found to exceed P. strobus in volume by

22 to 44 percent at ages 17-22 in progeny tests. These two hybrids

also exceed P. strobus in wood specific gravity. Selection and

breeding of P. strobus for blister rust resistance has been difficult

and has not yet yielded commercially useful resistant genotypes,

nor has selection and breeding for white pine weevil resistance been

successful. However, new approaches may overcome these problems. Air pollution tolerance varies widely in P. strobus; natural

selection in native stands has yielded highly tolerant progenies

while eliminating the most sensitive genotypes from the gene pool.

Future research directions are suggested.

Key words: Five-needle pines, eastern white pine, breeding, species incompatibility, genetic variability, growth rate,

species hybrids, resistance, white pine weevil, white

pine blister rust, air pollution tolerance.

Genetics and Breeding of Five-Needle Pines

in the Eastern United States

Howard B. Kriebel

Introduction ____________________

Research on the genetics and breeding of five-needle pines

in the eastern USA began about 1950 with species hybridization experiments and early tests for resistance to the white

pine weevil (Pissodes strobi Peck) and the white pine blister

rust (Cronartium ribicola J.C. Fischer). During the subsequent 30-year period, eastern white pine (Pinus strobus L.)

was probably the subject of more extensive cooperative tree

improvement research over the eastern half of the USA and

eastern Canada than was any other forest tree species, with

the exception of the southern pines. The greatest effort was

applied to the study of geographic variation over the entire

species distribution by replicated provenance tests, first

using seed collections made at the range-wide level, then

concentrating on the southern Appalachian part of the

species range. The results of this work by numerous state

university research stations, the USDA Forest Service and

Canadian research centers were combined in joint publications issued at various ages. They provide a very useful

knowledge base, especially on adaptability and growth traits

as related to seed origin interacting with plantation location.

These results have now been put into practice in the commercial planting of eastern white pine.

Studies also provided some measure of stand-to-stand and

within-stand variation. Controlled breeding research, although not as extensive as the common-garden testing of

open–pollinated seed, has provided estimates of the inheritance of growth rate and the potential for genetic gain from

half-sib and full-sib families. Efforts to develop weevil and

blister rust resistant eastern white pine have, on the other

hand, not yet been successful, although work continues.

Research on other species of five-needle pines has been

primarily limited to investigations of the potential usefulness of some of the more adaptable species and their hybrids

as a source of resistance to the weevil and blister rust. A later

research effort was the study of the genetic aspects of air

pollution tolerance in P. strobus, which was, prior to natural

selection for tolerance, highly sensitive to sulfur dioxide and

ozone, both major pollutants over much of the natural range

of the species.

Applied genetic research directed toward improved planting stock of five-needle pines has declined in the eastern

USA during the last 20 years as part of the general curtailment of public funding for tree improvement research and

low level of support from other sources. Thus available new

genetic information on five-needle pines published during

the last two decades is mainly concerned with 1) biochemical and molecular analysis of variability in natural stands,

and 2) continuing efforts to develop rust-resistant strains

of eastern white pine and to apply molecular technology to

the weevil resistance problem. This paper covers principal

results of fivc-needle pine breeding and improvement
USDA Forest Service Proceedings RMRS-P-32. 2004 21

Genetics and Breeding of Five-Needle Pines in the Eastern USA Kriebel

research in the USA; results of Canadian studies of stand

and provenance variation in field trials and the long-term

Canadian efforts to develop blister-rust resistant eastern

white pine are reported in the paper by Daoust and

Beaulieu in these proceedings.

Variability in Eastern White Pine ___

Geographic Variation in Growth Rate in

Test Plantations

Extensive range-wide provenance testing has shown that

P. strobus is a highly variable species, as compared, for

example, with the more range-restricted species Pinus

resinosa Ait. (Fowler 1965). Of particular importance for

tree improvement, eastern white pine includes a wide range

of genotypes with respect to rate of growth in height, diameter and stem volume, while at the same time retaining the

capacity for adaptation to a broad range of environmental

conditions. These conclusions are based on statistical analysis of the performance of trees in widely replicated rangewide seed source trials (Wright 1970). In trials at midlatitudes within the species distribution (Pennsylvania,

Maryland, Tennessee, Indiana, Ohio, southern Michigan,

Illinois, West Virginia, Nebraska), genotypes of western

North Carolina, Tennessee and northern Georgia origin

outgrew those of all other provenances by 70 to 80 percent in

tree height growth rate by the end of the first decade. The

same growth pattern was found in trials in Australia and

New Zealand. After the first decade, absolute differences

between fast-growing southern and slow-growing northern

seedlots increased but relative differences decreased. Diameter growth differences persisted, sustaining large differences in volume during the second decade (Funk 1979,

Kriebel 1982). Later trials concentrating on southern Appalachian seed origins confirmed the growth superiority, in all

tests, of seed sources in North Carolina, Tennessee and

Georgia over those from Virginia, West Virginia and Maryland. The fastest-growing seedlots commonly grew 40 percent faster than the slowest ones, translating into a 2 to 1

superiority in rate of volume growth (Wright and others

1979).

Population Structure

Progeny tests have shown that family differences in height

growth rate are commonly large within stands of eastern

white pine, the variance component for general combining

ability on a family mean basis averaging over several trials

about 25 percent of the mean variance over all plots (De

Vecchi Pellati 1967, Wright 1970, Kriebel 1978, Thor and

Gall 1978). This evidence of high genetic variability is

supported by more recent studies of gene diversity in natural

stands before and after thinning, facilitated by new technology, using isozyme and DNA analysis. In Wisconsin, using

simple sequence repeated satellite DNA markers, Echt

(2000) found that genetic diversity in eastern white pine was

the same in both 160-year-old trees and their natural regeneration after a shelterwood thinning that removed most

trees. Likewise, in Newfoundland, Rajora and others (1998)

were unable to show that genetic diversity had declined in

P. strobus populations, even after a century of decline in

population size. Allelic diversity in heavily thinned stands

was as high as it was in unthinned stands. In fact, the

Newfoundland populations were as genetically variable as

those from Ontario, near the center of the species range. On

the other hand, isozyme and microsatellite DNA analyses of

diversity in two old-growth Ontario stands showed that

harvesting caused a loss in genetic diversity (Buchert and

others 1997, Rajora and others 2000). Differences in silvicultural treatment of the old-growth stands may explain the

differences between the Wisconsin and Ontario results; the

forestry program on the Menominee Indian Reservation in

Wisconsin area is considered by many to be the finest

example of sustainable management of eastern white pine

on the continent (Echt 2000).

Genetics of Outbreeding and

Inbreeding _____________________

Genetic Control over Embryo Formation

and Cone Set

High yields of viable seed are usually obtained in eastern

white pine from both open and controlled crossing with other

individuals of the species. Hybrids can be obtained from

crosses with several other species of five-needle pines with

varying success in terms of seed yield (Wright 1953). Reported histogenetic and serological studies of fertilization

and embryogenesis in “soft” pines (Subgenus Strobus, Section Strobus) have shown that fertilization and early embryogenesis can occur normally in ovules of non-crossable

species combinations, i.e. those crosses never reported to

yield viable seed, with subsequent post-fertilization breakdown from embryo inviability. In fact, early development of

the true embryo has been observed in some of these crosses

that do not yield viable seed (Ueda and others 1961, Hagman

and Mikkola 1963, Hagman 1967, Kriebel 1972, 1981). In

contrast, incomplete pollen tube penetration and failure of

ovule fertilization have been the pattern in similar studies

of hard pines (McWilliam 1959, Hashizume and Kondo

1962a, 1962b, Chira and Berta 1965). The evidence from

these painstaking studies, therefore, usually based on daily

or-near daily ovule collections and sectioning over a period

of several weeks and some including more than one year’s

data, suggests a fundamental difference in species isolation

mechanisms between the soft and hard pines, the soft pines

being characterized by embryo inviability and the hard

pines by pollen tube incompatibility.

Soft pines and hard pines also differ in the way in which

interspecific hybridization affects cone retention. In soft

pines, non-crossable combinations consistently retain firstyear cones, whereas in hard pines they often do not. It is

possible, for example, to cross P. strobus with P. koraiensis

pollen without first-year cone abscission. Table 1 shows

the decrease in maternal control over cone abscission as

genetic divergence increases between parent species (left

column), for both pine subgenera. Subgenus Strobus seed

maturation is characteristically blocked by embryo inviability and subgenus Pinus by pollen incompatibility. In

comparisons of the effect of female parent, pollen species

and pollination year on premature cone drop in P. strobus,
22 USDA Forest Service Proceedings RMRS-P-32. 2004

Kriebel Genetics and Breeding of Five-Needle Pines in the Eastern USA

extensive histological analysis of developing ovules showed

that female parent exercised overriding genetic control in

crosses within and between species,. Pollen species had no

effect on cone abscission, even in the case of a totally noncrossable species combination, nor did year of pollination.

The year effect could be significant if, for example, prolonged

heavy rain seriously reduced the pollen supply (Kriebel 1981).

Practical Significance of Inbreeding in P.

strobus

P. strobus is more tolerant to inbreeding than most other

species of pines (Fowler 1965). Inbreeding is found in both

unmanaged and managed populations, and appears to be a

natural characteristic of the species (Echt 2000). Among

randomly-selected trees crossed in a small isolated Ohio

stand, six of 8 trees manually self-pollinated had no reduction in seed yield; yield of the other two averaged 22 percent

of outcross families. Reduction in growth rate in 5-10-year

old offspring resulting from controlled self-pollination was

variable compared to outcross progenies, averaging 18 percent on a good site and 38 percent on a poor site. Both openand control-pollinated progenies from this stand were as

vigorous as those from larger stands of comparable structure. (Kriebel 1975, 1982).

Growth Improvement in Eastern

White Pine _____________________

Heritability of Growth Rate

Estimates of narrow sense heritability of height growth

have been calculated from several progeny tests of eastern

white pine, and from these estimates, genetic gain has been

estimated using fixed assumptions. The heritability estimates have been fairly consistent when based either on

individual tree means or on family means. Table 2 summarizes early estimates for height growth from open-pollinated

(“half-sib”) and control-pollinated (full-sib) progeny tests,

and a later estimate of genetic gain in volume at age 13 in

Ohio. Height estimates based on individual trees are in

general agreement for both half-sibs and full-sibs, as are

those with a family mean basis (Adams and Joly 1977,

Kriebel and others 1972, Kriebel and others 1974, Thor and

Gall 1978).

Keathley (1977) estimated the heritability of volume growth

in an Ohio white pine half sib progeny test at age 13 as h2

(plot mean basis) = 0.45 ± 0.02. From this estimate, using a

selection differential of 1.3s and assuming family means to

be normally distributed around the plantation mean, estimated genetic gain in volume was about 22 percent (Table 3).

Table 1—Species isolation mechanism and extent of maternal control over cone

abscission in relation to degree of outcrossing in soft and hard pine

subgenera (Kriebel 1976b).

Subgenus Strobus Subgenus Pinus

Isolation mechanism fi Embryo inviability Pollen incompatibility

Type of cross Degree of maternal controlb

Intraspecific, outcross Strong Strong

Interspecific, viable seed Strong Strong

Interspecific, no viable seed Strong Usually weak

Intraspecific, limited pollen c Threshhold Threshhold

Unpollinated Almost nil Almost nil

The other subgenus Nil Nil

Another conifer genus Nil Nil

a

Summary constructed from research papers cited in Kriebel (1981), including chronological histogenetic studies of fertilization and embryogenesis, and breeding experiments. b

Strong = high level of cone retention.

c

Different species seem to have different threshold values for the number of collapsed

(unpollinated) ovules per cone above which abscission occurs.

Table 2—Estimates of heritability of tree height and stem volume in

eastern white pine (Keathley 1977).

Parameters for estimates TNa OHb NHc

Height, half-sib, individual tree 0.27 0.28 0.33

Height, half-sib, family mean — 0.59 0.59

Height, full-sib, individual tree — 0.32 —

Height, full-sib, family mean — 0.54 —

Volume, half-sib, family mean — 0.45 —

a

Age 7, Tennessee, Thor and Gall 1978

b

Age 13, Ohio, Kriebel 1978

c

Age 3. New Hampshire, Adams and Joly 1977
USDA Forest Service Proceedings RMRS-P-32. 2004 23

Genetics and Breeding of Five-Needle Pines in the Eastern USA Kriebel

Actually, thinning was not made until age 18, since selection

was a 2-stage process, combining a low-level individual tree

selection within family plots with a subsequent high-level

family selection. The realized gain in volume at age 18, based

on plot means, was about 40 percent (Kriebel 1983).

Species Trials and Species Hybridization

In addition to arboretum plantings of many of the fiveneedle pines, a number of species have been tested in the

eastern United States for their possible economic value as

commercial plantation trees. The two species of most interest have been the Himalayan pine (P. wallichiana A. B.

Jacks., syn. P. griffithii McClel.), and western white pine (P.

monticola Dougl. ex D. Don). Himalayan pine, or blue pine,

as it is known in its native region, has been tested on the

provenance level, including some families within provenances. It has fast growth and variable cold hardiness,

depending on the seed source and planting region. Although

it is cold-hardy in parts of the mid-Atlantic coastal plain, in

Ohio it suffers from late spring frost damage to new shoots,

resulting in deformity with multiple branching. Seed sources

in the eastern Himalayas (Nepal) do not survive winters in

this region (Kriebel 1976a, Kriebel and Dogra 1986). Tests

were made in Tennessee of some of the same provenances

tested in Ohio. Survival and height growth were probably

affected by a severe drought rather than cold winter temperatures. Families that had a high percentage of survival

and good growth at each plantation came from a wide

geographic spectrum (Schlarbaum and Cox 1990). Himalayan pine is generally susceptible to the white pine weevil

(Heimburger and Sullivan 1972) and variable in resistance

to white pine blister rust (Bingham 1972).

Western white pine has the necessary cold hardiness in

the eastern US and Canada, where it has a variable growth

rate and good form. It appears to suffer less weevil damage

than does eastern white pine, and this is probably its

principal value for planting in the these regions (Heimburger

and Sullivan 1972). It does not seems to be well adapted to

the warmer, drier interior part of the eastern USA within

the native range of P. strobus and is not recommended for

Ohio (Kriebel 1982).

Macedonian white pine (P. peuce Griseb.) has been reported to have some resistance to the white pine weevil. It

has good form but it has a slower growth rate in the eastern

US than eastern white pine (Wright and Gabriel 1959).

Species Hybrids

At least seven F1 hybrids and some of their reciprocals

frequently outgrow either parent during the first decade

(Wright 1959). The two of these hybrids that may have value

for forest planting are eastern white pine x Himalayan pine

and the reciprocal cross, and eastern white pine x western

white pine and its reciprocal (Kriebel 1972). The first of

these hybrids has had excellent growth and survival in Ohio

(Kriebel 1982). In a 43-family full-sib progeny test, the two

eastern white x Himalayan pine families were in the top 10

in volume growth at age 13, and one of these families

outranked all others in the plantation. Hybrids with western

white pine were more variable in volume growth; among the

43 families, two of the five eastern x western white pine

families ranked third and sixth. When wood specific gravity

of the hybrids P. strobus x wallichiana and P. strobus x

monticola was measured in the same test and compared

with that of eastern white pine, both hybrids clearly outranked P. strobus (Table 4). For the 152 trees tested for

specific gravity, the results were:

P. strobus x strobus, 120 trees, mean wood specific gravity =

0.266

P. strobus x wallichiana, 15 trees, mean wood specific

gravity = 0.295

P. strobus x monticola, 17 trees, mean wood specific

gravity = 0.312

Since eastern white pine wood fiber is now in use at some

paper mills, there could be a two-way gain from the use of

these two hybrids in plantations, both from wood volume

and wood specific gravity (Whitmore, F.W. and Kriebel,

H.B., unpublished data).

Weevil Resistance Breeding ______

Repeated destruction of terminal shoots by the weevil

larvae does not affect the health of white pines but it

Table 3—Estimated genetic gain in tree volume after thinning in a

half-sib progeny test of eastern white pine (Kriebel 1978).

Parameter Statistic

Initial spacing 1 m (within rows) x 2 m

Trees per linear plot 4

Plots per family 12

50% thinning to 2 best trees/plot Age 13 (to 2500 trees/ha)

h2 for volume (plot mean basis) 0.45 ± 0.02

Mean 2-tree plot volume 0.021 m3

Standard deviation (s) 0.008 m3

Selection intensity 6 families out of 48

Final stocking 313 trees/ha

Selection differential 1.3s

Genetic gain (DG) 0.223

Realized gain, thinning at age 18 0.40
24 USDA Forest Service Proceedings RMRS-P-32. 2004

Kriebel Genetics and Breeding of Five-Needle Pines in the Eastern USA

destroys their commercial value by causing permanent deformities in the main stem. Although significant progress

has been made in breeding for superior growth rate in

eastern white pine and hybrids, genetic improvement in

resistance to the white pine weevil and white pine blister

rust has so far been unattainable in eastern North America.

In geographic seed source trials, weevil damage was usually

heavy in trees from all sources. Although seed sources varied

in the degree of weevil injury, there was no total resistance,

i.e. complete absence of weevil attack, in trees of any origin

(Garrett 1972). However, since a few geographic provenances

have been identified that have low susceptibility to repeated

weevil attack, selection within stands based on relative

degree of susceptibility in provenance tests may be useful as

a first step in a selection program (Genys 1981, Wilkinson

1981). Noncrystallization of cortical oleoresins, earlier

thought to be a resistance factor (Santamour 1965, Van

Buijtenen and Santamour 1972) was subsequently found to

be unrelated to weevil susceptibility (Bridgen and others

1979, Wilkinson 1979a, 1979b). Later work indicated that

the concentrations of two monoterpenes may be useful as

selection criteria for reducing susceptibility to weevil attack

(Wilkinson 1980).

Two other species of five-needle pines have been considered in breeding for resistance to the white pine weevil. One

is P. peuce, which has variable resistance (Zsuffa 1979).

Although it has a moderate growth rate, its weevil resistance and crossability with P. strobus suggests its use as the

male parent in hybrids for the introduction of resistance into

eastern white pine. The hybrids could then be backcrossed to

P. strobus to improve the growth rate. The long-term nature

of this option, with low yields of hybrid seed, makes it

impractical. The second species is P. monticola, and in this

case instead of hybridization the species would be used

directly, since P. monticola appears to be more weevilresistant than P. strobus (Heimburger 1972). But western

white pine varies in vigor and adaptability in the eastern

United States, necessitating local progeny testing of trees

screened for weevil resistance. Since 1980, no studies have

been undertaken in the eastern USA to explore the feasibility of using hybrids or alternate species for weevil resistance. For a more detailed historical review of weevil resistance breeding, see Kriebel (1982).

Using Silviculture as an Alternative to

Breeding

An alternative strategy to minimize weevil damage is

particularly suitable for the northeastern and northern

Lake States (Michigan, Wisconsin, Minnesota). It integrates

genetics with silviculture by the planting of fast-growing

selections of eastern white pine in a mixture or under an

associated pioneer species such as aspen or birch, to provide

partial shade for weevil protection. There are two possibilities. One is to allow the pine to overtop the short-lived aspen

or birch, commonly in 20 years or less. The other is to remove

the aspen or birch, if economically feasible, after the pine has

developed one clear log that is free of weevil damage or

nearly so. This is based on the weevil preference for trees

growing in full sun over trees under shade or partial shade

(MacAloney 1952, Ledig and Smith 1981).

Breeding For Rust Resistance _____

In the eastern USA, the white pine blister rust is a serious

problem primarily in cool, humid regions, especially in the

northern Lake States and the northeastern states. The rust

is ubiquitous under these conditions and there are no reported unique populations that are threatened. The rust is

not a problem in warm and dry localities within these

regions and in low-elevation stands in the southern and

southwestern parts of the range of P. strobus. Resistance

appears to be polygenic in nature, thus requiring several

generations of breeding to build up a practical level of

resistance in offspring (Heimburger 1972). Artificial inoculation of eastern white pine alone does not accurately

identify breeding stock suitable for field planting, since

Table 4—Relative stem volume and wood specific gravity of the hybrids P. strobus x P. wallichiana and P. strobus x P. monticola , compared with

P. strobus; top 10 of 43 families in a full-sib progeny test at age 13 ranked by volume (Kriebel and Whitmore, unpublished, Kriebel 1982).

Hybrid family Crossa ➁ x ❹ Relative stem vol.b Rankc Relative specif. gr. Rank, sp. gr.d Rank, sp. gr. x vol.e

1430 st x wa 1278 x 1213 202 1 110 4 1

1375 st x st 1130 x 1279 171 2 100 4 4

1428 st x mo 1278 x 635 166 3 115 2 2

1408 st x st 1276 x 1277 155 4 93 8 6

1420 st x st 1278 x 1275 148 5 98 5 7

1429 st x mo 1287 x 645 143 6 124 1 3

1424 st x st 1278 x 1280 139 7 97 6 9

1393 st x wa 1275 x 1213 139 8 112 3 5

1448 st x st 1280 x 1279 139 9 94 710

1373 st x st 1130 x 1277 138 10 98 5 8

a

st = strobus, wa = wallichiana, mo = monticola. b

Mean stem volume in m3 at age 13 from seed, as a percentage of the mean of all 43 family means in the progeny test (0.0337 m3 per tree). The experiment included

33 full-sib P.strobus families, 5 P. strobus x monticola families, 2 P.strobus x wallichiana families, and 2 P. strobus x peuce families. c

Rank in volume among the 43 families in the progeny test.

d

Mean specific gravity of the stem wood at age 13 from seed, expressed as a percentage of the mean of all 43 familiy means (266 kg/m3). e

Rank in family mean specific gravity x mean volume.
USDA Forest Service Proceedings RMRS-P-32. 2004 25

Genetics and Breeding of Five-Needle Pines in the Eastern USA Kriebel

progenies succumbing to high concentrations of inoculum

may be tolerant to the levels to which they are actually

exposed in the field. Blister rust hazard is not high in all

regions of the northeastern US and Canada. In warm zones

and in cool zones with large openings to the sky, resistant

strains are not essential (Van Arsdel 1972, Zsuffa 1979).

Interspecific hybridization with other more rust-resistant

species of five-needle pines has so far not been an effective

breeding strategy (Zsuffa 1981).

Given these constraints on breeding eastern white pine for

resistance to the white pine blister rust, new technology is

now being applied to the problem by the US Forest Service

in forest genetics and forest pathology projects in the North

Central Region. Objectives are: (1) to develop a better understanding of the genetic structure of tree and pathogen

populations, using molecular markers; (2) to identify genes

for important quantitative traits; (3) to develop

micropropagation techniques for production of elite trees;

and (4) to develop gene transfer technologies for introduction of novel genetic constructs. Plantations of self- and

reciprocal-crosses of eastern white pine progenies are being

established for studying the genetics of blister rust resistance. The blister rust program has the objectives of determining rust variation in the Lake States and comparing it

with rust variation in the rest of North America, determining whether resistance can be reliably identified in inoculated seedlings at an early age and whether there is a racespecific component in seedling resistance. Although current

work involves inoculation of seedlings from clonal parents,

diallel crosses and selfs among selected parents have been

made for future studies. Sugar pines (Pinus lambertiana

Dougl.) with and without the MGR gene (Kinloch 2001) have

also been included in each inoculation for detection of one

form of race-specific virulence. Michler and Pijut have developed improved micropropagation technologies for eastern

white pine, and Michler and Davis have isolated eastern

white pine chitinase genes with the goal of developing

efficient genetic engineering protocols for pines using constructs with pine promoters. Eastern white pine seedlings

are currently being tested in greenhouse studies to determine stability of transgene insertion (Michler and Zambino,

personal communications).

Integrated Management for Rust and

Weevil Control

The feasibility of applying a silvicultural regime integrating management for growth, blister rust and weevil resistance is currently being studied in northern Wisconsin

(Ostry 2000). Variables in the study include survival, height,

rust infection and weeviling. Treatments include clearcut vs

shelterwood, nonselected stock vs selected stock, and no

pruning vs pruning. Age 10 analysis, although preliminary,

showed that:

1. Height growth was greater in clearcut than in

shelterwood plots.

2. Weevil attack was greater in clearcut than in

shelterwood plots.

3. Blister rust infection was higher in shelterwood than in

clearcut plots.

4. Selected planting stock outgrew nonselected stock.

5. Blister rust infection was higher in nonselected than

selected stock.

6. Blister rust infection was higher in unpruned than in

pruned trees.

Pruning of lower branches had double benefits, both in

correcting stem form for weevil damage, and also by removal

of the branches most susceptible to rust infection.

Genetics of Air Pollution

Tolerance ______________________

Because of inherent sensitivity and geographical distribution in relation to industry, population centers and prevailing winds, eastern white pine has probably been more

impacted by air pollution than any other tree species in

eastern North America (Gerhold 1977). Karnosky and Houston (1979) reviewed the genetics of air pollution tolerance of

eastern white pine in the northeastern US. White pines are

sensitive to both SO2 and O3, but the interaction of these

pollutants has more serious effects than either pollutant

alone (Houston 1974). There is significant tree-to-tree variation that has a strong genetic component (Houston and

Stairs 1973). The effect of this large genetic component has

been natural selection against sensitive trees in native

stands over a wide region of eastern North America, with

losses to the gene pool that may have included linked genes

of unknown biological or commercial value. It was estimated

that in northern Ohio stands of P. strobus, more than 40

percent of the potential seed-bearing trees had dropped out

of the breeding population. Only pollution-tolerant trees

survive to produce seed, and progenies from stands in

polluted regions were found to be almost totally free of air

pollution injury while retaining the vigor of the parent

stands (Kriebel and Leben 1981). Similar evidence of the

inheritance of pollution tolerance was obtained from the

progenies of healthy trees growing in Tennessee for many

years under polluted conditions. The progenies had darker

green needles than progenies from other southern Appalachian stands and were consistently faster-growing than

others in three polluted areas in Tennessee and in Ohio

(Thor and Gall 1978, Kriebel 1982).

As a result of this natural elimination of sensitive genotypes throughout the regions where eastern white pine is

native and planted, SO2 is not currently a serious problem.

Ozone injury tends to be more localized and is primarily a

problem where white pines are planted close to major highways with high emission levels from motor vehicles. However, the future effect of air pollution on eastern white pine

is uncertain. Industrial emissions of SO2 from coal-burning

power plants in the midwestern states continue to have an

impact on northeastern forest land and waters. Unless the

currently inadequate emission controls in the midwestern

region of the US are strengthened, increases in coal-burning

power plant outputs may further affect the survival and

growth of eastern white pine in the northeastern states.

Directions for the Future _________

The primary goal for future research on the genetics and

improvement of five-needle pines in the eastern United
26 USDA Forest Service Proceedings RMRS-P-32. 2004

Kriebel Genetics and Breeding of Five-Needle Pines in the Eastern USA

States should be the restoration of the superior position of

eastern white pine as a high-quality timber tree. The most

promising methods of achieving this goal through genetic

research and its applications are the following:

1. Ecology, management and resistance screening can be

integrated to achieve the objective of planting pest-free

eastern white pine planting stock in regions where white

pine blister rust and the white pine weevil are major deterrents to the commercial planting of this valuable native tree.

2. Existing older eastern white pine progeny tests can be

converted into seed orchards for rapid gain from the use of

open-pollinated seed of known parentage and proven potential. Seedlings from elite trees should be planted in rust-free

areas at close initial spacing to minimize weevil damage.

This procedure has already been applied in some progeny

tests.

3. Weevil resistance should be incorporated into eastern

white pine through genetic engineering to incorporate genes

for insect resistance into the species. A program has already

been initiated to develop the molecular technology that may

make possible the introduction of weevil resistance into

white pine.

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28 USDA Forest Service Proceedings RMRS-P-32. 2004

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

Geral McDonald and Paul Zambino are with the USDA Forest Service,

Rocky Mountain Research Station. Email: gimcdonald@fs.fed.us or

pzambino@fs.fed.us. Richard Sniezko is with the USDA Forest Service,

Dorena Genetic Resource Center. Email: rsniezko@fs.fed.us

Abstract—Introduction of Cronartium ribicola into the Western

United States created major disruptions in forests where fiveneedle pines were important components. In response, various

control measures were implemented on two commercially important species—western white pine (Pinus monticola) (WWP) and

sugar pine (P. lambertiana) (SP). The USDA Forest Service developed three programs to breed for resistance: one directed at northern Rocky Mountain WWP; a second for WWP and SP in Oregon and

Washington; and a third for SP in California. The Rocky Mountain

program developed a resistant population composed of durable or

multigenic resistance that shows no evidence to date of any R genes

(i.e., genes for “Major Gene Resistance”). The Cascade WWP program utilizes a mixture of an R gene and multigenes. Washington

populations correspond to the Rocky Mountain model, but resistance

of the Oregon populations seems to be partly due to an R gene. An

R gene is the threshold basis for selection of SP for the California

program. Progeny that carry R genes are screened for additional

resistance mechanisms to develop populations with multiple sources

of resistance. These breeding programs have created large seed

banks, several seed orchards, and numerous additional plantings of

pedigreed material. However, new concepts for examining genotype

by environment (G x E) interactions, recognition of phenotypic

plasticity of hosts and pathogens and of induced defenses, and

evidence of disease attenuation in the blister rust pathosystem may

have important consequences for future breeding, integrated management, and ecosystem restoration efforts in five-needle pine

ecosystems. Reanalysis of some old results and update of data from

some long-term plantings were coupled with current knowledge of

additional pathosystems to suggest an altered paradigm for white

pine blister rust in which complex interactions among the pine, rust,

and environmental components are the norm.

Key words: Induced defenses, ontogenic resistance, reaction norms,

phenotypic plasticity, disease genotype x environment

interaction, ecologic restoration

Breeding Rust-Resistant Five-Needle Pines

in the Western United States: Lessons from

the Past and a Look to the Future

Geral McDonald

Paul Zambino

Richard Sniezko

Introduction ____________________

Introduction and spread of the causal agent of white pine

blister rust (WPBR), Cronartium ribicola J. C. Fischer ex

Rabh., in western North America was recently reviewed

(McDonald and Hoff 2001). WPBR introduction precipitated a major reduction in populations of western white

pine (WP) (Pinus monticola Douglas ex. D. Don.) in the

northern Rocky Mountains (Neuenschwander and others

1999). In this region, WWP was a modifier keystone species

whose presence influenced many ecosystem processes

(McDonald and others 2000). Removal of WWP has also

altered patterns of mortality from Armillaria root rot on

replacement species and thus, of fire regimes that could

ultimately produce shortened fire return intervals and more

intense stand replacement fires (McDonald and others 2003).

Ecosystems where whitebark pine (WBP) (P. albicaulis

Engelmann) is a keystone species are also at risk of experiencing major perturbations (Tomback and others 2001).

The ecologic and economic importance of North American species of five-needle pines and their susceptibility to

WPBR have prompted most genetics work on western fiveneedle pines. Breeding work has centered on the two

important commercial species of WWP and sugar pine (SP)

(Pinus lambertiana Douglas). Programs were delineated

by geographic areas. The first, initiated in 1946, was

designed to produce WPBR resistant selections of WWP for

use in the “Inland Empire” (northeastern Washington,

northern Idaho, and western Montana) and utilized controlled crosses to obtain full-sib progeny for selection within

and among crosses (Bingham 1983). A program to develop

resistance in WWP and SP for Washington and Oregon was

started in 1956. This program switched to screening open

pollinated progeny of candidate trees in 1971, after initially using full-sib progeny (Sniezko 1996). A breeding

program designed to develop resistance in SP for California

was initiated in 1957 (Kitzmiller 1982).

Conditions influencing development of WPBR and types

and distributions of resistance mechanisms each vary

considerably among these western geographic regions. Differences in approaches among the breeding programs are

reflected in differences in kinds of seed orchards and other

breeding resources. Our intent in this paper is to briefly

describe each program, examine the development, deployment, and historical evidence of effectiveness of resistance to

WPBR, and provide insight into the development of management strategies for restoration of ecosystems. Some

recent literature will be reviewed. Results in existing
USDA Forest Service Proceedings RMRS-P-32. 2004 29

Breeding Rust-Resistant Five-Needle Pines in the Western United States: ... McDonald, Zambino, and Sniezko

literature will be examined and some previously unpublished results will be presented using new paradigms for

interpreting adaptation and epidemiological processes and

understanding the physiology of resistance. Finally, we will

discuss relevance of lessons learned from breeding WWP

and SP to new efforts directed at whitebark pine (WBP),

limber pine (LP) (P. flexilis James), southwestern white pine

(SWWP) (P. strobiformis Engleman), bristlecone pine (BP)

(P. aristata Engelmann), foxtail pine (FP) (P. balfouriana

Greville & Balfor.), and Great Basin bristlecone pine (GBBP)

(P. longaeva D. K. Bailey).

The Breeding Programs

Northern Rocky Mountain Western White Pine—

This breeding program has had two phases. The first phase

was based on about 400 phenotypically resistant (cankerfree to five cankers/tree) selections obtained from areas of

high rust severity (cankers/tree). This phase was initiated in

1946 when R. T. Bingham selected a full-crowned 60-yearold tree almost 30 meters tall as the only rust-free tree

within a northern Idaho stand of 380 trees (Bingham 1983).

By June of 1950, 58 trees had been selected from similar

areas of high rust severity. Counts of cankers from neighboring susceptible trees at the time of selection (data on file at

the Rocky Mountain Research Station, Moscow, Idaho)

allowed infection rates and probability of escape to be calculated. Under the levels of rust incidence and severity

observed for stands containing the 58 selections, only one

uninfected tree out of 143 million would be expected to be an

escape (McDonald and Hoff 1982). The full contingent of

400 candidate trees had been located by 1970 and the ratio

of resistant (rust-free to five cankers per tree) to susceptible

phenotypes in natural populations was established at

0.00001 (McDonald and Hoff 1982).

Beginning in 1950, selections were crossed to produce fullsib families whose seedlings could be artificially inoculated

to measure general combining ability (GCA) and specific

combining ability (SCA) (Bingham 1983). Wild inoculum

from two to three collection sites was used for each inoculation. Inoculations were conducted under a double-layered

tent (Bingham 1983). One fourth of the 400 parent trees

selected as phenotypically resistant proved to have GCA for

enhanced rust resistance. These 100 parents were ascribed

to three populations corresponding to elevation, with the

“low elevation” class originating below 1065 m, the “midelevation” class from 1066-1250 m, and the “high elevation”

class from over 1251 m. Since a balanced design of crossing

was desired and the smallest elevation class contained 24

GCA parent trees, 24 GCA trees were selected to represent

each elevation in subsequent controlled crosses. Foundation

stock for each of the three F2 seed orchards representing the

three elevation classes was of 12 unrelated GCA x GCA

families. Early artificial inoculation tests indicated that

about 65 percent of the seedlings of GCA x GCA F2 families

remained free of infection after intense rust exposure (Hoff

and others 1973).

The Phase I program was noted for selection of candidate

trees from stands under intense rust pressure that were

then used in various crossing schemes (Bingham and others

1969). Full-sib progeny were subjected to artificial inoculation in tests containing many thousands of seedlings

(Bingham 1972). Phase I generated many peer-reviewed

papers and left a legacy of nine well-documented plantations

and four seed orchards (Mahalovich and Eramian 1995).

Several are composed of well-marked pedigreed materials

suitable for new genetic research (records on file USFS

Region 1). A recently accepted paper uses these historical

records and materials to analyze the influence of blister rust

resistance breeding on the genetic structure of WWP as

revealed by AFLP DNA markers (Kim and others 2003).

Several plantings of Phase I materials have received repeated rust examinations and new data obtained from these

plantations will be discussed later in this report. The Phase

I program was also the source of descriptions of most of the

resistance mechanisms (see Hoff and McDonald 1980 for

descriptions) that have since been applied to the ongoing

breeding programs in the northern Rocky Mountains

(Mahalovich and Eramian 1995) and Oregon and Washington

(Sniezko 1996).

Northern Rocky Mountain WWP—Phase II Program—

Region 1 of the USDA Forest Service and the Inland Empire

Tree Improvement Cooperative (see Fins and others 2002)

administers the Phase II breeding program that was initiated in 1967 (Mahalovich and Eramian 1995). This program

is based on open pollinated (OP) selections screened for

inheritance of a set of resistance mechanisms (Hoff and

McDonald 1980, Mahalovich and Eramian 1995). The objective is to select for resistant progeny among seedlings of 3100

candidate WWP that have been chosen from stands at least

25 years in age. Maximum severity (cankers/tree) acceptable for select trees is set relative to severity in the surrounding stand as follows: 0 if severity was 10 to 20, 1 if severity

was 21 to 40, 2 if severity was 41-75, 3 if severity was 76-150

and 4 to 5 if severity was 150+. Severity on most selected

trees was less than three cankers. OP cones have been

collected from about 200 candidates each year for screening

at the USFS Region 1 Tree Nursery, Coeur d’Alene, Idaho.

Seedlings are grown under standard nursery regimes for

inoculation at the end of their second growing season.

Inoculum is generated in a disease garden located at the

Lone Mountain Tree Improvement Site, Spirit Lake, Idaho.

Aeciospores are collected each year at 10 sites covering a

target-breeding zone that includes the Idaho panhandle and

western Montana (Mahalovich and Eramian 1995). Collected spores are used to initiate a controlled epidemic on

R. nigrum L. and R. hudsonianum var. petiolare (Dougl.)

Jancz. Telia-bearing leaves are collected and transported to

the Coeur d’Alene nursery for use in controlled inoculations

as had been done for the Phase I program. Following September inoculations, seedlings are planted in beds outdoors

and inspected the following June for numbers of needle

lesions and the following September for presence/absence of

needle lesions, bark reactions, and cankers. The second and

third September after inoculation, seedlings are inspected

for presence/absence of bark reactions. Data are used to

select both families and individuals within families in an

attempt to accumulate resistance mechanisms (Mahalovich

and Eramian 1995). Families selected from the Phase II

program as having high proportions of rust-free seedlings at

the fourth inspection are rigorously outplanted to evaluate

long-term rust behavior and growth under operational conditions and natural levels of inoculum (Mahalovich and

Eramian 1995). Over 20 plantings have been established,
30 USDA Forest Service Proceedings RMRS-P-32. 2004

McDonald, Zambino, and Sniezko Breeding Rust-Resistant Five-Needle Pines in the Western United States: ...

using sound statistical designs, and plans are in place to

conduct regular inspections (Mahalovich and Eramian 1995).

Survival, damage to terminal stems, presence/absence of

bole and branch cankers, number of stem cankers, number

of branch cankers, and total tree height will each be tracked.

The planned assessment schedule is 5, 7, and 10 years after

planting, followed by 5-year intervals until one half of the

rotation age has been reached (Mahalovich and Eramian

1995).

Three additional testing regimes have been implemented

in the Phase II program. A crossing study with full-sibs and

selfs was initiated in 1993 to verify the genetic mechanisms

and inheritance for needle shed, short shoot, and bark

reaction traits, with crossing taking place at two field sites

and the Coeur d’Alene nursery. Secondly, realized gain

trials have been designed to test resistance of seed orchard

populations from both Phases under operational conditions.

For this study, F1, F2, B1 and several control lots will be

outplanted in 2004 at sites where infection levels have been

increasing in plantations of resistant material. Lastly, four

tests, each replicated at two locations per year beginning in

2001, are being planted to evaluate genotype by environment interactions and the idea that a single breeding zone

is sufficient for the northern Rockies. Results of the new

testing regimes will facilitate validation or revision of the

breeding strategy.

Additional breeding was initiated in 1995 using elite tree

selections from the Phase I and Phase II programs to

generate a second-generation program and obtain further

potential gains in resistance. Up to 360 selections will be

divided into 18 sublines that will ensure that seed from the

2nd generation program will minimize the potential for

inbreeding. Also, a WWP clone bank was established in 1999

at Dry Creek Tree Improvement Area, Clark Fork, Idaho to

protect unique trees, elite trees, and progeny of candidate

trees in the field that have been lost through timber removal,

road construction, and catastrophic fire. Entries are added

annually.

Oregon and Washington Sugar Pine and Western

White Pine—The Washington and Oregon breeding program is operated out of the Dorena Genetic Resource Center,

USFS, Region 6 and is located near Cottage Grove, Oregon

(Sniezko 1996). The USDI Bureau of Land Management,

Oregon Office, has been a major cooperator, particularly for

sugar pine, in the phenotypic selection of trees and development of seed orchards for many years. Other cooperators

represent a wide array of public agencies, Indian nations,

and other organizations.

Objectives of the resistance-breeding program for Oregon

and Washington include identifying the amount and type of

genetic resistance present in natural populations of WWP

and SP, selecting families and individuals within families

for resistance, and developing durable resistance to WPBR

while retaining broad genetic diversity and local adaptation

within both species. The program utilizes conventional breeding techniques to increase the durability of the partial

resistance currently available, while maintaining diverse

genetic populations. Patterns of genetic adaptation in SP

and WWP identified through growth responses in common

garden studies (Campbell and Sugano 1987, 1989) are the

primary basis for delineation of breeding zones.

The Dorena facility has screened seedlings of WWP or SP

from more than 9500 parent trees. The program has the

capability to inoculate more than 600 families per year and

evaluate over 100,000 seedlings annually with consistently

high seedling infection rates, as evidenced by development

of needle lesions and stem symptoms. Inoculations are

performed in a building that has been fitted with humidifiers and associated controls to act as a large, self-contained

incubation chamber (McDonald and others 1984). Seedlings

are inoculated after their second growing season and are

evaluated for five years thereafter to discern different resistance mechanisms. Historically, this program has used the

same set of definitions of resistance as the northern Rocky

Mountain Phase I and Phase II programs (see Sniezko

1996), although some re-evaluation of mechanisms is

underway. In general, 80 to 240 (often 120) families are

inoculated per ‘run’, with each family represented by 60

seedlings divided among six replications. Inoculum comes

from two sources: collections of telia produced in the wild

from natural inoculum under ambient conditions vs. in a

disease garden under more controlled conditions.

Until recently, conditions and techniques used in inoculation and evaluation of most forms of resistance had

changed only slightly since the 1960s. However, Dorena has

recently begun to use a modified screening technique to

differentiate families expressing an R gene (Cr2) specific to

WWP (Kinloch and others 1999) that develops a hypersensitive reaction as a barrier to colonization in infected needles

from other families that have a high incidence of canker-free

seedlings. Previously, disease-garden telia were generated

after Ribes were inoculated with bulked collections of aeciospores obtained from across Oregon and Washington.

Currently, Ribes in the disease garden become naturally

infected by aeciospores from WWP in the general vicinity.

Teliospore-bearing leaves from the disease garden are used

to represent a locally prevalent, virulent strain of rust (the

“Champion Mine Strain”) that can overcome Cr2 resistance

(McDonald and others 1984, Kinloch and others 1999). Since

wild-collected (wild-type race) and disease garden (virulent

race) Ribes leaves are each used in half of the inoculation

replications, differences in frequency of resistance between

inoculum sources identify resistance of families conferred by

the R gene, and differentiate them from forms of resistance

(partial resistance) that are not known to be overcome by

differences in rust race. Results from such inoculations

indicate that in Region 6, the frequency of the WWP R gene

is very low and that existing levels of partial resistance

are low, although a few outstanding parents exist (Kinloch

and others 1999, Sniezko 1996, Kegley and Sniezko this

proceedings).

Recent re-sowing of some previously tested families has

confirmed their resistance. Demonstration plantings at

Dorena that include representatives of such resistant families alongside susceptible families that develop extensive

cankering and mortality provide a dramatic visual demonstration of the effectiveness of the program to visitors.

Tentative discovery of a new resistant mechanism, termed

“mechanism X”, may cause further changes to the program.

One of us (RS) has observed that “X” is not Cr2 because of

resistance to the vcr2 race of the pathogen and that “X”

families do better than Cr2 families in field tests; one of us

(GM) has also observed that needle shed morphology of “X”
USDA Forest Service Proceedings RMRS-P-32. 2004 31

Breeding Rust-Resistant Five-Needle Pines in the Western United States: ... McDonald, Zambino, and Sniezko

is reminiscent of Idaho WWP needle shed. Only Washington

populations of WWP have been shown to express “X” but

further investigations of Oregon populations are underway

(Sniezko unpublished data).

Much of the early activity in the Oregon-Washington

white pine blister rust (WPBR) resistance program involved

selecting trees in natural stands in each of eight breeding

zones, inoculating and evaluating offspring of these trees for

resistance, and establishing seed orchards from the most

rust-resistant parents or progeny. Seed orchards have been

established for both WWP and SP in most breeding zones

and resistant seed is available for several zones. Orchards

include a diverse set of parents and an array of putative

resistance mechanisms.

Recently, an important, but long neglected, component

has received additional attention. Establishment of field

plantings is now under way to monitor resistance, validate

screening results, and demonstrate the potential for resistant seed in ecosystem restoration throughout Oregon and

Washington. Since 1996, families of WWP and SP have

been established at 20 and 10 sites, respectively, in conjunction with cooperators that include USDA Forest Service

Region 5, BLM, Confederated Tribes of Warm Springs, OR,

and Josephine County, OR. Additional trials, using control

crosses or open-pollinated seedlots from Dorena, have been

established in British Columbia and on WA Department of

Natural Resources lands (Rich Hunt, personal communication 2001). A planting in the Oregon coast range is planned

by Plum Creek Timber Company (Jim Smith personal communication). Full sib or OP families are utilized and have

been established at sites that differ in representation of the

presumed races of blister rust. These plantings will help

establish whether there are changes in rust virulence over

time, and will potentially give information on inheritance of

some of the resistance mechanisms (for example results,

see Sniezko and others 2000, Sniezko and others, these

proceedings). Information from a few older plantations has

also recently been collected, summarized, and presented at

technical meetings (Sniezko and others #2, these proceedings). In these older trials, infection levels after 15 or more

years in the field are high (>75 percent of trees infected). One

goal of these plantings is to determine the relative field

performance of WWP and SP lots that differed in levels of

rust resistance during screening, to calibrate by species and

resistance level the WPBR extension (McDonald and others

in preparation) of the Forest Vegetation Simulation Model

(Teck and others 1997) for use in Region 6.

California Sugar Pine—A program designed to identify

and utilize WPBR resistance in California sugar pine was

initiated in 1957 (Kitzmiller 1982) and is conducted out of

facilities located near Placerville, CA. The goal of this portion of the USDA Forest Service Region 5’s genetics program

is to maintain locally adapted SP having a mixture of

resistance mechanisms throughout sugar pine’s natural

range (Samman and Kitzmiller 1996). This is being achieved

through two approaches. First, resistant trees are identified

that can be used as seed sources for each of 27 California seed

zones (Kitzmiller 1976) where sugar pine occurs, with trees

additionally grouped according to 500 ft altitudinal bands.

Secondly, a subset of these and additional selected resistant

materials are being used to establish seed orchards and

breeding populations for each of seven breeding zones that

have been established based on physiographic and environmental parameters (Samman and Kitzmiller 1996).

Criteria for tree selection vary according to geographic

location and the frequencies at which resistance mechanisms

occur within local populations. The first-level criterion for

seed tree selection is the presence of the sugar pine R gene

(Cr1; Kinloch and Davis 1996). This was the first dominant

gene for resistance identified in a forest tree species (Kinloch

and Littlefield 1977), and like Cr2 described above, restricts

needle colonization and stem infection by C. ribicola by

development of a hypersensitive response (Kinloch and

Littlefield 1977). Assessment is based on symptom development in inoculated progeny seedlings. In regions where frequency of the R gene, and consequently, the occurrence of

disease free sugar pine are sufficiently high, timber and

growth traits are also considered. Conversely, in regions such

as the northern part of the range of sugar pine where the R

gene for sugar pine is low in frequency (Kinloch 1992), initial

tree selection is based solely on freedom of trees from disease

in stands with high incidence of blister rust. Selections are

protected from cutting until screened.

Open pollinated seed is collected and progeny seedlings

screened for the R gene at the Placerville facility using

artificial inoculations. Control lots are included that contain

several representative genotypes homozygous or heterozygous for Cr1 or homozygous for lack of the R gene. Telia used

as screening inoculum are produced by inoculating greenhouse-grown plants of susceptible cultivars of Ribes

nigrum L. with aeciospores of rust that lacks virulence to

Cr1 that have been collected from sugar pine in different

areas, or with urediniospores produced after previous inoculations. Inoculated plants are bagged for 24 hours until

infection has occurred, and then maintained in a cool,

partially shaded greenhouse for four to five weeks until

dense telia develop on leaves. Basidiospores are cast from

telia on trays of detached leaves onto 8-month-old seedlings

in three-layered, cloth-lined chambers in which temperature and humidity are kept at optimum levels for infection

by water flowing over the outer walls. Seedlings are each

evaluated three times approximately three weeks apart

after susceptible yellow or yellow-green needle spots can be

distinguished from necrotic spots or yellow spots with necrotic margins that signify resistance due to the sugar pine

R gene. Open pollinated families with approximately 50 and

100 percent resistant seedlings indicate heterozygous and

homozygous parent trees, respectively, whereas lower proportions indicate a non-Cr1 tree that has received pollen

from one or more Cr1 trees.

Those seedlings with the R gene are further screened for

slow rusting under natural conditions at a location (Happy

Camp, California) where a strain naturally occurs that can

overcome the sugar pine R gene (Kinloch and Davis 1996;

Samman and Kitzmiller 1996). Ribes sanguinium Prush. is

planted among the R-gene-resistant seedlings to produce

natural inoculum having a high proportion of vcr1 virulence

to the sugar pine R gene resistance. Seedlings are exposed to

a minimum of two, and usually three “wave” years of heavy

rust infection, with final slow rust determinations made for

seedlings and families a minimum of ten years after planting. Detection of slow-rusting resistance at the Happy Camp

site is based on observable low rates of increase in rust

incidence and severity, slow or abortive colonization, and
32 USDA Forest Service Proceedings RMRS-P-32. 2004

McDonald, Zambino, and Sniezko Breeding Rust-Resistant Five-Needle Pines in the Western United States: ...

reduced persistence of infections. Normal cankers and bark

reactions (corking out of branch and bole infections) in SP

appear similar to those in WWP in the other resistance

programs, although the Region 5 sugar pine program also

differentiates a “blight” reaction in which necrotic tissue

extends beyond the canker to the end of the branch proximally to the next branch whorl and distally to the end of the

branch (Kinloch and Davis 1996). Materials selected at

Happy Camp for having slow-rusting resistance in addition

to R gene resistance are then grafted and planted into seed

orchards and clone banks.

To date, 28654 candidate trees have been selected; 1395

living MGR trees identified (John Gleason, R5 Genetics,

pers. comm.); and approximately 2900 pounds of MGR seed

is available for restoration efforts. The three seed orchards

closest to completion represent the Sierra Nevada (i.e.,

Breeding zones 4, 5,and 3, in order of completeness). Each of

these orchards has separate but adjacent breeding blocks for

R gene only versus R gene plus slow-rusting resistance

mechanisms (durable resistance), and additional divisions

based on geographic and elevation differences. Duplicate

plantings of some of this material are being developed as

seed orchards by cooperators in industry, providing a buffer

against loss by fire or other causes. Although the numbers of

R-gene parent trees needed for the Sierra have been met and

exceeded (currently over 700 represented in the orchard

design), adequate numbers of selections have been difficult

for some extremes of elevation and for the northern range of

sugar pine. To increase the number of R-gene phenotypes

from such locations, the program is also identifying resistant MGR progeny of phenotypically resistant seed trees in

the northern range that lack the R gene. Lack of the R gene

in these phenotypically resistant trees may indicate high

levels of non-MGR resistance mechanisms. Fertilization by

R gene pollen may thus result in offspring that have several

resistance mechanisms. Pollen receptor, R-gene-carrying

progeny that are being used in the program are geographically distant from known R-gene trees, to maximize genetic

diversity. Alternative methods are being investigated that

may enable north zone candidate trees with potentially high

slow-rusting resistance to be identified during the initial

progeny screening for R gene resistance.

A network of widely dispersed plantations has been

established to evaluate silvicultural growth characteristics

and resistance of SP to both the wild type and the Cr1-

virulent races of WPBR, (Kitzmiller and Stover 1996). Eight

plantations established in 1983 utilized the same families at

each site, and included families that were homo- and heterozygous for Cr1 and susceptible families. Four plantations

established in 2000-2001 include homo- and heterozygous

Cr1 and susceptible families, as well as R gene plus slowrusting families. Within each category, some families have

demonstrably wide geographic adaptation, while other families are of local origin (P. Stover, personal communication).

Plots are monitored primarily for incidence of disease and

presence and numbers of cankers on branches and stems.

Thus far, monitoring after wave years has failed to detect the

race of WPBR virulent to the sugar pine R gene outside of the

immediate vicinities of its two known sites of occurrence at

Happy Camp and Mountain Home, California.

Other Five Needle Pine of the Western United

States—Some breeding work for other five needle pines is

just getting underway (Mahalovich and Dickerson these

proceedings). A common garden study designed to delineate

seed zones for northern Rocky Mountain WBP was planted

at the Priest River Experimental Forest in 2000 (D. Ferguson,

personal communication). Open pollinated seed, collected

from 10 trees in each of 45 stands, was planted in 2000.

Growth, development, and periodicity of shoot elongation

will be monitored using data collection methods patterned

after a previous WWP study (Rehfeldt and others 1984). A

second planting of most of the same sources was installed in

2001 on a dry site at the Priest River Forest to study drought

tolerance of WBP (Ferguson personal communication).

Seedlots from a number of national forests in OR and WA

were also included in a recent common garden study initiated at the University of British Columbia (Andy Bower,

personal communication). These studies should provide

insights into adaptive variation in WBP.

Evaluations of a small number of California WBP seedlots

for R genes (Kinloch and Dupper 2002) and OR/WA WBP

(unpublished data) have failed to find them. However, R

genes were only recently confirmed in LP and SWWP

(Kinloch and Dupper 2002). A recent sowing of WBP at

Dorena will increase the number of parents evaluated in

Region 6 for R genes and other resistance responses. Protocols will be adapted from the Region 6 WWP and SP resistance program and other efforts to screen for resistance in

WBP (Hoff and others 2001). Spore load for evaluation and

comparison of WBP sources will be a critical factor. An

inoculation trial in 2001 at Dorena has identified the spore

load needed for infection of this species under local conditions (Kegley and others in press). A recent preliminary

screening of phenotypically resistant WBP growing in the

northern Rocky Mountains demonstrated a resistance signature (low frequency and proportion of spotting among

progeny seedlings) very similar to that of northern Rockies

WWP (Hoff and others 2001). Early in the development of

WPBR in western North America, a study quantitatively

compared WPBR incidence and severity on WWP and WBP

at six sites in Washington, Oregon, and Idaho (Bedwell and

Childs 1943). Their data were used to compute infection

rates using the “monomolecular” or “monocyclic epidemic”

equation (see below) in WWP and WBP (McDonald and Hoff

2001). Comparison of these rates indicated WBP growing in

the northern Cascade Mountains of Washington and in the

northern Rocky Mountains in northern Idaho were about

70x more susceptible than contemporary WWP, while WBP

populations in the vicinity of Mount Hood, OR were only 4x

more susceptible (McDonald and Hoff 2001). Genetic

differentiation of populations was revealed as an explanation for differences in relative susceptibility by a recent

study of genetic structure of WBP (Richardson and others

2002). This study demonstrated distinct southern and northern populations of WBP that exhibited a distinct zone of

hybridization between Bedwell and Childs’ (1943) Mount

Hood and northern Washington Cascade populations of

WBP.

New Looks at Historic Issues

Separating Fact from Fiction—The WPBR pathosystem has been in North America for almost a century;

intense efforts to control the disease by minimizing the
USDA Forest Service Proceedings RMRS-P-32. 2004 33

Breeding Rust-Resistant Five-Needle Pines in the Western United States: ... McDonald, Zambino, and Sniezko

alternate host had been applied; and we have 50 years of

experience in developing resistance. Yet, detection of multiple forms of resistance, virulence that overcomes one form

of resistance in several hosts, observations on incidence and

severity of infection, and behavior of local epidemics all lead

us to ask critical questions of the breadth of our knowledge

of this system. Have we examined the blister rust

pathosystem in sufficient detail to predict whether disease

in stands genetically improved for resistance will perform

as predicted by our past experience with the epidemic on

susceptible plants, by artificial screening results, or by

models that predict disease incidence over time and space?

Have important assumptions about WPBR behavior remained unchallenged? Can new tools be employed to critically measure WPBR pathosystem dynamics in time and

space? What is the relative importance of classic genetic

trait-oriented selection vis-à-vis other sources of genetic

expression and change?

R genes and Resistance-Virulence Interaction—

The classic gene-for-gene system seems to play an important

role in WPBR. A series of papers has demonstrated that SP,

WWP, SWWP, and LP have R genes (major genes for resistance) at some frequency in their populations (Kinloch and

Comstock 1980, Kinloch and others 1999, Kinloch and Dupper

2002). Two virulence genes (vcr1 and vcr2) are known that

negate the resistance conferred by R genes in SP and WWP,

respectively. Virulence genes have not been found that

negate R genes in SWWP and LP, indicating that the three

genes are distinct (Kinloch and Dupper 2002). At least one

variant of C. ribicola is virulent on both SP and WWP R

genes (Kinloch and Dupper 2002). Some evidence indicates

that the C. ribicola gene for virulence to the sugar pine R

gene may be inherited by way of the cytoplasm (Kinloch and

Dupper 1999). The WWP R gene has not been found in

Northern Rocky Mountain populations but is found in the

Oregon Cascade Mountains (Kinloch and others 1999), with

additional occurrence in the northern Sierra (Kinloch and

others, unpublished). In some cases, expression of the WWP

R gene seems to be subject to maternal influences, which

may complicate its recognition (Kinloch and others 1999).

Distribution of the virulence gene that overcomes the WWP

R gene was tested using teliospore samples collected for

seedling inoculations, and was identified as prevalent in

recent years at Champion Mine (CM) and Grass Creek (GC)

(Kinloch and others 1999, Kinloch and Dupper 2002) in the

Oregon Cascade Mountains. Presence of the virulence gene

was signified by the development of typical susceptible

interactions in a group of full-sib families from Champion

Mine that carry the WWP R gene (Kinloch and others 1999).

Inoculations of some of these same full-sib WWP families

with rust collected in different ways at both CM and GC in

1984 and 2000 demonstrated an effect of Ribes source (or

environmental aspects correlated with occurrence of Ribes

species) and disease development. In their analysis,

McDonald and others (1984) used three different sources of

CM inoculum: telia collected directly at CM from R.

sanquineum or R. bracteosum (Dougl.), and telia produced

on R. bracteosum at Dorena, Oregon from aeciospores

collected from WWP at CM (McDonald and others 1984

table 3). The most striking difference among these sources

was the 4X higher rates of rust incidence (needle lesions) on

WWP per equivalent basidiospore density when telia had

been produced in situ at CM on R. bracteosum vs. the nearby

R. sanquineum. Telia produced at Dorena from CM aeciospores

behaved like the telia developed at CM on R. bracteosum

except that it took significantly longer to kill seedlings so

that significantly more stunted leaders of living plants were

noted at the time of assessment (McDonald and others

1984). These results strongly suggest that growth on different Ribes species at this location provides an important G x

E effect on ability of C. ribicola inoculum to subsequently

infect and develop on pine. These differences may be mediated through differential ability of a virulent race to utilize

local Ribes, or through more subtle interaction. Even though

WPBR from the GC site may now carry vcr2 virulence

(Kinlock and others 1999), in 1984 its apparent lack of

virulence to the WWP R gene was similar to WPBR obtained at the Still Creek (SC) site (see McDonald 2000) in

its apparent lack of virulence to the WWP R gene (McDonald

and others 1984). However, the GC inoculum produced

significantly more cankers that were delayed in their

development after needle infection than did the SC inoculum (McDonald and others 1984).

Ontogenic Resistance—An increase in ability of plants

or plant parts to resist a pathogen as they age and mature

and the physiological mechanisms by which this originates

are well documented in agricultural pathosystems (Ficke

and others 2002). However, in agricultural systems, plant

development is generally restricted to a single season. The

situation is much less clear for pathosystems involving

long-lived hosts that nonetheless have yearly production of

foliage and stems. Two types of ontogenic resistance have

been described for WPBR: resistance related to types and

maturity of needle tissues, and resistance related to physiological aging or other phenomena correlated with tree age.

Seedlings of the five-needle pines pass through four distinct maturation phases. Seeds germinate and produce

cotyledons, primary (simple) needles, and occasionally

secondary or fascicled needles during their first growing

season. In most seasons in subsequent years, only secondary

needles are produced, and these may be retained for multiple seasons. However, under certain environmental conditions, even older seedlings may produce primary needles in

varying numbers, as part of a variant shoot morphology

known as “lammas growth”.

Cotyledons and primary needles are extremely susceptible to infection and colonization and have been used for

early screening of R gene resistance in SP where resistant

and susceptible spots can be visually differentiated (Kinloch

and Comstock 1980). These juvenile needles have also been

used experimentally to assess rates of seedling colonization

and subsequent mortality in families of eastern white pine

(EWP) (P. strobus L.), since infection of both primary needles

and stems of such immature plants is uniformly high

(Zambino unpublished data). Conversely, the Phase I WWP

resistance-breeding program relied on the differential between needle lesions and stem canker incidence to signify

resistant families. An attempt to cut costs by inoculating

seedlings at the end of their first growing season nearly

eliminated the differentiation between resistant and susceptible seedling families (Bingham and others 1969). A

large-scale inoculation of WWP subsequently confirmed

that significant difference in needle lesion and canker incidence was associated with inoculation age and family
34 USDA Forest Service Proceedings RMRS-P-32. 2004

McDonald, Zambino, and Sniezko Breeding Rust-Resistant Five-Needle Pines in the Western United States: ...

(Bingham 1972). The same pattern was observed in an

interspecific cross of EWP with an Asian pine presumed to

have a well-evolved resistance to WPBR. Patton and Riker

(1966) inoculated two families of EWP x Himalayan white

pine (P.griffithii McClelland) hybrids at 4.5 months and 48

months, and obtained canker incidences of 0.997 and 0.4

respectively.

Ontogenesis and maturity of individual needles also has a

strong influence on resistance; lack of appreciation for this

fact may explain variability in screening success and in

ability to model the response of selected materials under

natural conditions that have repeated opportunities for

infection, versus controlled inoculations that are generally

exposed at a single time. In inoculation experiments, “year

old” secondary needles of EWP produced 3.7 times more

infection per linear distance of needle than “fully mature

current year” needles (Hirt 1938). In similarly inoculated

WWP, the ratio between “year-old” and “current year” needles

was 14.5 to 1 based on needle area (Pierson and Buchanan

1938). Most screening includes infection data on needles

within or at the end of the first year of development, even

though no studies have shown how this practice might

influence field performance of selected materials or the expression of canker resistance mechanisms. Timing for inoculation within the current year needle development may

also be critical as inoculation of WWP secondary needles

before they have completed development circumvents the

expression of resistance in needle tissues (Woo 2000). In

addition, stomatal density and shape and contact angle of

water droplets on needle surfaces showed significant differences in comparisons of resistant and susceptible WWP

(Woo and others 2001 and these proceedings).

Finally, branch and stem resistance appears to increase

with age: Comparisons among grafts obtained from susceptible and resistant ortets of various ages and susceptible

seedlings demonstrated that resistance increases with increasing age of the scion source for both resistant and

susceptible grafts of EWP (Patton 1961). In addition, the act

of grafting alone reduced incidence from .99 to .81 in 4-yearold seedlings (Patton 1961). In SP exposed to rust in the

Happy Camp disease garden, average incidence among

grafts of 17 ortets was 0.32, whereas seedling families

obtained from the same 17 candidates showed an incidence

of 0.92 while susceptible control seedlings had an incidence

of .98 (Kinloch and Byler 1981). This apparent increase in

a tree’s resistance with age must nonetheless be reconciled

with the fact that shortly after the introduction of WPBR,

canker incidence in some merchantable WWP stands reached

100 percent and was followed by high levels of mortality

(Buchanan 1936), indicating the ineffectiveness of the extant levels of ontogenic resistance at that time.

Ontogenic or Induced Resistance?—Defenses against

most enemies in most living organisms may be either constitutive or induced (Fluhr 2001). Most animals have both

kinds of defense, while plants may have evolved mostly

constitutive defenses in the form of R genes that stand as

sentinels on the lookout for virulent pathogens (Fluhr 2001).

These R genes have certain molecular signatures found

across many plant taxa (Fluhr 2001). Some of these signatures have been observed in both SP (Kinloch and Dupper

2002, Sheppard and others 2000) and north Idaho WWP

(Liu and Ekrammoddoulah 2004; M.-S Kim personal

communication). Perhaps these signatures are a sign of the

widespread occurrence of R genes in the five needle pines as

suggested by Kinloch and Dupper (2002).

Other recent reports suggest that induced defenses might

also be widespread in plants as well as animals. In the case

of the desert locust, Schistocerca gregaria, increased population density induced increased resistance to bacteria and

fungi (Wilson and others 2002). A large and rapidly developing literature is available for the topic of inducible (epigenetic) defenses in plants and animals that we will not

attempt to present here. Induced defenses vary from morphologic plasticity able to ward off predation or infection to

induced physiological resistance (Tollrian and Harvell 1999).

Epigenetic R-gene resistance that is expressed in subsequent

generations was demonstrated in Arabidopsis thaliana

(L.) Schur (Stokes and others 2002).

Since the topic of inducible defenses impinges on many

aspects of WPBR resistance breeding, it should be watched

closely. Impacts could range from understanding ontogenic

resistance through examination of phenotypically resistant

candidates, to improved selection and interpretation of performance of “control” lots used in progeny screening tests

and field plantings. Inducible defenses have already been

reported in both native (evolutionarily well-established)

conifer pathosystems such as fusiform rust-loblolly pine

(Enebak and Carey 2000), and Norway spruce (Picea abies

(L.) Karsten)-blue-stain-(Ceratocystis polonica) (Krokene

and others 2001, Franceschi and others 2002), as well as

recently established interactions such as Monterey pine

(P. radiata D. Don.) — pitch canker (Fusarium circinatum)

(Bonello and others 2001). Also, stilbenes — effective conifer

phytoalexins — are induced in various plant parts including

primary needles of Scots pine by such diverse challenges as

fungal attack in the phloem, UV light, and stress (Chiron

and others 2000).

Evidence for Induced Resistance to WPBR—In WWP,

36 phenotypically resistant ramets (clonal offspring) that

had been obtained by grafting from select trees from the field

(ortets) were established in plantations and exposed to

levels of field inoculation that produced 0.89 incidence of

cankering of control seedlings at the same sites after 13

years of exposure (Bingham 1966). Full-sib offspring were

obtained from 34 of the ortets that produced the tested

ramets. Controlled crosses were made on the 34 seed

parents using a common set of four pollen donors. When

11744 seedlings of these families were subjected to artificial inoculation, canker incidence was 0.84 (calculated

from table 4, Bingham 1966). Comparisons between infection of the ortets (plants in the field) and their ramets

should indicate presence or absence of induced resistance.

Bingham (1966) noted that ramets of all nine of the ortets

that were infected when selected did not themselves become infected; however, five of the 25 ortets that were

uninfected when selected had ramets that became infected.

Three kinds of phenotypically resistant parents (ortets) are

suggested. First, those in which a few cankers induced

resistance that prevented subsequent infection of their

ramets (i.e., the 9 infected ortets). Second, those that may

not have been resistant or may have been capable of induced

resistance that was not triggered (i.e., the 5 ortets whose

ramets became infected). The third class is composed of

those trees that possessed putative constitutive resistance
USDA Forest Service Proceedings RMRS-P-32. 2004 35

Breeding Rust-Resistant Five-Needle Pines in the Western United States: ... McDonald, Zambino, and Sniezko

(an additional 20 rust-free ortets that produced rust-free

ramets). This situation implies induced resistance.Furthermore, comparison of performance of seedling progeny of the

three classes may indicate the first evidence of

transgenerational induction of resistance in conifers: Logodds ratios (Sokal and Rohlf 1995) indicated significant

differences in rust incidence on seedlings that differentiated

the groups from one another. Rust incidence was lowest

(0.81) among 6863 seedlings representing the 20 “constitutive resistance” ortets, highest (0.92) among the 1612 seedlings representing the 5 “uninduced” ortets, and intermediate (0.88) among 13269 seedlings representing the 9

“induced” ortets.

G x E in Plantations of Rust Resistant WWP—Over

the years, WPBR-resistant stocks developed in the north

Idaho Phase I program were planted and/or inoculated in

many different environments. Differential responses in results illustrate gaps in our knowledge concerning the relative contributions of environment, rust genes, alternate host

diversity, pine diversity, and resistance genetics to the

behavior of WPBR. The most interesting of these tests

involve long-distance transfer of materials. For example,

susceptible controls and several full-sib F1 and F2 families

from the Phase I program were subjected to natural and

artificial inoculation at two sites in Japan where C. ribicola

basidiospores are produced on a host (Pedicularis resupinata

L.) in the Scrophulariaceae (Yokota 1983). The overall conclusion was that resistant material developed in north Idaho

was not resistant to the rust in Japan. Stock from the Phase I

program was also planted at several sites in British Columbia

(Bower 1987, Hunt and Meagher 1989). In a direct comparison after 11 years at a low elevation site (Mesachie), disease

incidence was higher (.75) for the Phase I F2 than for local

natural unimproved stock (0.52) (Hunt and Meagher 1989).

The pattern was reversed at a second site 45 km north of

Mesachie at Northwest Bay where a higher elevation site

produced incidences after 13 years of 0.12 for the F2 vs. 0.40

for local stock (Hunt and Meagher 1989). These authors

describe further significant G x E interactions for several

sources of resistant WWP as well as EWP. Significant G x E

constitutes a warning to pay close attention to inoculum

sources and geographic distributions of materials selected

for resistance. G x E was also observed in field plantings

of slash pine (P. elliottii Englm var. elliottii) resistant to C.

quercuum (Berk.) Miy. ex Shirai f. sp. fusiforme (Schmidt

and Allen 1998). Significant G x E in rust behavior signifies

that geographic partitions in addition to simple breeding

zones may be a required strategy in restoration programs.

New Tools and Concepts

Comparative Epidemiology—Plant disease epidemiologists have focused on modeling and disease management

from an endpoint perspective, while medical epidemiologists

have focused more on elucidating component parts, enabling

them to act as disease detectives who can reconstruct the

whole by understanding the interactions of the parts

(Waggoner and Aylor 2000). Subsequent to the inception of

pine breeding programs in western North America, advances

in epidemiological theory and evolutionary biology have added

several tools that should enhance our own capability to

become good disease detectives and to focus on how the

various parts of WPBR interact. Successful breeding and

deployment of resistance in a long-lived pathosystem requires significant understanding of how epidemics function

across time and space, which can lead to better integrated

management of blister rust and restoration of ecosystems

dominated by five needle pines.

Rust Progress Curves—Measuring disease is a significant problem often approached by determining incidence

(proportion of a population of plants or plant parts infected)

and severity (number of lesions or amount of area of

plants or plant parts infected) periodically during the

progress of the epidemic and estimating curves from the

plotted values. Nonlinear disease progress curves and their

associated linear incidence rate equations (Madden and

Campbell 1990) can be useful for comparing epidemics

and predicting outcomes in new times and places. Fracker

(1936), the first to apply nonlinear progress curves to plant

disease epidemics, was also first to apply them to WPBR.

Since then, epidemiological theory has matured into several

straightforward concepts (Zadoks and Schein 1979, Madden

and Campbell 1990). Almost all epidemics are described by

one of two basic forms. For most diseases and environments,

epidemics behave as a polycyclic process where diseased

plants contribute inoculum to increase local disease within

the same growth cycle. Such epidemics are represented as a

logistic curve where the inflection point indicates the point

at which the acceleration in rate of disease increase due to

increased inoculum is offset by the decline in healthy tissues

available for colonization. Other epidemics behave as a

monocyclic process where local diseased plants do not contribute to additional disease because inoculum is from relatively constant external sources. Because amounts and

infection efficiency of inoculum are relatively constant and

not affected by local feedback, curves that describe this

process have no inflection point. This “monomolecular” disease progress curve and its associated absolute infection

rate have been applied to WPBR (Kinloch and Byler 1981,

McDonald and Hoff 1982, Goddard and others 1985,

McDonald and others 1994, McDonald and Hoff 2001).

Presence or absence of an inflection point may provide

important clues about fundamental disease processes such

as relative importance of local vs. long-distance spread of

inoculum and degree of rust multiplication on alternate

hosts.

The epidemic asymptote K indicates maximum incidence

in an epidemic (Madden and Campbell 1990). The idea that

incidence may not reach 1.0 was anticipated by Fracker

(1936), who also introduced the concept of the multiple

infection transformation, an equation relating incidence to

severity. Agricultural epidemiologists have generalized K in

nonlinear disease progress curves (Madden and Campbell

1990) and the examination of K has been applied to WPBR

epidemics (McDonald and Dekker-Roberson 1998, McDonald

and Hoff 2001). Fracker’s (1936) ideas were combined with

those of Bald (1970) to develop the concept of a factor that

measures deviation in K over space for WPBR epidemics

(McDonald and others1991, McDonald and others 1994).

If one assumes that total incidence can theoretically

approach 1, then the difference between complete incidence

and predicted asymptote, 1-K, may be attributed to lack of

uniformity in distribution of resistance genes, microclimate,
36 USDA Forest Service Proceedings RMRS-P-32. 2004

McDonald, Zambino, and Sniezko Breeding Rust-Resistant Five-Needle Pines in the Western United States: ...

or composition or density of inoculum. Predictions of K in

different situations could be vitally important for breeding

and deployment programs alike. For a breeding program,

clumpy distribution of infection due to clumpy microclimate

and/or basidiospore distribution can significantly increase

the probability that rust-free candidate trees are susceptible

escapes whose processing reduces screening efficiencies.

However, asymptotes may also reflect effects of a major or

minor gene for resistance, or situations in which even susceptible materials will persist on a site, and changes in K

during an epidemic could indicate major changes in rust

and/or pine populations (Kinloch and Byler 1981, McDonald

and others 1994). Infection rates, especially comparative

rates, are useful for assessing durable resistance (McDonald

and Dekker-Robertson 1998).

Phenotypic Plasticity, Reaction Norms – Construction of Hypotheses, and Understanding G x E—Since a

central tenet of breeding for resistance in any pathosystem

is host/pathogen coevolution, it thus seems natural that

geneticists and phytopathologists engaged in development,

deployment, and maintenance of resistance should examine

theoretical interpretations utilized by other disciplines to

understand the implications of G x E in plant pathosystems.

Indeed, evolutionary biologists have developed very powerful concepts and tools – those of phenotypic plasticity and

reaction norms – not yet widely applied in phytopathology

and sparingly used in forest genetics. Phenotypic plasticity

is the ability of a genotype to express itself as different

phenotypes in different environments. A few notable applications of this concept in forest genetics were found (Wu

1998, Wu and Hinckley 2001). As a basis for the following

discussion, concepts from the excellent book by Pigliucci

(2001) are liberally presented. Central elements in the

concept of phenotypic plasticity are the reaction norm and

its connection to analysis of variance as applied in quantitative genetics. Reaction norms are functions that relate the

range of possible phenotypes a genotype can express across

an environmental gradient. They are generally used to

visualize genetic, environmental and G x E variance by

comparing at least two genotypes, families (any relationship), populations, species, or groups of species across an

environmental gradient. Any gradient of interest, e.g.,

light, temperature, moisture, nutrients, or host organisms,

can be displayed. However, if a true gradient is not present,

then sites or hosts must be compared in pairs or some

kind of artificial gradient needs to be assumed. Reaction

norms have a historic association with ANOVA as the

preferred method of analysis. Summary statistics are often

given in a suitable format such that all ANOVA elements

are present, to wit: differences among across-environment

means of the genotypes indicate genetic (G) variance; differences among across-genotype means of environments indicate environmental (E) variance; and when G and E interact

in ways not predicted by the combined influence of acrossenvironment and across-genotype means, there is plasticity

(P) variance. Further note that if means for an individual

genotype differ significantly across-environments, that genotype is plastic. This allows computation of the log-odds ratio

and its associated standard error for hypothesis testing. A

newly published book presents a multivariate approach to

the analysis of G x E in a crop-breeding context (Yan and

Kang 2003). Although examples of most types of organisms

are presented in Pigliucci’s (2001) book, fungi and plant

pathosystems are notably absent, arguably because

phytopathologists and mycologists have been slow to embrace the concepts and methodology of phenotypic plasticity

and reaction norms. Yan and Kang (2003) devote two chapters to the analysis of pest resistance G x E in the context of

crop resistance breeding rather than in terms used by

evolutionary biologists.

To foster a dialogue about these and other questions, we

will look at some blister rust pathosystem processes using

the concepts and tools just outlined. We will apply the

concepts of K, infection rate, inflection points, and reaction

norms in a critical assessment of published data as well as

updated data obtained from established plantings. Our

intent is to advance the idea that becoming better rust

detectives will lead to better development, deployment, and

management of resistance genes in western North American

5-needle pines.

Materials and Methods ___________

Data

To illustrate the utility of the reaction norm approach and

in keeping with the disease detective approach to the study

of WPBR epidemiology, reaction norms were constructed for

seven cases from previously collected or published data.

Case one examines time from inoculation to pustule development for inoculum sources from different geographic areas

inoculated onto individual Ribes clones; data was obtained

from a study (McDonald 2000) that was previously published using average responses over all clones. Reaction

norms (fig. 1-3) were selected to provide an elementary

lesson in recognizing types of trends that may be encountered. The second case identifies variation in three types of

pine response to the Champion Mine race of C. ribicola

obtained from different alternate hosts through a reexamination (fig. 4-5) of data from McDonald and others (1984).

Case three demonstrates the use of reaction norms (fig. 6-7)

to dissect effects of developmental stages, and possibly

ontogenic resistance, using developmental reaction norms

defined by periods of exposure to rust for WWP data published by Bingham (1966 and 1972). Case four uses reaction

norms to demonstrate a classic G x E interaction (fig. 8) for

WWP stocks grown at two ex situ locations in Japan (Yakota

1983). Case five demonstrates the fitting of incidence data

from various classes of resistance phenotypes grown at two

northern Idaho sites into disease progress curves (fig. 9);

further examination of differences in reaction norm parameters among resistance and site categories are presented

(fig. 10-12). Case six examines infection and disease progress

parameters in three northern Idaho full-sib test plantation

sites (Bingham and others 1973, McDonald and others 1994,

McDonald and Dekker-Robertson 1998, Fins and others

2002); reaction norms are set up to compare parameters that

were obtained for different resistance classes early versus

late in the epidemic (fig. 13-16). Case 7 is a detailed examination of infection severity and incidence to reveal clues

about the cause of unexpectedly high disease incidence on

resistant stocks at one of the three sites.
USDA Forest Service Proceedings RMRS-P-32. 2004 37

Breeding Rust-Resistant Five-Needle Pines in the Western United States: ... McDonald, Zambino, and Sniezko

Figure 1—Reaction norm demonstrating significant genetic variance in number of days from inoculation to first

appearance of urediniospores for two sources of

Cronartium ribicola (CM = Champion Mine, OR; SC = Still

Creek, OR) inoculated as aeciospores onto two clones of

Ribes bracteosum collected at the Champion Mine site

(Case 1 in text). ANOVA based on four replicated inoculations of 6 leaf disks for each Ribes-Rust combination.

Figure 2—Reaction norm demonstrating significant genetic and environmental variance in number of days from

inoculation to first appearance of urediniospores for two

sources of Cronartium ribicola (CM = Champion Mine,

OR; SC = Still Creek, OR) inoculated as aeciospores onto

two clones of Ribes lacustre collected at the Priest River

Experimental Forest, Idaho (Case 1 in text). ANOVA

based on four replicated inoculations of 6 leaf disks for

each Ribes-Rust combination.

0

2

4

6

8

10

12

14

Champion Mine Ribes bracteosum Clones

UAP (Days)

CM (12.9a) 12.9 12.9

SC (11. 0a) 11.3 11

CMB2 (12.1a) CMB5 (11.95a)

Two-Way ANOVA

Source df F Prob 

Reps 3 NS

Rust (G) 1 ****

Ribes (E) 1 NS

G x E 1 NS

Error 79

Plasticity Test: CM.CMB2 vs CM.CMB5 NS

 SC.CMB2 vs SC.CMB5 NS

0

2

4

6

8

10

12

14

16

Priest River Ribes lacustre Clones

UAP (days)

SC (13.2b) 11.9 14.5

CM (11.75a) 10.4 13.1

PRL1 (11.15a) PLR2 (13.8b)

Two-Way ANOVA

Source df F Prob 

Reps 3 NS

Rust (G) 1  

Ribes (E) 1 ** **

G x E 1 NS

Error 79

Plasticity Test: SC.PRL1 vs SC.PRL2  

 CM.PRL1 vs CM.PRL2 ** **

10.5

11

11.5

12

12.5

13

13.5

Champion Mine Ribes lacustre Clone

UAP (days)

SC (12.96 b) 12.6 13.3

CM (11.95 a) 12.4 11.5

CML1 (12.5a) CML2 (12.4 a)

Two-Way ANOVA

Source df F Prob 

Reps 3 *

Rust (G) 1 ****

Ribes (E) 1 NS

G x E 1  

Error 80

Plasticity Test: SC.CML1 vs SC.CML2 ** **

 CM.CML1 vs CM.CML2 *** *

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Pine Populaltions

Proportion of Seedlings

WT (.51) b 0.23 0.8

CM (.24) a 0.16 0.32

OP (.20) a SR (.56) b

Odds-Ratio Significance

Rust Genetic Variance: *

Pine Variance: *

Rust Plasticity Variance: *

Rust Plasticity Test: WTOP vs WTSR *

 CMOP vs CMSR *

Size and Significance

 WTOP = 35 a

 CMOP = 25 a

 WTSR = 76 b

 CMSR = 32 a 

Figure 3—Reaction norm demonstrating significant genetic, environmental and G x E variance in number of

days from inoculation to first appearance of urediniospores

for two sources of Cronartium ribicola (CM = Champion

Mine, OR; SC = Still Creek, OR) inoculated as aeciospores onto two clones of Ribes lacustre collected at

the Champion Mine site (Case 1 in text). ANOVA based

on four replicated inoculations of 6 leaf disks for each

Ribes-Rust combination

Figure 4—Incidence of premature needle shed resistance in Pinus monticola for two sources of Cronartium

ribicola basidiospores (CM = Champion Mine, OR; WT =

pooled Still Creek and Grass Creek, OR) inoculated onto

13 open pollinated pine families obtained from phenotypically resistant parents (OP) vs. seven full sib families

obtained from Champion Mine and specifically selected

for resistance to WT inoculum (SR) (Case 2 in text; data

from McDonald and others 1984).
38 USDA Forest Service Proceedings RMRS-P-32. 2004

McDonald, Zambino, and Sniezko Breeding Rust-Resistant Five-Needle Pines in the Western United States: ...

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Season of Growth upon Inoculation

Proportion with Needle Lesions

S (.90) b 0.95 0.84

R (.82) a 0.91 0.73

1st (.93) b 2nd (.78) a

Odds-Ratio Significance 

Pine Genetic Variance: *

Developmental Variance: *

Plasticity Variance: NS

Plasticity Test : S1st vs S2nd *

 R1st vs R2nd * Size and Significance

S1st = 1145 b

R1st = 11411 a

S2nd = 2130 b

R2nd =12075 a

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Season of Growth at Exposure

Proportion (Canekred/Spotted)

S (.42) b 0.16 0.68

R (.30) a 0.14 0.45

1st (.15) a 2nd (.57) b

Odds-Ratio Significance 

Pine Genetic Variance: *

Developmental Variance: *

Plasticity Variance: *

Plasticity Test : S1st vs S2nd *

 R1st vs R2nd * 

Size and Significance

S1st = 1085 a

R1st = 10354a

S2nd = 1797 b

R2nd = 8844 a

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

 Location of Inoculations in Japan

Rust Incidence (Proportion)

WP17 (.78) b 0.64 0.92

WPC (.75) b 0.82 0.68

WP22 (.74) b 0.54 0.94

WP58 (.74) b 0.60 0.88

WP19 (.62) a 0.55 0.70

Nakashibetsu (.63) a Hokkaido (.82) b

Odds-Ratio Significance

Genetic *

Environmental *

G x E *

Plasticity Test : WP17 -N vs WP17-H *

 WP C -N vs WP C-H NS

 WP22 -N vs WP22-H *

 WP58 -N vs WP58-H *

 WP19 -N vs WP19-H *

Numbers and Significance

 N H

WP17 28 a 26 b

WPC 11 b 19 a

WP22 26 a 18 b

WP58 25 a 19 b

WP19 44 a 27 a

Figure 5—Proportion of stunted leaders in Pinus monticola

for two sources of Cronartium ribicola basidiospores (CM

= Champion Mine, OR; WT = pooled Still Creek and Grass

Creek, OR) inoculated onto 13 open pollinated pine families obtained from phenotypically resistant parents (OP)

vs. seven full sib families obtained from Champion Mine

and selected for resistance to WT inoculum (SR) (Case 2

in text; data from McDonald and others 1984).

Figure 6—Effect of seedling development stage during

rust exposure on needle infection in Pinus monticola

(Case 3 in text): Incidence of infection for seedlings of

phenotypically resistant parents (R; full-sib progeny) vs.

susceptible infected parent trees (S; open pollinated progeny from high rust areas in northern Idaho) after exposure

to a bulked source of Cronartium ribicola basidiospores

from north Idaho at the end of first (1st) vs. second (2nd)

growing seasons (Data from Bingham 1972).

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

WWP Populations

Proportion Stunted Leaders

CM (.43) b 0.39 0.46

WT (.22) a 0.24 0.2

OP (.32) a SR (.33) a

Odds-Ratio Significance

Rust Genetic Variance: *

Pine Variance: NS

Rust Plasticity Variance: *

Rust Plasticity Test: CMOP vs CMSR *

 WTOP vs WTSR NS

Size and Significance

 WTOP = 301 b

 CMOP = 463 a

 WTSR = 85 b 

 CMSR = 221 a

Figure 7—Effect of seedling development stage during rust exposure on stem infection in Pinus monticola

(Case 3 in text; materials and definitions as in fig. 6).

Figure 8—Genetic x Environment interaction affecting

resistance in Pinus monticola (Case 4 in text): Incidence

of canker infections for five half-sib families from crosses

of north Idaho seed parents after inoculation with unique

sources of Cronartium ribicola basidiospores at two localities, i.e. natural inoculations at Nakashibetsu, Japan and

artificial inoculation at Hokkaido, Japan. (Data from Yokota

1983).
USDA Forest Service Proceedings RMRS-P-32. 2004 39

Breeding Rust-Resistant Five-Needle Pines in the Western United States: ... McDonald, Zambino, and Sniezko

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 5 10 15 20 25 30 35 40 45 50

Years of Exposure

Proportion of Stand infected

DC55cont Actual DC59cont Actual PR59ngca Actual

DC57gca Actual PR55ngca Actual PR57gca Actual

0

0.2

0.4

0.6

0.8

1

1.2

Planting Sites

Epidemic Asymptote (K at 40 years)

55C(.91) c 0.84 0.97

57C(.86) b 0.83 0.89

59C(.74) a 0.72 0.77

PR (.80) a DC (.87) b

Odds-Ratio Significance 

Genetic *

Environmental *

G x E *

Plasticity Test : 

 55CPR vs 55C-DC *

 57C-PR vs 57C-DC NS

 59C-PR vs 59C-DC NS

Numbers and Significance

 PR DC

 55C 23 b 34 c

 57C 59 b 87 b

 59C 31a 26 a 

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

Planting Sites

Epidemic Asymptote (K at 40 Years)

55G(.37) a 0.38 0.35

57G(.38) a 0.33 0.43

59G(.41) a 0.43 0.38

PR (.38) a D C (.39) a

Odds-Ratio Significance 

Genetic NS

Environmental NS

G x E *

Plasticity Test : 

 55G-PR vs 55G-DC NS

 57G-PR vs 57G-DC *

 59G-PR vs 59-DC NS

Numbers and Significance

 PR DC

55G 145 ab 159 a

57G 63 a 86 b

59G 90 b 86 ab 

0

0.1

0.2

0.3

0.4

0.5

0.6

PLANTING SITES 

Epidemic Asymptote (K at 40 years)

55GN(.39) a 0.35 0.42

57GN(.41) a 0.4 0.42

59GN(.49) b 0.51 0.46

PR (.42) a DC (.43) a 

Odds-Ratio Significance 

Genetic *

Environmental NS

G x E *

Plasticity Test : 

 55GN-PR vs 55GN-DC NS

 57GN-PR vs 57GN-DC NS

 59GN-PR vs 59GN-DC *

Numbers and Significance

 PR DC

55GN 103 a 128 a

57GN 107 a 155 a

59GN 116 b 102 a 

Figure 9—Disease progress curves for epidemics

caused by Cronartium ribicola showing differences in

disease incidence asymptotes (K) from fitting optimal

nonlinear equations (Gompertz or monomolecular) to

data from groups of Pinus monticola families representing three resistance pedigrees (GCA x GCA,

NonGCA x GCA, and OP Controls) grown at Priest

River (PR) and Deception Creek (DC) Experimental

Forests located in northern Idaho, USA (Case 5 in

text).

Figure 10—Disease incidence asymptotes (K) for

Cronartium ribicola epidemics in Pinus monticola open

pollinated control lots planted in 1955 and 1956 (55C),

1957 (57C) and 1959 (59C) at sites in northern Idaho on

the Priest River (PR) and Deception Creek (DC) Experimental Forests (Case 5 in text).

Figure 11—Disease incidence asymptotes (K) for

Cronartium ribicola epidemics in groups of Pinus monticola

full-sib families with a GCA x GCA pedigree planted in 1955

and 1956 (55G), 1957 (57G) and 1959 (59G) at sites in

northern Idaho on the Priest River (PR) and Deception

Creek (DC) Experimental Forests (Case 5 in text).

Figure 12—Disease incidence asymptotes (K) for

Cronartium ribicola epidemics in groups of Pinus

monticola full-sib families with a nonGCA x nonGCA

pedigree planted in 1955 and 1956 (55GN), 1957

(57GN) and 1959 (59GN) at sites in northern Idaho on

the Priest River (PR) and Deception Creek (DC) Experimental Forests (Case 5 in text).
40 USDA Forest Service Proceedings RMRS-P-32. 2004

McDonald, Zambino, and Sniezko Breeding Rust-Resistant Five-Needle Pines in the Western United States: ...

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Period of infection

Monomolecular Infection Rate

GC (0.23)c 0.171 0.293

JC (0.13)b 0.061 0.204

JF2 (0.016)a 0.008 0.024

GF2 (0.02)a 0.014 0.027

Period 1 (0.063)a Period 2 (0.137)b

Two-way ANOVA

Stock (G): ****

Periods (E)  

Interaction (P) ** **

Plasticity Test

 GC  

 JC ** **

 JF2 NS

 GF2 NS 

Numbers and Significance

 Period 1 Period 2

GC 216 c c

JC 434 b b 

JF2 163 a a

GF2 427 a a

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

0.400

0.450

Canker Accumulation Period and Mean

Cankers/tree/Year

GC (0.3)c 0.414 0.193

JC(0.12)b 0.072 0.169

JF2 (0.022)a 0.009 0.036

GF2 (0.029)a 0.039 0.02

Period 1 (0.133)a Period 2 (0.104)a

Two-way ANOVA

Stock (G):  

Periods (E) NS

Interaction (P) ** **

Plasticity Test

 GC  

 JC NS

 JF2 NS

 GF2 NS 

Numbers and Significance

 Period 1 Period 2

 GC 216 b b

 JC 434 a b 

 JF2 163 a a

 GF2 427 a a

0

0.5

1

1.5

2

2.5

3

Canker Severity Measurement Period

Canker/Tree/Year

F2-Forest (1.43) 0.135 2.73

F1-Forest (0.49) 0.12 0.858

F1 Brush (0.38) 0.121 0.64

F2 Brush (0.44) 0.078 0.8

!st 12 years (0.11) Next 14 years (1.26)

Number of Trees/Class

F2 Forest 68

F1 Forest 92

F1 Brush 54

F2 Brush 36

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

!st 12 years (0.09) Next 14 years (0.29)

Infection Incidence Measurement Periods Compared

Infection Rate ( Monomolecular Equation)

F2-Forest (0.13) F1-Forest (0.18) F1 Brush (0.22) F2 Brush (0.24) 

Gletty Cont (0.23)c Jaype Cont (0.13)b Jaype F2 (0.016)a Gletty F2 (0.02)a 

Figure 13—Cronartium ribicola infection incidence rate

constants computed using the monomolecular equation for the first 11 years (period 1) vs. the last 14 years

(period 2) during which infection was monitored in

Pinus monticola control (Cont) vs. second generation

resistant selections (F2) planted at Gletty Creek (G) in

northeastern Washington and at Jaype Mill (J) in northern Idaho (Case 6 in text).

Figure 14—Cronartium ribicola average canker accumulation rates for first 11 years (period 1) vs. last 14

years (period 2) during which infection was monitored in

Pinus monticola control (Cont) vs. second generation

resistant selections (F2) planted at Gletty Creek (G) in

northeastern Washington and at Jaype Mill (J) in northern Idaho (Case 6 in text).

Figure 15—Cronartium ribicola average canker accumulation rates for first 11 years (period 1) vs. last 14 years

(period 2) during which infection was monitored in Pinus

monticola first (F1) vs. second generation resistant selections (F2

) planted at Merry Creek in northern Idaho under

forest vs. brush site conditions (Severity data supplemental to study described by McDonald and Dekker-Robertson

1998) (Case 7 in text).

Figure 16—Cronartium ribicola infection incidence rate

constants computed using the monomolecular equation for

the first 11 years (period 1) vs. the last 14 years (period 2)

during which infection was monitored in Pinus monticola

control (C) vs. second generation resistant selections (F2)

planted at Gletty Creek (G) in northeastern Washington

and at Jaype Mill (J) in northern Idaho vs. Merry Creek

(Case 7 in text; based on data from McDonald and

Dekker-Robertson 1998).
USDA Forest Service Proceedings RMRS-P-32. 2004 41

Breeding Rust-Resistant Five-Needle Pines in the Western United States: ... McDonald, Zambino, and Sniezko

Statistical Methods

This paper utilized three main types of statistical tests:

ANOVA with multiple means testing, log-odds ratios applied to means of proportions, and nonlinear curve fitting.

Data were available for the Ribes-rust geographic variation

experiment (McDonald 2000) to conduct standard analysis

of variance (Systat 9.1 SPSS, Inc) for each set of four

comparisons in the reaction norm format. These data meet

the normal distribution assumption for ANOVA (see

McDonald 2000). This format and details of reaction norm

analysis can be found in Pigliucci (2001). Where multiple

means were compared in pairwise format the Bonferroni

correction was used (Systat 9.1). In the case of nonlinear

curve fits, the standard error of the parameter estimate

(disease asymptote) was computed by the curve fitting

software (Table Curve 3.0 SPSS, Inc).

Analyses based on published data and new analysis of the

vigor-quality plantings entailed comparisons of proportions;

log-odds ratios were used to determine if any particular pair

of proportions was significantly different (Sokal and Rohlf

1995). The standard error of the comparison was determined

by taking the square root of the sum of the reciprocals of the

number of observations in each cell of the comparison (Sokal

and Rohlf 1995). To obtain 95 percent confidence limits, the

standard error was doubled and then added and subtracted

from the natural log of the odds ratio (Sokal and Rohlf 1995).

When this range crossed zero, the proportions under comparison were considered not significantly different (Sokal

and Rohlf 1995). Reaction norms require comparison of

across-environment means of each family line or genotype,

as well as comparison of each family x environment proportion. Mean proportion of all families in an environment must

be compared across environments. To accomplish these

comparisons, we computed each relevant mean proportion

and estimated the standard error by computing the average

number of observations per cell of the four cells associated

with the proportions being compared.

Results and Discussion __________

Case 1: Uredinial Sorus Development Time

Data previously presented by McDonald (2000) but pooled

over five Ribes clones were separated by clone x WPBR

source and the period from inoculation to urediniospore

appearance. The number of days until urediniospores appeared (urediniospore appearance period; UAP) was then

subjected to a new ANOVA. Given that hosts of plant

pathogens are a significant component of a pathogen’s environment, our first example will include two WPBR populations (Champion Mine and Still Creek) compared on two R.

bracteosum Douglas clones from the Champion Mine site

(fig. 1) and two R. lacustre (Pers.) Poir. clones obtained from

the Priest River site (fig. 2). We also include two Champion

Mine R. lacustre clones (fig. 3). The important elements are

the mean UAP’s obtained from 24 leaf disks — four replications of inoculations of 6 disks each (McDonald 2000).

The across-environment means are important, as are the

slopes of the reaction norms. Defining the “genetic” component as differences due to rust source, and “environment” as

that due to rust clone in the first comparison (fig. 1), the

across-environment means differ significantly, indicating

significant genetic variance in the rust populations. There is

no environmental variance (means for the two Ribes clones

across rust populations do not differ), no plasticity variance

(no G x E interaction), and no plasticity for either population

(the individual environment means for each rust population

do not differ). There would be no genetic variance if means

for the two rusts were not significantly different. Next, we

illustrate sloping parallel reaction norms and associated

ANOVA (fig. 2). In this specific combination, the rust populations showed significant genetic variance (across-environment means differ), environmental variance (environmental means differ), and both rust populations show plasticity

because means within a rust population differ across environments, but there is no plasticity variance (no G x E).

Crossing or divergent reaction norms (fig. 3) also yield

specific genetic information about R. lacustre clones CML1

and CML2: Genetic variance is present since the acrossenvironment means differ, but environmental variance is

not present since the mean performance of the two rust

populations on Ribes clones, CML1 and CML2 do not differ.

Variance for plasticity is present (significant G x E). If we

were comparing rust clones derived from single spores, we

could make the case that rust clones have heritable plasticity. Since we are comparing rust populations, we can only

argue that the two populations are genetically different.

Both populations are exhibiting plasticity, since their performances differ by Ribes clone.

The real power in such specific G x E comparisons resides

in the fact that specific hypotheses can be created and tested.

These hypotheses can then form the basis for designing

specific experiments, which can include application of molecular techniques (Wu 1998, Pigliucci 2001). For example,

figure 3 might suggest that genetic variation in C. ribicola is

caused by allelic differences within mixed populations, variation in gene expression, or some other factor such as conditionally dispensable chromosomes or transposon activity.

Conditionally dispensable chromosomes are small chromosomes that often contain genes coding for pathogencity that

can be conditionally removed from the genome (Hatta and

others 2002, Covert 1998). Transposons — transportable

genetic elements — are known in the basidiomycotina (Fowler

and Mitton 2000) and are known to influence the expression

of host resistance genes in a rust pathosystem (Luck and

others 1998). Another potential role for utilizing reaction

norms for understanding pathosystems is to examine quantitative geographic and host differential responses beyond

the qualitative virulent – avirulent dichotomy based on R

genes, as discussed above.

Case 2: R Gene-Virulence Interaction

We will briefly discuss aspects of the CM (Champion Mine)

race in regard to qualitative vs. quantitative differential

reactions. The CM race was first described in 1984 (McDonald

and others 1984) using a series of quantitative pine host

responses relating mostly to reduction in frequency of

expression of several resistance traits. Open pollinated

families obtained from phenotypically resistant candidate

trees growing in Oregon and Idaho were compared to a group
42 USDA Forest Service Proceedings RMRS-P-32. 2004

McDonald, Zambino, and Sniezko Breeding Rust-Resistant Five-Needle Pines in the Western United States: ...

of full-sib families obtained from selection and breeding

candidates obtained from the CM site (Kinloch and others

1999, McDonald and others 1984). Trees were inoculated

with CM and wild-type inoculum and expression of various

symptoms was recorded monthly for 24 months. Data were

published (tables 4 and 5 in McDonald and others 1984) that

now allow reaction norms to be developed for genetic interpretations. Responses of the OP and selected-resistant families with regard to premature needle-shed resistance

(McDonald and Hoff 1970) yielded significant genetic and

environmental (host genetic) variance when genotype is

defined by host and environment is defined by kind of rust

inoculum (fig. 4). Plasticity variance was indicated by the

combination of nonsignificant (NS) and significant differences in the E means. The reaction norm interpretation is

that both sources of blister rust carry genetic variation for

plasticity since means from their cross-environment plasticity tests were significantly different. This analysis shows

that the OP candidate families carried some premature

needle shed resistance that was of equal effectiveness to

both populations of the rust; i.e., they did not differentiate

the rust populations. However, the resistant population

selected by screening and breeding for resistance to inoculum collected at CM dropped from 80 percent expression of

resistance to 32 percent. This is still an important level of

resistance, which suggests some mechanism other than

simple negation of an R gene may underlie susceptibility to

rust variants capable of neutralizing R genes. An alternative

explanation for the observed pattern in the R-gene-containing/R-gene-lacking couplet is variation in expression of R

genes associated with host maternal cytoplasm (Kinloch and

others 1999).

Before we leave the CM race, we should investigate the

effect of this rust population on frequency of stunted leaders

(McDonald and others 1984). Reaction norms were constructed (fig. 5) showing significantly greater incidence of

stunted leaders by CM rust across host environments. There

was no significant response of this trait to selection for

resistance when inoculated with wild-type rust, but significant genetic and plasticity variances in the rust. The pattern

of these norms indicates experimental conditions that could

facilitate the study of physiological aspects of WPBR. This is

especially interesting since the CM inoculum grown at the

site of inoculation exhibited a significantly higher incidence

of stunted leaders than did the two sources obtained at the

CM site. In closing our discussion about CM rust, evidence

of many kinds of differential interactions involving CM on

both Ribes and pine hosts (fig. 1 to 5) sends a message that

there is much more to host interactions with WPBR than

simple virulence / avirulence alleles in the rust and R genes

in the pine hosts. An initial step toward understanding

these complexities should be to reanalyze the initial test

(McDonald and others 1984) in light of the current knowledge about Cr2 genes in WWP (Kinloch and others 1999).

Case 3: Ontogenic Resistance

In Bingham’s (1972) large-scale screenings of 1966 and

1967, many thousands of seedlings in a number of control

lots and full-sib tester families germinated one year late.

Thus, seedlings belonging to the same seedlots were simultaneously inoculated at 1 and 2 years of age. Incidence of

needle infection and subsequent incidence of cankers were

analyzed for each seedling. For incidence of needle lesions,

WWP clearly showed genetic variance, environmental (developmental) variance, and plasticity in both resistant and

susceptible populations (fig. 6). No variation for plasticity

was evident because both populations moved in the same

direction. Thus, we can argue that age (primary needles vs.

secondary needles) has an equal influence in both resistant

and susceptible populations. This leads to the conclusion

that screening efficiency could have been enhanced by early

inoculation. As pointed out by Bingham (1972) the lower

needle lesion incidence in older seedlings is surprising given

that target increases about 4x by the end of the second

season. Take note that almost 30,000 seedlings were included in this test (fig. 6).

Analysis of canker incidence clearly illustrates why primary needle inoculation did not work (fig. 7). Incidence of

cankers does not differentiate populations under first season inoculation (within E1 NS). Meanwhile populations

were clearly separable when secondary needles were inoculated. This result also indicates a large amount of resistance

(spots but no cankers) in the control populations used in the

1964 and 1965 screenings. Bingham (1972) noted the essential differences from his perspective. So what, if anything,

does the reaction norm perspective add? In this case, two

obvious advantages are to focus attention on interactions

and development of questions from “outside the box” by

forcing consideration of a wider viewpoint. For example,

comparative incidence of both needle and stem infections

brings into question just how much and what kind of resistance was present in materials selected for “susceptible

controls” in the 1950s after just 25 years exposure of the

WWP to WPBR. During the Phase I screenings at Moscow,

Idaho, incidence of combined infection (spots and cankers)

varied from 0.5 to 1.0 for individual control lots, and no two

inoculations utilized the same control lots (Bingham 1972).

It appears that few of the WWP control lots supported

cankering incidences > 0.99, as had five of eight control lots

of EWP (Patton 1961).

Case 4: Analysis of Pine Rust G x E in

Japan

The western breeding programs have been very aware of

growth G x E and in some cases have gone to great lengths

to capture its potential in selection programs (Kitzmiller

and Stover 1996). In our introduction, we discussed indications gleaned from the literature that both rust-pine and

rust-Ribes G x E may be of great import to understanding

WPBR. We have just seen how reaction norms are constructed and interpreted, and from other sources (McDonald

and Andrews 1981, McDonald 2000), that WPBR-Ribes G x

E might play an important role in WPBR dynamics. Now we

will take a closer look at WPBR-pine G x E.

Yokota (1983) provided sufficient data and descriptions of

his experiments for us to apply a reaction norm analysis.

Several full-sib F1 families created within the north Idaho

Phase I breeding program and two lots of susceptible north

Idaho WWP were inoculated in Japan at two sites. Potted

seedlings were transported from Hokkaido to Nakashibetsu

440 km eastward where they were exposed to basidiospores

produced on local Pedicularis resupinata L. under natural
USDA Forest Service Proceedings RMRS-P-32. 2004 43

Breeding Rust-Resistant Five-Needle Pines in the Western United States: ... McDonald, Zambino, and Sniezko

conditions. The stressful nature of the latter site is indicated

by Yokota’s statement that in a related test conducted the

previous year, many WWP seedlings were killed by winter

exposure to cold temperatures. In the experiment we are

analyzing, seedlings were exposed from August 22 to October 16, 1978, and then transported back to Hokkaido. Identical families were exposed to artificial inoculation September 18 to 20, 1978, and again September 27 to 30, 1979, at

Hokkaido to basidiospores that developed naturally on local

P. resupinata. Mean spore cast for the two inoculations were

90 and 80/cm2 (Yokota 1983). Both naturally and artificially

inoculated seedlings were placed in the same nursery to

allow development of symptoms. Needle lesions were not

observed, but incidence of cankering was recorded based on

continuous observations for 5 years (Yokota 1983). Reaction

norms were constructed from Yokota’s canker incidence

data.

Our example is a bit light in numbers since the control at

one site included only 11 seedlings, but features of the

reaction norm facilitate the extraction of useful information.

One might say that the differences noted resulted from

different rust populations or inoculation procedures or both.

At one site the seedlings received a two-month natural

exposure, while at the other site they received two shortterm artificial exposures that delivered only about 85 spores/

cm2

. This is far short of the 2,000 spores/cm2 or more that are

delivered in artificial inoculations from Ribes leaves during

screening and experimentation (McDonald and others 1984).

The fact that one family group (WP22) went from having the

lowest to the highest incidence upon change of location,

while another went from highest to lowest (this change was

not significant due to low numbers) argues that the comparison may have a message, despite the aforementioned shortcomings. For one thing, the families tested generally gave

about 30 percent canker-free individuals after artificial

inoculation in Idaho and in the Nakashibetsu test so that

they still had 30 percent less incidence than controls (fig. 8).

However, almost no resistance was expressed at Hokkaido

(fig. 8). Significant differences were seen even though the

trees spent only two months at Nakashibetsu. There were

significant differences associated with family groups and

lots of G x E expressed in families WP19 and WP 22. Is this

complex picture caused by variation of virulence or aggressiveness on the part of the rust, or by environment acting on

gene expression in the pine or the rust? We have seen that

planting northern Rockies F2 WWP at a low elevation

(warm) coastal site increases susceptibility while planting

the same material nearby at a higher and more northerly

site resulted in expected performance (Hunt and Meagher

1989). If the Nakashibetsu site was consistently cold during

initial colonization (as mentioned, WP seedlings died of cold

exposure the previous winter) and the Hokkaido site was

warmer during initial colonization, then cool temperatures

during inoculation and early colonization is a common element that may be related to activation of resistance genes.

Expression of resistance genes in some pathosystems is

influenced by temperature (Pérez-Garcia and others 2001).

Another possible source of G x E is needle physiology. In

EWP, certain trees showing one-season needle retention

also exhibited low WPBR canker incidence (Hirt 1944).

Trees retaining one season’s needles became more normal

in their retention of three seasons of needles after transplantation to a new site, and there exhibited normal rates of

infection (Hirt 1944).

Case 5: Vigor-Quality Western White Pine

Plantations

Repeated measurements of experimental material [e.g.,

resistant material having or lacking general combining

ability (GCA) as well as susceptible materials] growing in

natural environments are a powerful source of information

about the dynamics of WPBR epidemics. In 1955, 1957, and

1959, outplantings of early generational materials from the

Phase I program (full-sib GCA x GCA and GCA x Non GCA,

open pollinated GCA, and Non GCA) were made at three

locations in north Idaho to monitor the vigor and quality of

the resistant families (Steinhoff 1971, Goddard and others

1985). Control lots arising from seed collected from infected

members of the same cohorts as the resistant selections were

also planted. In 1953 and 1955, OP seed was collected from

infected members of the cohorts of phenotypically resistant

trees at the same five locations to serve as control lots for the

1957 and 1959 plantings respectively (tree location data on

file). Controls for the 1955/1956 planting were collected, in

1951, from infected trees located outside of resistant-tree

selection sites. Plantings were replicated at Priest River,

Deception Creek Experimental Forest, and at Emerald Creek

on the Saint Joe National Forest, all in northern Idaho, USA.

Poor survival at Emerald Creek precluded further consideration of that site. Plantations were inspected for rust incidence in 1970, 1975, 1980, and 1997. The 38 to 42 year-old

trees were inspected from the ground for canker incidence in

1997. Records of individual trees were checked for continuity

across all dates and records of unknown mortality were

removed. Any record of rust infection during the life of a tree

placed it in the infected category. A few records lost continuity between the 1980 and 1997 inspections because specific

trees could not be relocated. The 1980 data were published

(Goddard and others 1985) and data for the other years are

on file (Moscow Forestry Sciences Laboratory). Reaction

norms were constructed using as data estimates of the

asymptote (K) of canker incidence obtained by fitting nonlinear functions to rust incidence data recorded at about 13,

18, 23, and 40 years. Equations (Madden and Campbell

1990) fitting expectations of the monomolecular (monocyclic

disease assumption), the logistic (polycyclic disease assumption with inflection assumed at 0.5 K), and Gompertz

models (polycyclic disease assumption with variable inflection point) were calculated with Table Curve software

(SPSS Inc). Each site initially received equal numbers of

seedlings from the same families at each planting, although

uneven numbers of losses due to planting occurred at each

site. Each of the three years contained a unique mix of fullsib families for each resistance category. Pivotal to this

discussion is the fact that the same mix of full-sib families

was planted at the two sites for each planting time.

Estimated K and rate parameters of the best fitting

equation, planting site, family groups, and standard errors

of the estimated K and rate values are given in table 1. Out

of nine combinations of site, family group, and planting

year at Priest River, eight of the best fits (highest R2 and

lowest SE for K and infection rate) were derived using the

Gompertz and, out of the nine at Deception Creek, eight were
44 USDA Forest Service Proceedings RMRS-P-32. 2004

McDonald, Zambino, and Sniezko Breeding Rust-Resistant Five-Needle Pines in the Western United States: ...

derived using the monomolecular model. This indicates the

rust was behaving in a fashion expected by a monocylic

disease cycle at Deception Creek and in a polycyclic fashion

at Priest River (fig. 9). The fit of these standard disease

progress curves to the long-term WPBR canker incidence

data was generally excellent for 18 combinations of families x site (table 1). Each genetic group seems to have its own

unique path (fig. 9). However, some common patterns are

evident. All attained their unique asymptote at about 25

years. At these sites and with these materials, once the

plateau was reached, it has been stable. Although a stable K

value at 25 years may not be obtained for epidemics at all

sites, as demonstrated in Kinloch and Byler’s (1981) longterm data collected at Happy Camp during the onset of the

outbreak of the Happy Camp race in resistant SP, it is

nonetheless true that when a stable predicted K value is

obtainable, it can be an excellent trait for reaction norm

analysis. If our plantings were composed of clones, or even

full-sib families, instead of groups of full-sib families, we

would have some very robust genetic information for drawing hypotheses about G x E interactions, as has already been

demonstrated with poplars (Wu 1998, Wu and Hinckley

2001).

In these plantings, significant differences in K parameter could be due to clumpy distribution of basidiospores,

clumpy microclimate, or variation in resistance among

trees within a resistance category (McDonald and Hoff

2001). Ribes were eradicated from the vicinity of the plantations prior to establishment (Steinhoff 1972), and few

bushes can be found in the local vicinity today. If a susceptible control lot can be shown to approach an incidence of

one, then we can assume that departures from unity are

due to resistance (McDonald and Dekker-Robertson 1998).

When K is not constant throughout an epidemic, rust

incidence alone is an inadequate measure of disease behavior. Under such cases, considering canker counts may be

required to augment the use of incidence data; a multiple

infection transformation function can then be used to

generate predictability and for understanding irregular

rust behavior.

We begin our analysis of the vigor quality (VQ) plantings

by inspecting the reaction norms of the susceptible controls

(fig. 10). Rust incidence of the 1955 OP controls at Deception

Creek reached 0.97 after 42 years of exposure (fig. 10). Even

through the 1955 control lots exhibited significantly lower

rust incidence at Priest River than at Deception Creek (fig.

10) we will assume that potential K = 1 at both sites. The

significant across-environment means for all three populations [significance estimated by log-odds ratio 95 percent

confidence limit according to Sokal and Rohlf (1995)] is

taken as evidence of a genetic difference among the OP

seedlots. This difference presumably arose from changes in

pollen cloud during the 4 years (1951 to 1955) over which the

OP seedlots were being collected. Further, the significant

difference between the within-environment K means suggests either that expression of the putative accumulated

resistance was affected by the environment or that the rust

populations differ. The significant interaction among 1955

vs. 1959 controls across environments argues in favor of

heritable variation in plasticity of the pine but against

variation in virulence among these rust populations.

Table 1—Fit of nonlinear equations to white pine blister rust canker incidence on full-sibs

western white pine growing in the vigor-quality plantations located on northern Idaho

Experimental Forest.

Full-sib Planting Planting Nonlineara Infection

group year location equation K ± SE rate ± SE

OP Cont 1955 Priest River Gomp 0.84 ± 0.025 0.16 ± 0.030

OP Cont 1957 Priest River Mono 0.83 ± 0.017 0.08 ± 0.005

OP Cont 1959 Priest River Gomp 0.72 ± 0.027 0.22 ± 0.058

GCA x GCA 1955 Priest River Gomp 0.38 ± 0.018 0.10 ± 0.017

GCA x GCA 1957 Priest River Gomp 0.33 ± 0.009 0.20 ± 0.046

GCA x GCA 1959 Priest River Gomp 0.43 ± 0.011 0.25 ± 0.031

GCA x N 1955 Priest River Gomp 0.35 ± 0.003 0.14 ± 0.007

GCA x N 1957 Priest River Gomp 0.40 ± 0.009 0.18 ± 0.028

GCA x N 1959 Priest River Gomp 0.51 ± 0.003 0.32 ± 0.011

OP Cont 1955 Deception Mono 0.97 ± 0.024 0.12 ± 0.013

OP Cont 1957 Deception Mono 0.89 ± 0.032 0.11 ± 0.015

OP Cont 1959 Deception Mono 0.77 ± 0.046 0.10 ± 0.017

GCA x GCA 1955 Deception Gomp 0.35 ± 0.008 0.14 ± 0.016

GCA x GCA 1957 Deception Mono 0.43 ± 0.005 0.12 ± 0.007

GCA x GCA 1959 Deception Mono 0.38 ± 0.015 0.11 ± 0.015

GCA x N 1955 Deception Mono 0.42 ± 0.007 0.11 ± 0.008

GCA x N 1957 Deception Mono 0.42 ± 0.018 0.12 ± 0.022

GCA x N 1959 Deception Mono 0.46 ± 0.055 0.06 ± 0.016

aGomp = Gompertz and Mono = Monomolecular equations (Madden and Campbell 1990).
USDA Forest Service Proceedings RMRS-P-32. 2004 45

Breeding Rust-Resistant Five-Needle Pines in the Western United States: ... McDonald, Zambino, and Sniezko

The full-sib GCA x GCA analysis was similar to the GCA

x nonGCA resistance in that E was not significant and P was

significant. These groups of families exhibited a nonsignificant G variance. Nevertheless, the downward slopes between the 1955 and 1959 plantings indicate the potential for

a significant G (fig. 11). Full-sib crosses between GCA and

other parents (mostly nonGCA) exhibited variable behavior

across the sites (fig. 12) that are probably due to differential

expression of genes. The families included in the 1955 and

1957 plantings show no distinctions at either site (fig. 12).

The site-mean Ks do not differ (no E variance) but the K for

the 1959 planting at PR is high and performance of the 1959

planting at PR and DC is significantly different, leading to

significant G and P variances. The absence of a difference

among sites for two family groups argues for similar rust at

both sites.

An over-all conclusion from this analysis of K based on

evidence of an overall reduction of the asymptote of disease

incidence at the end of the epidemic of 0.16 in the OP

controls in just 4 years (fig. 10) is that the WWP population

in north Idaho may have the natural capacity to respond

very quickly to rust pressure. Possible explanations are

rapid and significant changes in frequency of resistant genes

or expression of induced resistance that could even be

partially transgenerational in areas of extreme WPBR impact. However, we also need to remember that the WPBR

pathosystem is composed of other factors, such as

epiparasites, that may have been subject to change, so that

some alternative sort of disease attenuation or damping of

the ability of C. ribicola to damage pine might be in play

(Pfennig 2001, Ebert 1998, Levin and Bull 1994, Roy and

Kirchner 2000).

Long-term performance of the vigor-quality plantations

also indicates that overall early resistant populations obtained from the Phase I program performed equally well at

both sites in that both kinds of crosses resulted in about 60

percent clean trees. Individual groups of families showed

significant G x E for expression of resistance as well as

differences among the groups. These differences are more

than likely traceable to individual full-sib families that made

up the groups. Results from the plantings also strongly

indicate that perhaps site is more important than genetic

background in determining whether local epidemics will

behave in characteristically monocylic or polycyclic fashions.

An explanation for the existence of two kinds of epidemics

would add much insight to our understanding of rust epidemics. It is important to remember that well-characterized

materials (full-sibs given 50 years of natural rust exposure

and subjected to frequent examinations for rust behavior) are

an experimental and developmental treasure that we should

make every effort to protect and continue to develop.

Case 6: Changes in Rate “Constants” at

Full-Sib Resistance Evaluation Plantations

in Northern Idaho

One of the final tasks associated with completing the

Phase I program was establishment of three test plantations: Merry Creek (MC), Gletty Creek (GLC) and Jaype Mill

(JM) (Bingham and others 1973, McDonald and others 1994,

McDonald and Dekker-Robertson 1998, Fins and others

2002). MC and GLC are replicate sites, each consisting of

36, 0.4 ha plantings laid out in a randomized complete block

design for four stock types (Bingham and others1973). Each

contains full-sib F2, F1, B1 (backcross from F1 to original

parents), and local OP controls (that is, different seedlots

representing different genetic backgrounds). MC, established in 1970, is located about 82 km east of Moscow, Idaho.

GLC, established in 1972, is about 205 km northwest of

Moscow near the town of Newport, Washington. JM, established in 1971, is about 125 km southeast of Moscow and

contains only control and full-sib F2 stock. At MC, a 225-tree

subset of permanent sample trees from each resident stock

had been selected, tagged, and inspected for rust incidence

and severity at 2, 4, 6, 12, and 26 years. Many of the original

225 selected plants at MC have since been lost to animals

and other unknown causes to the extent that the original

design was destroyed (McDonald and Dekker-Robertson

1998). A hot site-preparation burn that stimulated a heavy

stand of evergreen ceanothus (Ceanothus velutinus Douglas)

further compromised the design. In response, the stand

was divided for analysis into two site classes: brush and

forest. This partition produced two classes of about equal

size for all stock types, as described in an earlier report

(McDonald and Dekker-Robertson 1998). The GLC and JM

plantations remained largely intact, but each experienced

lower rust incidence than MC and were inspected only 3

times (1973, 1983, and 1996). Incidence in 1973 was too low

for analysis. The JM layout paired F2 and control plots

having 64 planting spots. In each plot, WWP seedlings

alternated with those of grand fir (Abies grandis (Dougl.)

Lindl.) and Douglas-fir (Pseudotsuga menziesii (Mirb.)

Franco.). All 32 WWP on each plot were inspected each time.

Blocking at these plantations enabled use of ANOVA to

construct reaction norms of infection rate and canker accumulation rate for two periods: plantation establishment to

1982, and 1982 to 1996. Infection rate was computed using

Madden and Campbell’s (1990) equation 18 – the monomolecular equation where K is assumed to equal one and initial

incidence is set to zero. Rate of infection from 1982 to 1996

was computed using the same equation with initial incidence set equal to the 1982 value. Canker accumulation rate

was initially computed by dividing the average number of

cankers in 1982 by the number of years of exposure, but for

the final period, the number of cankers in 1982 was subtracted from the 1996 number and the result divided by 14

years. These values were subjected to ANOVA (Systat 9.1,

SPSS) and reaction norms constructed to compare stocks

growing at JM and GLC.

All variances for comparison of WPBR annual infection

rate between the two periods of assessment were highly

significant. The across-environment mean indicated that

the local GLC control was almost 2 times more susceptible

than the Jaype local control growing at Jaype, yet examination of F2 material that was of the same genetic background

at both sites indicated that the sites were of equal hazard

(fig. 13). Could an explanation be found in natural selection

operating at different levels of selection in different natural

stands? The expectation is that WPBR had a much larger

impact on WWP near the JM site on the Clearwater National

Forest than in the GLC vicinity on the Colville National

Forest. The significant interaction term is also of interest.

The controls show no differential interaction as materials at
46 USDA Forest Service Proceedings RMRS-P-32. 2004

McDonald, Zambino, and Sniezko Breeding Rust-Resistant Five-Needle Pines in the Western United States: ...

both sites showed a proportional gain in infection rate for the

second period. The interaction is generated by the nonsignificant plasticity test for the F2 at both sites (fig. 13).

Cankering rate provides a different and unexpected result

(fig. 14). In contrast with the increasing parallel reaction

norms for infection rate in the controls over the two periods

(fig. 13), cankering rate moved from a difference of 6x to a

nonsignificant difference. While values were much smaller

and differences not significant, the trend for the F2 was the

same. In addition, the JM cankering rate appeared flat and

was not significantly different between periods. The GLC

control illustrated that infection rate can increase while

cankering rate is simultaneously decreasing. Does the JM

control represent a pathosystem that is coming to a stable

asymptote and therefore a stable and predicable K parameter? On the other hand, could the increasing cankering rate

coupled with an increasing infection rate signify an impending unstable K?

Case 7: Anatomy of an Unexpected

Epidemic—The Merry Creek Plantation

An expected epidemic at the MC site provides some insight

into stability or instability of K, although the relatively

small numbers of designated “sample” trees remaining from

plantation establishment to the end of sampling removed

any chance of computing significances in the standard fashion. The good news is that enough of the sampled population

remained in records and on the ground to establish patterns

and generate hypotheses. Infection rate and cankering rate

were computed as for GLC except that the F1 was used as a

substitute control because none of the highly susceptible

“true” control seedlings survived to the second period (fig.

15 and 16). All resistant stocks exhibited nearly equal

infection rates during the first period when all the controls

were becoming infected and dying (fig. 16) (McDonald and

Dekker-Robertson 1998). Most infection rates increased 7 to

8x with very little differentiation between the brush and

forest sites for the F1 (fig. 16). In striking contrast, the F2

stock growing in the forest site-type had rates of canker

incidence increase by 20x (fig. 15). Infection rate at MC

shows interesting patterns when displayed with the F2 data

from JM and GLC in a common format (fig. 16). Of note is the

almost parallel nature of most reaction norms. The MC

forest-site-type F2 is most similar to the F2 growing on the

low hazard GLC and JM sites and is not distinguishable

from the JM control in spite of the huge increase in cankering rate. This argues that the entire increase in number of

cankers occurred on already infected trees! How can such an

increase in amount of inoculum not result in an increase in

number of previously clean trees becoming infected? An

additional observation is that the highest overall rate increase (F2 brush-site-type) was closely followed by the F1

brush-site-type. Did the presumed large increase in aeciospore production by F2 forest-site-type trees result in

increased infection rate on F1 and F2-brush trees? Allowing

ourselves to consider even “outside the box” possibilities,

could this pattern signify spontaneous local appearance of a

microcyclic form of WPBR capable of infecting pine to pine,

and if so, might it recently have evolved, or might it be a new

arrival? WPBR is known to encompass microcyclic forms or

species (Imazu and others 1991, Imazu and Kakishima

1995). An intercontinental “river of dust” has been demonstrated to allow viable transport of spores of some fungi

(Griffin and others 2002); could microcyclic aeciospores from

Japan occasionally have survived such a journey?

These relationships can be studied in other ways. It has

been argued (McDonald and Dekker-Robertson 1998) that

the increase in infection at MC above the expected K of 0.34

in the F2 was just the continuation of a constant rate that

after 26 years resulted in the observed 0.89 incidence of

infection. Since 7 out of 26 plantings of F2 stock in north

Idaho have exceeded the expected asymptote of 0.34

(McDonald and others 1994, Fins and others 2002) in the 12

years or less it took MC F2 to exceed the threshold, a

definitive answer about the causal dynamics is demanded.

As a focal point for this discussion (not intended to convey

statistical significance), we present a hypothetical rust progress

curve for the MC F2 based on some of the tools we have been

discussing (fig. 17). By fitting the monomolecular equation

(McDonald and Dekker-Robertson 1998 equation 1) using

the first four data points from MC (Table Curve 2D, SPSS),

a K of 0.5 is obtained. If we next assume the appearance of

a variant that can overcome the resistance that was responsible for the K = 0.5 of the first 10 years of the epidemic

and then simulate the behavior based on a new timeline,

with the 10th year as year 0, then incidence in year 10 = 0,

12th = .5 and 26th

year = .89. The perfect fit with K = .89 that

these three points allow (fig. 17), illustrate the limitations of

fitting a multiparameter function to a small data set but is

also intriguing. This figure illustrates some important points.

Control and F2 stock are clearly delineated and the hypothetical curve is similar to the actual curve shown for SP at

the Happy Camp site (Kinloch and Byler 1981). Interruption

of the established cycle of inspections at MC by a bureaucratic decision to stop WPBR research in northern Rocky

Mountains in 1983 precluded knowing the exact shape of

Figure 17—Expected rust progress curve for Merry Creek

site (north Idaho) second generation resistant families

(F2

) if epidemic asymptote were breached by Cronartium

ribicola adjustment (plasticity or evolution?) compared

with control lot. Both populations growing on forest sitetype (Case 7 in text; data from McDonald and DekkerRobertson 1998).

-0.1

0.1

0.3

0.5

0.7

0.9

1.1

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Years of Rust Exposure

Rust Incidence (Proportion Infected)

Full Fit Actual F2 1st Stage 2nd Stage Cont Actual C 
USDA Forest Service Proceedings RMRS-P-32. 2004 47

Breeding Rust-Resistant Five-Needle Pines in the Western United States: ... McDonald, Zambino, and Sniezko

the MC progress curve. However, the hypothesized hump in

the rust progress curve (fig. 17) might even now be verified

or rejected by aging cankers at MC after the fact, in order to

construct a reasonable progress curve.

Summary and Conclusions _______

The above discussion reinforces several significant lessons relating to resistance breeding, deployment, and management of all western five-needle pines. Of primary importance, pathosystems of long-lived plants demand long-term

commitment in an atmosphere that fosters freethinking as

well as commitment and focus to getting a big job accomplished efficiently. This probably means that lasting and

workable relationships need to be forged between research

and the practical breeding programs. Efforts in the past

(Kinloch and Byler 1981, McDonald and others 1984,

McDonald and others 1991, Kinloch and others 1999) exemplify such cooperation and sharing of data, and should be

encouraged. The breeding programs generate large amounts

of reliable data that are seldom subjected to peer review but

could be of inestimable value, provided that such programs

ensure that uniform and “adequate” control crosses are

routinely included in artificial inoculations. Greater interregional cooperation may also help elucidate new resistance

mechanism, for example mechanism “X” that USDA Forest

Service Region 6’s breeding and screening program at Dorena

has observed in northern Cascade Mountain populations

and that could potentially be related to resistance mechanisms in the northern Idaho WWP populations. Examination of this mechanism could be of critical importance in

delineating seed zones and understanding the genetic structure of host populations. Recent studies of migration patterns of whitebark pine strongly suggest a north-south

dichotomy whose boundary is in the southern Washington

Cascades (Richardson and others 2002). This boundary

could also be characterized by a significant change in the

kinds and numbers of resistance genes in both WBP and

WWP. Tools that are presently available to assess field

performance are powerful, useful, and relatively inexpensive, if appropriate experimental designs and inspection

intervals are maintained. Parting guidelines are as follows:

Establish permanent plots so that adequate numbers of

individuals can be tracked for at least 50 years. Ensure that

repeated and blocked designs will provide sufficient numbers over time. Define measurement protocols to ensure

continuity of data quality through changes in personnel.

Ensure that canker incidence and severity, age, and size are

recorded for whole trees to provide data appropriate for

epidemiological analysis. Embrace the potential discriminatory power of pathosystem G x E by ensuring that genetic

groupings to be tested (clones, full-sib families, half sib

families and etc) are replicated in at least two environments.

Finally, and perhaps most importantly, create customized

control lots having known behavioral specifications and use

them to link plantings in different geographic regions.

When dealing with long-term pathosystems, refrain from

thinking “inside the box”. For example, a strong correlation

had been expected between density of Ribes populations and

resulting WBBP impact (McDonald and Hoff 2001). Quite to

the contrary, evidence is accumulating that such correlations are weak in the northern Rockies (Toko and others

1967, McDonald unpublished data) and southwestern Oregon (McDonald unpublished data). Two explanations are

evident. First, western basidiospores may be more robust or

travel further than assumed so that far fewer telial infections are needed to cause a corresponding amount of pine

damage expected under the 300m “limit” of basidiospore

spread. Second, other hosts could be involved; forms of C.

ribicola are known in Japan and South Korea that alternate

to Ribes spp and Pedicularis; in Japan and Germany, a form

cycles to Ribes only; in South Korea, a form cycles only to

Pedicularis; and in Canada it cycles to Ribes (Stephan and

Hyun 1983). There is one report of a single branch of one

plant of common red paintbrush (Castillia miniata Douglas,

a North American Scropulariaceae) inoculated with WPBR

that produced teliospores (Hiratsuka and Maruyama, 1976),

and another report of artificial infection of this host but

without spore production (Patton and Spear 1989). However, infections of artificially inoculated plants of this and

other Scrophulariaceae were not obtained at multiple field

sites by Hunt (1984), despite successful infection of “appropriate” Castilleja hosts after inoculation with stalactiform

rust. Another potential problem with measuring WPBR

susceptibility is the assumption that any old woods run

collection of the pine host will make an adequate control.

Custom controls should be developed and maintained for use

by all breeding programs. This paper presented initial evidence that unknown mechanisms may cause rapid accumulation of “resistant” WPP. Theoretical discussions about

disease attenuation are beginning to appear that invoke

various kinds of evolutionary and plasticity (polymorphisms

and polyphenisms) adjustments (Pfennig 2001, Ebert 1998,

Levin and Bull 1994, Roy and Kirchner 2000). If the WPBR

pathosystem is as dynamic as we have suggested, then an

entirely new plan of attack may be needed to ensure that we

can successfully restore and/or maintain ecosystems containing or requiring a five-needle pine component.

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Abstract—An overview of genetic research on native and exotic

five-needle pines (Pinus subgenus Strobus) in Europe, including the

impact of white pine blister rust (Cronartium ribicola), is presented.

The natural populations of Pinus cembra from the Alps and

Carpathians are free from blister rust; even though the rust occurs

throughout Europe on other five-needle pines and Ribes species.

Pinus strobus was once considered to be an important exotic species

for timber production in Europe, but plantations have been abandoned due to the high susceptibility to blister rust. Blister rust

resistance has now been transferred to P. strobus through hybridization with Eurasian five-needle pines, and the potential for

successful utilization of the species in Europe now exists. Other fiveneedle pine species are not of major interest for European forestry

operations.

Key words: five-needle pines, Europe, genetics, Cronartium ribicola,

genetic resistance, provenance, breeding, hybrid, heritability, genetic gain

Introduction ____________________

Only two species of five-needle pines (Pinus L. subgenus

Strobus Lemm.) grow naturally in Europe: Cembran pine

(Pinus cembra L.) and Balkan pine (P. peuceGris.) (Critchfield

and Little 1966). Exotic five-needle pine species have been

used to enrich European forests due to the relatively low

number of native species and have been the subject of much

research and development. For this paper, we prepared a

survey on genetic research and use of five-needle pine

species in European countries (table 1). We used the responses of the survey in conjunction with published literature and personal communications to compile the following

overview on five-needle research and development in Europe.

Cembran pine is distributed in the high-altitude forests in

the Alps and the Carpathian region, including the Tatra

Mts. (Georgescu and Ionescu-Barlad 1932, Critchfield and

Genetic Research and Development of FiveNeedle Pines (Pinus subgenus Strobus) in

Europe: An Overview

Ioan Blada

Flaviu Popescu

Little 1966, Holzer 1975, Contini and Lavarelo 1982). It is

naturally distributed in the following countries: Austria,

France, Germany, Italy, Poland, Romania, Slovakia, Switzerland, and Ukraine. Cembran pine is important for reforestation of subalpine forests, including restoration of forests

near timberline to stabilize watersheds and reduce the risk

of avalanches and flash floods (Holzer 1975). The species is

used to create mixed Norway spruce (Picea abies (L.) Karst)

– European larch (Larix decidua Mill)-Cembran pine stands

at high elevations for increased wind resistance (Blada

1996). Cembran pine also contains high genetic resistance to

white pine blister rust caused by Cronartium ribicola J.C.

Fisch in Rabenh. (Bingham 1972a,b, Soegaard 1972, Holzer

1975, Hoff and others 1980, Blada 1987, 1994a) Cembran

pine produces a dense-brown-reddish wood useful for handicrafts (Contini and Lavarelo 1982), and is an excellent

landscaping tree due to the crown color, density, and a

conical-oval shape (Blada 1997b).

Balkan pine naturally occurs in Albania, Bulgaria, Greece,

and Macedonia and is confined to higher elevations in the

Balkan and Macedonian regions (Nedjalkov 1963, Fukarek

1970, Mitruchi 1955, Popnikola and others 1978). Balkan

pine is important for planting in severe mountain climates

to prevent soil erosion, as well as timber production for

furniture, barrels, and other purposes (Figala 1927, Nedjalkov

and Krastanov 1962, Nedjalkov 1963, Popnikola and others

1978). The species has good tolerance to SO2 pollution

(Enderlein and Vogl 1966) and has shown high blister rust

resistance in genetic tests containing both European and

North American species (Delatour and Birot 1982, Blada

1987, 2000a, 2000b, Heimburger 1972, Hoff and others

1980). Balkan pine has been used in crossing with other

white pines and is considered a good bridging species (Righter

and Duffield 1951, Kriebel 1963, Patton 1966, Nikota and

others 1970, Heimburger 1972, Blada 1987).

The Cembran and Balkan pines have and are still planted

most countries where they naturally occur (table 1, columns

9, 10, 12, and 13). Cembran pine, however, has been less used

in France, Germany, and Italy. These species are relatively

slow growing and as more five-needle pine species from

around the world became known to Europeans, foresters

began to experiment with plantations of exotic species.

Exotic five-needle pine species have had a long history in

Europe. The following five-needle pine species have been

more frequently planted: P. strobus L., P. monticola Dougl.,

P wallichiana A. B. Jacks., P. sibiricaDu Tour, P. koraiensis

Sieb & Zucc., P. armandii Franch., P. flexilis James, and P.

lambertiana Dougl. (Schmitt 1972, Soegaard 1972). Eastern white pine (P. strobus) was one of the first five-needle

pines to be introduced to Europe. With minor exceptions,
52 USDA Forest Service Proceedings RMRS-P-32. 2004

Blada and Popescu Genetic Research and Development of Five-Needle Pines (Pinus subgenera Strobus) in Europe: An Overview

eastern white pine was introduced in all European countries (table 1, column 5) and planted for economic reasons

(table 1, column 11) (Radu 1974). The species has demonstrated good qualities for timber production, is well adapted

to the European climate (Schmitt 1972, Kriebel 1983) and

has proven to be resistant to SO2 pollution (Enderlein and

Vogl 1966). The other exotic five-needle pine species have

shown to be of minor importance, being used primarily for

landscaping and in arboretums. Some Asiatic species have

been introduced as genetic sources for blister rust resistance. However, the vast majority of research and development efforts on exotic five-needle pines in Europe have

been conducted on eastern white pine

In Europe, eastern white pine was first recorded in 1553,

in the Royal Gardens of Fontainebleau, France (Lanier

1961), followed by introduction at Badminton, Great Britain

(MacDonald and others 1957) and in Germany in 1770

(Schenck 1949). The first records of the eastern white pine in

other European countries were: Switzerland 1850 (Litscher

1908), Poland 1876 (Bialobok 1960), Slovakia 1773 (Musil

1969), Austria 1886 (Cieslar 1901), Romania 1894 (Davidescu

1894) and Bulgaria 1903 (Rusakoff 1936).

Small plantations of eastern white pine were established

at intervals through the late 18th and 19th centuries, as the

species exhibited good growth. This was especially true in

Germany during the great reforestation period that took

place through the 18th and 19th centuries (Borchers 1952,

Schmitt 1972). Initially, it appeared that eastern white pine

would become one of the most important trees of the European forests. In west central Germany, eastern white pine

could outgrow all European coniferous species and keep a

dominant position in stands for more than 80 years (Schmitt

1972). The species’ growth performance also was remarkable under various site conditions in other countries such as

Poland, Czechoslovakia, Romania, and Russia (Radu 1974).

However, white pine blister rust has seriously impacted the

survival and growth of this species. Blister rust attack has

halted planting of eastern white pine for wood production in

all countries in the last two decades (see table 2, column 14).

Table 1—Survey results for native white pine and Pinus strobus presence, past or present occurrence of white pine blister rust, past planting or

breeding work, and current planting or breeding work in European countries. (Y= yes; N=no; ?=unknown)

Native Introd. PPBROa PPb PPBWc

Rank Country P.c P.p P.s P.c P.p P.s P.c P.p P.s P.c P.p P.s

1 2 3 4 5 6 7 8 9 10 11 12 13 14

1 Albania N Y ??????????

2 Austria Y N Y N N Y Y N Y Y N N

3 Belarus N N Y N N Y N N Y NNN

4 Belgium N N Y N N Y N N Y NNN

5 Bulgaria N Y Y N N Y N Y Y N Y N

6 Croatia N Y? Y N N Y N N Y N ? Y

7 Czech Republic N N Y N N Y N N Y ?NN

8 Denmark N N Y N N Y N Y Y NNN

9 England N N Y N N Y N N Y NNN

10 Estonia N N Y N N Y N N Y NNN

11 Finland N N Y N N Y N N Y NNN

12 France Y N Y N N Y N? N Y N? N N

13 Germany Y N Y N N Y N? N Y N? N Y

14 Greece N Y Y NNNNNNNNN

15 Hungary NNNNN Y N N Y NNN

16 Ireland N N Y N N Y N N Y NNN

17 Italy Y N Y N N Y Y? N Y Y N N

18 Latvia N N Y N N Y N N Y NNN

19 Lithuania N N Y N N Y N N Y NNN

20 Luxembourg N N ? N N ? ? N ? N N ?

21 Macedonia N Y Y N N Y N Y Y N Y N

22 Moldova NNNNNNNNNNNN

23 Netherlands N N Y N N Y N N Y NNN

24 Norway N N Y N N Y NNNNNN

25 Poland Y N Y N N Y Y N Y Y ? N

26 Portugal NNNNN?NNNNN?

27 Romania Y N Y N N YYYYYYY

28 Slovakia Y N Y N N Y Y N Y Y N N

29 Slovenia N N Y N N Y N N Y N?N

30 Spain N N ? N N ? ? ? Y? N N ?

31 Sweden NNNNN Y NNNNNN

32 Switzerland Y N Y N N Y Y N Y Y N N

33 Ukraine Y N Y N N Y Y N Y Y N N

34 Yugoslavia N Y? Y N N Y N Y Y N Y? N

a

PPBRO = past and present blister rust occurrence.

b

PP = past planting or breeding work.

c

PPBW = present planting or breeding work; P.c, P.p, P.s = P. cembra, P. peuce, P.strobus.
USDA Forest Service Proceedings RMRS-P-32. 2004 53

Genetic Research and Development of Five-Needle Pines (Pinus subgenera Strobus) in Europe: An Overview Blada and Popescu

According to our survey (table 1 column 11), eastern white

pine was planted over almost all Europe. Germany contains

the largest planted area, approximately. 25,000 ha, of eastern white pine (Stephan, personal communication). In France,

plantations with eastern white pine were established during

the 19th century. The first plantations for wood production in

Romania were established at the beginning of the 20th

century, but much larger areas (about 1,360 ha) were established after 1960 (Radu 1974). Based on the initial results of

provenance trials, Croatia planted approximately 1,500 ha

(Gracan, personal communication).

White pine blister rust began to cause severe problems as

early as 1865, when the rust was first noticed by H. A.

Dietrich in Estonia (Leppik 1934). The rust spread throughout Europe by 1900 via the alternate host Ribes nigrum L.

and susceptible P. strobus genotypes (Georgerscu and others

1957, Bingham and Gremmen 1971). It was not until 1926,

however, that Germany recognized high mortality in eastern white pine due to the disease (compare Schmitt 1972).

Correspondingly, planting eastern white pine in Germany

was prohibited (Tubeuf 1927). Unfortunately, the species

began to be planted again in Germany after 1935 (Wappes

1935). Other European countries followed the German lead

(Radu 1974), and suffered serious economic losses due to

blister rust. For example, the rust invaded almost all young

stands in Romania after 1970, promoted by the simultaneous culture of eastern white pine and Ribes nigrum (Blada

1982). Blister rust attacks eastern white pine in all countries

where the species has been planted (table 1, column 8). The

rust does not attack P. cembra nor P. peuce in their natural

habitats, however, in spite of concurrence with susceptible

P. strobus and Ribes species (table 1, columns 6 and 7).

Research Studies _______________

Provenance Trials

Once widespread planting with eastern white pine stopped,

genetic research activities began to slow. Despite the problem of blister rust, Germany and Croatia still continue to

make measurements in their provenance trials (table 1,

column 14).

Provenance testing of eastern white pine has been conducted in Germany, where two trials with 69 provenances

were established in 1966 and 1967, respectively (Stephan

1974). All provenances were originated from the natural

range of the species. Growth rate, mortality, and infections

by blister rust were assessed at age 11. (Stephan 1974).

Height at age 11 was negatively correlated with provenance

latitude (r = -0.40 to r = -0.51). Provenances from southern

Appalachian mountain States of North Carolina, South

Carolina, and Virginia (south of the 39th degree of latitude)

showed better growth than the average in these trials.

Provenances from areas north of the 45th degree of latitude

(Manitoba, Quebec, Ontario, New Brunswick, Minnesota,

and Wisconsin) had poor growth. Significant (p less than 0.5)

and highly significant (p less than 0.1) height-height correlations among provenances at different ages were found. The

correlations were not strong when the differences in age

were great, but particularly strong between heights from the

age of five onward. A high number of dwarfed trees from one

provenance from Illinois were noticed. Under natural conditions, differences among provenances could be observed for

mortality and infection by blister rust. Pinus monticola (one

provenance) and P. wallichiana (two provenances), tested in

the same German trial, showed only 85 percent and 40

percent, respectively, of the average height of P. strobus. In

addition, high mortality could be observed in P. wallichiana.

The Croatian research program included 10 eastern white

pine provenances, of which six from North America and four

from established plantations in Croatia. The tests were

established in 1970 and were measured at age 18 (Orlic

1993). For American provenances, the average values for

survival, height, and diameter were 70.7 percent, 12.8 m and

20.7 cm, respectively. Similarly, the survival and growth

estimates for local provenances were 73.2 percent, 13.7 m

and 22.2 cm, respectively, which were higher (3.5, 7.0, and

7.2 percent, respectively) than the American provenances.

In term of survival, the local source Hrvatska ranked first

and the American provenance New Hampshire ranked second. The New York (USA) and the other two local sources

exhibited the poorest survival. The average of total height

ranged from 11.9 m to 14.0 m, with an average of 13.3 m,

whereas the diameter from 17.7 cm to 23.2 cm with an

average of 21.5 cm. The best and the poorest provenance in

diameter were Georgia and Wisconsin, respectively. No

information was given about blister rust resistance.

Croatia also conducted a species comparison test with

eastern white pine, Scots pine (P. sylvestris L.), black pine

(P. nigra Arnold), European larch, and Douglas-fir

(Pseudotsuga menziesii (Mirb). Franco) was established in

Tocak area. After 23 years of testing, eastern white pine was

found to have produced the greatest about of volume per

hectare (479 cu. m), in comparison to 214, 209, and 164 cu. m/ha

produce by Scots pine, European larch and Douglas-fir,

respectively (Orlic and Ocvirek 1993). In a 26 year-old trial

at Slatki Potok, eastern white pine generated 549.1 cu. m/ha

in comparison to only 270 cu. m/ha produced by black pine

(Orlic and others 1997). These results demonstrate the

superior performance in wood production of eastern white

pine in European site conditions.

In Romania, a nursery provenance test of eastern white

pine was planted with 45 provenances: 19 from North

America and 26 of unknown origin taken from old stands

planted across Europe. The weight of 1,000 seeds per provenance was assessed before sowing (Radu 1974). Seed weight

means ranged from 11.4 g to 23.2 g, with a mean of 16.9 g.

The North American provenances averaged 15.9 g, whereas

European provenances weighted 8.8 percent more (17.3 g).

Twelve traits including dry matter were measured at age 2.

The total height ranged from 8.9 cm to 15.9 cm, with

Romanian local seed sources surpassing the other groups

of provenances. Significant differences were found among

provenances for dry matter content. The provenances from

northern Minnesota (USA) had the lowest average height,

but the highest proportion of dry matter. In contrast, the

fastest growing provenances (North Carolina, Kentucky and

Ohio, all from the USA) had the lowest dry matter content

even though their height was greater.

A nursery provenance test of Cembran pine was established in Romania. The test consisted of 12 provenances

including seven from the Carpathian Mountains and five

from the Alps. Blada (1997a) found significant differences
54 USDA Forest Service Proceedings RMRS-P-32. 2004

Blada and Popescu Genetic Research and Development of Five-Needle Pines (Pinus subgenera Strobus) in Europe: An Overview

among provenances for total height growth, annual height

growth and root collar diameter. The top four provenances

were Pietrele, Gemenele and Calimani from the Carpathians

and Blunbach Grunalpe from the Austrian Alps, which

exhibited faster height, height incremental growth, and root

collar diameter than other provenances at age 6. Duncan

multiple range tests (1955) for these traits suggested that

major gaps separated provenances within the natural range

of the species; that is, genetically distinct populations could

be found in both the Alps and Carpathian Mountains. The

same data suggested a discontinuous pattern of distribution

suggesting the absence of a gene flow among populations.

There were highly significant positive correlations between

all traits, suggesting that indirect selection can be applicable. No significant correlations were found between any

growth trait and geographic coordinates.

Provenance tests of Balkan pine were investigated in

Bulgaria. Thirteen provenances, originating from 1,700 m –

2,100 m in the Pirin and Rila Mountains, were represented

in each of the two tests. At age 3, it was found that the Balkan

pine provenances from high elevations initiated growth

earlier than the provenances from middle elevations (Dobrev

1997a). Approximately 90 percent of the seedlings had

lammas shoots at the end of the August of the third growing

season. Significant correlations were found between seed

size with provenance latitude and elevation. Also, a significant positive correlation was found between height growth

and needle length. No significant correlations were found

between height growth and latitude or with elevation of

provenances.

Dobrev (1997b) investigated the concentration of macroand microelements in the needles of the 3-year-old seedlings

from different Balkan pine provenances. Variation of concentration in macro- and microelements of needles was

discontinuous among provenances. Provenances from the

northern Pirin Mountains greatly differed in their relative

concentration of nitrogen and calcium and copper as compared with provenances from southern Pirin and Rila and

Central Balkan Mountains. A geographic differentiation in

magnesium concentration of the needles was found. The

Pirin Mountains provenances had a higher concentration of

magnesium than provenances from Rila and Central Balkan

Mountains. A significant positive correlation was found

between phosphorus concentration and seedling height.

About three decades ago, various provenance trials were

established in Ukraine (Yatsyk and Volosyanchuk, personal

communication). A P. cembra trial with six provenances

originating from Ukrainian Carpathian Mountains was

established on 0.5 ha. At age 28, the mean height growth,

diameter and volume were 5.0 m, 9.3 cm and 31 cu m/ha were

achieved. Two tests of P. koraiensis comprising 19 provenances of unknown origin were planted on 2.4 ha. The

latest measurement took place at age 22 and 30. The mean

height growth, diameter, and volume in the first and second

tests were 6.7 m, 11.8 cm, and 8.1 cu m/ha, and 3.9 m, 7.3 cm,

and 8.5 cu m/ha, respectively. A trial with four P. pumila

Regel provenances of Russian origin was established on

0.25 ha. The mean height growth and diameter at the

ground level at age 30 were 11.3 m and 3.6 cm, respectively.

A 2.5 ha test of P. sibirica with 36 provenances of Russian

origin was planted. The height growth, diameter and volume

at age 23 were 5.4 m, 10.9 cm and 2.4 cubic meters per

hectare, respectively. A second trial with 31 provenances, of

the same origin, was established on 13 ha. Until now, no

other measurements were made. Two P. peuce provenances

of unknown origin were planted on 0.1 ha. Height, diameter

and volume at 22 years of age, measured 7.0 m, 12.5 cm and

13.2 cubic meters per ha, respectively. All provenances of the

above species proved to be resistant to blister rust.

The Ukrainians also planted tests of North American

species. A 0.1 ha P. flexilis plantation with one provenance

of unknown origin was made about 33 years ago. The height

growth and diameter at age 21 were 6.3 m and 10.8 cm,

respectively. All provenances exhibited high susceptibility

to blister rust and only 10 percent of trees survived after 21

years of testing. In the same timeframe, a P. strobus trial

with three provenances of unknown origin was planted on

0.1 ha. After 23 years of testing, the height growth, diameter

and volume were 9.7 m, 23.4 cm and 56.3 cu m/ha, respectively. Heavy blister rust attack occurred and only 1.5

percent of the trees survived.

Open-Pollinated (Half-Sibling) Progeny

Tests

Blue pine—In 1971 an international program for testing

white pines of known origin for resistance against blister

rust was proposed (Bingham and Gremmen 1971). Another

program dealt with seed collection and exchange (Kriebel

1976). As part of this program, Romania received 36 openpollinated families of blue pine (P. wallichiana) from 16

provenances originated from Pakistani Himalayan Mountains. This material was tested for growth and blister rust

resistance by artificial inoculation. Eight traits, including

blister rust resistance and height growth were measured at

age 11, and the results reported by Blada (1994b). Highly

significant differences were observed among families for

blister rust resistance (BR1), percentage of trees free of

blister rust (BR2), tree survivors (BR3), total height growth

(HT), and stem volume (V). BR1, BR2, BR3, HT, and V

averaged 3.2 points (10 = the highest resistance), 9.7 percent, 49 percent, 10.4 dm, and 0.458 dm3 respectively.

Genetic variance estimates for BR1, BR2, BR3, HT, and V

accounted for 91, 99, 96, 79, and 38 percent, respectively, of

phenotypic variance. Therefore, this high amount of genetic

variance could be used in a blue pine breeding program.

Narrow-sense heritability estimates at the family level for

BR1, BR2, BR3, and HT were high: 0.909, 0.998, 0.960, and

0.974, respectively. Heritability for V was much lower (0.380).

These estimates, coupled with the large amount of variation

observed within blue pine population suggest a two-way

selection program for rust resistance and growth would be

rewarding. If the best 5, 10, or 15 families were selected and

planted on sites more or less similar to that used in this trial,

a genetic gain of 67, 51, and 40 percent in BR1 and 18, 14, and

11 percent in V, respectively, could be achieved.

A highly significant positive phenotypic correlation was

found between blister rust resistance and latitude. All families that originated from above 35∞ N latitude exhibited a

higher resistance to rust than families from lower latitudes.

No significant phenotypic correlations were found among
USDA Forest Service Proceedings RMRS-P-32. 2004 55

Genetic Research and Development of Five-Needle Pines (Pinus subgenera Strobus) in Europe: An Overview Blada and Popescu

growth traits and latitude or between growth traits and

blister rust resistance. Therefore, growth and blister rust

resistances are independent traits, indicating that improvement using indirect selection is not possible.

Cembran pine—As part of the Romanian five-needle

pine breeding program, a nursery test with 136 open pollinated families of P. cembra was established. Highly significant differences among families were found for height,

root collar diameter (RCD), and total number of branches

(TNB) (Blada in preparation). The genetic coefficient of

variation for height, RCD, and TNB were 22, 14.6, and 26.3

percent, respectively, and the narrow-sense heritability

estimates for height, RCD and TNB were 0.968, 0.938, and

0.966, respectively. Consequently, an improvement program with stone pine would be yield positive results.

Genetic correlations among height with TNB, RCD, and

TNB were high: 0.881, 0.571 and 0.713, respectively. Selection in height or in RCD should lead to an indirect increasing

of the TNB. However, an increased number of branches is a

negative characteristic, as it lowers the quality of wood.

Therefore, the number of branches in the next generation

should be minimized by selecting fast growing trees with

small number of branches.

If selecting the best 30, 35, 40, or 45 of the 136 families,

genetic gains in height of 28.8, 26.8, 25.1, and 23.4 percent,

and in RCD of 18.8, 17.6, 16.4, and 15.3 percent, respectively, could be expected. By using these early test results to

guide an operational improvement program, two types of

production seed orchards were planned: a seedling seed

orchard using the fastest growing seedlings in the best 45

families and a clonal seed orchard using ortets from the best

45 trees.

Breeding and Seed Orchards

A few countries reported clonal seed orchards, such as:

Cembran in Austria, Slovakia Romania and Ukraine; Balkan

pine in Romania; and eastern white pine in Croatia and

Romania.

A genetic improvement program was started in Romania

in 1977 due to the potential importance of eastern white pine

in consideration of blister rust. This program included both

intra- and interspecific hybridization and has a final objective of establishing seed orchards composed of selections

with high general combining ability for resistance to blister

rust (Blada 1982). Due to the lack of official interest in fiveneedle pines, the scope of the initial program was restricted

to continuing the measurements in the already established

trials. These hybrid trials perform well, and additional

details will be given elsewhere in these proceedings.

Another Romanian breeding program under way is concerned with P. cembra. The program was initiated in 1989

with the following objectives: (a) phenotypic selection of

parents in natural populations; (b) provenance and halfsibling family testing; (c) full-sibling family (both from intraand inter-specific crossing) testing and genetic parameters

estimation; and (d) seed orchards establishment with the

best combiners for both improved mass seed production and

as a base population for advanced-breeding population (Blada

1990).

Full-Sibling Progeny Tests

Romaina was the only country that reported full sibling

progeny tests in response to the survey. A 10 x 10 full diallel

crossing experiment was conducted in Gemenele on native

populations of P. cembra from Romanian Carpathian Mountains. The experiment was conducted to provide information

on the genetic variation and inheritance of important breeding traits. Cotyledon number, total height, annual height

increment, RCD, TNB, and lammas shoots formation were

measured from age 2 to 6 (Blada 1999). The most prominent

result from this experiment was that significant general

combining ability (GCA), specific combining ability (SCA),

and reciprocal effects for all traits were found. Also significant maternal effects occurred in number of traits, suggesting control by nuclear and extranuclear genes and by nuclear

x extranuclear gene interactions.

Growth measurements indicated a progressive increase

with age of the GCA variance within phenotypic variance.

The GCA variance of the total height growth, increased from

2 percent at age 2 to 25 percent at age 6, while the GCA

variance of the root collar diameter increased from 8 percent

at age 4 to 14 percent at age 6. Similarly, the SCA variance

of the total height growth ranged from 15 percent at age 2 to

27 percent at age 6. The diallel analysis showed that both

GCA and SCA variances were important sources of variation. Dominance variance exerted a greater influence on 10

out of 17 traits as evidenced by SCA / GCA variance ratios.

However, the magnitude of these ratios suggested that

additive effects might be almost as important as nonadditive

effects in the study. Consequently, the breeding strategy can

employ both additive as well as nonadditive variations,

indicating that considerable progress under direct selection

is possible. If two out of 10 randomly selected parent trees

exhibited significant GCA effects for total height, then it can

be estimated that 20 percent of trees within the basic

natural population could be selected as good combiners.

Heritability estimates were high enough to ensure genetic

progress in improving growth and other traits. For height

growth, heritabilities for both family and single tree level

increased from age 2 (0.065 and 0.021 respectively) to age 6

(0.453 and 0.366 respectively). In the same manner, heritabilities for root collar diameter, for both family and individual level, increased from 0.228 to 0.321 and from 0.126 to

0.157, respectively.

Interspecific Hybridization

P. strobus x P. peuce hybrids—In Romania a 7 x 4

factorial crossing was conducted between eastern white pine

(female) and Balkan pine (male) to combine the rapid growth

of eastern white pine with high resistance to blister rust of

Balkan pine (Blada 2000a). The resulting families were

artificially inoculated at age 2 and planted in the field at age

6. Blister rust resistance (BRR), trees free of blister rust

(TFBR), tree survival (TS), tree height (H), diameter at 1.30

m (D), basal area (BA), stem volume (V), stem straightness

(SS), and branch thickness (BT) were measured at age 17.

Highly significant differences among hybrid families were

found for all traits except stem straightness. Selection at
56 USDA Forest Service Proceedings RMRS-P-32. 2004

Blada and Popescu Genetic Research and Development of Five-Needle Pines (Pinus subgenera Strobus) in Europe: An Overview

family level, therefore, can be carried out for the most

economically important traits, including BRR and V.

There was large genetic variation among the parents for

all traits examined. The effects of eastern white pine female

parents were significant not only for growth traits but for

BRR, TFBR, and TS as well. This suggests that there were

(1) additive genetic control for all traits and (2) parents with

high GCA could be selected for breeding. The existence of

resistance to the blister rust within eastern white pine (as a

female parent) agreed with results found by Riker and

others (1943), Riker and Patton (1954), Patton and Riker

(1958), and Patton (1966) and were contrary to Heimburger

(1972). Balkan pine (as a male parent) had significant effects

on growth traits but no significant effects on BRR, TFBR,

and TS. Therefore, all male Balkan pine parents exhibited

the same level of resistance to blister rust in this study, as

found by Blada (1989) at an earlier age of the study. This was

contrary to Patton’s study (1966), which found differences in

blister rust resistance among his P. peuce selected parents.

Male x female interaction effects were significant and highly

significant for all traits except for stem straightness, suggesting a nonadditive gene action on most traits.

Significant, positive phenotypic correlations were found

among growth traits. Such correlations imply significant

genetic gains in these traits even if selection was practiced

on only one trait. However, correlations between stem

straightness and growth traits were low, ranging from 0.30

to 0.32. Low phenotypic correlations (0.01 to 0.33) were

obtained between blister rust resistance and growth traits,

thereby suggesting that the two traits were inherited independently and tandem selection cannot be applied.

Mid- and high-parent heterosis was calculated (MacKey

1976, Halauer and Miranda 1981). Balkan pine was found to

be the best parental species for blister rust resistance and

stem straightness, whereas eastern white pine was the best

parent species for all growth traits. Mid-parent heterosis

was positive for all but one trait and accounted for 34 percent

for BBR, 55 percent for TFBR, and 53 percent for TS.

Substantial mid-parent heterosis was also found in most

growth traits, such as 26 percent in volume growth. The total

height growth had the lowest (13 percent) positive midparent heterosis, while the stem straightness was the only

trait displaying a negative mid-parent heterosis. Highparent heterosis was negative for all traits. For example, at

age 17, this heterosis accounted for -5 percent for blister rust

resistance, -8 percent for trees free from blister rust, and -9

percent for total height growth. Generally, hybrids were

intermediate between the two parental species over all

characteristics and incorporated desired characteristics from

both parent species.

Genetic gain using the average of breeding values of the

best parents was calculated. Selecting the best three eastern

white pines (females) for blister rust resistance (average

breeding value was 0.833) would result an increase of 9.5

percent for blister rust resistance. Similarly, using the best

five Balkan pines (males - average breeding value was 21.6

dm3

) to cross with the above females for volume growth rate

would result in a genetic gain of 18.3 percent in the overall

mean (118.0 dm3

).

P. strobus x P. wallichiana hybrids—In Romania, a

factorial crossing was conducted among seven female trees

of eastern white pine and four male trees of blue pine to

combine the rapid growth of former species with high resistance to blister rust of the latter species (Blada 2000c). The

hybrid families were artificially inoculated at age 2 and

planted at age 6. Blister rust resistance (BRR), TS, H,

annual height growth, D, BA, V, SS, and BT were the

measured traits at age 17.

Factorial analysis indicated significant differences among

hybrid families for all traits except branch thickness. The

effects of eastern white pine (female) were significant not

only for the growth traits, but for BRR and TS, again

suggesting an additive genetic control in all growth traits

and blister rust resistance and that high GCA parents could

be selected for breeding. Blue pine (male) had significant

effects on TS, H, tree survivors, annual height growth, BA,

and SS, but no significant effects on BRR, D, V, and BT. It is

important to note that blue pine male parents exhibited the

same level of blister rust resistance. Male x female interaction effects were significant except for TS and SS, suggesting

that nonadditive gene action had an influence on all economically important traits.

The contribution of GCA variance to the phenotypic variance was 87 percent for H, 53 percent for D, and 77 percent

for V; whereas the contribution of SCA variance to the same

traits was lower, that is, 10, 41, and 22 percent, respectively.

Both additive and dominance variances could be used for

improvement in wood production. The contribution of GCA

variance to the phenotypic variance was 73 percent for BRR

and 53 percent for TS, while the contribution of the SCA

variance for the same traits was only 9 percent for the former

and 0 percent for the latter trait. The GCA / SCA variance

ratios demonstrated that there was additive genetic variation for all traits. The GCA-F / GCA-M variance ratios

revealed that estimates of GCA variance of females were

much greater than estimates of males for all traits, except

BT. These results suggested that the greatest amount of

additive variance associated with both blister rust resistance and growth traits was found within eastern white pine

parent population. The blue pine male parent contribution

to the additive variance was insignificant for blister rust

resistance but significant for some growth traits.

The narrow-sense heritability estimates at the family

level were high for all traits. For example, the estimates of

0.828 and 0.885 and 0.777 for BRR, H, and V, respectively,

were obtained. The magnitude of heritability estimates was

due to the high level of additive variance attributable to the

female parents. In general, the individual-tree narrow sense

heritabilities appeared to be high for blister rust resistance

(h2

w = 0.421), moderately high for total height growth (h2w=

0.327), low for diameter (h2

w= 0.122), and very low for branch

thickness (h2

w= 0.085). In conclusion, heritability estimates

were high enough to ensure progress in improving genetic

blister rust resistance and growth traits by using P.

strobus x P. wallichiana F1 hybrids.

Both positive and negative GCA effects, which significantly (p<0.05) differed from zero, were generally found for

both male and female parents for most traits. None of the

blue pine male parents had significant GCA effects on BRR

as these parents exhibited the same level of resistance. The

range of estimated GCA effects among parents suggested

that it may be possible to select parents with superior

breeding values for BRR and growth traits.
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Genetic Research and Development of Five-Needle Pines (Pinus subgenera Strobus) in Europe: An Overview Blada and Popescu

Genetic and phenotypic correlations between SS and

growth traits were relatively low, ranging between 0.143

and 0.288. Both types of correlations between BT and total

height were high and negative (-0.543 and -0.533), indicating that larger trees produced thinner branches. High,

positive genetic and phenotypic correlations were obtained

between these two traits with BRR, rG = 0.928 and rp = 0.916

respectively. In addition, high, positive genetic correlations

were obtained between BBR and D (rG = 0.680), BA (rG =

0.655), and V (rG =. 608), suggesting that tandem selection

can be applied.

Estimates of high-parent heterosis were positive only for

TS and height and negative for all other traits. From these

estimates, it is evident that hybrids combined their parental

genes for both rapid growth and blister-rust resistance.

These results may justify the use of F1 P. strobus x P.

wallichiana hybrid production. Genetic gains could be realized in increasing in both blister-rust resistance and timber

production. Even smaller increases in resistance and volume growth would give appreciable improvement in yield,

especially when considered in relation to large-scale plantation programs.

Host-Parasite Investigations

In order to investigate the pathogenic variation of C.

ribicola a German-Korean joint inoculation experiment was

conducted (Stephan and Hyun 1983, also in Stephan, these

proceedings). Cronartium ribicola strains used in the German experiment were able to infect only Ribes nigrum, but

not Pedicularis resupinata L., the alternate host species in

Korea. Therefore, this host plant species cannot be considered as a host for the German fungus material. Cronartium

ribicola strains used in the Korean experiment could infect

only P. resupinata, thereby confirming pathogenic variation

in C. ribicola.

The same joint experiment corroborated other reports,

such as La and Yi (1976), that demonstrated a wider pathogenic variation of C. ribicola in eastern Asia. There is an

increase of blister rust strains with a stronger virulence

than had been observed in Korea since 1963 (La and Yi

1976)) and Japan since 1972 (Yokota and others 1975).

Apparently, only the Ribes host-strain of C. ribicola had

invaded Europe in the last century, and from Europe was

subsequently introduced to North America. Introduction of

the Pedicularis-host strain of C. ribicola into North America

would be a potential disaster for endemic five-needle species

(Stephan and Hyun 1983), as this strain is very virulent.

In 1971, the International Union of Forest Research

Organizations (IUFRO) proposed an international program

for testing white pine blister rust resistance. Parallel trials

were carried out in the Western and Eastern United States,

in France, South Korea, Japan and Germany (Bingham and

Gremmeen 1971, Stephan 1986). In Germany, 14 species

with a total of 63 provenances/progenies have been tested by

artificial inoculation. After 10 years, Stephan (1986) found

significant differences among the species investigated. Asian

five-needle pines are generally more resistant to the blister

rust than North American species. No or relatively weak

symptoms were observed in P. pumila, P. parviflora Zieb. &

Zucc., P. sibirica, and P. koraiensis. Surprisingly, a few

provenances of P. wallichiana showed high infection rates.

Most of native North American white pines showed a very

high infection rate of more than 90 percent. Pinus albicaulis

Engelm., P. flexilis, P. lambertiana and P. monticola were

severely damage, although P. aristata Engelm. was relatively less infected. The fungus infected nearly 100 percent

of the tested P. strobus provenances. This partially can be

explained by the theory that the gene center of blister rust

and blister rust resistance is in north central Asia (Bingham

and others 1971).

There were no differences among provenances within

species. The experiment also included a few F1 and F2

progenies between P. lambertiana and P. monticola, which

possessed a certain level of improved resistance to blister

rust under North American conditions. Only one provenance

of P. lambertiana showed 20 percent less attack than in

unimproved provenances. In P. monticola, the F1 and F2

progenies selected for rust resistance were severely attacked

7 years after artificial inoculation. The comparison of the

German results with those obtained in France (Delatour and

Birot 1982), Japan (Yokota 1983), and North American tests

were interesting. Three years after inoculation there were

significant correlations between the German and French

results with respect to the percentage of rust-free trees in the

seedlot BR n∞ 41 (P. lambertiana) and seedlot BR n∞ 43 (P.

monticola). There were no significant correlations with the

American results and the French seedlot BR n∞ 46 results,

nor were there correlations with the Japanese results

(Stephan 1986).

A similar IUFRO test was carried out in France. This

French test demonstrated that the species from Europe and

Asia proved to be less susceptible to rust than the American

species. (Delatour and Birot 1982).

A survey on blister rust resistance in native (Cembran

pine) and introduced five-needle pine species was conducted

in Romania (Blada 1982, 1990). After 1970, blister rust had

caused severe attacks to all young stands of eastern white

and western white pines. This severe outbreak was promoted by simultaneous culture of the five-needle pine species with Ribes nigrum. Mature populations and young

seedlings from natural regeneration of P. cembra distributed throughout the Carpathian Mountains were free from

blister rust. Ribes alpinum L. and R. petraeum Wulf. Populations coexist at high altitude with Cembran pine and were

also free from blister rust, although they have been shown to

be susceptible (Georgescu and others 1957, Blada, unpublished data). After approximately two decades of survey,

blister rust still could not be found on Cembran pine nor the

Ribes species (Blada, unpublished data). The absence of the

infection on both Cembran pine and natural Ribes populations indicates that the Romanian Carpathian Mountains

do not represent a gene center for C. ribicola, as suggested

by Leppik (1967). The rust was recently introduced via

eastern white pine seedlings from Germany and Ribes sp.

collections from elsewhere (Blada, in preparation).

Research at Molecular Level

Studies concerning genetic differentiation and phylogeny

of stone pine species based on isozyme loci are summarized

in a previous publication and in this volume (Krutovskii and
58 USDA Forest Service Proceedings RMRS-P-32. 2004

Blada and Popescu Genetic Research and Development of Five-Needle Pines (Pinus subgenera Strobus) in Europe: An Overview

others 1992, Politov and Krutovskii in these proceedings).

Another molecular study of a Russian five-needle pine

(P. sibirica) was conducted by Goncharenko and others

(1992). Enzyme systems in the seeds of natural populations

from various parts of Siberia were analyzed by starch gel

electrophoresis, and 36 alleles at 20 loci were defined. Of

the genes controlling these enzyme systems, 55 percent

proved to be polymorphic, with an average 17.6 percent of

gene/tree being heterozygous. Interpopulation genetic diversity accounted for a little over 4 percent of the total

genetic diversity. The Ney distance coefficient ranged from

0.008 to 0.051, with an average of 0.023. The data obtained

suggested lack of any marked genetic differences between

central and marginal Siberian populations.

Genetic diversity and differentiation among five populations of Cembran pine from the Italian Alps were studied by

means of isoenzyme variation at 15 loci and contrasted with

five Scots pine populations (Bulletti and Gullace 1999). The

two species showed similar values for the mean number of

alleles per locus and percentage of polymorphic loci, while

the expected heterozygozity for Scots pine was higher than

that for Cembran pine (0.332 vs. 0.281). All the populations

studied showed an excess of homozygotes; the Allevet population of Cembran pine had the highest value of fixation

index (0.206). Furthermore, the latter stand exhibited the

lowest allelic richness index value. Only 2.7 and 3.5 percent,

respectively, of the observed genetic diversity in Cembran

and Scots pines was due to differentiation among populations. Therefore, the populations for each species studied

share similar respective gene pools, and there were no

barriers hampering gene flow. The results of the study

provide useful information for in situ conservation of genetic

variability. Moreover, the data obtained can also be used for

the identification of the most valuable stands for the production of high quality seeds.

A comprehensive study concerning phylogenetic relationship among P. cembra, P. sibirica and P. pumila, using

microsatellites and mitochondrial nad1 intron 2 sequences

was recently completed (Gugerli and others 2001). The

three-chloroplast microsatellite loci combined into a total of

18 haplotypes. Fourteen haplotypes were detected in 15

populations of P. cembra and one of P. sibirica, five of which

were shared between the two species, and the two populations of P. pumilacomprised four species-specific haplotypes.

Mitochondrial intron sequences confirmed grouping of the

species. Sequences of P. cembra and P. sibirica were completely identical, but P. pumila differed by several mutations and insertions/deletions. A repeat region found in the

former two species showed no intraspecific variation. These

results indicate a relatively recent evolutionary separation

of P. cembra and P sibirica, despite their presently distinct

distributions. The species-specific chloroplast and mitochondrial markers of P. sibirica and P. pumila should help

to trace the hybridization in their overlapping distribution

area and to possibly identify fossil remains with respect to

the still unresolved postglacial recolonization history of

these two species.

Acknowledgments ______________

The authors express their gratitude to all colleagues from

several countries who supplied information concerning genetic activities in five needle pines in their own countries.

Particularly, our thanks are due to Drs. R. Stephan (Germany),

R. Yatsyk and R. Volosyanchuk (Ukraine), J. Gracan

(Croatia), A. Nanson (Belgium) and R. Longauer (Slovakia).

The thanks are extended to our technician, Cristiana Dinu,

for her help during the preparation of this report.

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In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

The authors are with the Forest Research Institute, 132, St. Kliment

Ohridski Blvd. 1756 Sofia, Bulgaria. E-mail: forestin@bulnet.bg

Abstract—Macedonian (Balkan or Roumelian) pine (Pinus peuce

Griseb.) is a five-needle pine native to the Balkan Peninsula,

occupying in Bulgaria an area of 14,223 ha. Genetic investigations

made in Bulgaria include determination of the monoterpene composition of oleoresins, the delineation of geographic and ecological

races, detailed analysis of progeny tests and other genetic studies.

Many of the natural stands have the status of national parks and

reserves with a total area of 5,250 ha, including 65 seed stands with

an area of 709 ha. In addition, 152 candidate-elite trees have been

selected. Ex situ methods for conservation of the genetic resources

of this species include 40 clones in seed orchards (10 ha), six half-sib

progeny trial plantations (5.6 ha), five provenance trial plantations

(7.2 ha), and a forest seed bank. The indigenous populations of

Macedonian pine in Pirin are a valuable genetic resource available

for the introduction of this species into other countries of Europe,

and also North America and Asia.

Key words: Pinus peuce Griseb., genetic resources, in situ

conservation and ex situ conservation.

Species Distribution _____________

Pinus peuce Griseb.is found only in the Balkan Peninsula,

occurring in some of the high mountains of Bulgaria, Serbia,

Macedonia, Montenegro, Albania and Greece in the range

between 41∞ and 43∞ northern latitudes. In Bulgaria, the

natural range of this species consists of two parts separated

by the valley of the Vardar River. The eastern part is in

southwestern Bulgaria and includes Pirin Mountain,

Slavyanka Mountain (Ali Botush), Rila Mountain, the western Rhodopes, Vitosha Mountain, and the Central Balkan

Range. The western part includes Macedonia, southwestern

Serbia, southeastern Montenegro, eastern Albania, northeastern Greece and some spurs of the Dinar Alps, including

Prokletija, Kom, Sekiritsa, Sar, Pelister, Kozhuh, Nidje,

Korab, Rudoka, and Tsena (Dimitrov 1963).

Genetic and Conservation Research on

Pinus peuce in Bulgaria

Alexander H. Alexandrov

Roumen Dobrev

Hristo Tsakov

Distribution in Bulgaria __________

The easternmost occurrence of Macedonian pine is in the

Central Balkan Range. The westernmost, which is also the

northernmost population, is on Sekiritsa Mountain, and the

southernmost is in the Pelister, Nidje and Tsena Mountains

(Dimitrov 1963). The areas occupied by the species in Bulgaria, listed by mountain, are as follows: Pirin 7,175 ha, Rila

6,230 ha, Central Balkan Range 193 ha, Western Rhodopes

170 ha, Vitosha 104 ha and Slavyanka 57 ha. Within these

areas, P. peuce stands are scattered like islands, the most

compact ones being those in the Pirin, Rila, Prokletija and

Pelister Mountains.

There are two complexes of the species on Pirin Mountain,

one in the northeast with an area of 3,775 ha, where the

altitudinal distribution of the trees ranges from 1,600 to

2,200 m, and one in the southwest with an area of 3,400 ha

with an elevation range from 1,700 to 2,200 m.

On Rila Mountain there are three P. peuce complexes, one

in the southern part (1,635 ha in area and 1,700 to 2,000 m

in elevation), one in the central part along the Rilska river

(911 ha and up to 2,100 m elevation), and one in the northern

part (3,684 ha with a 1,600 to 2,100 m range in elevation).

In the Central Balkan Range, two populations separated

by the main ridge have been differentiated, one of 188 ha on

the northern slope, from 1,500 m to 1,900 m elevation, and

the other on the southern slope with an area of 5 ha and from

1,300 m to 1,400 m elevation.

On Slavyanka Mountain, P. peuce occurs in groups and as

solitary trees, while on Vitosha Mountain and in the Western Rhodopes the species occurs mainly in plantations. On

Sredna Gora it occurs only in plantations totalling 88 ha

(Alexandrov 1998).

In 2000, the total wood volume of the 14,223 ha of

Macedonian pine stands in Bulgaria was 4,198 000 m3

,

distributed by age class from I (1-20 years) to VIII (141-160

years) and following approximately the normal curve. The

stands of age classes V (81-100 years) and VI (101-120 years)

had the largest area, totalling 6,037 ha (42.5 percent of all

stands) with a growing stock of 2,160 000 m3 (51.5 percent of

the total wood volume).

The overall average volume of the Macedonian pine forests in Bulgaria is 295 m3

/ha, the average quality class is III

(medium) and the rotation period is 160 years (Tsakov 2001).

The average stand volume exceeds that of Picea abies (L.)

Karst and is considerably higher than that of Pinus silvestris

L. (Krastanov 1970).
62 USDA Forest Service Proceedings RMRS-P-32. 2004

Alexandrov, Dobrev, and Tsakov Genetic and Conservation Research on Pinus peuce in Bulgaria

Genetic Research _______________

Genetic studies of the Macedonian pine in Bulgaria, which

were performed during the last 10 years, included a seed

stand in each of the following areas, except for the larger

number of stands in the Pirin and Rila regions, as indicated:

1. Pirin (3 Forestry Estates) – 1,900 m altitude, 10 seed

stands

2. Gotse Delchev Forestry Estate – 1,800 m alt.

3. Bansko Forestry Estate – 1,700, 1,800, 1,900, 2,000 m alt.

4. Razlog Forestry Estate – 1,700, 1,900, 2,000, 2,100 m

alt.

5. Rila (7 Forestry Estates) – 1,800, 2,000 m alt., 9 seed

stands

6. Belitsa Forestry Estate – 1,900 m alt.

7. Yakoruda Forestry Estate – 2,000 m alt.

8. Rila Monastery Forestry Estate – 1,800 m alt.

9. Kostenets Forestry Estate – 1,900 m alt.

10. Samokov Forestry Estate – 1,800 m alt.

11. Doupnitsa Forestry Estate – 1,800, 1,900 m alt.

12. Central Balkan (Ribaritsa Forestry Estate – 1,700 m alt.

Analyses were made of variation in monoterpene composition and in morphological and physiological characteristics.

Monoterpene Variation

Monoterpene composition was determined from apical

buds, 2-year-old needles, wood samples and bark from 2-yearold branches collected from representative Macedonian pine

populations in the northern Pirin Mountains, the northern

Rila Mountains and the northern slopes of the Central

Balkan Mountain. Twelve monoterpenes were identified,

eight of them (a-pinene, camphene, b-pinene, D-3-carene,

myrcene, limonene, b-phellandrene and terpinolene) having

relative proportions above 0.5 percent, regardless the origin

of the samples or the investigated tissue. It was shown that

the populations studied differ statistically in their monoterpene compositions. This made possible the division of the

Macedonian pine populations from Northern Pirin, Rila and

Central Balkan into separate geographical races based on

the monoterpene composition of the oleoresins (Dobrev 1992).

Variation in Morphological and

Phenological Characteristics

Measurements were made on cones, seeds and seedlings.

On the basis of these results, the Macedonian pine population from Southern Pirin could be distinguished as a separate geographic race. It was established that the repeatability coefficients for these traits, i.e. repeatability in different

areas, are relatively high for the origins from the central

parts of the species natural range in Bulgaria, whereas the

reproductive materials (cones and seeds) from the marginal

parts of the natural range have lower coefficient values

(Dobrev 1995).

With respect to 20 characteristics reflecting the morphology of the cones, cone scales, seeds and the sizes, morphology

and phenology of 1-year-old seedlings in half-sib progeny

trials, a phenotypic similarity was established between mature

trees of 13 representative provenances of Macedonian pine

from Pirin, Rila and Central Balkan Range. Based on of the

calculated similarity matrix and the dendrogram of grouping pattern of populations from these mountains, the taxonomic distances were shown to be large and not proportional to the geographic distances between them. The

results were comparable with those from preliminary studies of monoterpenes from sample trees in several P. peuce

populations in Bulgaria (Dobrev 1996).

Identification of Geographic/Ecological

Races

From analysis of 44 morphological, growth, phenological

and chemical traits characterizing the populations of

Macedonian pine and their progenies, it was possible to

distinguish five geographic and ecological races of this species in Bulgaria, as follows: Central Balkan, Rila, Southern

Pirin, and Northern Pirin (where one middle mountain and

one high mountain ecotype could be separated).

In an evaluation of growth rate differences, as determined

from total tree height, statistically significant differences

(p = <0.01) were found among six-year-old families from 13

Bulgarian seed sources in five half-sib progeny trials distributed over a diversity of sites. Test locations included Stara

Reka Forestry Estate at 1000 m elevation, Sliven Forestry

Estate at 1000 m elevation, Yakoruda Forestry Estate at

1450 m elevation, Belitsa Forestry Estate at 1650 m elevation and Kostenets Forestry Estate at 1850 m elevation

Family heritability estimates for height growth were statistically significant, varying with site from 0.220 to 0.574

(Dobrev 1998).

A 10th-year evaluation of these 13 progenies growing in

four of these locations (excluding Kostenets) showed that the

fastest-growing trees came from the Northern Pirin region

at 1,900 m elevation. This population was consistently

superior in growth in different tests at elevations from 1,000

to 1,450 m.

Genetic Considerations in Reforestation

and Afforestation

Macedonian pine is one of the species most suitable for

restoring the upper forest zone below the tree limit, which,

in many mountains, has been moved down as a result of

human interference. The trial plantations of P. peuce, which

have been successfully established in the high parts of the

mountains, provide a reason for expanding these plantings.

However, satisfactory growth is possible only if transfer of

genotypes is from lower to higher altitudes, with a maximum

vertical seed transfer distance of 300 m (Alexandrov 1998).

Conservation of Genetic

Material________________________

In situ Conservation

National parks, nature parks, reserves, seed stands and

plus trees provide in situ conservation of genetic resources of

Macedonian pine. The total area of the natural forests,
USDA Forest Service Proceedings RMRS-P-32. 2004 63

Genetic and Conservation Research on Pinus peuce in Bulgaria Alexandrov, Dobrev, and Tsakov

which are included in Pirin, Rila and Central Balkan National Parks and Bayuvi Dupki – Djindjiritsa, Yulen,

Rilomanastirska Gora, Parangalitsa, Ibar, Tsarichina and

Central Rila Reserves, amounts to 5,250 ha. The gene pool

of this species as a whole is preserved through the genetic

diversity inherent in these different ecological regions.

The preservation of gene resources in permanent seed

stands provides the basis for implementing a breeding

programme. There are 65 P. peuce seed stands in Bulgaria,

totalling 709 ha, or approximately five percent of the

Macedonian pine forests. These seed stands are sufficient to

meet anticipated needs for the species (Alexandrov 2000). In

addition, the 152 candidate-elite Macedonian pine trees

showing phenotypically superior growth, form and resistance (trees showing no damage from insects and diseases)

have been selected by the Forest Seed Testing Stations in

Sofia and Plovdiv. This in situ selection in natural stands

provides a base of information and material for future

genetic investigations, breeding improvement and greater

utilization of P. peuce for reforestation and afforestation.

Ex situ Conservation

The genetic resources of Macedonian pine are also being

preserved ex situ in Bulgaria through provenance testing

plantations, progeny trial plantations, seed orchards and

gene banks for seeds. Provenance trials at ages of 28-31

years include plantations on Rila Mountain at 2,050 m, the

Western Balkan Range at 1,650 m and 1,700 m, and the

Rhodopes at 2,050 m and 2,100 m, with a total area of 7.2 ha

(Dakov and others 1980).

There are six half-sib progeny trial plantations, age 12 at

the time of writing of this paper, distributed in various parts

of the species range at elevations from 1,000 to 1,850 m. In

all, 170 half-sib families from 13 provenances are being

tested in these plantations on a total of 5.6 ha.

A 32-year-old, 10 ha clonal seed orchard in the Western

Rhodopes (1450 m elevation) includes 40 clones (Bogdanov

1970).

Assessing the relative advantages of in situ and ex situ

conservation of the genetic resources of Macedonian pine,

the first seems to be a more reliable method for Bulgaria,

because of the growth and health of the species under a

diversity of ecological conditions. Our results indicate

that the native populations of Macedonian pine in Pirin

are an especially valuable genetic resource for the introduction of this species into many countries of the Northern Hemisphere.

References _____________________

Alexandrov, A. 1998. Pinus peuce Grisb. – In Enzyklopädie der

Holzgewächse, ECOMED, Landsberg, Germany, III-1:1-10.

Alexandrov, A. 2000. Genetic conservation of conifers in Bulgaria –

In: The Balkan Ecology 3:5-10.

Bogdanov, B. 1970. Seed Orchards ? from Pinus peuce. -In: Proc.

Symp. on the Macedonian pine, Skopje, 211-220 (in Serb.).

Dakov, M., I. Dobrinov, A. Iliev, V. Donov, S. Dimitrov. 1980.

Raising the upper forest limit. Zemizdat, Sofia, 220 pp (in Bulg.).

Dimitrov, T. 1963. Roumelian pine (Pinus peuceGrisb.), Sofia, 116 pp

(in Bulg.).

Dimitrov, M. 1980. The Macedonian pine (Pinus peuce Grisb.).

Zemizdat, Sofia, 180 pp (In Bulg).

Dobrev, R. 1992. Monoterpene composition of the essential oil of

some populations of Macedonian pine (Pinus peuce Grisb.) in

Bulgaria. Forest Science (Sofia) 2:8-16 (In Bulg.).

Dobrev, R. 1995. Intraspecific variation in Pinus peuce in Bulgaria:

cone characteristics. In: Carying for the Forest: Research in a

Changing World. Abstracts of Invited Papers. IUFRO XX World

Congress, Tampere, Finland, p. 146.

Dobrev, R. 1996. Phenotypic similarities of representative populations of Macedonian pine (Pinus peuce Grisb.) in Bulgaria. Forest

Science (Sofia) 4:16-23 (in Bulg).

Dobrev, R. 1998. Variance, genotypic stability and family mean

heritability of the growth in height of 6-year-old seedlings of

Macedonian pine (Pinus peuce Grisb.) in a series of half-sib

progeny trial plantations. Forest Science (Sofia)1-2:5-23. (in

Bulg).

Krastanov, K. 1970. Wachstum, Leistung und Technisches

Haubarkeitsalter von Pinus peuce - Bestände in Bulgarien.

Berichte. Symposium über Pinus peuce, Skopje, pp 277-289 (In

Serb.).

Tsakov, H. 2001. Composition and productivity of natural

Macedonian Pine endrocoenoses in Northern Pirin Mountain and

created anthropogenic ones in Vitosha and Sredna Gora Mountains. D.Sc. Thesis, Sofia, 137 pp. (in Bulg.).
64 USDA Forest Service Proceedings RMRS-P-32. 2004

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

Anatoly I. Iroshnikov is with the Research Institute of Forest Genetics and

Breeding, Voronezh, Russia. Dmitri V. Politov is Senior Research Associate,

Laboratory of Population Genetics, N.I.Vavilov Institute of General Genetics,

Russian Academy of Sciences, 3 Gubkin St., GSP-1, Moscow 119991, Russia.

Phone: 7 (095) 135-5067. Fax : 7 (095) 132-8962. E-mail: dvp@vigg.ru.

Abstract—Pinus sibirica Du Tour, P. pumila (Pall.) Regel and P.

koraiensis Sieb. et Zucc. are primarily located in Russia and occupy

about 36.69, 38.30 and 2.87 million ha in the Asian part of Russia,

respectively. These species, together with P. strobus L., P. peuce

Gris., P. cembra L. and P. monticola Doug. ex D. Don, have been

introduced in the European part of Russia. In all genetic tests P.

strobus was found to have a high growth rate, but it was severely

affected by white pine blister rust (Cronartium ribicola J.C. Fisch.).

An intensive breeding program that would incorporate disease

resistance genes from P. peuce is suggested to address this problem.

P. sibirica is resistant to blister rust and has highly nutritious edible

seeds and, thereby, is considered the most promising five-needle

pine species for propagation in the European part of Russia. Its

disease resistance and high intrapopulation variation in reproductive and growth traits provide a good basis for selecting new

varieties. More than 2,500 plus trees have been selected throughout

the entire P. sibirica area, and 6,100 ha of genetic reserves have

been established. Growth traits of 12 to 32 year old plantations were

studied on six common garden test sites in Siberia and on three sites

in the European part of Russia. Seed sources that exhibited optimum growth were from the Altai and Sayan Mountains in southern

Siberia and the southern taiga in western Siberia and are considered to contain the most valuable gene pool. Progeny from northern

and sub-alpine populations had growth rates 20-40 percent lower

than average, and progeny from transitional sub-zones and elevations had intermediate growth rates. There was no significant eastto-west difference in the growth rate. The similarity in growth traits

corresponds well with the relatively low interpopulation differentiation demonstrated by isozyme data. The existing network of

common garden tests of P. sibirica is insufficient for complete

characterization of the gene pool and revision of existing seed zones.

Certain adjustments, such as merging of some seed zones, can be

suggested.

Key words: Pinus, P. sibirica, P. koraiensis, P. pumila, P. strobus

five-needle pines, stone pines, introduction, breeding,

Russia

Five-Needle Pines in Russia: Introduction

and Breeding

Anatoly I. Iroshnikov

Dmitri V. Politov

Background ____________________

Three species of five-needle pines (Pinus L. subsection

CembraeLoudon) are naturally distributed in Russia: Pinus

sibirica Du Tour, P. koraiensis Sieb. et Zucc. and P. pumila

(Pall.) Regel. Each species has distinctive morphological,

genetic, and, sometimes, biological traits and are adapted to

specific ecological niches. P. sibirica naturally occurs in

areas with a humid continental climate: northeastern European Russia, the Urals Mountains, Western Siberia taiga,

and the northern macroslopes of mountains in southern

Siberia and Transbaikalia. In these forests P. sibirica occupies a total area of 36.69 million ha. Forests containing P.

koraiensis occupy a much smaller area (2.87 million ha) and

occur in a monsoon climate, predominantly in the Primorskii

territory and the southern part of the Chabarovskii territory. Thickets formed by P. pumila occupy a total area of 38.3

million ha and are located in the cold climate zones of eastern

Siberia and the Far East. This species is usually found in

localities characterized by a high level of snow cover.

Total stem volumes for P. sibirica and P. koraiensis are

estimated as 7.4 billion m3

. Total stem volume of P. pumila

is estimated at 1.1 billion m3. Specific traits of these Russian

five-needle pines, site descriptions, and relationships with

other forest tree species have been reviewed in numerous

publications (Tikhomirov 1949; Iroshnikov 1974; Pravdin

and Iroshnikov 1982; Krilov and others 1983; Semechkin

and others 1985; Kolesnikov 1954, 1966).

Under optimal site conditions, the high productivity of P.

sibirica and P. koraiensis stands resulted in clearcutting of

many large and valuable stone pine forests between the

1930s and the 1980s. Forests in the Altai, Western Sayan,

and Sikhote-Alin Mountains, southern taiga in Western

Siberia, and lower Amur basin were subjected to intensive

exploitation. The cutting of these forests caused widespread

changes in species composition, soil erosion, and declines in

harvests of stone pine seeds (= nuts) and commercial fur

hunting. Due to public efforts, stone pine forests were

recognized for their exceptional environmental values and

have been protected in Russia since 1989. Clearcutting was

prohibited, and management of these forests has since been

oriented to provide multiple values. Intensive reforestation

efforts for these species have occurred including introduction in regions outside the respective natural ranges.

Early efforts in the breeding and introduction of native

and exotic Pinus section Strobus species and other economically valuable conifers in the former Soviet Union occurred

in the 1920s through 1950s (Kern 1934; Georgievski 1931,

1941; Nesterov 1935; Pogrebnjak 1938; Eitingen 1938, 1946;

Tikhomirov 1949; Grozdov 1952; Girgidov 1955). Specific

studies evaluated the acclimatization of different species in
USDA Forest Service Proceedings RMRS-P-32. 2004 65

Five-Needle Pines in Russia: Introduction and Breeding Iroshnikov and Politov

different environments (Sukachev 1922; Maleev 1933; Gurski

1957). Throughout the 1960s through 1980s, several important studies evaluated the introduction of coniferous species

in botanical gardens, arboreta, experimental forests, and

private estates during second half of 1800s (Shin 1961;

Vekhov and Vekhov 1962; Timofeev 1965; Maurin 1970;

Shkutko 1970; Mashkin 1971; Nekrasov 1980; Redko and

Fedorov 1982; Ignatenko 1988; Logginov 1988; Plotnikova

1988; Lapin 1971). Unfortunately, these materials were

often of unknown geographic origin, which limited the value

of the studies’ conclusions.

In the 1960s and 1970s, broad-scale experimental studies

on the introduction and breeding of prospective conifer

species were undertaken by the Institute of Forestry in the

Siberian Branch of the Russian Academy of Sciences, AllUnion Research Association “Soyuzlesselektsia,” and Moscow State Forest University. These studies were designed to:

(1) review results from past plantings and the current state

of introductions of section Strobus, subsection Strobi pines

to Russia; and (2) evaluate introduction and breeding experiments on P. sibirica conducted in different regions of

Russia.

Introduction of Subsection Strobi

Species________________________

Four of the 17 pine species classified in subsection Strobi,

P. flexilis James, P. monticola Douglas ex D. Don, P. peuce

Grisebach and P. strobus L., have been introduced, over

time, into arboreta and botanical gardens in European

Russia. Among these species, P. flexilis and P. monticola

were found to be unfit for planting in Russia and are not

widely distributed. Since the early 19th century, most introduction efforts were of P. strobus, initially as ornamental

tree and later as a forest tree that could rapidly produce high

quality wood. Between 1860 and 1890, P. strobuswas planted

in the Baranovichi forest management unit of Byelorussia

(Shkutko 1970), Forest Experimental Station of PetrovskoRazumovski Academy of Agroforestry in Moscow region

(Nesterov 1935; Eitingen 1938, 1946), and in Shatilov’s

estate (now the Mokhovskoi forest management unit of

Orlovskaia region) (Pankov and others 2000; Kapper 1954).

P. strobus was widely planted in Byelorussia, Latvia,

Lithuania, Ukraine and several regions of Russia in the late

1800s and early 1900s (Grozdov 1952; Girgidov 1955; Shin

1961; Sevalnev 1966; Fedoruk 1969, 1980; Maurin 1970;

Shkutko 1970; Redko and Fedorov 1982; Usachev 1983;

Sirotkin and Gvozdev 1987; Logginov 1988; Kapper 1954).

Later in the 20th century, the species was evaluated in

Kazakhstan (Rubanik 1974), the Far East (Samoilova 1972),

Georgia, the northern Caucasus Mountains (Holjavko 1981),

and Estonia (Kasesalu 2000).

Recommendations for the introduction of P. strobus have

been adjusted in recent years, as new information on productivity and disease resistance was accumulated for different

vegetation zones of the former USSR. Initially, it was recommended that the species be planted in forests, steppeforests, and the northern part of steppe zones eastwards up

to the Yenissey River. These recommendations were altered

to advise planting of P. strobus in the following regions:

northwestern Ukraine, southern Moscow region, Kurskaia,

Voronezhskaia, Orlovskaia, Belgorodskaia, Lipetskaia,

Tambovskaia, western Saratovskaia region, and the Republic of Moldova. It was specified that P. strobus should be

planted only in areas where the species would be higher in

growth and value than P. sylvestris L.

It is noteworthy that Byelorussia, Latvia, Lithuania,

Estonia and the northwestern regions of Russia were not

designated for planting P. strobus. These regions have a

relatively wetter climate where extensive blister rust

(Cronartium ribicola J.C. Fisch) infection has been observed

in both pure and mixed stands of P. strobus at different ages

(Nesterov 1935; Eitingen 1946; Girgidov 1955; Maurin 1970;

Shkutko 1970; Grozdova 1975; Potapova 1984; Kasesalu

2000). However, there are environments in these regions

where P. strobus can be successfully grown. In Byelorussia,

stands with different levels of tolerance to this pathogen

have been noted (Shkutko 1970; Fedoruk 1980; Sirotkin and

Gvozdev 1987). In the central Chernozem regions of Russia

(Kurskaia, Voronezhskaia, Orlovskaia, Belgorodskaia,

Lipetskaia, Tambovskaia), blister rust causes only minor

damage to P. strobus (Shin 1961; Pismenni 1967; Kapper

1954). P. strobus plantings in the Ukrainian steppe zone are

rarely attacked by blister rust (Logginov 1988). Pismenni

(1967) concluded that the threat of C. ribicola to P. strobus

plantations is largely overestimated, based on observations

of 30 plantings established throughout European part of the

former USSR. Despite the wide distribution of alternate

hosts – black currant (Ribes nigrum L.) and gooseberry

(Grossularia Mill.) – in this area, Pismenni concluded that

there was a higher degree of damage associated with soil pH,

relative air humidity (at 1 p.m.), mean air temperature, and

number of days with cloudy weather in August in the regions

of growing than with proximity of alternate hosts (table 1).

Additionally, Pismenni found that stands with faster growing trees and a higher productivity class were more susceptible to the pathogen. There are some problems, however,

with Pismenni’s (1967) conclusions. Pismenni was not aware

that diseased trees had been removed in sanitation cuttings

and that there were well-known cases of extensive blister

rust infections (especially on thin sandy soils) and even

mortality in some of the plantations (Nesterov 1935; Grozdov

1952; Kapper 1954).

Valuable information on the intensity and dynamics of P.

strobus mortality due to blister rust has been obtained from

long-term observations (1880 to 1920) on P. sylvestris L.

plantations established at the Forest Experimental Station

in the vicinity of Moscow (Nesterov 1935, Eitingen 1946).

These studies revealed extensive damage of Scots pine in

several regions of Russia (Rudzski 1874, Sobichevski 1875).

Long-term (130 plus years) investigations of the dynamics of

Scots pine populations, in different parts of the natural

range, showed populations with a high incidence of

Peridermium pini (Pers.) Lev., and Cronartium flaccidum

(Alb. et Schw.) Wint. had periodic epidemics caused by these

pathogens in association with solar activity. The studies also

revealed different types of interactions between the pathogen and trees, the influence of growth conditions on the

metabolism of each organism, and respective levels of tree

resistance and pathogen virulence. These studies suggested

a similar mode of interaction between the host-pathogen

pair of P. strobus and C. ribicola. Definitive conclusions,

however, can not be made as P. strobus – C. ribicola interac-
66 USDA Forest Service Proceedings RMRS-P-32. 2004

Iroshnikov and Politov Five-Needle Pines in Russia: Introduction and Breeding

tions have been viewed for only a relatively short time in

Russia, and there is a lack of comparative studies of homogenous progeny over different sites. In addition, Minkevich’s

(1986) observations on the lack of correlation between solar

activity and C. ribicola outbreaks in Europe (r=0.09±0.18)

over a 31-year period suggests a different interaction between P. strobus – C. ribicola than P. sylvestris and its

pathogens.

The low resistance of P. strobus plantations to C. ribicola

in different regions of the former USSR stimulated selection

of individuals resistant to the pathogen and the introduction

of P. peuce Grisebach, as a species highly tolerant to blister

rust (Eitingen 1946; Grozdov 1952; Maurin 1970; Shkutko

1970). However, the limited availability of seeds of this

Balkan species has prevented widespread field testing.

Tables 2 and 3 present growth data of different subsection

Strobi species in plantations throughout the country. Plantations older than 20 to 30 years are reviewed; that is, those

that have reached the “critical age when fitness of exotics at

particular conditions is finally elucidating” (Maleev 1933, p.

108).

Toward the end of the 20th century, the total area occupied

by P. strobus plantations reached 259 ha (Beloborodov and

others 1992). Some stands were 60 to 120 years old, highly

Table 1—Damage of P. strobus stands by white pine blister rust Cronartium ribicola (Cr.r%) in regions differing by air humidity (W, %), mean

air temperature (T, ∞C) and percentage of cloudy days (V,%) in August (Pismenni 1967)

Location of plantations: republic, region (forest management unit) Age, years Cr.r, % W,% T ∞C V,%

Lithuania (Valkinskii, Smalininskii) 48; 45 14; 24 68 15 55

Byelorussia (Bobruiskii, Uzdenskii, Prilukskaia Dacha) 50; 56; 48 14; 12; 8 65 16 55

Ukraine, Sumskaia (Trostianetskii) 69; 52; 28 6; 4; 1 55 16.4 40

Russia, Orlovskaia (Mokhovskoi) 10; 28; 80 0; 6; 9 54 17 39

«-», Lipetskaia (LOSS, Leninskii) 28; 28 3.2; 1 54; 52 17.2 39

«-», Kurskaia (Rylskii) 16; 54; 54 0; 1; 1.5 52 17.2 39

«-», Penzenskaia (Yursovskii) 67 0,5 51 17.6 38

«-», Voronezhskaia (Vorontsovskii, Savalskii) 30; 32 0 50 18; 18.2 38

Moldova (Kalarashskii) 24 0 49 19 30

Table 2—Growth indices of some five-needle pine species in the points of their introduction in Russia.

Age Height, Diameter,

Region year m cm Source

Pinus peuce

Moscow. LOD MSHA 53 19 28 Eitingen, 1946

Moscow. Ivanteevka 33 9.8 13 Grozdova, 1975

Lipetsk. LOSS 31 11 15 Kuzmin, 1969

Orjol. Mokhovoe 70-80 22-24 40-46 Vekhov, Vekhov, 1962

Orjol. Shestakovo 75-80 26 50 Mashkin, 1971

P. flexilis

Lipetsk. LOSS 44a 14.2 31 Vekhov, Vekhov, 1962; Kuzmin, 1969

P. monticola

Lipetsk. LOSS 40a 11.4 26.8 Kuzmin, 1969

P. koraiensis

Altai. G.-Altaisk 32 6-10 11-15 Luchnik, 1970

Lipetsk. LOSS 40 9.4 20.2 Kuzmin, 1969

Mari. Ioshkar Ola 30 7-8 10-12 Alimbek, 1991

Moskow. Ivanteevka 35-38 6-8 6-9.5 Yablokov, Dokuchaeva, 1976

P. cembra

Lipetsk. LOSS 30 5.4 8 Vekhov, Vekhov, 1962

Moscow. Chlebnikovo 80 16.6 25.5 Ignatenko, 1988

Smolensk. Dugino 35 11 6 Grozdov, 1952

P. pumila

Altai. G.-Altajsk 30 3-4 2-6 Luchnik, 1970

Lipetsk. LOSS 36 3.3 -b Kuzmin, 1969

a

Very sensitive to fungal disease Cronartium ribicola b

Not measured
USDA Forest Service Proceedings RMRS-P-32. 2004 67

Five-Needle Pines in Russia: Introduction and Breeding Iroshnikov and Politov

productive, free from blister rust, and produced large seed

yields with high quality (Kapper 1954; Maurin 1970). In

order to conserve gene pools and create a permanent seed

base of P. strobus in the USSR, a gene reserve (1.6 ha), plus

stands (5 ha), 180 plus trees, 17 ha of permanent seed

orchards, and 2.5 ha of clonal plantations were established

in the early 1990s (Beloborodov and others 1992). In addition experiments on hybridization of this species with P.

wallichiana A.B. Jackson and P. ayacahuite Ehrenberg ex

Schlechtendahl, were initiated in the Ukraine, and 11 candidates for varieties were selected (Patlaj and others 1994).

Currently, a spontaneous hybrid between P. wallichiana

and P. strobus is under observation at the Sochi Arboretum,

Institute of Mountain Forestry and Ecology in the northern

Caucasus Mountains. The hybrid tree measured 23 m in

height and 42 cm in diameter at the age of 39 years (Soltani

2001).

Genetic plantings and orchards of P. strobus have been

established at different locations. Since the 1980s, the Research Institute of Forest Genetics and Breeding in Voronezh

has been evaluating seed and clonal progenies of plus and

phenotypically superior P. strobus trees selected in old

plantations in three forests from the Kaliningrad region,

Borskoie forests of Voronezh Natural State Reserve,

Glushkovskoie forests of Lipetskaia region, and Mokhovskoie

forests of Orlovskaia region (Beloborodov and others 1993,

1994). Testing of 29 families was initiated in 1984 in a 1.2 ha

area within the Homutovskoie forest, and 96 families were

established in 3.8 ha planting at Mezen Pedagogical College

in Orlovskaia region in 1986 and 1987. An additional 33

families are being evaluated in a 1.2 ha planting that was

established in 1989 in the Davydovskoie forest (Voronezh

area). An archive (0.7 ha) of 41 clones was established in the

Gremyachenskoie forest within the Voronezhskaia region,

and a clonal seed orchard consisting of 25 plus trees (1.4 ha)

has been established in Zagon forest (Smolenskaia region).

The 10 year-old results of these progeny testing experiments have shown superior growth of eight families from

the Mokhovskoie and Glushkovskoye forests, while progenies from Kaliningradskaia region and Voronezh State

Reserve show significant variability in growth rate. Only 25

percent of the tested families have shown resistance to

white pine blister rust (Beloborodov and others 1993,

1994). Further studies of these plantations and clonal

archives indicate that the stands become more susceptible

to blister rust with advancing age. At age 16, only 7.7

percent of families and 36 percent of clones were free from

blister rust. In some families, all trees are infected, and up

Table 3—Growth indices of Pinus strobus artificial stands on the territory of the former USSR.

Number Growing

Republic Age, Height Diameter of trees stock,

region year m cm per ha m-3/ha Source

The Ukraine

Ivano-Frankovsk 70 30.5 37.6 700 960 Usachev, 1983

Trans-Carpathians 68 38.5 35.3 633 915 ’’ - ’’

Right-bank forest steppe 60 27 28 -a 570 Logginov, 1988

Byelorussia

Brest 54 25 42.2 429 650 Usachev, 1983

Minsk, Uzdenski 66b 25.5 32.5 550 386 Shkutko, 1970

Latvia

Shkedovskoe 70b 23.9 39.7 - 348 Maurin, 1970

Skriverskaya 70b 24 36.8 - 360 ’’ - ’’

Lithuania 60 25 40 - - Jankauskas, 1969

Estonia

Agali arboretum 33b 16.6 22 900 282 Kasesalu, 2000

Kazakhstan

Alma-Ata 23b 5 7 - - Rubanik, 1974

Russia

Bryansk 84 22.3 23.1 940 468 Smirnova, 1997

Kaliningrad, Nagornoe 67 29.8 37.2 699 1199 Redko, Fedorov, 1982

Kaliningrad, Novo- 93 29.3 41.8 294 630 ’’ - ’’

Bobruyskoe

Kursk, Rilsky 55 22 18 - 250 Sevalnev, 1966

Leningrad, Viborgsky 45b 14.9 26 - - Girgidov, 1955

Lipetsk, LOSS 41 15.6 37 - - Kuzmin, 1969

Moskow, LOD MSHA 47b 16.5 20 - - Eitingen, 1946

’’ - ’’ 45b 14 14 - - Timofeev, 1965

Moskow, Ivanteevka 40b 15.5 19.2 - - Grozdova, 1975

Orjol, Mokhovoe 125 31.4 35.2 103 383 Pankov et al., 2000

Penza 67 18.4 28 750 425 Usachev, 1983

Voronezh 40 18 28 - - Dorofeeva, Sinitsin, 1996

a

Not measured

b

Very sensitive to fungal disease Cronartium ribicola
68 USDA Forest Service Proceedings RMRS-P-32. 2004

Iroshnikov and Politov Five-Needle Pines in Russia: Introduction and Breeding

to 67 percent of the ramets were susceptible in the clonal

plantings (Shirnina and Beloborodov 1999).

A blister rust resistance study on subsection Strobi species was conducted by Bsaibes (2000) in northwest Russia

(St. Petersburg and Leningradskaia region) and in CentralChernozem regions. This study confirmed the high resistance of P. peuce to C. ribicola and the extremely high

susceptibility of P. monticola and P. strobus to the pathogen.

To ensure resistance of P. strobus, Arefiev and Bsaibes

(2000) recommended combining selection of resistant provenances and progenies with site selection and silvicultural

practices to form plantations with a genetic composition and

environmental conditions not favorable for the pathogen. To

some extent, these plantations represent a synthesis of

earlier recommendations (Maleev 1933; Nesterov 1935) but

do not assume blister rust resistance through interspecific

hybridization, which was shown to be effective in the case of

P. strobus and P. peuce in Romania (Blada 1994, 2000a,b), or

inoculation with pathogen spores at early stages of pine

ontogenesis that became a common practice in North America

(McDonald and Hoff 2001).

Strategies for establishment of future P. strobus plantations will benefit from knowledge of the modes and mechanisms for coadaptation of P. strobus and C. ribicola (Millar

and Kinloch 1991). Understanding host-blister rust interactions in spatially and temporarily heterogeneous environments will provide a significant contribution to the theory

and practice of species introduction, as well as to forecasting

and development of preventive measures for decreasing the

negative effects of pathogen epidemics.

Introduction of Stone Pines,

Subsection Cembrae Loudon _____

Stone pines with all uncertainty about their phylogeny

and taxonomy have always been attractive objects for

botanists and foresters. Concern about protection of P.

sibirica stands against fire and unwarranted clear-cutting is

reflected in early publications, such as before the 19th century, which also discussed their diversity and biological, and

aesthetical resources (Pallas 1786; Dmitriev 1818). In the

19th century, data on testing of stone pines in botanical

gardens, arboreta, and various experiment stations

(Lisinskoie forest near St. Petersburg and the PetrovskoPazumoskaia Forest Station in Moscow) stimulated a broader

introduction of Siberian stone pine in parks and orchards in

European Russia. These plantings were studied to evaluate

species variability and ecology (Gomilevski 1909). At the

same time, there was a shift from seed collections in natural

stands in remote taiga regions to seed production and

collection in seed orchards formed from progenies of highly

productive trees of foothill (low mountain) populations in

optimal growth conditions (Barishentsev 1917).

Numerous publications and documents have been focused

on broad-scale popularization of seed production in seed

orchards (Georgievski 1932; Vekhov and Vekhov 1962; Yablokov

1962; Nekrasov and Tvelenev 1970; Shkutko 1970; Potapova

and Potapova 1984; Ignatenko 1988; Usmanov and Korolkova

1997; Drozdov and Drozdov 2002; Titov 1999; Iroshnikov and

Titov 2000). A number of methods have been developed for seed

and vegetative reproduction of stone pines.

The limited number and area occupied by old plantations

of P. sibirica in European Russia, as well as the unexplained

mortality of 55 to 68 year-old stands in Lisino and PetrovskoRazumovskaya Experiment Station has hindered planting

of the species outside of its native range. Establishment of P.

sibirica plantings also would be hindered by competitive

ability in sites outside of the native range. Comparison of

growth between 22 and 32 year-old stone pines with local

forest tree species in common garden tests in the

Leningradskaia region (forest-steppe site), Moscow region

(subtaiga site), and Yaroslavskaia region (foothill site) has

shown inferior growth of P. sibirica by 20 to 40 percent

(Iroshnikov 2000).

The results discussed above as well as data from subsequent studies (summarized in tables 4 and 5) show that

there is substantial interspecific variation in P. sibirica. The

tests show that the best growing seed sources are from low

and middle mountain belts in the south Siberia mountain

ridges and the sub-taiga and southern taiga zones in western Siberia. Progenies from northern and subalpine regions

had growth rates 20 to 30 percent lower. Meridian (westeast) differences in origin within corresponding zonal and

altitudinal complexes of stone pine forests had little effect on

growth of progenies.

Common garden and test plantations of P. sibirica have

been established in different vegetation zones, as well as a

study of variation in natural populations of P. sibirica

(Iroshnikov 1974). These tests confirmed the high variability of the species and also revealed unique morphological

tree forms and variation in reproductive processes. Significant

variation was detected in needle size, shape, and position on

shoots, duration of juvenile phase, long-term dynamics of

macrostrobili formation, macrostrobili maturation dates, and

in premature ripening of cones within the first year after

“flowering” with formation of 40 to 60 percent of unsound

seeds.

Investigations on natural populations of P. sibirica have

been limited to Altai and Sayan Mountains and do not allow

conclusions about the distribution of blister rust across the

species range. Lebkova (1964, 1967) found a high frequency

of Cronartium species, including C. ribicola in a 10 to 15

year-old stone pine understory in the subalpine and middle

mountain zone in the western Sayan Mountains and the

northeastern Altai Mountains. Tovkach (1968) also reported

severe damage to the P. sibirica understory by C. ribicola

(up to 36 percent of trees) in the eastern Sayan Mountains

(Nizhneudinskii forest management unit of Irkutskaia region). Ulcerous cankers putatively caused by Biatoridinia

pinastri Colov. et Stzedr. were also detected in the P. sibirica

understory in the Shestakovskii and Kyrenskii forest management units of the Irkutskaia region (Osipova 1968).

The only P. sibirica common garden study damaged by C.

ribicola was in the Dmitrovskii forest management unit in

the the Moscow region (table 5). Aeciospores were detected

in 1993 and 2001 on seedlings of 17 of the 24 families.

Approximately 2 to 11 percent of the trees were infected.

After 40 years of observation, C. ribicola damage was rarely

detected in Siberian plantations or the seed orchards and tests

planted at the Yemelyanovskii, Ermakovskii and Uzhurskii

forest management units of the Krasnoyarskii territory.

The introduction of P. cembra and P. sibirica occurred in

the 19th century in botanical gardens in the Ukraine, Latvia,
USDA Forest Service Proceedings RMRS-P-32. 2004 69

Five-Needle Pines in Russia: Introduction and Breeding Iroshnikov and Politov

Table 5—Indices of growth and damage with Cronartium ribicola of Siberian and Korean pine geographic cultures in

Dimitrovsky Forest in Moscow Region (Iroshnikov and Tvelenev 2002).

Origin of seeds Heighta

Forest Latitude/longitude % of Diameter,b

Region management unit ∞N ∞E cm control mm Stem rustb, %

Siberian pine

Komi Troitsko-Pechersky 63 57 522±16 71 99±4 8.9

Sverdlovsk Novo-Lyalinsky 59 60 533±18 68 110±5 0.0

N.-Tagiljsky 58 60 572±17 73 125±4 0.0

Tomsk Tymsky 60 80 651±18 84 123±5 2.4

Chainsky 58 83 671±18 86 118±4 0.0

Shegarsky 57 84 644±22 83 119±7 3.2

Zyryansky 57 86 705±18 90 141±5 2.4

Tver Kalininsky 57 36 605±16 78 118±5 11.1

Elevation above Sea Level, m

Altai Baygolsky 1300-1800 655±17 84 124±7 2.3

Kyga 430 779±24 100 146±6 0.0

’’ - ’’ 1250 730±20 94 150±6 2.1

’’ - ’’ 1500 635±16 82 129±5 2.4

Krasnoyarsk Shushensky 550 745±26 96 146±8 0.0

’’ - ’’ 800 816±18 105 170±4 2.4

’’ - ’’ 1300 621±16 80 113±5 0.0

Irkutsk Slyudyansky 1000 722±13 93 160±3 2.0

Buryatia Dzhidinsky 1000 612±19 79 115±5 0.0

Bichursky 1000 678±16 87 146±4 5.1

Korean pine ∞N ∞E

Khabarovsk Bikinsky 47 134 804±23 103 159±8 0.0

a

At 32-year age (2001)

b

At 30-year age (1999)

Table 4—Growth indices of 37-year-old descendants of Siberian pine of various origins in

Krasnoyarsk forest-steppe (Emelyanovsky Forest of Krasnoyarskii Territoty). (Iroshnikov

A.I.).

Origin of seeds Height Diameter

Forest Altitude above sea % of

Region management unit level, m, zone cm control cm

E. Kazakhstan Leninogorsky 1200-1500 731±37 83 8.3±0.9

Altai G.-Altajsky 1200-1500 712±55 81 8.7±0.8

Kemerovo Kuzedeevsky 300-700 930±27 106 11.8±0.8

Myskovsky 1000-1300 856±40 98 9.9±0.9

Tisuljsky 700-1000 897±17 102 10.6±0.4

Mariinsky subtaiga 876±24 100 10.0±0.6

Yurginsky subtaiga 875±19 100 10.3±0.6

Khakasia Tashtypsky 900-1000 931±14 106 10.9±0.4

Birikchuljsky 1000-1300 885±17 101 11.9±0.6

Balyksinsky 900-1000 877±38 100 9.4±0.6

Khakassky 900-1000 900±14 103 11.4±0.4

Oktyabrsky 1100-1300 822±26 94 12.6±1.1

Sonsky 1100-1400 891±23 102 12.6±1.2

Krasnoyarsk Ermakovsky 400-500 877±57 100 13.1±1.3

’’ - ’’ 1500-1600 594±53 68 7.8±1.3

Irkutsk Cheremkhovsky 1200-1300 893±24 102 10.8±0.7

Ikejsky 900-1000 932±33 106 13.7±0.8

Slyudyansky 600-900 930±29 106 11.8±0.8

Oljkhonsky 1000-1300 768±22 88 10.3±0.6

Buryatia Zakamensky 1100-1300 800±19 91 12.5±0.6

Dzhidinsky 1300-1400 809±21 92 13.4±0.9

Chita Kr.-Chikojsky 900-1100 807±40 92 11.4±1.1

Khiloksky 900-1100 888±20 101 10.9±0.4
70 USDA Forest Service Proceedings RMRS-P-32. 2004

Iroshnikov and Politov Five-Needle Pines in Russia: Introduction and Breeding

Lithuania, and Central-Chernozem regions of Russia (table

2). Due to lack of information about geographic origin,

combined data on the state of these stands has been often

presented in literature (Maurin 1970; Shkutko 1970; Fedoruk

1980). Plantations established in Khlebnikovskii Park near

Moscow in 19th century (described by Ignatenko as P. sibirica),

evidently originated from seeds obtained from the Balkans,

as the planting also contains several Abies alba Mill. trees

and three cone-bearing Pinus peuce individuals. Single trees

were observed to be infected by C. ribicola, although natural

stands of P. cembra in eastern Carpathia have been reported

to be highly resistant to pathogens up to age 400 to 500 years

(Smagljuk 1969).

Progenies of two P. cembra clones originating from the

Carpathians were planted in the forest-steppe zone in

Krasnoyarsk territory. Testing of 40-year-old clones grafted

in 1963 onto P. sylvestris stocks showed the same growth

characteristics as even-aged P. sibirica grafts originating

from the western Sayan Mountains and bore few cones

(10 to 50 per tree). Grafts of two other Carpathian clones

(20 to 28 years-old) planted in the Dmitrovskii forest management unit (Moscow region) are characterized by exclusively high level of micro- and macrostrobili formation, up to

300 to 500 per each tree with a well-developed crown.

P. koraiensis was introduced in Russia after P. sibirica

and P. cembra. In many regions of European Russia, Korean

pine grows quickly during the first few decades (table 2) and

has early formation of female cones (macrostrobili). For

instance, in the Dmitrovskii forest management unit, 32

year-old plantations originating from seeds (brought from

the Khabarovskii territory) grew as fast as the best P.

sibirica stands (table 5). However, some trees were infected

by C. ribicola. Azbukina (1974, 1984) describes white pine

blister rust outbreaks across the whole Korean pine range.

Additional studies are required to evaluate the different

perspectives of broad-scale introduction of this species in

Russia.

Dwarf Siberian Pine _____________

P. pumila is relatively rare in botanical gardens and

arboreta. Tikhomirov (1949) concludes that cultivation is

possible, as there has been successful cultivation in the

subalpine zone of in the Urals and at other locations. However, the species has been somewhat ignored due to a

comparatively low economic value. Semechkin and

Semechkina (1964) concluded that P. pumila is sensitive to

mechanical squeezing when planted in loamy and loose

soils, and sown seeds are often destroyed by rodents and

nutcrackers (Nucifraga caryocatactes L.). In our (A. I.

Iroshnikov) experiments in the western Sayan foothills, P.

pumila did not survive longer than 15 years. Low survival of

this species was also observed in Khakasia (Likhovid 1994)

and in the Lipetsk region (Vekhov and Vekhov 1962). Additionally, grafts of P. pumila to P. sylvestris and to P. sibirica

at the Dmitrovskii forest management unit did not survive

past 20 to 25 years.

Susceptibility to blister rust is another factor that should

be considered when planting P. pumila. Azbukina (1974,

1984) observed intense blister rust damage of P. pumila in

the Far East. A subsequent study (Azbukina and others

1999) demonstrated that Cronartium kamtschaticum Joerst.,

which had been described as a pathogen of P. pumila, is

really C. ribicola.

In conclusion, it appears that successful introduction of

P. pumila, as well as other stone pines, is largely dependent

of number and origin of individuals. Genetic differentiation

within the range of pines of subsection Cembrae is reviewed

in this volume (Politov and Krutovskii, this proceedings).

Conclusions____________________

Effectiveness of using exotic woody plants for increasing

forest biodiversity, productivity, quality and sustainability

to biotic and abiotic factors depends on genotype, adaptive

potential, and competitiveness. Approximately 200 years

of studies in Russia suggest that the success of an introduced

species can be evaluated only through long-term testing of

progenies representing a broad spectrum of provenances

and tested in different ecological conditions. Information from

such studies increases when more genotypes are involved.

Pinus sibirica progeny tests established in diverse ecological conditions, inside and outside the species’ natural range,

have revealed intra- and interpopulation diversity, high

breeding potential, and have helped to specify a geographic

zone that is optimal for P. sibirica growth with maximal

genetic variation. Long-term experiments with P. sibirica

and P. strobus have allowed for development of current

forest seed zone recommendations for P. sibirica (in 1982)

and help direct the planting program for introduction of

P. strobus.

To increase successful introduction of exotic five-needle

pine species requires the development of international collaboration, coordination of corresponding studies within

Russia, wider publication of results in peer-reviewed journals and monographs, maintenance of available and establishment of new experimental objects, and in situ and ex situ

conservation of genetic resources.

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USDA Forest Service Proceedings RMRS-P-32. 2004 73

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

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Abstract—East Asia is very rich in the genetic resources of fiveneedle pines, including 11 species and three varieties. Of these taxa,

Pinus armandii is the most widely distributed, ranging from Taiwan and Korea to central and western China. The natural range of

P. koraiensis includes northwestern China, North and South Korea,

Japan, inland Siberia and the Russian Far East. Because they are

the most commercially important pines for wood and nut production, most genetic improvement research has been carried out with

these two species. Pinus fenzeliana and P. dabeshanensis are used

in plantations in some areas. However, along with the other species

of five-needle pines, they are mainly of importance in studies of

taxonomy and ecophytogeography. In addition to a review of genetic

resources, this review paper also gives a brief overview of tree

improvement and disease and insect pests of the five-needle pines

in east Asia.

Key words: East Asian pines, five-needle pines, gene resources,

tree improvement, tree pathogens, tree insects

Introduction ____________________

This paper attempts to give a brief review of the status of

genetic resources, tree improvement and breeding programs

and gene conservation of five-needle pines in east Asia,

including China, Japan, the Democratic People’s Republic of

Korea (North Korea), Republic of Korea (South Korea),

inland Siberia and the Russian Far East region.

The genus Pinus L. is generally divided into two subgenera, Subgenus Strobus (Haploxylon pines), and Subgenus

Pinus (Diploxylon pines), commonly known as soft pines and

hard pines respectively. Of the soft or five-needle pines

native to east Asia, P. armandii Franch. and P. koraiensis

Sieb. & Zucc. are as important for timber production as such

hard pines as P. massoniana Lamb. and P. yunnanensis

Franch. in China and P. densiflora Sieb.& Zucc. in Japan.

The rest of the five-needle pines are of more limited economic

importance due to their restricted gene resources. In this

paper, most emphasis is placed on the research in the genetic

Genetic Resources, Tree Improvement and

Gene Conservation of Five-Needle Pines in

East Asia

Huoran Wang

Jusheng Hong

resources, tree improvement and gene conservation of the

two most important species, P. armandii and P. koraiensis.

In fact, very little information other than that on taxonomy

and ecology is available for other species.

Genetic Resources ______________

Of 24 species of Pinus in East Asia, 11 including three

varieties of P. armandii are five-needle pines (Wu 1956;

Mirov 1967; AASE 1978; Zheng 1983, Price and others

1998). They are listed here in conformity with the system

standardized for all papers presented at this conference

(Price and others 1998) and so listed in the conference

program, which differs in a few cases from the taxonomic

designations used in China. There is no universal agreement

on the taxonomy of east Asian five-needle pines. In Chinese

literature, for example, kwangtungensis is a separate species morphologically related to P. wangii.The east Asian list

follows, with more detail in table 1:

P. armandiiFranchet var. armandii, widely distributed and

planted in China;

P. armandii Franch. var. mastersiana (Hayata) Hayata, in

Taiwan of China;

P. armandii Franch. var. amamiana (Koidzumi) Hatusima,

includes isolated populations in Japan;

P. dabeshanensis Cheng & Law, very restricted in a small

area in central south China;

P. dalatensis de Ferré, central Vietnam;

P. fenzelianaHandel-Mazzetti, (including P. kwangtungensis

Chun & Tsiang) southern China to northern Vietnam;

P. wallichiana Jackson (P. griffithii McClellan), Himalayan

chains;

P. koraiensis Siebold & Zuccarini, China, Japan, Korea,

Siberia and Russian Far East;

P. morrisonicola Hayata, central Taiwan;

P. parviflora Siebold & Zuccarini, native to Japan and exotic

in China and Korea;

P. pumila (Pallas) Regel, China, Japan, Korea and Russian

Far East;

P. sibiricaDu Tour, northwestern China, Siberia and Russian

Far East;

P. wangii Hu & Cheng, scattered and limited populations in

southwestern China.
74 USDA Forest Service Proceedings RMRS-P-32. 2004

Wang and Hong Genetic Resources, Tree Improvement, and Gene Conservation of Five-Needle Pines in East Asia

Table 1—Classification and phytogeography of five-needle pines in east Asia.

Species Natural distribution Characterization

Pinus armandii China in Shanxi, Henan, Shaanxi, Tree up to 35 m high and 1 m dbh. Widely planted for forestry and

var. armandii Franch. Gansu, Sichuan, Hubei, Guizhou landscape. Flowers April- May; cones mature September-October

(Armand pine, and Yunnan provinces and Tibet; of the following year; cone size 10-20 cm x 5-8 cm; seed 1.0-1.5 cm

Huashan Mountain pine) elevation range 1000-3300 m. long,edible; 3 medial resin canals; wood density 0.43-0.48.

P. armandii var. Central Taiwan at 1800-2800 m Tree up to 20 m high and 100 cm dbh; leaves 15 cm. Mature cones

mastersiana (Hayata) elevation. peduncled, ovoid, up to 10-20 cm long and 8 cm in diameter. Seeds

Hayata (Taiwan ovoid, compressed, wingless, with a sharp edge all around, 8-12 mm

Armand pine) long. Wood density 0.46.

P. armandii Franch. var. On two isolated islands, Tree up to 25 m height, 1 m dbh; dark gray shoots; leaves 5-8 cm

amamiana (Koidz.) Tanegashima and Yakushima, Japan. long; cones short stalked, oblong-ovoid, 5-8 cm long; seeds wingless,

Hatusima (Japanese Ecologically and genetically isolated about 12 mm long.

Armand pine) populations, vulnerable.

P. dabeshanenesis Anhui and Hubei provinces of China Tree over 20 m, dbh 50 cm. Wood similar to that of P. armandii,

Cheng & Law at elevations between 900 and 2 external resin canals. Wood density 0.43.

(Dabeshan Mountain 1400 m; range very restricted.

white pine)

P. dalatensis de Ferré Very restricted range in evergreen Tree to 15-25 m, 60-100+ cm dbh, crown conical, somewhat open.

Dalat or Vietnamese subtropical forests of Vietnam at Female cone has 20-30 scales, yellowish-brown maturing to dark

white pine elevations of over 1500 m. Often gray. Cones mature October-December. No data on resin canals or

in mixed stands, very sparsely wood density.

distrubuted; species surtvival threatened.

P. fenzeliana Hand.-Mzt. South China in Hainan, Guangxi, Tree to 50 m in height, 2 m in dbh; leaves 10-28 cm long. Seed

(Hainan white pine) and Guizhou provinces; central cones ovoid-ellipsoid, 6-9 cm long; seeds chestnut brown with a wing

Vietnam; elevation range 1000-1600 m. 2-4 mm long. No data on resin canals .Wood density 0.55-0.59.

P. koraiensis Sieb. & Northeast China, also in Korea, Tree to to 50 m and over 1 m dbh; flowering in June; cones mature

Zucc. (Korean pine) Japan and Russian Far East, September-October in the following year, seed edible; 3 medial resin

elevation range 150-1800 m. canals; wood density 0.38-0.46; major timber species in northeast

Dominant species in mixed forest region of China.

stands with broadleaved trees,

pure stands can be found.

P. kwangtungensis China, geographically disjunct from Tree to 30 m high and 1.5 m dbh; flowers April-May, cones mature

Chun & Tsiang (South P. fenzeliana proper; found in in October of the following year; seed 8-12 mm long. Planted trees

China white pine). In southern Hunan, northern Guangxi grow fastest 10-30 years after planting, reaching 25 m in height and

China considered a and Guangdong provinces, with 45 cm dbh at about age 60; 2-3 resin canals; wood density 0.50.

separate species; in outliers in Guizhou and Hainan

this conference, a part provinces. Elevation range 700-

of P. fenzeliana (Price 1600 m. Forms pure stands or mixed

et al. 1998). stands with Tsuga, Fagus, Quercus.

P. morrisonicola Central mountains in Taiwan, no Tree up to 30 m tall and 1.2 m in dbh.; 2 dorsal resin canals.

Hayata pure stands; mixed with other

(Taiwan white pine) conifers or hardwoods

P. parviflora Sieb. & Naturally distributed in Japan and A tree up to 25 m tall and 1 m in dbh, 2 external resin canals.

Zucc. (Japanese white introduced to China. Ornamental tree, often grafted as bonsai

pine)

P. pumila (Pall.) Regel Northeastern China with altitudes Shrub or small tree 2-8 m, often multi-stemmed; 2 dorsal resin

(Japanese stone pine) ranged 1 000-1 800m and extended canals. Good for ground cover and ornamental

in Russia, Japan and Korea; forms

dense and low community on top of

mountains and exposure sites.

P. sibirica Du Tour Northwestern Altai in Xinjiang and the A tall tree, up to 35 m and over 1.8 m in dbh; 3 medial resin canals;

(Siberian stone pine) Great Xingan Range in China and flowering in May and cones mature in September-October in the

extended to Sibirica, ranged 66∞ 25'- following year. Wood density 0.45. Timber is as good as that of

46∞ 40'N in latitude and 49∞ 40'-127∞ P. koraiensis, an important forest tree in Siberia, Russia.

20'E in longitude, altitudinaly 1,600-

2,350 m; dominated in the stands

mixed with Larix sibirica

(con.)
USDA Forest Service Proceedings RMRS-P-32. 2004 75

Genetic Resources, Tree Improvement, and Gene Conservation of Five-Needle Pines in East Asia Wang and Hong

From an ecological standpoint, five-needle pines are, in

contrast to hard pines, mostly adapted to cold or temperate

and moist environments. There exists a latitudinal gradient

and a trend of species replacement from north to south

within their natural occurrences in East Asia (Wu 1956; Wu

1980; Kuan 1982; Zhao, G. 1991; Ma 1992). In the north, P.

sibirica, P. pumila and P. koraiensis occur at relatively lower

elevations; southward in the temperate and subtropical

zones, they are replaced by P. armandii; still farther south,

P. fenzeliana and P. dalatensis occur discontinuously at

higher elevations in subtropical and tropical areas. P.

dabeshanensisand P. wangii occur as relicts on difficult sites

for tree growth and survival in central south and southwestern China respectively as restricted populations or scattered

individuals.

From a geographical standpoint, all the five-needle pines

in east Asia are discontinuously distributed. P. sibirica

(Zhao, G. 1991) and P. armandii (Ma 1989,1992) are typical

examples. P. armandii is extensively distributed on the

mainland of China from temperate to subtropical regions,

with its two varieties, P. armandii var. mastersiana extending to Taiwan Island and P. armandii var. amamiana

appearing in Japan (Nakashima and Kanazashi 2000). The

populations of P. fenzeliana and its form recognized in China

as P. kwangtungensis Chun & Tsiang are also geographically isolated from each other. The ecological and geographic

patterns have introduced great genetic variability into these

five-needle pines, making some of them very difficult to

classify taxonomically. For instance, P. koraiensis and P.

sibirica were confused with each other in taxonomic status

for long time (Zhao 1991).

Tree Breeding and

Improvement ___________________

Tree improvement in East Asian countries is mainly

focused on commercially important species. Five-needle

pine research activities are mostly undertaken with P.

koraiensis and P. armandii, although P. fenzeliana and P.

dabeshanensis have been used locally in planting programs.

Pinus koraiensis

Pinus koraiensis is by far the most important species in

conifer tree improvement programs in northeast China,

North Korea and South Korea. Kim and others (1994a)

estimated that more than 250,000 ha of plantations had

been established with this species in South Korea by 1994.

In North Korea 305,000 ha were established with this

species for wood and nut production. For nut production, 250

clones were selected. It is estimated that the plantation area

of Korean pine in North Korea is increasing at a rate of about

30,000 ha per year.

In China, tree improvement and breeding research programs were launched in the early 1980s with emphasis on

provenance trials, plus tree selection and gene conservation.

A provenance trial was established in 1986 with 12 seedlots

collected from natural stands throughout the range and one

seedlot from a plantation. Tenth-year results showed that

there were significant differences in growth rates between

provenances, and that the seedlots collected from areas

around Changbaishan Mountain performed best (Zhang

and Wang 2000).

Wang and others (2000a) reported tenth-year results of

progeny tests established on three sites using open-pollinated seeds of 557 parents from natural stands. Significant

differences were found in growth performance between

individual families and between provenance zones, and

there were also large genotype x environment (GxE) interactions. Based on the progeny testing, a genetically improved

seed orchard was established by grafting (Wang and others

2000b). Observations indicated that the average interval

between every two good seed crops was five years. Strobilus

abortion rate reached 46.5 percent in the seed orchard, but

cone yield could be increased by 20 percent through crown

pruning and controlled pollination (Wang and others 1992).

Although seed orchard clones varied in fertility level,

leading to an increased level of relatedness in the offspring,

genetic diversity in seed orchard progenies was nevertheless

only slightly depressed compared with that of the reference

populations from which the plus trees were selected (Kang

and Lindgren 1998).

Variation in effective number of clones in the seed orchards of P. koraiensis was examined. The mean number of

clones averaged about 70 in each orchard, but the average

effective number (Nc) was 43 (Kang and others 2001).

Pinus armandii

P. armandii is the most widely and discontinuously distributed of the five-needle pines in China, occurring in 12

P. wallichiana Jacks. Southern Tibet and northwestern Tree to 50+ m with straight trunk and short, down-curved branches.

(Himalayan white pine) Yunnan in China, Bhutan, Burma, Branches longer in solitary trees, creating a dome-like crown. Leaves

Nepal, India, Pakistan and Afghanistan. 15-20 cm, usually pendant but in some trees spreading. Cones 20-

at 1600-3300 m elevation. 30 cm, bluish-green when young, maturing to light brown.

P. wangii Hu & Cheng Restricted area in southeastern Tree to 20 m high and 60 cm dbh; 3 medial resin canals.

(Yunnan white pine) Yunnan Province in China altitude

1100-2 000 m. Occurs in or mixed

stands with oaks on limestone

slopes. Endangered status.

Table 1—(Con.)

Species Natural distribution Characterization
76 USDA Forest Service Proceedings RMRS-P-32. 2004

Wang and Hong Genetic Resources, Tree Improvement, and Gene Conservation of Five-Needle Pines in East Asia

provinces ranging in latitude from 23∞30’ to 36∞30’ N, in

longitude from 85∞ 30’ to 113∞ 00’ E and in altitude from 1,000 to

3,500 m, which suggests that a large amount of geographicallyrelated genetic variation may exist within the species.

Range-wide provenance trials of P. armandii were established on 9 experimental sites in 1980. The trials were

coordinated by the Research Institute of Forestry of the

Chinese Academy of Forestry, using seed collected from 30

provenances. Because the seedlings of southern seedlots

were all killed by frost at northern experimental sites,

successive trials were established on 12 sites in the following

year using all northern seedlots. The provenance trial results indicated that not only did GxE interactions exist but

also that the differences in morphological characteristics

and growth rates among provenances were so significant

that two provenance zones, southern and northern, could be

clearly distinguished (Ma and others 1992a,b; Cooperative

1992). Moreover, Ma (1992) was of the opinion that these two

population groups should be considered for recognition as

two varieties, namely, P. armandii var. armandii and P.

armandii var. yunnanensis. Ma noted that many plantations of P. armandii have failed, especially in central China

in the 1960s and 1970s, due to the wrong provenances being

used in afforestation programs. He cautioned that great

attention must be paid in plantation forestry to provenance

selection, and P. armandii should not be grown for commercial purposes north of 40∞N and beyond its natural altitudinal limits in central subtropical China.

To establish clonal seed orchards by grafting, 850 superior

trees were selected in the southern provenance zone, covering Yunnan, Sichuan and Guizhou provinces. Research in

reproductive biology showed that P. armandii starts flowering at 5-7 years of age, but over 70 percent of female strobili

abort (Wu 1992; Zhang and others 1992). Generally, seed

yields of P. armandii are very low, averaging 15kg/ha for the

133 ha of seed orchards in the whole of China. In addition to

much rain and wind during flowering in May, genetic variation in reproductive ability between clones has been recorded (Liao and others 1998).

Genetic Diversity and Gene

Conservation ___________________

Genetic diversity has been studied in recent years using

analysis of isoenzyme gene markers. Kim and Lee (1995)

found that the overall mean proportion of polymorphic loci

was 66.7 percent in P. koraiensis compared with 86.2 percent

in P. densiflora , the most widely distributed native pine in

South Korea. They also found that although many populations of P. koraiensis were small in size, distributed at high

elevations and composed of closely related individuals, gene

flow between these isolated populations still remained high

in this species. To study genetic variation, eight populations

of P. koraiensis were sampled within its range in South

Korea. Research results suggested that seven of the eight

populations should be included in gene conservation programs (Kim and others 1994b; Kim and Lee 1998).

Politov and others (1999) reported genetic evidence of

natural hybridization and possible gene exchange in Siberia

between P. sibirica and P. pumila.

In northeastern China, where the natural forest resources

were over-exploited in the last several decades, the establishment of natural reserves with total area of 56,000 has

protected the remaining forest of P. koraiensis ha. In addition, 88 natural stands totaling over 40,000 ha have been

identified and conserved in situ as gene resources for sustainable forest management (Li 1997). However, due to the

increasing number of plantations of P. koraiensis being

established, necessitating the proper seed sources, more

than 30 ha of seed stands in the natural forest were documented for seed supply on several locations, with about

1,000 individuals selected for ex situ gene conservation (Niu

and others 1992).

In South Korea, three outstanding stands of P. koraiensis

and one of P. pumila, with areas of 55 ha and 2 ha respectively, have been identified and reserved for in situ gene

conservation. In the last 30 years, 91 ha of Korean pine seed

orchards have been established, the seed orchards also

serving as ex situ gene conservation (Lee 1997).

Diseases and Insect Pests ________

A survey of the literature indicates that little breeding

for resistance to diseases and insect pests has been done on

any species of five-needle pine in east Asia. However, major

pathogen and insect pests attacking P. koraiensis and P.

armandii have been investigated.

Histopathological research has shown that the stem rust

disease of P. koraiensis is caused by Cronartium ribicola J.C.

Fischer in Raben. (Xue and others 1995). Occurrence of C.

ribicola attack on P. koraiensis depends on the coexistence

of Pedicularis sp. (lousewort), the alternate host of the

pathogen (Jia and others 2000).

A dieback fungus isolated from P. koraiensis was identified as Cenangium abietis (Pers.) Duby (Cenangium

ferruginosum Fr.). When it was inoculated on five-year-old

seedlings of P. koraiensis, 80 percent of the seedlings were

infected with the same symptoms and signs as those of

naturally infected trees (Lee and others 1998)

P. armandii in the Qinling Range and Dabashan Mountains have been attacked since 1956 by Dendroctonus

armandii Tsai & Li and also by 20 other species of beetles,

causing mortality in many trees over 30 years old in the

natural forest (Tang and Chen 1999). Ophiostoma sp. and

Leptographium sp., which are fungi symbiotic with the

insects, attack the host prior to the beetle invasion.

Chen and others (1999) studied the ecology of the insect

pests in natural stands of P. armandii at middle elevations

(1,600-2,000 m) in the Qinling Range. In this study, 19

species of beetles were listed as follows:

Dendroctonus armandii Tsai & Li

Xyloterus lineatum Olivier

Hylurgopus longipilis Reitter

Polygraphus sinensis Eggers

Pityogenes sp.

Ips acuminatus Gyllenhal

Ips sexdentatus Borner

Tomicus piniperda Linnaeus

Cryphalus lipingensis Tsai & Li

Cryphalus chinlingensis Tsai & Li

Cryphalus pseudochinlingensis Tsai & Li
USDA Forest Service Proceedings RMRS-P-32. 2004 77

Genetic Resources, Tree Improvement, and Gene Conservation of Five-Needle Pines in East Asia Wang and Hong

Hylastes paralellus Chapuis

Hylastes techangenesis Tsai & Huang

Hylurgopus major Eggers

Polygraphus rudis Eggers

Polygraphus verrucifrons Tsai & Yin

Dryocoetes luteus Blandford

Dryocoetes uniseriatus Eggers

Dryocoetes autographus Ratzeburg

Of these listed species, the first 11 coexist, since they

possess different spatial and temporal niches in the stand,

enabling an equilibrium between living space and nutrient

availability among these insects.

Cone damage in seed orchards of P. koraiensis caused by

Dioryctria mendacella Staudinger andRhyacionia pinicolana

Doubleday was reported by Liao and others (1998).

Another type of injury to P. koraiensis is terminal shoot

attack leading to forking of the main stem (Zhao and others

1999). Pissodes nitidus Roelofs and Diorictria splendidella

H.S. are the main agents responsible for the damage.

Blister rust usually occurs in association with Pineus

armandicola Zhang on P. armandii, necessitating control of

both the pathogen and the insect (Li and others 2000). Other

diseases found by Liao et al .(1998) in a study of seed

orchards include Pestalotia funereal Desm., Hypoderma

desmazieriDuby, Coleosporium solidaginies (Schw.) Thum.,

Capnodium sp. and Lophodermium pinastri (Schrad. ex Fr.)

Conclusions____________________

P. koraiensis andP. armandii are important forest species

for wood and nut production. Traditional breeding programs

for advanced generations should be maintained and strengthened by using molecular genetic markers, especially for P.

armandii, which has such a wide geographic range. No

resistance breeding research is yet under way for any species

of five-needle pine in east Asia.

Currently genetic structure and genetic diversity in these

five-needle pine species in China is not well understood.

Therefore priority in research programs should be given to

studies of genetic variation, a critical prerequisite for longterm breeding and gene conservation of the two most important species. There is a trend, however, to use more Japanese

larch, [Larix kaempferi (Lamb.) Carr.], to establish plantations in China, since Japanese larch grows much faster in

early years than P. armandii growing under similar ecological conditions.

Biochemical and molecular genetic markers have proven

to be a powerful tool in biosystematic and biogeographic

studies of forest trees (Adams 1992; Strauss and others 1992).

Molecular biology can also help with understanding of the

scattered patterns of occurrence of the less-known five-needle

pines, providing information on evolutionary history and

intraspecific variation, fields of investigation not yet undertaken in China. Without this information, conservation

strategy cannot be established on a sound scientific basis.

In China, the remaining genetic resources of P. koraiensis

are mostly protected in nature reserves, whereas the outstanding seed stands of P. armandii identified in earlier

provenance trials have not yet been integrated into forest

management activities. Gene conservation is a crucial component of sustainable forest management (Eriksson and

others 1993; Palmberg-Lerch 1999). Therefore, a strategy

for gene conservation and utilization of five-needle pines

should be developed and integrated into a regional action

plan for the conservation of the forest genetic resources in

east Asia, as recommended by the FAO and other concerned

organizations (Palmberg-Lerch 2000; Sigaud and others

2000).

Acknowledgments ______________

The senior author wishes to sincerely thank Professor

Scott Schlarbaum of the University of Tennessee, Coordinator of five-needle Pines Working Party, IUFRO, for his kind

invitation and Dr. Safiya Samman of Forest Service, USDA,

for making all the arrangements for his attention to the

conference, and also thanks the USDA/FS for their generous

support. The authors also thank Professor Zhao Guangyi,

Northeast Forestry University, and Professor Ma Changgeng,

Chinese Academy of Forestry, for useful discussions during

the preparation of this paper.

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Abstract—Stands of blue pine (P. wallichiana A.B. Jacks. syn. P.

griffithii McClelland) are highly diverse throughout its range of

distribution in the Himalayan Mountains where the species grows

under varying geographic, climatic, and edaphic conditions. The

species occurs in two distinctly different ecotypes (mesic monsoon

and dry nonmonsoon), and strict avoidance of germplasm transfer

between the ecotypes is necessary for survival and productivity in

Pakistan, India, and Nepal. The role of these ecotypes in enhancing

productivity and in establishing large-scale plantations resistant to

blister rust is presented and compared with plantations in India and

Bhutan. An alternate management strategy to establishing a pure

species stand is to interplant with other native conifers. Testing of

blue pine in other countries is discussed, notably the superior

performance of blue pine hybrids in the USA at specific sites, which

indicates its breeding value as a parent for volume growth rate. In

Germany and Japan, the species does not appear to be useful for

forest plantations. Further international cooperation in testing of

blue pine for rust resistance is proposed, given the variable resistance to blister rust within the species. Research needs to be

conducted, especially in neighboring countries, to further assess the

extent, nature, and pattern of geographic variability.

Key words: genetic diversity, blue pine, Himalayan pine, P.

wallichiana, blister rust, pine hybrids, ecotypes

Introduction ____________________

Blue pine (Pinus wallichiana) is a highly variable species

that occurs in the mountainous region of lower Asia. Natural

stands of blue pine, generally known outside of southern

Asia as Himalayan pine, are distributed in Afghanistan,

Pakistan, India, Nepal, Bhutan, Tibet, China, and Burma.

The species spans a longitudinal range between 68∞ and 100∞

E, a latitudinal range between 25∞ and 37∞ N, and an

altitudinal range between 1,500 and 3,800 m (Critchfield

and Little 1966, Khan, 1986). In the Himalayan Mountains,

blue pine occurs under two rainfall regimes, within and

outside the monsoon region. Dogra (1972) and Ahsan and

Genetic Variation in Blue Pine and

Applications for Tree Improvement in

Pakistan, Europe and North America

Shams R. Khan

Khan (1972), along with several earlier investigators including Brandis (1906), Osmaston (1927), and Shebbeare (1934),

have recognized this variable site distribution of the species

occurring in several countries of the region. Pure and mixed

patches at varying altitudes are found, but the species grows

well at an optimum elevation of 2,000-2,500 M. Although

this pine occurs over a wide altitudinal range, there is no

evidence of altitudinal races that could be given subspecific

or specific taxonomic ranking.

This species has been known by a number of scientific

names since first described. The taxonomy of blue pine has

been a subject of controversy, probably corresponding to

the diversity in the species on the wide range of ecotypes

where it occurs. The older scientific names of blue pine were

P. excelsa Wall. ex Lamb. (1824), P. chylla Lodd. (1836), P.

nepalensisDe Chambr. (1845), P. griffithiiMcClelland (1854),

and P. dicksonii Hort. ex Carriere (1855). Pinus griffithii

persisted in international botanical acceptance until the

mid-twentieth century (Rehder 1940, Little and Critchfield

1969), and in China it is still the accepted taxonomic name

(Wang, pers. comm.). Elsewhere, P. wallichiana A.B. Jacks.

(1938) is the latest revision and is now the internationally

accepted name (see citation in the list of five-needle pine

species in this volume).

Although ecotypic variation has been observed (Khan

1986, 1994, 1995a), no detailed taxonomic studies of blue

pine have been made at the ecotypic level. Patschke (1913),

Wilson (1916), Kew Bulletin (1938), Dallimore and Jackson

(1961), Ouden and Boom (1965) have mentioned varieties/

forms of blue pine. Grierson and others (1980) reported “var.

parra” as a blue pine variety. Critchfield and Little (1966)

also made this distinction and described “var. parra” from

Arunachal Pradesh (India) as the mesic monsoon zone

ecotype of blue pine.

Blue pine has also been found to occur at high altitudes in

low rainfall areas and at low altitudes (less than 2,800 m) in

high rainfall areas (Khosla and Raina 1995). The soils of

mesic habitat were found to be different from those of xeric

sites in pH, maximum moisture holding capacity, and Ca

and Mg concentrations throughout the species’ distribution

in Pakistan (Khan 1986). There were also differences exhibited in the phenology of male and female flowers in the two

distinct ecological zones of the species (Khan 1995b).

In view of the importance of geographic variability to any

tree improvement program, provenance studies were undertaken in Nepal, India, Bhutan, and Pakistan. In Pakistan,

the species has been extensively studied for genetic variability and timber utilization, as well as the species’ role in

protection of the fragile ecosystems in the Himalayan Mountains. Such studies have further led to the delineation of
80 USDA Forest Service Proceedings RMRS-P-32. 2004

Khan Genetic Variation in Blue Pine and Applications for Tree Improvement in Pakistan, Europe and North America

seed zones with strict prohibition of transfer of germplasm

between ecological zones (Khan 1991, 1994). Two geographic

ecotypes were identified on the basis of differences in mean

annual increment and growth in Pakistan, accounting for 56

percent of the variation among stands (Khan 1997, 2000).

These two ecotypes also occur in India, Nepal, and Bhutan

under similar geoclimatic conditions and consequently have

similar ecological requirements and distribution patterns.

The present paper outlines the extent to which genetic

diversity in blue pine could be utilized for forest plantings in

these countries.

Genetic Research on Blue Pine in

Pakistan _______________________

Pakistan has sampled the genetic variation in blue pine

more extensively than has any of the other countries in a

cooperative project with the USDA Forest Service. Detailed

studies of genetic diversity and its utilization in the natural

stands of blue pine in Pakistan were conducted in several

phases during the past 25 years. Studies of 32 provenances

sampled throughout its range of distribution in Pakistan

were undertaken to assess genetic variation in several

morphological and anatomical traits of the needles, cones,

and seeds.

Seed Movement

The potential for further genetic improvement in blue pine

was shown by studies that showed evidence of ecotypic

differentiation and suggested strict avoidance of transfer of

germplasm from xeric to mesic habitat and vice versa (Khan

1991, 1994).

Comparison of Blue Pine with Other

Species

Paudel and others (1996) tested P. patula Schiede &

Deppe ex Schlechtendal & Chamisso and blue pine in Nepal.

Of these species, blue pine had better survival at age six.

Wallace (1989) compared the growth of P. strobusL. and blue

pine in eastern Nepal and observed little difference in

survival, growth, and form. However, he suggested blue pine

was a better species overall. Siddiqui and others (1989)

explored variability in the wood characteristics of three

forest tree species, including blue pine, and found marked

differences in percentage of late wood, smaller tracheid

dimensions, higher density, and better strength in wood of

drier areas when compared to wood from moist areas. Ashley

and Fisk (1980) tested 45 species in 15 trial plots and

observed that blue pine growing well in Kathmandu Valley,

Nepal. Of several exotic temperate pines tested in the past

two decades, none seems to thrive better than native blue

pine in Pakistan (Annual Progress Reports, Pakistan Forest

Institute, Peshawar). The consensus from these reports is

that exotic pines have little potential in large-scale plantations in the native habitat of blue pine.

Interplanting Blue Pine to Increase Overall

Yield

In Pakistan, interplanted mixtures of blue pine and other

coniferous species in different stands have led to studies that

identified the best combinations of associated conifers for

high productivity, in addition to further quantifying genetic

diversity in blue pine. Differences in mean annual increment between two species habitats were compared with

those of the highly associated conifer Cedrus deodara (D.

Don.) G. Don (Khan 1997, 2000). These studies suggest that

blue pine should be planted as a monoculture in the xeric

areas (less than 750 ± mm per annum), whereas it should be

planted in mixtures with C. deodara in mesic habitats.

Similar recommendations were also made by Khan (1979)

for moist coniferous forests of northern Pakistan, although

he did not report on the dry, nonmonsoon zone. Further

research is under way on growth differences of the two

ecotypes of blue pine in combination with other associated

conifers, for example, Abies pindrow (Lamb.) Royle and

Picea smithiana (Wall.) Boiss., to assess the possibility of

improving productivity of mixed stand plantations in the

Himalayan Mountains.

Performance of Blue Pine as an Exotic

Species

Blue pine has been tested for variation in growth rate and

in breeding for blister rust resistance (Cronartium ribicola

Fisch. ex Rabenh.) in a few countries of Asia, Europe, and the

USA. Stephan (1974) observed differences in rust resistance

between trees of two provenances of blue pine in Lower

Saxony, Germany at age 11, although there was a high level

of mortality in trees from both provenances. In a species

comparison test, he found that only 40 percent of the blue

pine seedlings were infected by blister rust compared with

an infection rate of 100 percent in P. strobus, P. monticola

Dougl. ex D. Don, and P. flexilis James (Stephan 1985).

Borlea (1992) and Blada (1994) have also used blue pine in

Romania in experimental breeding for growth and blister

rust resistance. These authors found that blue pine has

potential value for genetic improvement in rust resistance.

The growth and blister rust data recorded in Romania

indicated that half-sib families from low rainfall areas in

Pakistan were more resistant to blister rust at age 11,

compared with those originating from high rainfall areas

(Blada 1994). Blada reported a correlation between latitude

and blister rust (r=0.66** *), but could not include rainfall

data of the native habitat in his studies, as they were in

remote locations. To aid in interpretation of results, rainfall

of the nearest meteorological station was included. Two of

the most important phenomena affecting rainfall in the

Himalayas, namely inversion of temperature and rainshadow

effect, were also not taken into consideration, as no reliable

data were available for such sites. In spite of these limitations, the moderately high correlation between blister rust

and rainfall suggests the adaptive potential of the two

ecotypes of this pine. The correlation would have been more

reliable, however, if the annual rainfall data had been
USDA Forest Service Proceedings RMRS-P-32. 2004 81

Genetic Variation in Blue Pine and Applications for Tree Improvement in Pakistan, Europe and North America Khan

included from the actual collection sites, as the distribution

of rainfall is highly erratic in the Hindukush-Karakorum

region. It was concluded that differences in the adaptation of

the species to varying moisture regimes in its native habitat,

along with morphological and phenological differences between the two ecotypes, might be casually associated with

corresponding differences in resistance to blister rust in blue

pine. These results, therefore, suggest that the selection of

trees/stands for rust resistance should be primarily based

upon the origin of seed lots of the native habitat of this pine.

Comparative studies on genetic diversity in natural stands

for various traits and the degree of resistance involving blue

pine and other five-needle pines have also been reported by

Dogra (1972), Ahsan and Khan (1972), and Bakshi (1972).

The comparative growth of blue pine as an exotic in Europe,

USA and Canada has been studied by Gremmen (1972),

Kriebel (1983), Heimburger (1972), and other scientists.

These studies emphasized mass production of rust resistant

stands through hybridization. However, this approach can

be expensive, cumbersome and time-consuming and may

not be practical for developing countries. A more feasible

strategy would be to collect seed from the xeric habitat of the

species range for mass production of desirable trees in

Pakistan and Romania. Insufficient cold hardiness is a

deterrent in the USA and Canada, where hybridization

might be feasible. The authors cited agree that conventional

intraspecific breeding may not be the best method due to

long generation time, mode of pollination, highly diverse

populations due to cross pollination, haploid nature of infecting rust and the presence of an alternate five-needle pine

host(s).

A two-way seed exchange of plant material was conducted

between Pakistan and the USA. This exchange was part of

a worldwide program to test haploxylon pines for resistance

to the white pine blister rust and to test other five-needle

pine species in Pakistan. This project yielded useful information on the nature of resistance, growth potential of some

white pines, inoculation techniques, and breeding schemes

for mass production of resistant trees.

Garrett (1992) reported that Himalayan white pine was

not resistant to the white pine weevil (Pissodes strobi Peck)

and suggested that it should only be planted in weevil-free

regions of the USA. However, the species has not performed

well where tested in weevil-free regions (Ohio and Tennessee), and commercial planting cannot be recommended anywhere in the USA on the basis of present knowledge. In 20-

year-old provenance tests in Ohio of trees from India,

Pakistan, and Nepal, new terminal shoots on trees of all seed

sources were repeatedly killed back by late spring frosts. No

trees from Nepal survived the winter climate. Field testing

of P. strobus x wallichiana hybrids in Ohio over two decades

demonstrated superiority in wood volume growth over most

P. strobus genetic selections (Kriebel 1983). The hybrids also

have a higher wood specific gravity than P. strobus (Kriebel,

pers. comm.). In Tennessee, initial survival of drought was

very low (Schlarbaum, pers. comm,). Genys (1979) compared

variability among 21 seedlings of blue pine originating from

India, Pakistan, and Bhutan. Strains from Pakistan were

hardier than those of other countries in Maryland. However,

the performance of the species has not been satisfactory in

Germany and Japan (Takahashi and others 1974, Stephan

1985). From these studies, it appears that blue pine as a

hybrid parent has a potential for better growth and resistance against blister rust in the USA, but its performance in

Europe and Asia has yet to be ascertained.

Conclusions and Recommendations

Seed origin of blue pine is critical to survival, growth, and

perhaps blister rust resistance. Two general ecotypes are

known and careful attention should be paid to which ecotype

is represented in a seed collection. Selections and collection

of blue pine seed in native stands from trees growing near

glaciers or river banks as well as from buffer zone should be

avoided as these sites may represent atypical habitat in blue

pine. Efforts should be made to include meteorological data

from actual sites to minimize biased estimates derived from

the nearest weather stations. Because of dysgenic selection

in several countries of the Himalayan region, including

Pakistan, the establishment of in situconservation stands in

the xeric habitat could assist in mass-producing desirable

individuals resistant to blister rust. These efforts should be

supported by further research on genetic diversity in this

widespread and phenotypically variable species.

Research has shown that blue pine often thrives in planting of mixtures with other coniferous species in certain

environments. Additional research should be conducted to

better delineate the effects of mixture composition, blue pine

genetics, and site variation to maximize productivity.

Establishment of further blue pine provenance tests in the

USA and Canada does not appear to be practical because of

the demonstrated insufficiency of winter-hardiness in regions where the species might be useful. In contrast, cooperative international trials should be conducted in Pakistan

and Romania to continue research and establish seed orchards and seed production areas from ecotypes showing

higher degree of blister rust resistance. The prevalence of

field races of the rust, as reported by Patton (1972), should

also be explored in this species. Multiclonal hybrids, particularly of blue pine and P. strobus, could be developed for rust

resistance as a cooperative effort of the USA, Canada,

Romania, and Pakistan to achieve desirable objectives in

breeding both species.

References _____________________

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Shahzad and others Proc. IFS/IUFRO Workshop, Hyytyala, Finland,

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strategies for the natural stands of Pinus wallichiana A.B.

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Cedrus deodara and Pinus wallichiana from dry and wet temperate forests. Pak. Jour. For. 39:27-33.

Stephan, B.R. 1974. Geographic variation in Pinus strobus on the

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Genet. 23:214-220.

Stephan, B.R. 1985. Resistance of 5-needled pines to blister rust.

Allgemeine Forstzeitschrift 28:695-697.

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growth of young samples stands of introduced species in the

Tokyo University Forests in Hokkaido. Tokyo University Forests

18:29-66.

Wallace, D. 1989. Report on provenance trial of Pinus maximinoi/

pseudostrobus. PAC Tech. Paper, Pakhribas Agr. Centre No. 102,

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University Press; 85 pp.
USDA Forest Service Proceedings RMRS-P-32. 2004 83

Part II: Genetics, Genecology, and Breeding

Pinus lambertiana (sugar pine)

photo courtesy of B. Danchok
84 USDA Forest Service Proceedings RMRS-P-32. 2004
USDA Forest Service Proceedings RMRS-P-32. 2004 85

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

Dmitri V. Politov is Senior Research Associate, Laboratory of Population

Genetics, N.I.Vavilov Institute of General Genetics, Russian Academy of

Sciences, 3 Gubkin St., GSP-1, Moscow 119991, Russia. Phone: 7 (095) 135-

5067. Fax : 7 (095) 132-8962. E-mail: dvp@vigg.ru. Konstantin V. Krutovsky

(author for correspondence) is Research Plant Molecular Geneticist, Institute

of Forest Genetics, USDA Forest Service, Pacific Southwest Research Station, Environmental Horticulture Department, University of California,

Davis, One Shields Avenue, Davis, CA 95616 USA. Phone: 530-752-8412. Fax:

530-754-9366. E-mail: kkroutovski@ucdavis.edu

Abstract—Phylogenetic and population genetic studies of native

five-needle pines growing in Russia and neighboring countries were

reviewed. Four species, Pinus cembra, P. sibirica, P. pumila and P.

koraiensis, together with North American species P. albicaulis,

comprise the subsection Cembrae (stone pines) of the section Strobus

(white pines). They share bird-dispersal related traits such as seed

winglessness and cone indehiscence that differentiate them from

most other white pines and related section Parrya. Phylogenetic

analysis showed that P. cembra, P. sibirica and P. albicaulis

represented a close group based on isozyme loci and other molecular

genetic markers. Pinus pumila and P. koraiensis also clustered

together in phylogenetic trees, but they were closer to P. parviflora

and other East Asian pines of the subsection Strobi than to the

cembra-sibirica-albicaulis group. Therefore, we hypothesized that

seed winglessness and other bird-dispersal related traits could

either arise independently in these lineages or could have been

introduced via occasional hybridization. Natural hybridization between P. sibirica and P. pumila in their zone of sympatry in the

Baikal region was confirmed by isozyme methods, but no significant

introgression was revealed. The possibility of producing hybrids in

artificial crosses between P. sibirica and P. cembra, and P. sibirica

with P. koraiensis was also confirmed by isozyme analysis. Intrapopulation genetic variation, measured as expected heterozygosity of

isozyme loci (He), was relatively high in P. koraiensis (He = 0.130)

and P. sibirica (0.106) and similar to the average for other soft pines,

but significantly higher in P. pumila (0.198) and lower in P. cembra

(0.082). There was no obvious difference in the level of heterozygosity between bird-dispersed pines of the subsection Cembrae and

wind-dispersed pines of the closely related subsection Strobi. Relatively low inbreeding was observed in embryos in all five-needle

pines and was primarily caused by self-pollination. However, Hardy

– Weinberg equilibrium or even a slight excess of heterozygosity was

usually observed among mature trees, apparently as a result of

selection against inbred progeny and in favor of heterozygotes. Two

Phylogenetics, Genogeography and

Hybridization of Five-Needle Pines in Russia

and Neighboring Countries

Dmitri V. Politov

Konstantin V. Krutovsky

types of polyembryony, monozygotic and polyzygotic, were found in

P. sibirica. Gene geography studies in P. sibirica and P. pumila

populations based on multivariate analysis of isozyme data was also

discussed.

Key words: Cembrae, genogeography, isozyme, phylogenetics,

Strobi, stone pines, white pines

Introduction ____________________

Four species of five-needle pines, also known as haploxylon

or soft pines (genus Pinus L., subgenus Strobus Lemm.), are

native in Russia and neighboring countries (fig. 1). These

four species - P. cembra L. (Swiss stone pine), Pinus sibirica

Du Tour (Siberian stone pine), P. pumila (Pall.) Regel

(Siberian dwarf pine) and P. koraiensis Sieb. et Zucc. (Korean pine) - are classified with section Strobus Loud. (white

pines) and subsection Cembrae Loud. (stone pines). Pinus

sibirica forms forests of great economical importance in

Siberia and the Far East, while P. pumila occupies vast

territory in East Siberia and along the Asian Pacific coast.

Pinus koraiensis is distributed in the Russian Far East,

including the Amur Region, Khabarovsk and Primorskii

Territories, and the Island of Sakhalin. Pinus cembra is

scattered in the East Carpathian Mountains in Ukraine.

Figure 1—Geographic distribution of five-needle pines in

Russia and neighboring countries.
86 USDA Forest Service Proceedings RMRS-P-32. 2004

Politov and Krutovskii Phylogenetics, Genogeography and Hybridization of Five-Needle Pines in Russia and Neighboring Countries

The Cembrae species have large edible wingless seeds that

are dispersed by birds and play a key role in Siberian taiga

(P. sibirica, P. koraiensis) and subalpine (P. cembra, P.

pumila) ecosystems. Although P. parviflora is a five-needle

pine species that occurs in Russia, it is also endemic to

Japanese islands and occurs in a few isolated populations on

the Kuril Islands along the Russian Pacific coast.

Evolutionary relationships and intraspecific population

genetic structure of Eurasian Cembrae pines have been

studied since early 1900s, primarily by traditional morphological methods (compare Bobrov 1978; Iroshnikov 1974;

Lanner 1990, 1996, 1998; Smolonogov 1994). However, the

levels of intrapopulation, intraspecific and interspecific genetic variation have been quantitatively estimated only

after development of reliable molecular genetic markers.

This paper presents an overview of genetic studies of the

above-mentioned five-needle pines. We have concentrated

on results obtained using molecular genetic markers, but

also included karyological and morphological data when

needed.

Phylogenetics __________________

All four Cembrae pine species produce functionally indehiscent cones and large wingless seeds. Based on those

traits, these species and the North American whitebark pine

(P. albicaulis Engelm.) are traditionally included in the

subsection Cembrae (stone pines) within section Strobus

(white pines). This section together with the species of the

section Parrya Mayr. comprise the group of soft pines (subgenus Strobus, or Haploxylon) of the genus Pinus. We used

the most widely accepted classification by Critchfield and

Little (1966) with minor modifications and additions (Price

and others 1998). According to this classification section

Strobus is subdivided into two subsections Cembrae and

Strobi Loud. that both have representatives in Eurasia and

North America (fig. 2).

This subdivision is based on the occurrence of large wingless seeds and indehiscent (not opening upon ripening)

macrostrobili (cones) in Cembrae pines. It has been widely

accepted that both these traits evolved as a result of the

Section Strobus

Subsection Strobi

winged seeds

dehiscent cones

Subsection Cembrae

wingless seeds

indehiscent cones

Europe 

P. monticola Douglas ex D. Don

P. lambertiana Douglas

P. flexilis James

P. strobiformis Engelmann

P. strobus Linnaeus

P. ayacahuite Ehrenberg ex Schlechtendahl Mexico, Central America

P. chiapensis (Martínez) Andresen

P. parviflora Siebold & Zuccarini

P. morrisonicola Hayata

P. dalatensis de Ferré

P. fenzeliana Handel-Mazzetti

P. wangii Hu & Cheng

P. dabeshanensis Cheng & Law

P. wallichiana A.B. Jackson

P. bhutanica Grierson, Long & Page

P. armandii Franchet China, Tibet, Burma

Japan, Russia

Bhutan, India

P. peuce Grisebach Southeastern Europe

Vietnam

Taiwan

China

China, Vietnam

Pakistan, India

Eastern and Central China

Himalayan Mountains, China,

Tibet, Afganistan, Burma

Eastern North America

Northwestern North America

Southwestern USA, Mexico

Northwestern North America,

Western North America

Mexico, Guatemala

P. sibirica Du Tour Siberia, Mongolia

P. pumila (Pallas) Regel Northwestern Asia,

Northern Far East,

Korea, Japan

P. koraiensis Siebold &

Zuccarini Southeastern Siberia,

Northern Far East,

Korea, Japan

P. albicaulis Engelmann Western North America

P. cembra Linnaeus Europe (Carpathian

Asia Mountains, Alps)

North America

Europe 

Asia 

North America

Figure 2—Taxonomic classification and generalized distribution of white pines (Critchfield and Little 1966; Price and

others 1998). Underlined are species with wingless or almost wingless seeds (Lanner 1996, 1998).
USDA Forest Service Proceedings RMRS-P-32. 2004 87

Phylogenetics, Genogeography and Hybridization of Five-Needle Pines in Russia and Neighboring Countries Politov and Krutovskii

adaptation to dispersal of their seeds by corvid birds (for

example, Lanner 1996; Tomback and Linhart 1990), and

particularly nutcrackers (Nucifraga spp.). The closely related subsection Strobi includes mainly typical wind-dispersed species with small winged seeds released from the

cones upon ripening. However, seeds of some of Strobi

species (underlined in fig. 2) are large and virtually maladapted to dispersal by wind. Moreover, traits that are

normally characteristic of Cembrae pines (especially winglessness) sporadically occur also in Strobi pines as rare

abnormalities or as within-population polymorphism. Reproductive barriers are incomplete between these two subsections, and there are numerous examples of artificial and

natural interspecific hybridization both within and between

subsections (Critchfield 1986; Blada 1994). The occurrence

of intermediate forms and documented hybridization between species of different subsections make the hypothesis

of monophyletic origin of Cembrae and Strobi subsections

controversial. This problem stimulated repeated attempts

to revise the taxonomy of the group and to rearrange the

composition of the subsections. However, until recently, the

problem was studied mainly using morphological comparisons (for example, Kupriyanova and Litvinceva 1974).

Muratova (1980) compared karyotypes of three Eurasian

Cembrae pine species and found only slight differences in

chromosome size and morphology, as well as in number and

localization of nucleoli organizers. Pinus cembra and P.

sibirica karyotypes were the most similar, although there

were slight differences. The karyotype of P. pumila was

closer to these species than to P. koraiensis, which partially

contradicts molecular genetic data presented below.

The first molecular genetic evidence of great similarity

between P. cembra and P. sibirica was provided with isozyme

loci (Krutovsky and others 1990). This study also showed

that P. pumila and P. koraiensis comprise the next closely

related pair. These relationships among Eurasian Cembrae

pines were confirmed later by other scientists (for example,

Goncharenko and others 1991; Shurkhal and others 1992).

Pinus albicaulis is the only North American Cembrae pine.

This species together with all four Eurasian Cembrae pines

were studied by Krutovsky and others (1994, 1995), which

were the first studies where phylogenetic relationships were

analyzed within the entire subsection Cembrae using molecular genetic markers. Two lineages were found again in

Cembrae pines; P. cembra and P. sibirica formed a pair of the

most closely related Cembrae species, while another lineage

was represented by two Far Eastern species P. pumila and

P. koraiensis. The position of P. albicaulis was not fully

resolved at that time, but it was found to be closer to the

cembra–sibirica group.

Krutovsky and others (1994, 1995) studied chloroplast

DNA (cpDNA) restriction fragment length polymorphisms

(RFLPs) in all five Cembrae pine species, as well as in four

representatives of North American pines from subsection

Strobi (P. strobus L., P. lambertiana Dougl., P. monticola

Dougl., and P. flexilis James). Species of Strobi were close to

Cembrae species, but they did not form a separate cluster in

the dendrogram. A similar pattern was also observed in

other studies based on cpDNA markers, although unfortunately neither of those studies included all representatives

of Cembrae pines species (for example, Wang and Szmidt

1993; Wang and others 1999). For instance, Wang and

Szmidt (1993) used cpDNA RFLP markers to study phylogenetic relationships in several Asian pine species including

one Cembrae pine (P. sibirica) and five Strobi pines (P.

armandii Franch., P. griffithii McClelland (syn. P.

wallichianaA. B. Jacks.), P. kwangtungensisChun & Tsiang,

P. parviflora Sib. & Zucc., and P. peuce Griseb.). Pinus

sibirica (Cembrae) clustered very closely with P. parviflora

(Strobi) with 95 percent of the bootstrap support within the

following topology in the maximum parsimony consensus

tree: (peuce ((parviflora, sibirica) (armandii (griffithii,

kwangtungensis)))). However, the absence of Cembrae species other than P. sibirica in the analysis made it impossible

to draw any conclusions about phylogenetic relationships

between Cembrae and Strobi pines. In a more recent study

based on cpDNA markers (Wang and others 1999), the

topology of the resulting consensus tree was different: (peuce

(strobus (monticola (parviflora (koraiensis, armandii,

griffithii, cembra, kwangtungensis, pumila))))). However,

resolution within a group of six species belonging to both

subsections Strobi (P. armandii, P. griffithii and P.

kwangtungensis) and Cembrae (P. cembra, P. koraiensis and

P. pumila) was insufficient to make any strong conclusions

on the phylogeny of these species. The parsimony tree

indicated that two North American (P. strobus and P.

monticola) and one European (P. peuce) species of subsection

Strobi appeared to be ancestral to all Asian white pine

species including P. cembra. These results contradict our

point of view that Asian pine species are ancestral to North

American pines, which is supported by Belokon and others

(1998) study based on more numerous nuclear markers and

more representative set of white pine species. Liston and

others (1999) used ribosomal internal transcribed spacer

(ITS) DNA markers to study phylogeny of the genus Pinus.

White pines of the section Strobus formed a separate cluster,

but relationships within the section were unclear. The only

clade well supported by bootstrap was comprised of P.

cembra and P. albicaulis, which agreed with our earlier

allozyme and cpDNA data (Krutovsky and others 1994,

1995). Belokon and others (1998) studied 13 white pine

species using isozyme markers coupled with Principal Component Analysis (PCA) and also did not find a strong support

for dividing section Strobus into the two traditional subsections Cembrae and Strobi. Instead, the study found even

more evident differentiation between North American and

Eurasian white pines. The updated results from the Belokon

and others (1998) study with increased sample size and two

additional species (P. lambertiana and P. strobiformis) are

presented in figure 3 (Politov and others, unpublished).

Cembrae pines were separated from Strobi pines along the

first dimension on the two-dimensional (2D) plot obtained by

multidimensional scaling of Nei’s (1972) genetic distance

matrix based on isozyme data, while the second dimension

differentiated North American and Eurasian species. Among

them, P. peuce of southern Europe was located (genetically)

as the nearest species to North American Strobi pines (fig.

3). In general, Eurasian Strobi species were more differentiated, and P. griffithii (as P. wallichiana in the paper) was

the nearest to the Asian P. pumila and North American P.

albicaulis (Cembrae pines).

A representative of the related section Parrya (pinyons),

Pinus edulis Engelm., was included as an outgroup in

Belokon and others (1998) study. The dendrogram of white
88 USDA Forest Service Proceedings RMRS-P-32. 2004

Politov and Krutovskii Phylogenetics, Genogeography and Hybridization of Five-Needle Pines in Russia and Neighboring Countries

cembra 

sibirica

koraiensis 

pumila

albicaulis 

wallichiana 

parviflora 

peuce 

lambertiana 

strobus 

flexilis

monticola strobiformis 

armandii 

morrisonicola

Cembrae Strobi 

N o r t h A m e r i c a and E u r o p e 

Eurasia 

Figure 3—Two-dimensional (2D) plot of 13 white pine species (section Strobus)

obtained by multidimensional scaling of Nei’s (1972) genetic distance matrix based

on isozyme loci.  - Eurasian Cembrae;  - North American Cembrae; , Eurasian

Strobi;  - North American Strobi.

pine species and P. edulis obtained in this study (fig. 1 in

Belokon and others 1998) indicated that Asian Strobi pines

are evolutionarily older than both American Strobi and all

Cembrae pines. There is other evidence also supporting the

hypothesis that subsection Cembrae is an evolutionarily

younger taxonomic group. It is based on the relatively recent

origin of nutcracker birds (Nucifraga spp.) that disperse

seeds of Cembrae pines.

However, despite indicating evolutionary trends in section Strobus, none of the above mentioned phylogenetic

studies was able to unambiguously confirm the validity of

the subsection Cembrae. We believe that there can be several explanations for the controversial origin of Cembrae

pines. Their wingless seeds and indehiscence could have

been indeed inherited from a common ancestor (true monophyly), but sufficiently so long ago that relatedness is obscured by millions of years of evolution in diverse habitats.

Allozyme variation is not necessarily neutral, at least in

part, and may be shaped by selection of particular alleles or

genotypes advantageous in particular environment reflecting local adaptation rather than phylogenetic relationships.

The monophyly of subsection Cembrae assumed under this

scenario does not necessarily mean monophyly of subsection

Strobi. Cembrae pines could have arisen within wind-dispersed Strobi, but these two subsections are not necessarily

two sister groups.

The second scenario assumes polyphyletic origin of

Cembrae pines. Genetic divergence between P. sibirica – P.

cembra (and perhaps P. albicaulis), from one side, and P.

pumila – P. koraiensis, from another side, supports this

hypothesis. It is relatively high and comparable to the level

of divergence between species from different subsections,

Cembrae and Strobi. This could mean an independent and

multiple (polyphyletic) origins of wingless seeds and indehiscent cones in these two lineages. The wingless seeds occur

also in several white pines of subsection Strobi. As to cone

indehiscence, this trait is underlain by a simple mechanism;

Cembrae cones lack specific tracheid fascicles (coarse fibers)

in the scales. In other pine species, the fascicles contract,

when a cone dries, causing scales to bend outward (Lanner

1990). The critical question is whether the absence of this

tissue, a homologous trait, synapomorphic for all five Cembrae

species. We think that this is not necessarily the case, since

the lack of this tissue could arise independently in two

evolutionary lineages (P. sibirica - P. cembra - P. albicaulis

and P. pumila - P. koraiensis) as a result of nonhomologous

mutations. Indehiscence then could be fixed via coevolution

of pines and their dispersers, corvid birds (Tomback and

Lindhart 1990), as this trait helps to hold the seeds in the

cones making them more easily available for birds.

There is also a possibility of interspecific gene flow and

multiple cases of gene exchange between different white

pine species (Critchfield 1986). Interspecific hybridization

and occurrence of natural interspecific hybrids were documented between P. sibirica and P. pumila within subsection

Cembrae (Politov and others 1999), and between P. pumila

and P. parviflora from different subsections Cembrae and

Strobi, respectively (Watano and others 1995, 1996). Once

indehiscence appeared, it could have crossed species borders

as a result of sporadic hybridization events. Therefore,

indehiscence could be a result of a “monophyletic” event

(appearing once in the evolution). However, genes responsible for indehiscent cones could share the same ancestor

gene or genes, while other genes in the genome may have a

quite different evolutionary history. Past or present gene

introgression may also be responsible for similarity of ge-
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Phylogenetics, Genogeography and Hybridization of Five-Needle Pines in Russia and Neighboring Countries Politov and Krutovskii

netic markers in sympatric or nearly sympatric species, such

as western North American white pines P. monticola, P.

flexilis and P. strobiformis Engelm., or white pines from

Taiwan, P. morrisonicola Hayata and P. armandii (which

was represented by the Taiwanese variety in our material).

If introgressive hybridization indeed took place in the

white pine evolutionary history, this could violate major

principles of phylogenetic analysis, such as independence of

compared operational taxonomic units (OTUs), and therefore, white pine evolution would be better described as

reticulate evolution. Even a large number of molecular

markers could be insufficient to resolve complicated reticulate patterns. Although molecular genetic markers have

already provided valuable information for the understanding of genetic relationships in the section Strobus, their

value for phylogeny is still not fully understood.

Genogeography_________________

Pinus sibirica

Pinus sibirica was the first Cembrae pine in Russia that

was studied by isozyme loci. Eleven isozyme systems coded

by 19 loci were described more than a decade ago (Krutovsky

and others 1987), and genetic differentiation among populations was estimated (Krutovsky and others 1989; Politov

and others 1989; Politov and others 1992; Krutovsky and

others 1994, 1995). The proportion of interpopulation variation in total species variation measured as FST value was

relatively low (2.5 percent) in this species; nevertheless

cluster analysis of 11 populations based on isozyme genetic

distances showed good correspondence to their geographic

origin. Populations from major regions that were substantially different in habitat types formed separate clusters:

South Siberian Mountains, Western Siberian Plain, and

Northern Siberia.

Using practically the same set of isozyme loci, Goncharenko

and others (1993b) reported FST in P. sibirica to be 3.9

percent. Despite broader representation of samples (from

northwestern Siberia to eastern Kazakhstan and to Lake

Baikal Region in East Siberia), the concordance of UPGMA

clustering to geographic origin was less pronounced in their

study. We believe that this could have been caused by biased

allele frequency estimations due to relatively low sample

size, 12.9 trees on average per population as compared to

38.9 trees in Politov and others (1992) and Krutovsky and

others (1994, 1995). Using seven isozyme loci analyzed in the

needle tissue, 41 samples were studied by Podogas (1993),

and the position of samples on the resulting PCA plot

generally corresponded to the geographic location of populations while the FST value was also relatively low (3 percent).

Using 31 allozyme loci, Politov (1998) studied genetic

differentiation among 15 populations in Lake Baikal region

in East Siberia. A higher level of interpopulation diversity

(FST=6.3 percent) was revealed in these populations, and

genetic differentiation studied by PCA was in good concordance with geography (fig. 4). Comparative analysis of

Baikalian populations together with earlier studied populations from West and Middle Siberia using 10 common loci

showed that Baikalian populations were different from

other major provinces (Politov 1998).

Pinus cembra

Allozyme variation in P. cembra populations was first

studied by Szmidt (1982). The estimated population genetic

differentiation (FST = 0.313) was unusually high compared

to other pine species. In part, the high FST value could be

explained by greater isolation among P. cembrapopulations.

There are reasons, however, other than isolation that can

explain that high FST. Based on the description of the

geographic origin provided by the author, one of the samples

appeared to be collected from a P. sibirica population in

Chitinskaya Region in East Siberia. This area is sympatric

for both P. sibirica and P. pumila. We compared genetic data

obtained for East Siberian populations of P. sibirica and P.

pumila, which revealed that this sample represented neither P. cembra nor P. sibirica. Several alleles in this sample

are species specific for P. pumila and were never found in

either P. cembra or P. sibirica. Correspondingly, we believe

that this sample actually represented P. pumila, which is

highly diverged from P. sibirica in isozyme loci. Subsequently, only a few single populations of P. cembra have been

studied by isozymes (Krutovsky and others 1990, 1994,

1995; Goncharenko and others 1991; Politov and others

1992; Pirko 2001). However, recently Belokon, Belokon and

Politov (in preparation) estimated genetic diversity in five P.

cembra samples collected from the Alps (Switzerland and

Austria) and the Carpathian Mountains populations in

western Ukraine. The FST value was not exceptionally high

(0.047). Therefore either stand isolation did not drastically

affect population differentiation of this species or this isolation is possibly a recent event, and the stands were much

more dense and widespread in the recent past. We conducted

Correspondence Analysis (StatSoft 1998) on these five populations, and its results are shown in figure 5. Samples from

the Alps and the Carpathian Mountains were well separated

along the first dimension, while the second dimension differentiated northern and southern macroslopes of the eastern

Carpathian Mountains. This differentiation showed that

Alpine and Carpathian populations as well as populations

from northern and southern macroslopes of the eastern

Carpathian Mountains have different genetic constitutions,

although the southern macroslopes were represented by

only a single population in our study.

Gugerli and others (2001) studied 15 populations in P.

cembra, one in P. sibirica and two in P. pumila using a few

DNA markers of several different types, but no data on

intraspecific variation were reported. The authors confirmed

allozyme data that P. pumila was the most divergent species

among these three species. However, they failed to differentiate P. cembra from P. sibirica, which clustered among P.

cembra populations.

Pinus pumila

Genetic structure of P. pumila was first analyzed in

studies on three populations from the north region of

Kamchatka Peninsula (Krutovsky and others 1990; Politov

and others 1992; Krutovsky and others 1994, 1995). FST

value based on 22 allozyme loci was relatively low (2.1

percent), but dendrograms based on genetic distances corresponded to the geographic localization of the populations.
90 USDA Forest Service Proceedings RMRS-P-32. 2004

Politov and Krutovskii Phylogenetics, Genogeography and Hybridization of Five-Needle Pines in Russia and Neighboring Countries

Figure 4—Sampling site locations (A) and results of Principal Component Analysis of 19 populations of

P. sibirica based on allelic variation in 31 isozyme loci (B) studied in Baikal Lake region (Politov 1998).

PC I

PC II
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Phylogenetics, Genogeography and Hybridization of Five-Needle Pines in Russia and Neighboring Countries Politov and Krutovskii

Figure 5—Sampling site locations (A) and results of Correspondence Analysis (B) of five populations of P. cembra

based on allelic variation in 31 isozyme loci (Belokon, Belokon and Politov, unpublished). Eigenvalue 1 = 0.02466

(54.46 percent of inertia), eigenvalue 2 = 0.00891 (19,68 percent of inertia). Empty circles represent isolated

populations or relatively small stands of P. cembra in the Carpathian Mountains that were not sampled.
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Politov and Krutovskii Phylogenetics, Genogeography and Hybridization of Five-Needle Pines in Russia and Neighboring Countries

Goncharenko and others (1993a) reported slightly higher

differentiation (FST=4.3 percent) in five populations from

two regions, the Chukotka Peninsula and Island of Sakhalin.

However, the authors failed to find clear pattern in differentiation among populations. Similar to their

abovementioned study of P. sibirica, this study has the

same problem of the limited sample sizes (12.6 trees per

population on average), which could bias estimation of

allele frequencies in populations. Substantial genetic differentiation (FST=7.3 percent) was found in recent, more

representative and wide ranging geographic studies of 22

populations of P. pumila sampled from the Lake Baikal

region through the inland mountain ridges to the Pacific

coast uncluding the Sakhalin Island (Maluchenko and

others 1998; Politov 1998). The distribution of populations

on a PCA plot based on 26 allozyme loci was in good

correspondence to their geographic origin (fig. 6).

Pinus koraiensis

Early studies of three populations of P. koraiensis (Politov

and others 1992; Krutovsky and others 1994, 1995) revealed

a relatively low level of genetic differentiation (FST=4.0

percent). The FST value was even lower (1.5 percent) in a

more representative study of 19 populations collected from

most of P. koraiensis’ range in Russia (Potenko and Velikov

1998), but the authors did not use any clustering technique

to analyze genogeography. Belokon and Politov (2000) analyzed nine populations that included seven populations from

three regions of Russian Far East (Primorski Territory,

Amurskaya Oblast and Khabarovsk Territory) and two

samples from northeastern China. However, populations

did not cluster according to their geographic origin on the

PCA plots. It appears that studies on genetic differentiation

in P. koraiensis will require combined efforts of different

scientists and more samples from the entire range (see also

accompanying paper by V. V. Potenko).

Intra- and Interpopulation Genetic

Variation in Cembrae Pines _______

The different number and type of isozyme loci or other

genetic markers and the different size and origin of samples

used in various studies make comparison among studies of

levels of genetic variation between species and even within

species problematic if not impossible. Estimates of genetic

variation greatly varied in different studies of Cembrae

pines. Expected heterozygosity (He) calculated from allele

frequencies by assuming Hardy - Weinberg equilibrium in

population and FST or GST parameters are the most common

estimates of intrapopulation genetic variation and interpopulation genetic differentiation, respectively. We have

summarized these estimates obtained in Russian populations of Cembrae pines (table 1). To make comparisons more

accurate we presented only those He values that were based

on the same set of loci. The FST/GST values based on the same

or almost the same loci and obtained within a geographic

range of the same or similar scale were also calculated and

presented. Although these adjustments do not necessarily

ensure correct comparisons, we believe that such compariFigure 6—Sampling site locations (A) and results of Correspondence Analysis (B) of 23 populations of P. pumila based

on allelic variation in 31 isozyme loci (Maluchenko and others

1998; Politov 1998).  - Southeastern Baikal (Hamar-Daban

Range), - Northern Baikal,  Inland Ridges,  Sakhalin

Island,  - Northeastern coast of Sea of Okhotsk.

sons are more objective than direct comparison of estimates

based on unbalanced data. The He values increased in our

study in the following order: P. cembra > P. sibirica > P.

koraiensis> P. pumila. FST was also the greatest in P. pumila

followed by P. cembra, P. koraiensis, and P. sibirica (fig. 7).

The latter two species had similar FST values. We also

calculated He values for 15 white pine species based on the
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Phylogenetics, Genogeography and Hybridization of Five-Needle Pines in Russia and Neighboring Countries Politov and Krutovskii

same set of 16 isozyme loci (table 2). There was considerable

variation in He values among species, but differences were

associated neither with subsections nor with subdivision of

species into bird-dispersed versus wind-dispersed species

(table 2). Birds are known as efficient dispersers that ensure

substantial gene flow within Cembrae pine populations

(Furnier and others 1987). It is commonly believed that

nutcrackers may also be responsible for extensive migration

among populations of pine species, with which they are

associated (Bruederle and others 2001). However, although

table 3 shows that FST/GST values were lower in bird-dispersed than in wind-dispersed pines, the variance of estimates was very high. Difference between mean values (0.057

± 0.045 vs. 0.103 ± 0.082) for the two groups did not generally

exceed the differences that often observed between different

species within each group or between several independent

studies conducted for the same species, but using different

loci sets and/or different population range. Therefore, additional studies that are based on the same set of genetic

markers and the same geographic and ecological range are

Figure 7—Comparison of intrapopulation (He) and interpopulation (FST) genetic diversity between four Cembrae

pine species.

Table 1—Genetic diversity in Eurasian stone pine species (subsection Cembrae) estimated as expected heterozygosity (He) and interpopulation

genetic differentiation (FST or GST) parameters using isozyme loci.

Species Populations Loci (mean) FST/GST FST a He Reference

P. cembra 5 30 0.047 0.065 0.093 Belokon, Belokon and Politov, unpublished

P. sibirica 32 19-31 0.025-0.063 b 0.035 0.124 Politov 1998

(25)

P. koraiensis 5 30 0.015-0.040 0.035 0.128 Belokon and Politov 2000

19 26 - - 0.182 Potenko and Velikov 1998

P. pumila 29 22-32 0.021-0.073 0.070 0.234 Politov 1998; Maluchenko and others 1998

(30)

a

 Calculated for populations within the same scale of geographic range and using the same set of loci for comparison.

b The range of estimates from different studies.

0.00

0.05

0.10

0.15

0.20

0.25

P. cembra P. sibirica P. koraiensis P. pumila

FST

He

needed to calculate FST/GST values that would allow a strong

conclusion as to whether migration is higher in bird-dispersed pines.

In general, such factors as high gene flow (due to efficient

mechanisms of seed and pollen dispersal) along with a high

effective population size, a high stand density, and a predominantly outcrossing mating system should lead to a

higher intrapopulation component of genetic variation and

a lower differentiation among populations. This is a typical

pattern of genetic structure observed in widely distributed

coniferous species (Ledig 1998). However, the fact that FST

values significantly vary among loci contradicts the common

opinion that migration plays a major role in differentiation

and possibly indicates a nonneutral nature of at least some

of loci. Loci with low FST values can be subjected to balancing

selection that equalize gene frequencies, while loci with

higher than average values can be affected by diversifying

selection, and loci with intermediate FST values can be

neutral (Altukhov 1991). The introduction of new genetic

markers that are more neutral than isozyme loci will provide

an opportunity for testing this hypothesis and estimating

the role of different factors in maintaining of genetic structure of Cembrae pines.

Heterozygosity Dynamics and

Mating System __________________

The combination of a haploid endosperm (megagametophyte) that represents a segregating maternal gamete with

a diploid embryo in conifer seeds allows researchers to

genotype two consecutive generations in seeds collected

from individual trees. Genotypes of maternal trees can be

inferred from segregation of isozyme alleles in megagametophytes. At the same time embryos of these seeds represent

open-pollinated progenies (half- or full-siblings) that can be

considered as next generation of individuals in a particular

tree stand. Comparisons of observed genotype distributions

in two generations with those that are expected assuming

Hardy - Weinberg equilibrium revealed interesting and

important trends of temporal changes in the level of heterozygosity in all Eurasian Cembrae pines. Embryos typically demonstrate slight to moderate deficiency of homozy-
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Politov and Krutovskii Phylogenetics, Genogeography and Hybridization of Five-Needle Pines in Russia and Neighboring Countries

Table 3—Level of genetic differentiation between populations in bird- versus wind-dispersed white pines estimated as

FST or GST parameters using isozyme loci.

Species Populations Loci Geographic scale FST or GST Reference

Bird-dispersed

P. albicaulis 3 21 Local 0.004 Rogers and others 1999

9 19 Local 0.025 Bruederle and others 1998

14 Mediumwide 0.088 Yandell 1992

30 17 Rangewide 0.034 Jorgensen and Hamrick 1997

P. sibirica 8 20 Mediumwide 0.039 Goncharenko and others 1993b

11 17 Mediumwide 0.025 Krutovskii and others 1994, 1995

P. pumila 3 18 Local 0.021 Krutovskii and others 1994, 1995

5 22 Mediumwide 0.043 Goncharenko and others 1993a

18 19 Mediumwide 0.170 Tani and others 1996

12 32 Rangewide 0.073 Maluchenko and others 1998

P. koraiensis 3 16 Mediumwide 0.040 Krutovskii and others 1995

8 23 Mediumwide 0.059 Kim and others 1994

10 26 Rangewide 0.063 Belokon and others unpublished

P. flexilis 8 10 Mediumwide 0.022 Schuster and others 1989

30 24 Rangewide 0.101 Jorgensen and Hamrick 1997

16 27 Rangewide 0.149 Hamrick and others 1994

7 10 Rangewide 0.016 Latta and Mitton 1997

P. strobiformis 8 11 Rangewide 0.047 Ledig, pers. comm.

Mean 0.057 ± 0.045

Wind-dispersed

P. ayacahuite 2 23 Local 0.047 Ledig 1998

14 23 Rangewide 0.222 Ledig 1998

P. monticola 28 12 Rangewide 0.148 Steinhoff and others 1983

P. strobus 27 12 Rangewide 0.080 Ryu and Eckert 1983

10 18 Mediumwide 0.019 Beaulieu and Simon 1994

Mean 0.103 ± 0.082

Table 2—Genetic diversity in white pines

(section Strobus) estimated as

expected heterozygosity (He)

parameter using the same 16

isozyme loci.

Species a He

Subsection Cembrae

P. cembra 0.082

P. sibirica 0.106

P. albicaulis 0.113

P. koraiensis 0.130

P. pumila 0.198

Mean 0.126 ± 0.020

Subsection Strobi

P. armandii 0.080

P. wallichiana 0.091

P. strobus 0.093

P. parviflora 0.109

P. monticola 0.119

P. strobiformis 0.122

P. peuce 0.125

P. flexilis 0.128

P. morrisonicola 0.132

P. lambertiana 0.142

Mean 0.114 ± 0.006

Mean for bird-dispersed 0.123 ± 0.012

Mean for wind-dispersed 0.112 ± 0.009

a Underlined are species for which there is some

evidence of bird-dispersal of seeds (Lanner 1996,

1998).
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gotes (Politov and others 1992; Politov and Krutovsky 1994;

Krutovsky and others 1994, 1995; Politov 1998). Estimates

of mating system parameters have shown this deficiency is

caused by partial self-pollination that can occur with up to 15

percent in P. sibirica populations, 8 percent in P. koraiensis,

and can be as high as 31 percent in P. cembra. The latter

species exists in small isolated stands that can have a

limited pollen flow distance, which often promotes selfpollination.

In contrast to embryos, heterozygote deficiency was not

observed in mature trees (Politov and Krutovsky 1994;

Krutovsky and others 1995). They typically demonstrate

either Hardy – Weinberg equilibrium or slight heterozygote

excess that likely indicates the selective elimination of

progeny originated from self-pollination during early stages

of life and probably, also the balancing selection in favor of

heterozygotes in some loci (Politov and Krutovsky 1994;

Krutovsky and others 1995). Assortative mating of parents

with different genotypes and/or preferential fertilization of

gametes with different haplotypes are mating system mechanisms that may potentially also contribute to the excess of

heterozygosity, but it also would cause an excess of heterozygosity in both embryos and mature trees and would not

explain the increase of heterozygosity with age. We are also

unaware of any evidence or data that would prove that

assortative mating of such kind occurs in pines or other

conifers.

Interspecific Hybridization ________

Critchfield (1986) summarized data on interspecific crosses

among white pines of the section Strobus. Successful crosses

between pines within the subsection Cembrae such as P.

sibirica x P. cembra were also mentioned in that review,

although this successful hybridization is not surprising due

to the phylogenetic proximity of the parental species. Titov

(1988) obtained sound seeds from the cross between P.

sibirica x P. koraiensis; however, these were never tested by

genetic markers to confirm hybridity. Successful crosses

between Cembrae and Strobi pines, such as P. koraiensis x

P. lambertiana were also reported earlier (Bingham and

others 1972).

Interspecific crosses of P. sibirica x P. koraiensis were

conducted in Ivanteevka Arboretum (Moscow Region, Russia) in the late 1960s. Needles and buds from 27 putative

“hybrid” mature trees were tested by 18 allozyme loci (Politov,

Belokon and Belokon, unpublished). Only two trees were

unambiguously identified as hybrids based on species specific alleles in loci Gdh, Adh-1, and Lap-3, while the other

trees were apparently P. sibirica. It is worth noting that

these hybrids were superior in growth and showed resistance to the pest Pineus cembrae Cholodkovsky 1888

(Adelgidae: Chermes).

Natural hybridization between P. sibirica and P. pumila

in Lake Baikal region was confirmed genetically using

isozyme analysis (Politov and others 1999). Both species

were thoroughly studied in their allopatric areas and across

a wide zone of their sympatric distribution. Putative natural

hybrids were identified using 28 allozyme loci controlling 14

enzyme systems. The Adh-1, Fe-2, and Lap-3 loci in the

hybrids had genotypes that were typical for P. sibirica, but

did not occur or were unlikely in P. pumila, while five other

loci carried alleles and genotypes that are unknown in P.

sibirica, but common in P. pumila. The Skdh-2 locus was

heterozygous for alleles, one of which was specific for P.

sibirica, but another for P. pumila. Some embryos from the

seeds of the hybrid were likely resulted from self-pollination

while others from backcrosses with parental species. This

was the first genetic evidence of natural hybridization and

potential gene exchange between P. sibirica and P. pumila.

Hypothetically, gene exchange between P. sibirica and P.

pumila may play a significant adaptive role. The zone of

sympatry in the Baikal Region and Southern Yakutia is not

optimal for both species and is intrinsically occupied by

marginal populations. P. sibirica and P. pumila are adapted

to different environments, and survival outside of their

respective optimal environments may be promoted by genes

from related species coming from another side of the sympatry zone with different environmental gradients. The frequency and distribution of hybrids in the sympatry zone of

P. sibirica and P. pumila and their possible role in the

species adaptation and evolution are still largely unknown.

It is still questionable whether observed hybridization leads

to extensive gene introgression, but if it is so the gene

exchange could be a mechanism of increasing of total population fitness. Despite intercrossing among Cembrae species, we do not consider this fact alone as an evidence of their

closer relationships to each other than to other white pines.

Crossability and closeness of relationship can be correlated,

but the lack of crossability is not convincing of a more distant

relationship. Blada (1994) reported a number of successful

artificial crosses between P. cembra and pines of subsection

Strobi. Watano and others (1995, 1996) studied trees with

morphological traits that were “intermediate” when compared to traits in “pure” P. pumila and P. parviflora using

DNA markers, and proved the trees to be interspecific

hybrids. High conservatism in the number and morphology

of chromosomes probably facilitates hybridization of white

pines as well as other Pinaceae, and interspecific gene

exchange might be an important (although usually underestimated) factor of population genetic structure dynamics.

Acknowledgments ______________

We thank Yuri Altukhov, Yuri Belokon, Maryana Belokon,

Oleg Maluchenko, Alexey Podogas, and Natalia Gordon

(Vavilov Institute of General Genetics, Moscow), Ekaterina

Titorenko and German Ross (Moscow State University), and

Le Thi Kinh (Cantho University) for their help in the study.

This study was supported in part by grants from Russian

Academy of Sciences (Cooperative Program “Plant Genome”),

Russian Foundation for Basic Research (Program for Support of Scientific Schools), Russian Ministry of Science

(Programs “Genetic Monitoring of Population Genetic Diversity” and “Development of Databases for Genetic Variability in Plants and Animals”) and by Federal Program

“State Support for Integration of Higher Education and

Fundamental Research” (Educational and Scientific Center

for Genetics).
96 USDA Forest Service Proceedings RMRS-P-32. 2004

Politov and Krutovskii Phylogenetics, Genogeography and Hybridization of Five-Needle Pines in Russia and Neighboring Countries

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98 USDA Forest Service Proceedings RMRS-P-32. 2004

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

The author is with the Institute for Forest Genetics and Forest Tree

Breeding, Siekerlandstrasse 2, D-22927 Grosshansdorf, Germany. Telephone and fax no.: + 49 - 4102 – 63555. E-mail: r-c.stephan@t-online.de

Abstract—After 30 years, a field trial with 65 seed samples of

eastern white pine (Pinus strobus) showed that the best growing

provenances have their origin in the Appalachians south of latitude

39∞, and provenances with the slowest growth in regions north of

latitude 45∞. Provenances from regions between 39∞ and 45∞ latitude

varied greatly in their growth, even when their origins were from

adjacent locations. The great interspecific and intraspecific differences of five-needle pines in resistance to the blister rust fungus

Cronartium ribicola was demonstrated by resistance tests. Obvious

racial variation in the blister rust fungus was found by a joint

inoculation experiment with alternate hosts (Ribesand Pedicularis).

Key words: Eastern white pine, Pinus strobus, provenance trial,

blister rust, growth performance, Pinus cembra, P.

wallichiana, P. peuce, P. parviflora

Introduction ____________________

Europe has in contrast to North America only two native

five-needle pine species. Only Swiss stone pine (Pinus cembra

L.) is native in Germany. This species occurs in small

populations in the Alpine regions of southern Germany at

elevations up to 1867 m.

The second European species is the Macedonian pine

(Pinus peuce Griseb.) of the mountainous Balkans. This

species is rather slow-growing and in general of less interest

for forestry practice. However, because of a relatively high

tolerance against air pollutants, it is suitable for afforestation in southeastern Germany (Lattke and others 1987,

Lattke 1998), where other tree species (for example Norway

spruce) were severely damaged or even eliminated during

recent decades.

In addition to these native species, several other fiveneedle pine species were introduced, mainly for ornamental

purposes: The Himalayan white pine (Pinus wallichiana A.

B. Jacks.) from Pakistan and India is grown in parks and

larger gardens in the warmer climate of southwestern Germany. In other regions the trees are subject to damage from

late frost, as shown in some provenance trials (for example

Stephan 1974).

Studies of Genetic Variation with FiveNeedle Pines in Germany

Bruno Richard Stephan

The Japanese white pine (Pinus parviflora Sieb. et Zucc.)

is a common ornamental tree species in parks, arboreta,

gardens and cemeteries, where it is of interest because of its

slow growth and attractive blue needle colour.

Pinus strobus L., the eastern white pine from North

America, is the only five-needle pine with extensive silvicultural use in Germany. The species was introduced in Europe

in 1605. It can be grown successfully under various environmental site conditions and shows good natural regeneration.

The main disadvantage is the high susceptibility to blister

rust disease caused by Cronartium ribicola J.C. Fischer.

In the following paper some results of a provenance trial

with P. strobus will be presented. In addtion, results are

summarized from resistance tests with several five-needle

pine species and studies of variation in Cronartium ribicola.

Materials and Methods ___________

Provenance Trials

Provenance trials with 65 seed samples from the natural

range of P. strobus were started in 1963. Geographical data

for the provenances are given in table 1. Field trials were

established with 3-year-old plants on two sites in northern

Germany in 1966 and 1967. Measurements and assessments of growth performance, mortality (rust and non-rust

related) and the presence of stem infections as well as branch

infections by blister rust were conducted in the following

years. Detailed information is given in earlier papers (Stephan

1974, 1986a). The last evaluations were carried out in 1994

when the trees were 32 years old, and these data are

presented here. Because of the design of the trial and the

narrow space within the plots, further exact evaluations are

not possible.

Resistance Test with Five-Needle Pines

The Institute for Forest Genetics at Grosshansdorf participated in the international IUFRO experiment testing

resistance of white pines to Cronartium ribicola. This joint

experiment was initiated by Bingham and Gremmen (1971).

A total of 17 five-needle pine species including 76 provenances and progenies were tested by artificial inoculation

(Stephan 1986b). About 10,000 pine plants were grown in

containers under greenhouse conditions at temperatures

above 0 ∞C to avoid frost damage of the frost sensitive

species. For artificial inoculations, rust-infected leaves of

the alternate host Ribes nigrum L. were used. The pine

seedlings were inoculated at an age of two years. Each plant

was assessed annually for the rust symptoms, beginning

with needle lesions or spots, appearance of spermogonia,

normal cankers and/or bark reactions, and blisters with
USDA Forest Service Proceedings RMRS-P-32. 2004 99

Studies of Genetic Variation with Five-Needle Pines in Germany Stephan

Table 1—Provenance trial with Pinus strobus at Forest District Nordhorn (Wielen Ki 26) in northwestern Germany.

Provenances are listed in order of their volume under bark per ha at age 27.

Seed-book Latitude Longitude Altitude Height (m) Dbh (cm) m3/ha

no. County, state N W (m) age 27 age 32 age 27

3862 Newaygo, MI 43∞43' 85∞55' 262 13.30 26.00 321.8

3851 South Carolina 34∞39' 82∞55' 533 13.50 30.35 319.7

3874 Clearfield, PA 41∞00' 78∞27' — 12.80 — 307.7

3800 Henderson, NC 35∞20' 82∞30' 671 13.03 22.00 304.9

3850 Kentucky 37∞00' 87∞00' — 13.25 29.00 285.9

3812 Garrett, MD 39∞30' 79∞25' 707 12.95 21.40 279.7

3871 Garrett, MD 39∞30' 79∞25' 707 12.70 27.67 281.2

3829 Carroll, VA 36∞42' 80∞52' 780 13.13 29.00 275.7

3803 Schoharie, NY 42∞45' 74∞25' 274 12.60 — 272.8

3839 Buncombe, NC 35∞28' 82∞32' 655 13.00 28.50 274.4

3861 Manistee, MI 44∞16' 86∞03' 213 13.05 26.70 269.9

3857 McKean, PA 41∞42' 78∞55' 457 12.78 22.35 272.0

3818 Washington, MD 39∞41' 78∞14' 194 12.78 27.25 269.0

3875 Warren, NY 43∞37' 73∞44' 305 13.35 21.85 267.2

3825 Saratoga, NY 43∞00' 73∞43' 152 12.95 28.50 260.8

3827 Dunn/Polk, WI 45∞00' 91∞19' 366 12.35 26.10 260.1

3804 Berkshire, MA 42∞30' 73∞14' 274 12.80 24.40 258.0

3872 Pike, PA 41∞10' 75∞00' 335 12.38 27.50 251.5

3837 Oconee, SC 34∞50' 83∞10' 457 12.95 21.75 246.5

3876 Warren, NY 43∞41' 73∞41' 396 12.55 26.25 244.4

3810 Middlesex, CT 41∞38' 72∞30' — 13.03 26.00 240.8

3842 Quebec, QC 46∞55' 71∞31' 168 12.95 24.50 239.9

3823 Litchfield, CT 41∞58' 73∞13' 390 12.58 26.25 238.8

3809 Hillsborough, NH 43∞06' 71∞55' 262 12.05 23.30 241.3

3838 Wytha, VA 37∞00' 81∞15' 762 12.65 19.25 237.5

3801 Greenbrier,WV 38∞59' 80∞09' 686 12.50 25.15 231.8

3808 Chittenden, VT 44∞27' 73∞12' 91 12.28 25.50 236.1

3802 Strafford, NH 43∞08' 70∞57' 31 12.53 25.07 232.5

3821 Somerset, PA 39∞47' 79∞02' 640 12.95 24.00 228.9

3820 Somerset, PA 39∞47' 79∞02' 640 12.08 26.00 234.2

3824 Litchfield, CT 41∞58' 73∞13' 408 12.40 25.75 234.2

3815 Allegany, MD 39∞40' 78∞28' 239 12.50 23.40 226.9

3853 Schoharie, NY 42∞45' 74∞25' 305 12.63 24.00 226.8

3870 Strafford, NH 43∞08' 70∞56' 18 12.10 23.05 227.7

3822 Tucker, WV 39∞10' 79∞35' 503 12.38 26.57 227.6

3819 Garrett, MD 39∞42' 79∞08' 678 13.23 24.93 228.5

3830 Carroll, VA 36∞37' 80∞53' 780 12.75 22.90 233.0

3835 Sauk, WI 43∞30' 89∞55' 305 12.18 25.15 227.4

3805 Juneau, WI 43∞35' 90∞00' 210 12.00 23.00 217.7

3806 Chittenden, VT 44∞28' 73∞09' 290 12.15 24.67 216.6

3814 Preston, WV 39∞33' 79∞29' 777 11.63 22.00 208.9

3833 Garrett, MD 39∞33' 79∞21' 756 12.48 16.00 213.3

3828 Coerthier, QC 46∞17' 73∞25' 213 12.30 25.00 207.4

3855 Addison, VT 44∞07' 73∞13' 122 12.30 24.03 206.4

3834 Garrett, MD 39∞25' 79∞24' 701 12.00 23.50 201.8

3811 Garrett, MD 39∞30' 79∞25' 707 12.38 24.20 201.7

3836 Sauk, WI 43∞30' 89∞55' 305 12.25 22.15 199.0

3843 Sawyer, WI 46∞00' 91∞25' — 12.95 22.25 194.3

3844 Renfrew, ON 45∞57' 77∞27' 160 11.50 23.00 194.2

3807 Chittenden, VT 44∞28' 73∞09' 122 12.23 23.85 195.0

3841 Lake, MN 48∞02' 91∞36' 402 12.15 22.50 188.4

3877 Essex, NY 44∞20' 73∞46' 229 12.05 21.70 181.7

3845 Sunbury, N.B. 46∞22' 66∞11' 122 11.50 26.75 175.5

3848 LaSalle, IL 41∞19' 88∞59' 155 11.90 23.73 173.4

3840 York, ME 43∞22' 70∞53' 122 11.83 20.33 169.8

3854 Todd, MN 46∞21' 94∞12' 405 12.58 21.70 170.1

3826 Itasca, MN 47∞19' 93∞34' 397 12.13 21.10 166.0

3846 Quebec, QC 46∞57' 71∞31' 305 12.25 24.00 147.5

3813 Preston, WV 39∞33' 79∞29' 786 11.75 21.50 139.7

3847 Manitoba, MN 54∞00' 100∞00' — 11.40 20.90 130.1

3856 Ogle, IL 41∞57' 89∞23' 221 11.00 20.00 119.5

3831 North Carolina — — — 12.75 27.00 —

3852 Wisconsin — — — 12.73 26.90 —

3849 Michigan — — — 12.50 26.33 —

3832 North Carolina — — — 11.70 21.37 —

Average 12.47 24.23 228.4
100 USDA Forest Service Proceedings RMRS-P-32. 2004

Stephan Studies of Genetic Variation with Five-Needle Pines in Germany

aeciospores. Rust related and non-rust related mortality

was recorded. During the experiment all plants were kept in

greenhouses until they reached the age of eight to nine

years. The white pine plants were not exposed to further

natural infections.

Studies on Race Differences of C. ribicola

The objective of these studies was to investigate the extent

of pathogenic variation in C. ribicola in ability to infect

alternate host species. Therefore, joint inoculation experiments were carried out with Ribes nigrumL. (Saxifragaceae)

and Pedicularis resupinata L. (Scrophulariaceae) in Germany and South Korea. Various cultivars of R. nigrum are

grown in Germany and are the main alternate hosts of the

white pine blister rust fungus. Pedicularis resupinata is

native in eastern Asia and is also an alternate host plant of

C. ribicola. Both alternate host species or cultivars were

grown in Germany as well as in South Korea, and inoculated

with the respective C. ribicola aeciospores in both countries.

Further details of the materials and methods are given in

the paper of Stephan and Hyun (1983).

Results ________________________

Provenance Trial with Pinus strobus

Height growth (age 27), stem diameter at breast height

(1.3 m) (age 32) and calculated total volume under bark

(m3/ha) (age 27) are shown as an example for the trial at

Wielen (Ki 26), northwestern Germany, in table 1. The

averages for height growth at age 27 varied between 11 m

and 13.5 m, for diameter at age 32 from 16 cm to about 30 cm,

and for volume at age 27 from 119.5 m3/ha to 321.8 m3/ha.

The performance of the provenances at the two test sites

was very similar. The provenances differed significantly in

their growth performance at different ages. Traits are correlated negatively with provenance latitude (for correlation

coefficients see Stephan 1974). Trees from provenances in

the southern Appalachians south of 39∞ latitude grew well

under the conditions in northern Germany (fig. 1). Provenance samples from the regions north of latitude 45∞ grew

poorly. Provenance samples from areas between 39∞ and 45∞

latitude showed great variation in growth rate.

Differences between provenances in blister rust infection

could be observed. As the trees were mostly infected at the

lower part of the stems or branches, it could be assumed that

they were obviously infected already as young plants in the

nursery. The range in rust infection was relatively low and

varied from 2 percent and 7 percent between the two test

sites, but from 0 percent to 25 percent between provenances.

There was a weak correlation (r = 0.32) among provenances

over the two test sites. A correlation between origin of the

provenances and infection could not be found. One provenance from Maryland and one from Quebec had no rust

infected trees after seven years in the field. On the contrary,

25 percent trees of a provenance from Wisconsin were rust

infected.

Wood density of the provenances was investigated

separately and varied only slightly, ranging from 0.257 to

0.290 g/cm3 with an average of 0.271 g/cm3.

Figure 1—Volume under bark (m3/ha) at 27 years of 61

provenances of eastern white pine in relation to geographic

latitude at one provenance trial (Ki 26) site location at

Wielen, northwestern Germany.

Differences Among Five-Needle Pines in

Blister Rust Resistance

There was wide genetic variation among pine species,

provenances and progenies in the reaction after artificial

inoculation with basidiospores of C. ribicola (table 2). Generally, European and Asian pines remained uninfected or

were less infected by the rust fungus on the basis of needle

lesions, percent of trees with stem symptoms and mortality

rate, than the extremely susceptible North American pines.

Particularly P. cembra, P. armandii and P. pumila showed

neither needle symptoms nor stem symptoms. Large differences of percent rust infected trees existed among seed

samples within the Asian species P. parviflora and P.

wallichiana (table 2). Among the North American pine

species, four P. aristata provenance samples had the lowest

percentage of tree with stem cankers (mean of 66 percent).

Progenies from crosses between selected rust-free parents

from two of the North American species had some trees with

heavy infection, but in general they had fewer infected trees

than did trees in provenances of their respective pine species

six and a half years after inoculation (see P. lambertiana and

P. monticola in table 2). A few years later, however, these

canker-free progenies were also heavily infected by blister

Trial Wielen Ki 26

at age 27

100

150

200

250

300

350

30° 35° 40° 45° 50° 55°

latitude

Volume m3/ha
USDA Forest Service Proceedings RMRS-P-32. 2004 101

Studies of Genetic Variation with Five-Needle Pines in Germany Stephan

Table 2—Blister rust infection (percent of trees with cankers) of 8- and 9-year-old white pines about 6

years after artificial inoculation with Cronartium ribicola.

No. of Blister rust attack (%)

White pine species provenances/progenies Mean (provenances) Mean (species)

Europe

Pinus cembra 1 00

P. peuce 6 0-30 22

Asia

P. armandii 2 00

P. pumila 1 00

P. sibirica 1 — 17

P. parviflora 3 0-67 22

P. koraiensis 3 18-29 23

P. wallichiana 5 17-60 40

P. morrisonicola 1 — 40

North America

P. aristata 4 50-94 66

P. strobiformis 4 75-95 88

P. balfouriana 4 74-100 90

P. lambertiana 6 88-100 97

— R-progeny a) 1 — 76

P. albicaulis 2 94-100 97

P. flexilis 4 97-100 98

P. monticola 6 93-100 99

— R-progenies a) 10 85-100 97

P. strobus 8 98-100 100

P. strobus (Germany) 4 60-88 71

a) R-progenies = F1 and F2 progenies from controlled crosses between parent trees resistant in North America

rust and subsequently died. Canker development in these

progenies seemed to require more time. Natural infection

was excluded as all trees were grown in greenhouses far

away from alternate host plants.

Interestingly, the four seed samples of P. strobus populations grown in Germany and used in the inoculation

experiment were obviously more tolerant to the German

blister rust race than were autochthonous samples of

North American P. strobus (table 2).

Genetic Variation Within Cronartium

ribicola

Alternate hosts of the white pine blister rust fungus were

inoculated simultaneously with aeciospores of the fungus in

a joint experiment in Germany and South Korea. In the

German trial only Ribes nigrum and in the Korean trial only

Pedicularis resupinatawere infected, although in both countries the respective other alternate host was also inoculated.

Urediniospores and teliospores were formed after infection only on the leaves of R. nigrum in Germany and P.

resupinata in South Korea. The respective other host

plant species remained uninfected. Therefore, one can

assume that the C. ribicola types used in both countries

differed in their pathogenicity. Differences between various C. ribicola samples regarding the size of aeciospores

and urediniospores could not be found.

Discussion _____________________

Pinus strobus is the most important species among the

white pines of interest for forestry uses in Germany (Ritter

1978, Stratmann 1988, Waldherr 2000). The first plantation

was established in southwestern Germany around 1770

(Stratmann 1988). Growth performance and natural regeneration are superior compared to the native Scots pine (P.

sylvestris L.). The main problem is its high susceptibility to

blister rust, presenting an obstacle to its otherwise desirable

use as a main tree species for silviculture. First observations

of the blister rust disease are known from Estonia (northeastern Europe) around 1854. Thirty years later the fungus

had reached the Atlantic Ocean in western Europe and had

caused tremendous losses of P. strobusafforestations. Therefore, around 1930 growing of eastern white pine was prohibited. Later the prohibition was again canceled and instructions for the afforestation of P. strobus were given. To avoid

most severe losses it is recommended that eastern white

pines be planted in mixture with other tree species and at a

greater distance than at present from villages and plantations, where the alternate host Ribes nigrum is cultivated.

As evidenced by the lower blister rust infection of the

German land race (compared to the North America provenances) some natural selection for C. ribicola resistance

may be occurring. Further investigation of the potential for

developing more resistance in the German land race may be

warranted.
102 USDA Forest Service Proceedings RMRS-P-32. 2004

Stephan Studies of Genetic Variation with Five-Needle Pines in Germany

There is a wide intraspecific variation in P. strobus, as

shown by provenance trials. The results of the German trials

agreed very well with those in the United States of America,

Australia and New Zealand (Genys and others 1978). In

Germany, southern provenances from the Appalachians are

of particular interest. They seem to be very well adapted to

climatic and other site conditions, but, unfortunately, resistant progenies of P. strobus are not available yet.

The resistance tests clearly showed that the German

blister rust race used for artificial inoculations was more

aggressive than the race used in western North America

(Idaho), since progenies of rust resistant parents of P.

lambertiana and P. monticola were also heavily infected

(table 2). Our results were in a generally good agreement

with French results, but differed from the American test

results (Delatour and Birot 1982, Stephan 1986a). This may

demonstrate similarity within the European blister rust

fungus, but differences from the North American fungal

type.

Race differences have also been found between the German

and South Korean blister rust fungus, and a wider pathogenic variation of C. ribicola can be assumed in eastern

Asia, for example in Korea and Japan (Stephan and Hyun

1983). These areas can be considered as the main gene

centers, where host and pathogen coexisted during long

periods. Because of the common coevolution tolerance of the

host as well as virulence of the parasite are there in a

dynamic equilibrium (Leppik 1970).

Acknowledgments ______________

The author is indebted to Professor Howard B. Kriebel and

an anonymous reviewer for help with the manuscript and

valuable comments.

References _____________________

Bingham, R.T. and Gremmen, J. 1971. A proposed international

program for testing white pine blister rust resistance. Eur. J. For.

Path. 1: 93-100.

Delatour, C. and Birot, Y. 1982. The international IUFRO experiment on resistance of white pines to blister rust. The French trial.

In Heybroek, H.M., Stephan, B.R. and von Weissenberg, K.

(Eds.). Resistance to diseases and pests in forest trees. PUDOC,

Wageningen, pp. 412-414.

Genys, J.B., Canavera, D., Gerhold, H.D., Jokela, J.J., Stephan,

B.R., Thulin, I.J., Westfall, R. and Wright, J.W. 1978. Intraspecific variation of eastern white pine studied in U.S.A., Germany,

Australia and New Zealand. Center for Environmental and

Estuarine Studies, Univ. of Maryland, College Park. Special

Report No. 8: 27 pp.

Lattke, H. 1998. Kiefern für die Immissionsschadgebiete der

Mittelgebirge - züchterische Ergebnisse und Perspektiven.

Schriftenreihe der Sächsischen Landesanstalt für Forsten,

Graupa, Germany, Heft 13/98: 24-35.

Lattke, H., Braun, H. and Richter, G. 1987. Pinus peuceGriseb., eine

erfolgversprechende Alternativbaumart für die Schadgebiete des

oberen Erzgebirges. Sozial. Forstwirtschaft 37: 279-282.

Leppik, E.E. 1970. Gene centers of plants as sources of disease

resistance. Ann. Rev. Phytopath. 8: 323-344.

Ritter, H. 1978. Die Weymouthskiefer – eine verlorene Holzart?

Holz-Zentralblatt 104: 1287-1288.

Stephan, B.R. 1974. Zur geographischen Variation von Pinus strobus

aufgrund erster Ergebnisse von Versuchsflächen in

Niedersachsen. Silvae Genetica 23: 214-220.

Stephan, B.R. 1985. Zur Blasenrostresistenz von fünfnadeligen

Kiefernarten. Allgemeine Forstzeitschrift 40: 695-697.

Stephan, B.R. 1986 a. Results of a 22-year old provenance trial with

eastern white pines in northern Germany. In Proc. 18th IUFRO

World Congress, Ljubljana, September 1986. Division II, Vol. II,

p. 859 (Abstract).

Stephan, B.R. 1986 b. The IUFRO experiment on resistance of white

pines to blister rust (Cronartium ribicola) in northern Germany.

In Proc. 18th IUFRO World Congress, Ljubljana, September

1986. Division II, Vol. I, p. 80-89.

Stephan, B.R. and Hyun, S.K. 1983. Studies on the specialization of

Cronartium ribicola and its differentiation on the alternate hosts

Ribes and Pedicularis. Zeitschr. f. Pflanzenkrankh. und

Pflanzensch. 90: 670-678.

Stratmann J. 1988. Ausländeranbau in Niedersachsen und den

angrenzenden Gebieten. Schriften aus der Forstl. Fak. der Univ.

Göttingen und der Niedersächs. Forstl. Versuchsanst., Band 91:

131 pp.

Waldherr, M. 2000. Die Strobe in Ostbayern (Niederbayern -

Oberpfalz). Wachstum und waldbauliche Erfahrungen. Forst

und Holz 55: 35-39.
USDA Forest Service Proceedings RMRS-P-32. 2004 103

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

The author is Regional Geneticist, USDA Forest Service, Pacific Southwest Region, Genetic Resource and Conservation Center, 2741 Cramer Lane,

Chico, CA 95928. Telephone: (530) 879-6607. Fax: (530) 879-6610. E-mail:

jkitzmiller@fs.fed.us.

Abstract—Sugar pines from range-wide provenances displayed a

complex genetic structure in adaptive traits in four contrasting

common gardens. Strong, rank-changing G-x-E interactions for 10-

to 17-year survival and growth require restrictions in seed transfer

across altitudes and coastal-interior transects. Geographic and

altitudinal subdivisions on a regional scale correspond with major

changes in climate, soil, and vegetation. Altitude of origin was the

primary geographic variable influencing growth potential; coastal

proximity also affected it. Genetic sources accounted for about 40

percent of the variability in growth. Central Sierra Nevada tests at

contrasting low and high altitudes exhibited better growth for seed

sources from altitudes similar to the test sites. However, distant

sources from high elevation, southern and eastern origins had

higher survival at the low (840 m) altitude site. Best survival (and

growth) at the high (1860 m) altitude test was attained by sources

from matching altitudes in the Sierra Nevada, peaking for 1850 m

origins, and diminishing rapidly below 1530 m and above 2030 m.

In Oregon tests, the local region had best growth, southern regions

had least, and sources within region were interactive between the

two contrasting coastal and interior tests. Seed transfer from

southern Sierra Nevada northward is desirable to boost rust resistance (also growth and genetic diversity) in northern populations,

where natural resistance is rare. Seed transfer up to 220 km

northward along the Sierra Nevada west-slope may be advantageous and safe, if temperature regimes of seed source and planting

site are matched, and if proven-superior sources are used.

Key words: sugar pine, tree genetics

Introduction ____________________

Sugar pine (Pinus lambertiana Dougl.) is often described

as the largest, noblest, and most beautiful of pines in the

world. With long life and a remarkable capacity to sustain

growth into old age, not reaching culmination of mean

annual increment until 400 years, individual trees may

reach enormous size, some over 76 m tall, 305 cm dbh

(Larson and Woodbury 1916, Oliver 1996). Excellent wood

qualities that include uniformity, stability, workability, and

Adaptive Genetic Variation in Sugar Pine

Jay H. Kitzmiller

finish ability, elevate the commercial value of sugar pine to

most preferred species for paneling, cabinetry, and moulding (Willits and Fahey 1991).

Sugar pine ranges widely in latitude (over 14∞) and in

altitude (2750 m) from Baja California to northern Oregon

(Critchfield and Little 1966). Sugar pine possesses wide

ecological amplitude, having adapted to different temperature, humidity, and soil environments from coastal to inland

montane forests. This late seral species is adapted to high

elevations because of its short period of height growth, and

to low elevations due to its tolerance of hot, dry sites.

Distribution is nearly continuous through the Siskiyou and

Klamath Mountains and along the western slopes of the

Cascade and Sierra Nevada Ranges (Oliver 1996).

Rarely forming pure stands, sugar pine is typically a

minor (less than 25 percent of canopy cover) but highly

consistent (more than 70 percent constancy) component

with high ecological value in mixed-conifer forests (Fites

1996). These majestic pines protect watersheds, provide

large, edible seeds for wildlife forage and large snags for

cavity nesting birds, enhance ecological diversity, and improve recreation. Best development has been observed in

virgin stands on deep soils in the Stanislaus-Toulumne

Experimental Forest, and Yosemite, Kings Canyon, and

Sequoia National Parks.

Unfortunately, the high resource values and future of

sugar pine are seriously threatened by its extreme susceptibility to an introduced disease, white pine blister rust

(WPBR) caused by Cronartium ribicola Fisch. Natural regeneration is further impeded by poor seed dispersal, heavy

seed predation, low tolerance of competition, and the lack of

recurrent, non-stand-replacing fires (Oliver 1996). Needed

are proper pest management, genetic conservation, and

silviculture, using group selection harvest and artificial

regeneration with resistant stock.

Genetic conservation programs in both California and

Oregon aim to improve genetic resistance and to restore

sugar pine in its native habitats (Samman and Kitzmiller

1996, Sniezko 1996). A major (dominant) gene (R) conveys

resistance in natural populations. Natural resistance is rare

(R frequency is less than 0.01) in northern populations,

where the disease entered 75 years ago and spread southward in sporadic “wave years”. Resistance is highest, but

still very low, in southern Sierra populations (R<0.09).

WPBR is absent below 35∞ N. “Slow rusting” and ontogenetic

resistance mechanisms also exist (Kinloch and Davis 1996).

Management policy seeks to maintain the viability of

sugar pine in mixed-conifer forests. Restoration will be

needed most in the north, because of the greater impact of

WPBR on local occurrence. Likewise, an adequate genetic

base of durable resistant genotypes for restoration will be

extremely difficult to find in northern populations. Moving

resistant genotypes from southern populations to northern

planting sites could help solve this problem.
104 USDA Forest Service Proceedings RMRS-P-32. 2004

Kitzmiller Adaptive Genetic Variation in Sugar Pine

If sugar pine is closely adapted to regional or local environments, long-distance seed transfer could have adverse effects, e.g. maladaptation to “foreign” restoration sites or

genetic “contamination” of local gene pools. If sugar pine is

not closely adapted, then long-distance transfers would

infuse vitally needed genetic diversity to maintain species

viability and restore it towards pre-WPBR distribution.

Management policy in California aims to protect the natural genetic structure of sugar pine and all woody plant species

(Kitzmiller 1976, 1990). The “default” policy restricts movement of seed (genes) to relatively short distances, defined by

standard seed zones (fig. 1) and transfer guides (Buck and

others 1970) that apply to all species (“one size fits all”).

Transfer is allowed within the local seed zone (152 m elevation

band and ca 80 km), and, in certain cases, across climatically

similar seed zones (305 m elevation band and ca 200 km).

Developed 30 years ago, this general forest seed policy assumes highly structured sub-populations within species, which

theoretically form as a result of high environmental heterogeneity, strong natural selection, and limited gene flow.

Natural selection promotes adaptation of populations to

local environments. Climatic, edaphic, and biotic factors

affect survival, health, and growth. These traits measure

adaptability of seed sources to their transplant environment. Vigorous, healthy trees are expected when natural

selection at test and seed origin environments sufficiently

Figure 1—California tree seed zone map.
USDA Forest Service Proceedings RMRS-P-32. 2004 105

Adaptive Genetic Variation in Sugar Pine Kitzmiller

corresponds to favor similar genotypes. When test and seed

origin environments differ widely, maladaptation is expected. Seed zones restrict seed transfer to similar origin

and planting environments.

Genetic Studies of Sugar Pine

Both conservation goals, maintaining species viability

and protecting adaptive genetic structure, require knowledge of the nature and pattern of adaptive genetic variation.

Together, allozyme and common garden studies provide this

knowledge. Conkle (1996) reported strong allozyme differentiation into at least four groups (southern CaliforniaBaja, southern Sierra Nevada, northern Sierra Nevada,

northern California-Oregon) with a north-south cline in

gene frequency. Seed zones with steeper genetic gradients

(more complex topography and climate) reflect higher genetic heterogeneity and transfer risk. Heterogeneity of

allozyme multi-locus genotypes is a transfer risk parameter

that estimates the proportion of genotypes that do not

“match” the group (zone) and presumably are maladapted

(Westfall 1991). Within Sierra Nevada seed zones, heterogeneity varied in a north-south clinal pattern (Kitzmiller, data

filed 1995). The most heterogeneous was 24 percent within

the southern-most seed zone 540 (fig. 1), and the least was

13 percent within the northern-most zone 524. Mendocino

interior coast zones ranged between 8 to 13 percent, while

California North Coast, Klamath, and Southern Cascade

zones were 2 to 8 percent. Thus, heterogeneity and transfer

risk within geographic seed zones was lower in northwestern California than in southern Sierra Nevada.”

In three separate nursery common gardens, sugar pine

showed marked geographic variation in early growth and

phenology. Altitudinal provenances (or the original geographic source of seed) from a central Sierra Nevada transect

were highly differentiated (Harry and others 1983). Regional patterns were evident for 1185 families representing

27 California seed zones (Kitzmiller and Stover 1996).

Campbell and Sugano (1987) found about 50 percent of the

adaptive genetic variability in Oregon occurred among local

populations. They developed larger and very different zones

from the standard Oregon seed zones delineated in 1966

without the aid of genecological data.

Ideally, seed zones should be defined from long-term field

trials. Such studies with associated species, ponderosa pine

(Pinus ponderosa Dougl.) (Conkle 1973) and white fir (Abies

concolor (Gordon & Glend.) Lind.) (Jenkinson data filed,

1985) have shown strong adaptation to altitudinal climatic

gradients. In a 9-year California field study (Kitzmiller and

Stover 1996), 37 sugar pine families at seven common

garden sites expressed Genotype x Environment (G-x-E)

interaction for height. Families originating in coastal influence belts, North Coast-Klamath Mountains (NC-KM), grew

faster at three NC-KM sites than families from the northern

Sierra Nevada (NSN). NSN families grew faster at the two

coldest NSN sites, but no regional differences were expressed at the two NSN sites with long, warm growing

seasons. Close inspection revealed that about 30 percent of

the families were stable, with half ranking consistently high

across all sites. Liberal transfer of seed was suggested for

stable families if trends continued.

This paper presents recent results of the most comprehensive study of adaptation and G-x-E interaction in sugar pine

established by the Institute of Forest Genetics, PSW Research Station, and the Siskiyou and Eldorado National

Forests. This range-wide provenance trial was planted in

1984 in paired low and high elevation Sierra Nevada sites in

northern California, and in 1988 in paired coastal and

inland sites in southwest Oregon. Jenkinson (1996) reported

early results (4- to 8-year). Seed source elevation was significant for 8-year growth of Sierra Nevada sources at the low

elevation Sierra Nevada site. There, height was reduced by

10 percent and volume by 26 percent per 300 m rise in source

elevation. At the high elevation Sierra Nevada site, where

annual winter damage from wet, wind-driven snows became evident at 7-years, high sources were beginning to

excel. Source elevation was less important for northern

sources at both 4-year-old Oregon sites. Best early growth

in all plantations was attained by sources from the middle

to upper elevations of the most productive center of the

species range.

My objectives are to further assess adaptation and G-x-E

interactions in sugar pine for changes in early trends, and to

determine the current results and their implications for seed

transfer. This paper reports for the first time: (1) comparisons among all four tests for 10-year survival and growth,

(2) paired comparisons for survival and growth after 13-

years (Oregon) and 17-years (California), and (3) rust infection and health of rust-free trees.

Materials and Methods ___________

Study Design and Establishment

Jenkinson (1996) detailed selection of seed sources, nursery and planting procedures, and early results. Seed sources

represent 69 wild stands extending across environmental

gradients associated with elevation, latitude, and distance

from the Pacific Ocean (fig. 2, table 1). Provenance (seed

source) samples were well distributed, except in California

for the southern Cascades and interior north coast. Each

was represented by offspring from 4 to 20 (predominantly

10) healthy, vigorous natural seed parents occurring 0.8 km

to 3.2 km apart. The 69 sources were grouped by region

(physiographic province) of origin.

Four contrasting common garden test locations were selected to represent and compare two main areas within the

ecologically-variable species range: (1) compare range-wide

provenance performance in coastal (Sundown) versus inland

(Burnt Timber) forest environments within southwestern

Oregon, and (2) compare range-wide provenance performance at low (Cannon) vshigh elevation (Fitch-Rantz) forest

environments within the Sierra Nevada.

Bayleton systemic fungicide was sprayed annually in

autumn for 5-years to reduce blister rust infection. California

tests were lightly irrigated the first two summers to ensure

initial survival. Herbaceous vegetation was controlled intensively for 2-years using chemical and mechanical methods.

Woody vegetation (including Ribes) was controlled every

3- to 5-years.
106 USDA Forest Service Proceedings RMRS-P-32. 2004

Kitzmiller Adaptive Genetic Variation in Sugar Pine

Seed Elev Seed Seed Elev

 Plantation zone ft source zone ft

N Sierra Nevada North Coast Range

Cannon 526 2750 18 094 1500

Fitch-Rantz 526 6100 19 371 4500

S OR Coast Range N Sierra Nevada

Sundown 081 2400 20 524 2330

N Klamath Mtns 21 524 3775

Burnt Timber 512 1500 22 524 5250

23 524 5500

Seed Seed Elev 24 523 6100

source zone ft 25 525 4380

26 525 3965

W OR Cascade Range 27 772 5880

1 462 3000 28 772 6475

2 491 2495 29 772 6400

3 491 2065 30 526 2530

4 491 3500 31 526 2800

5 491 3500 32 525 3000

6 491 4500 33 526 3400

7 492 2395 34 526 4000

8 501 3080 35 526 4500

9 502 3500 36 526 4650

10 502 3855 37 525 5000

S OR Coast Range 38 526 5100

11 081 3050 39 526 5265

Klamath Mtns 40 526 5600

12 512 3660 41 526 5925

13 511 4500 42 526 6600

14 321 4000 43 525 7000

15 311 3940 44 531 3160

E Cascade Ranges 45 531 4000

16 703 5300 46 531 4720

17 741 5500 47 531 5450

48 531 6250

49 531 6950

S Sierra Nevada

50 532 6070

51 532 6055

52 533 5860

53 534 5565

54 534 5710

55 534 6565

56 534 6660

57 534 7615

58 534 6725

59 540 6265

60 540 5915

61 540 6100

62 540 6265

63 540 6300

South Coast Range

64 120 5800

Transverse-

 Peninsular Ranges

65 993 6950

66 994 6720

67 997 5950

68 997 7500

Sa San Pedro Martir

69 — 8200

Figure 2—Sugar pine seed sources and locations of common garden tests.
USDA Forest Service Proceedings RMRS-P-32. 2004 107

Adaptive Genetic Variation in Sugar Pine Kitzmiller

Table 1—Seed sources of sugar pine evaluated in common gardens in northern California and southwest Oregon. Sources are listed by

latitude and elevation.

Seed Elevation Lat Lon Tested No. Map

Region a Locale County Zone b (ft) (m) ∞N ∞W In c Fam Code

WOC Breitenbush R Marion 462 3000 915 44.80 122.03 C,F,_,_ 4 1

WOC Steelhead Ck Douglas 491 2495 761 43.40 122.66 C,F,S,B 7 2

WOC Grass Ck Douglas 491 2065 595 43.35 122.70 C,F,S,B 10 3

WOC Limpy Rock NE Douglas 491 3500 1067 43.33 122.62 C,F,_,_ 7 4

WOC Limpy Rock SW Douglas 491 3500 1067 43.33 122.62 C,F,S,B 10 5

WOC OK Butte Douglas 491 4500 1372 43.22 122.65 C,_,S,B 10 6

WOC Camp Comfort Douglas 492 2395 730 43.12 122.58 C,F,S,B 10 7

WOC Woodruff Flat Jackson 501 3080 939 42.87 122.49 C,F,S,B 10 8

WOC Elk Ck Jackson 502 3500 1067 42.85 122.68 C,_,S,B 10 9

WOC Camp Ck Jackson 502 3855 1175 42.60 122.38 C,F,S,B 10 10

SOC Gold Beach Curry 81 3050 930 42.60 124.15 C,_,S,B 10 11

KM Pea Soup Josephine 512 3660 1116 42.40 123.63 C,_,S,B 10 12

KM Bolan Josephine 511 4500 1372 42.05 123.43 C,F,_,_ 10 13

KM Dutch Ck Siskiyou 321 4000 1220 41.98 123.07 C,F,S,B 10 14

KM Salmon R Siskiyou 311 3940 1201 41.08 123.10 C,F,S,B 10 15

EC Black Hills Klamath 703 5300 1616 42.64 121.20 C,F,_,_ 10 16

EC Glass Mtn Siskiyou 741 5500 1677 41.60 121.50 C,F,_,_ 20 17

NC Fish Rock Rd Mendocino 94 1500 457 38.87 123.46 C,F,S,B 10 18

NC S Fk Elk Ck Glenn 371 4500 1372 39.56 122.65 C,F,S,B 10 19

NSN Forest Ranch Butte 524 2330 710 39.93 121.65 C,F,S,B 10 20

NSN Diamond I Butte 524 3775 1151 40.02 121.63 C,F,S,B 10 21

NSN Jonesville Butte 524 5250 1601 40.12 121.48 C,_,S,B 10 22

NSN Colby Mtn Tehama 524 5500 1677 40.13 121.52 C,F,S,B 10 23

NSN Stover Mtn Plumas 523 6100 1860 40.28 121.30 C,F,S,B 10 24

NSN Cal-Ida Sierra 525 4380 1335 39.54 121.01 C,F,_,_ 6 25

NSN N Shirttail Cyn Placer 525 3965 1209 39.18 120.75 C,F,_,_ 6 26

NSN Sierraville Sierra 772 5880 1793 39.55 120.34 C,F,_,_ 6 27

NSN Little Truckee Sierra 772 6475 1974 39.48 120.24 C,F,_,_ 5 28

NSN Upper Truckee Placer 772 6400 1951 39.27 120.21 C,F,_,_ 5 29

NSN Pleasant Valley Eldorado 526 2530 771 38.68 120.67 C,F,S,B 5 30

NSN Crozier Loop Eldorado 526 2800 854 38.82 120.72 C,F,S,B 10 31

NSN Breedlove Eldorado 525 3000 915 38.98 120.78 C,F,S,B 10 32

NSN Big X Mtn Eldorado 526 3400 1037 38.79 120.63 C,F,S,B 10 33

NSN Big Mtn Eldorado 526 4000 1220 38.57 120.52 C,F,S,B 10 34

NSN Snow Mill Rd Eldorado 526 4500 1372 38.71 120.46 C,F,S,B 10 35

NSN Caldor Rd Eldorado 526 4650 1418 38.62 120.47 C,F,S,B 10 36

NSN Uncle Tom’s Eldorado 525 5000 1524 38.93 120.48 C,F,S,B 10 37

NSN Tells Creek Eldorado 526 5100 1555 38.89 120.37 C,F,S,B 10 38

NSN Alder Ck Eldorado 526 5265 1605 38.69 120.30 C,F,S,B 10 39

NSN Pilliken Eldorado 526 5600 1707 38.69 120.35 C,F,S,B 10 40

NSN Sugarloaf Eldorado 526 5925 1806 38.79 120.28 C,F,S,B 10 41

NSN Mule Cyn Eldorado 526 6600 2012 38.72 120.18 C,F,S,B 10 42

NSN Bunker Hill Placer 525 7000 2134 39.04 120.39 C,F,S,B 10 43

NSN Groveland Stn Tuolumne 531 3160 963 37.82 120.11 C,F,_,_ 10 44

NSN Sugar Pine Tuolumne 531 4000 1220 38.08 120.20 C,F,S,B 10 45

NSN Lyons Reservoir Tuolumne 531 4720 1439 38.10 120.17 C,F,S,B 10 46

NSN Summit Stn Tuolumne 531 5450 1662 38.20 120.03 C,F,S,B 10 47

NSN Pinecrest Tuolumne 531 6250 1905 38.19 119.98 C,F,S,B 10 48

NSN Dodge Ridge Tuolumne 531 6950 2119 38.18 119.97 C,F,S,B 10 49

SSN Chowchilla Mtns Mariposa 532 6070 1851 37.53 119.72 C,F,S,B 10 50

SSN N Fk Willow Ck Madera 532 6055 1846 37.41 119.53 C,F,S,B 7 51

SSN Shaver Lake Fresno 533 5860 1787 37.12 119.26 C,F,S,B 8 52

SSN Landslide Fresno 534 5565 1697 36.77 118.88 C,F,S,B 10 53

SSN Happy Gap Fresno 534 5710 1741 36.73 119.00 C,F,S,B 10 54

SSN Lockwood Grove Fresno 534 6565 2002 36.80 118.87 C,F,S,B 10 55

SSN Hume Lake Rd Tulare 534 6660 2030 36.73 118.88 C,F,S,B 10 56

SSN Burton Mdws Fresno 534 7615 2322 36.78 118.83 C,_,S,B 10 57

SSN Hossack Mdw Tulare 534 6725 2050 36.17 118.62 C,F,_,_ 5 58

SSN Black Mtn Grove Tulare 540 6265 1910 36.11 118.65 C,F,_,_ 9 59

SSN Peyrone Camp Tulare 540 5915 1803 36.03 118.62 C,F,_,_ 6 60

SSN Cunningham Grove Tulare 540 6100 1860 36.02 118.58 C,F,_,_ 10 61

SSN Bull Run Basin Tulare 540 6265 1910 35.81 118.54 C,F,_,_ 6 62

SSN Greenhorn Sum Kern 540 6300 1921 35.74 118.56 C,F,_,_ 9 63

SC Junipero Serra Monterey 120 5800 1768 36.15 121.42 C,F,S,B 10 64

T-P San Gabriel Los Angeles 993 6950 2119 34.35 117.92 C,F,_,_ 10 65

T-P San Bernardino San Bernardino 994 6720 2049 34.24 117.10 C,F,_,_ 10 66

T-P San Jacinto Riverside 997 5950 1814 33.80 116.75 C,F,S,B 10 67

T-P San Jacinto Riverside 997 7500 2287 33.83 116.75 C,F,S,B 10 68

SSPM Parque Nacional Baja CA 8200 2500 31.00 115.57 C,F,_,_ 10 69

a

WOC: Western Oregon Cascades; SOC: South Oregon Coast; KM: Klamath Mt; NC: North Coast; NSN: Northern Sierra Nevada; SSN: Southern Sierra

Nevada; SC: South Coast; T-P: Transverse- Peninsular. b

 Buck et al 1970, Kitzmiller 1976, 1990.

c

C=Cannon, F=Fitch-Rantz, S=Sundown, B=Burnt Timber.
108 USDA Forest Service Proceedings RMRS-P-32. 2004

Kitzmiller Adaptive Genetic Variation in Sugar Pine

Measurements

Survival, height, and basal diameter at ground level (dg)

through age 10 were taken in all four tests. In fall of 2000,

at age 13, these same measurements were repeated at Sundown and Burnt Timber. At Cannon and Fitch-Rantz, survival,

height, and diameter at breast height (db) were recorded in the

fall of 2000 at age 17. Stem volume (dm 3

) of individual trees

was calculated using the formula: v= 0.1 p d 2 h, where d=db

or dg and h=height, in dm. At Sundown and Burnt Timber,

13-year volumes were based on dg. In contrast, 17-year

volumes at Cannon and Fitch-Rantz were based on db. At

California sites, tree injuries caused by blister rust, insects,

weather, and stem defects (including forking) were recorded.

A different crew measured Oregon tests; damage codes

consisted mostly of blister rust (infected or not), which

became readily apparent after 10-years. Other causes of

injuries were difficult to evaluate on rust-infected trees. So,

rust-free trees formed the base for assessing freedom from

other injuries. For seed sources, rust infection and mortality

were expressed as percent of surviving trees and of total

planted trees, respectively.

Design Differences between California

and Oregon Tests

The study is really composed of two pairs of different

experiments in terms of: year of establishment, provenance

and family representation, number and size of block replications and of trees per source-plot, and measurement schedule (table 2). Cannon (CAN) and Fitch-Rantz (FIT) had 63

sources (provenances) in common. Sundown (SUN) and

Burnt Timber (BUR) had 46 common sources. All four tests

shared 42 sources and 10-year data in common for a combined analysis. An important difference in experimental

design was family representation within source-plots. CAN/

FIT source-plots consisted of 10 families each with one tree.

SUN/BUR source-plots had 8 families with two contiguous

trees each. The effect of families within source could be

evaluated accurately only for SUN/BUR tests due to missing

family-plots at CAN/FIT.

Statistical Analysis

Statistical Analysis System (SAS) software (v. 8.1) was

used exclusively. PROC SUMMARY computed simple means,

within family plot variances, and correlations. Means were

built sequentially starting at the family-plot level (that is,

“mean of means” method). ANOVAs were based on familyplot (SUN/BUR pair) or source-plot means (CAN/FIT pair).

INSIGHT computed simple and canonical correlations,

simple and polynomial regressions, and contour-plot graphs

to relate adaptability traits to two geographic variables

using test-source means. PROC MIXED (method=REML,

option=satterth) estimated variance components, least

square means, and standard errors of means and of differences between two means using appropriate degrees of

freedom for unbalanced subclasses. PROC GLM (option=test)

generated accurate expected mean squares and synthesized

the proper F-tests.

The analysis consisted of three parts: (1) 42 common

sources for all four tests, (2) 63 common sources for the CAN/

FIT pair, and (3) 46 common sources for the SUN/BUR pair.

Analyses were made both across plantations and by individual test plantation. Models were developed using two sets

of assumptions regarding fixed and random effects. One

model assumed all factors random (REM) to estimate variance components for all factors studied (table 3a). Since test

plantations and regions of seed origins were not actually

selected at random, and since least square means are computed only for fixed effects, a more realistic mixed effects

model (MEM) was also used. For this MEM, Plantations,

Regions, and P-x-R interaction were assumed fixed, while

Sources within Region and P-x-S(R) interaction were random (table 3b). Lastly, to compute least square means for

S(R) and P-x-S(R) interaction, a third model was used where

only Blocks and Error were assumed random. Least squares

means were considered most useful for unbalanced data (e.g.

missing source plots, unequal replication among tests, unequal sources within regions). G-x-E interactions were examined for cause attributable to scale effects or true rank

changes according to Surles and others (1995).

The general run sequence was: (1) PROC MIXED for the

REM to estimate variance components; (2) PROC GLM for

Table 2—General description contrasting the four common garden tests.a

Description Cannon Fitch-Rantz Sundown Burnt Timber

State California California Oregon Oregon

Year Planted 1984 1984 1988 1988

Blocks 6 6 5 5

Sources 68 (14OR53CA1M) 64 (10OR53CA1M) 46 (8OR38CA) 46 (8OR38CA)

Trees/Plot 10 10 16 16

Spacing 2.25 m 1.83 m 1.83 m 1.83 m

Elevation 838 m 1860 m 732 m 457 m

Latitude 38.73 38.68 42.55 42.52

Longitude 120.75 120.21 124.13 123.58

Aspect south east south south

Climate warm, dry cool, moist cool, moist warm, dry

Soil PM volcanic basalt granite schist metavolcanic

Forest Type Lower M-C Upper M-C Coastal M-C Inland M-C

Irrigate 2-Yrs 3 week interval 3 week interval none none

Measure Age 2,3,4,6,8,9,10,17 2,3,4,6,8,9,10,17 3,4,5,6,8,10,13 3,4,5,6,8,10,13

a

(14OR53CA1M) indicates 14 Oregon, 53 California, and 1 Mexico seed sources in test.
USDA Forest Service Proceedings RMRS-P-32. 2004 109

Adaptive Genetic Variation in Sugar Pine Kitzmiller

the REM to make F-tests; (3) PROC GLM for a MEM to make

F-tests for Plantations, Regions, and P-x-R, (4) PROC MIXED

on the MEM to estimate and test differences between least

square means of significant factors; (5) repeat 3 and 4 above

for the Sources(R) level. The random vs fixed assumption

affected the F-test outcome for the main effects: Plantations

and Regions, because P-x-R was part of the error term in the

REM but was not in the MEM. Similarly, the assumption for

S(R) and P-x-S(R) affected the F-test outcome for P-x-R and

for S(R) (table 3b).

Trees shorter than breast-height (BUR:132, FIT: 62) were

discarded from growth analyses. All other trees were used,

even rusted and injured trees. Results were similar whether

rusted/injured trees were included or not. Rust on limbs had

no effect on current height or diameter. Rust on stem

reduced height by 4 to 5 percent, but had no effect on

diameter. Since this effect was rather small, and preservation of plot sample size was needed to prevent missing plots,

all except severely stunted trees were analyzed. I also

excluded trees that died after 10 years in my 10-year analysis across all tests.

Results ________________________

Survival, Rust Infection, and General

Health

Survival was highest at SUN and BUR (73.3 and 72.5

percent, respectively) followed closely by FIT (69.3 percent).

Survival was lowest at CAN (38.3 percent) due to progressive mortality from charcoal root rot (Macrophomina

Table 3a—ANOVA structure for the random model based on family plot means.a

Source of

 variation DF Expected mean square (EMS)

Plantations p-1 s2

 + 4 sPF2 + 32 sPS2 + 100 sPR2 + 148 sB

2

 + 740 sP

2

Blocks (P) p(b-1) s2 + 313 sB

2

Regions r-1 s2 + 4 sPF2 + 8 sF

2

 + 33 sPS2 + 66 sS

2

 + 147 sPR2 + 293 sR

2

PxR (p-1)(r-1) s2 + 4 s PF2 + 33 sPS2 + 146 sPR2

Sources (R) r(s-1) s2 + 4 s PF2 + 9 sF

2

 + 34 sPS2 + 68 sS

2

PxS(R) (p-1)r(s-1) s2 + 4 s PF2 + 34 sPS2

Families (SxR) rs(f-1) s2 + 4 s PF2 + 9 sF

2

PxF(SxR) (p-1)rs(f-1) s2 + 4 s PF2

Error b s2

a Includes family effect; coefficients are rounded to nearest whole number; p=number of plantations, b=number of

blocks or replications within plantation, r=number of regions, s=number of sources within region, and f=number of

families within source. b

 Error degrees of freedom = p(b-1)(r-1) + p(b-1)r(s-1) + p(b-1) rs(f-1).

Synthesized Error Terms (computed by GLM):

Plantation: 0.47*MS Blk(P) + 0.68*MS PxR + 0.29*MS PxS(R) + 0.01*MS PxF(SxR) – 0.46*MS Error;

Region: MS PxR + 0.97*MS S(R) – 0.96*MS PxS(R) + MS F(SxR) – 0.01*MS PxF(SxR) – MS Error;

PxR: 0.96*MS PxS(R) + 0.02*MS PxF(SxR) + 0.02*MS Error;

Sources (R): MS PxS(R) + 0.99*MS F(SxR) – 0.99*MS PxF(SxR) + MS Error;

PxS(R): 0.98*MS PxF(SxR) + 0.02*MS Error.

Table 3b – ANOVA structure for a mixed model based on source plot means.a

Source of

 variation DF Expected mean square (EMS)

Plantations p-1 s2

 + 5 sPS2 + _ + 21 sB

2

 + QP

Blocks (P) p(b-1) s2 + 46 sB

2

Regions r-1 s2 + 5 sPS2 + 10 sS

2 + _ + QR

PxR (p-1)(r-1) s2 + 5 sPS2 + QPR

Sources (R) r(s-1) s2 + 5 s PS2 + 10 sS

2

PxS(R) (p-1)r(s-1) s2 + 5 sPS2

Error p(b-1)(r-1) +p(b-1) r(s-1) s2

a P, R, and P-x-R are assumed fixed effects; “_” denotes their absence relative to random

model; bold components would be absent from a mixed model with S(R) and PxS(R) also

assumed fixed; coefficients are rounded to nearest whole number; p=number of plantations,

b=number of blocks or replications within plantation, r=number of regions, s=number of

sources within region, and f=number of families within source.

Synthesized Error Terms (computed by GLM):

Plantation: 0.47*MS Blk(P) + 0.96*MS PxS(R) – 0.42*MS Error;

Region: 0.97*MS S(R) + 0.03*MS Error
110 USDA Forest Service Proceedings RMRS-P-32. 2004

Kitzmiller Adaptive Genetic Variation in Sugar Pine

phaseolina [Tassi] Goid). During the last nine years, mortality was 29 percent at CAN, 9 percent at FIT, 15 percent at

SUN, and 8 percent at BUR. Note: Mortality the first 3-years

was 23 (CAN), 16 (FIT), 2 (SUN), and 18 (BUR) percent.

Trees living with rust infection were very low at CAN (0.1

percent), much higher at FIT (19.3 percent) and BUR (20

percent), and highest at SUN (44 percent). No trees were

recorded as dead from rust at CAN, SUN, or BUR, but 40

trees were rust-killed at FIT. The high post-establishment

mortality at SUN was probably rust-related, though it was

not so recorded. Of rust-free trees, 95 percent were healthy

and without major stem defects at CAN, 65 percent at FIT,

98 percent at SUN, and 99 percent at BUR. Of surviving

trees, 52 percent (in other words, 0.65*(1-0.19)) were healthy

at FIT and 95 percent were healthy at CAN. Only 36 percent

of total trees planted at both California tests were healthy at

17-years (table 4).

The widest range in survival among sources (table 4) was

at FIT (18 to 95 percent). At FIT, survival (and health) varied

in a curvilinear pattern with elevation of origin (R2

=0.57,

n=64, (R2

=0.40, n=64, respectively)), with highest survival

(and health) occurring for sources that match the test site

elevation (fig. 3). Survival and health of living trees was

directly correlated at FIT (r=0.70, n=63); sources with higher

survival were also healthier. Among tests, current survival

is significantly correlated only for CAN x FIT (r=0.44, n=63).

At CAN, survival based on regional means was highly

correlated with elevation of origin (R2

=0.88, n=10); TransTable 4—Survival, health, and rust infection by seed source and test plantation.a

Seed Origin:b Cannon Fitch-Rantz Sundown Burnt Timber

Region Locale MC Sdz Elev Surv Heal Surv Heal LR DR Surv Heal LR Surv Heal LR

m - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - % - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

WOC Breitenbush R 1 462 915 24 92 48 20 13 2 np np np np np np

WOC Steelhead Ck 2 491 761 38 91 45 50 11 83 96 20 84 100 18

WOC Grass Ck 3 491 595 30 89 58 48 17 2 74 98 29 79 96 13

WOC Limpy Rock NE 4 491 1067 10 83 72 55 28 np np np np np np

WOC Limpy Roc SW 5 491 1067 20 100 50 33 20 78 100 37 75 100 20

WOC OK Butte 6 491 1372 40 100 np np np np np np np np np

WOC Camp Comfort 7 492 730 45 96 43 42 8 86 98 36 73 100 31

WOC Woodruff Flat 8 501 939 48 90 43 65 23 2 56 100 38 75 100 17

WOC Elk Ckn 9 502 1067 27 94 np np np np np np np np np

WOC Camp Ck 10 502 1175 32 100 65 70 15 3 80 90 52 60 100 35

SOC Gold Beach 11 81 930 22 100 np np np 74 97 36 59 100 23

KM Pea Soup 12 512 1116 27 94 np np np 70 97 39 79 98 19

KM Bolan 13 511 1372 45 89 50 40 7 4 np np np np np np

KM Dutch Ck 14 321 1220 12 86 43 40 23 2 68 100 59 81 100 18

KM Salmon R 15 311 1201 53 91 88 65 13 65 100 63 56 100 22

EC Black Hills 16 703 1616 43 100 92 69 18 np np np np np np

EC Glass Mtn 17 741 1677 43 100 55 72 24 np np np np np np

NC Fish Rock Rd 18 94 457 50 90 18 0 0 84 100 33 64 100 14

NC S Fk Elk Ck 19 371 1372 12 86 55 48 12 74 100 66 61 100 8

NSN Forest Ranch 20 524 710 38 96 27 46 19 86 98 28 78 100 16

NSN Diamond I 21 524 1151 38 96 67 54 30 83 100 27 79 100 8

NSN Jonesville 22 524 1601 43 96 np np np 75 100 65 63 100 44

NSN Colby Mtn 23 524 1677 12 86 78 95 21 3 55 100 50 70 100 34

NSN Stover Mtn 24 523 1860 32 100 85 86 14 3 75 100 43 79 100 17

NSN Cal-Ida 25 525 1335 32 95 60 46 28 2 np np np np np np

NSN N Shirttail Cyn 26 525 1209 23 93 73 64 25 np np np np np np

NSN Sierraville 27 772 1793 48 100 93 84 20 np np np np np np

NSN Little Truckee 28 772 1974 47 100 83 90 16 np np np np np np

NSN Upper Truckee 29 772 1951 50 100 83 76 16 3 np np np np np np

NSN Pleasant Valley 30 526 771 45 93 47 71 14 81 100 35 75 98 18

NSN Crozier Loop 31 526 854 42 84 70 63 17 73 97 50 83 98 20

NSN Breedlove 32 525 915 33 90 48 48 14 90 100 39 86 100 14

NSN Big X Mtn 33 526 1037 20 100 53 52 22 88 100 43 64 100 25

NSN Big Mtn 34 526 1220 25 100 77 77 15 75 100 32 90 100 28

NSN Snow Mill Rd 35 526 1372 25 93 68 63 15 68 100 48 79 100 24

NSN Caldor Rd 36 526 1418 35 90 62 78 38 78 91 45 58 100 15

NSN Uncle Tom’s 37 525 1524 37 91 77 62 15 73 100 52 79 100 22

NSN Tells Creek 38 526 1555 30 100 92 65 22 78 100 53 80 100 23

NSN Alder Ck 39 526 1605 42 100 92 80 16 83 100 48 85 100 19

NSN Pilliken 40 526 1707 47 96 68 93 27 73 97 38 74 100 37

(con.)
USDA Forest Service Proceedings RMRS-P-32. 2004 111

Adaptive Genetic Variation in Sugar Pine Kitzmiller

verse-Peninsular and Sierra San Pedro Martir sources had

highest survival (fig. 4a). In contrast, based on regional

means at FIT, survival varied in a curvilinear pattern with

elevation of origin ((R2

=0.68, n=9); southern Sierra Nevada

sources had highest survival (fig. 4b). Contour plots for

survival pattern by latitude and elevation of origin likewise

revealed distinctions between test sites.

Generally, sources with higher survival and health at

SUN had lower survival and health at FIT (r=-0.40, r=-0.35,

n=42). At SUN, highest survival was associated with lower

elevation of source origin (r=0.44, n=46). At BUR, survival

was not associated with source origin. Rust infection of

sources was not consistent (not correlated) across test sites.

Survival and rust infection of sources at FIT were weakly

correlated (r=0.35, quadratic R2

=0.18, n=63); sources with

11 percent or lower infection had less than average survival.

Figure 3—Association between 17-year survival and origin

elevation for all provenances at Fitch-Rantz.

NSN Sugarloaf 41 526 1806 48 93 66 82 24 2 80 100 55 76 100 15

NSN Mule Cyn 42 526 2012 50 97 85 81 29 3 60 86 54 68 97 30

NSN Bunker Hill 43 525 2134 33 90 70 87 10 2 74 100 36 69 100 13

NSN Groveland Stn 44 531 963 23 100 58 68 3 np np np np np np

NSN Sugar Pine 45 531 1220 37 100 68 70 27 81 97 45 71 100 21

NSN Lyons Reservoi 46 531 1439 43 96 75 68 18 66 100 49 68 100 22

NSN Summit Stn 47 531 1662 28 82 75 82 24 5 78 100 55 75 100 28

NSN Pinecrest 48 531 1905 33 95 83 76 24 3 69 100 55 66 100 15

NSN Dodge Ridge 49 531 2119 33 100 93 76 13 76 100 43 79 98 13

SSN Chowchilla Mtn 50 532 1851 33 90 77 77 33 2 71 100 35 64 100 20

SSN N Fk Willow Ck 51 532 1846 53 97 83 77 30 59 100 40 74 100 12

SSN Shaver Lake 52 533 1787 65 97 87 80 23 88 100 44 86 100 10

SSN Landslide 53 534 1697 43 100 95 61 23 3 63 95 56 68 100 33

SSN Happy Gap 54 534 1741 42 100 77 76 28 2 65 95 58 80 100 27

SSN Lockwood Grov 55 534 2002 42 100 83 69 28 2 64 100 61 74 100 20

SSN Hume Lake Rd 56 534 2030 53 100 83 69 28 2 48 100 34 76 100 21

SSN Burton Mdws 57 534 2322 np np 73 63 14 2 56 94 64 56 100 18

SSN Hossack Mdw 58 534 2050 32 95 75 59 36 np np np np np np

SSN Black Mtn Grov 59 540 1910 65 92 77 71 26 np np np np np np

SSN Peyrone Camp 60 540 1803 42 96 83 83 20 3 np np np np np np

SSN Cunningham G 61 540 1860 52 100 87 72 31 np np np np np np

SSN Bull Run Basin 62 540 1910 45 93 92 86 24 np np np np np np

SSN Greenhorn Su 63 540 1921 38 91 83 91 12 3 np np np np np np

SC Junipero Serra 64 120 1768 37 95 58 32 20 71 100 42 61 100 6

T-P San Gabriels 65 993 2119 67 95 62 65 8 3 np np np np np np

T-P San Bernardino 66 994 2049 42 100 67 65 15 3 np np np np np np

T-P San Jacinto 67 997 1814 45 89 67 45 18 np np np np np np

T-P San Jacinto 68 997 2287 63 100 68 45 2 85 100 29 64 100 8

SSPM Parque Nacion 69 2500 63 95 52 59 6 3 np np np np np np

a np signifies not planted. Map Code 54 at Cannon had 4% LR.

Surv=Survival as % of trees planted. Heal= Healthy trees as percent of living rust-free trees.

LR= Trees living with rust infection as % of living trees. DR= Trees dead from rust as % of total trees planted b WOC: Western Oregon Cascades; SOC: South Oregon Coast; KM: Klamath Mts; NC: North Coast;

NSN: Northern Sierra Nevada; SSN: Southern Sierra Nevada; SC: South Coast; T-P: Transverse- Peninsular.

Table 4 (Con.)

Seed Origin:b Cannon Fitch-Rantz Sundown Burnt Timber

Region Locale MC Sdz Elev Surv Heal Surv Heal LR DR Surv Heal LR Surv Heal LR

m - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - % - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
112 USDA Forest Service Proceedings RMRS-P-32. 2004

Kitzmiller Adaptive Genetic Variation in Sugar Pine

Figure 4—Association between 17-year survival and origin elevation for regions at: a. Cannon and b. Fitch-Rantz.

All 4 Test Plantations: 42 Common

Sources

Genetic Structure in Adaptive Traits: ANOVA: 10-

Year Growth—All factors except Regions reflected significant differences for 10-year height across plantations

(table 5). Plantations accounted for most of the variability in

growth; trees at CAN and SUN grew much faster than trees

at FIT and BUR (table 6). All pair-wise differences between

test means were significant (p<0.05), except 10-year height

at CAN and SUN were similar. The G-x-E interactions: P-xR and P-x-S(R) were about twice the magnitude of their

genetic main effects (Regions and Sources within Region)

(table 5). Thus, height means for regions and sources within

region changed ranking across test plantations. Together,

genetic effects and their interactions accounted for about 11

percent of the total variability in height and volume (8

percent for diameter). Comparing across all four plantations, Regions and Sources(R) were about equal for height

and diameter, but for volume, Sources(R) far exceeded

Regions.

Analysis by plantation revealed that BUR, a xeric site,

was a relatively poor site for expression of genetic differences in growth (table 5). At BUR, diameter and volume did

not vary significantly by genetic source, and differences

were relatively weak for height. CAN was a good site for

genetic expression, but only at the Sources(R) level. Genetic

differences for all traits were expressed best at FIT, even

with its short growing season; Regions were 2.5 to 4 times

more variable than Sources(R). Together, genetic effects

accounted for: 39, 65, and 30 percent in height, diameter,

and volume, respectively. SUN also was excellent for total

genetic expression for height (65 percent of total variability),

and, at the Sources(R) level, for diameter and volume (35

percent).

The region effect was similar and small for growth at

CAN and BUR, but it was very different at FIT and SUN,

where Regions were about twice the magnitude of Sources(R)

for height. Both sites expressing strong regional differences have low moisture stress and productive soils. But

SUN has a strong maritime influence and long growing

season without snow pack, while FIT has a short growing

season with deep, persistent snow pack. Trees at SUN grew

twice as much in height and three times as much in volume

as trees at FIT (table 6). At SUN the region effect was due

most notably to superiority of the western Oregon Cascade

and North Coast sources, with Klamath Mts. and Sierra

Nevada sources intermediate, and South Coast and Transverse-peninsular sources were distinctly inferior in growth

(table 7). In contrast, at FIT, Sierra Nevada trees grew

most, and North Coast and Transverse-Peninsular trees

grew least. The Plantation-x-Region interaction was caused

by region height differences between SUN and FIT, the two

most climatically distinct tests.

Seed Source Correlations Among All Test Sites (10,

13-, and 17-Year Data)—Simple correlations between tests

were consistently strongest for height, and consistently

lowest when FIT was involved. Simple correlations between

test-source means for growth traits among low altitude tests

(CAN, SUN, BUR) were positive and mostly significant (r =

0.21 to 0.65, n=42, p <0.05 when r > 0.31). But r-values are

relatively low and show evidence for G-x-E interaction.

Correlations between CAN and SUN source growth means

were highest (0.48 to 0.65). CAN x BUR (0.34 to 0.48)

followed by SUN x BUR (0.21 to 0.46) were lower still. Height

at FIT was inversely related to height at SUN (r = 0.36), but

all other growth trait correlations between FIT and other

sites were non-significant and generally negative. Survival

was significantly correlated only for CAN x FIT (r = 0.44, p<0.01).

Overall, source means were generally correlated between

low altitude tests, but not impressively so. Ranking among

sources at FIT were most different from ranking in other

tests. These results imply relatively large genetic source x

test environment interaction.

Geographic Trends in Seed Source Means—Simple

correlations by test site between source mean growth traits

and their geographic origin variables (elevation, latitude,

longitude, and distance from test site to seed origin) were

relatively high (table 8). Highest correlations were inverse

associations between growth and source elevation, except at

FIT, the high elevation test, where growth-source elevation

correlations were always positive. Also, correlations at FIT

were opposite in sign for source latitude and longitude from

the other three tests. SUN exhibited the strongest correlations of all tests, with over 60 percent of the variation in height

a

b
USDA Forest Service Proceedings RMRS-P-32. 2004 113

Adaptive Genetic Variation in Sugar Pine Kitzmiller

Table 6—Least square means for test plantations by

growth traits.a

Plantation Trait - Least Square Mean

cm mm dm3 cm

46 sources h13 db13 v13 hgr3

Sundown 466 130 72.8 146

Burnt Timber 284 76 17.8 95

Std Error 17.1 4.4 5.2 5.4

63 sources h17 db17 v17 hgr7

Cannon 714 154 52.4 378

Fitch-Rantz 458 90 11.1 234

Std Error 14.4 3.1 2.4 9.8

42 sources h10 dg10 v10

Sundown 320 95.6 11.07

Burnt Timber 188 49.8 2.19

Cannon 325 108.9 14.31

Fitch-Rantz 158 74.1 3.36

Std Error 10 3.3 0.9

a h=height, db=diameter breast height, db=diameter ground

level, v=volume, hgr=recent 3- or 7-yr height growth increment;

Based on three analyses: 10-yr-all plantations-42 common seed

sources; 13-yr-SUN/BUR-46 common sources; 17-yr-CAN/FIT63 common sources.

Table 5—Variance component analyses combining all test plantations with 42 common seed sources for 10-yr height

(h10), diameter (dg10), and volume (v10) across and by plantation.

Source of h10 dg10 v10

 Variation DF VarC % PR > F VarC % PR > F VarC % PR > F

Plantations 3 7463 69 0.0001 680 69 0.0001 38 60 0.0001

Blocks(P) 18 444 4 0.0001 46 5 0.0001 3 5 0.0001

Regions 6 196 2 0.2325 32 3 0.0161 0 0 0.1891

Sources(R) 35 172 2 0.0061 17 2 0.0020 2 3 0.0028

P*R 18 376 3 0.0002 5 1 0.1995 0 1 0.2382

P*S(R) 105 395 4 0.0001 24 2 0.0001 5 7 0.0001

Error 680 1772 16 186 19 16 24

865 10818 100 990 100 64 100

Cannon

Regions 6 0 0 0.3607 3 1 0.2684 0 0 0.5206

Sources(R) 35 1012 20 0.0001 76 15 0.0021 16 24 0.0001

Blocks 5 756 15 0.0001 75 15 0.0001 8 12 0.0001

Error 157 3246 65 338 69 42 64

203 5014 100 492 100 66 100

Fitch-Rantz

Regions 6 507 28 0.0001 52 26 0.0001 1 23 0.0001

Sources(R) 35 200 11 0.0004 13 6 0.0205 0 6 0.0174

Blocks 5 124 7 0.0001 22 11 0.0001 1 13 0.0001

Error 196 969 54 112 57 3 57

242 1801 100 198 100 6 100

Burnt Timber

Regions 6 199 6 0.0569 15 5 0.0613 0 1 0.3639

Sources(R) 35 204 7 0.0344 6 2 0.2597 0 5 0.0963

Blocks 4 840 27 0.0001 72 26 0.0001 2 20 0.0001

Error 163 1877 60 190 67 6 74

208 3120 100 283 100 8 100

Sundown

Regions 6 1535 42 0.0008 17 7 0.0484 3 9 0.0677

Sources(R) 35 843 23 0.0001 82 35 0.0001 10 35 0.0001

Blocks 4 50 1 0.0298 14 6 0.0002 1 5 0.0006

Error 164 1197 33 121 52 14 51

209 3625 100 234 28 100

and 50 percent in volume associated with source elevation.

Weakest correlations between growth and geographic variables other than elevation were for CAN.

Distance was expressed both as simple kilometer distance

irrespective of direction (“Dist”) and as north vs south

direction-dependent (sources north of test latitude were

given positive kilometer values, and sources south of test

were given negative values). All test plantations except CAN

expressed an association for better growth for sources closer

in distance to the test site,”i.e.”correlations between growth

and “Dist” were negative (table 8). Close examination revealed one local source (Big X Mtn) with vastly superior

growth at CAN, and some superior sources at FIT were

transferred 250 km. For Oregon tests, nearly all sources

originated southward, so both variables gave the same

result. However, at FIT, better growth was associated with

closer source distance, and southern sources tended to outgrow northern sources (“Dist1” negative r-values, table 8).

Canonical correlations (1st variates) between growth and

geographic variables were higher for SUN (r=0.89), and FIT

(r=0.86), and lower for CAN (r=0.80) and BUR (r=0.77).

Height and elevation were weighted most. Sixty-three percent of the variance in the derived geographic origin variable

was associated with the variance in the derived growth trait

variable.
114 USDA Forest Service Proceedings RMRS-P-32. 2004

Kitzmiller Adaptive Genetic Variation in Sugar Pine

Table 7—Least square means and ranks by test, region and 10-yr height (h10), diameter (dg10), and volume

(v10).

Sundown Burnt Timber Cannon Fitch-Rantz

Region and Trait Rank LSM Rank LSM Rank LSM Rank LSM

h10

W Oregon Cascade 1 375 1 224 3 338 4 153

Klamath Mts 3 348 2 197 1 369 3 158

North Coast 2 372 4 189 2 360 7 129

N Sierra Nevada 4 322 3 194 4 330 2 184

S Sierra Nevada 5 307 5 183 5 328 1 195

South Coast 6 258 6 170 6 277 5 151

Transverse-Peninsular 7 254 7 160 7 274 6 133

 Plantation Mean 320 188 325 158

dg10

W Oregon Cascade 1 106.4 1 58.4 5 109.4 5 73.0

Klamath Mts 4 100.1 4 49.9 3 116.0 4 73.5

North Coast 3 103.2 5 46.9 4 111.2 6 68.2

N Sierra Nevada 2 103.5 2 55.8 1 117.0 2 82.5

S Sierra Nevada 5 100.0 3 51.3 2 116.1 1 84.7

South Coast 6 79.1 6 46.3 6 100.7 3 76.5

Transverse-Peninsular 7 76.9 7 40.0 7 92.2 7 60.5

 Plantation Mean 95.6 49.8 108.9 74.1

v10

W Oregon Cascade 1 15.24 1 3.29 5 14.21 5 3.07

Klamath Mts 4 12.28 3 2.16 1 17.86 4 3.20

North Coast 2 14.61 5 1.91 3 16.66 6 2.19

N Sierra Nevada 3 12.47 2 3.01 2 16.67 2 4.69

S Sierra Nevada 5 11.33 4 2.16 4 15.88 1 5.27

South Coast 6 6.07 6 1.74 6 9.91 3 3.30

Transverse-Peninsular 7 5.47 7 1.10 7 8.96 7 1.80

 Plantation Mean 11.07 2.19 14.31 3.36

Seed Source Rank Changes Across Test Plantations—Rank comparisons for the same 42 seed sources on

the same four test sites for 10-year growth traits revealed

clear genetic source interactions across tests (table 9). These

interactions were not due to scale effects; rather they were

determined to be true rank change interactions (Surles and

others 1995). Major rank changes among tests for the same

sources were evident between: Oregon sites, California sites,

and states. Patterns of rank changes may reflect adaptive

responses to different climates. Based on the subjective

criterion of at least one quartile rank change (11+) in both

height and volume, within the Oregon pair (coastal vs inland

tests) 12 sources were interactive, five of which (Map Codes:

18, 23, 41, 45, 50) were highly so. Within the California pair

(low vs high elevation tests), 18 sources were interactive, 12

of which (5, 7, 8, 14, 18, 24, 32, 33, 37, 39, 42, 51) were highly

so. Some sources displayed specific preference for their

“home” state (2, 3, 8, 50, 56). Among the four test sites, 18

sources (43 percent) were considered highly interactive.

Some 13 sources were interactive only at FIT, being stable

across all other tests; five low elevation sources were especially maladapted to FIT; eight mid to high elevation sources

were especially well-adapted. Only 10 sources (24 percent)

were relatively stable in rank across all tests (15, 19, 31, 34,

43, 46, 52, 55, 64, 68).

Growth Trends Over Time—Within-test simple correlations between 10-year total growth and subsequent 3- or 7-

year growth increment were made to monitor potential

trend changes among sources over time. Most recent 7-year

height increment at FIT was highly correlated with height

at age 10 (r=0.94, p<0.0001), but much less so at CAN

(r=0.62, p<0.0001). Corresponding values for recent 3-year

increment and 10-year total were identical at both SUN and

BUR (r=0.87, p<0.0001). Thus, the pattern of growth differences among sources at CAN changed to a greater degree

during recent years compared to the other tests, which

remain very stable.

Cannon and Fitch-Rantz: 63 Common

Seed Sources: 17-Year Data

Genetic Structure in Adaptive Traits: ANOVA

Variance Components and Means—Analysis of 17-year

data for CAN/FIT provided 21 additional sources to better

represent regions and sources within regions. Forty-two of

the 63 common sources were from the Sierra Nevada.

Plantations accounted for the majority of variability in all

traits (66 to 74 percent for growth, and 50 percent for form,

Table 10). Trees at CAN grew 1.6 times more height and 4.7

times more volume as trees at FIT (table 6). Genetic sources

accounted for 10 to 23 percent of total variability. Significant G-x-E interactions (P*R, P*S(R)) imply that growth

trait differences among Regions and Sources(R) must be

interpreted separately for each test plantation. G-x-E interactions (composed mainly of P*R for height, and mainly

P*S(R) for volume) were 3 times the magnitude of genetic

main effects (table 10).
USDA Forest Service Proceedings RMRS-P-32. 2004 115

Adaptive Genetic Variation in Sugar Pine Kitzmiller

Table 8—Simple correlations between seed source growth trait means and

geographic origin variables at each test site.a

Test_Trait b Elev Lat Lon Dist Dist1

Sun_h10 –0.808 0.624 0.638 –0.637 –0.637

Sun_dg10 –0.565 0.330 0.290 –0.347 –0.347

Sun_v10 –0.698 0.424 0.423 –0.436 –0.436

Sun_h13 –0.792 0.638 0.653 –0.652 –0.652

Sun_dg13 –0.599 0.370 0.327 –0.378 –0.378

Sun_v13 –0.712 0.478 0.469 –0.484 –0.484

Sun_hgr3 –0.685 0.610 0.626 –0.624 –0.624

Sun_dggr3 –0.559 0.396 0.355 –0.379 –0.379

Sun_vgr3 –0.709 0.504 0.490 –0.507 –0.507

Bur_h10 –0.571 0.550 0.441 –0.517 –0.517

Bur_dg10 –0.413 0.362 0.216 –0.342 –0.342

Bur_v10 –0.398 0.275 0.187 –0.264 –0.264

Bur_h13 –0.580 0.590 0.495 –0.556 –0.556

Bur_dg13 –0.360 0.355 0.207 –0.339 –0.339

Bur_v13 –0.396 0.319 0.224 –0.302 –0.302

Bur_hgr3 –0.546 0.615 0.554 –0.582 –0.582

Bur_dggr3 –0.155 0.256 0.138 –0.250 –0.250

Bur_vgr3 –0.388 0.340 0.242 –0.320 –0.320

Can_h10 –0.633 0.284 0.367 –0.045 0.324

Can_dg10 –0.371 0.017 0.050 –0.378 0.028

Can_v10 –0.475 0.064 0.134 –0.298 0.089

Can_h17 –0.583 0.302 0.345 –0.007 0.333

Can_db17 –0.383 0.052 0.094 –0.315 0.071

Can_v17 –0.465 0.101 0.161 –0.242 0.129

Can_hgr7 –0.377 0.256 0.233 0.048 0.268

Fit_h10 0.551 –0.292 –0.460 –0.559 –0.360

Fit_dg10 0.389 –0.276 –0.351 –0.632 –0.314

Fit_v10 0.472 –0.323 –0.441 –0.603 –0.372

Fit_h17 0.556 –0.268 –0.463 –0.547 –0.339

Fit_db17 0.525 –0.272 –0.471 –0.609 –0.347

Fit_v17 0.475 –0.279 –0.426 –0.611 –0.331

Fit_hgr7 0.547 –0.249 –0.455 –0.529 –0.321

a Italics: p<.05, n=42 Bold: p<.01, n=42. Elev: elevation, Lat: latitude, Lon: longitude, Dist:

seed origin distance from test site, Dist1: distance of northern (+) or southern (–) origins from

test site. b Sun,Bur,Can,Fit refers to test: Sundown, Burnt Timber, Cannon, Fitch-Rantz. h, db, dg,

v, hgr, dggr, and vgr refers to trait: height, diameter breast height, diameter ground level,

volume, recent 3- or 7-yr growth increment for height, diameter ground level, and volume

respectively.

Analyses by test revealed large genetic source effects at

both plantations, accounting for 38 to 40 percent for height

and 32 to 40 percent for diameter and volume (table 10).

Region of origin contributed most to growth variability at

CAN/FIT, but Sources(R) was a highly significant contributor to growth in each test. Regions accounted for four times

that of Sources(R) at FIT and 1.5 to two times Sources(R) at

CAN. Regions were less interactive with plantations than

Sources(R), perhaps because Regions were more highly

buffered with broad genetic diversity. The P*R interaction

involved regions with high elevation persistent snow-packs,

Eastern Cascade and Sierra Nevada Ranges grew more at

FIT than coastal and milder regions which grew more at

CAN (fig. 5). Seed source means were highly variable between tests (tables 10, 12).

Growth and Geographic Origin Factors: Correlation

and Regression—Region height means at CAN decreased

with origin elevation (R2

=0.76, p=0.001, n=10). At the source

level, height of Sierra Nevada sources decreased linearly

with increase in elevation (R2

=0.37, p<0.0001, n=42, fig. 6a).

At FIT, growth of Sierra Nevada sources increased with

source elevation in a curvilinear pattern peaking at 1860 m

and then decreasing (R2

=0.44, p<0.0001, n=42, fig. 6b).

For 63 sources at both test sites, volume (v17) and previous 7-year height growth (hgr7) varied in curvilinear patterns with distance in miles north (+) or south (-) from test

to seed origin (dist1), with R2

=0.22, p=0.0006 at CAN (fig. 7a)

and R2=0.38, p<0.0001 at FIT (fig. 7b) for v17. Corresponding values for hgr7 were only slightly lower. Best hgr7

growth occurred for sources nearest CAN. At FIT best

growth occurred for sources near the site and up to 240 km

south. Recent height growth (hgr7) was correlated with 10-

year height more closely at FIT (r=0.93) than at CAN

(r=0.68), just as it was in the 10-year 42-source analysis.
116 USDA Forest Service Proceedings RMRS-P-32. 2004

Kitzmiller Adaptive Genetic Variation in Sugar Pine

Table 9—Ranking among 42 common sources within test for 10-yr height (h10), diameter (dg10), and volume (v10).

Seed Origin: Elev Map SUN BUR CAN FIT SUN BUR CAN FIT SUN BUR CAN FIT

Regn Locale Sdz m Code h10 h10 h10 h10 dg10 dg10 dg10 dg10 v10 v10 v10 v10

WOC Steelhead Ck 491 761 2 5 8 29 38 18 19 36 40 11 19 30 38

WOC Grass Ck 491 595 3 7 4 14 26 8 8 26 32 7 5 25 32

WOC Limpy Rock SW 491 1067 5 17 2 13 40 34 2 33 36 32 2 29 39

WOC Camp Comfort 492 730 7 1 14 8 36 3 22 23 33 2 24 18 35

WOC Woodruff Flat 501 939 8 3 6 19 34 2 12 17 35 3 12 17 37

WOC Camp Ck 502 1175 10 16 7 17 31 31 18 21 26 27 15 23 30

KM Dutch Ck 321 1220 14 11 23 5 39 29 34 11 39 25 35 7 40

KM Salmon R 311 1201 15 15 19 15 23 23 23 25 24 22 18 19 21

NC Fish Rock Rd 94 457 18 2 32 4 42 9 41 15 41 4 39 9 42

NC S Fk Elk Ck 371 1372 19 19 15 26 28 30 28 34 37 28 22 24 34

NSN Forest Ranch 524 710 20 13 20 9 33 14 16 16 23 14 17 14 31

NSN Diamond I 524 1151 21 12 3 3 25 12 3 3 16 10 3 3 18

NSN Colby Mtn 524 1677 23 33 17 35 16 37 13 39 31 36 13 40 29

NSN Stover Mtn 523 1860 24 14 24 31 3 20 25 40 11 20 27 39 5

NSN Pleasant Valley 526 771 30 24 10 27 37 19 7 30 38 16 7 28 36

NSN Crozier Loop 526 854 31 6 11 6 17 6 5 1 21 6 11 2 16

NSN Breedlove 525 915 32 10 1 1 32 17 1 2 30 12 1 1 28

NSN Big X Mtn 526 1037 33 8 21 2 30 4 14 5 25 5 16 4 22

NSN Big Mtno 526 1220 34 9 16 10 12 59448859

NSN Snow Mill Rd 526 1372 35 22 12 22 22 15 11 7 6 21 6 11 12

NSN Caldor Rd 526 1418 36 23 28 16 29 16 27 12 19 18 29 12 26

NSN Uncle Tom’s 525 1524 37 31 30 37 14 24 33 22 9 26 36 32 10

NSN Tells Creek 526 1555 38 38 22 20 7 39 10 13 8 39 14 15 7

NSN Alder Ck 526 1605 39 29 42 32 4 26 40 20 3 29 41 22 3

NSN Pilliken 526 1707 40 28 27 23 11 22 24 18 14 24 25 20 13

NSN Sugarloaf 526 1806 41 26 9 38 15 25 4 37 15 23 4 35 19

NSN Mule Cyn 526 2012 42 37 39 39 1 35 31 32 2 35 26 38 4

NSN Bunker Hill 525 2134 43 34 31 36 27 33 35 28 27 34 32 36 23

NSN Sugar Pine 531 1220 45 4 33 12 24 1 32 8 28 1 34 10 25

NSN Lyons Reservoi 531 1439 46 25 18 25 13 13 17 27 17 17 9 26 15

NSN Summit Stn 531 1662 47 30 35 24 5 27 29 14 5 30 31 16 2

NSN Pinecrest 531 1905 48 39 41 42 18 36 30 41 22 38 30 37 24

NSN Dodge Ridge 531 2119 49 32 26 34 19 28 20 31 18 31 21 34 17

SSN Chowchilla Mtn 532 1851 50 21 37 11 2 10 39 9 10 13 40 8 8

SSN N Fk Willow Ck 532 1846 51 18 29 30 6 7 26 24 1 9 28 27 1

SSN Shaver Lake 533 1787 52 20 5 7 10 11 6 6 12 15 10 6 11

SSN Landslide 534 1697 53 40 25 28 20 40 21 29 20 40 20 33 20

SSN Happy Gap 534 1741 54 27 13 21 8 21 15 19 7 19 23 21 6

SSN Lockwood Grov 534 2002 55 35 38 33 21 38 38 35 34 37 38 31 27

SSN Hume Lake Rd 534 2030 56 36 36 18 9 32 36 10 13 33 37 13 14

SC Junipero Serra 120 1768 64 41 34 40 35 41 37 38 29 41 33 41 33

T-P San Jacintos 997 2287 68 42 40 41 41 42 42 42 42 42 42 42 41

SUN and BUR: 46 Common Seed Sources:

13-Year Data

Genetic Structure in Adaptive Traits: Variance Components and Means—The family-plot structure at Oregon

test plantations allowed estimation of variance components

that included among-families and within-family plots

(table 11). Plantations accounted for 42 to 51 percent of the

total variability in growth traits. Trees at SUN grew 1.64

times taller and 4 times more volume than BUR at 13-years

(table 6). Regions, Families, and Plantations-x-Sources(R)

were highly significant for 13-year height. Applying a fixed

assumption for P-x-R interaction (MEM) resulted in significance for Regions main effect for all traits (not shown). About

10 percent of all variability in 13-year height was attributable to genetic main effects and their interactions with

plantations (table 11). The within-family plot variance is

composed of confounded genetic and environmental factors.

Within-family plot variance was similar but slightly smaller

than the among-family plot (experimental error).

Genetic sources at SUN accounted for 25 percent of the

total variability in 13-year height including the within-plot

component (or 38 percent excluding it). Corresponding values at BUR were only 11 percent (or 16 percent) and about

the same magnitude as Blocks. Although Regions and

Sources(R) were significant for 13-year height, Regions was

larger at SUN, while Sources(R) was larger at BUR. Effect

of Regions was not significant for 13-year diameter and
USDA Forest Service Proceedings RMRS-P-32. 2004 117

Adaptive Genetic Variation in Sugar Pine Kitzmiller

Table 10—Variance component percent of total variability and F-tests for growth traits of 63 common seed sources at Cannon

and Fitch-Rantz.a

Source of h17 dbh17 v17 h17/dbh17 hgr7

Variation DF % Pr > F % Pr > F % Pr > F % Pr > F % Pr > F

Across Tests:

Plantations 1 73 0.0001 74 0.0001 68 0.0001 48 0.0002 67 0.0001

Blocks(P) 10 2 0.0001 2 0.0001 2 0.0001 2 0.0001 3 0.0001

Regions 8 2 0.1630 3 0.0399 3 0.1088 2 0.2808 3 0.2038

Sources(R) 54 0 0.8324 0 0.5797 0 0.5967 2 0.0897 0 0.9388

P*R 8 5 0.0016 3 0.0051 3 0.0532 15 0.0001 6 0.0005

P*S(R) 54 3 0.0001 3 0.0001 6 0.0001 4 0.0024 3 0.0001

Error 537 14 15 20 28 18

By Test: Cannon

Regions 8 25 0.0003 21 0.0001 15 0.0031 12 0.0001 18 0.0009

Sources(R) 54 15 0.0001 11 0.0012 17 0.0001 0 0.5933 8 0.0040

Blocks 5 7 0.0001 3 0.0043 4 0.0020 7 0.0001 5 0.0001

Error 243 52 64 64 80 69

By Test: Fitch-Rantz

Regions 8 31 0.0001 33 0.0001 29 0.0001 35 0.0001 32 0.0001

Sources(R) 54 7 0.0007 7 0.0002 9 0.0001 12 0.0001 11 0.0001

Blocks 5 11 0.0001 11 0.0001 12 0.0001 3 0.0012 12 0.0001

Error 243 51 48 50 50 45

a h, dbh, v, hgr: height, diameter breast height, volume, and recent periodic height increment. Random Effects Model was assumed to estimate

variance components and F-tests. W Oregon Cascade

E Cascades

Klamath Mts

North Coast

N Sierra Nevada

S Sierra Nevada

South Coast

Transverse-Penis

Sierra San Pedro

FIT_H17

CAN_H17

0

100

200

300

400

500

600

700

800

Region

17-Yr HT cm Cannon and Fitch-Rantz

FIT_H17

CAN_H17

Figure 5—Mean 17-year height for regions at Cannon and Fitch-Rantz.
118 USDA Forest Service Proceedings RMRS-P-32. 2004

Kitzmiller Adaptive Genetic Variation in Sugar Pine

Figure 6—Association between 17-year height and origin

elevation for Sierra Nevada provenances at: a. Cannon

and b. Fitch-Rantz.

Figure 7—Association between 17-year volume and

provenance distance north(+) or south(-) of test locations

for: a. Cannon and b. Fitch-Rantz.

volume in either test. Effect of families was relatively small

for all traits but was significant at SUN (table 11).

Ranking of regions for h13 was: WOC > NC > KM > SOC

> NSN > SSN > SC > T-P. Significant (p<.05) differences

between Region means across tests for h13 were: WOC >

NSN, SSN, SC, T-P; SOC > T-P; KM > SSN, SC, T-P; NC

> SSN, SC, T-P; NSN > T-P; where: WOC = Western Oregon

Cascade, SOC = South Oregon Coast, KM = Klamath

Mountains, NC = North Coast, NSN = Northern Sierra

Nevada, SSN = Southern Sierra Nevada, SC = South Coast,

and T-P = Transverse-Peninsular. Seed sources within region varied greatly between test sites (table12).

Stability in Performance Across Plantations: Correlations and Regressions—Simple correlations between

test plantation-seed source means, a measure of G-x-E

interaction, were significant only for height (r=0.53 for h10,

r=0.51 for h13, r=0.37 for hgr3, n=46). This indicates substantial differences in performance ranking of sources between SUN and BUR that are increasing with age.

Growth and Geographic Origin Factors: Correlation and Regression—Growth was inversely correlated

with elevation of origin for region means (fig. 8a, 8b) and for

source means (fig. 8c, 8d). Among traits, height was most

highly correlated with seed origin. Among seed origin variables, elevation was most highly correlated with growth.

SUN provided the strongest pattern of growth with seed

origin for source means: 64 percent of the variation in 13-

year height was associated with elevation. The corresponding value at BUR was 40 percent. Based on regional means,

85-86 percent of the variation in height was associated with

region mean elevation at both sites. The dominant pattern

was that growth decreased linearly with increasing elevation and with decreasing latitude and longitude of seed

origin. Using canonical correlation, the derived seed origin

variable accounted for 79 percent of the variation in the

derived growth variable for SUN, and 61 percent for BUR.

Further, the derived growth variable for SUN accounted for

62 percent of the variation in the derived growth variable for

BUR. So, the implied adaptive pattern of growth response

was somewhat different at these Oregon sites.

Discussion _____________________

Sugar pine displayed a complex genetic structure in adaptive traits. Strong G-x-E interactions were expressed for

a

b

a

b
USDA Forest Service Proceedings RMRS-P-32. 2004 119

Adaptive Genetic Variation in Sugar Pine Kitzmiller

Table 11—Variance components, percent of total variability, and F-tests for 13-yr height diameter, and volume for 46

common seed sources at Sundown and Burnt Timber across and by plantation.

Source of h13 dg13 v13

Variation DF VarC % PR > F VarC % PR > F VarC % PR > F

Plantations 1 15964 49 0.0001 1464 51 0.0001 262 42 0.0001

Blocks(P) 8 975 3 0.0001 59 2 0.0001 9 1 0.0001

Regions 7 1548 5 0.0050 47 2 0.0590 11 2 0.1489

Sources(R) 38 337 1 0.1244 15 1 0.3925 2 0 0.3397

Family(R S) 321 151 0 0.0001 11 0 0.0004 3 1 0.0158

P*R 7 0 0 0.5367 0 0 0.9389 0 0 0.3053

P*S(R) 38 1092 3 0.0001 84 3 0.0001 33 5 0.0001

P*F(R S) 320 0 0 0.9996 0 0 0.9982 0 0 0.9031

Block * (R,S,F) 2495 6983 21 653 23 161 26

Within Fam Plot 2094 5472 17 556 19 140 22

32522 100 2889 100 622 100

Burnt Timber

Regions 7 570 4 0.0180 21 2 0.1053 1 1 0.4006

Sources(R) 38 1031 7 0.0001 70 6 0.0001 10 7 0.0001

Families(R S) 320 0 0 1.0000 0 0 0.9999 0 0 1.0000

Blocks 4 1722 12 0.0001 107 9 0.0001 11 9 0.0001

Blocks*R,S,F 1219 6738 46 602 49 67 51

Within Fam Plot 1062 4450 31 435 35 42 32

14510 100 1236 100 130 100

Sundown

Regions 7 2460 13 0.0013 15 1 0.1054 19 3 0.0602

Sources(R) 38 1852 10 0.0001 148 9 0.0001 62 10 0.0001

Families(R S) 320 359 2 0.0078 26 2 0.0224 11 2 0.0194

Blocks 4 235 1 0.0001 11 1 0.0002 6 1 0.0001

Blocks*R,S,F 1276 7166 39 699 44 249 42

Within Fam Plot 1032 6528 35 682 43 240 41

18600 100 1581 100 587 100

Table 12—Least square means for growth traits by test plantation and seed source.a

 Seed Origin: Cannon Fitch-Rantz Sundown Burnt Timber

 Locale MC Sdz Elev h17 db17 v17 h17 db17 v17 h13 dg13 v13 h13 dg13 v13

m cm mm dm3 cm mm dm3 cm mm dm3 cm mm dm3

Breitenbush R 1 462 915 637 133 42.1 364 68 7.9 np np np np np np

Steelhead Ck 2 491 761 716 144 51.6 398 69 7.8 532 144 95.3 321 82 21.7

Grass Ck 3 491 595 740 163 66.0 444 85 13.1 548 149 106.3 359 94 32.2

Limpy Rock NE 4 491 1067 799 154 61.4 374 65 4.9 np np np np np np

Limpy Rock SW 5 491 1067 736 142 53.0 388 70 6.6 485 125 69.2 386 99 38.4

OK Butte 6 491 1372 np np np np np np np np np np np np

Camp Comfort 7 492 730 822 157 68.2 420 81 9.7 609 157 127.8 316 80 19.6

Woodruff Flat 8 501 939 705 152 55.6 423 77 8.4 543 153 120.4 330 91 26.7

Elk Ckn 9 502 1067 np np np np np np np np np np np np

Camp Ck 10 502 1175 733 152 56.5 450 85 13.4 485 129 71.3 320 82 20.7

Gold Beach 11 81 930 np np np np np np 487 129 69.9 299 75 16.5

Pea Soup 12 512 1116 np np np np np np 495 131 73.7 318 81 23.2

Bolan 13 511 1372 793 171 75.3 374 66 4.3 np np np np np np

Dutch Ck 14 321 1220 859 174 85.8 349 61 5.4 499 132 77.2 294 73 14.3

Salmon R 15 311 1201 714 141 51.4 464 87 15.6 516 144 92.6 304 80 20.5

Black Hills 16 703 1616 726 141 50.0 587 106 22.5 np np np np np np

Glass Mtn 17 741 1677 616 123 37.2 452 86 12.8 np np np np np np

Fish Rock Rd 18 94 457 828 161 72.4 263 33 0.0 587 148 116.5 278 71 14.6

S Fk Elk Ck 19 371 1372 707 158 63.8 429 83 9.9 486 133 76.0 306 77 19.9

Forest Ranch 20 524 710 762 170 78.0 469 103 13.9 504 143 90.0 284 83 19.7

Diamond I 21 524 1151 834 182 89.7 457 89 17.6 504 143 93.5 345 96 31.5

(con.)
120 USDA Forest Service Proceedings RMRS-P-32. 2004

Kitzmiller Adaptive Genetic Variation in Sugar Pine

Table 12 (Con.)

 Seed Origin: Cannon Fitch-Rantz Sundown Burnt Timber

 Locale MC Sdz Elev h17 db17 v17 h17 db17 v17 h13 dg13 v13 h13 dg13 v13

m cm mm dm3 cm mm dm3 cm mm dm3 cm mm dm3

Jonesville 22 524 1601 np np np np np np 475 132 70.9 269 72 15.9

Colby Mtn 23 524 1677 642 142 44.2 487 96 17.6 412 119 51.8 300 89 24.5

Stover Mtn 24 523 1860 706 151 52.6 548 109 25.6 492 140 82.8 287 80 19.4

Cal-Ida 25 525 1335 756 164 67.4 434 92 15.3 np np np np np np

N Shirttail Cyn 26 525 1209 746 188 102.5 486 106 20.6 np np np np np np

Sierraville 27 772 1793 686 158 59.0 526 102 20.3 np np np np np np

Little Truckee 28 772 1974 664 144 45.3 462 91 15.5 np np np np np np

Upper Truckee 29 772 1951 595 129 34.1 460 99 17.2 np np np np np np

Pleasant Valley 30 526 771 690 140 47.3 424 78 9.6 454 136 78.1 306 89 27.2

Crozier Loop 31 526 854 812 185 95.7 455 92 17.9 538 157 111.4 307 91 25.6

Breedlove 32 525 915 815 179 87.3 457 93 14.3 516 145 98.1 383 105 56.1

Big X Mtn 33 526 1037 908 189 107.0 448 90 14.5 517 152 101.9 287 88 22.3

Big Mtn 34 526 1220 743 170 74.7 476 102 20.4 515 155 104.3 288 86 24.9

Snow Mill Rd 35 526 1372 712 165 66.2 461 98 18.8 465 147 85.2 301 88 27.2

Caldor Rd 36 526 1418 735 153 61.5 452 92 13.0 458 142 83.5 272 81 17.9

Uncle Tom’s 37 525 1524 640 148 47.1 502 113 24.8 437 139 75.5 246 72 12.8

Tells Creek 38 526 1555 723 176 81.4 518 109 25.8 406 121 52.3 296 95 26.7

Alder Ck 39 526 1605 685 150 56.2 512 105 22.9 443 136 70.3 228 69 10.7

Pilliken 40 526 1707 682 151 56.3 485 102 18.3 455 138 79.9 270 85 18.7

Sugarloaf 41 526 1806 655 146 51.6 483 106 19.6 464 136 77.8 316 97 30.7

Mule Cyn 42 526 2012 701 146 49.6 547 109 24.7 399 123 53.2 242 75 17.4

Bunker Hill 43 525 2134 678 143 45.4 456 92 14.8 422 127 60.0 258 75 17.0

Groveland Stn 44 531 963 829 187 95.6 448 91 16.8 np np np np np np

Sugar Pine 45 531 1220 763 166 71.9 410 84 11.8 544 166 127.2 249 72 13.6

Lyons Reservoir 46 531 1439 726 159 59.7 483 95 17.0 449 141 81.4 311 86 29.1

Summit Stn 47 531 1662 705 162 62.6 542 112 26.6 427 136 70.0 249 75 14.8

Pinecrest 48 531 1905 601 138 42.5 467 94 15.9 385 119 47.3 239 80 17.9

Dodge Ridge 49 531 2119 686 151 54.5 461 97 16.3 439 134 68.9 271 82 19.8

Chowchilla Mtn 50 532 1851 769 168 72.8 544 113 25.0 503 150 98.3 238 72 12.5

N Fk Willow Ck 51 532 1846 683 155 57.6 546 115 26.6 500 152 99.3 249 78 15.4

Shaver Lake 52 533 1787 769 167 70.1 536 112 24.6 473 142 81.5 324 92 27.7

Landslide 53 534 1697 713 150 55.5 465 94 15.5 381 112 44.6 292 82 22.0

Happy Gap 54 534 1741 772 165 69.8 492 93 16.9 451 140 78.8 299 84 18.3

Lockwood Grove 55 534 2002 690 152 54.8 485 90 16.0 393 118 49.0 237 66 11.3

Hume Lake Rd 56 534 2030 746 165 70.5 499 98 18.7 399 125 57.7 251 77 15.8

Burton Mdws 57 534 2322 np np np np np np 392 120 54.7 234 66 11.4

Hossack Mdw 58 534 2050 693 159 64.8 477 94 15.7 np np np np np np

Black Mtn Grove 59 540 1910 731 152 58.1 500 105 19.7 np np np np np np

Peyrone Camp 60 540 1803 673 147 51.6 463 91 16.5 np np np np np np

Cunningham Gr 61 540 1860 761 165 72.2 488 95 17.6 np np np np np np

Bull Run Basin 62 540 1910 674 141 47.6 578 118 28.8 np np np np np np

Greenhorn Sum 63 540 1921 737 157 59.3 559 109 24.3 np np np np np np

Junipero Serra 64 120 1768 626 142 44.9 378 70 8.0 379 109 42.5 261 73 15.3

San Gabriels 65 993 2119 604 128 33.4 423 81 8.9 np np np np np np

San Bernardino 66 994 2049 517 108 24.2 353 63 5.0 np np np np np np

San Jacintos 67 997 1814 662 143 47.4 343 59 4.8 np np np np np np

San Jacintos 68 997 2287 578 121 29.9 340 58 3.9 367 110 38.8 230 61 9.3

Parque Nacional 69 2500 571 114 26.5 404 72 8.1 np np np np np np

a

MC=mapcode. Sdz=Seed Zone. h17,h13=total height at 17-,13- yrs; v17,v13=total volume at 17-,13-yrs; db17=diameter breast height at 17-yrs; dg=diameter at

ground at 13-yrs.

Standard errors of means: Sundown and Burnt Timber: h13=28.3 cm, dg13=8.0 mm, v13=9.5 dm3

; Cannon and Fitch-Rantz: h17=37.9 cm, db17=8.3 mm,

v17=34.9 dm3.
USDA Forest Service Proceedings RMRS-P-32. 2004 121

Adaptive Genetic Variation in Sugar Pine Kitzmiller

Figure 8—Association between 13-year height and origin elevation for: a. regions at Sundown, b. regions at Burnt Timber,

c. provenances at Sundown, and d. provenances at Burnt Timber.

survival and growth among four contrasting test environments. These were true rank-change interactions that require restrictions in seed transfer within the species range.

Only 24 percent of the sources were stable in performance

across four tests, and 43 percent were highly unstable.

Regions of seed origin were defined to represent broad

climatic zones that might prove useful for guiding seed

transfer. Region means were also interactive with test

environments. Although Regions ranked similarly at the

three low elevation tests, ranking changed at the FIT environment. Therefore, both regions and sources within regions

with high performance on one or more test sites often had

low performance on another.

Common garden test environments greatly affected performance of sugar pine. These four test environments represent a temperature and moisture gradient from the very

mild, maritime climate (SUN), to moderate-stress summer,

mild winter climate (BUR and CAN), to cool, short, dry

summers and long, cold winters with heavy snow-packs

(FIT).

Source-Test Response Patterns

SUN represents mild, humid, and productive northern

maritime environments for sugar pine having low diurnal

temperature fluctuation. Although high rust infection will

greatly reduce survival at SUN, previous and current results show a strong regional geographic genetic variation

pattern that is increasing with time. Best growth occurred

for sources originating below 1220 m and most commonly

from the local region. Growth at SUN was highest with

mesic, mild-site western Oregon Cascade (WOC) and north

coastal sources (NC). Height based on region means at SUN

(as well as at BUR and CAN) followed an inverse linear

elevation cline.

BUR represents warm, dry, very low elevation, southfacing environments in the interior Klamath Mountains

with moderately high moisture stress for sugar pine, even

though survival was high. Trees at BUR expressed weaker

geographic patterns than those at SUN. Like SUN, and

CAN, best growth occurred for sources originating below

a

b

c

d
122 USDA Forest Service Proceedings RMRS-P-32. 2004

Kitzmiller Adaptive Genetic Variation in Sugar Pine

1220 m. But in contrast to SUN, the more inland BUR site

favored sources from moderate moisture stress sites farther

inland in the western Oregon Cascades (WOC), Klamath

Mountains (KM), and some Northern Sierra (NSN) sources.

CAN represents the lowest elevations for sugar pine in the

Sierra Nevada, where growing seasons are long, winters are

wet and mild, and summers are hot and dry. Survival at

CAN was higher for southern California sources (below 38∞

N and above 1830 m elevation), but growth was better for

sources originating above 38∞ N between 850 m and 1160 m

elevation in the northern Sierra Nevada (up to 330 m higher

than CAN). Survival based on region means at CAN closely

follow a positive elevation cline. High survival of southern

California sources suggests they may have higher resistance

to charcoal root rot and/or higher drought resistance. Northern low elevation sources may have higher shoot growth

potential due to natural selection in mesic, stable temperature climates with long growing seasons, rich, deep soils, and

high competition for rapid height growth. Sites with long

growing seasons and mild winters favor sources from low to

middle elevation that are genetically flexible to extend their

growth periods.

FIT is unique in this study in representing typical upper

mixed-conifer forest sites with short, cool growing seasons

and long, cold winters with persistent snow-packs. Adaptive

genetic variation in survival and growth was most pronounced at FIT, and displayed opposite geographic patterns

from those at the other test locations. Short-growing seasons

and harsh cold winters with persistent snow packs favor

trees that cease growth and enter dormancy early enough to

escape freeze damage and those that heal stem injuries from

snow bend and tearing of primary branches. Winter damage

occurred annually, but reduced growth of low elevation

sources was detected only after 7 years at FIT (Jenkinson

1996). This environment presented strong selection pressure against sources from distant origins at low or high

elevations relative to the test site. Best survival, health, and

growth developed for certain sources originating from 1550

m to 2020 m in the Sierra Nevada, with a higher likelihood

of success for sources originating within 150 m elevation of

the site. At FIT, southern Sierra Nevada sources (and 3

northern, eastside sources: Black Hills, Stover Mt, and

Sierraville) tended to exhibit greater diameter and volume

than others.

Seed Transfer Considerations: Matching

Seed Source and Planting Site

Clearly, different seed sources should be used in mild,

coastal Oregon sites like SUN than in upper mixed-conifer

sites like FIT in the Sierra Nevada. Also, different seed

sources should be used at low elevation sites like CAN than

at middle to upper altitudes in the Sierra Nevada. Moisturestressed sites (like CAN and BUR) at very low elevations in

the Klamath and Sierra Nevada Mountains may require

more site-specific matching of seed source to local site conditions, because regional effects were not as strongly expressed

as they were for the two mesic sites. Such site-specific

matching would be safest within local region, but some

transfers across regions with known superior provenances

may be successful (for example, Diamond and Breedlove to

BUR, and Dutch Ck to CAN).

Extrapolation to predict safe seed transfers to other planting sites not represented in this study appears risky due to

the high G-x-E interactions. Each test site displayed some

selective advantage for the local climatic region and/or

certain local sources. Local sources appear “safest though

not necessarily best”. Even so, certain long distant transfers

within a broad altitudinal band were among the best performers. The growth-elevation response pattern showed

that the best source elevation was higher south of the site

and was lower north of the site across a wide geographic

distance. This suggests that elevation may be a “surrogate”

for mean annual temperature (MAT).

MAT changes across gradients in elevation, latitude, and

distance from the ocean. From long-term temperature and

precipitation data recorded at 41 climate stations, MAT

lapse rate was determined to be ca 1.62∞ C per 300 m rise in

elevation and ca 0.55∞ C per 1∞ rise in latitude (ca 110 km)

along the Sierra Nevada western slope (Ledig, data filed

1995). A general guide is to use provenances from ca 1 m

higher in elevation than the plantation site per 1 km transfer

northward. Applying this guide to the nearly 600 km span

along the west-side Sierra Nevada predicts that a northernmost site should have a similar MAT as a southernmost site

at 600 m higher elevation.

In general, seed transfer should be most restricted at

higher altitudes. When geographic distances for transfer are

shorter, the elevation range can be somewhat broader. As

the distance of transfer increases, the range in acceptable

elevation decreases. For high altitudes like FIT, in central

Sierra Nevada, seed from northern or southern Sierra Nevada origins could be used provided source mean annual

temperature (MAT), and perhaps even diurnal temperature

variation (DT) during elongation, are matched closely with

the temperature regime at the planting site. Transfer risk

might be substantially reduced if provenance and restoration site temperatures are harmonized, and if stable, provensuperior provenances are used.

Sugar pine at mid to upper elevations has a comparatively

short period of shoot elongation (51 days), only about 60

percent as long as and beginning later than ponderosa pine

(81 days) and incense-cedar (Calocedrus decurrensTorr.) (91

days), but is similar to white fir (46 days) (Fowells 1941).

Thus, sugar pine shoot growth is very rapid during a time

(late May to mid July) when the temperature range is rather

narrow and soil moisture is high. This suggests that sugar

pine may be closely adapted to a specific and fairly narrow

range of temperature for optimum growth at upper elevations. This delayed and brief growth period is still early

enough to avoid drought and to allow “hardening” prior to

onset of harsh weather. The temperature regime during

elongation may be important to adaptation of sugar pine.

Growth of ponderosa pine was inversely related to the

diurnal range of temperature (DT) during elongation at the

planting site (Church and others, data filed 2000). Sites with

wide diurnal temperature fluctuation may selectively favor

sources that grow slower (have lower energy efficiency) in a

given narrow temperature range, but grow faster (have

higher energy efficiency) over broader temperature ranges.
USDA Forest Service Proceedings RMRS-P-32. 2004 123

Adaptive Genetic Variation in Sugar Pine Kitzmiller

Transfer Northward to Enhance Rust

Resistance

Seed transfer from south to north is the most desirable

direction of movement to boost rust resistance in northern

populations, where natural resistance is extremely low.

Furthermore, many southern populations had higher survival potential in the low elevation plantation and high

growth potential at the upper elevation site, in addition to

having highest rust resistance. These results suggest significant potential advantages of seed transfer northward.

For northern (or central) Sierra Nevada sites, seed transfer northward about 220 km from central (or southern)

Sierra Nevada zones may be done with success by adjusting

elevation to match temperature. And, to avoid significant

growth losses relative to local sources, proven (tested) highgrowth provenances should be transferred. Before transfer,

rust resistant families should be tested for stable high

performance in contrasting environments. Similarly, transfer of northern Sierra Nevada provenances to California

Klamath Mountain sites might be done successfully. Other

transfers such as: Sierra Nevada or Klamath Mountains to

the Cascade Range, southern California to Sierra Nevada, or

elsewhere are not recommended.

References _____________________

Buck, J. M., Adams, R. S., Cone, J., Conkle, M. T. Libby, W. J., Eden,

C. J., and Knight, M. J. 1970. California Tree Seed Zones. Forest

Service, California Region and Forestry, Misc. Pub. 5 pp.

Campbell, R. K., and A. I. Sugano. 1987. Seed zones and breeding

zones for sugar pine in southwestern Oregon. Res. Pap. PNW-RP383. Portland, OR. USDA Forest Service, Pacific Northwest Res.

Sta. 18 p.

Church, J. N., Kitzmiller, J. H., Boom, B. A., Lunak, G. A., Greenwood, K. L., and R. S. Criddle. 2002. 15-year height of Pinus

ponderosa is correlated with mean diurnal temperature variation

during bud elongation. Data filed at UC Davis Dept Environ

Hort., Davis, CA.

Conkle, M.T. 1973. Growth data for 29 years from the California

elevational transect study of ponderosa pine. Forest Science

19:31-39.

Critchfield, W. B. and E. L. Little, Jr. 1966. Geographic distribution

of the pines of the world. USDA Forest Service Misc. Publ. 991.

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Cascades (Abstract). In: Kinloch, Bohun B., Jr., Mellisa Marosy,

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and Roles in Ecosystems. Proceedings of a Symposium presented

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Davis, California. Publication 3362. p. 38.

Fowells, H. A. 1941. The period of seasonal growth of ponderosa pine

and associated species. Journal of Forestry 39(7):601-608.

Harry, D. E., J. L. Jenkinson, and B. B. Kinloch. 1983. Early growth of

sugar pine from an elevational transect. Forest Science 29:660-669.

Jenkinson, J. L. 1996. Genotype-environment interaction in common garden tests of sugar pine. In: Kinloch, Bohun B., Jr., Mellisa

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Kinloch, B. B., and D. Davis. 1996. Mechanisms and inheritance of

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virulence in Cronartium ribicola: what is the threat? In: Kinloch,

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of Agriculture and Natural Resources, Davis, California. Publication 3362. pp. 133-136.

Kitzmiller, J. H., and P. Stover. 1996. Genetic variation in the

growth of sugar pine progenies in California plantations. In:

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(eds.). 1996. Sugar Pine: Status, Values, and Roles in Ecosystems.

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of Agriculture and Natural Resources, Davis, California. Publication 3362. pp. 83-98

Kitzmiller, J. H. 1976. Tree improvement master plan for the

California region. USDA Forest Service, Pacific Southwest Region. San Francisco. California. 96p.

Kitzmiller, J. H. 1990. Managing genetic diversity in a tree improvement program. For. Ecol. Manage. 35:131-249.

Larson, L.T., and T.D. Woodbury. 1916. Sugar Pine. USDA Forest

Service Bull. 426. Washington, DC.

Ledig, T. 1995. Data filed at Institute of Forest Genetics, Placerville, CA.

Oliver, W. W. 1996. Silvics of sugar pine: clues to distribution and

management. In: Kinloch, Bohun B., Jr., Mellisa Marosy, and

May E. Huddleson (eds.). 1996. Sugar Pine: Status, Values, and

Roles in Ecosystems. Proceedings of a Symposium presented by

the California Sugar Pine Management Committee. University of

California, Division of Agriculture and Natural Resources, Davis,

California. Publication 3362. pp. 28-33.

Samman, S. and J. H. Kitzmiller. 1996. The sugar pine program for

development of resistance to blister rust in the Pacific Southwest

Region. In: Kinloch, Bohun B., Jr., Mellisa Marosy, and May E.

Huddleson (eds.). 1996. Sugar Pine: Status, Values, and Roles in

Ecosystems. Proceedings of a Symposium presented by the California Sugar Pine Management Committee. University of California, Division of Agriculture and Natural Resources, Davis,

California. Publication 3362. pp. 162-170.

Sniezko, R. A. 1996. Developing resistance to white pine blister rust

in sugar pine in Oregon. In: Kinloch, Bohun B., Jr., Mellisa

Marosy, and May E. Huddleson (eds.). 1996. Sugar Pine: Status,

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Resources, Davis, California. Publication 3362. pp. 171-178.

Surles, S. E., T. L. White, and G. R. Hodge. 1995. Genetic parameter

estimates for seedling dry weight traits and their relationships

with parental breeding values in slash pine. Forest Science

41(3):546-563.

Westfall, R. D. 1991. Developing seed transfer zones. In: Fins, L. and

Friedman, S. T. (Eds) Handbook of Quantitative Forest Genetics.

pp. 313-395.

Willits, S, and T. D. Fahey. 1991. Sugar pine utilization: a 30-year

transition. Res. Pap. PNW-RP-438. Portland, OR. USDA Forest

Service, Pacific Northwest Res. Sta. 21 p.
124 USDA Forest Service Proceedings RMRS-P-32. 2004

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

The author is with the USDA Forest Service, Rocky Mountain Research

Station, 240 West Prospect Road, Fort Collins, CO 80526 USA. Phone:

970 498-1333. FAX: 970 498-1010. E-mail: aschoettle@fs.fed.us.

Abstract—Limber pine (Pinus flexilis James) and Rocky Mountain

bristlecone pine (Pinus aristata Engelm.) are two white pines that

grow in Colorado. Limber pine has a broad distribution throughout

western North America while bristlecone pine’s distribution is

almost entirely within the state of Colorado. White pine blister rust

(Cronartium ribicola J. C. Fisch.) was discovered in Colorado in

1998 and threatens populations of both species. Available information suggests that these species have several important ecological

roles, such as (1) occupying and stabilizing dry habitats not likely to

be occupied by other, less drought tolerant tree species, (2) defining

ecosystem boundaries (treelines), (3) being among the first to

colonize a site after fire, especially fires that cover large areas, (4)

facilitating the establishment of high elevation late successional

species such as Engelmann spruce and subalpine fir and (5) providing diet and habitat for animals. While the rust is not likely to

eliminate five-needle pines from Colorado ecosystems, it is likely to

impact species’ distributions, population dynamics and the functioning of the ecosystems. These changes may well affect (1) the

distribution of forested land on the landscape, (2) the reforestation

dynamics after fire, (3) the rate and possibly fate of forest succession, and (4) habitat for wildlife. Our incomplete understanding of

the ecology, genetic structure and adaptive variation of limber pine

and Rocky Mountain bristlecone pine constrain our ability to rapidly develop and implement conservation programs.

Key words: Limber pine, Pinus flexilis James, Rocky Mountain

bristlecone pine, Pinus aristata Engelm., Cronartium

ribicola J. C. Fisch., regeneration, fire

Limber pine (Pinus flexilis James) and Rocky Mountain

bristlecone (Pinus aristata Engelm.) are two white pine

species that grow in Colorado. Limber pine’s distribution

includes habitats throughout the Rocky Mountains while

the distribution of Rocky Mountain bristlecone pine is almost entirely within the state of Colorado. In southern

Colorado, it is speculated that a limber pine - southwestern

white pine (Pinus strobiformis) complex exists. The distribution of southwestern white pine extends south into New

Ecological Roles of Five-Needle Pines in

Colorado: Potential Consequences of Their

Loss

A.W. Schoettle

Mexico. This paper will focus on limber pine and Rocky

Mountain bristlecone pine. These species are white pines

(subgenus Strobus) yet limber pine is in section Strobus,

subsection Strobi and Rocky Mountain bristlecone pine is in

subgenus Parrya, subsection Balfournianae (Lanner 1990).

Their often bushy growth form (fig. 1) and slow growth rate

combined with the inaccessibility of the rocky sites that they

dominate make them poor timber species and ones that have

long been overlooked by the forestry community. The most

basic ecological information, such as the forest cover, has not

been quantified for these species in Colorado or throughout

their ranges.

The impact of white pine blister rust (Cronartium ribicola

J. C. Fisch.) on commercial North American white pines has

been a focus of attention since its introduction from Europe

in the early 1900s. In the mid-1980s, the focus expanded to

Figure 1—Limber pine on a dry site with a bushy growth form

with upward reaching branches.
USDA Forest Service Proceedings RMRS-P-32. 2004 125

Ecological Roles of Five-Needle Pines in Colorado: Potential Consequences of Their Loss Schoettle

impacts of the disease to the non-commercial whitebark pine

(Pinus albicaulis Engelm.) as forest practices shifted toward

management of ecosystems. White pine blister rust’s threat

to whitebark pine and the resultant impacts to the habitat

of the endangered grizzly bear (Ursus arctos horribilis) have

brought whitebark pine ecosystems into view by the management and research community (for example, Schmidt

and McDonald 1990, Tomback and others 2001). With the

recent discovery of white pine blister rust in Colorado on

limber pine in 1998 (Johnson and Jacobi 2000) and Rocky

Mountain bristlecone pine in 2003 (Blodgett and Sullivan

2004), both limber pine and Rocky Mountain bristlecone

pine populations are threatened. To predict the impacts of

white pine blister rust on Colorado ecosystems, we must first

understand the role of these five-needle pines in the absence

of the rust. It is not clear how similar the ecological roles of

limber pine and bristlecone pine are to the more studied

whitebark pine. Therefore, in the interest of brevity, this

paper will focus on research conducted on limber pine and

Rocky Mountain bristlecone pine, recognizing that some

information from other species may be applicable but will

not be summarized here. This paper will discuss what is

currently known about the ecology of limber pine and Rocky

Mountain bristlecone pine in the central Rocky Mountains

and the possible repercussions of white pine blister rust on

these ecosystems.

Limber Pine ____________________

Limber pine is a species whose distribution has changed

from continuous to patchy and presently displays

metapopulation dynamics (Webster and Johnson 2000,

Antolin and Schoettle 2001). Approximately 14,000 years

ago, at the last glacial maximum, limber pine was widespread along the eastern slope of the Colorado Front Range

in the central Rocky Mountains (Wells and Stewart 1987).

Currently limber pine is characterized by a patchy distribution, spanning a broad latitudinal and elevational range

(Burns and Honkala 1990) (fig. 2). In the central Rocky

Mountains limber pine grows from below the lower tree line

up to the upper tree line, from ~ 1600 m in the short grass

steppe to > 3300 m at Rollins Pass near the continental

divide (Schoettle and Rochelle 2000). Limber pine’s

elevational range is wider than any of its co-occurring tree

species in this region (table 1). In the northern Rocky

Mountains and west, limber pine is generally found at lower

elevations with whitebark pine occupying the higher elevations. In the southern mountains limber pine grows at highelevation sites with the lower elevations occupied by southwestern white pine (Pinus strobiformis Engelm.).

Limber pine is similar to the stone pines (subsection

Cembrae) in so much as it has large wingless (or near

wingless) seeds that depend on corvid species (for example,

Clark’s nutcracker, Nucifraga columbiana Wilson) for dispersal (Lanner and Vander Wall 1980). In contrast to the

stone pines, which have indehiscent cones necessitating

animals to extract the seed, limber pine cones open when

dry. As for whitebark pine, seeds of limber pine can be an

important food source for corvids (Tomback and Kramer

1980), black and grizzly bears (Ursus spp.; Kendell 1983,

McCutchen 1996), red squirrels (Tamaisciurus hudsonicus;

Hutchins and Lanner 1982) and other small rodents. The

role of limber pine forests as habitat for wildlife species is

unknown. The phloem, cones and seeds all provide habitat

and diet for arthropod fauna (Hedlin and others 1981,

Cerezke 1995, Schoettle and Négron 2001).

Limber Pine Stand Dynamics

Limber pine is often the first species to colonize an area

after fire (Donnegan and Rebertus 1999). Clark’s nutcrackers can cache seed many kilometers from the parent tree

(Vander Wall and Balda 1977), enhancing seed dispersal

across the landscape as well as into the central areas of large

burns where wind-dispersed seeds of other conifer species

are scarce (Tomback and others 1993). The germination of

multiple seeds from one cache results in a cluster of seedlings that are often related (Carsey and Tomback 1994). The

clustered distribution of seedlings facilitates successful establishment of limber pine (Donnegan and Rebertus 1999).

However, as the trees mature, the clustered distribution

may reduce the reproductive output (Feldman and others

1999) and lifespan of the individuals (Donnegan and Rebertus

1999) compared to trees growing singly.

The dynamic of stands containing limber pine depends on

the site; limber pine form sustainable stands on dry rocky

sites and tend to be limited to early succession on more mesic

sites. Dry sites can be occupied by limber pine at any

elevation within the species range and are often windswept

and accumulate little snow. Limber pine dominates xeric

sites not because they provide the optimal physical environment for limber pine growth (Lepper 1974, Schoettle and

Rochelle 2000) but because the conditions are not suitable

for the growth of other species and therefore competition is

minimal. Competition is likely to be the largest limitation

defining the realized niche of limber pine and the location of

sustainable limber pine stands. On dry sites, maximum tree

ages have been reported of more than 1500 years for limber

pine in Colorado (Schuster and others 1995) and over 2000

years for individuals in Nevada and California (Lanner

1984). The stands tend to be low density, open, and support

continual recruitment of limber pine (Knowles and Grant

1983, Stohlgren and Bachand 1997). Upon sexual maturity,

which may take over 50 years (personal observation), limber

pine on dry sites can produce large cone crops. Loss of apical

dominance due to leader damage provides many cone bearing branches per tree. The frequency of mast years, the

environmental factors that affect their periodicity, and the

repercussions of them on the population dynamics of animal

species deserve research attention. In addition to the extreme longevity of individuals, the lack of competing tree

species and sustained regeneration, the persistence of these

limber pine stands is also possible because catastrophic

disturbance (i.e. wildfire) is rare on dry, rocky sites.

While rocky ridges and dry slopes are the most obvious

habitat occupied by limber pine, scattered occurrence of

limber pine throughout the forested region of the Colorado

Front Range is typical (Marr 1961, Schoettle and Rochelle

2000). On these more mesic sites, limber pine’s early postdisturbance dominance succeeds over time to other conifer

species (Rebertus and others 1991). Limber pine acts as a

nurse tree, mitigating the harsh open environment after

disturbances and facilitating the establishment of Engelmann spruce and subalpine fir (Rebertus and others 1991,
126 USDA Forest Service Proceedings RMRS-P-32. 2004

Schoettle Ecological Roles of Five-Needle Pines in Colorado: Potential Consequences of Their Loss

Figure 2—Distribution of limber pine (Pinus flexilis James). (From Burns and Honkala 1990)
USDA Forest Service Proceedings RMRS-P-32. 2004 127

Ecological Roles of Five-Needle Pines in Colorado: Potential Consequences of Their Loss Schoettle

Donnegan and Rebertus 1999). Such facilitation accelerates

limber pine’s mortality due to the close proximity of, and

competition by, the succeeding species. Seedlings of limber

pine occur frequently throughout all stand types along the

elevational gradient, yet successful establishment in late

successional stands on mesic sites is rare (Stohlgren and

others 1998).

Seral limber pine is likely to maintain apical dominance

and retain an erect forest tree form and is suspected to

produce fewer cones per tree than those trees on drier sites

(Lepper 1974). Seed yields for limber pine can also be

reduced by some of the same cone and seed insects that affect

co-occurring conifer species (Hedlin and others 1981,

Schoettle and Négron 2001). Due to the lower seed yields of

successional stands, it is unclear what proportion of seed

from these sites is consumed on site by animals versus

dispersed and cached. Therefore, the relative contribution of

progeny from seral compared to persistent limber pine

stands to the recolonization of nearby disturbances has not

yet been established.

Limber Pine Population Genetics

Despite limber pine’s wide range and patchy distribution,

it shows little genetic differentiation related to elevational

changes (Latta and Mitton 1997, Schuster and others 1989,

Schuster and Mitton 1991, 2000). Other species with long

distance dispersal of seed by birds show similar apparent

lack of genetic structure (Bruederle and others 1998). This

is in contrast to species that depend on the wind for dispersal

of seed; these species show not only local genetic differentiation, but also differentiation within local populations (see

Rehfeldt 1997). Genetic studies of limber pine indicate that

within local populations, pollen is dispersed evenly among

trees (Schuster and Mitton 2000) but that seed dispersal

patterns result in local clusters of related individuals

(Schuster and Mitton 1991). Differences in pollen phenology

along elevation gradients could limit gene flow via pollen

between local populations (Schuster and others 1989), but

low between-population differentiation suggests gene flow

by stepping-stone pollination across intermediate populations. Long-distance seed dispersal by birds (Lanner and

Vander Wall 1980) also contributes to gene flow across the

elevation gradient. Currently, the only large genetic differences in limber pine that have been identified are on a

regional geographic scale that may reflect isolation in Pleistocene refugia on the Great Plains east of the Rocky Mountains and in the Great Basin west of the Rocky Mountains

(Latta and Mitton 1997; Mitton and others 2000).

Limber pine appears to be a genetic generalist based on

presumably selectively neutral genetic markers, yet extensive common garden and genetic by environment interaction

experiments have not been conducted to evaluate local

adaptation. One common garden study of several seed sources

for limber pine suggests some geographic variation in seedling growth characteristics (Heit 1973). Seed transfer rules

for limber pine have not been established.

Limber Pine Adaptive Variation

Despite living in metapopulations along a broad elevational

gradient, limber pine shows remarkably low morphological

variation (Schoettle and Rochelle 2000). The genetic basis

for the morphological variation or lack thereof has not yet

been assessed. Schoettle and Rochelle (2000) hypothesized

that if limber pine lacked elevational races, the environmental effect of elevation on growth and resultant phenotype

would be greater for limber pine than for species that have

undergone adaptations to local environments. Contrary to

this hypothesis, the environmental stress of increasing elevation that is apparent in the growth patterns of other tree

species was less obvious for limber pine (fig. 3). Leaf longevity, ranging from 4 to 10 years, was one of the few characteristics to vary along an elevational gradient (Schoettle and

Rochelle 2000). Limber pine appears less stressed than

other species by the environmental gradients associated

with elevation (Schoettle and Rochelle 2000).

How can limber pine uncouple its growth from the environmental differences from the upper to the lower tree line?

The rates of most physiological and biochemical processes

are a function of temperature. Limber pine seedlings from

four of five populations from Wyoming, Nevada and California revealed a typical photosynthetic temperature optimum

(15 ∞C) but an unusually broad response curve with a

variation in photosynthetic rate of only 12 percent from the

maximum over the temperature range of 10-35 ∞C (Lepper

1980). This is in contrast to the sharper temperature response of photosynthesis of balsam fir (Abies balsamea (L.)

Mill., Fryer and Ledig 1972) and Great Basin bristlecone

pine (then called Pinus aristata Engelm. but now recognized

Table 1—Elevation ranges of tree species in Colorado. Data from Peet (1981) and Baker (1992).

Scientific name Common name Elevation range (m)

Pinus flexilis James Limber pine 1600-3400

Juniperus scopulorum Sarg. Rocky Mountain juniper 1600-2800

Pinus ponderosa Dougl. ex Laws. Ponderosa pine 1700-2800

Pseudotsuga menziesii (Mirb.) Franco Douglas-fir 1700-3000

Populus tremuloides Michx. Quaking aspen 2000-3400

Pinus contorta Dougl. ssp. latifolia Bailey Lodgepole pine 2300-3300

Picea engelmannii Perry ex Engelm. Engelmann spruce 2400-3500

Pinus aristata Engelm. Rocky Mountain bristlecone pine 2750-3670

Abies lasiocarpa (Hook.) Nutt. Subalpine fir 2500-3500
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Schoettle Ecological Roles of Five-Needle Pines in Colorado: Potential Consequences of Their Loss

as Pinus longaeva Bailey) according to Bailey (1970) Mooney

and others (1964) where photosynthesis fell 63 percent and

87 percent, respectively, below the maximum rates within

the range of 5∞C below and 20∞C above the optimum temperature for photosynthesis (fig. 4). Strong variation in

photosynthetic capacity between mature trees at the

elevational extremes (Schoettle, unpublished data) also suggests considerable adaptive physiological variation for limber pine. Limber pine also has a high degree of variation in

other physiological traits, both among individuals as well as

within individuals (Barrick and Schoettle 1996, Schoettle

and Rochelle 1996). Therefore physiological plasticity or

broad physiological tolerances appear to contribute to limber

pine’s wide fundamental niche with respect to temperature.

Limber pine seedlings, similar to the stone pines, have

large root to shoot ratios. How or if this allocation pattern

varies among habitats hasn’t been studied. This pattern of

carbon allocation is often associated with shade intolerance

as well as drought tolerance and avoidance. Both limber pine

seedlings and mature trees demonstrate drought tolerant

behavior, compared to co-occurring species, by maintaining

leaf gas exchange even under severe soil drying (Lepper

1980; Pataki and others 2000). The hypothesis that, on xeric

sites, the long roots of limber pine are able to access ground

water sources not within reach of other conifer species has

not been tested. Mature limber pine also demonstrates

drought avoidance behavior by closing its stomata more

readily than associated species during periods of atmospheric dryness (high vapor pressure deficit) (McNaughton

1984, Pataki and others 2000). Stomatal closure may prevent xylem cavitation but also sacrifices photosynthetic

carbon gain; this pattern of water conservation at the expense of carbon assimilation may contribute to limber pine’s

poor competitive abilities.

Limber pine may be a case where turnover of local populations, combined with high dispersal and gene flow, results

in evolution of a generalist lifestyle capable of tolerating a

wide variety of environmental circumstances (Schoettle and

Rochelle 2000; Antolin and Schoettle 2001). It is unclear at

this time if being a poor competitor is the “cost” associated

with the generalist lifestyle for limber pine.

Rocky Mountain Bristlecone

Pine___________________________

In 1970, Bailey (1970) split the North American bristlecone pine (Pinus aristata Engelm.) into two species, the

Rocky Mountain bristlecone pine (retaining the name Pinus

aristata Engelm.) and Great Basin bristlecone pine (newly

named Pinus longaeva Bailey). Most of the research on

bristlecone pines before 1970 was conducted on Great Basin

bristlecone pine; very little research has been conducted on

Rocky Mountain bristlecone pine. Both species are recognized as charismatic and are appreciated by the public for

their majestic and artistic tree form and their extreme

longevity (fig. 5). Great Basin bristlecone pine can reach

ages in excess of 4,000 years (Schulman 1958, Curry 1965),

while the oldest Rocky Mountain bristlecone pine is just over

2,400 years of age (Brunstein and Yamaguchi 1992). Both

species of bristlecone pine have been utilized in dendrochronology studies (such as Kreb 1973, LaMarche and Stockton 1974).

The distribution of Rocky Mountain bristlecone pine is

primarily in Colorado and extends south into New Mexico

along the Sangre de Cristo Mountains and includes a disjunct population on the San Francisco Peaks in central

Arizona (fig. 6). It is thought that during the Pleistocene

glacial periods there was nearly continuous habitat for

bristlecone pine between the New Mexico and Arizona stands,

suggesting that the Arizona stand is a relic of a formerly

larger distribution (Bailey 1970). The current southern

distribution of bristlecone pine appears limited by suitable

habitat, however it is not known what limits bristlecone pine

Figure 3—Effect of elevation on the annual twig growth of

mature conifers. Data for Engelmann spruce and subalpine fir are from Hansen-Bristow (1986), limber pine are

from Schoettle and Rochelle (2000), and lodgepole and

whitebark pine are from Schoettle (unpublished data).

(Adapted from table 7 of Schoettle and Rochelle, 2000)

Figure 4—Relative temperature response of net photosynthesis of seedlings of three conifer species. The optimum

temperature for photosynthesis for each species is that

temperature that the maximum rate of photosynthesis was

recorded. To enable comparison among species, photosynthesis is expressed as a percentage reduction from the

maximum rate.
USDA Forest Service Proceedings RMRS-P-32. 2004 129

Ecological Roles of Five-Needle Pines in Colorado: Potential Consequences of Their Loss Schoettle

from occupying apparently suitable habitat to its north. The

distribution of this species may reflect a dependence on

summer monsoons, restricting it from occupying higher

elevation sites in northern Colorado. Rocky Mountain

bristlecone pine (referred to as bristlecone pine hereafter)

has a narrow elevation range and is primarily a high elevation species occupying dry sites from 2750 to 3670 m elevation (Baker 1992). Bristlecone pine forests may contain

limber pine, Engelmann spruce, subalpine fir, quaking aspen, and Douglas fir.

Bristlecone Pine Stand Dynamics

The origin of bristlecone pine stands throughout Colorado

is related to episodes of drought and presumably peak fire

occurrence (Baker 1992). Bristlecone pine is a long-lived

species that regenerates well after fires. Bristlecone pine

has been identified as a component of two climax vegetation

types (DeVelice and others 1986). The first is dominated by

bristlecone pine with or without Engelmann spruce with an

understory of Festuca. These sites are open and park-like.

This habitat type transitions into one where bristlecone pine

succeeds to the more shade tolerant spruce’s competitive

edge on moister sites (Moir and Ludwig 1979). On lower

elevation sites, bristlecone pine dominates or co-dominates

stands with Douglas fir. Using a different approach based on

environmental variables and species distributions, Baker

(1992) characterized sixty-five bristlecone pine stands into 6

forest structures that are distinguished by (1) the time since

the last disturbance (age of the oldest tree in the stand),

(2) presence of young quaking aspen, and (3) relative

amounts and sizes of Engelmann spruce and subalpine fir

(Baker 1992). Baker (1992) reports that bristlecone pine

regenerates well only on recently burned sites and therefore

attributes the persistence of old stands of bristlecone not to

climax stand dynamics but to the long lifespan of the individual pioneer trees in the absence of competition and fire.

However, Baker’s data reveal some bristlecone pine regeneration in most of the sampled bristlecone pine stands. This

raises the question of how much regeneration is necessary to

sustain bristlecone pine on sites with little to no competition.

Regardless of whether one subscribes to climax vegetation

theory or not for very long-lived species, it is clear that the

rate of succession from bristlecone pine to other species

varies with site and the transition may proceed very slowly

(>1000 yrs) on dry high elevation sites and may be preempted by disturbance. Ranne and others (1997) followed up

on Baker’s work and characterized the vegetation characteristics of the six bristlecone pine forest groups. Vegetation in

bristlecone forests is influenced primarily by elevation and

soil pH and secondarily by substrate, soil texture, topographic

position, and geographic location (Ranne and others 1997).

The relative role of wind versus animal-dispersal of seeds

for bristlecone pine regeneration within existing stands and

colonization of burned areas is not known. Bird-dispersal of

seeds appears common at higher elevations while winddispersal may predominate at lower elevations for Great

Basin bristlecone pine (Lanner 1988). Clustered individuals, indicative of animal-mediated seed dispersal, are apparent in mature high elevation Rocky Mountain bristlecone

pine stands in central Colorado (Torick and others 1996), as

well as in the young seedlings establishing in those stands

(personal observation, 2001). The frequency of clustered

individuals on sites that have been recently burned, those at

lower elevation stands or those in southern Colorado has

not been assessed. Therefore it is not clear if long-distance

animal-mediated seed dispersal of bristlecone pine plays a

major role in recolonization of disturbed areas.

Although bristlecone pine is a pioneer species after fire, its

role in mediating the environment to facilitate the establishment of late successional species has not been fully explored.

At the forest - alpine ecotone, bristlecone pine growing in the

krummholz form facilitate the establishment of Engelmann

spruce and subalpine fir (personal observation). In the

subalpine zone, bristlecone pine forests tend to have relatively clear boundaries with bristlecone pine densities

abruptly falling as elevation decreases and moisture regimes change.

Although bristlecone pine has delayed sexual maturity,

its extreme longevity enables each tree to be a seed source for

many years. During a good cone year, cone production per

tree appears to increase with increasing elevation within a

stand, including good production by krummholz trees at tree

line (Schoettle, unpublished data, 2001). The gradient in

cone production may be a function of differences in the

number of cones initiated or rates of cone damage or abortion. Cone insects were common on low elevation trees and

absent from trees growing at the higher elevations (personal

observation, 2001), similar to the findings for limber pine

(Schoettle and Négron 2001). As with limber pine, squirrels

are very efficient at harvesting bristlecone pine cones and

create large cone caches within the forests. Again, similar to

Figure 5—Rocky Mountain bristlecone pine near treeline

in central Colorado. Note partial cambial dieback (see

fig. 7).
130 USDA Forest Service Proceedings RMRS-P-32. 2004

Schoettle Ecological Roles of Five-Needle Pines in Colorado: Potential Consequences of Their Loss

Figure 6—Perimeter of the distribution of Rocky Mountain bristlecone pine (Pinus aristata Engelm.) based on data

from Bailey (1970), Brunstein and Yamaguchi (1992) and Ranne and others (1997).
USDA Forest Service Proceedings RMRS-P-32. 2004 131

Ecological Roles of Five-Needle Pines in Colorado: Potential Consequences of Their Loss Schoettle

limber pine, the frequency of mast years, the environmental

factors that affect their periodicity, and the repercussions of

them on the population dynamics of animal species deserve

research attention.

Bristlecone Pine Population Genetics

Very little is known about the population genetics of

bristlecone pine. Recent research has shown that stands as

close as 11 km from one another near the northern extreme

of the species distribution differed from one another in allele

frequencies and the distribution and presence of certain

alleles, suggesting a strong founders effect (Oline 2001).

This pattern may suggest that long-distance transport of

seed by birds for this otherwise wind-dispersed species may

play a significant role in the establishment of bristlecone

pine stands. As mentioned above, the caching behavior of

birds also results in fine scale genetic structure for bristlecone pine, similar to that of the other bird-dispersed pines

(Torick and others 1996). As with limber pine, common

garden and genetic by environment interaction experiments

have not been conducted for bristlecone pine.

Bristlecone Pine Adaptive Variation

Phenotypic variation associated with elevation has been

observed for bristlecone pine (Ewers and Schmid 1981) yet

the genetic basis for the differences has not been studied.

Bristlecone pine has several traits that may contribute to its

longevity. This species has considerable plasticity with respect to leaf longevity, ranging from 7 to over 15 years, and

has the unusual ability to maintain high physiological function of leaves as they age. Both of these traits may contribute to the absence of growth declines in aging bristlecone

pine trees that are commonly observed in other species

(Schoettle 1994). Bristlecone pine and limber pine both

express partial cambial dieback, resulting in a strip of dead

bark extending from dead roots to dead branches (fig. 7)

(Schauer and others 2001). It is speculated that partial

cambial dieback contributes to the exceptional longevity of

individuals by effectively isolating damaged roots, stem or

branches from remaining healthy tissues and thereby maintaining a favorable photosynthetic to non-photosynthetic

tissue ratio (Schulman 1954, LaMarche 1969).

Similar to limber pine, bristlecone pine seedlings allocate

a large amount of resources below ground. How this allocation pattern affects the performance of seedlings regarding

stress tolerance or competitive abilities has not been studied, yet this pattern is usually reflective of poor shade

tolerance (Tilman 1988).

Threat of White Pine Blister

Rust __________________________

The most immediate threat to limber pine and bristlecone

pine is the exotic disease white pine blister rust caused by

the fungus Cronartium ribicola J.C. Fisch. This pathogen

was introduced into North America in the early 1900s and

has caused significant impacts to white pines throughout

North America. For a summary of the biology of the rust and

Figure 7—Schematic of partial cambial dieback. Note that

the dead cambial strip connects a dead root with a dead

branch.

the impacts of this disease to white pines, see McDonald and

Hoff (2001). The rust has been affecting limber pine since

1945 in the Northern Rocky Mountains and down into

southern Wyoming since the 1970s (Brown 1978) and was

identified in Colorado in 1998 (Johnson and Jacobi 2000).

White pine blister rust was first reported on Rocky Mountain bristlecone pine in 2003 in the Sangre De Cristo Mountains of Colorado (Blodgett and Sullivan 2004).

The white pine blister rust spores enter trees through the

stomatal openings of young leaves (McDonald and Hoff

2001). The effectiveness of older leaves as infection sites

needs to be assessed for Colorado white pines since more

than 90 percent of their foliage is greater than 1 year old

(Schoettle 1994). The rust causes cankers that girdle the

infected branch or stem killing the distal tissue. Cankers on

the main stem of a tree will usually kill the individual.

Branch cankers often will not kill the tree until the reduction

in leaf area is so great that the tree cannot survive or the

canker grows to affect the main stem. The contribution of

rust-caused branch mortality to an increase in sensitivity of

the tree to other stresses such as drought, competition, and

bark beetle attacks deserves research attention to fully

assess the impacts of the disease. Very old trees that have

significant partial cambial dieback, such that all of the tree’s

surviving foliage is supported on a few branches, may be

rapidly killed by white pine blister rust. Alternatively, it is

possible that those trees that support foliage on many
132 USDA Forest Service Proceedings RMRS-P-32. 2004

Schoettle Ecological Roles of Five-Needle Pines in Colorado: Potential Consequences of Their Loss

upwards-reaching branches may prolong the time between

canker formation and tree mortality.

Effects of white pine blister rust on recolonization of

disturbed areas may well precede the mortality of existing,

mature white pine trees (fig. 8). While a tree may survive

with white pine blister rust cankers it is likely to experience

substantial branch mortality and reduced cone and seed

production. If seed yields are low, it is unclear if Clark’s

nutcrackers will visit and cache seeds from these stands. In

addition, even if seed is available for colonization and regeneration, white pine blister rust exerts strong selective pressure at the seedling – sapling stage and can cause high rates

of seedling mortality within several years of infection.

White pine blister rust has its own set of environmental

constraints as influenced by the tolerances of its biology as

well as the distribution of its two hosts, the five-needle pines

and Ribes spp. The degree of overlap between the rust’s

potential habitat with that of limber pine and bristlecone

pine’s distributions has not been fully defined. While the

selective pressure exerted by the rust on these five-needle

pines will not be uniform across their distribution, existing

information on Ribes distributions suggests that it may be

extensive; three-fourths of the limber pine sites sampled

along the elevation gradient of Colorado’s Front Range

contained Ribes spp. (8 of 12 stands; Schoettle and Rochelle

2000) and more than half of the bristlecone pine sites

evaluated by Ranne and others (1997) contained Ribes spp.

(27 of 50 stands). Many of these stands support Ribes cereum

Douglas, a species that has been thought to be a poor

alternate host for white pine blister rust in other parts of

North America (Van Arsdel and others 1998), yet it may

serve as a host for the disease in Colorado, southern Wyoming and South Dakota (Lundquist and others 1992, Johnson

and Jacobi 2000). Ribes spp. may also be present and be

potential sources of blister rust spores near white pine

stands that do not support it directly. Long-distance dispersal of white pine blister rust spores needs research

attention before it will be possible to assess the risk to white

pine patches based on the spatial relationships among hosts

and the rust.

The white pine populations in other parts of North America

that have been severely affected by white pine blister rust

have all shown some level of genetic resistance to the disease

(e.g. Hoff and others 1980, Kinloch and Dupper 2002, Sniezko

and others this proceedings). A bulk seed lot from one

Colorado Front Range limber pine population showed evidence of the presence of a hypersensitive reaction to the rust

at moderate frequencies, although the bulk seed lot precluded an estimation of the incidence or inheritance of the

resistance mechanism within the population (Kinloch and

Dupper 2002). No data is currently available on the presence

of other resistance mechanisms in limber pine. The loss or

near loss of limber pine on xeric sites will likely transition

the sites to treeless vegetation communities with currently

unknown implications on slope stability, hydrology and

wildlife. The impact of the loss of nurse trees on the establishment success of late successional species on mesic sites

has yet to be understood. Exclusion of limber pine from some

Figure 8—Schematic of potential effects of white pine blister rust on limber pine and bristlecone

pine populations. The rust may cause extinction of some stands and isolation of others while also

affecting reforestation of disturbed sites. See text for further discussion.
USDA Forest Service Proceedings RMRS-P-32. 2004 133

Ecological Roles of Five-Needle Pines in Colorado: Potential Consequences of Their Loss Schoettle

habitats by the selective pressure of the blister rust may

isolate surviving patches with implications on gene flow

among patches and recolonization success of forest disturbances (fig. 8). As a result, the extinction rate of limber pine

patches, although already different for persistent and seral

patches, is likely to be disrupted by this exotic disease with

implications on the genetic structure of the limber pine

metapopulation. The impacts of white pine blister rust on

high elevation stands of bristlecone pine will likely lower

treeline in those locations and transition the cover to a

subalpine understory/alpine species mixture. The presence,

nature, and geographic distribution of resistance mechanisms in bristlecone pine have not been studied.

In addition to the obvious population effects of rust-caused

tree mortality, the rust may also affect the environmental

tolerances of the future rust-resistant population. It is well

known in plant ecology that the allocation of resources to

defense, be it from herbivory or pests and pathogens, diverts

resources from other plant functions. It is not known if the

physiological cost on the part of the white pines associated

with expressing resistance to white pine blister rust may

alter a tree’s sensitivity to environmental stresses, potentially causing the rust-resistant trees to have a different

fundamental niche from that of the original population.

After being challenged by white pine blister rust, the resultant populations of limber pine and bristlecone pine may

have a different suite of environmental tolerance and competitive abilities than we see today.

Interaction of Five-Needle Pines, White

Pine Blister Rust, and a Changing Fire

Regime

The effects of white pine blister rust on five-needle pines

will interact with the changing fire regimes in the Rocky

Mountains. As fire regimes get more frequent and unpredictable due to past fire suppression and forest practices,

large wildfires may jeopardize the usually less-flammable

five-needle pine ecosystems on dry sites. In addition, branch

and tree mortality caused by white pine blister rust may

contribute to fuel loading in white pine stands, increasing

the susceptibility of these stands to sustain and be consumed

by fire. In the event of larger fires, especially those covering

a larger area than can be seeded effectively by wind dispersal mechanisms, the loss of bird-dispersed pines as colonizers may be especially pronounced. Alternatively, if fires

do not burn five-needle pine dominated stands and white

pine blister rust does not affect Clark’s nutcracker dispersal

and caching behavior, burned areas offer recolonization

opportunities for the establishment and natural selection of

rust resistant pine genotypes (fig. 8).

Fire regimes may also change as a result of climatic

changes in temperature and precipitation patterns. Again

depending on the availability of seed and the scale and

location of the fires, this may isolate stands or provide

colonization opportunities. However, because persistence of

limber pine stands is so sensitive to the competitive ability

of co-occurring species, the indirect effects of climatic change

on the performance of other species may alter the distribution of persistent versus seral limber pine.

Conservation Strategies __________

In the case of Colorado white pines, there are at least two

possible conservation goals: (1) conservation of the genetic

diversity within each species and (2) attempt to maintain the

species’ existing distribution by accelerating the establishment of white pine blister rust resistant genotypes across

the landscape. It is unclear if selection for rust resistance

will result in the loss of some physiological traits from these

species; as a result conservation of genetic diversity of each

species may be critical for future breeding stock to attempt

to restore the traits that confer stress tolerance in these

species in the future. Both bristlecone pine and limber pine

have the extraordinary capability of surviving in very harsh

environments and it is not known if the selective pressure of

the blister rust may cause the loss of any of these traits from

the surviving populations. Because white pine blister rust

has only just entered Colorado and has contributed little to

mortality at this time, the opportunity to conserve the full

genetic diversity of Colorado limber pine and bristlecone

pine populations exists. However, the feasibility of this task

is another matter. Until the genetic structures of the natural

populations and seed transfer rules have been defined, the

only option is to immediately collect and archive seed and

pollen from throughout each species geographic range. Concurrent with this approach, seed storage protocols for the

species will need to be developed.

Management to accelerate the establishment of white

pine blister rust resistant genotypes across the landscape

may require silvicultural treatment and identifying resistant individuals and collecting and planting the seed or

seedlings from those individuals in disturbed areas (Schoettle,

in press). Identifying resistant individuals can be done, as

has been done for other white pines, by field assessment in

areas already challenged by white pine blister rust or by

screening seedlings in nursery trials with artificial inoculations (Sniezko and Kegley, this proceedings).

Summary ______________________

In summary, both limber pine and bristlecone pine are

long-lived species that regenerate well after fires. They can

persist on xeric sites and may facilitate establishment of late

successional species on more mesic sites. Disturbances

throughout the elevational gradient of forested lands open

habitat for limber pine and are recolonized by the effective

bird dispersal of limber pine seed. Disturbances in the

higher elevations open possible habitat for bristlecone pine.

The genetic structure has not been defined for either species,

yet limber pine may be more of a genetic generalist than

bristlecone pine, and displays metapopulation dynamics.

Both species are poor competitors and dominate sites that

are not suitable for other species. It is unclear at this time if

being a poor competitor is the “cost” associated with the

stress tolerant behavior of both species and the generalist

lifestyle for limber pine.

The currently available information on limber pine and

bristlecone pine suggests that these species have several

important ecological roles in Colorado ecosystems. (1) These

white pines are exceptionally stress tolerant and occupy and
134 USDA Forest Service Proceedings RMRS-P-32. 2004

Schoettle Ecological Roles of Five-Needle Pines in Colorado: Potential Consequences of Their Loss

stabilize habitats not likely to be occupied by other, less

tolerant tree species. (2) Often these species define ecosystem boundaries (treelines). (3) These species are among the

first to colonize a site after fire, especially fires that cover

large areas. (4) Limber pine, and possibly bristlecone pine,

facilitate the establishment of high elevation late successional species such as Engelmann spruce. (5) The seeds of

both five-needle pines provide a dietary component for

several animal species, and the stands likely also provide

habitat for other species.

The recent discovery of white pine blister rust in Colorado

threatens limber pine and bristlecone pine populations.

While the rust is not likely to eliminate the five-needle pines

from Colorado ecosystems, it is likely to impact species’

distributions, population dynamics and the functioning of

the ecosystems. The rust may cause local population extinctions as well as greatly reduce genetic diversity and alter

environmental tolerances of the species. The reduction in

effective population numbers may hinder the evolutionary

development or render local populations even more subject

to risk. Changing fire regimes resulting from management

or climatic changes will contribute to determining the future

importance of the ecological role of white pines. In addition,

change in the competitive interactions among Rocky Mountain conifers as a result of climatic changes may affect the

future of these landscapes. The interaction of these factors

with the stress of this exotic pathogen may well affect (1) the

distribution of forested land on the landscape, (2) the reforestation dynamics after fire, (3) the rate and possibly fate of

forest succession, and (4) habitat for wildlife. Our incomplete understanding of the ecology, genetic structure and

adaptive variation of limber pine and bristlecone pine constrain our ability to rapidly develop and implement conservation programs.

References _____________________

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136 USDA Forest Service Proceedings RMRS-P-32. 2004

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Raphael Thomas Klumpp and Marcus Stefsky are with the Institute of

Silviculture, University of Natural Resources and Applied Life Sciences,

Peter Jordan Str. 70 A-1190, Vienna, Austria. Contact Klumpp: Phone 0043

1 47654 4063. Fax 0043 147654 4092. Email: raphael.klumpp@boku.ac.at

Abstract—The genetic variation of Pinus cembra was analyzed by

means of isozyme gene markers along an elevational transect at

Koetschach Valley (Salzburg State/Austria). Mature stands and

their natural recruitment were studied at three elevation levels:

subalpine zone, high montane zone, and middle montane zone.

Sample included juvenile and mature individuals. Thirteen enzyme

systems encoding for 22 gene loci were scored. The results showed

increasing allelic multiplicity with increasing altitude in mature

stands and decreasing polymorphism with increasing altitude in

juvenile populations. Surprisingly high values of allelic multiplicity

and hypothetic gametic multilocus diversity were found in middle

elevation populations, which have potential for generating genetically diverse gametes in future generations. Seed dispersal by a

nutcracker bird species, as well as gene flow by local wind systems,

may be the reason for this phenomenon, which is obviously strengthened by strong selection forces.

Key words: Pinus cembra, isozyme, elevation, population, genetic

diversity, seed dispersal, Nucifraga caryocatactes.

Introduction ____________________

Pinus cembra L., a locally important species in Europe,

has had multiple uses over centuries. The species has been

used for timber, when indoor use for country style furniture

and wall ornaments were in fashion, and the large seeds

(nuts) were used to improve the diets of farmers living in

alpine pastures. Presently, the blue cones are harvested for

preparing liquor, which leads to crown damage and problems for species that depend on the seeds for food like

nutcracker birds (Nucifraga caryocatactes L.).

It is well known that P. cembra was eliminated in lower

elevations by competition. It is less commonly known that

virgin forests at the timberline in alpine mountains were

harvested in order to extend alpine pastures, even up to the

20th century (Fromme 1957). Furthermore, large amounts

of timber from these forests were needed and utilized.

Genetic Variation of Pinus cembra Along an

Elevational Transect in Austria

Raphael Thomas Klumpp

Marcus Stefsky

Hence, distortion of genetic architecture is anticipated in all

natural populations. In spite of these facts, we hypothesize

that there is still a relatively high level of genetic variation

in high elevations, as those forests have not been regularly

managed. Furthermore, the competitiveness of P. cembra,

as well as the mutualism with nutcracker birds at these

elevations, may lead to more effective preservation of the

gene pool. In contrast, populations at lower elevation may

have suffered a reduction of genetic diversity due to regular

management activities and limited activity of nutcrackers

in such dense forests. Isoenzyme studies in P. cembra and

related species have been conducted by different authors.

Heterozygosity is low in comparison to other pine species

(Szmidt 1982), and species can be easily differentiated

(Goncharenko and others 1992, Politov and Krutovskii 1994).

Different numbers of gene loci have been found to be encoding the same enzyme systems of different pine species

(Bergmann and Hattemer 1995), but P. cembra and its

relatives from subgenus Strobus Lemm. section Strobus

exhibit the same number of gene loci controlling the enzyme

system of 6PGDH (Bergmann and Gillet 1997).

Genetic architecture in conifer populations can be affected

by the age of the population. Investigations on the natural

recruitment in P. sylvestris L. revealed that there is an

excess of homozygotes in the embryo stage, which decrease

as age advances according to the species’ life cycle (Muona

and others 1987, Yazdani and others 1985). Similar results

have been found in other coniferous species, such as P.

radiata D. Don. (Plessas and Strauss 1986), Pseudotsuga

menziesii [Mirb.] Franco (Shaw and Allard 1982), and Abies

alba Mill. (Hussendoerfer 1998).

Variation along elevational transects has been observed

by means of isozyme gene markers in several species. Mitton

and others (1980) found clinal variation at two gene loci in

ponderosa pine (P. ponderosa Laws.). Ruetz and Bergmann

(1989) found variation of allelic structures along one

elevational transect in Norway spruce (Picea abies (L.)

Karst.). However, other studies, for example Neale and

Adams (1985) in balsam fir (Abies balsamea (L.) Mill.), could

not confirm this finding. Similarly, some publications on

Norway spruce (Konnert 1991) and European beech (Fagus

sylvatica L.) (Loechelt and Franke 1995) did not show clinal

variation along elevational gradients. These studies were

conducted on populations in southwestern Germany, where

silvicultural activities by humans over centuries may have

lead to distortion of natural variation.

The reproductive potential of this species is currently in

danger, as the local forest authority is unable to control the

harvesting of cones in Austria. As this tree species is less

competitive than other species, we initiated an investigation

on genetic diversity along 11 elevational transects in the
USDA Forest Service Proceedings RMRS-P-32. 2004 137

Genetic Variation of Pinus cembra Along an Elevational Transect in Austria Klumpp and Stefsky

Table 1—Characteristics of the sampling area.

Austrian Alps (Alpine Mountains). This study will provide

information to guide gene conservation/restoration efforts

and can be used as well to indicate changes in genetic

diversity due to global warming over time. In this paper,

initial results will be reported using isozyme gene markers

to study diversity along a single Austrian transect.

Materials and Methods ___________

The Koetschach Valley in the State of Salzburg, Austria,

is found in the eastern part of the central Alps, which

exhibits a subcontinental, cold winter climate. Sampling

areas were designated on a single slope with northern

exposition at three different elevation levels (table 1). The

transect number is AT1, and the sampling area was designated as “U” for the subalpine zone, “M” for the high

montane zone, and “L” for the middle-montane zone. Samples

were taken from adult and juvenile trees (51 to 149 individuals per population) and recorded by age class.

Isoenzyme analyses were conducted following the methods described by Cheliak and Pitel (1984) and Hertl (1997).

The analyses used 13 enzyme systems, which encode for 22

gene loci (table 2). Data were scored using the GSED proTable 2—Analysed enzyme systems and gene loci.

Nomenclature Enzyme system Analysed loci

Alaninaminopeptidase (E.C. 3.4.11.2) AAP AAP-A

AAP-B

Aspartataminatransferase (E. C. 2.6.1.1) AAT AAT-A

AAT-B

AAT-C

Aconitase (E. C. 4.2.1.3) ACO ACO-A

Diaphorase (E.C. 1.6.4.3) DIA DIA-A

DIA-B

Glutamatdehydrogenase (E. C. 1.4.1.2) GDH GDH-A

Isocitrat-Dehydrogenase (E. C. 1.1.1.42) IDH IDH-B

Leucin-Aminopeptidase (E. C. 3.4.11.1) LAP LAP-A

LAP-B

Malat-Dehydrogenase (E. C. 1.1.1.37) MDH MDH-A

MDH-B

MDH-C

Menadion-Reduktase (E. C. 1.6.99.2) MNR MNR-A

6-Phosphogluconic-Dehydrogenase 6-PGDH 6-PGDH-A

(E. C. 1.1.1.44) 6-PGDH-C

Phosphoglucose-Isomerase (E. C. 5.3.1.9) PGI PGI-A

PGI-B

Phosphoglucomutase (E. C. 2.7.5.1) PGM PGM-A

Shikimat-Dehydrogenase (E. C. 1.1.1.25) SKDH SKDH-A

Transect AT 1 

Altitudinal zone Subalpine (U) High montane (M) Middle montane

(L)

Elevation 1900 – 2100 m 1600 – 1700 m 1200 – 1300 m

Forest community P. cembra – larch

forest with Pinus

mugo Turra

Larch – Norway

spruce forest with

P. cembra

Spruce forest with

some P. cembra and

larch

Soil Semipodsol

Precipitation 900 – 1100 mm

Total number of

individuals:

Adult population

Juvenile population

149

99

49

148

100

48

51

34

17
138 USDA Forest Service Proceedings RMRS-P-32. 2004

Klumpp and Stefsky Genetic Variation of Pinus cembra Along an Elevational Transect in Austria

gram (Gillet 1994). We used allelic multiplicity (number of

alleles M), relative allelic multiplicity (M/Mmax) and the

average number of alleles per locus (A/L) for describing

allelic variation (Hattemer and others 1993). Genetic diversity was described using average (observed) heterozygosity

(Ho), diversity of the gene pool (V) and hypothetic gametic

multilocus diversity (Vgam) (Gregorius 1978, 1987). The

genetic variation within each population was quantified by

calculating the percentage of polymorphic loci (P95), where

the frequency of the most common allele does not exceed 95

percent.

Results and Discussion __________

The P. cembra subpopulation from the middle montane

zone on the valley floor is scattered in a spruce-dominated

forest that also includes larch. Correspondingly, only 34

adult and 17 young individuals were found, which represented approximately 80 percent of the whole population in

this area. This bias in sampling was considered when drawing some conclusions from this study.

The number of observed alleles was found to obviously

increase with elevation in the adult populations. However,

the highest value (M=36) was found at medium elevation in

juvenile populations (table 3). In the high montane zone, 75

percent of the known variants were represented in the

juvenile population, as a total of 48 alleles over 22 gene loci,

were found in this valley. Closer inspection revealed that

variants with allele frequencies of more than 5 percent were

found to comprise between 58.3 and 64.6 percent of the total

known variants. Adult populations obviously possessed

more rare variants with an allele frequency of 1 percent,

such as AT1-U: M/Mmax=83.3 and M(f>1)/Mmax=77.1, than

in young populations, such as AT1-U: 70.8: 70.8 (table 3).

Surprisingly, the populations of the middle elevation

exhibited not only the highest values of observed alleles,

but also high values of relative allelic multiplicity (M/Max),

which was 75 percent of the known variants (table 3). Stone

pine appears to be different from other species, where clinal

variation was not found in allozyme gene markers (see for

example Moran and Adams 1989), or was only found only

in certain loci (for example Mitton and others 1980). This

may be due to strong selective forces such as the harsh

climate at the timberline, or competition with spruce in the

valley floor may influence the gene pool composition of this

species. Moreover, trees resulting from movement of seed

by birds (compare Marzluff and Balda 1992) may have

enhanced the existing gene flow (primarily by pollen transport up and down the slopes), which leads to high level of

genetic multiplicity.

A comparison of selected parameters of genetic variation

in mature stands from different elevations shows a slight

increase of multiplicity (M) with elevation but nearly no

trends in the other parameters (fig. 1). In contrast, no trend

was found in the heterozygosity in the juvenile stands, and

polymorphism decreased with elevation. Multiplicity as well

as hypothetic gametic multilocus diversity (the potential

for generating genetically diverse gametes in future generations) is highest in the middle elevation (fig. 2). This indicates that the genetic architecture in the juvenile populations at the middle elevation has been preserved better than

at other locations/age classes (fig. 3; table 3).

Conclusions____________________

The higher allelic multiplicity in juvenile populations at

the mid-montane zone is due to a combination of birds and

wind. Nutcracker birds transport seed to the middle slopes

from higher and lower elevations, as the middle slopes

provide less snow cover and easy access. Snow cover at the

timberline and the dense forest in the valley are not as

attractive for habitats for the bird, and allelic multiplicity is

correspondingly lower. Pollen transport by local wind systems cause gene flow among populations up and down

slopes, thereby the mid-montane zone has an influx of genes

from populations at both higher and lower elevations. Selection forces are obviously active, which to a certain extent

keeps the gene pool of the timberline population different

Table 3—Genetic variation at transect AT1a in the Koetschach valley, Salzburg State / Austria

AT1 - U AT1 – M AT1 - L

Ab Jc A J A J Average

Number of individuals 100 49 100 48 34 17 —

Number of loci 22 22 22 22 22 22 22

Number of observed alleles (M) 40 34 38 36 34 32 35.7

M/Mmax (%) 83.3 70.8 79.2 75.0 70.8 66.7 74.3

A/L 1.82 1.54 1.73 1.64 1.54 1.45 1.62

A/L ≥ 5 % 1.27 (58.3) 1.27 (58.3) 1.32 (60.4) 1.32 (60.4) 1.36 (62.5) 1.41 (64.6) 1.33 (60.6)

A/L ≥ 1 % 1.68 (77.1) 1.55 (70.8) 1.64 (75.0) 1.64 (75.0) 1.55 (70.8) 1.41 (64.6) 1.58 (72.2)

P95 in % 23 18 18 27 32 32 25

Ho (observed) 0.095 0.081 0.083 0.083 0.083 0.086 0.085

Gene pool diversity (V) 1.10 1.09 1.12 1.13 1.13 1.12 1.12

Hyp. gam. diversity (Vgam) 28.3 24.6 27.3 37.1 26.4 24.8 28.1

a

Where U refers to the subalpine zone, M refers to the high montane zone, and L refers to the middle montane zone of transect AT1. bA - Adult population

c

J - Juvenile population
USDA Forest Service Proceedings RMRS-P-32. 2004 139

Genetic Variation of Pinus cembra Along an Elevational Transect in Austria Klumpp and Stefsky

from that of the valley population. Strong competition with

the dominating spruce at the ground of the valley reduces P.

cembra populations to the extent that the opportunity for

rare variants in the gene pool is limited.

Acknowledgments ______________

This study was funded by the European Union as part of

the project on “biodiversity of alpine forest ecosystems” (EU

CT96-1949). We thank Eva, Tajana, Herwig, and Wolfgang

for technical assistance in collecting the samples as well as

0

10

20

30

40

50

M P95 Ho Vgam

AT1 - U

AT1 - M

AT1 - L

Figure 1—Average genetic variation for selected parameters in mature stands from different elevation.

0

5

10

15

20

25

30

35

40

M P95 Ho Vgam

AT1 - U

AT1 - M

AT1 - L

0

5

10

15

20

25

30

35

40

M P95 Ho Vgam

adult

juvenile

Figure 3—Average genetic variation for selected parameters in adult and juvenile populations at sample area

AT1- M (high montane zone).

Figure 2—Average genetic variation for selected parameters in the juvenile population from different elevation.

for laboratory assistance. The advice and many helpful

comments by Prof. Gerhard Mueller-Starck (Munich/Germany) is greatly appreciated. Furthermore, we thank Scott

Schlarbaum and Richard Sniezko for helpful comments on

an earlier draft.

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USDA Forest Service Proceedings RMRS-P-32. 2004 141

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

The authors are with the New Zealand Forest Research Institute, Private

Bag 3020, Rotorua, New Zealand. J.T. Miller’s current address is Regency

Park Estate, Te Ngae Road, Rotorua, New Zealand. F.B. Knowles’ current

address is Ridge Road, Lake Okareka, Rotorua, New Zealand. R.D. Burdon,

the corresponding author, may be reached at phone +64 7 343 5742; fax +64

7 348 0952; e-mail Rowland.burdon@forestrearch.co.nz.

Abstract—Five-needle pines that have been tried as plantation

crops in New Zealand are: Pinus strobus L., P lambertiana Dougl.,

P. monticola Dougl. ex. D. Don., and P. wallichiana A.B. Jacks. Total

plantation areas have reached approximately 1,300 ha, 75 ha, 20 ha

and 10 ha, respectively but have generally dropped in recent years.

A partial picture of provenance variation in performance has been

obtained. These species have grown well on a range of sites but have

tended to be affected by drought and exposure. While they are

sometimes affected by out-of-season frosts, the tolerance of partly

continental conditions is generally good. Disease has been little

problem, except for root disease in P. strobus on poorly drained soils

and Dothistroma needle blight on P. lambertiana at some sites.

However, animal damage (mainly deer and Australian possums)

has often been troublesome and has led to much malformation.

Productivity can be high, with mean annual increments of approximately 25 m3

/ha/year stemwood in P. strobus and P. lambertiana,

and equal to or greater than 30 m3/ha/year in P. monticola, given

favourable sites and appropriate provenances. One other species,

P. ayacahuite, has shown promise; while fairly frost-tender and

prone to animal damage, it can have the fastest height growth and

produce 24 m3

/ha/year or so in stem volume. Eight other species are

known to have grown successfully in New Zealand without arousing

commercial interest, and a few others have failed to grow. Despite

some good performances, five-needle pines are eclipsed in New

Zealand by Monterey pine (P. radiata Don.), Douglas-fir and some

eucalypts. However, there has been a recent reintroduction of a

provenance collection of P. lambertiana.

Key words: Pinus strobus, Pinus monticola, Pinus lambertiana,

Pinus wallichiana, Pinus ayacahuite, provenance,

genecology, growth, yield, site adaptation, browse

damage, fungal diseases.

Five-Needle Pines in New Zealand: Plantings

and Experience

J.T. Miller

F.B. Knowles

R.D. Burdon

History in New Zealand___________

Five-needle pines were first introduced into New Zealand

well over 100 years ago, including: P. strobus in 1866, P.

wallichiana in 1868, P. lambertiana in 1869; P. monticola in

1905 and 1907, and P. ayacahuite in 1915.

The first plantings were often few trees and mostly on

private estates. Later plantings were more extensive, and

predominantly within state forests, either as part of routine

plantings or in designated research trials. Plantings have

been mostly in the central North Island (Lats 37-39∞S, Long.

176∞E), but plantations have been established as far as 46∞S.

Elevations of planting have varied from near sea level to

around 600 m, mainly in areas of 1,000 to 1,600 mm annual

precipitation.

In the case of P. strobus, state plantings between 1931

and 1935 comprised more than 50 percent of the eventual

total area. By 1957 there were 1,282 ha in pure stands (1210

ha in the North Island and 73 ha in the South Island), with 39

ha in mixture with other species. By 1960 there were estimated to be 1,307 ha under all ownerships, over 1,130 ha

being in the central North Island. Pinus strobus has proved

to be vigorously invasive in some localities. It has regenerated freely around Rotorua, spreading up to 3 km from the

parent source into scrubland. It has also become naturalised

in the South Island in Nelson, North Canterbury, and at Mt

Linton (Lat. 46∞S).

Plantings of P. monticola were small by comparison. State

trials were planted in the North Island about 1910 and in the

South Island in 1932 and 1933. Further experimental plantings

occurred in the central and lower North Island between 1947

and 1958, and by 1961 the total area had increased to 21 ha

of which 98 percent was in the North Island.

The main plantings of P. lambertiana took place in the

central North Island between 1928 and 1931, with small

areas being added in the South Island about the same time.

By 1957 the total area of P. lambertiana in State Forests

was approximately 74 ha, of which only 5 ha were in the

South Island. By 1961 this was down to 68 ha, over 80

percent of which was in the North Island.

Small areas of P. ayacahuite were planted by the state

forestry agency in 1915, and in species trials between 1957

and 1959. Further plantings were confined to provenance

trials established between 1959 and 1963 at nine locations

throughout the country.

Pinus wallichiana has been fairly widely planted in New

Zealand but usually singly, as small groups as an ornamental, or with in species trials. By 1956 there were approximately 9 ha in State Forests, nearly all in Golden Downs

Forest in the north of the South Island.
142 USDA Forest Service Proceedings RMRS-P-32. 2004

Miller, Knowles, and Burdon Five-Needle Pines in New Zealand: Plantings and Experience

Other five-needle pines occasionally found growing satisfactorily in New Zealand arboreta include P. flexilis James,

P. aristata Engelm., P. peuce Griseb., P. koraiensis Sieb. &

Zucc., P. cembra L., P. maximartinezii Rzedowski, and P.

morrisonicola Hayata.

A species that failed was P. chiapensis (Martinez) Andresen.

Planted in two North Island trials, it suffered heavy initial

losses from frost, and then succumbed to browsing by the

Australian brush-tailed possum (Trichosurus vulpecula).

Little record exists of the seed origins of New Zealand

white pine imports. Seed for some of the P. strobus plantings

of the 1920s came from Ontario in Canada. Seed of P. monticola was imported from interior British Columbia in 1927

and from a Washington, USA, supplier in 1941. A small

amount of P. lambertiana seed came in 1869 from a San

Francisco supplier, but the sources of other early importations are unknown. Origins of seed imported by the Forest

Service between 1927 and 1930 included Butte, Eldorado,

and Placer Counties in California, and, in 1947 and 1948,

from the northern Sierra Nevada of California and the

southern Cascades into Oregon.

In recent years the areas of plantings of five-needle pines

have declined sharply, largely through widespread replacement with the Monterey pine (P. radiata).

Site Tolerances _________________

Most of the five-needled pines grown in New Zealand can

tolerate a wide range of local soil types. However, they prefer

fertile, moderately deep and well-drained soils, with poor

drainage favouring root disease. While fastest growth, particularly in early years, tends to occur on warm, mild sites,

and out-of-season frosts can be damaging, the temperate

species tolerate well the partly continental conditions of the

central South Island. Drought, particularly on poor soils,

can be associated with mortality, and tolerance of exposure

tends to be limited.

Pinus monticola, P. lambertiana, P. flexilis, P. aristata,

P. cembra andP. peuce have been planted in the Craigieburn

Ranges (Lat. 43∞S) in Canterbury. Although growth over

early years was often slow, on the better sites below 1,000 m

elevation (above sea level) they have generally been healthy,

of good form and have performed consistently. At higher

altitudes and on depleted soils there has been poor survival.

Here it should be noted that timberline in New Zealand is

low in relation to latitude.

Pinus strobus

In New Zealand P. strobus tolerates a fairly wide range of

sites and climatic conditions including poorer dry sites but

grows best on moderately fertile, deep, fresh soils. It has

grown particularly well in damp, sandy, deep soils adjacent

to streams in Canterbury. Reasonably good shelter is preferred and P. strobus is generally unsuitable for too exposed

sites. It has grown successfully at elevations of 60 to 900 m

in the North Island and 60 to 600 m in the South Island.

Early growth is generally quite slow, requiring control of

weed growth on fertile sites. It grows best in full light when

young but some shade is tolerated.

Pinus monticola

Pinus monticola has been tried mostly in cooler parts of the

North Island and northern South Island with annual rainfall of 760 to 2,030 mm, and appears to be the least demanding species. It is hardy and has withstood –14∞C of frost. It

has grown well on pumice soils in the central North Island

and will also tolerate fairly poor drainage and rather sour

clay soils. It has succeeded at elevations of 60 to 670 m in the

North Island and 275 to 410 m in the South Island.

Pinus lambertiana

This species has been planted in the central North Island,

Wellington, Canterbury, and Otago in localities with annual

rainfall of 760 to 1,650 mm. Establishment has often been

slow and variable. Young trees need shelter for successful

establishment and on exposed sites they can be checked or

killed by frosts. It failed at Hanmer (Lat. 43∞S, elevation

approximately 400 m) due to frost; however, older trees have

withstood frosts of –14∞C. Pinus lambertiana has succeeded

on well-drained pumice soils in the central North Island

where optimum growth and form occurred at 300 m elevation. In Canterbury it has grown strongly in well-drained,

sandy or silty sites but cannot tolerate waterlogging, while

older trees growing on dry shingly soil at Greendale in

Canterbury have died, probably due to drought. It has been

reported to favor alkaline soils.

Pinus wallichiana

Pinus wallichiana has been tried in Wellington, Nelson,

and Canterbury within a rainfall range of 760 to 1,400 mm

and has grown well on fertile, alluvial soils and light loams

with adequate moisture. It is relatively wind-tolerant, but

growth is poor on exposed sites, and trees in a trial at 490 m

in Kaingaroa Forest were badly malformed. The species is

hardy and has withstood frosts of –14∞C.

Pinus ayacahuite

In most New Zealand trials of P. ayacahuite growth and

survival have been poor. Frost damage occurred when the

trees were young, and they proved susceptible to possum

damage. On some sites, however, growth is excellent.

Pests and Diseases______________

Five-needle pines grown in New Zealand have generally

been free of serious pests and diseases. The white pine

blister rust fungus (Cronartium ribicola J.C. Fisch.) is absent. Even if it did arrive, the absence in forests of naturally

occurring alternate hosts (Ribes spp.) should make it unimportant, although the genus Ribes does occur as some orchards and a few naturalised populations. Poorly drained

soils tend to be associated with attack by root pathogens.

Among other pathogens, the root-rot fungi, Armillaria spp.,

can affect most species, Diplodia sp. can kill occasional

shoots, and Dothistroma pini can cause severe needle cast,

depending on species and site. Various insect pests have
USDA Forest Service Proceedings RMRS-P-32. 2004 143

Five-Needle Pines in New Zealand: Plantings and Experience Miller, Knowles, and Burdon

been recorded on five-needle pines; they are usually unimportant, but the wood wasp Sirex noctilio can sometimes

cause significant mortality. Browsing damage by mammals

(especially deer, goats, and possums) has tended to be

troublesome on almost all species, but the phenomenon of

preferential attack of minority species may well have been

an important contributing factor.

Pinus strobus

The root-disease fungus Leptographium procerum (previously Verticicladiella procera) has been involved in considerable mortality of P. strobus in New Zealand. However, it is

mostly associated with sticky, poorly drained soils and

sometimes with root damage associated with road and

access track construction. The foliage of infected trees becomes light green before wilting and turning rusty brown,

and a black stain appears in the roots and the wood at the

base of the trunk. The root-rot fungi, Armillaria spp., have

been recorded from the roots and butts of P. strobus. The

needle-cast fungi, Dothistroma pini and Cyclaneusma minus, have both been recorded from the foliage but not as

significant pathogens. Other minor disease-causing fungi

recorded for P. strobus include Diplodia spp., Hypoderma

sp., and Lophodermium sp.

In New Zealand various insect pests have been recorded as

present but not usually troublesome on P. strobus. Sirex

noctilio, with its fungal symbiont, has caused damage in

thinned stands.

Pinus monticola

In New Zealand P. monticola has generally been healthy

and there are few recorded pest and disease problems.

Disease-causing fungi recorded include Armillaria spp.

(apparently troublesome in provenance trials but not in

plantations), and Dothistroma pini, Diplodia sp.,

Leptographium sp., Lophodermium sp. and Rhizosphaera sp.,

generally as minor pathogens.

Pinus lambertiana

Dothistroma needle blight can cause severe defoliation on

trees of P. lambertiana on New Zealand sites with summer

humidity. Cylindrocladium scoparium has caused troublesome root rot in 2-year-old nursery stock. Otherwise, fungal

pathogens and insect pests are generally unimportant.

Growth and Yield ________________

Available growth data from a selection of measurement

plots are summarized for different species in tables 1 and 2.

They cover a range of latitudes, although latitude is not

critical in itself. Elevations varied but none were extreme.

Stem volume production could be high, despite modest

height growth. Data from other sources are also considered

below.

Pinus strobus

In New Zealand early growth generally rates as slow, but

P. strobus is ultimately capable of high stem volume production with mean annual increments (m.a.i.) at 35 to 40 years

ranging up to over 24 m3

/ha/year, depending much on site

quality. Near Rotorua (Lat. 38∞S), 50-year-old unthinned

stands on good sites planted at 2,224 stems/ha had mean top

height (m.t.h.) of 30 m, a mean diameter at breast height

(d.b.h.) of 25 cm, a volume of 1,220 m3

/ha and a m.a.i. of

24.4 m3/ha/year (Weston 1957). Mean top height is defined

as the mean height of the 100 largest diameter trees per

hectare. Small piece sizes can limit recovery rates: the

39-year-old stand with a total volume of 927 m3

/ha (table 1)

had a recovered volume of 795 m3

/ha. Higher productivity

could be expected with optimal provenances (see later).

Pinus monticola

In New Zealand P. monticola has, on the whole, grown

slightly faster than P. strobus, with observed m.a.i. ranging

up to equal or greater than 30 m3

/ha/year (table 1). In the

early years annual height growth is generally 0.3 to 0.6 m.

At Patunamu (Lat. 39∞S), a fertile, low-elevation site, mean

top height of 16-year-old trees was 15.8 m and the largest

tree was 32 cm in diameter at breast height. In species trials

in inland Canterbury (Lat. 44∞S), a few 17-year-old trees

averaged 7.9 m in height. In Kaingaroa Forest (Lat 381

/2∞S,

elevation 488 m), a 22-year-old unthinned stand carried a

total volume of 108 m3/ha (m.a.i.= 5 m3/ha/year) in 587 stems

with m.t.h. of 15 m and a mean d.b.h. of 19.6 cm.

Pinus lambertiana

In New Zealand, on favorable sites, height growth of P.

lambertiana in early years is 0.3 to 0.6 m annually. In wellstocked stands, trees in the upper crown classes are of good

form; however, the proportion of double-leadered trees tends

to be high, greater than 50 percent in some areas. At low

stockings, especially on fertile sites, stem taper can be

severe. On a site of moderate quality in Kaingaroa Forest

(Lat 381

/2∞S), a 26-year-old stand (containing about 40 percent of malformed stems) has produced an estimated total

volume of 452 m3

/ha (m.a.i.= 17.4 m3/ha/year) in 1268 stems/

ha averaging 13 m tall and 29.5 cm d.b.h. In a small plot on

a good site nearby, the best dominant trees grew to about

27.4 m in height and 63 cm d.b.h. in 48 years (Weston 1957);

at age 59 the same stand had a m.t.h. of 39.6 m, and a mean

d.b.h. of 38 cm with 30 percent malformed, the largest tree

being 89 cm d.b.h. A stand in lowland Canterbury (Lat. 44∞S)

on relatively dry fluvial gravels (precipitation approximately 700 mm/annum), planted in 1931 at 400 stems/ha,

with stocking 260 stems/ha remaining, had a basal area of

12.5 m2

, a mean d.b.h. of 39 cm and a mean height of 11.9 m.

Height growth was uniform but the trees were rough at this

low stocking. Individual trees over 60 years old have reached

35 m height and around 100 cm d.b.h.
144 USDA Forest Service Proceedings RMRS-P-32. 2004

Miller, Knowles, and Burdon Five-Needle Pines in New Zealand: Plantings and Experience

Table 1—Growth and yield of—Pinus strobus, P. monticola, P. ayacahuite and P. lambertiana in New Zealanda.

Age No. of D.B.H. BA Volume MAI/yr Latitude

Species (yrs) plots Stems/ha (cm) Ht (m) (m2/ha) (m3/ha) (m3/ha) (∞S)

Pinus strobus

Age 20-29 yrs 22 6 350 30.2 14.6 19.8 124 5.6 40

27 4 920 33.1 19.4 41.7 331 12.3 38

28 1 459 48.8 20.3 46.4 316 11.2 38

28 1 1156 36.4 20.0 79.4 557 19.9 391/2

mean 721 37.1 18.6 332 12.2

Age 30-39 yrs 34 1 1416 29.2 17.8 51.9 251 7.4 46

36 1 287 48.7 30.3 42.7 513 14.2 39

38 1 1211 34.0 17.3 65.6 515 13.5 46

39 1 1538 - - 88.3 927 23.8 38

mean 1113 37.3 21.8 62.1 551 14.7

Age 40-49 yrs 41 1 1800 32.0 18.5 90.4 772 18.8 46

42 1 2743 29.5 21.3 102.4 861 20.5 46

43 1 1495 40.4 23.5 99.6 923 21.5 46

44 1 1424 42.8 28.5 88.2 999 22.7 38

45 3 626 44.7 28.5 56.3 638 14.2 381/2

46 4 644 46.0 27.2 66.2 666 13.9 381/2

48 1 1139 40.0 25.3 88.1 810 16.9 38

48 1 941 45.1 25.4 78.3 833 17.3 38

mean 1351 32.2 21.2 83.7 813 18.2

Age 50-59 yrs) 52 2 607 46.5 27.1 63.9 673 12.9 38

53 3 909 41.9 26.0 60.0 622 11.7 38

53 3 946 38.1 22.1 67.4 947 17.9 43

56 1 356 48.5 31.0 47.4 580 10.4 38

mean 704 43.7 26.5 59.7 705 13.2

Age 60 yrs + 62 3 784 51.8 35.1 93.3 960 15.5 381/2

61-65 1 593 49.5 28.3 80.1 720 11.4 46

69 4 694 50.5 34.8 75.7 1035 15.0 381/2

mean 690 50.6 32.7 60.4 905 14.0

P. monticola

Age 30-39 yrs 34 1 949 39.5 17.7 53.6 371 10.9 38

34 1 1480 36.3 18.5 86.6 624 18.4 391/2

mean 1215 37.9 18.1 70.1 497 14.6

Age 40-49 yrs 49 1 476 49.7 33.8 63.1 1560 31.8 38

P. ayacahuite

Age 30-39 yrs 34 1 1400 45.3 23.4 116.4 863 25.4 391

/2

36 1 536 55.6 33.8 73.0 792 21.9 38

mean 968 50.4 28.6 94.7 827 23.6

P. lambertiana

Age 30-39 yrs 36 1 536 53.4 20.5 62.5 406 19.2 38

39 1 1183 63.2 21.2 156.4 1068 27.4 391/2

mean 58.3 20.8 109.4 737 23.3

a

 Source: New Zealand Forest Research Institute Ltd permanent sample plot system.

Table 2—Summary figures (mainly from table 1) for mean annual volume increment

and mean height growth, by species.

Mean annual increment—

Total stemwood (m3/ha/yr) (>30 years old) Height growth

Species plots Range Mean (m/yr) (Range)

P. strobus 30 7-24 15.5 0.42-0.84

P. lambertiana 2 19-27 23.3 0.54-0.57

P. monticola 4 11-32 20.4 0.52-0.99

P. wallichiana 2 - - 0.42-0.48

P. ayacahuite 2 22-25 23.6 0.69-0.94
USDA Forest Service Proceedings RMRS-P-32. 2004 145

Five-Needle Pines in New Zealand: Plantings and Experience Miller, Knowles, and Burdon

Pinus wallichiana

At Golden Downs Forest (Lat. 411/2∞S), a 21-year-old

unthinned stand with about 1,000 stems/ha developed evenly,

with tree heights 9 to 12 m and average diameter about 15.2

cm. In lowland Canterbury (Lat. 44∞S) trees measured at 17

years averaged 8.2 m tall, while farther inland a few trees in

species trials averaged 7.6 m in height at 18 years. An

exceptional specimen, in the North Island, was 38.6 m tall

with a d.b.h. of 105 cm when felled.

Wood Properties and Uses________

New Zealand-grown timber of P. strobus tends to be

slightly less dense than that from its native habitat, but

otherwise the properties are similar. The relatively low

density (370 kg/m3 at 12 percent moisture content), low

strength, and poor nail-holding ability make P. strobus

unsuitable for structural purposes. Indications are that end

joints machine poorly. Uniform texture and dimensional

stability allow certain specialist uses, however, including ice

cream spatulas and similar products, and for toy making,

shoe heels, picture frames, and rulers. Its good woodworking

properties make it useful for panelling. Timber assessed in

pilot studies was seriously degraded by the frequent small

encased knots, with insufficient lengths free of defects; this

largely reflects the relatively short rotations that are deemed

to be economic in New Zealand.

Pinus strobus has a large percentage of heartwood that

has a high moisture content, leading to drying and seasoning

difficulties, requiring relatively long drying times in either

the kiln or open air. Drying is further complicated by the

development of an irregular brownish stain resulting from

enzymes acting on the sugars in the wood. Compared with P.

radiata, the high proportion of heartwood in P. strobus

renders it unattractive for pulping and also makes the

product difficult to season and preserve for use as posts.

Timber tests on P. monticola (Harris and Kripas 1959)

have indicated a general similarity to imported American

material, including early formation of heartwood (34 percent

at 24 years) and a high moisture content in the heartwood.

Establishment and

Silviculture _____________________

Five-needle pines, while often being vulnerable to exposure, often need vigorous weed control for satisfactory establishment and good early growth in New Zealand. Usually the

preferred way to achieve this is by the use of chemical

sprays, best practice depending very much on local conditions. Good control of browsing animals is also important.

In general, five-needle pine stands in New Zealand have

tolerated high stockings, with little difference between

heights of dominant and other trees, yet only moderate

mortality from mutual suppression. However, in dense stands

the crowns are small, leaving long lengths of stem with

persistent dead branches. Response to delayed thinning

appears to be rather slow.

Although the five-needle pines in New Zealand are monocyclic or ‘uninodal’, the knot clusters are typically too closely

spaced and far too persistent to yield the relatively long’

‘internodal’ clear-cuttings grades that have come readily

from P. radiata. Because of this and their low wood density,

standard silvicultural regimes do not exist. Appropriate

regimes, if the species are to be grown further, would be

governed by some largely conflicting considerations, including the desirability of early pruning to produce clear timber;

the desirability of maintaining high stockings to realize the

inherent productivity; and the time required to obtain clear

timber from pruning, particularly if stockings are kept high.

Stem forking has been notorious in P. strobus in Southland.

This is assumed to be due to wind, snow, and unseasonable

frosts, but it could also be attributable in part to both animal

damage and genetic origin.

High initial stocking for P. lambertiana also allows for

selection of good-form trees at mean top height 15 m or later.

The unusual ability of this species to sustain a high rate of

growth to advanced ages argues for long rotations that,

however, would make effective growing costs high by local

standards.

Provenance Variation and Genetic

Improvement ___________________

Provenance testing is the only level at which withinspecies genetic variation has been studied. Some limited

cross-referencing of species has been done within provenance trials. Tree-to-tree genetic variation is not obvious in

the way that it is in P. radiata.

Pinus strobus

Provenance trials were planted in 1970 with 77 seedlots of

P. strobus on three New Zealand sites: Rotoehu (Lat. 38∞S)

and Gwavas (Lat. 393

/4∞S) in the North Island, and Golden

Downs (Lat. 411

/2∞S) in the South Island. Seed was provided

by various suppliers in the United States and represented a

fairly complete sample of the geographic range of the species

(Lats 341

/2-48∞N), albeit weighted toward the southern part.

(Note: One lot was recorded as having been collected from

54∞N in Manitoba, outside the limit of 52∞N shown by

Critchfield and Little (1966), but without appearing to be an

outlier for growth rate). Ten-tree row-plots were used. Heights

were measured at ages 3 and 5 years from planting

(Shelbourne and Thulin 1976), while diameters and incidence

of malformation were assessed at Rotoehu and Gwavas at

18 years, in 1988 (Chen 1989). Six seedlots of P. monticola

and one of P. wallichiana were also included.

The early heights were closely correlated (negatively)

with latitude of origin, the southernmost provenances being

almost twice as tall as the northerly ones (Lat. ≥45∞N)

(Shelbourne and Thulin 1976). This was essentially irrespective of planting site, provenance x site interaction being

only minor despite marked differences among sites at that

stage. In fact, the estimated genetic correlation (compare

Burdon 1977) between year-18 diameter across the two sites

and latitude of origin was equal to or greater than 0.9. Thus

most of the best provenances were from the south of the

species’ range; from the southern Appalachian region, northern Georgia, western North Carolina, West Virginia, and

eastern Tennessee. These trees at 18 years averaged up to 30

percent greater in diameter than the overall mean, and had
146 USDA Forest Service Proceedings RMRS-P-32. 2004

Miller, Knowles, and Burdon Five-Needle Pines in New Zealand: Plantings and Experience

higher survival and less forking. This pattern matched

closely the results of Sluder and Dorman (1971) in a trial in

the southern Appalachians. However, heights of provenances

from the New England States were about the same as those

from Virginia, Maryland, and Pennsylvania – that is, 7 to 10

percent above the overall mean for provenances from the

same latitudinal range.

Consistency of provenance rankings within the three

assessments suggested that early selection of P. strobus is

possible at ages 3 to 5 years from planting, at least at the

provenance level. Pinus monticola, and P. wallichiana had

the poorest growth over all sites.

Some of the results remain puzzling. While the southern

Appalachian provenances promised significantly greater

productivity in New Zealand than Ontario material, the

local seedlots from Kaingaroa Forest performed markedly

better than their reported origins in Ontario would suggest,

even allowing for any likely effects of both release from the

neighbourhood inbreeding of natural stands and natural

selection in the New Zealand environment. Thus, the reported origins from the Ontario region come into question.

Generally, growth was best at two milder North Island trial

sites, suggesting by itself that a higher altitude and warmer

climate are more suited to this species.

The relatively poor showing of the P. monticola provenances included in these trials was also puzzling, in the

light of stand growth data (tables 1 and 2). However, the lack

of 18-year height data prevented rigorous testing of the

hypothesis that the slower early growth of the P. monticola

was a transient phenomenon.

Pinus monticola

Provenance trials were established using seven seedlots of

P. monticola, comprising five native provenances collected

by a New Zealand operator in 1956, plus one from the

Institute of Forest Genetics at Placerville and another from

a stand in Kaingaroa Forest. The native provenances came

from the Sierra Nevada (as far south as 381

/2∞N), and the

Southern Cascades, extending as far north as Lat. 451/2∞,

elevations ranging from 425 m to 2,300 m. They were

planted out at three South Island sites in 1963 and four

North Island sites in 1964. In addition four demonstration

rows were planted out at Rotorua. The provenance from

Kaingaroa Forest, derived from selected parent trees within

an approved seed stand (N.Z. Forest Service), which reportedly originated in interior British Columbia, grew best

among the tested origins, and was obviously well adapted.

No formal assessments have been carried out, but inspection

notes and observation of the demonstration planting revealed a strong pattern of inverse relationship between

height and elevation of origin, within that part of the natural

range. This pattern contrasts strongly with the weak elevational differentiation reported for the species in Idaho (Anon.

2000). Also surprising, in the light of provenance tests for

various other species, were the indications that material

originating from British Columbia was among the most

vigorous samples. A suspicion arises that material from the

part of the native range sampled for the provenance trials

may be relatively prone to Armillaria root rot. Pinus monticola

in general maintained better stem form than P. strobus.

Pinus lambertiana

Seven provenances were tested in trials established in

1960 to 1961 at nine New Zealand sites (two North Island,

seven South Island; Lats 38-46∞S). The seedlots used were

collected from localities in California ranging in mean elevation from 850 m (1.6 km southeast of Koberg) to 2,000 m at

Jordan Peak in Sequoia National Park.. Five of the trials

remained current by 1999, showing, as expected, considerable within-population variation in tree size. Inspection

notes from well-grown trials in each island showed that the

relatively low elevation (760 to 1,030 m) seedlot from Lassen

National Forest in northern California yielded trees with

the best height and diameter growth.

In 1997 a wide provenance range of P. lambertiana seed

was obtained from the United States. A total of 279 singletree progenies representing eight provenance groups were

sown in the research nursery at Rotorua in September 1977.

Coastal Californian, and certain central Sierra lots showed

vigorous early growth and good survival in the first 18

months in the nursery bed. However, other lots from the

central Sierra area were among the poorest in growth and

survival among this material, pointing to the necessity of

further testing over time.

Pinus wallichiana

Little information is available on genetic variation in this

species, and it has not been planted in provenance trials in

New Zealand. One seedlot of P. wallichiana from a single

tree was included in the provenance trials of P. strobus

planted in 1970. At age 18 years, survival of this lot was

comparable to P. strobus (about 70 percent), but diameter

was 18 percent less than the mean of the 77 P. strobus lots

in the trials. The only other known planting in New Zealand

of P. wallichiana is as a forest stand of 9 ha planted in

1932 through 1938 at Golden Downs Forest (Lat 411

/2∞S)

and in arboretum-scale plantings in inland Canterbury in

the South Island (Lat. 431

/2∞S).

Pinus ayacahuite

Ten provenances of P. ayacahuite from locations in Mexico

and Guatemala were planted at each of nine New Zealand

locations between 1962 and 1968. Most plantings survived

poorly and grew slowly, with high malformation rates mainly

due to browsing by possums, and most were abandoned.

Exceptions were two trials at Kaingaroa Forest and two at

Gwavas Forest (Lats 38-391

/2∞S). Survival at the best of

these were over 90 percent and the average diameter in

1983, at age 15 years, was 20 cm. Although the incidence of

multileadering was high, much of this could have been

corrected by thinning. There was little provenance variation

in growth.

Two plots of P. koraiensis were incorporated in a trial in

Karioi Forest, at over 700 m elevation, in 1968, but all trees

were killed by frost.
USDA Forest Service Proceedings RMRS-P-32. 2004 147

Five-Needle Pines in New Zealand: Plantings and Experience Miller, Knowles, and Burdon

Future Roles in New Zealand ______

Despite often excellent growth by standards of natural

ranges, ability to thrive on a range of sites, and resistance to

certain diseases, five-needle pines are not foreseen as having

any major role in New Zealand, because they suffer so much

by comparison with the preferred species P. radiata, Douglasfir, two cypresses, and various eucalypts. The required long

rotations, plus some other factors that increase effective

growing costs, in conjunction with limitations of timber

quality and limited site tolerance all mitigate against any

important role, even with use of better provenances.

Pinus monticola, despite lesser heights in some trials,

emerges preferred over P. strobus in New Zealand because

of its generally good growth and form (less stem forking) and

its tolerance of difficult sites. Pinus lambertiana, despite its

attractions as a virgin timber in its native sites and its high

long-term productivity, is seen as seriously disadvantaged

by a need for long rotations. Pinus ayacahuite, despite its

growth rate, appears to be to site-demanding, apart from its

timber being unproven.

A major collection of P. strobus remains, but the ‘land

races’ represented in the commercial plantings have largely

disappeared. However, a significant genetic collection of

P. lambertiana has recently been established.

References _____________________

Anon. 2000. Forestry Compendium Global Module. CD Publication.

CAB International, Wallingford, U.K.

Burdon, RD 1977. Genetic correlation as a concept for studying

genotype-environment interaction in forest tree breeding. Silvae

Genetica. 26: 168-175.

Chen, Jianxin 1989. Provenance selection of Pinus strobus in New

Zealand. Ministry of Forestry Forest Research Institute, Project

Record No. 2339, 22 pp. (unpubl.).

Critchfield, WB; Little, EL 1966. Geographic distribution of the

pines of the world. USDA Forest Service Miscellaneous Publication No. 991, v + 97 pp.

Harris JM, Kripas S 1959. Notes on the physical properties of

ponderosa pine, monticola pine western red cedar, and Lawson

cypress grown in New Zealand. NZ Forest Service, FRI Research

Note No. 16, 24 pp.

Shelbourne, CJA; Thulin, IJ 1976. Provenance variation in Pinus

strobus; heights five years after planting in New Zealand. New

Zealand Forest Service, FRI, Genetics & Tree Improvement

Internal Report No. 100, 23 pp. (unpubl.).

Sluder, ER 1963. A white pine provenance study in the Southern

Appalachians. USDA Forest Service Southeastern Forest Experiment Station Research Paper SE-2, 16 pp.

Weston, GC. 1957. Exotic Forest Trees of New Zealand. New

Zealand Forest Service, FRI Bulletin No. 13, 103 pp.
148 USDA Forest Service Proceedings RMRS-P-32. 2004

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

Kwan-Soo Woo is with the Department of Forest Genetic Resources, KFRI,

Korea Forest Service, Suwon, Kyonggido 441-350, South Korea. Telephone:

(031) 290-1106, fax: (031) 292-4458, e-mail: woo9431@yahoo.co.kr. Lauren

Fins is with the Department of Forest Resources, University of Idaho,

Moscow, ID 83844-1133, U.S.A. Telephone: (208) 885-7920, fax: (208) 885-

6226, e-mail: lfins@uidaho.edu. Geral I. McDonald is with the USDA Forest

Service, Rocky Mountain Research Station, 1221 South Main Street, Moscow,

ID 83843, U.S.A. Telephone: (208) 883-2343, fax: (208) 883-2318, e-mail:

gimcdonald@fs.fed.us.

Abstract—This paper compares the results of two studies (differences related to genotype versus differences related to growing

environment) that have been reported in previous publications

(Woo and others 2001; Woo and others 2002) and highlight information that may be useful to tree breeders in refining rust resistance

evaluation procedures. The objectives of these studies were to

assess genetic and environmentally related variation in needle

surface traits in rust–resistant western white pine (Pinus monticola

Dougl.). Statistically significant differences were found in 14 needle

traits (needle length and width, number of stomatal rows, number

of stomata per row, total stomata per needle, adaxial surface area,

stomatal density, major axes of stomata, stomatal shape, stomatal

area, stomatal occlusion, epistomatal wax degradation, weight of

wax per dry weight of needle, and the contact angles of water

droplets) of western white pine seedlings grown from the same seed

orchard source in three nurseries in northern Idaho. Waxes on

needle surfaces were tubular in structure and the amount of surface

wax appeared to be associated with surface wettability. In a separate study, stomata on needles from susceptible families were found

to be significantly wider and larger than those from genetically

resistant families and from genetically improved bulked lots from

the seed orchard. Neither the percent of stomatal occlusion nor the

amounts of degraded epistomatal wax were statistically different

among the seed sources. Contact angles of water droplets on needles

of the resistant families were significantly larger than those of the

susceptible families and the seed orchard lots. Results of both the

genetic study and the nursery study should be more broadly tested

to determine their generality and applicability to refining rust

screening procedures. Information from this study may be useful

in refining protocols for selecting genotypes for tree improvement

Genetic and Environmentally Related

Variation in Needle Morphology of

Blister Rust Resistant and Nonresistant

Pinus monticola

Kwan-Soo Woo

Lauren Fins

Geral I. McDonald

programs and/or for quantifying levels of rust resistance in selectively bred western white pine stocks. Rust screening protocols may

be made more efficient if any of the traits prove to be reliable

indicators of resistance.

Key words: Epistomatal wax, Pinus monticola Dougl., wettability,

stomatal occlusion, blister rust, needle morphology,

rust resistance

Introduction ____________________

Routine tests of western white pine (Pinus monticola

Dougl.) for blister rust resistance rely on successful inoculation of seedlings with spores of the fungus Cronartium

ribicola J. C. Fisch. in Rabenh., and subsequent ocular

evaluations for rust symptoms and expression of the resistance mechanisms. Presumably, phenotypic variation that

has a genetic basis will play a critical role in infection

success; for example, a specific resistance type may prevent

entry of germinating rust spores into needle tissue. However, phenotypic variation caused by a particular nursery

regime that temporarily prevents or diminishes successful

inoculation of seedlings may hamper attempts to identify

genetically resistant stocks for tree improvement programs

or attempts to quantify realized levels of resistance in

selectively bred stock. The studies reported here were designed to assess and quantify phenotypic variation in a

variety of needle surface traits that may be associated with

genetic differences in resistant versus susceptible genotypes

and which may also be affected by differences associated

with nursery growing regimes and or their environments.

Results of the individual studies (differences related to

genotype versus differences related to growing environment) have been reported in previous publications (Woo and

others 2001; Woo and others 2002). The purpose of this paper

is to compare the results of the studies and highlight information that may be useful to tree breeders in refining rust

resistance evaluation procedures.

Background ____________________

White pine blister rust, a devastating disease caused by

the fungus Cronartium ribicola J. C. Fisch. in Rabenh., was

introduced into western North America in 1910 on eastern
USDA Forest Service Proceedings RMRS-P-32. 2004 149

Genetic and Environmentally Related Variation in Needle Morphology of Blister Rust... Woo, Fins, and McDonald

white pine seedlings imported from Ussy, France, to Point

Grey, near Vancouver, British Columbia. The rust appears

to have reached Idaho about 1923 (Mielke 1943). Blister rust

is largely responsible for the drastic reduction in white pine

cover type (greater than 90 percent) in the inland northwestern United States (eastern Washington, northeastern

Oregon, northern Idaho, and northwestern Montana) since

1923 (Fins and others 2001). A breeding program designed

to increase white pine’s resistance to blister rust was initiated in the late 1940s as part of an effort to restore white pine

to inland Northwestern ecosystems (Bingham 1983). The

program produces genetically improved stocks that outperform unimproved (susceptible) stocks in operational

plantings and field tests throughout the region (Fins and

others 2002).

Rust Screening

As a key component of the ongoing breeding program,

white pine seedlings are routinely evaluated for rust resistance after inoculation with C. ribicola basidiospores. Although most seedlings subjected to the screening process are

grown at the USDA Forest Service Coeur d’Alene (Idaho)

nursery, occasionally seedlings grown elsewhere have been

included in the rust inoculations. At these times, differences

in infection levels, mortality, variation in needle thickness,

and the amount of water that collects on needle surfaces

were observed between seedlings grown in different nurseries, suggesting that inoculation success may vary not only

with the genetic backgrounds of the seedlings, but also with

the nursery in which the seedlings are grown.

The relative number of needle spots that appear on white

pine seedlings after exposure to blister rust has been shown

to be under genetic control (Hoff and McDonald 1980; Meagher

and Hunt 1996) and it appears to be a type of “rate reducing

resistance” (Rossi and others 1999) that reduces infection

efficiency by 10 times (Hoff and McDonald 1980).

At least two blister rust resistance types, “no spot” and

“reduced needle lesion frequency,” are recognized by the lack

of or low numbers of needle spots that appear on needles

after exposure to blister rust spores. “No spot,” which occurs

in high frequencies in Eurasian white pine species and

occasionally in North American white pine species, is rare in

western white pine, but the “reduced needle frequency” type

of resistance can be found in higher frequencies (Hoff and

others 1980). Although the exact mechanisms by which

seedlings avoid or reduce infection are not known, it is

known that C. ribicola germ tubes enter needles through

their stomata (Patton and Johnson 1970), indicating that

stomatal features are likely to be important to the infection

process. Whether or how specific variations in stomatal

features of western white pine needles are critical to

infectability by blister rust is also not known.

Surface Wettability

“Wettability” refers to the tendency of a surface to retain

water. Water beads up and remains on nonwettable surfaces

longer than on wettable surfaces. Because water droplets

take different shapes on wettable versus nonwettable surfaces, the angles they make with the surfaces on which they

rest can be used as a measure of surface “wettability” (see

Woo and others 2002 for images). A higher contact angle

(produced by a more globular, rounder bead) indicates lower

wettability of the surface (Cape 1983; Leyton and Juniper

1963). This phenomenon may be important in blister rust

infection because water collection on needle surfaces is

essential for fungal infections (Huttunen 1984), and the

amount and distribution of free moisture have been reported

to be the most important factors for germination of C.

ribicola basidiospores and subsequent infection of eastern

white pine needles (Spaulding and Rathbun-Gravatt 1926;

Hansen and Patton 1977).

Study Objectives

The objectives of these studies were to describe and compare differences in needle surface traits, including surface

wettability, stomatal size, frequency and distribution, and

other needle traits that may be influenced by differences in

nursery environment and/or genetic constitution. Our hypothesis was that both nursery environment and genetic

differences affect needle surface traits that are related to

infectability by C. ribicola.

Materials and Methods ___________

Nursery Study

Seeds of western white pine were sown in 1997 and grown

for 2 years (1997 and 1998) at three nurseries in northern

Idaho, USA: the USDA Forest Service nursery in Coeur

d’Alene, Potlatch Corporation’s nursery in Lewiston, and

the University of Idaho Forest Research nursery in Moscow.

All seedlings used for the comparisons between nurseries

originated from F2 open pollinated seeds collected from the

R.T. Bingham White Pine Seed Orchard in Moscow, ID. This

stock has been reported as approximately 66 percent resistant to blister rust at 2.5 years after inoculation (Hoff and

others 1973).

Morphometric traits were measured on three needles per

seedling collected from each of 30 seedlings per nursery, one

needle from each of three directions on the current stem of

each seedling. Stomatal size (major and minor axes) and

area were assessed on additional collections of one needle

from each of two fascicles per seedling from 25 seedlings per

nursery. Details on field, laboratory and statistical methods

can be found in Woo (2000) and Woo and others (2002).

Genetics Study

For the genetics study, needle samples were collected from

western white pine seedlings grown at the USDA Forest

Service nursery in Coeur d’Alene as part of a routine rust

screening operation of 271 entries that included open-pollinated families from phenotypic selections in natural stands,

woods-run check lots, and open-pollinated bulk F2 seed

orchard lots from the R.T. Bingham White Pine Seed Orchard in Moscow. The seeds were sown in 164 cm3 single

super cells in spring 1993 and remained in a greenhouse for

two growing seasons. They were inoculated in August 1994

by suspending blister rust-infected Ribes leaves over them

in an inoculation chamber at 100 percent relative humidity.
150 USDA Forest Service Proceedings RMRS-P-32. 2004

Woo, Fins, and McDonald Genetic and Environmentally Related Variation in Needle Morphology of Blister Rust...

In May 1995, the seedlings were transplanted to five

outdoor nursery beds where they remained for the next 3

years (Mahalovich and Eramian 1995). Four families with

low spot frequency (3062, 3653, 4437, and 4922), four families with high spot frequency (3162, 3233, 4110, and 4778),

and two open-pollinated F2 resistant bulked lots from the

R.T. Bingham White Pine Seed Orchard (lots 4815 and 4816)

were selected for this study (mean spot frequencies: 0.065,

3.32, and 0.20) (Woo and others 2001). One family in the

“resistant” group (4437) also had 73 percent zero spot individuals 1 year after inoculation. Of the two seed orchard lots,

lot 4815 was a general seed orchard collection, and lot 4816

was collected from parent trees that had been selected for

the “short shoot” type of resistance (needle lesions appear

after inoculation but rust infection is stopped between the

needle and the branch), but seedlings from both lots exhibit

a variety of rust resistance mechanisms (Rust 1998). Needle

samples consisted of one needle from the current stem from

each of three fascicles from each of 21 seedlings per source

(seven seedlings per source per replication). Details on field,

laboratory and statistical methods used can be found in Woo

(2000) and Woo and others (2001).

Results ________________________

Nursery Study

Significant differences were found among the three nurseries (l=5.67; P<0.0001) and among seedlings within nurseries (l=4.17; P<0.0001) for the eight measured needle characteristics and three of the four stomatal measurements; that

is, major axes, stomatal shape, and mean stomatal area

(P<0.001) (table 1). Minor axes of stomata (stomatal width)

were nearly identical among the three nurseries (P=0.94).

Relatively larger deposits of waxes were commonly distributed along the stomatal rows and over epistomatal

chambers. Stomata with severely degraded epistomatal

waxes were found side by side with stomata whose waxes

were completely intact. Percent of occluded stomata was

similar on needles from the Coeur d’Alene (86 percent) and

Moscow nurseries (90 percent), and both had significantly

more occluded stomata than needles from the Lewiston

nursery (56 percent, P=0.0001). Needles from the Lewiston

nursery produced more wax per dry weight of needle than

those from either the Coeur d’Alene or the Moscow nursery,

but those from the Moscow nursery had statistically higher

levels of degraded wax than either of the other two (table 1).

Mean contact angles of water droplets on the needle

surfaces differed significantly among the three nurseries

and were highest on needles from the Lewiston nursery,

both with and without the presence of surface waxes (table

1). Needles from the Moscow nursery had the smallest

contact angles.

Genetics Study

In the genetics study, we found significant differences

among the 10 seed sources (P<0.10) for nearly all of the traits

(Woo and others 2002). When grouped by resistance type

(resistant, susceptible, and Moscow Seed Orchard), most of

the comparisons were no longer statistically significant.

Several exceptions stood out, however, including needles

from the four resistant families were significantly shorter

than those from the seed orchard lots (P=0.015), and the

stomata of the susceptible families were significantly wider

and greater in area than stomata on the seed orchard lots

and the resistant families. The stomata of the susceptible

families were also “rounder” in shape (smallest ratio of

Table 1—Means and standard errors of western white pine needle traits (from Woo and others 2002).

Nursery

No. of Coeur d’Alene Lewiston Moscow

Variables needles (mean ± SE) (mean ± SE) (mean ± SE) P-value

Needle length (mm) 270 54.3 ± 0.78 65.7 ± 1.08 81.2 ± 1.12 0.0001

Needle width 1 (mm) 270 0.8 ± 0.006 0.7 ± 0.008 0.9 ± 0.01 0.0001

Needle width 2 (mm) 270 0.8 ± 0.006 0.7 ± 0.008 0.9 ± 0.01 0.0001

Stomatal rows/plot 270 2.4 ± 0.05 3.3 ± 0.07 3.6 ± 0.07 0.0001

Stomata/row 270 13.1 ± 0.11 13.5 ± 0.13 13.8 ± 0.15 0.0228

Stomata/needle 270 3449 ± 76.9 5811 ± 158.7 8048 ± 246.7 0.0001

Adaxial surface area (mm2) 270 82.8 ± 1.46 93.2 ± 1.92 146.2 ± 3.58 0.0001

Stomatal density 270 41.8 ± 0.63 62.2 ± 0.97 55.0 ± 0.92 0.0001

Major stomatal axes (mm) 150 59.5 ± 0.61 54.8 ± 0.55 57.9 ± 0.66 0.0001

Minor stomatal axes (mm) 150 33.2 ± 0.27 33.1 ± 0.31 33.0 ± 0.28 0.9369

Stomatal shape:

Major axes/Minor axes 150 1.80 ± 0.03 1.66 ± 0.02 1.76 ± 0.02 0.0001

Mean stomatal area (mm2) 150 1549 ± 19.41 1426 ± 24.41 1502 ± 23.73 0.001

Mean wax degradation 150 2.46 ± 0.06 2.58 ± 0.06 3.27 ± 0.05 0.0003

Wax per dry weight (mg/mg) 75 6.26 8.48 4.38 0.0002

Wax per surface area (mg/mm2) 75 9.48 7.76 6.07 0.0719

Contact angles with wax 270 90.8 ± 0.79 105.4 ± 0.74 62.9 ± 1.68 0.0001

Contact angles, no wax 30 94.7 ± 1.31 101.0 ± 0.93 55.3 ± 2.3 0.0051
USDA Forest Service Proceedings RMRS-P-32. 2004 151

Genetic and Environmentally Related Variation in Needle Morphology of Blister Rust... Woo, Fins, and McDonald

major to minor axes) than stomata on the seed orchard lots,

and contact angle of water droplets on needles surfaces was

significantly larger on resistant families than on susceptible

families and Moscow Seed Orchard lots.

Discussion _____________________

Nursery Study

Growth Environment and Nursery Regimes—The

differences we found in needle traits of the same seed source

grown in three northern Idaho nurseries likely reflect differences in nursery growth regimes, temperature levels, container size, and/or sowing dates. Some of these nongenetic

differences may be associated with variation in blister rust

infection levels. For example, differences among nursery

samples in stomatal traits are potential candidates as predictors of differences in initial infection levels because the

blister rust fungus enters white pine needles through their

stomata (Patton and Johnson 1970). We did not test this

hypothesis in our current study. However, incidental evidence suggests a link between nursery growth regimes and

rust infection following artificial inoculation, as on several

occasions, attempts to inoculate seedlings grown in the

Lewiston nursery had resulted in low infection levels compared to seedlings grown in Coeur d’Alene and inoculated at

the same time.

Different growth regimes in each nursery (amounts and

timing of water, temperature, light, growth medium and

fertilization) likely contribute to differences in needle morphology and possible differences in infectability by rust

infection. Fertilization, for example, may generally increase

seedling growth and vigor but also increases susceptibility of

southern pines to fusiform rust (Schmidt and others 1972;

Blair and Cowling 1974; Rowan and Steinbeck 1977). Another potential association links differences in infection to a

“functional resistance” associated with stomatal behavior

(Hart 1929; Hirt 1938). If, for example, wider and larger

stomata close slowly or incompletely compared to small

narrow stomata, fungal germ tubes may more easily invade

the larger ones.

Surface Waxes—Other, less obvious, surface traits may

be implicated in differences in infection. For example, the

high proportion of degraded waxes on seedlings from the

Moscow nursery, which may be related to the use of surfactants and other treatments, such as the use of an acid rinse

following fertilization, may be associated with rust infection. This relationship is suggested by a study showing that

fungal hyphae penetrated Norway spruce needles more

easily when surface or epistomatal waxes were degraded

compared to when the wax structures were well-preserved

(Huttunen 1984; Elstner and others 1985).

Stomatal Occlusion—Previous researchers have suggested a possible link between reduced blister rust infection

and occlusion of the stomatal antechamber on needles of

Pinus strobus L. (Patton and Johnson 1970; Patton and

Spear 1980). However, seedlings from the Coeur d’Alene

nursery (86 percent occluded) have historically been more

easily infected than those from the Lewiston nursery (56

percent occluded). If previous infection patterns hold true, it

would suggest that factors other than stomatal occlusion

may be relevant to infectability, but this relationship was

not tested in the current study.

Needle Wettability—The distribution and amount of

water on a needle is important for basidiospore germination

(Spaulding and Rathbun-Gravatt 1926; Hansen and Patton

1977). Thus, the wettability of a needle will likely affect

infectability because “nonwettable” surfaces hold more water and for a longer time than “wettable” surfaces (Leyton

and Juniper 1963; Cape 1983; Haines and others 1985). In

routine rust screenings in 1989 and 1992, water tended to

bead up more and remain longer on seedlings from the Coeur

d’Alene nursery compared to seedlings from the Lewiston

nursery; they also contracted higher infections (Eramian,

personal communication). In our study, however, the contact angles on needles from the Lewiston nursery were

significantly higher than those from the Coeur d’Alene

nursery suggesting either that nursery regimes have changed

since the mid 1990s or that is the contact angle of water

droplets is not informative with regard to predicting levels

of infection with blister rust fungus. We did not, however,

test these hypotheses in this study.

We hypothesized that the amount of wax on the needle

surface would be associated with needle wettability. Surface

waxes on needles of Scots pine are more hydrophobic than

the cuticle itself (Cape 1983), but the amount of wax is not

critical to the hydrophobic properties of a leaf surface (Silva

Fernandes 1965; Holloway 1969a and 1969b). In our study,

the amount of extracted surface wax, expressed over needle

dry weight, varied among nurseries and did appear to be

associated with differences in needle wettability.

Genetics Study

Perhaps the most noticeable (and potentially the most

important) finding came from the genetics study, in which

the susceptible families had larger and rounder stomata and

smaller contact angles of surface water droplets than either

or both the resistant families and the seed orchard bulk lots.

Gansel (1956) investigated seven white pine needle traits

based on the hypothesis of direct penetration of C. ribicola

germ tubes through the epidermis but found no differences

in the traits between four uninfected (but untested) and four

susceptible individual trees growing in natural stands. Our

results are generally consistent with Gansel’s findings for

needle traits other than stomatal size.

Stomatal Traits—Stomatal width and area may be important and potentially definitive traits that distinguish

trees with “reduced needle lesion frequency” from those that

are genetically susceptible. It has been suggested that the

reduced needle lesion frequency may be related to occlusion

of stomata by wax, which was hypothesized to reduce the

chance of infection of Pinus strobus L. by C. ribicola (Patton

and Johnson 1970; Patton and Spear 1980). However, we

found no evidence that resistant families had different

proportions of stomatal occlusion compared to susceptible

families. Most of the observed stomatal chambers were

occluded in all sampled families and lots.

Surface Waxes—At the time we sampled the seedlings,

which was 3 years after inoculation, we found no differences among families and bulked lots in the proportions of
152 USDA Forest Service Proceedings RMRS-P-32. 2004

Woo, Fins, and McDonald Genetic and Environmentally Related Variation in Needle Morphology of Blister Rust...

degraded epistomatal wax on needles. It is possible that

there were differences among families in the amounts of

degraded wax and/or the proportions of occluded stomata

at the time the seedlings were inoculated, possibly due to

differences in wax chemistry or different melting temperatures. Alternatively, environmental factors may play a

greater role than genetic differences in epistomatal wax

degradation and stomatal occlusion (Cape 1983).

Needle Wettability—Previous research has indicated

that the most important factor in basidiospore germination

and needle infection of Pinus strobusL. with C. ribicolais the

distribution and amount of free moisture on the needle

surface during incubation (Spaulding and Rathbun-Gravatt

1926; Hansen and Patton 1977). However, our finding of

larger contact angles of the water droplets on needles of the

resistant families compared to both the susceptible families

and the R.T. Bingham Seed Orchard bulked lots, whose

contact angles were similar to each other, suggests relatively more moisture was retained on needles of the resistant families. Unless wettability changed differentially

among the families from the time of inoculation to the time

they were sampled, infectability does not appear to be a

simple function of needle wettability. We note too that trees

in the R.T. Bingham Seed Orchard were selected for resistance mechanisms other than low needle lesion frequency

(although many of the trees exhibit this resistance trait as

well) (Bingham 1983). Thus, if there were a relationship

between needle wettability and specific resistance mechanisms, it would not be surprising to find differences in

wettability between the Moscow bulks and the resistant

families.

Summary and Conclusions _______

Most of the needle traits examined in these studies exhibited significant phenotypic plasticity, varying across samples

of the same genetic stock grown in three nurseries. But only

a few of the traits varied genetically (that is, across stocks of

different rust resistance types grown in a single nursery).

Although not tested in this study, our results suggest that

variation in stomatal traits and/or the characteristics of

surface water on pine needles may be critical features in the

dynamics of blister rust infection. If some nursery regimes

produce seedlings with needle surface characteristics similar to those of resistant genotypes, such seedlings may

exhibit lower initial infection rates when planted under field

conditions, even in “high rust hazard” areas. Nonetheless,

such an effect would likely be short-lived as the needles

produced in the nursery senesced and new ones were produced under field conditions. Furthermore, seedlings produced under these nursery regimes would be unsuitable for

use in rust screening procedures, as initial infection is

required for detection of most of the rust resistance traits.

Whether the variations in needle surface properties we

observed are associated with differences in infectability by

white pine blister rust has yet to be determined. Differences

in infectability are not likely to be associated with stomatal

occlusion, which did not vary among nurseries or between

resistant and susceptible families. Larger wax deposits were

associated with higher contact angles and lower wettability

on western white pine needle surfaces. This apparent

relationship may reflect a broader or more even distribution

of wax as the quantities increased. Factors such as chemical

composition of the waxes or surface roughness may play an

important role in surface wettability and may be related to

fungal spore attachment and germination. The physiological condition of seedlings may also be related to infectability

because C. ribicola absorbs nutrients from its hosts.

Further exploration of nursery environments and growth

regimes for effects on the morphology of seedling needles is

warranted. Additional studies are needed to explore the

relationships between the chemical and physical nature of

needle surfaces (particularly at the time of inoculation) and

their infectability by the blister rust fungus. Finally, the

initial finding of differences in stomatal size and shape

between resistant and susceptible families should be more

fully explored and verified using a larger number of families.

References _____________________

Bingham, R.T. 1983. Blister rust resistant western white pine for

the Inland Empire: The story of the first 25 years of the research

and development program. General Technical Report INT-146,

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Ogden, Utah. 45 p.

Blair, R.L., and Cowling, E.B. 1974. Effect of fertilization, site, and

vertical position on the susceptibility of loblolly pine seedlings to

fusiform rust. Phytopathology 64:761-762.

Cape, J.N. 1983. Contact angles of water droplets on needles of Scots

pine (Pinus sylvestris) growing in polluted atmospheres. New

Phytol. 93:293-299.

Elstner, E.F., Osswald, W., and Youngman, R.J. 1985. Basic mechanisms of pigment bleaching and loss of structural resistance in

spruce (Picea abies) needles: Advances in phytomedical diagnostics. Experientia 41:591-596.

Eramian, A. Personal communication. 1997. USDA Forest Service,

Coeur d’Alene nursery, Coeur d’Alene, Idaho. U.S.A.

Fins, L., Byler, J., Ferguson, D., Harvey, A., Mahalovich, M.F.,

McDonald, G., Miller, D., Schwandt, J. and Zack, A. 2001. Return

of the Giants. Idaho Forest, Wildlife, and Range Experiment

Station Bulletin 72, University of Idaho, College of Natural

Resources, Moscow, ID. 20p.

Fins, L., Byler, J., Ferguson, D., Harvey, A., Mahalovich, M.F.,

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needles from blister rust- resistant and non-resistant western

white pine. M.S. Thesis, University of Idaho, 24 p.

Haines, B.L., Jernstedt, J.A., and Neufeld, H.S. 1985. Direct foliar

effects of simulated acid rain. II. Leaf surface characteristics.

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inoculation of Pinus strobus with Cronartium ribicola. Phytopathology 67:1108-1112.

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in wheat. J. Agr. Res. 39:929-948.

Hirt, R.R. 1938. Relation of stomata to infection of Pinus strobus by

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rust resistance of white pines. Eur. J. For. Path. 10:307-316.

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resistance in the second generation. USDA For. Serv. Res. Note

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with atmospheric pollution. p. 321-356. In Treshow, M. (ed.). Air

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California. 921 p.

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relations of pine needles. Nature 198:770-771.

Mahalovich, M.F., and Eramian, A. 1995. Breeding and seed orchard plan for the development of blister rust resistant white pine

for the Northern Rockies. Draft tree improvement plan. USDA

Forest Service Northern Region. 59 p.

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reduced spotting vs. blister rust on western white pine in British

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needles of eastern white pine by Cronartium ribicola. Phytopathology 60:977-982.

Patton, R.F., and Spear, R.N. 1980. Stomatal influences on white

pine blister rust infection. Phytopathologia Mediterranea 19:1-7.

Rossi, V., Giosue, S., and Racca, P. 1999. A model integrating

components of rate-reducing resistance to Cercospora leaf spot in

sugar beet. J. Phytopathology 147:339-346.

Rowan, S.J., and Steinbeck, K. 1977. Seedling age and fertilization

affect susceptibility of loblolly pine to fusiform rust. Phytopathology 67: 242-246.

Rust, M. (1998). Performance of Moscow Seed Orchard lots in

artificial inoculation trials. Twenty-second Progress Report of

the Inland Empire Tree Improvement Cooperative. University of

Idaho, Moscow, Idaho 83843. 130p.

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SAS Institute Inc., Cary, N.C.

Schmidt, R.A., Foxe, M.J., Hollis, C.A., and Smith, W.H. 1972. Effect

of N, P, and K on the incidence of fusiform rust galls on greenhouse-grown seedlings of slash pine. Phytopathology 62: 788

(Abstr.).

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Surface waxes in relation to water-repellency. Ann. Appl. Biol. 56:

297-304.

Spaulding, P.C., and Rathbun-Gravatt, A. 1926. The influence of

physical factors on the viability of sporidia of Cronartium ribicola

Fischer. J. Agric. Res. 33:397-433.

Trimbacher, C., and Eckmullner, O. 1997. A method for quantifying

changes in the epicuticular wax structure of Norway spruce

needles. Eur. J. For. Path. 27:83-93.

Woo, K-S. 2000. Variation in needle morphology of blister rust

susceptible and resistant western white pine. Ph.D. dissertation,

University of Idaho, Moscow. 161 p.

Woo, K-S., Fins, L., McDonald, G.I, Wenny, D.L. and Eramian, A.

(2002). Effects of nursery environment on needles morphology of

Pinus monticolaDougl. and implications for tree improvement

programs. New Forests 24:113-129.

Woo, K-S., Fins, L., McDonald, G.I, and Wiese, M.V. 2001. Differences in needle morphology between blister rust resistant and

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154 USDA Forest Service Proceedings RMRS-P-32. 2004

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

The authors are with the USDA Forest Service, Dorena Tree Improvement Center, 34963 Shoreview Road, Cottage Grove, Oregon, 97424, USA.

Andrew Bower’s current address is Forest Sciences Department, University

of British Columbia, Vancouver, B.C. V6T 1Z4 CANADA, Phone: (604) 822-

1951, Fax: (604) 822-9102, Email: adbower@interchange.ubc.ca.

Abstract—An evaluation plantation of western white pine (Pinus

monticola) was established in southwestern Oregon in 1991 using

3-year-old seedlings. The planting was comprised of 98 full-sib

families from Dorena seed orchards and 42 wind-pollinated families

from parents in natural stands from a wide range in elevation,

latitude, and longitude. All parent trees had previously been selected for above average resistance to white pine blister rust (caused

by Cronartium ribicola) in seedling testing at Dorena. Growth,

survival, and level of blister rust infection were assessed in 1993 at

age 5 and in 1998 at age 10. Overall survival in 1993 and 1998 was

72.1 and 66.7 percent, respectively, and 7.5 percent of the trees were

infected with blister rust by 1998. Significant differences were

found between families within each seed type (both seed orchard

and natural stand seed collections) for mean height increment and

survival percentage, but not for rust infection. Within a subset of

families from the Rogue River National Forest, significant differences were found between seed types for both height increment and

survival percentage, and differences in infection percentage were

nearly significant. Families from this forest originating from orchard seed were found have larger height increment (93.03 vs. 70.71

cm), higher survival (73.8 vs. 61.4 percent), and lower infection (6.0

vs. 9.6 percent) than trees from wild seed. In a stepwise regression,

height increment of trees from orchard seed was only associated

with seed weight, but trees from wild seed sources local to the

planting site (when northerly and easterly sources were excluded)

were only associated with latitude of the female parent. Large

differences in height among families provide good potential for

future selection in growth. Despite very slow initial growth, the

plantation is progressing on a trajectory that suggests that we have

successfully regenerated the site. Recent site visits indicate little

additional mortality, low rust infection and increasing annual

growth as well as some cone production on this site which was

problematic for previous Douglas-fir (Pseudotsuga menziesii) reforestation efforts.

Key words: western white pine, Pinus monticola, white pine

blister rust, Cronartium ribicola, height growth,

survival, geographic location

Eight-Year Growth and Survival of a Western

White Pine Evaluation Plantation in the

Southwestern Oregon Cascades

Andrew D. Bower

Richard A. Sniezko

Introduction ____________________

Western white pine (Pinus monticola Dougl.) was once a

much larger component of forests in the Pacific Northwest

than it is today. Historically and ecologically, western white

pine has been considered important in the Pacific Northwest, yet information on its status is limited (Goheen 2000).

In areas of Idaho and the Inland Empire, the white pine

cover type spanned an area of five million acres (Fins and

others 2001). However, due to logging, white pine blister

rust (caused by Cronartium ribicola J.C. Fisch. in Rabenh.),

mountain pine beetle (Dendroctonus ponderosae Hopk.) attack, and the exclusion of fire, it now is a minor component

throughout its range with only 5 to 10 percent of the original

five million acres of white pine cover type in the inland

northwest still carrying a significant component of white

pine (Fins and others 2001). A comparison of surveys done in

1957 and the mid-1990s in southwest Oregon showed a drop

from 60 to 40 percent in the number of plots with 5-needle

pines, and a drop in pine cover of five percent in 10 years

from the 1980s into the 1990s (Goheen 2000).

Many areas where western white pine dominated historically are now populated by stands of Douglas-fir (Pseudotsuga

menziesii (Mirb.) Franco.), grand fir (Abies grandis (Dougl.)

Lindl.), and western hemlock (Tsuga heterophylla (Rafn.)

Sarg.). Western white pine is more suitable for wetter, root

disease-prone sites (Harrington and Wingfield, 1998) because it is less susceptible to laminated root rot (Phellinus

weirii), Annosus root rot (Heterobasion annosum) (Hadfield

and others 1986), and insect attacks than Douglas-fir and

grand fir (Fins and others 2001). Western white pine is also

more tolerant to drought than western hemlock (Fins and

others 2001) and is among the most tolerant conifers to frost

(Burns and Honkala 1990). Despite these adaptive advantages, western white pine has been largely ignored by

managers in Oregon and Washington as a species to plant in

burned and harvested areas because of white pine blister

rust. Despite the availability of resistant stock, western

white pine has not achieved its reforestation potential or

been used in restoration plantings, even on lands where

historically it was present.

White pine blister rust has caused widespread mortality,

but even in areas of heavy infection, a low percentage of trees

remained disease free (Bingham and others 1953). The

presence of naturally resistant stock formed the basis for an

operational breeding program to produce genetically resistant western white pine that began in the USDA Forest

Service Region 6 (Oregon and Washington) in the late 1950s,

and has been based at the Dorena Genetic Resource Center
USDA Forest Service Proceedings RMRS-P-32. 2004 155

Eight-Year Growth and Survival of a Western White Pine Evaluation Plantation in the Southwestern Oregon Cascades Bower and Sniezko

(Dorena) since 1966 (Sniezko 1996, Sniezko and others these

proceedings).

Screening for rust resistance with artificial inoculation

has revealed several different resistant mechanisms in

western white pine (Struckmeyer and Riker 1951, Hoff

1984, 1986, Hoff and McDonald 1971, 1980, McDonald and

Hoff 1970, 1971, McDonald 1979; Kinloch and others 1999).

Field plantings have also shown differences in rust infection

among families (Sniezko and others 2000, Sniezko and

others, this proceedings), and breeding programs have resulted in offspring with increased resistance to white pine

blister rust. The ultimate goal of these breeding programs is

to produce stocks for reforestation that will be able to survive

despite the presence of white pine blister rust.

The site described in this paper was planted with the

purpose of assessing growth as well as rust resistance for

families from a range of geographic origins in Oregon and

Washington (fig. 1). Little information is available on family

variation in growth and survival of western white pine in

southwestern Oregon. Most of the seedlots included in this

field planting represent the part of southwestern Oregon in

which the planting occurs. The additional seedlots from

throughout Oregon and Washington will provide basic information on adaptability of the species.

This report has two objectives: to present our results

regarding the magnitude and potential sources of genetic

variation in growth, survival, and blister rust infection in a

field planting, and to use these results to illustrate that

despite high rust hazard in many localities, there are areas

in which western white pine is a suitable choice for reforestation, especially where other species may be unsuccessful.

Materials and Methods ___________

Study Site Description

A 10-acre site in southwestern Oregon that had been

logged in the early 1980s was chosen for the evaluation

planting. The site is located on the Butte Falls Ranger

District on the Rogue River National Forest at an elevation

of approximately 1230 m (4035 ft) (fig. 1). The site is located

in a white fir (Abies concolor (Gord. & Glend.) Lindl.)/Shasta

red fir (Abies magnifica A. Murr.) mixed conifer plant association with an ENE aspect and gentle slope (10 percent).

The soil is a sandy clay grading from relatively deep on the

south and east to very rocky on the north half of the unit.

The surrounding overstory is composed of Douglas-fir,

white fir, englemann spruce (Picea englemannii Parry),

Shasta red fir, and western white pine. Understory regeneration includes golden chinquapin (Chrysolepsis

chrysophylla (Dougl.) A. DC.), pacific yew (Taxus brevifolia

Nutt.), white fir, Shasta red fir, Douglas-fir, englemann

spruce, western white pine, and some ponderosa pine

(Pinus ponderosa Laws), with Ribes (the alternate host of

C. ribicola in N. America) present in the shrub population.

Although the site had been cleared for several years, there

was little grass or brush growing before the site was

planted with white pine in 1991. The location of the site and

the surrounding canopies of mature trees make the site

susceptible to frost. This is illustrated by the nearly complete mortality of the original Douglas-fir evaluation

Figure 1—Map of parent test site and parent tree locations.

planting in the mid-1980s due to frost (Jim Hamlin pers.

comm.). The local plant associations along with the environmental conditions made this site a good candidate for

regeneration with western white pine.

Seed from 140 western white pine families were sown in

containers at Dorena in spring of 1988. The population

consisted of 42 wind-pollinated families from phenotypically

rust-resistant trees selected in natural stands (“wild”) from

ten national forests (table 1), and 98 controlled cross families

of clonal grafts growing in the Dorena orchards. All trees in

the Dorena orchards had previously been screened for blister rust resistance at Dorena. Resistant individuals within

families were then selected for inclusion in the orchards for

future seed production of resistant stock. Selection was

based entirely on the presence of one or more resistance

mechanisms, without regard to growth. The 42 ‘wild’ parents had also been selected as above average for rust resistance using artificial inoculation of progeny. Most seedlots

(126) represented the southwestern portion of Oregon in

which the planting site is located, but a small number (14)

of seedlots representing a wider range of Oregon and

Washington were also used. In all, 106 different female

parents were used in at least one (but up to four) different

crosses, and 66 male parents were used in usually one (but

up to five) crosses. Forty-four trees were used as both male

and female parents. Seedlings were transplanted from containers to 100 cm x 115 cm boxes with two families per box

and 45-50 trees per family after the first growing season.

They were grown outdoors for an additional two-and-one-
156 USDA Forest Service Proceedings RMRS-P-32. 2004

Bower and Sniezko Eight-Year Growth and Survival of a Western White Pine Evaluation Plantation in the Southwestern Oregon Cascades

half years. In February of 1991 seedlings were lifted and

stored near 0∞ C. until planting. Seedlings were planted in

April and May of 1991 at 3 m x 3 m spacing in a randomized

complete block design with seven replications (reps). Families were represented by four-tree or three-tree (when seedlings were limited) row plots. Weight per 100 seed (in grams)

was available for 111 of 140 families.

The trees were assessed in 1993 for survival (S5), height

growth (HT5) and percentage of trees with blister rust

infection (RUST5) after two years in the field when trees

were five years old from seed. A more comprehensive assessment was done in 1998 at age 10, with growth measurements including survival (S10), incidence of rust infection

(RUST10) and tree height (HT10), and status/health measurements including the height, number, and type of blister

rust cankers, canker activity (active or inactive), damage

and severity of damage. To help remove differences in early

height growth that may have been influenced by differences

in seed weight, height increment from 1993 to 1998 was used

to test for differences in growth. Only results on height

increment from age 5 to age 10 (HTINCR), survival, and

infection are presented here.

Statistical Analysis

The GLM, REG and CORR procedures of the SAS system

were used for all statistical analyses (SAS Institute 1989).

For height increment, individual tree data were used in an

analysis of variance (ANOVA) to test for differences between

reps and families for each seed type (orchard origin and wild

stand origin) separately with the following model:

Yijk = m + Ri + Fj

 + RFij + eijk

Where m is the overall mean, Ri is the effect of the ith

replication, Fj is the effect of the jth family, RFij is the

interaction of rep by family, and eijk is the error term. The rep

and family effects were both tested using the rep-by-family

interaction as the error term.

Plot means (using family rep means) were used in a

separate ANOVA to test for differences in survival and rust

infection using a similar model.

Due to the differences in distribution between orchard and

wild families from different forests (see Figures 4 and 5, and

Table 1), we could not test the significance of the differences

in height growth, survival, and infection between seed types

using data from all families. The Rogue River National

Forest was the only seed origin location that included both

orchard and wild seed types (table 1), so this subset of

families was used to test for differences between the two seed

types. For testing height increment, an additional ANOVA

was performed using the following model:

Yijk = m + Ri + Sj + RSij + F(S)jk + R(F(S))ijk + eijk

Where m is the overall mean, Ri is the effect of the ith

replication, Sj is the effect of the jth seed type (orchard vs.

wild stand origin), RSij is the interaction of rep by seed type,

F(S)jk is the effect of the kth family within the jth seed type,

R(F(S))ijk is the interaction of rep by family within seed type,

and eijk is the error term, with appropriate error terms used

as needed to test each effect. Seed type was tested with a

composite error term as determined by SAS that was approximately (RSij + F(S)jk - R(F(S))ijk - eijk). For survival and

infection percentage, the same model was used but without

the rep x family within seed type interaction and the family

within seed type term used as the error for testing the source

main effect.

Pearson correlations were calculated using family means

for five-year height increment (from 1993 to 1998) with

seed weight, as well as between height increment, survival,

infection percentages and seed weight with latitude, longitude, and elevation of both the female and male parent

(when available). These correlations were used to examine

the relationship of source location of the mother tree with

seed weight, and the relationship of height growth with both

mother tree location and seed weight for all orchard and wild

seedlots. Infection and survival percentages were calculated

on a family mean basis by rep, then averaged for each family.

All trees killed by something other than blister rust were

excluded when calculating infection percentage.

Families from the Colville and Wenatchee National Forests are from the eastern side of the Cascade Range, and the

Colville families are also geographically disjunct from the

Table 1—Number of parent trees by source and elevation range (in meters).

Map #a Source Seed type No. of females Elevation mean Elevation range

3 Gifford Pinchot NF Orchard 1 1325

5 Mt. Baker-Snoqualmie NF Orchard 2 1250 1140-1355

6 Mt. Hood NF Orchard 1 1200

10 Rogue River NF Orchard 23 1150 800-1463

Wild 26 1450 900-1900

11 Siskiyou NF Wild 6 1150 985-1290

14 Umatilla NFb Wild 2 n/a

15 Umpqua NF Orchard 34 1390 1310-1480

17 Wenatchee NF Wild 2 990 930-1050

18 Willamette NF Orchard 32 1075 645-1385

21 Colville NF Wild 6 1060 800-1415

Roseburg BLM Orchard 3 n/a

Eugene BLM Orchard 2 n/a

a

 Refers to national forest number as shown in figure 1.

b

 Latitude and longitude data not available.
USDA Forest Service Proceedings RMRS-P-32. 2004 157

Eight-Year Growth and Survival of a Western White Pine Evaluation Plantation in the Southwestern Oregon Cascades Bower and Sniezko

other families in this test which are on the western side of the

Cascades (see fig. 1). Geographic and environmental differences of the source locations of these families may strongly

influence the correlations of height growth with seed weight,

latitude, longitude, and elevation. Therefore, these correlations were recalculated excluding these families.

To better understand the relationship between height and

seed weight and parental location, regression was used

twice; once for all families and once excluding the families

from the Colville and Wenatchee National Forests. Variables with moderate correlations were included in stepwise

simple regressions with an alpha = 0.05 level used for

inclusion and retention in the model. PROC REG (SAS 1989)

with height increment for both seed types as the independent variable and latitude, longitude, and elevation of the

female parent, and seed weight as the independent variables

was used in the regression analyses.

Results and Discussion __________

Overall Infection and Survival

Mean survival over all families in 1998 (S10) was 66.7

percent (a slight decrease from 72.1 percent in 1993) with a

range in family means from 28.6 to 96.4 percent (fig. 2).

Figure 2—Distribution of survival percent in 1998 for 140

families.

Survival by seed type (orchard (S10o) and wild (S10w)) in

1998 was 69.5 and 60.2 percent, respectively, down from

1993 when it was 74.9 and 65.7 percent (table 2). Most of this

early mortality appears to be due to gophers (Marc Ellis,

pers. comm.) rather than blister rust. Survival at this site is

within the range of western white pine survival reported

elsewhere. (Parent 1998 and 1999, Bower 1987, Harrington

and others 2003, Steinhoff 1981)

Significant differences (p < 0.01) were found between reps

and families for orchard seed (S10o) and between families

only for wild seed (S10w) for survival percentage. Rep mean

survival percentage for orchard seed ranged from 61.7 to

73.5 percent with a general trend of the reps lower on the

slope having lower survival than the higher reps. For wild

seed, mean rep survival percentage ranged from 56.0 to 69.0

percent with a similar but weaker trend. The mean infection

percentage for both orchard and wild seed was approximately 7.5 percent, with a range in family means from 0 to

31.25 percent. Significant differences in infection percentage were found only between reps for orchard seed. Neither

reps nor families were significantly different for wild seed.

Although reps were different for orchard seed, there was no

clear pattern among reps that indicated that infection was

higher or lower at a specific position on the slope. The lack

of differentiation for infection is most likely due to the low

level of infection present on this site.

Overall Height Growth

Mean height in 1993 (two years after planting and five

years from seed) and in 1998 for both seed types is presented

in table 2. When all families are included, overall mean

height increment (HTINCR) was 87.2 cm, with a mean for

the orchard families (HTINCRo) of 92.2 cm and a mean for

the wild families (HTINCRw) of 75.7 cm. Height increment

ranged from 72.6 cm in rep 1 to 106.9 cm in rep 7 for orchard

seed and from 65.5 cm in rep 1 to 90.7 cm in rep 7 for wild

seed. Height increment was significantly different (p<0.01)

for rep, family, and the rep x family interaction for both

orchard and wild seed. Clear differences in height growth

were visible between reps with height increment increasing

from bottom to top of the slope. This would be expected due

to the pooling of cold air lower on the slope. Tree heights at

the time of planting are unavailable, so height increment

Table 2—Mean survival percent, height growth, and seed weight by seed

type.

Orchard Seed Wild Seed

Variable n mean n mean

Survival 1993 98 74.9% (1.12) b 42 65.7% (2.18)b

Survival 1998 98 69.5% (1.17) 42 60.2% (2.28)

Height 1993 (cm) 98 36.78 (0.53) 42 30.42 (0.86

Height 1998 (cm) 98 128.93 (1.88) 42 106.65 (3.07)

Height Increment 98 92.2 (1.48) 42 75.73 (2.45)

Seed Weighta 70 2.376 (0.046) 41 2.13 (0.075)

a

 Seed weight in grams, weights were not available for all seed lots.

b

 Standard errors are in brackets

60

50

40

30

20

10

0

0 20 30 4 10 0 50 60 70 80 90 100

Survival %

Number of Families
158 USDA Forest Service Proceedings RMRS-P-32. 2004

Bower and Sniezko Eight-Year Growth and Survival of a Western White Pine Evaluation Plantation in the Southwestern Oregon Cascades

during the first two years in the field cannot be compared

with height increment during the next five years. Although

growth has been slow, site visits in summer of 2001 showed

that growth had begun to accelerate, and many trees appeared to be at the age where rapid growth was commencing.

Early height growth of western white pine is relatively slow

compared with other white pine species until about age 10 to

15 when it begins to accelerate rapidly. This onset of rapid

growth is usually later in natural stands than in plantations, but growth in both can continue at 30 – 90 cm per year

for more than 100 years (Bingham and others 1972). The

range among families in height increment was 50.27 to

138.25 cm which supports previously reported results showing considerable genetic variation in height growth of this

species. Significant differences between family mean height

have been reported in several studies for both controlledcross and open-pollinated western white pines <15-years-old

(Rehfeldt and Steinhoff 1970, Steinhoff 1979, Bower and

Yeh 1988). These reports agree with earlier results that

showed that although western white pine is highly variable,

most of the variation is related to individual trees within a

family or stand (Rehfeldt and Steinhoff 1970, Hanover and

Barnes 1969). The range in height growth that was observed

on this site indicates a good potential for selection opportunities in the future for growth, in addition to the rust

resistance for which these families were originally selected.

Orchard vs. Wild Seed Source Type

Comparison

Height Growth and Survival—Using only the subset of

families from the Rogue River National Forest, significant

differences were detected between orchard and wild seed for

both height increment (p = 0.0002) and survival percentage

(p = 0.0017). The orchard seedlots had greater height increment (93.0 vs. 71.6 cm) and higher survival (73.8 vs. 61.4

percent) than the wild seedlots. The differences between

orchard and wild seedlots could be the result of a number of

factors, including differences in the geographic ranges (even

within the Rogue River N.F.) of the parents of these families

(see Figures 4 and 5). Sites in southwestern Oregon are very

diverse, covering a wide range of elevations, rainfall, and soil

types, all of which most likely differ from the more stable

environmental conditions experienced by the families from

the orchard. In addition, orchard seed are generally harvested at the point of optimum ripeness before the cone

scales begin to flare. Cones collected from wild stands may

have been harvested at a point when they were accessible

and available but may not have achieved the same ripeness.

Potential differences in seed maturity may also contribute to

the differences between orchard and wild seed (Jerry Berdeen,

pers. comm.)

Infection—Using the same subset of families from the

Rogue River National Forest, differences between seed types

in infection percentage approached significance (p = 0.0854)

with the wild seed having a higher level of infection than the

orchard seed (15.9 vs. 9.3 percent). The seedlots from the

orchard would be expected to show higher rust resistance

since both the male and female contribution to the seed

would be from resistant parents.

Seed Source Location Effects

Height growth—Previous reports of ecotypic variation

in western white pine have been inconsistent. Squillace and

Bingham (1958) reported that progeny from high elevation

parents were shorter at four years than progeny from lower

elevation parents within the same watershed when grown

together at a low elevation site, but they were taller when

grown on a high elevation site. Evidence of local differentiation at this small scale has not been substantiated by other

research (Rehfeldt 1979); however, differentiation has been

found on a larger scale. Steinhoff (1979) found that seedlings

from higher elevation parents grew slower than seedlings

from lower elevation parents at low- and mid-elevation sites,

but he did not find them to be taller at high elevation sites.

Townsend and others (1972) found no evidence of racial

differentiation in monoterpenes, photosynthesis, or growth

for 4-year-old seedlings, despite marked differences in elevation and geographic separation of certain sources.

In contrast, other studies have shown patterns of differentiation among western white pine populations. Results from

these studies indicate that differences in height growth,

isozymes, and cold hardiness have separated the western

white pine range into a relatively small southern population

(restricted to the Sierra Nevada mountains in California)

and a broad northern population (covering the northern part

of the species distribution, including the Washington Cascades), with a transition zone in the Southern Cascades and

Warner Mountains in Oregon, but with no differentiation

related to the elevation of the seed source (Steinhoff and

others 1983, Rehfeldt and others 1984, and Meagher and

Hunt 1998). The northern populations are characterized by

relatively high growth potential and low cold hardiness,

while the southern populations have lower growth potential

and higher hardiness, with the transition zone population

from southern Oregon (Cascades and Warner Mountains)

arranged along a steep latitudinal gradient linking the two

populations. In a western white pine provenance test in

western Washington, the more southerly high elevation

Figure 3—Scatterplot of latitude vs. family mean height

increment by forest* for 44 families from wild seed.

*see table 1 for abbreviations

40

60

80

100

120

41 43 45 47 49

Latitude (degrees N.)

Height Increment (cm)

WEN. COL

GP MTB-5; MTH

RR SIS; UMP; WIL
USDA Forest Service Proceedings RMRS-P-32. 2004 159

Eight-Year Growth and Survival of a Western White Pine Evaluation Plantation in the Southwestern Oregon Cascades Bower and Sniezko

sources were dramatically shorter than both northern and

inland sources (Richard Sniezko, pers. comm.). Rehfeldt and

others (1984) reported that an apparent relationship between seedling height and elevation for populations from

this transition zone is derived from the strong correlation of

latitude and elevation. Campbell and Sugano (1989) found

similar trends across populations, as well as for families

from within the transition zone, which they attributed to

steep precipitation gradients. They also found, as others

have, that most of the variation in western white pine is

among individuals within a population, with only small

amounts occurring between populations. Similarly, when

our data is separated by seed type and forest of origin, for the

four groups with more than six female parents represented

(table 1), we found that families were significantly different

in all cases.

Our correlations of growth with latitude and elevation

reflect a similar relationship across populations, especially

for the wild seed (table 4), even when the more northerly and

easterly populations are excluded (table 5), (so that the

remaining families fall within the “transition zone” described above). A plot of seed source elevation vs. latitude

Table 3—Correlation of height growth with seed weight for 111 families.

Seed Weight

Overall Orchard Seed (n = 70) Wild Seed (n = 41) Wild Seed* (n = 33)

Height 1993 –0.07 –0.13 –0.37b –0.14

Height 1998 –0.17a –0.26b –0.53c –0.34a

Height Increment –0.19b –0.28b –0.51c –0.33a

* Correlation excluding Colville and Wenatchee families

a

 significant at 10% level

b

 significant at 5% level

c

 significant at 1% level

Table 5—Family mean correlations of survival, infection,

growth and seed weight with maternal location

for wild seed, excluding Wenatchee NF and

Colville NF.

Wild Seed

Lat. Long. Elev.

Survival % 0.25 0.55b 0.28

Infection % –0.08 0.13 0.07

Height Increment 0.25 0.40a –0.19

Seed Weight –0.06 0.01 0.01

a

 significant at 5% level

b

 significant at 1% level

Table 4—Family mean correlations of survival, growth and seed weight with location, by parent and

seed source type for 133 Families*.

Orchard Seed (n=93*) Wild Seed (n=40*)

Lat. Long. Elev. Lat. Long. Elev.

Survival % – F** –0.06 0.06 –0.06 0.29 0.40b 0.07

Height Incr. – F 0.08 0.22a –0.11 0.71b 0.76b –0.41

Height Incr. – M** 0.13 0.23a –0.06 N/a n/a n/a

1993 Height – F 0.23a 0.40b –0.14 0.62b 0.64b –0.48b

1993 Height – M 0.27b 0.36b –0.10 N/a n/a n/a

1998 Height – F 0.13 0.28b –0.12 0.75b 0.79b –0.46b

1998 Height – M 0.18 0.28b –0.07 N/a n/a n/a

Seed Weight – F –0.18 –0.24 –0.12 –0.53b –0.49b 0.26

Seed Weight – M –0.11 –0.15 –0.14 N/a n/a n/a

* Latitude, longitude, elevation, and seed weight not available for all seedlots

** F = correlation with female location, M = correlation with male location

a

 significant at 5% level

b significant at 1% level
160 USDA Forest Service Proceedings RMRS-P-32. 2004

Bower and Sniezko Eight-Year Growth and Survival of a Western White Pine Evaluation Plantation in the Southwestern Oregon Cascades

Figure 5—Latitude vs. longitude of female parents of

orchard and wild seed families.

Figure 4—Latitude vs. elevation of female parents by

seed type.

(fig. 4) shows a significant negative relationship for wild

seed (r = –0.49, p< 0.01) indicating that the northern

sources in this study are generally from lower elevations

than southern sources. With the Colville and Wenatchee

National Forest sources excluded, the relationship is weaker

and still nearly statistically significant (r = –0.35, p = 0.051).

The plot also shows that when the more northerly sources

are excluded, the remaining families come from a range of

latitudes that is much more limited than the orchard seed,

and these two groups should not be viewed as paired samples

since they represent different parts of the ranges with only

partial overlap.

An examination of a plot of the origin of the seedlots

(fig. 5) shows that orchard and wild parents cover different

ranges in geographic location. There are strong relationships between latitude and longitude for both orchard families (r = 0.79, p<0.01) and wild families (r = 0.92, p<0.01).

When the northerly families are excluded, the correlation for

the wild families drops dramatically (r = 0.39), although it is

still significant (p = 0.02). It is likely that the significant

correlations between growth traits and longitude and

elevation actually can be explained by differences associated

with latitude, and our results suggest that height growth

may be associated with latitude, at least within families

from the Cascade Range in Oregon and Washington.

Seed Weight Effects—Previous studies have reported

that total height for western white pine in the first several

years of seedling growth is positively correlated with seed

weight (Squillace and Bingham 1958, Squillace and others

1967). Squillace and Bingham (1958) found that seed weight

had a variable effect on parent-progeny growth correlations.

In our study, HT10o and HTINCRo both had significant but

low negative correlations with seed weight (r = –0.26 and –

0.28; p = 0.033 and 0.020, respectively, Table 3). The correlation for HT5o was also negative, but weaker and nonsignificant (r = –0.13; p = 0.273). All three height growth

variables for the wild seed had significant negative correlations with seed weight with moderate r-values (r-values

ranged from –0.37 to –0.53).

However, using all families for each seed type, seed weight

was also negatively correlated with latitude and longitude of

the female parent, significantly so for the wild seed. In a

stepwise regression of family mean height increment on

seed weight, latitude, longitude, and elevation of the female

parent for orchard seed, only seed weight was significant in

the model (p = 0.023), while for the wild seed, only longitude

was significant (p<0.01) in the model. Latitude and longitude of the wild seed are highly correlated (r = 0.922), and the

Colville families are more disjunct in longitude than latitude

compared to the other wild families (see Forest 21 on fig. 1).

When the regression was redone excluding the families from

the Wenatchee and Colville National Forests, only latitude

was significant in the model (p< 0.01). Although seed weight

is correlated with height growth, the correlation is probably

a function of maternal origin which is reflected by latitude of

the mother tree. Heavier (larger) seed would be expected to

produce larger seedlings, therefore the correlations of seed

weight and height increment should be positive, as has been

reported previously (Squillace and Bingham 1958, Squillace

and others 1967). However, the correlation we observed was

negative. This would indicate that wild seed from higher

latitudes is smaller but the seedlings grow faster. Therefore,

we feel that it is likely that the difference in height growth

between orchard families and wild families is not due to

differences in seed weight, but due to genetic effects associated with the source of origin of the parents.

Conclusions____________________

Despite its desirable growth and adaptive advantages,

white pine is underutilized for reforestation or restoration in

Oregon and Washington due to potential losses from white

pine blister rust and the limited availability of resistant

seedlings. Our results from a young (10-year-old) western

white pine plantation on a site with low rust-hazard (but

high frost-hazard) show moderate to high survival among

families, with significant differences between families, and

low rust infection through age 10. Even though rust infection levels were low, orchard seedlots showed lower infection

500

1000

1500

2000

41 43 45 47 49

Latitude (degrees N.)

Elevation (meters)

Orchard

Wild

41

42

43

44

45

46

47

48

49

125 123 121 119 117

Longitude (degrees W.)

Latitude (degrees N.)

Wild Seed

Orchard Seed

WASHINGTON

OREGON
USDA Forest Service Proceedings RMRS-P-32. 2004 161

Eight-Year Growth and Survival of a Western White Pine Evaluation Plantation in the Southwestern Oregon Cascades Bower and Sniezko

than wind-pollinated wild seedlots from resistant parents,

as expected. Although growth has initially been slow, observations in summer 2001 by the authors indicate that many

trees appear to be entering the rapid phase of growth typical

of western white pine. We found strong family differences in

height growth that appear to be associated with seed weight

and latitude of the parent trees, and also found differences

between progeny from orchard and wild trees from similar

geographic origins. Families from parents originating from

the more northerly latitudes show higher average growth

potential than families from more southerly latitudes, although the best families from southerly latitudes are comparable. These findings correspond with other results in the

literature, which distinguish three zones for western white

pine: (1) A broad zone north from northern Oregon; (2) A

southern zone in California; (3) A distinct transition zone in

the southern Oregon cascades. Current plans are to follow

this planting over time to see if the growth differences that

are apparent among sources from different latitudes will

remain and/or increase as the trees age during the period of

rapid growth, and to see if sources originating furthest from

the planting site remain vigorous. In addition, the early

results on this site show that with careful selection of

appropriate sites and utilization of resistant planting stock,

western white pine can be a viable choice for use in reforestation or restoration.

Acknowledgments ______________

The authors would like to acknowledge the work of several

past and present employees of the Dorena Genetic Resource

Center: Rob Mangold and Jude Danielson for selecting and

growing the seedlings, Bob Danchok and Sally Long for

taking the measurements on the trees, and all of the Dorena

personnel for their assistance in planning, preparation, and

planting of the site. Thanks also to Jeremy Kaufman for

construction of the map, Angelia Kegley for her careful

review of an early draft of this paper and Randy Johnson for

his review and suggestions on this manuscript.

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In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

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Danilo D. Fernando is with the Faculty of Environmental and Forest

Biology, SUNY College of Environmental Science and Forestry, 461 Illick

Hall, 1 Forestry Drive, Syracuse, NY 13210. E-mail: fernando@esf.edu.

Phone: (315) 470-6746. Fax: (315) 470-6934. John N. Owens is with the Centre

for Forest Biology, University of Victoria, Victoria, BC V8W 3N5, Canada.

Abstract—In spite of the progress made towards isolating blister

rust resistant white pines, there is still a threat from the evolution

of new pathogenic strains of blister rust in North America and/or

introduction of new virulent strains from Asia. Through interspecific hybridization with the most resistant Eurasian white pines,

resistance genes may be passed on to North American white pines,

and the gene pool for rust resistance in North America diversified.

An earlier approach using wide crosses with Eurasian white pines

was abandoned because of failure to obtain viable seeds. We believe

that wide hybridizations are possible through the removal of the

nucellus which is a probable site of incompatibility reactions. This

study aims to develop a novel approach to hybridization through in

vitro fertilization (IVF). Our work involves co-culture of Pinus

aristata female gametophytes with P. monticola pollen tubes (and

vice versa). Female gametophytes were isolated and introduced to

pollen tubes grown in culture for 2 to 3 days. Pollen tubes and female

gametophytes were then co-cultured for 6 to 10 days. Histological

analysis showed that in both types of interspecific crosses, pollen

tubes did not only penetrate the female gametophytes through the

neck cells of the archegonia, but also release their contents into the

egg cytoplasm. The inability to maintain viability of female gametophytes in culture presently precludes successful IVF. Our results

on the in vitro interspecific cross between P. aristata and P. strobus

showed the same interaction as the reciprocal cross between P.

aristata and P. monticola.

Key words: in vitro fertilization, Pinus aristata, P. monticola,

P. strobus, pollen tube, female gametophyte, interspecific hybridization

Introduction ____________________

Blister rust (Cronartium ribicola) is one of the most

destructive forest pathogens, and it affects all native North

American white pines. Infection caused by this fungus

results in the formation of large blister-like cankers on

Development of an In Vitro Technology for

White Pine Blister Rust Resistance

Danilo D. Fernando

John N. Owens

branches and the main stem leading to stunted growth and

eventually death of trees. This disease has resulted in the

significant loss of white pine timber values, but according to

Kinloch (2000), the ecological damage may even be worse.

Since the unwanted introduction of the fungus to North

America in the early 1900s, white pine breeders have been

concerned with isolating resistant trees through selection,

screening and intraspecific breeding. As a result, blister rust

resistant stocks are available (Kinloch and others 1970,

Bingham 1983, Kinloch 1992, Blada 1994, Kinloch and

others 1999). However, as new pathogenic strains develop

and/or new races of wider virulence are reintroduced from

Asia (Kinloch and Comstock 1981, MacDonald and others

1984, Kinloch and others 1996, Kinloch and Dupper 1999,

Kinloch 2000), the rust problem remains a constant threat.

Therefore, it is necessary to develop new strategies that can

be incorporated into the current breeding programs to serve

as insurance against new or different pathogenic races of

blister rust. The need to widen the spectrum of rust resistance is imperative and one such strategy is in vitro fertilization (IVF) coupled with interspecific hybridization and/or

genetic transformation (Fernando and others 1998).

The present “resistant” selection process that is underway

in North America may not impart long-term resistance.

Widening the spectrum of resistance in North American

white pines entails interspecific hybridizations with the

most resistant Eurasian white pines (Spaulding 1929,

Bingham 1972). Interspecific hybridization may not only

impart resistance genes, but may also diversify the gene pool

for rust resistance. Of the species ranked by Bingham

(1972), Pinus armandii is considered the most resistant,

followed by P. cembra and P. aristata. These species constitute a repository of resistance genes that seem advisable to

exploit in white pine breeding programs. In fact, hybrids

have been formed between P. armandii and P. lambertiana

(Stone and Duffield 1950, Heimburger 1972), and P. cembra

and two of the most susceptible but economically important

white pines, P. monticola and P. strobus (Blada 1994).

Unfortunately, P. armandii or P. aristata crossed with P.

monticola or P. strobus were all unsuccessful (Wright 1959,

Patton 1964, Bingham 1972, Bingham 1983). The cross

between P. armandii and P. monticola did not even produce

any cone (Wright 1959), and while cones were produced

between P. armandii and P. strobus, no filled seeds developed (Patton 1964).

One of the important features of IVF is its capability to bypass prefertilization incompatibility barriers (Fernando and

others 1998), and through IVF, species that do not normally

hybridize in nature may be hybridized in culture. The

ultimate aim of this project is to develop rust resistant white

pines through interspecific hybridization in vitro. Because
164 USDA Forest Service Proceedings RMRS-P-32. 2004

Fernando and Owens Development of an In Vitro Technology for White Pine Blister Rust Resistance

there are no previous works on the culture of reproductive

structures of any species of white pine that can be directly

used, the current and immediate concerns of this research

are basic. What are the nutrient and cultural requirements

for growing pollen tubes and female gametophytes of pines

in vitro? How long can pollen tubes and female gametophytes remain viable in culture? Will pollen tubes penetrate

the archegonia of female gametophytes? Will in vitro fusion

occur between two different pine species?

Materials and Methods ___________

Plant Materials

Pollen and seed cones of P. aristatawere obtained from the

University of Victoria, British Columbia, while pollen and

seed cones of P. monticola were obtained from Saanich Seed

Orchard, Saanich, British Columbia. All crosses involving

female gametophytes of P. aristata and P. monticola were

done at the Centre for Forest Biology, University of Victoria,

Victoria, British Columbia, Canada.

Pollen cones of P. strobus were collected from the SUNYESF Lafayette Experimental Station, Syracuse, New York,

while seed cones were obtained from the SUNY-ESF Heiberg

Memorial Forest, Tully, New York. Crosses involving female

gametophytes of P. strobus were done at the Department of

Environmental and Forest Biology, SUNY College of Environmental Science and Forestry, Syracuse, New York USA.

Surface Sterilization of Pollen and Seed

Cones

Pollen cones of Pinus aristata, P. monticola and P. strobus

were collected 2 to 3 days before dehiscence while seed cones

were collected at central cell stage (Fernando and others

1997). Pollen and seed cones were surface-sterilized by

washing in 70 percent ethanol, sterile distilled water, and 1

percent sodium hypochlorite for 30 seconds each step. They

were rinsed three times with sterile distilled water for 10

seconds each time, blotted dry on sterile paper towels, and

left in Petri dishes covered with sterile filter paper for 48 to

72 hours at 27∞C. Dried sterile pollen grains were collected

in sterile vials and stored at 4 ∞C for short-term or –20 ∞C for

long-term storage.

After surface sterilization of seed cones, ovuliferous scales

were separated individually using sterile forceps. Ovules

were dissected under a stereomicroscope, and the female

gametophytes were mechanically isolated and placed in

culture. Representative female gametophytes from each

seed cone used in culture were fixed in formalin-aceticalcohol. These were used to monitor the initial stage of

development and also serve as the control.

Media Composition and Co-Culture

Conditions

The basal medium contained macro- and micronutrients

and vitamins as described by Murashige and Skoog (1962),

supplemented with boric acid and calcium nitrate following

Brewbaker and Kwack (1963). The working solution was

half-strength diluted with deionized distilled water, and

supplemented with 15 percent sucrose and 0.4 percent

phytagel. The pH was adjusted to 6.0 with KOH. This

medium is referred to as MSBK.

Pollen grains were grown on MSBK and after 2 to 3 days,

freshly isolated female gametophytes were introduced at

the tips of growing pollen tubes. Viability of pollen tubes

(table 1) was based on whether they had collapsed or not,

while viability of female gametophytes (table 2) was based

on whether the central cell had undergone plasmolysis or not

(Fernando and others 1997). The co-cultures were incubated

in the dark at 23 ∞C. Several intraspecific and interspecific

crosses were done and a total of 1,200 female gametophytes

were used (table 3).

Histological Analysis

Pollen grains and tubes were examined at various stages

of development by fixing in 4 percent paraformaldehyde in

saline phosphate buffer and staining with DAPI (4’,6-

diamidino-2-phenylindole). The specimens were examined

using a Leica DMLB fluorescence microscope. A total of

1,200 female gametophytes were co-cultured with pollen

tubes. After the co-cultures were incubated for 6 to 10 days,

female gametophytes which when lifted, had firmly attached pollen tubes were fixed in 4 percent glutaraldehyde

in phosphate buffer. Specimens were rinsed with phosphate

buffer and dehydrated through a graded series of ethanol.

Table 1—Length and longevity of pollen tubes in culture (n = 50).

Species Days in culture Length of pollen tubes (mm) Viability (%)

Mean (range)

P. aristata 15 650 (530-850) 100

20 750 (690-980) 100

30 920 (840-1090) 98

P. monticola 15 600 (460-800) 100

20 760 (540-920) 98

30 800 (630-990) 95

P. strobus 15 580 (340-700) 95

20 620 (440-880) 88

30 770 (650-910) 85
USDA Forest Service Proceedings RMRS-P-32. 2004 165

Development of an In Vitro Technology for White Pine Blister Rust Resistance Fernando and Owens

Table 2—Viability of female gametophytes in culture (n = 100).

Number of viable female gametophytes

Number of days in culture P. aristata P. monticola P. strobus

2 92 85 70

4 70 61 54

6 58 49 37

8 34 23 12

10 19 12 05

Table 3—Intraspecific and interspecific crosses in vitro (n = 200).

Pollen Female gametophytes

tubes P. aristata P. monticola P. strobus

P. aristata xx x

P. monticola xx -

P. strobus -- x

x indicates intraspecific or interspecific cross; - indicates no cross was made

Specimens were gradually infiltrated with a solution containing hydroxyethyl methacrylate (Technovit 7100 embedding kit, Energy Beam Sciences Inc., MA). Sections (8 to 10

mm) were cut using a JB4 ultramicrotome, mounted on glass

slides and stained with Toluidine Blue O. Specimens were

examined under a brightfield microscope and images captured using a digital video camera (Optronics, CA).

Results and Discussion __________

Viability and Longevity of Pollen Tubes

and Female Gametophytes

Percentage pollen viability in P. aristata,P. monticola and

P. strobus were very high (table 1). Growth of pollen tubes

was maintained in vitro for 30 days with at least 85 percent

viability (table 1). Of the three species of pine examined, P.

aristata appears to be the most vigorous because of relatively greater longevity and length of pollen tubes. Our

results also show that pollen tubes of all three species

continue to elongate under in vitro conditions. This shows

that in vitro, there is no stage that corresponds to the resting

stage that occurs in vivo (Gifford and Foster 1989). Under

our in vitro conditions, the average length of pollen tubes

(table 1) attained by all three species of white pines (that is,

830 mm) is much longer than the average length of the

nucellus (that is, 700 mm) that the pollen tubes traverse

prior to reaching the archegonia in vivo.

The longevity of pollen tubes in culture makes them

suitable as targets for genetic transformation. In fact, pollen

grains are natural vectors for delivering foreign DNA since

they are involved in the normal fertilization process. Transformed pollen grains are being used to artificially pollinate

flowers in several plants and these have resulted in the

formation and recovery of transgenic progenies (Häggman

and others 1997, Aronen and others 1998). This technique is

very promising because it avoids the use of elaborate and

time-consuming tissue culture steps. In white pines, the

protocols for the transformation of pollen grains and tubes

have already been optimized for P. aristata and P. monticola

(Fernando and others 2000). There are several broad-spectrum pathogenesis related genes that are available for

flowering plants (Shewry and Lucas 1997, Osusky and

others 2000, Powell and others 2000), and these need to be

tested in white pines.

In culture, the viability of female gametophytes declined

very rapidly reaching very low numbers after 10 days in

culture (table 2). The decline in the viability of female

gametophytes in P. aristata appears less drastic when compared to those of P. monticola or P. strobus (table 2). It has

long been known that unlike some other conifers, unpollinated

ovules in pine do not develop into maturity. In vivo, development of pine ovules proceeds only in the presence of germinated pollen (McWilliam 1959). This suggests that the

presence of developing pollen tubes on the nucellus provides

some stimulatory factors that are required for the maturity

of the female gametophytes. Apparently, the co-culture of

pollen tubes and female gametophytes does not have the

same effect. It will be interesting to find out if pollen tube

extracts added to the culture medium will improve the

response of female gametophytes in culture.

Interactions Between Pollen Tubes and

Female Gametophytes

When freshly isolated female gametophytes of P. monticola

were co-cultured with 2 to 3 day old P. aristata pollen tubes

(and vice versa), the pollen tubes continued to elongate

resulting in the penetration of the female gametophytes. In

several instances, pollen tubes of P. aristata entered the

canal leading to the neck cells of the archegonia in P.

monticola (fig. 1). This is similar to what has been reported

to happen in nature under intraspecific crosses (Owens and

Morris 1990). This type of penetration was also observed

between P. monticola pollen tubes and P. aristata female

gametophytes. In both types of interspecific crosses, some

pollen tubes also penetrated the female gametophytes

through the prothallial cells far from the neck cells of the

archegonia (fig. 2). Only in one instance was a pollen tube

observed to reach the neck cells of viable female gametophytes (fig. 3).

It is interesting to note that in vitro, pollen tubes formed

minute projections to penetrate between neck cells (fig. 4), as
166 USDA Forest Service Proceedings RMRS-P-32. 2004

Fernando and Owens Development of an In Vitro Technology for White Pine Blister Rust Resistance

happens when pollen tubes make contact with the neck cells

in vivo (Owens and Morris 1990). In culture, however,

formation of minute projections appeared to form not only

from the tips of pollen tubes but also from the lateral walls

as seen in P. aristata.

During co-culture, the elongating pollen tubes could have

all passed under or over the female gametophytes, but

instead many penetrated the archegonia through the neck

cells. This suggests that some sort of cellular recognition do

exists under in vitro conditions. Furthermore, some pollen

tubes that penetrated the archegonia released their contents into the egg cytoplasm (fig. 5). This suggests not only

that in vitro pollen tubes recognize their target destination,

Figure 1—Pollen tube penetrating female gametophyte

through neck cells. Figures 1-5 – Acronyms are: EC egg

cell, EN egg nucleus, FG female gametophyte, NC neck

cells, PC prothallial cells, PT pollen tube, SC starch

grains.

Figure 2—Pollen tube penetrating prothallial cells of female

gametophyte.

Figure 3—Female gametophyte with pollen tube

and unplasmolyzed egg.

Figure 4—Pollen tube tip inside plasmolyzed egg

cell; pollen tube containing starch grains.
USDA Forest Service Proceedings RMRS-P-32. 2004 167

Development of an In Vitro Technology for White Pine Blister Rust Resistance Fernando and Owens

cytoplasm. Therefore, there is a need to develop a culture

medium that is suitable to sustain growth and development

of female gametophytes, and at the same time allow the

sperm cells that are released in the egg cytoplasm to fuse

with the egg nucleus and develop into embryo.

Although in vitro fertilization was not achieved, our results are promising. If we succeed in sustaining the growth

of female gametophytes in culture, this IVF technology can

offer several novel alternative approaches such as interspecific hybridization and imparting resistance genes into pollen tubes or archegonia followed by IVF. Another benefit of

this work applies to all pines and their breeding system. The

normal life cycle from pollination to mature embryos takes

about 15 months. Through IVF, the time from “pollination”

to development of mature embryos could be shortened to 3 to

4 months.

References _____________________

Aronen, T.S., Nikkanen, T.O., Häggman, H.M. 1998. Compatibility

of different pollination techniques with microprojectile bombardment of Norway spruce and Scots pine pollen. Can J. For. Res.

28:79-86.

Bingham, R.T. 1972. Taxonomy, crossability and relative blister

rust resistance of 5-needled white pines, pages 271-280. In:

Biology of Rust Resistance in Forest Trees, Bingham, R.T., Hoff,

R.J. and McDonald, G.I. Miscellaneous Publication No 1221.

USDA, Washington DC.

Bingham, R.T. 1983. Blister rust resistant western white pine for

the inland empire: the story of the first 25 years of the research

and development program. USDA General Tech Rep INT-146.

Blada, I. 1994. Interspecific hybridization of Swiss stone pine (Pinus

cembra L.). Silvae Genet. 43:14-20.

Fernando, D.D., Owens, J.N. and Misra S. 2000. Transient gene

expression in pine pollen tubes following particle bombardment.

Plant Cell Rep. 19:224-228.

Fernando, D.D., Owens, J.N. and von Aderkas, P. 1998. In vitro

fertilization from co-cultured pollen tubes and female gametophytes of Douglas fir (Pseudotsuga menziesii). Theor. Appl. Genet.

96:1057-1063.

Fernando, D.D., Owens, J.N., von Aderkas, P. and Takaso, T. 1997.

In vitro pollen tube growth and penetration of female gametophyte in Douglas fir (Pseudotsuga menziesii). Sex. Plant Reprod.

10:209-216.

Gifford, E.M. and Foster, A.S. 1989. Morphology and Evolution of

Vascular Plants. W.H. Freeman Company, NY.

Häggman, H.M., Aronen, T.S. and Nikkanen, T.O. 1997. Gene

transfer by particle bombardment to Norway spruce and Scots

pine pollen. Can. J. For. Res. 27:928-935.

Heimburger C. 1972. Breeding of white pine for resistance to

blister rust at the interspecies level, pages 541-549. In: Biology

of Rust Resistance in Forest Trees, Bingham, R.T., Hoff, R.J.

and McDonald, G.I. Miscellaneous Publication No 1221. USDA,

Washington DC.

Kinloch, B.B. Jr. 1992. Distribution and frequency of a gene for

resistance to white pine blister rust in natural populations of

sugar pine. Can. J. Bot. 70:1319-1323.

Kinloch, B.B. Jr. and Comstock, M. 1981. Race of Cronartium

ribicolavirulent to major gene resistance in sugar pine. Plant Dis.

Rep. 65:604-605.

Kinloch, B.B. Jr. and Dupper, D.E. 1999. Evidence of cytoplasmic

inheritance of virulence in Cronartium ribicola to major gene

resistance in sugar pine. Phytopathology 89:192-196.

Kinloch, B.B. Jr., Davis, D. and Dupper, G.E. 1996. Variation in

virulence in Cronartiumribicola: What is the threat? pages 133-

136. In: Kinloch, B.B. Jr., Marosy, M. and Huddleston, M.E.

Proceedings of a Symposium presented by the California Sugar

Pine Management Committee: Sugar pine: status, values and

roles in ecosystems. Publication No. 3362, University of California, Division of Agriculture and Natural Resources, Davis,

California.

but they also react in the same way as in vivo. None of the

pollen tubes that penetrated the prothallial cells of the

female gametophytes released their contents.

Summary ______________________

Our results show that our current culture conditions are

optimum for growth and development of P. aristata, P.

monticola, and P. strobus pollen tubes. The activities of

pollen tubes as they penetrate the archegonia of the female

gametophytes in culture resemble those that have been

reported to occur in vivo. Our results on histological analysis

show similar pattern for intraspecific and interspecific crosses

(table 3). It is also important to note that the activities of

pollen tubes are not hindered by the source of the co-cultured

female gametophytes, suggesting that in vitro, no incompatibility reaction is manifested.

Sustaining growth and development of female gametophytes in culture is extremely difficult. Although we have

tried different media and supplements without success

(unpublished data), there are still countless options to try.

Because the culture medium is not optimized for female

gametophyte development, no interaction occurred after

pollen tube penetration and release of gametes into the egg

Figure 5—Contents of pollen tube such as starch grains

are in the egg cytoplasm.
168 USDA Forest Service Proceedings RMRS-P-32. 2004

Fernando and Owens Development of an In Vitro Technology for White Pine Blister Rust Resistance

Kinloch, B.B. Jr., Parks, G.K. and Fowler, C.W. 1970. White pine

blister rust: simply inherited resistance in sugar pine. Science

167:193-195.

Kinloch, B.B. Jr., Sniezko, R.A., Barnes, G.D. and Greathouse, T.E.

1999. A major gene for resistance to white pine blister rust in

western white pine from the western cascade range. Phytopathology 89:861-867.

McDonald, G.I., Hansen, E.M., Osterhaus, C.A. and Samman, S.

1984. Initial characterization of a new strain of Cronartium

ribicola from the Cascade Mountains of Oregon. Plant Dis.

68:800-804.

Osusky, M., Zhou, G.Q., Hancock, R.E.W., Kay, W.W. and Misra, S.

2000. Transgenic potatoes expressing cationic peptides confers

broad-spectrum resistance to phytopathogens. Nature Biotech.

18:1162-1166.

Owens, J.N. and Morris, S.J. 1990. Cytoplasmic basis for cytoplasmic inheritance in Pseudotsuga menziesii. II. Pollen tube and

archegonial development. Amer. J. Bot. 78:1515-1527.

Patton, R.F. 1964. Interspecific hybridization in breeding for white

pine blister rust resistance. 367-376.

Powell, W.A., Catranis, C.M. and Maynard, C.A. 2000. Design of

self-processing antimicrobial peptides for plant protection. Let.

Appl. Microbio. 31:163-168.

Shewry, P.R. and Lucas, J.A. 1997. Plant proteins that confer

resistance to pests and pathogens. Adv. Bot. Res. 26:135-192.

Spaulding, P. 1929. White pine blister rust: a comparison of European

with North American conditions: USDA Technical Bulletin 87.

Stone, E.C. and Duffield, J.W. 1950. Hybrids of sugar pine by

embryo culture. J. Forestry 48:200-201.

Wright, J.W. 1959. Species hybridization in the white pines. Forest

Science 5:210-222.
USDA Forest Service Proceedings RMRS-P-32. 2004 169

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

Sergej N. Goroshkevich is with the Russian Academy of Sciences, Siberian

Branch, Filial of the Forest Institute,Academichesky pr., 2, Tomsk, Russia,

634021. Telephone 3822-258680, Fax 3822-258855, e-mail

gorosh@forest.tsc.ru.

Abstract—A study was conducted on phenology and reproductive

characteristics of Siberian stone pine (Pinus pumila), Japanese

stone pine (Pinus pumila), and their putative hybrids in high

altitudes. The study demonstrated that the species were not reproductively isolated, and putative hybrids were identified. Seed development and production were very poor in the putative hybrids

indicating that introgression is very slowly or not occurring beyond

the first filial generation.

Key words: Pinus siberica, Pinus pumila, stone pines, reproductive biology, natural hybridization

Introduction ____________________

In the Baikal region of Siberia, the natural ranges of

Siberian stone pine (Pinus sibirica Du Tour) and Japanese

stone pine (P. pumila Regel) overlap. Siberian stone pine

grows to become a large tree and occurs in pure stands

between 600 and 1,600 m in elevation but becomes increasingly scarce as altitude increases. Japanese stone pine

generally grows as a shrub (1 to 4 m high) that can generate

adventitious roots. A single individual can often form vast

clones, up to 50 m in diameter. Japanese stone pine is an

occasional understory species or occurs in pure populations

on steep, stony slopes at altitudes between 1,000 and 1.600

m. The occurrence of this species in forest composition

increases with altitude. At altitudes of 1,600 to 2,000 m, it

forms a thick brushwood. At altitudes of 2,000 to 2,100 m

(timberline), Japanese stone pine and dwarfed, sterile Siberian stone pine occur together.

The occurrence of natural hybridization between these

species has been the subject of debate since putative hybrids

were described 70 years ago (Pozdnjakov 1952; Galazij 1954;

Molodjnikov 1975). Although natural hybridization has been

refuted in many recent reviews (Bobrov 1978; Lanner 1990;

Homentovski 1995), individual trees were recently found

north of Khamar-Daban (15 km southwest of Baikal), which

Natural Hybridization between

Russian Stone Pine (Pinus siberica)

and Japanese Stone Pine (Pinus pumila)

Sergej N. Goroshkevich

were intermediate between the species in a number of traits

including: form, growth, needle and shoot structure, and

cone color (Goroshkevich 1999). The purpose of this research

was to determine where this natural hybridization occurs

and to study reproductive characteristics and seed production in these two pine species and their putative hybrids.

Material and Methods ____________

Samples from trees were collected north of KhamarDaban, 30 to 35 km southwest of Baikal, where Siberian

stone and Japanese stone pines can be found growing together. Putative hybrids are occasionally found in intermixed populations. An area of approximately 5 by 5 km in

the region of Cherskij peak, Serdze Lake, and the

Podkomarnaja riverhead was inspected for putative hybrids. Putative hybrids were distinguished by a combination

of characteristics from the parental species: multiple crooks

in the stem (Japanese stone pine characteristic) and the

violet color of 2-year-old cones (Siberian stone pine characteristic).

Studies on reproductive characteristics were conducted in

populations at an altitude of approximately 1,500 m from

June 30 to July 10, 1998, according to methodology of Titov

(1982) and Nekrasova (1983). At this altitude, Siberian

stone pine was common (approximately 200 cone-bearing

trees per ha), but Japanese stone pine was less frequently

encountered (approximately 10 clones per ha). In development of pollen cones and pollen dispersal, successive stages

were distinguished as follows: (1) initiation of pollen shedding, (2) abundant pollen shedding, and (3) residual pollen

shedding. In development of the female cone, successive

stages of receptivity for pollination were distinguished by

changes in the ovuliferous scales as follows: (1) onset of

receptivity, (2) optimal receptivity, and (3) declining receptivity.

Cone samples were made from 10 Siberian stone pines, 10

Japanese stone pines, and 10 putative hybrids. Samples of

cones (10 per individual) were collected at the late August

1998 from 106 individuals (27 Siberian stone pines, 59

Japanese stone pines, and 20 putative hybrids). Seed quality

was studied by X-ray photography. Different characteristics

were assessed including: cone length (cm), cone width (cm),

average number of scales/cone, average number of initial

ovules/cone, ovules/cone lost in the early stages of development (percentage), average number of seeds/cone, poorly

developed seeds/cone (percentage), average number of filled

seeds/cone, hollow seeds/cone (percentage), average number

of seeds/cone with endosperm, seeds/cone with imperfect

endosperm (percentage), average number of seeds/cone with
170 USDA Forest Service Proceedings RMRS-P-32. 2004

Goroshkevich Natural Hybridization between Russian Stone Pine (Pinus siberica) and Japanese Stone Pine (Pinus pumila)

perfect embryos, seeds/cone without embryos (percentage),

average number of seeds /cone with embryo, seeds/cone with

differentiated embryos (percentage), average number of

seeds/cone with differentiated embryos, seeds/cones with

undifferentiated embryos (percentage), and average weight

of a single seed with complete endosperm (mg).

Significant differences (p<0.05) among means were determined using an analysis of variance (ANOVA), and mean

separation was by Duncan’s multiple range test (Steel and

Torrie 1980).

Results ________________________

The phenology studies indicated that putative hybrids

were occasionally found in populations below 1,990 m in

elevation (one clone per ha). In contrast, the number of

hybrids increased with elevation with a density of two to

three clones per ha found above 1,900 m in elevation. The

observations on reproductive characteristics showed that

timing of reproductive maturation of the two species and

putative hybrids practically coincide. Reproductive maturation generally occurred during a 2 to 3 week period, which

precludes reproductive isolation of either species, given that

hybridization is possible.

The results of cones and seeds morphological analysis are

presented in table 1. The seed analysis showed that Japanese stone pine was fertile up timberline, and Siberian stone

pine became increasingly infertile above altitude of 1,750 m.

Siberian stone pine was significantly different from Japanese stone pine in most of the cone and seed traits studied.

Putative hybrids were found to differ from either Siberian

pine or both of the parental species in all characteristics,

except for the initial number of ovules. Putative hybrids had

intermediate values between both parents for cone length,

cone diameter, average number of scales per cone, and

average weight of seed. For characteristics related to seed

quality (with the exception of seed weight), the putative

hybrids had significantly higher poorly developed seeds/

cone (percentage), hollow seeds/cone (percentage), and seeds/

cone without embryos (percentage), and significantly lower

seeds/cone with differentiated embryos (percentage), when

compared to both parents. Differences of putative hybrids

only from Siberian pine were apparent in a number of

characteristics.

Discussion _____________________

It is known that taxonomically close pine species can be

often crossed to form fertile hybrids (Critchfield and Little

1966) and that species with sympatric ranges can form

hybrid swarms (Wright 1975). Siberian and Japanese stone

pines belong to the same Pinus subsection (Cembrae Lond.),

but their natural distribution overlaps only by 5 to 10

percent. This study shows that no reproductive isolation

between the species occurs due to the timing of pollen flow

and female cone receptivity, and occasionally morphologically intermediate individuals occur, that is, putative hybrids. Further introgression, however, does not appear to be

rapidly occurring, if at all. Production of viable seed in the

putative hybrids was proportionally lower than in the parental species. Additionally, the number of seed/cone in the

putative hybrids was very low (on average one filled seed per

cone), thereby indicating little introgression between these

species beyond the first filial generation. Future studies

using various combinations of controlled pollinations between Siberian and Japanese stone pines with corresponding genetic analyses could further corroborate the origin of

the naturally occurring hybrids and better delineate limitations for introgression between these two species.

Table 1—Cones and seed characteristics in Siberian stone pine, Japanese stone pine, and putative hybrids.

Characteristic Siberian stone pine Putative hybrid Japanese stone pine

Cone length (cm) 5.0a1 4.0b 3.0c

Cone diameter (cm) 4.3a 3.0b 2.1c

Average number of scales/cone 73.6a 55.9b 37.6c

Average number of initial ovules/cone 68.3a 57.0ab 35.9b

Ovules/cone lost in the early stages of development (%) 15.5a 32.2b 43.9b

Average seed number/cone 58.3a 38.6b 20.7b

Poorly developed seeds/cone (%) 14.6a 52.0b 27.9a

Average number of filled seeds/cone 49.5a 19.6b 14.1b

Hollow seeds/cone (%) 3.2a 24.6b 5.3a

Average number of seeds/cone with endosperm 48.0a 14.9b 13.5b

Seeds/cone with imperfect endosperm (%) 32.3a 79.2b 63.0b

Average number of seeds/cone with perfect endosperm 32.5a 3.6b 5.0b

Seeds/cone without embryos (%) 0.2a 48.5b 5.9a

Average number of seeds/cone with embryo 32.4a 1.6b 4.7b

Seeds/cone with undifferentiated embryo (%) 5.8a 35.3b 17.8b

Average number of seeds/cone with differentiated embryo 30.5a 1.0b 3.9b

Seeds/cone with differentiated embryo (%) 44.7a 1.8c 10.9b

Average weight of a single seed with complete endosperm (mg) 216.2a 142.4b 72.3c

1

Significant differences (p<0,05) between means were determined using Duncan’s multiple range test; values with different letters within a line differ significantly.
USDA Forest Service Proceedings RMRS-P-32. 2004 171

Natural Hybridization between Russian Stone Pine (Pinus siberica) and Japanese Stone Pine (Pinus pumila) Goroshkevich

References _____________________

Bobrov, E.G. 1978. Forest-creating conifers of the USSR. Nauka.

Leningrad. 189 p. (In Russian).

Critchfield, W.B. and Little, E. L., Jr. 1966. Geographic distribution

of the pines of the world. USDA Forest Service. Misc. Publ. 991,

97 p.

Galazij, G.I. 1954. Timberline flora in the mountains of Eastern

Siberia and their dynamics. Proceedings of Botanical Institute,

USSR Academy of Sciences. 3 (9): 210-329. (In Russian).

Goroshkevich, S.N. 1999. On the possibility of natural hybridization

between Pinus sibirica and Pinus pumila in the Baikal Region.

Botanicheskij Journal. 84(9): 48-57. (In Russian).

Homentovskij P.A. 1995. Japanese stone pine ecology on Kamchatka.

Dalnauka. Vladivostok. 227 p. (In Russian).

Lanner, R.M. 1990. Biology, taxonomy, evolution, and geography of

stone pines of the world. Proceedings of the Symposium on

whitebark pine ecosystems: ecology and management of a highmountain-resource; 1989 March 29-31; Bozeman, MT. Ogden,

UT: USDA Forest Service Gen. Tech. Rep. INT-270, pp. 14-22.

Molodjnikov, V.N. 1975_ Japanese stone pine of mountain landscapes of Northern Baikal Region. Nauka. Moscow. 203 p. (In

Russian).

Nekrasova, T.P. 1983. Pollen and pollination in Siberian conifers

trees. Nauka. Novosibirsk. 168 p. (In Russian).

Pozdnjakov, L.K. 1952. Tree form of Japanese stone pine.

Botanicheskij jurnal. 37: 688-691. (In Russian).

Steel, R.G.D. and Torrie, J.H. 1980. Principles and procedures of

statistics. 2nd ed. McGraw-Hill. New York. 633 p.

Titov E.V. Seed and cone development in Siberian stone pine.

Lesnaja geobotanica i biologija drevesnyh rastenij. 18: 136-140.

(In Russian).

Wright, J.W. 1975. Introduction in forest genetics. Academic Press.

New York, San Francisco, London. 470 p.
172 USDA Forest Service Proceedings RMRS-P-32. 2004

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

Wan-Yong Choi, Kyu-Suk Kang, and Sange-Urk Han are with the Tree

Breeding Division, Korea Forest Research Institute, 44-3 Omokchun, Suwon

441-350, Republic of Korea. Seong-Doo Hur is with the Sobu Forest Experiment Station, Korea Forest Research Institute, 670-4 Sangmo, Chungjoo 380-

940, Republic of Korea. Correspondence should be addressed to e-mail

wychoi@foa.go.kr.

Estimation of Heritabilities and Clonal

Contribution Based on the Flowering

Assessment in Two Clone Banks of Pinus

koraiensis Sieb. et Zucc.

Wan-Yong Choi

Kyu-Suk Kang

Sang-Urk Han

Seong-Doo Hur

Abstract—Reproductive characteristics of 161 Korean pine (Pinus

koraiensisSieb. etZucc.) clones were surveyed at two clone banks for

3 years. These clone banks were established at Yongin and Chunchon

(mid-Korea) in 1983. Characteristics in female and male strobili

were spatial (between locations) and temporal (among investigated

times) variables. Broad sense heritabilities were found to vary

between 0.20 - 0.46 in females and between 0.34 to 0.56 in males.

Among 161 clones, 32 clones (20 percent of the total clones) accounted for 42 to 54 percent of clonal contribution in female strobili

and 83 to 96 percent in male strobili, suggesting that the clonal

contribution for male parents was severely unbalanced compared to

that for female parents. The effective population numbers varied

depending on time (year), location and sex. The mean values of

relative effective population numbers at gamete levels were 0.56 in

females and 0.09 in males, respectively, and that value at the clonal

level was 0.27 (0.25 at Yongin and 0.29 at Chunchon). The degree of

sexual asymmetry (As) varied with a range of 0.03 to 0.24 at

Chunchon and 0.07 to 0.44 at Yongin. The pattern of gamete

production within clones was highly asymmetrical as compared to

that of other conifers. This indicates that P. koraiensis is extremely

low in male gamete production compared to female gamete production.

Key words: Pinus koraiensis, strobili, clone bank, clonal

contribution, broad sense heritability, effective

population number, sexual asymmetry

Introduction ____________________

Korean pine (Pinus koraiensis Sieb. et Zucc.) is a fiveneedle pine (Pinus subgenus Strobus) belonging to subsection Cembrae. The species has a wide natural distribution in

the northeastern part of Eurasia. It usually occurs as a

mixed forest stand consisting of various broad-leaved tree

species and other conifers. Korean pine has been widely

planted as a pure stand accounting for about 30 percent of

the yearly planting areas in Korea due to its high-quality

timber and edible seeds. A breeding program for this species

has been conducted since 1959 and has resulted in the

establishment of 98 ha seed orchards (Mirov 1967, Chun

1992, Choi 1993, Wang 2001).

The main goal for seed orchards is large-scale production

of genetically improved seeds that maintain genetic diversity to prevent inbreeding depression. Thus, the maintenance of random mating among clones is one of the key

elements to successful management of seed orchards (Roberds

and others 1991, Chaisursri and El-Kassaby 1993, Matziris

1993, El-Kassaby and Cook 1994, Burczyk and Chalupka

1997, Han and others 1999).

The clonal contribution to seed production in a seed

orchard is one of the most important factors; genetic composition of the seed produced is determined by the contributions of each clone. Differences in clonal contribution have

been previously reported in several studies and have been

attributed to genetic rather than environmental factors

(Griffin 1982, Schmidtling 1983, Askew 1988, Brunet and

Charlesworth 1995, Kjaer 1996, Han and others 1999,

Nikkanen and Ruotsalainen 2000).

To date, numerous studies have been conducted to obtain

information related to reproductive processes such as flowering characteristics, clonal contribution, and sexual asymmetry in seed orchards. Clonal contribution within a seed

orchard is commonly depicted by a flowering or cone yield

curve. In this method, the clones are ranked from high to low

in flower production, and cumulative contribution (in percent) is plotted against the proportion of the clones. Additionally, the concept of effective population number has been

recently applied to the estimation of clonal contribution

(Griffin 1982, Kjaer 1996, Choi and others 1999, Han and

others 2001a, 2001b, Kang 2001).

Our major interest in this study is to quantify the reproductive processes using empirical data from two Pinus

koraiensis clone banks, to survey the differences of clonal

contribution by means of flowering assessments, and to

monitor the genetic diversity measured by effective population sizes. These include estimating heritability, gamete

contribution, and sexual asymmetry.
USDA Forest Service Proceedings RMRS-P-32. 2004 173

Estimation of Heritabilities and Clonal Contribution Based on the Flowering Assessment in Two Clone Banks... Choi, Kang, Han, and Hur

Materials and Methods ___________

Reproductive characteristics such as number of male and

female strobili were surveyed in the two clone banks of P.

koraiensis. The two clone banks were established at Chunchon

(lat. 37∞55', long. 120∞46') and Yongin (lat. 37∞30', long.

127∞20') in 1983. A total of 167 clones were grafted at

Chunchon and 180 clones at Yongin with a space of 4mx4m.

Reproductive characteristics of 161 clones, which the two

clone banks have in common, were investigated for 3 consecutive years (1998 to approximately 2000).

The clone banks were not considered as fully mature

populations when the numbers of female and male strobili

were counted. Generally, Korean pine begins to show strobili

at age of 12 or 15 in natural stands. Grafted clones, however,

produce strobili earlier than natural stands. In these clone

banks, there is not much difference in height (4 to 5m) and

DBH. Five ramets per clone were chosen for assessment in

early June. The number of female strobili was counted

individually from a whole tree. The total number of male

strobili was estimated by multiplying the average number of

strobili per branch by the total number of branch bearing

male strobili.

Analysis of variance (ANOVA) tests and heritability estimates were conducted based on the data for female and male

strobili production. The ANOVA was performed using a

logarithmic transformation of the original data to normalize

the distribution of variances (Steel and Torrie 1980). SAS

program (ver 6.12; SAS Institute Inc., 1996) was used for

ANOVA tests and heritability estimation. Broad-sense heritabilities (H2) were estimated on the basis of individual trees

(Schmidtling 1983) as:

H c

c e

2

2

2 2 = +

s

s s

.

Parental balance was assessed using a cumulative gamete

contribution curve (Griffin 1982). The numbers of female

and male strobili were ordered by clone from high to low

strobilus production, and the cumulative contribution percentages were plotted against the proportion of the clones

(Kang 2000).

The maleness index (Ai) is defined as the proportion of a

clone’s reproductive success that is transmitted through its

pollen (Kang 2000). Maleness index based on strobilus

production was estimated as follows:

gi ai

ai Ai

+ =

where ai and gi are the proportions of i

th clone of which male

and female strobili contribute to the whole population. A

high maleness index of a clone indicates that the clone is

contributing more as a paternal, rather than maternal

parent.

The effective population numbers at gamete level (Eq. 1

and Eq. 2) and clonal level (Eq. 3 and Eq. 4) and the sexual

asymmetry (Eq. 5) were estimated using Choi and others’

(1999) methods as follows:

m➁ = (

i

n

=

Â

1

xi(➁)

2)–1 (1)

m❹ = {

i

n

=

Â

1

xi(❹)

2}

–1 (2)

ma = 1⁄

2 (m ➁+ m ❹) (3)

mb = {

i

n

=

Â

1

(

1⁄

2(xi(➁)+xi(❹))

2}–1 (4)

As = ma /|mb – ma|, 0 £ As £ 1 (5)

where n is the total number of clones, m ➁ is the female

effective population number, and xi (➁) is the proportion of

the female strobili of the i

th clone to the whole production of

females. m ❹ and xi (❹) in males correspond to those for

females. ma is the arithmetic mean of the two measures (m➁

and m ❹) and mb is based on the relative frequency of xi (➁) and

xi (❹). In this study, we used the relative effective population

number instead of effective population number for easy

comparison with those of other studies.

Results and Discussion __________

Reproductive Characteristics and

Heritability

Large variations in both female and male strobilus production among clones were observed at both Yongin and

Chunchon (table 1). The differences of male strobilus production among clones were far more extreme than that of

female strobilus production. It seems that this phenomenon

is a typical character of Korean pine from our experience of

Table 1—Mean, standard deviation (S.D.) and coefficient variation (C.V.) for the number of female and male strobili at Yongin and Chunchon during

the period of 1998 to 2000.

Yongin Chunchon

1998 1999 2000 1998 1999 2000

Female Male Female Male Female Male Female Male Female Male Female Male

Mean 5.3 64 13.6 240 9.1 36 2.2 146 12.5 393 5.8 307

S.D.(±) 4.6 184 12.8 975 9.2 111 2.1 514 9.3 988 5.1 982

C.V.( percent) 87 289 94 406 101 305 97 353 75 251 87 320
174 USDA Forest Service Proceedings RMRS-P-32. 2004

Choi, Kang, Han, and Hur Estimation of Heritabilities and Clonal Contribution Based on the Flowering Assessment in Two Clone Banks...

orchard management. The average female strobilus productions per clone ranged between 0 and 46.7 at Yongin and

between 0.1 and 63.5 at Chunchon. The production of female

strobili in Yongin was consistently greater than that in

Chunchon, while male strobili production showed an opposite trend. During this study, the production of female and

male strobili was most abundant in 1999.

The ANOVA results and broad sense heritabilities for

reproductive characteristics are presented in table 2. The

number of female and male flowers was significantly different among clones within a clone bank, while those for ramets

within a clone did not show any significant differences.

These results showed that the reproductive characteristics

are under genetic influences rather than environmental

influences. Similar results have been reported in other

conifers such as P. taeda (Byram and others 1986), P.

densiflora (Han and others 1999), P. thunbergii (Han and

others 2001b) and Picea abies (Nikkanen and Ruotsalainen

2000).

The values of broad sense heritabilities for female strobili

ranged from 0.21 to 0.20 in a poor flowering year (1998) and

they varied between 0.46 and 0.27 in a good flowering year

(1999). Temporally those values for male strobili varied

between 0.21 in 1999 and 0.42 in 1998 at Chunchon and 0.20

in 1998 and 0.34 in 2000 at Yongin. The values for males

(0.22 to 0.56) were higher than females (0.20 to 0.51) for all

years studied. This indicates that the genetic influence

determining the reproductive characteristics is stronger in

males than in females.

The two-way ANOVA results and estimated heritabilities for reproductive characteristics in the two clone banks

are presented in table 3. The differences in the number of

female and male strobili among clones were statistically

significant for 3 years excluding that of males in 1998 and

that of females in 2000. Significant differences in reproductive characteristics between the two locations were observed for females in 1998, and for females and males in

2000. In 1999, the flowering characteristics for both sexes

were significantly different among clones. The interaction

of clone and location effects was significant in all years,

implying that clones should be selectively chosen when

production (in other words, seed orchards) and/or breeding

populations are established at the different sites.

The heritabilities for female and male strobili in each

year showed maximum values of 0.59 and 0.77, respectively. The minimum values for heritabilities were 0.02 for

female in 2000 and 0.23 for male in 1998.

Clonal Contribution

We used two types of measures, cumulative contribution

curves and relative effective population number, for demonstrating the clonal contribution. The cumulative contribution curves of 161 clones for female and male strobili are

presented in figure 1. Thirty-two clones (20 percent of the

total clones investigated were at both locations) accounted

for 42-54 percent of clonal contribution in female and 83 to

Table 2—Analysis of variance and broad sense heritability (H2) for the number of female and male strobili at Yongin and Chunchon

during the period of 1998 to 2000.

1998 1999 2000

Location Female Male Female Male Female Male

Yongin Among clones 0.37** 2.06** 0.69** 3.50** 0.43** 1.23**

Within clones 0.17 0.49 0.15 0.56 0.25 0.28

H2 0.21 0.42 0.46 0.56 0.24 0.45

Chunchon Among clones 0.46** 5.99** 0.60** 5.47** 0.68** 4.24**

Within clones 0.12 0.47 0.17 0.92 0.24 0.96

H2 0.20 0.51 0.27 0.42 0.22 0.34

**: Significant at 1 percent level.

Table 3—Two-way ANOVA and broad sense heritabilities—(H2) for the number of female

and male strobili at Yongin and Chunchon during the period of 1998 to 2000.

1998 1999 2000

Female Male Female Male Female Male

Clone 0.48* 3.40 0.90** 7.03** 0.50 2.92**

Location 33.55** 0.04 0.33 4.94 8.59** 46.72**

Clone x Location 0.31** 3.03** 0.37** 1.34** 0.49** 1.07**

Error 0.13 0.47 0.17 0.79 0.24 0.83

H2 0.38 0.23 0.59 0.77 0.02 0.52

**,* Significant at 1 percent and 5 percent level, respectively.
USDA Forest Service Proceedings RMRS-P-32. 2004 175

Estimation of Heritabilities and Clonal Contribution Based on the Flowering Assessment in Two Clone Banks... Choi, Kang, Han, and Hur

96 percent in male strobili. The curves for male strobili were

severely distorted compared to those for female strobili.

Alternately, the clonal contributions of female and male

strobili for each year were 49 percent and 89 percent in 1998,

44 percent and 92 percent in 1999, and 54 percent and 96

percent in 2000, respectively at Yongin, while those for

Chunchon were 51 percent and 92 percent in 1998, 42

percent and 86 percent in 1999, and 47 percent and 83

percent in 2000, respectively. The biased contribution of a

small number of clones to the whole clonal contribution was

greater for pollen parents than female parents.

Park and others (1987) reported that 19 percent of the

total clones in a P. koraiensis clone bank accounted for 63

percent of male strobili production and 58 percent of female

strobili production. This study was conducted at Chunchon

where our study was also conducted. However, they studied

a 4 to 5 year old clone bank. Alternately, Han and others

(1997) conducted a similar study in a P. koraiensis clone

bank at Yongin, our other study site. In that study, they

reported that 20 percent of the total clones investigated

accounted for 49 to 65 percent of female strobili production,

while 8 to 15 percent of the total clones accounted for over 80

percent of male strobili production. The differences in results between the above studies and our study might be due

to plantation age. Regardless,these comparisons show that

clonal contribution to strobili production is more balanced in

female than that in male reproduction.

When compared to other conifers, Korean pine appears

to have a more unbalanced clonal contribution in seed

production. Han and others (1999) observed that the contribution of 33 percent of 99 P. densiflora Ait. clones varied

between 46 percent and 70 percent in female and 40 percent

and 87 percent in male, and the degree of contribution

increased with age. In P. radiata D. Don, 23 percent of the

total clones accounted for 50 percent of seed production

(Griffin 1982). Adams and Kunze (1996) found that 49

percent of the total clones in Picea mariana (Mill.) B.S.P.

and 43 percent of the clones in P. glauca (Moench.) Voss.

accounted for a total of 80 percent of seed production.

The relative effective population numbers estimated at

gamete and clonal levels are shown in table 4. The relative

effective population number for sexes were extremely different with m➁ = 0.56 and m❹☎= 0.09. The values of relative

effective population number at the gamete level did not

differ significantly by year or location. In a Pinus sylvestris

L. clonal seed orchard at the age of 17-19, Burczyk and

Chaluka (1997) found that the effective population number

(0.76) in males was only slightly lower than that (0.96) in

females. In contrast, Han and others (2001a) observed

slightly higher values in males (mean 0.63 with a range of

0.24 - 0.94) than those in females (mean 0.55 with a range of

0.28 - 0.83) in a P. densiflora clonal seed orchard.

The values at the clonal level (mb) ranged from 0.19 - 0.38.

The values of mb between the two locations ranged from 0.24

in 1998 to 0.38 (mean: 0.29) in 1999 at Chunchon and from

0.19 in 1999 to 0.28 (0.25) in 2000 at Yongin. Interestingly,

the values of mb are lower than those of m➁ and ma in all

observations. It is generally known that the values of mb are

Female

0

25

50

75

100

0 30 60 90 120 150

Number of clones

Percent of flowers(%)

1998 

1999 

2000 

Male

0

25

50

75

100

0 30 60 90 120 150

Number of clones

Percent of flowers(%)

1998

1999

2000

Female

0

25

50

75

100

0 30 60 90 120 150

Number of clones

Percent of flowers(%)

1998

1999

2000

Male

0

25

50

75

100

0 30 60 90 120 150

Number of clones

Percent of flowers(%)

1998

1999

2000

Yongin

Chunchon

Figure 1—Cumulative female and male strobilus production curves of

clones at Yongin and Chunchon during the period of 1998 to 2000.
176 USDA Forest Service Proceedings RMRS-P-32. 2004

Choi, Kang, Han, and Hur Estimation of Heritabilities and Clonal Contribution Based on the Flowering Assessment in Two Clone Banks...

Table 4—Relative effective population number at the gamete level and the clonal level in Pinus koraiensis clone banks

investigated for 3 consecutive years.

Yongin Chunchon

N 1998 1999 2000 1998 1999 2000

m➁ 161 0.52 (83.7)a 0.62 (99.8) 0.50 (80.5) 0.52 (83.7) 0.64(103.0) 0.57 (91.8)

m❹ 161 0.10 (16.1) 0.06 ( 9.7) 0.10 (16.2) 0.07 (11.3) 0.14 (22.5) 0.09 (14.5)

ma 161 0.31 (49.9) 0.34 (54.7) 0.30 (48.3) 0.30 (48.4) 0.39 (62.8) 0.33 (53.1)

mb 161 0.28 (45.1) 0.19 (30.6) 0.28 (45.2) 0.24 (38.6) 0.38 (61.2) 0.25 (40.3)

a

 Effective population number in parenthesis

always larger than those of ma and similar to or larger than

those for m❹ and m➁. For instance, Han and others (2001a)

showed that the value for mb (0.69) was higher than those for

ma (0.58) in a P. densiflora seed orchard. The reason for the

contrary tendency as shown in this study was explained in

elsewhere (Choi and others 1999).

Sexual Asymmetry

The degrees of sexual asymmetry between female and

male strobili were shown in table 5. The degree of sexual

asymmetry (0.03 to approximately 0.24 with a mean of

0.17) for Chunchon was lower than that of Yongin (0.0 to

approximately 0.44 with a mean of 0.20). The degree of

sexual asymmetry (As) was variable depending on time and

location.

Table 5—Estimation of the degree of sexual asymmetry (As) at

Pinus koraiensis clone banks for 3 years.

Yongin Chunchon

1998 1999 2000 1998 1999 2000

| m a – mb | 4.84 24.16 4.83 9.66 1.61 12.87

As 0.10 0.44 0.07 0.20 0.03 0.24

Figure 2—Maleness index curves for two different P. koraiensis clone banks estimated during the period

of 1998 to 2000.

1998 1998

1.00

.75

0.50

0.25

0.00

1 41 81 121 161

Number of clones

Maleness index

Yongin

1.00

0.75

0.50

0.25

0.00

1 41 81 121 161

Number of clones

Chunchon

2000 2000

Average Average

1999 1999

The degree of sexual asymmetry in this study was higher

especially when it was compared to that of P. densiflora

(Han and others 2001a). In this species, the difference

between two types of effective population number at clonal

levels ma and mb were large because a majority of clones did

not bear male flowers while most of them bore female

flowers, therefore contributing to sexual asymmetry (see

also Choi and others 1999). In contrast, most conifer species

(P. densiflora and P. thunbergii) had similar effective population numbers between sexes.

Male index estimates are showed in figure 2. The distribution pattern of maleness indices in the present study

deviated from the normal distribution pattern found in

other pine trees (Burczyk and Chalupka 1997). Generally,

most clones in other pines such as P. densiflora, P.thunbergii

and P. sylvestris had maleness index of 0.8 to 0.2. Our study

demonstrated a bimodal distribution, with the majority of

Korean pine clones maleness indices above 0.8 or below 0.2.

For instance, more than 80 percent of clones had values

above 0.9 or below 0.1 regardless of year or location. In 2000

at Yongin and in 1998 at Chunchun, more than 95 percent

of clones had maleness indices above 0.9 or below 0.1. On

the other hand, the sexual balance within clones was highly

asymmetrical in P. koraiensis as compared to that of other

pine species such as Pinus densiflora, P. thunbergii Parl.

and P. sylvestris (Han and others 2001a, 2001b, Burczyk

and Chalupka 1997). This tendency is due to the extreme
USDA Forest Service Proceedings RMRS-P-32. 2004 177

Estimation of Heritabilities and Clonal Contribution Based on the Flowering Assessment in Two Clone Banks... Choi, Kang, Han, and Hur

difference in effective population number between sexes

and high degree of sexual asymmetry as already shown

above.

Our study indicates potential problems in the seed

orchard management of P. koraiensis. These problems

are: 1) differential fertility variation, 2) inadequate pollen

supply, 3) panmictic disequilibria, and 4) parental unbalance. Such problems relate to both the amount of seed

produced and the genetic diversity of seed crops (Kang

2001). Thus, some management options, such as supplemental mass pollination, flower stimulation and equal seed

harvest, should be considered in the clonal seed orchard of

P. koraiensis.

References _____________________

Adams, G.W. and Kunze, H.A. 1996. Clonal variation in cone and

seed production in black and white spruce seed orchards and

management implications. For. Chron. 72: 475-480.

Askew, G.R. 1988. Estimation of gamete pool compositions in clonal

seed orchard. Silvae Genet. 37: 227-232.

Brunet, J. and Charlesworth, D. 1995. Floral sex allocation in

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178 USDA Forest Service Proceedings RMRS-P-32. 2004

Choi, Kang, Han, and Hur Estimation of Heritabilities and Clonal Contribution Based on the Flowering Assessment in Two Clone Banks...
USDA Forest Service Proceedings RMRS-P-32. 2004 179

Part III: Genetic Diversity and Conservation

Collage by R. Berdeen
180 USDA Forest Service Proceedings RMRS-P-32. 2004

1.

2.

3.

4.

5.

6.

7. Infected pine seedling exhibiting

8.

Collage by: R. Berdeen

Underside of Ribes bracteosum leaf exhibiting

blister rust infection.

Dorena crew members laying out Ribes leaves

in preparation for inoculation.

Pine seedlings under racks of Ribes leaves

during inoculation.

Inoculation chamber at 100% RH with mist

system engaged.

Infected pine seedling exhibiting numerous

needle lesions.

Infected pine seedling with several stem

cankers.

a) a needle lesion

b) an incipient stem canker at a needle fascicle

c) a bark reaction

Frames of pine seedlings showing a high rate

of mortality due to blister rust infection.

1

5

2 3

4

67 8
USDA Forest Service Proceedings RMRS-P-32. 2004 181

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

Mary F. Mahalovich is a Geneticist, USDA Forest Service, Northern,

Rocky Mountain, Southwestern, and Intermountain Regions, Forestry Sciences Lab, 1221 S. Main Street, Moscow, ID, USA 83843. Office phone (208)

883-2350, Fax (208) 883-2318, e-mail mmahalovich@fs.fed.us. Gary A.

Dickerson is Acting Budget Director, USDA Forest Service, Northern Region,

P.O. Box 7669, 200 E Broadway, Missoula, MT, USA 59807. Office phone (406)

329-3352, Fax (406) 329-3132, e-mail gdickerson@fs.fed.us.

Abstract—A strategy to restore whitebark pine communities is

presented that emphasizes genetic resistance to white pine blister

rust (Cronartium ribicola Fisch.) and mountain pine beetle

(Dendroctonus ponderosae Hopkins), in combination with an active

tree planting program. Early and active intervention may prevent

listing of whitebark pine under the Endangered Species Act and

further aid in the successful recovery of the grizzly bear (Ursus

arctos horribilis). The restoration program initiated in 2001 includes a multi-State effort (Idaho, Montana, Oregon, Nevada, Wyoming, and Washington) designating permanent leave trees, emphasizing clean trees in high blister rust areas or areas with a high

incidence of mountain pine beetle, or areas where both conditions

are present. Cone collections from these trees will provide an

immediate seed source for fire restoration, reforestation, ex situ

genetic conservation, and seedlings to be screened for blister rust

resistance. Pollen will be collected for genetic conservation and to

advance blister rust resistance in seed and breeding orchards. Data

generated from the rust screenings will identify whitebark pine

seed sources that provide high levels of blister rust resistance and

provide information needed to refine seed transfer guidelines.

Leave trees elevated to elite-tree status, as identified by their rustresistant progeny in the rust screenings, will serve as a seed source

for operational collections and seed trees for natural regeneration.

Survivors from the blister rust screening will be planted in clone

banks for genetic conservation purposes, to serve as donors for

future seed orchard establishment, and to facilitate selective breeding for blister rust resistance.

Key words: white pine blister rust resistance, fire restoration,

genetic conservation, seed transfer guidelines.

Whitebark Pine Genetic Restoration Program

for the Intermountain West (United States)

M.F. Mahalovich

G. A. Dickerson

Introduction ____________________

Whitebark pine, a keystone species in upper and subalpine ecosystems, provides a food source for grizzly bear,

Clark’s nutcracker (Nucifraga columbiana), and red squirrels (Tamiasciurus hudsonicus). It is also a foundation

species for protecting watersheds as it tolerates harsh, windswept sites that other conifers cannot, the shade of its

canopy regulates snowmelt runoff and soil erosion, and its

roots stabilize rocky and poorly developed soils (Tomback

and Kendall 2001).

The native pathogen, limber pine dwarf mistletoe

(Arceuthobium cyanocarpum (A. Nelson ex Rydberg) Coulter

& Nelson) and the exotic pathogen, white pine blister rust,

are contributing to the overall decline of the species. The

parasitism of dwarf mistletoe impacts cone and seed production, reducing the reproduction potential of whitebark pine

in severely infested stands (Taylor and Mathiason 1999).

White pine blister rust rapidly kills small trees, impeding

successful regeneration. Blister rust infections in larger

trees can persist a long time and are frequently found in the

upper crown, reducing a tree’s cone-bearing potential.

Whitebark pine trees that survive blister rust infections are

further threatened by mountain pine beetle attacks.

Wildfire occurrence aids in the preparation of a seed bed

for natural regeneration. Fire suppression has reduced the

role of fire in regeneration of pure whitebark pine stands and

has allowed successional replacement of subalpine fir (Abies

lasiocarpa (Hook.) Nutt.), lodgepole pine (Pinus contorta

Dougl. ex Loud.) and Engelmann spruce (Picea engelmanni

Parry ex Engelm.) in mixed-conifer stands. Careful control

is needed to reintroduce fire into high elevation ecosystems.

Uncontrolled wildfire can destroy young whitebark pine

regeneration and kill trees of cone-bearing age, which will

limit the food supply for dependent wildlife and cause loss of

future seed sources for restoration purposes.

High elevation ecosystems are at high risk because one or

two species of white pines are usually dominant (McDonald

and Hoff 2001). Because the loss of mature whitebark pine

is occurring so rapidly, often in the absence of successful

regeneration, there has been a pronounced loss of whitebark

pine cover type. When only thinning and prescribed fire are

utilized to promote vigorous stands of western white pine

(Pinus monticola Dougl. ex D. Don), this has led to increased blister rust infection levels by opening up stands

and encouraging Ribes spp. establishment (Schwandt and

others 1994). Successful natural regeneration is dependent

upon sufficient blister rust resistant seed available on site.

This is due to the unique seed dispersal and seed caching by

Clark’s nutcrackers (Tomback and Schuster 1994) and red

squirrels.
182 USDA Forest Service Proceedings RMRS-P-32. 2004

Mahalovich and Dickerson Whitebark Pine Genetic Restoration Program for the Intermountain West (USA)

The 2000 fire season burned 929,000 ha on USDA National Forest System lands. Much of the fire devastation

occurred in high elevation ecosystems, resulting in the

destruction of both diseased and healthy whitebark pine

trees.

Emergency National Fire Plan funding was made available in 2001 to initiate a landscape-level approach to restoring whitebark pine over the next 5 years on National Forest

System lands in Idaho, Montana, Nevada, and Wyoming.

Adjacent National Forests in Washington and Oregon were

invited to participate. Glacier, Grand Teton, and Yellowstone

National Parks, facing similar management challenges and

stringent restoration policies (Kendall 1994), were also

invited to participate. The scope of the program is based on

cooperators whose landholdings are high elevation sites

typically found in Federal ownership. The multi-State,

multiagency collaboration forged in this endeavor provides

a unified front to increase the likelihood of favorable outcomes in our restoration efforts, and a synergy that has been

difficult to achieve by any one administrative unit or special

project in the past.

Project Goals ___________________

The short-term goals over the next 5-year period are:

(1) operational cone collections for planting burned areas,

and (2) plus-tree identification and individual-tree cone

collections for rust screenings and genetic conservation.

These activities will facilitate identification of whitebark

pine populations at most risk due to blister rust (more

than 70 percent infection), which may require additional

intervention to stabilize their survival. Field personnel

will also become more familiar with the distribution of

whitebark pine, which will provide land managers current

information on the species distribution (Little 1971) and

associated blister rust infection levels and mountain pine

beetle infestations across the landscape. These data will also

be used to adjust the number of plus-trees needed per zone

and to develop a database for a seed transfer expert system.

Over the long-term, seedlings from the plus-tree selections will reveal patterns of genetic variation in survival,

blister rust resistance, and early growth in rust screening

trials. Data obtained from the rust screenings will help

identify the presence or absence of various blister rust

resistance mechanisms (Mahalovich and Eramian 1995)

and their relative frequency among populations. The performance of the rust-resistant progeny will also be used to rank

the original plus-trees. Those with high rankings (elite

trees) will be identified as scion and pollen donors for seed

orchard and clone bank establishment.

Implementation Plan _____________

Cone Collections for Fire Rehabilitation

National Forests and Parks with immediate restoration

needs should use the current seed zone boundaries to estimate their seed needs (fig. 1). There are no elevational

restrictions on seed transfer within a seed zone. When

blister rust infection levels vary within a zone, seeds collected for immediate rehabilitation efforts should not be

moved from areas with low (less than 49 percent) to moderate (50 to 70 percent) infection levels to planting sites with

higher infection levels (more than 70 percent). Seeds collected from phenotypically resistant trees in areas with high

infection levels are suitable for planting on sites with low,

moderate or high infection levels (Mahalovich and Hoff

2000).

Operational cone collections should be from no fewer than

20 individuals separated by 67 m within a zone to ensure a

broad genetic base in the seed lot. This bulked seed lot

collected from similar rust infection sites is referred to as a

tree-seed zone or bulked collection.

Additional improvement in insect and disease resistance

and growth can be achieved by collecting from above-average stands with more than 50 reproductively mature trees

per 0.5 ha, emphasizing collections from a minimum of the

20 best trees. This bulked seed lot is referred to as a seed

collection stand.

Moreover, communities with high blister rust infection or

mountain pine beetle infestations, with at least 50 clean,

reproductively mature trees per 0.5 ha, could be cultivated

as a seed production area. This concept offers even more

improvement, by first selecting an above-average stand,

followed by removal of undesirable trees with insect and

disease problems and poor growth and form, improving the

genetic base of both the seed and pollen parents. These

potential seed production areas will provide the most promising seed source for immediate cone collections until a

grafted seed orchard of proven rust-resistant donors can be

established and cultured for cone production.

Identifying Phenotypically Superior

Individuals

An effective restoration strategy in whitebark pine includes components related to patterns of genetic variation,

particularly to blister rust. Restoration efforts may be hampered if the assumption is made that whitebark pine and

western white pine have a similar genetic response to blister

rust. One key difference is that percent infection is higher in

whitebark than western white pine (Bingham 1972, Hoff

and others 1994, McDonald and Hoff 2001). Until more

information becomes available on the biology and genetics of

whitebark pine and blister rust in the Inland West, the best

model to develop blister rust improvement in whitebark pine

is the western white pine protocol (Mahalovich and Eramian

1995). Several modifications have recently emerged regarding the western white pine protocol and in the recommended

breeding plan to develop resistance in whitebark pine put

forth by Hoff and others (1994). The revised protocol follows.

Plus-tree selections (that is, designation of permanent

leave-trees) are based on existing seed zones (fig. 1). Assignments within zones facilitates broad sampling among National Forests and Parks, emphasizing broadly adaptable

populations for blister rust resistance development and

isolated populations supporting unique gene frequencies or

adapted gene complexes for gene conservation. If the target

seed orchard size is 30 unrelated individuals, sufficient

candidate trees must be identified within a zone to assure

finding several genes for blister rust resistance in the rust

screenings.
USDA Forest Service Proceedings RMRS-P-32. 2004 183

Whitebark Pine Genetic Restoration Program for the Intermountain West (USA) Mahalovich and Dickerson

Figure 1—Whitebark pine seed zones for the Intermountain West, USA.

Approximately 100 plus-trees are assigned in each seed

zone relative to the number of hectares of whitebark pine

occurring on National Forests and Parks (Little 1971). The

state of Nevada is comprised primarily of isolated populations with an expectation of 50 plus-trees for that zone. The

outlier populations in northeastern Oregon are typically

considered as part of the western range of whitebark pine

(McCaughey and Schmidt 2001); however, these populations are also in proximity to the Bitterroots/Idaho Plateau

seed zone boundary (fig. 1). Until more information becomes

available on these populations, progeny from northeastern

Oregon should be evaluated in rust screenings alongside

progeny from both the Bitterroots/Idaho Plateau and the

Nevada seed zones.

The total base population across all zones is 650 trees.

The base population may seem small as compared to the

3,100 plus-trees in the western white pine tree improvement program (Mahalovich and Eramian 1995). The effective population size in western white pine is actually less

than 3,100 plus-trees, as field validation has shown some

trees separated by as little as 10 m will increase the probability that they are related. The goal is to have a moderate

number of trees per zone to assure finding several genes for

resistance. Problems in too small a population size within a

zone may arise if 30 rust-resistant elite trees are not identified in a rust screening.
184 USDA Forest Service Proceedings RMRS-P-32. 2004

Mahalovich and Dickerson Whitebark Pine Genetic Restoration Program for the Intermountain West (USA)

The western white pine program required 900 sound

seeds per plus-tree; 300 to be set aside for gene conservation and the remaining to be sown to provide 144, 2-year-old

container seedlings for rust screenings (Mahalovich and

Eramian 1995). For whitebark pine, field units have been

asked to collect 1,800 wind-pollinated seed per tree, 300 for

gene conservation, and the remaining for rust screenings.

Wire cages are recommended to protect the cones from bird

and squirrel predation to achieve the target number of

seeds per tree. The wire cages should be installed during

June, on branches bearing second-year conelets. The increased number of seeds per tree are needed to compensate

for low germination rates from sowing seedlots that have

been in extended cold storage from the early 1990s (Burr and

others 2001). Efforts are under way with the USDA Forest

Service National Tree Seed Laboratory to study the special

germination and seed storage requirements of whitebark

pine, to make a seed bank a more promising gene conservation tool in the future.

Whitebark Pine Plus-Tree Selection

Criteria

Stand-Level Selection Criteria—The stand selection

criteria were relaxed for whitebark pine, emphasizing blister rust infection levels instead of mortality levels (table 1).

If average mortality levels were followed, as was recommended for western white pine, almost no whitebark pine

stands would qualify for plus-tree selections. Mortality levels

can reach upwards of 90 percent or higher in whitebark pine

stands in the Selkirk-Cabinet seed zone. Where field units

do not support stands of whitebark pine (for example, more

than 50 trees per 0.5 ha) and have dispersed trees in

mixed-conifer settings, field personnel should move forward

to the individual-tree selection criteria.

The average infection level for the target stand is determined by carefully counting both live and dead cankers on a

representative sample of 100 living or dead trees. Presence

or absence of cankers (bole and branch) from the 100-tree

survey is used to determine the overall stand infection level.

Actual counts should be made for main-bole cankers, whereas

branch cankers can be estimated and grouped in the following categories: 0=no cankers present, 1 to 9 cankers, 10 to 20,

21 to 40, 41 to 75, 76 to 150, 150+ cankers. The combined

total of main bole cankers and estimated branch cankers is

equal to the number of cankers per tree. The average

number of cankers per tree for the 100-tree survey then

yields the stand average. When rust infection is heavy

(some 90 percent), allowances are made for the possible

presence of difficult-to-see or undetectable cankers (for

example, flagging, dead tops, dead branches, and animal

damage with extensive sap on the main bole are assumed to

be due to a canker).

Each area should be more than 25 years of age and the

average tree height around 10 to 35 m. This will increase

the likelihood that the stand will have had at least 25 years

of exposure to blister rust, be of cone bearing age, be producing pollen, and be climbable. A moderately open stand

density is desirable so the target plus-trees are easy to

examine from the ground, have persistent branches at

ground level to facilitate climbing, and have full crowns for

better cone-bearing potential (Hoff and McDonald 1980).

When rust infection levels are low (less than 50 percent)

and whitebark pine grows in either a mixed- or pure-stand

setting, field units should proportionally balance the number

Table 1—Whitebark pine plus-tree selection criteria.

Stand level criteria Individual-tree level

Vigorous and representative of the species Dominant or co-dominant trees

Habitat type where species normally occurs Minimum of 100 1

 m between selected trees

to avoid relatedness

Provide a broad sample of both the Free of insects and diseases

geography and range of elevations

Overall composition has a high proportion Have a history or the potential to bear

of living or dead whitebark pine, well cones

represented throughout the stand

Uniformly and heavily infected with blister Be within 100 to 200 m from the nearest

rust (10 or more cankers per tree on the road or trail

average)

Confirmed blister rust infection of 90 No more than three of the best candidates

percent or higher in uniform stands in any given stand

Stands with 50 to 90 percent rust infection, No squirrel cache cone collections

limit selected trees to no more than five

cankers

1 Spacing between plus-trees (100 m) differs from spacing requirements in operational cone collections

(67 m).
USDA Forest Service Proceedings RMRS-P-32. 2004 185

Whitebark Pine Genetic Restoration Program for the Intermountain West (USA) Mahalovich and Dickerson

of selections between the two stand types. Likewise, if field

units have both concentrated stands and sparsely distributed whitebark pine, plus-tree collections should be proportionally balanced based on the number of hectares occurring

in both types of tree densities.

Individual-Tree Selection Criteria—Each plus-tree

should be relatively free of blister rust when compared to the

overall infection level in the stand. Allowable infection levels

for each plus-tree (table 2) are modeled after Hoff and

McDonald (1977, 1980). The presence or absence of cankers

is determined by examining each tree both from the ground

with binoculars and by climbing the tree and examining

each individual whorl. Though desirable, based on the preliminary field reports, accurate canker counts are difficult in

whitebark pine because of the high levels of infection, sap

weeping from cankers and animal damage (chewing), as

compared to western white pine.

Three growth forms are acceptable in whitebark pine:

single-stem, erect; multiple-stem, erect; and krummholz.

Dominant or co-dominant trees are preferable, but the

multiple-stem, erect or krummholz categories may lend

themselves more to the intermediate or suppressed crown

classes. In contrast to western white pine, the acceptable

growth form is the single-stem, erect form, or the timber

archetype in the dominant or co-dominant crown class.

Each tree should be free of insects, particularly mountain

pine beetle, and other diseases such as limber pine dwarf

mistletoe, as these characteristics are likely inherited and

passed onto their progeny. Squirrel-cache cone collections

should be avoided because of unknown parentage and because seeds have come in contact with forest litter and soils,

increasing the likelihood of seed-borne fungi Fusarium spp.,

Sirococcus strobilinus, and the snow bank or cold fungus,

Calocypha fulgens (Kolotelo and others 2001, Hoff and

Hagle 1990).

Each tree should be within 100 to 200 m from the nearest

road or trail, unless intervening vegetation is sparse enough

so that longer lines of sight are possible, to facilitate caging

of branches to protect cone crops from bird predation and for

ease of relocation. When plus-trees are easily accessible by

road or trail, the possibility exists to use cherry pickers or

man-lifts to collect cones from the upper portion of the crown.

Care should be taken to avoid collections from limber pine,

when whitebark and limber pine are intermixed on the same

national forest or park. The operational cone collection

guidelines for whitebark pine (Mahalovich and Hoff 2000)

provide additional information on how to distinguish the two

species by cone morphology, strobilus color, and pollen

catkin color.

Blister Rust Screening Trials

A rust screening will let us know how successful our

restoration efforts may be by identifying the amount of

genetic variation present in survival and disease resistance

and by quantifying how much of that variation occurs among

or within stands.

The progeny of 200 plus-trees can be reliably handled in a

rust screening, allowing approximately two seed zones to be

tested at a time. A bulked check lot of untested seed from

existing whitebark pine seed lots will need to be constructed

upfront, to facilitate comparisons among the plus-trees.

Rust screening scheduling will depend on how quickly each

field unit completes its plus-tree selections within a zone.

The goal is to sow a rust screening trial by 2005.

Modifications in the composition of aeciospore samples

are recommended as a conservative course of action for

inoculating Ribes spp. in the rust screening trials. Low levels

of genetic differentiation exist among samples of C. ribicola

collected from eastern white pine (Pinus strobus L.) in

eastern North America (Et-touil and others 1999) and among

C. ribicola samples collected from western white pine in

western North America (Kinloch and others 1998). Little

is known however, about specific races of blister rust in the

Inland West in western white or whitebark pine. One

exception is the identification of yellow and red-spotting

races occurring on western white pine (McDonald 1978),

with one type not necessarily more virulent than the other.

Ribes spp. leaves used in the inoculations should be treated

with aeciospores collected from cankers on whitebark pine,

in the event there are different rust populations in whitebark and western white pine communities. Aeciospores will

be collected 1 to 2 years prior to rust screening from a

representative sample across all seed zones. State-to-state

plant inspection regulations may prohibit the transfer of

spore collections across state lines, so further modifications

in the rust screening protocol may be warranted in the future.

This conservative approach is also appropriate when considering the alternate host, because a different mix of Ribes

spp. occurs in whitebark pine communities (for example,

Ribes lacustre, R. viscosissimum, and R. montigenum) than

in western white pine (for example, R. cereum, R. nigrum

and R. hudsonianum var. petiolare). A Ribes garden for

whitebark pine inoculations was established at Lone Mountain Tree Improvement Area, Idaho Panhandle National

Forests in 2000.

Hoff and others (1994) recommended inoculating 2-year

old whitebark pine seedlings. Due to the slower growth rates

of whitebark pine as compared to western white pine, these

rust screenings will use 3-year old container seedlings in

order to have enough top shoot and secondary needles to be

challenged with inoculum.

During the inoculation procedure, basidiospores will

be delivered at a target rate of 3,500 spores per cm2.

Previous rust screenings of whitebark pine using a rate

recommended for western white pine have shown a delivery

of 6,000 spores per cm2 to be too high, killing most of the

seedlings in a given block (Mahalovich unpublished data).

Four rust inspections will be performed in each trial. The

first and second inspections will occur 9 months and 12

months, respectively, after inoculation. The third and fourth

inspections will occur during September in subsequent

Table 2—Acceptable canker limits for individual plus-trees based

on stand averages.

Stand average (cankers/tree) Plus-tree limits

10 to 20 No cankers

21 to 40 1 canker

41 to 75 2 cankers

76 to 150 3 cankers

151+ 4 or 5 cankers
186 USDA Forest Service Proceedings RMRS-P-32. 2004

Mahalovich and Dickerson Whitebark Pine Genetic Restoration Program for the Intermountain West (USA)

years. Overall, the four rust inspections span a 3-year

period. Data collected during each inspection will be the

same as data acquisition for western white pine trials

(Mahalovich and Eramian 1995).

Last, to minimize cross-contamination of susceptible

seedlings and inoculated Ribes spp. leaves, and the possible

introduction of virulent rust races between species, a

recommended quarantine procedure is to avoid inoculating

western white and whitebark pine seedlings in the same

calendar year at the same location (Coeur d’Alene Nursery,

Coeur d’Alene, Idaho).

Data Applications

Refine Seed Transfer Guidelines—Seed transfer

(Mahalovich and Hoff 2000) is currently based on seed

zones (fig. 1) driven by major mountain ranges and existing

knowledge of blister rust infection levels in populations of

whitebark pine (Hoff and others 1994). A better approach

to seed transfer is to develop guidelines based on phenological and blister rust resistance data. Early genetic studies

using isozymes point to low levels of genetic variation among

and within-stands of whitebark pine (Lanner 1982, Jorgensen

and Hamrick 1997, Bruederle and others 1998). Richardson

(2001) examined uniparentally inherited mitochondrial

(mt)DNA and chloroplast cp(DNA) microsatellites (cpSSRs)

to examine population genetic structure from 38 coastal and

interior populations of whitebark pine. Analysis of Molecular Variance (AMOVA) groups based on an exact test suggested four zones among Inland West populations: Sierra

Nevada Mountains, Yellowstone, central Idaho, and northern Idaho. Data obtained from the sites sampled for plustrees (blister rust infection levels) and the rust screening

trials will validate whether the existing seed zones could be

combined into four zones, determine where the geographic

boundaries should be drawn, and provide a model for predicting safe seed transfer for individual seed lots using a

seed transfer expert system. Zone boundaries will be revised

before proceeding with the establishment of seed orchards

and clone banks.

Seed Orchard Establishment and Design—Each

plus-tree will be ranked based on the performance of its

progeny in the rust screening trials using the same evaluation criteria established in western white pine (Mahalovich

and Eramian 1995). Preliminary rust screenings have shown

whitebark pine seedlings to exhibit rust resistance responses

much like the other five-needle pines but at different frequencies (Hoff and others 1980, Hoff and Hagle 1990). The

higher-ranking parent trees will be revisited to collect scion

for establishing production seed orchards within each zone.

Sowing and growing of rootstock will be coordinated with the

completion of each rust screening. Until these orchards

reach reproductive maturity, the rankings of the plus-trees

can be matched to their native stands to identify promising

cone collecting areas (seed collection stand or seed production area) not previously identified during 2001 through

2005, to meet more immediate seed needs for resistant

planting stock.

Data collected from the rust screenings will also be used to

facilitate seed orchard design and seed deployment strategies by resistance mechanism(s). This strategy of using

patterns of genetic variation and deploying more than one

resistance mechanism on any given hectare makes it unlikely a new, more virulent race of rust will develop in

planted stock (Mahalovich and Eramian 1995).

Pollen can be a limiting factor in immature pine orchards,

when the goal is to obtain enough sound seed from a broad

genetic base as quickly as possible. A practical application of

collecting whitebark pine pollen will be supplemental mass

pollination in the grafted seed orchard(s) to promote an

earlier cone crop rather than relying on wind pollination.

Unlike long-term storage of whitebark pine seed, there are no

major pollen viability problems over the long-term with Pinus

spp., as long as the pollen is properly extracted and stored.

Additional Gene Conservation Measures—Pollen will

also be collected to establish a pollen bank as part of the ex

situ strategy and to advance blister rust resistance in seed

and breeding orchards.

The surviving progeny in each rust screening will be used

to establish clone banks. Though not in our life times, these

clone banks could serve as an operational cone collection

site if they are designed by zone, concentrating the better

performers in the interior core to enhance gain and in

grouping trees by resistance mechanism, as is done in the

Phase II western white pine seed orchards (Mahalovich and

Eramian 1995).

Last, this information can be cross-referenced with field

inventories to prioritize those communities that are good

candidates to stabilize their numbers by active intervention

involving prescribed fire to promote natural regeneration

and by removal of encroaching species such as subalpine fir,

lodgepole pine and Engelmann spruce.

Summary ______________________

This restoration strategy highlights the need to incorporate genetic considerations into a comprehensive strategy to

restore whitebark pine. It emphasizes the biology and

genecology of the host species, with a modest emphasis on

the biology and ecology of the rust. The amount of gain

achieved in blister rust and mountain pine beetle resistance

will be determined by how many cones are collected from

presumably rust-free and insect-free trees in areas with a

high frequency of blister rust and insect populations. Meaningful levels of genetic variation are needed in adaptive

traits (for example, survival, growth, insect and disease

resistance) to develop seed transfer guidelines and improved

planting stock.

Acknowledgments ______________

We thank Robert L. Schrenk and Michael J. Paterni for

their leadership and support for a landscape approach to

whitebark pine restoration and the many silviculturists,

wildlife biologists, pathologists, and fire fighters on National Forest System and National Park lands involved in

cone and spore collections. We also thank Drs. Sue Hagle,

Mee-Sook Kim, Ned Klopfenstein, Gerald E. Rehfeldt, and

John Schwandt, and Ms. Maridel Merritt for their comments

on an earlier version of this manuscript. This work is funded

in part by USDA Forest Service Title IV Wildland Fire

Emergency, Rehabilitation and Restoration Appropriations.
USDA Forest Service Proceedings RMRS-P-32. 2004 187

Whitebark Pine Genetic Restoration Program for the Intermountain West (USA) Mahalovich and Dickerson

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albicaulis (Pinaceae). Can. J. Bot. 76:83-90.

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pine seedlings for restoration. In Whitebark pine communities,

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R.C. 1999. Genetic structure of Cronartium ribicola populations

in eastern Canada. Phytopathology 89:915-919.

Hoff, R.J., Bingham, R.T., McDonald, G.I. 1980. Relative blister rust

resistance of white pines. European Jounral of Forest Pathology.

10:307-316.

Hoff, R.J., Hagle, S. 1990. Disease of whitebark pine with special

emphasis on white pine blister rust. In Proc.—Symposium on

Whitebark Pine Ecosystems: Ecology and Management of a HighMountain Resource, March 29-31, 1989, Bozeman, MT, USDA

Forest Service, Intermountain Research Station, Gen. Tech. Rep.

INT-GTR-270, Ogden, Utah, pp 179-190.

Hoff, R.J., Hagle, S.K., Krebill, R.G. 1994. Genetic consequences

and research challenges of blister rust in whitebark pine forests.

In Proc. International Workshop on Subalpine Stone Pines and

Their Environment: the Status of Our Knowledge, September 5-

11, 1992, St. Moritz, Switzerland, USDA Forest Service, Intermountain Research Station, Gen. Tech. Rep. INT-GTR-309, Ogden,

Utah, pp 118-135.

Hoff, R.J., McDonald, G.I. 1980. Improving rust-resistant strains of

Inland Western white pine. USDA Forest Service, Intermountain

Forest and Range Experiment Station, Ogden, Utah, Research

Paper INT-245, 13 p.

Hoff, R.J., McDonald, G.I. 1977. Selecting western white pine leavetrees. USDA Forest Service, Intermountain Forest and Range

Experiment Station, Ogden, Utah, Research Note INT-218, 2p.

Jorgenson, S.M., Hamrick, J.L. 1997. Biogeography and population

genetics of whitebark pine, Pinus albicaulis. Can. J. For. Res.

27:1574-1585.

Kendall, K. 1994. Whitebark pine conservation in North American

National Parks. In Proc. International Workshop on Subalpine

Stone Pines and Their Environment: the Status of Our Knowledge, September 5-11, 1992, St. Moritz, Switzerland, USDA

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INT-GTR-309, Ogden, Utah, pp 302-305.

Kinloch,B.B., Jr., Westfall, R.D., White, E.E., Gitzendanner, M.A.,

Dupper, G.E., Foord, B.M, Hodgskiss, P.D. 1998. Genetics of

Cronartium ribicola. IV. Population structure in western North

America. Can. J. Bot. 6:91-98.

Kolotelo, D., Van Steenis, E., Peterson, M., Bennett, R., Trotter, D.,

Dennis, J. 2001. Seed Handling Guidebook. B.C. Ministry of

Forests, Tree Improvement Branch, 106 pp.

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by Clark’s nutcracker. Can. J. For. Res. 12: 391-402.

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and important hardwoods. USDA Forest Service Misc. Pub. No.

1146, Washington, DC, p 43-W.

Mahalovich, M.F., Hoff, R.J. 2000. Whitebark pine operational cone

collection instructions and seed transfer guidelines. Nutcracker

Notes No. 11, pp 10-13.

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restoration plan for the development of blister rust resistant

white pine for the northern Rockies. USDA Forest Service Northern Region and Inland Empire Tree Improvement Cooperative,

60 pp.

McCaughey, W.W., Schmidt, W.C. 2001. Taxonomy, distribution,

and history. In Whitebark pine communities, Island Press, Washington, D.C., pp 29-40.

McDonald, G.I. 1978. Segregation of “red” and “yellow” needle

lesion types among monoaeciospore lines of Cronartium

ribicola. Can. J. Genet. Cytol. 20:313-324.

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In Whitebark pine communities, Island Press, Washington, D.C.,

pp 193-220.

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movement and biogeography. Unpublished Master’s Thesis,

University of Idaho, College of Forest Resources, Moscow, ID,

55pp.

Schwandt, J.W., Marsden, M.A., McDonald, G.I. 1994. Pruning and

thinning effects on white pine survival and volume in northern

Idaho. In Proc. Interior Cedar-Hemlock-White Pine Forests:

Ecology and Management, March 2-4, 1993, Spokane, WA. Edited

by Baumgartner, D.M., J.E. Lotan, and J.R. Tonn,Washington

State University, Department of Natural Resources, Cooperative

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Northern Region, Missoula, MT.

Tomback, D.F., Kendall, K.C. 2001. Biodiversity losses: the

downward spiral. In Whitebark pine communities, Island Press,

Washington, D.C., pp 243-262.

Tomback, D.F., Schuster, W.S.F. 1994. Genetic population structure and growth form distribution in bird-dispersed pines. In

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188 USDA Forest Service Proceedings RMRS-P-32. 2004

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

Sei-ichi Kanetani, Ayako Kanazashi, and Hiroshi Yoshimaruare with the

Ecological Genetics Laboratory, Department of Forest Genetics, Forestry

and Forest Products Research Institute, 1 Matsunosato, Tsukuba, Ibaraki

305-8687, Japan. Phone: +81- 298-73-3211 ext. 445; FAX: +81- 298-74-3720;

e-mail: kanekane@ffpri.affrc.go.jp. Takayuki Kawahara is with the Hokkaido

Research Center, Forestry and Forest Products Research Institute, Japan.

Abstract—Pinus armandii var. amamiana is endemic to two small

islands in the southern region of Japan and listed as a vulnerable

species. The large size and high wood quality of the species have

caused extensive harvesting, resulting in small population size and

isolated solitary trees. Genetic diversity of the P. armandii complex

was studied using allozyme analyses. Genetic distances between

(1) P. armandii var. amamiana and P. armandii var. armandii and

(2) P. armandii var. amamiana and P. armandii var. mastersiana

were 0.488 and 0.238, respectively. These genetic differences were

comparable with congeneric species level (0.4) and much greater

than conspecific population level (less than 0.1) that occur in Pinus

species in general. No differences in diversity were recognized

between populations of P. armandii var. amamiana from each

island. The impact of human activity on the endangered status of P.

armandii var. amamiana in Tane-ga-shima Island was demonstrated by inspecting historical records, starting in the 16th century. A strategy for conservation of P. armandii var. amamiana was

discussed in consideration of sparse distribution, pollen flow, and

the effects of pine wilt disease, caused by Bursaphelenchus xylophilus.

Key words: Pinus armandii var. amamiana, endangered species,

genetic diversity, pollen flow, conservation strategy

Introduction ____________________

Pinus armandii Franch. var. amamiana (Koidz.)

Hatusima, is an endangered pine species endemic to Tanega-shima and Yaku-shima Islands, southern Japan (Yahara

and others 1987). The species is closely related toP. armandii

var. armandii that is distributed in the western part of

continental China andP. armandii var. mastersianaHayata

from the highlands of Taiwan. The wood of P. armandii var.

Diversity and Conservation of Genetic

Resources of an Endangered Five-Needle

Pine Species, Pinus armandii Franch. var.

amamiana (Koidz.) Hatusima

Sei-ichi Kanetani

Takayuki Kawahara

Ayako Kanazashi

Hiroshi Yoshimaru

amamiana was traditionally used for making fishing canoes

and also used in house construction (Kanetani and others

2001). Consequently, large numbers of P. armandii var.

amamiana trees have been harvested and populations have

dwindled on both islands. Currently, the estimated number

of surviving P. armandii var. amamiana trees in natural

populations are 100 and 1,000 to 1,500 on Tane-ga-shima

and Yaku-shima Islands, respectively (Yamamoto and Akasi

1994).

In recent years, the number of P. armandii var. amamiana

trees has rapidly declined, with dead trees frequently observed (Hayashi 1988; Yamamoto and Akashi 1994; Kanetani

and others 2002). Several factors are responsible for the

recent decline, including inbreeding depression (Hayashi

1988; Kanazashi and others 1998), reduced natural regeneration (Chigira 1995; Kanetani and others 1998), and pine

wilt disease (Hayashi 1988; Yamamoto and Akashi 1994;

Nakamura and others 2002). Pinus armandii var. amamiana

has been classified as an “Endangered” species in the Japanese Red List, a compilation of endangered Japanese species,

due to the rapid decrease in population size and the isolation

of small populations (Environment Agency of Japan 2000).

In order to establish an in situ conservation scheme for an

endangered species, it is important to collect information on

the species in natural habitats, such as decline and genetic

variation (compare Primack 1995; Meffe and Carroll 1997).

In this study, we clarified the genetic diversity and phylogenetic relationship of P. armandii var. amamiana with other

P. armandii varities, researched the historical distribution

of the species on Tane-ga-shima Island, and propose a

strategy to conserve the genetic resources of this species.

Materials and Methods ___________

Study Site

Tane-ga-shima and Yaku-shima Islands are located about

60 km south of Kyushu Island in southern Japan (fig.1).

These islands are approximately 20 km apart. Tane-gashima Island is relatively flat (elevation to 282 m), 58 km

long (north to south), and 10 km wide (west to east). In

contrast, Yaku-shima Island is round with a diameter of

about 30 km and dominated by a series of peaks over 1,800 m

in height. About 21 percent of the island has been registered

as a World Natural Heritage Area in 1993.
USDA Forest Service Proceedings RMRS-P-32. 2004 189

Figure 1—Distribution of Pinus armandii vars. amamiana,

armandii and mastersiana populations.

Surviving P. armandii var. amamiana trees are located in

the center of Tane-ga-shima Island, while three local populations of this species are distributed from 300 to 800 m

elevation on Yaku-shima Island (Yamamoto and Akashi

1994; Kanetani and others 1997).

Genetic Variation

We collected 88 samples of P. armandii var. amamiana

from three populations, Segire, Takahira and Hirauchi, on

Yaku-shima Island, and 17 samples from Tane-ga-shima

Island. The trees from Tane-ga-shima Island were treated as

one population because most trees are solitary or occur in

small groups. We collected 24 samples of P. armandii var.

armandii from one natural population near Dali, Yunnan,

China, and 12 samples of P. armandii var. mastersiana from

seedlings originating from a population near Musha, Taiwan.

Crude extracts from inner bark of twigs were prepared

according to Yahara and others (1989) for allozyme analyses. The starch gel electrophoretic system with tris-borate

buffer (#8 in Soltis and others 1983) was used for allozyme

analysis on mannose phosphate isomerase (MPI; 1),

phosphoglucoisomerase (PGI; 2) and triose-phosphate

isomerase (TPI; 2). The numbers in parentheses refer to the

numbers of allozyme loci employed in the following analyses.

Another starch gel electrophretic system with histidine and

citric acid buffer (Cardy and others 1981) was used for

isocitrate dehydrogenase (IDH; 1), malate dehydrogenase

(MDH; 2), 6-phosphogluconate dehydrogenase (6PGD; 1),

phosphoglucomutase (PGM; 2), shikimate dehydrogenase

(SkDH; 1) and UDP glucose pyrophosphorylase (UGP; 1).

Acrylamide gel electrophoresis (Tsumura and others 1990)

were used for asparatate aminotransferase (AAT; 2), amylase (AMY; 1), esterase (EST; 1), glutamate dehydrogenase

(GDH; 1) and leucine aminopeptidase (LAP; 2).

Calculations of Genetic Parameters

Three kinds of genetic diversity (P, rate of polymorphic

loci; A, mean number of alleles; He, expected mean heterozygosity) and fixation index for the populations of P. armandii

var. amamiana were calculated. For calculations of Nei’s

genetic distance and genetic identity (Nei 1972), the Segire

population was excluded, since the number of examined loci

was different from those of the other populations. F-tests

were used to detect significant differences.

Historical Distribution of P. armandii var.

amamiana on Tane-ga-shima Island

We surveyed historical records owned by Kagoshima Prefectural Museum of Culture, Reimei-kan for all descriptions

on surviving number and size of “Goyo-matsu (5-needlepine; var. amamiana)” and the number of “Maruki-bune”

(canoes) constructed.

Results ________________________

Genetic Variation

The three populations on Yaku-shima Island showed

values of 0.069-0.131 (mean: 0.100) for He, while the He

value for the Tane-ga-shima population was 0.112 (table 1).

Although the population of surviving trees in Tane-gashima Island is much less than populations on Yaku-shima

Island, it contains the nearly same amount of genetic diversity. The Fixation Index (F) in the Tane-ga-shima population, 0.198 (not significant), is larger than those of three

populations in Yaku-shima Island, 0.012-0.074 (mean: 0.051).

Genetic identity and Nei’s standard genetic distance among

the populations of P. armandii vars. amamiana, armandii,

and mastersiana were shown in table 2. Mean genetic distance among P. armandii var. amamiana populations was

ranged from 0.003 to 0.029 (mean: 0.017). Genetic distances

between P. armandii var. amamiana vs. var. mastersiana, P.

armandii var. amamiana vs. var. armandii and P. armandii

var. armandii vs. var. mastersiana were 0.460-0.510 (mean:

0.488), 0.220-0.250 (mean: 0.238) and 0.137, respectively.

Historical Distribution on Tane-ga-shima

Island

In Tane-ga-shima Island, harvest of large P. armandii

var. amamiana populations was regulated by the Shimadzu

local government from the 16th through the 19th century

(Kanetani and others 2001). Descriptions of the number

and stem girth of P. armandii var. amamiana trees were

found from 1685 to 1782. In particularly, 428 P. armandii

var. amamiana trees with stem girth above 150 cm were

recorded in 1755. Table 3 is a summary of the number of
190 USDA Forest Service Proceedings RMRS-P-32. 2004

Kanetani, Kawahara, Kanazashi, and Yoshimaru Diversity and Conservation of Genetic Resources of an Endangered Five-Needle Pine Species,...

trees and stem girth of P. armandii var. amamiana found in

the historical record.

It is known large numbers of P. armandii var. amamiana

were harvested for making fishing canoes and house construction during late 19th and early 20th century (Kanetani

and others 2001). In 1918, 455 canoes were made probably

from P. armandii var. amamiana. Canoes were used for

fishing until twenty years ago in Tane-ga-shima Island.

Discussion _____________________

The genetic diversity level of P. armandii var. amamiana

(He: 0.069-0.131) is a little lower than the mean value of

the genus Pinus (He: 0.136) (Hamrick and others 1992), in

which Hamrick and Godt (1996) recognized wide variation of

genetic diversity and population structure. In comparison,

Pinus torrayana, an endemic species in California with an

extremely restricted distribution, has a low genetic diversity of 0.017 (Ledig and Conkle 1983). P. armandii var.

amamiana also has a limited distribution, but maintains a

more or less high genetic diversity level. In Tane-ga-shima

Island, the trees are separated each other but still retain a

level of genetic diversity as populations on Yaku-shima

Island. This indicates that the serious decrease of tree

number in Tane-ga-shima Island occurred in the near past.

Fixation indices are almost zero in Yaku-shima Island

populations and 0.198 in Tane-ga-shima Island. This value

shows that adult P. armandii var. amamiana trees in Yakushima Island have been produced from random mating. The

high value in Tane-ga-shima Island may be explained by the

Wahlund effect.

The genetic difference among the three conspecific taxa of

P. armandii is large. Hamrick and Godt (1996) reviewed

genetic heterogeneity of pine populations and introduced

some species with disjunct distributions that have high gene

diversity levels. The genetic difference of the varieties in P.

armandii is at the congeneric species level (0.4) and much

greater than conspecific population level (less than 0.1)

according to Gottlieb (1977, 1981) and Crawford (1983).

Therefore genetic conservation on P. armandii should be

conducted at least for varieties amamiana, armandii and

mastersiana, respectively.

A bibliographical study demonstrated the destructive

cuttings of P. armandii var. amamiana in Tane-ga-shima

Island until the early 20th century. The species was preferred for making canoes because of a greater amount of

resin than in other tree species in Tane-ga-shima Island and

only large trees, 2.7m - 6.3 m were used. The historical record

indicates that 400 trees were preserved about 250 years ago

(table 3). In 1918, however, harvesting must have increased,

as 455 canoes made of P. armandii var. amamiana, were

counted. Recently, we have discovered that a fewP. armandii

var. amamiana trees were harvested for use in quarries

(Kanetani and others 2001). Therefore, the human impact

on this species is still continuing.

As a consequent of human impacts, the reproductive

potential of the surviving trees on Tane-ga-shima Island

Table 2—Genetic identity (upper triangle) and Nei’s standard genetic distance (lower triangle) among Pinus armandii

varieties.

Population (variety) 1 2 3 4 5

1. Takahira (var. amamiana) .980 .998 .631 .803

2. Hirauchi (var. amamiana) .020 .971 .600 .779

3. Tane-ga-shima Island (var. amamiana) .003 .029 .610 .784

4. Dali, Yunnan, China (var. armandii) .460 .510 .495 .872

5. Musha, Taiwan (var. mastersiana) .220 .250 .244 .137

Table 1—Genetic diversity and fixation index of Pinus armandii var. amamiana populations.

Population Number of loci Pa Ab Hec Fd

Segire (Yaku-shima Island) 13 0.31 1.31 0.069 0.067ns

Takahira (Yaku-shima Island) 20 0.50 1.60 0.131 0.074ns

Hirauchi (Yaku-shima Island) 20 0.40 1.45 0.101 0.012ns

Tane-ga-shima Island 20 0.45 1.55 0.112 0.198ns

a: rate of polymorphic loci

b: mean number of alleles per locus

c: mean heterozygosity

d: fixation index

ns: not significantly different from zero

Table 3—Historical record of stem girth and number of trees of

Pinus armandii var. amamiana growing on Tanega-shima Island.

Year Stem girth (cm) Number of trees

1685 — 247

1748 210 - 420 355

1755 210 - 420 218

150 - 180 210

1782 — 28
USDA Forest Service Proceedings RMRS-P-32. 2004 191

Diversity and Conservation of Genetic Resources of an Endangered Five-Needle Pine Species,... Kanetani, Kawahara, Kanazashi, and Yoshimaru

may be limited. Kanazashi and others (1998) and Nakashima

and Kanazashi (2000) compared cone yields from artificial

pollinations to natural (open) pollinations on several isolated trees on Tane-ga-shima Island. The percentage of filled

seeds per cone from natural pollinations (15.4 to 37.7 percent) was less than artificial cross-pollinations, which ranged

from 66.8 to 97.3 percent. However, there was no significant

difference (F-test) in the percentage of filled seeds after

artificial self-pollination (34.3 percent) and after open-pollination (34.6 percent). This suggests that natural pollination

among Tane-ga-shima Island trees could be primarily selfpollination, which will promote inbreeding depression.

P. armandii var. amamiana are now threatened by pine

wilt disease, an epidemic disease of the genus Pinus in

Japan caused by the nematode Bursaphelenchus xylophilus

(Steiner and Buhrer) Nickle (Kiyohara and Tokushige 1971;

Kishi 1995). This disease has been inferred to be a major

mortality factor of P. armandii var. amamiana in natural

populations (Hayashi 1988; Yamamoto and Akashi 1994).

Recently, the nematode’s presence was confirmed through

detection in dead P. armandii var. amamiana trees in Tanega-shima Island (Nakamura and others 2002). Pine wilt

disease, therefore, is a significant threat to the continued

existence of natural populations of P. armandii var. amamiana.

The serious decline of P. armandii var. amamiana necessitates the formation of a strategy for genetic conservation.

Protection and management should be required for in situ

populations on both islands, and demise from pine wilt

disease should be carefully monitored. The small, diffuse

population on Tane-ga-shima Island requires artificial cross

pollinations for restoration of seed fertility and successful

reproduction. Additionally, the establishment of ex situ

plantations containing grafts of mature trees of P. armandii

var. amamiana (for ex situ conservation) is needed to ensure

conservation of genetic diversity of Tane-ga-shima Island

populations.

Acknowledgments ______________

We are very grateful to K. Nakashima for his valuable

contributions to our research. Special thanks are due to the

Yaku-shima Management Office of the Environment Agency,

the Yaku-shima Forest Environment Conservation Center

and the Kagoshima Forest Office of the Forest Agency for

permission to conduct this study. We express our gratitude

to H. Iwatsubo, M. Nao, Y. Sameshima, M. Okumura, Y,

Furuichi, K. Tokunaga and Y. Oguchi for their kind help

with bibliographical and field studies.

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192 USDA Forest Service Proceedings RMRS-P-32. 2004

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

Vladimir Potenko is head of the Department of Genetics and Breeding,

Breeding and Seed Growing Forestry Center, Nagornaya 12, Sosnovka

680555, Khabarovsk Territory, Russia. Tel./fax: +7-4212-922-445, e-mail:

forestry@mail.kht.ru

Abstract—Based on the analysis of 26 allozyme loci, levels of

genetic variation were ascertained in 25 natural populations of

Korean pine. On average, 58.0 percent of the loci were polymorphic;

the number of alleles per locus was 1.92; the expected heterozygosity was 0.182, and the observed heterozygosity was 0.180. On

average, the heterozygote deficiency was characteristic of Korean

pine populations (FIS=0.013). The most diversity was found within

populations (FST=0.018). Genetic distances between populations

were small (on average, DN=0.003). Level of gene flow was 10.98

migrants per generation. Multilocus outcrossing estimates ranged

from 0.751 to 1.031, indicating mating system differences. Results

of this study lead to the assumption that the genetic structure of

Korean pine populations is under the influence of a complex combination of microevolution factors, including genetic drift, gene flow

and natural selection.

Key words: Korean pine, allozymes, genetic variation, differentiation, gene flow, mating system.

Introduction ____________________

The Korean pine, Pinus koraiensis Sieb. & Zucc., occurs in

natural and artificial stands in Russia, China, Korea, and

Japan. In the Russian Far East, P. koraiensis is distributed

in the Primorski Territory, in the southern part of Khabarovsk

Territory, in the Jewish Autonomous Region and at the

southeast end of the Amur Territory (fig. 1). Usually, P.

koraiensis grows in mixed stands with broadleaf tree species.

The Korean pine-broadleaf forests occupy low and middle

altitude zones growing in a wide range of relief and soil

conditions. In the south Sikhote Alin mountain range they

occur up to 900 m above sea level, while in the north, Korean

pine reaches only to 500 m (Usenko 1969). Selective harvesting and fires have repeatedly stressed most of the forests. At

present, clear cuttings of broad-leaved Korean pine mixed

forests are illegal. However, the harvest of the broad-leaved

Korean pine forests is occurring without authorization because of demand for pine and hardwood timber. For this

reason, the broad-leaved Korean pine forestlands are decreasing (Koryakin and Romanova 1996). Thus there is a

Genetic Diversity and Mating System of

Korean Pine in Russia

Vladimir Potenko

need to emphasize conservation of Korean pine genetic

resources.

Knowledge of the level and distribution of genetic variation, both within and among populations, facilitates the

conservation of gene resources (Brown 1978; Millar and

Libby 1991). Recently, the results of genetic variation studies of Korean pine populations in the Russian Far East have

been reported (Krutovskii and others 1995; Potenko and

Velikov 1998) and South Korea (Kim and others 1994).

Differences in levels of genetic variation were observed

within and among the populations in different parts of

Korean pine’s natural range (Potenko and Velikov 1998).

Greater variation was found in South Korean populations,

with less variation occurring in the northwestern part of

the natural range in Russia. Additionally, the measurements of mating systems showed a high proportion of outbred progeny in an earlier study of three Korean pine

populations (Politov and Krutovskii 1994; Krutovskii and

others 1995).

The primary objectives of this study were to analyze the

genetic diversity and mating system of Korean pine throughout the natural range in Russia and to describe geographical

patterns of genetic variation.

Materials and Methods ___________

Seeds for electrophoresis were collected in 25 native populations from 43∞ to 51∞ latitude north (fig. 2). In 17 populations the collection of seeds was performed on individual

trees. The remaining eight populations were represented by

bulked seed lots that were collected from native populations

by state forest farms for artificial reforestation (table 1).

More details about seed samples and characteristics of the

sampled populations can be found elsewhere (Potenko and

Velikov 1998, 2001).

Six megagametophytes and ten embryos per tree were

subjected to horizontal starch gel electrophoresis. Details of

laboratory procedures are described in Potenko and Velikov

(1998). Seed tissues were analyzed for 15 enzyme systems:

aspartate aminotransferase (AAT), alcohol dehydrogenase

(ADH), aconitase (ACO), diaphorase (DIA), fluorescent esterase (Fl-EST), formate dehydrogenase (FDH), glutamate

dehydrogenase (GDH), glutamate pyruvate transaminase

(GPT), isocitrate dehydrogenase (IDH), leucine aminopeptidase (LAP), malate dehydrogenase (MDH), phosphoglucomutase (PGM), 6-phosphogluconate dehydrogenase (6-PGD),

shikimate dehydrogenase (SkDH) and sorbitol dehydrogenase (SDH). In total, 26 loci were scored for genetic variation

analysis (Aat-1, Aat-2, Aat-3, Adh-1, Adh-2, Aco, Gdh, Dia1, Dia-3, Idh, Lap-1, Lap-2, Mdh-1, Mdh-2, Mdh-3, Mdh-4,

Gpt, Sdh, Fl-Est, Fdh, Pgm-1, Pgm-2, 6-Pgd-1, 6-Pgd-2,

Skdh-1 and Skdh-2). For mating system analysis, four loci

(Aat-3, Dia-1, Pgm-1 and Skdh-1) were used.
USDA Forest Service Proceedings RMRS-P-32. 2004 193

Genetic Diversity and Mating System of Korean Pine in Russia Potenko

Figure 1—Distribution of Korean pine (modified from Schmidt 1994). Nos: 1 - Amur Territory;

2 - Jewish Autonomus Region; 3 - Khabarovsk Territory; 4 - Primorski Territory.
194 USDA Forest Service Proceedings RMRS-P-32. 2004

Potenko Genetic Diversity and Mating System of Korean Pine in Russia

Figure 2—Location of the sampled populations: ▲ - seed collection was conducted from individual

tree; D- seed lot was sampled. Nos: population numbers shown in Table 1. Solid line: limit of distribution

of P. koraiensis in Russia. Dotted line: northern limit of the Korean pine-broadleaf and Korean pinespruce-larch mixed forests in the Holocene climate optimum (modified from Korotkii and others 1997).
USDA Forest Service Proceedings RMRS-P-32. 2004 195

Genetic Diversity and Mating System of Korean Pine in Russia Potenko

Allele frequencies were analyzed using the BIOSYS-1

computer program (Swofford and Selander 1989). For each

population, mean number of alleles per locus (A), percentage

of polymorphic loci (P0.95 and P0.99) and expected heterozygosity (He) were computed. In addition, Nei’s genetic distances (DN) were calculated (Nei 1978).

For assaying the population genetic structure, the fixation

indices (FIS, FIT and FST) were used (Nei 1977). FIS and FIT

measure the deviation of genotype frequencies from HardyWeinberg proportions in the populations and in the total

population respectively, whereas FST measures the degree of

genetic differentiation among populations. The FST values

were used to calculate interpopulational gene flow (Nm) as

follows: FST=1/(4Nma+1), where a=(n/n-1)2, and n is the

number of populations (Govindaraju 1989).

The expected fixation index at inbreeding equilibrium was

computed as Fe=(1-tm)/(1+tm), where tm is the multilocus

outcrossing rate (Allard and others 1968).

Single locus (ts) and multilocus (tm) estimates of the

proportion of progeny resulting from outcrossing in a population were determined using the MLT computer program

(Ritland 1990). Maternal genotypes, assessed from megagametophyte segregations, were taken into account. The confidence intervals of the outcrossing rates were estimated

after 100 boot-straps. At Dia-1, the 4th allele Dia-10.60 with

the lowest frequency was combined with the allele Dia-11.37

having the nearest frequency because the computer program can only process a maximum of three alleles per locus.

Both ts and tm estimates are based on the mixed mating

model, which assumes (1) that each viable offspring is the

result of a random outcross (with probability t) or a selffertilization (with probability s=1-t), (2) that the probability

of an offspring being an outcross is independent of the

genotype of the maternal parent, (3) that outcross pollen

pool allele frequencies are homogeneous over space and over

time, and (4) that there is no selection between pollination

Table 1—Genetic variability at 26 loci in 25 populations of P. koraiensis (standard errors in parentheses).

Mean No.

of alleles Percentage of

Number of per locus, polymorphic loci Mean heterozygosity

Population trees A P95 P99 Observed, Ho Expected, He

1. Obluchie 21 1.85 46.2 61.5 0.170 0.171

2. Sutaraa 70b 1.69 42.3 57.7 - 0.169

3. Dogordon 52 1.92 50.0 53.8 0.164 0.171

4. Kukana 70b 1.65 46.2 50.0 - 0.170

5. Nirana 70b 1.88 46.2 61.5 - 0.165

6. Selgona 70b 1.96 42.3 53.8 - 0.154

7. Galichnoe 72 2.12 57.7 69.2 0.193 0.204

8. Pivana 70b 1.85 53.8 53.8 - 0.194

9. Innokentievkaa 70b 1.77 46.2 53.8 - 0.174

10. Burga 53 1.92 50.0 53.8 0.174 0.187

11. Mulcha 62 2.08 46.2 57.7 0.191 0.189

12. Sukpaya 70b 1.96 50.0 61.5 - 0.181

13. Obora 70b 2.00 53.8 61.5 - 0.183

14. Medvezhy 49 1.88 53.8 57.7 0.180 0.194

15. Boicovo 24 1.81 46.2 53.8 0.164 0.181

16. Pokrovka 73 2.19 50.0 61.5 0.188 0.193

17. Mel’nichnoe 73 2.15 46.2 57.7 0.180 0.180

18. Malinovo 50 2.08 53.8 69.2 0.189 0.218

19. Kedrovaya Pad’ 38 1.85 50.0 61.5 0.186 0.187

20. Arkhipovka 51 2.08 50.0 61.5 0.199 0.197

21. Kievka 68 1.85 50.0 53.9 0.172 0.178

22. Petrov‘s Island 21 1.73 42.3 50.0 0.177 0.161

23. Ustinovka 63 2.00 50.0 61.5 0.193 0.187

24. Ternei 61 1.85 46.2 53.9 0.192 0.190

25. Lesnoi 58 1.92 50.0 57.7 0.156 0.163

Mean for populations Nos. 1-6

Maliy Khingan - Kukan mountain 1.83 45.5 56.4 0.167 0.167

ranges (0.13) (2.9) (4.6) (0.004) (0.007)

Mean for populations Nos. 7-25

Sikhote Alin - Low Amur mountain 1.95 49.8 58.5 0.182 0.186

ranges (0.14) (3.7) (5.2) (0.012) (0.013)

Mean 1.92 48.8 58.0 0.180 0.182

(0.14) (3.9) (5.1) (0.012) (0.015)

a Bulked seed lot analyzed. Total weight of any seed lot was 500 kg.

b Number of analyzed seeds per seed lot.
196 USDA Forest Service Proceedings RMRS-P-32. 2004

Potenko Genetic Diversity and Mating System of Korean Pine in Russia

and the time that seeds or seedlings are sampled (Shaw and

others 1981). Multilocus estimations require the additional

assumption of independence among loci in the outcross

pollen pool. Conkle (1981), Politov and others (1989) and

Goncharenko and others (1998) showed that the gene arrangement is highly conservative in the pines and found no

linkage among loci Aat-3, Dia-1, Pgm-1 and Skdh-1.

Results and Discussion __________

Genetic Diversity and Differentiation

Parameters of genetic variation (table 1) were calculated

on the basis of allele frequencies of 26 loci. In Korean pine

populations, the mean number of alleles per locus ranged

from 1.65 to 2.19, with an average of 1.92. The proportion of

polymorphic loci (P0.99) ranged from 50.0 to 69.2 percent,

with an average of 58.0 percent. The observed heterozygosity was from 0.156 to 0.199, with an average of 0.180. The

expected heterozygosity ranged from 0.154 to 0.218, with an

average of 0.182. The genetic variation was lower than in

Korean pine populations of South Korea (on average,

P99=69.0, A=2.0, H0=0.200, He=0.208; Kim and others 1994).

The results seem to support the hypothesis that Korean pine

expanded to the far eastern region of Russia from the south

in the Holocene. The studies of fossil conifer pollen (Korotkii

and others 1997) indicate that 18,000 to 20,000 years ago the

vegetation of Sikhote Alin was similar to that of the contemporary northwest coast of the Sea of Okhotsk. After climatic

cooling, the Korean pine appeared among mountain vegetation approximately 9,500 years ago, in the Holocene period,

and in the middle Holocene the northern border of its area

had spread to the Selemdja, Tugur and Nimaelen rivers. As

can be seen, the range of P. koraiensis was previously much

wider than at present (fig. 2). Southward decline of Korean

pine occurred because of the cooler climate periods in the

middle and late Holocene, resulting in the expansion of taiga

boreal forests with Picea, Abies, and Larix species.

Natural populations of Korean pine in Russia contain an

appreciable amount of genetic variation comparable to the

mean value for the genus Pinus (on average, P99=52.0,

H0=0.159, He=0.159; Goncharenko and others 1989). The

average values for genetic variation of P. koraiensis are

intermediate among pine species of subsection Cembrae. In

particular, the values of expected heterozygosity for these

species are: for P. cembra – 0.109 and 0.118, P. sibirica –

0.158 and 0.169, and P. pumila – 0.249 and 0.271. These

values are in agreement with those of Politov and Krutovskii

(1994) and Goncharenko and Silin (1997). A higher heterozygosity level (He=0.204) was also found in the only

population of P. albicaulis studied (Politov and Krutovskii

1994).

Geographical patterns of the distribution of expected

heterozygosity (fig. 3) and mean number of alleles per locus

(fig. 4) show that Korean pine has a small number of centers

of genetic variation. The largest of them is situated in the

south of Sikhote Alin. Two small centers are located at the

northwestern limit of natural range of Korean pine and the

middle part of Sikhote Alin. As the Sikhote Alin and Low

Amur mountain range populations have appreciable levels

of genetic variation, this may serve as confirmation of the

hypothesis of the long-term existence of Korean pine within

these areas. In that region, the mean expected heterozygosity was higher than in populations of the Maliy Khingan -

Kukan mountain ranges (table 1).

In the coastal region, the peripheral population Lesnoi

possesses a lower heterozygosity. This can be explained by

genetic drift due to the founder effect of populating a territory by a small number of individuals in recent history,

possibly the result of the northward migration of the Korean

pine during the Holocene along the narrow coastline (fig. 1,

2). Lower estimates of genetic variation in the Petrov’s

Island population can also be attributed to a founder effect

of Korean pine colonizing the island. Apparently the population was established 9,500 to 9,800 years ago, during the

Holocene period, when Korean pine appeared as a member

of the mountain vegetation complex of Sikhote-Alin (Golubeva

and Karaulova 1983; Korotkii and others 1997). At present,

the Petrov’s Island area encompasses 36 hectares, on which

grow a few hundred Korean pine trees. Heterozygosity

decrease has also been found in peripheral populations of

other conifers, including Pinus contorta Dougl. ex Loud.

(Yeh and Layton 1979), Pinus rigida Mill. (Guries and Ledig

1982), Picea abies (L.) Karst. (Bergmann and Gregorius

1979) and Picea rubens Sarg. (Hawley and DeHayes 1994).

More intense selection in marginal environments, genetic

drift, greater inbreeding in small populations, or migration

from different glacial refugia explained the heterozygosity

differences between central and peripheral populations in

several studies (Yeh and Layton 1979; Guries and Ledig

1982; Hawley and DeHayes 1994).

Positive FIS and FIT values indicate that a deficiency of

heterozygotes is typical for P. koraiensis populations and

for the whole species (table 2). The deficiency of heterozygotes was also found in the south Korean populations, where

the FIS and FIT values were 0.007 and 0.066, respectively

(Kim and others 1994). For pines, this deficiency was attributed to mating among closely adjacent individuals within a

stand, partial self-pollination, pooling of individuals (during

sampling) from different family groups within populations,

and selection against heterozygotes (Guries and Ledig 1982;

Dancik and Yeh 1983; Kim and others 1994; Politov and

Krutovskii 1994; Changtragoon and Finkeldey 1995; Lee

and others 1998).

The mean FST value (FST=0.018) was lower than the mean

GST estimate for genus Pinus (GST=0.065; Hamrick and

others 1992). The value indicates that 1.8 percent of the

genetic variation is distributed among the Korean pine

populations; in other words, the majority of the variation

resides within populations and any prominent differentiation processes are absent between populations.

The estimates of Nm, averaged over all populations per

locus, were well above 1.0 (ranged from 5.00 at Skdh-2 and

Mdh-3 to 26.51 at Pgm-2 and 6-Pgd-2) with a mean of 10.98

migrants per generation (table 2). The gene exchange exceeded those of P. koraiensis in South Korea (Nm=3.987;

Kim and others 1994) and most of the coniferous tree species

(Govindaraju 1989; Goncharenko and Silin 1997). Animal

dispersing of Korean pine seeds may explain the large values

of Nm. Tomback and Schuster (1994) noted that dispersal of

pine seeds by nutcrackers, Nucifraga (Corvidae), which

occurs routinely over large distances, might result in higher
USDA Forest Service Proceedings RMRS-P-32. 2004 197

Genetic Diversity and Mating System of Korean Pine in Russia Potenko

Figure 3—Geographical patterns of the distribution of expected heterozygosity. Grey scale gradient

show the most variable parts of Korean pine natural range. Five levels of expected heterozygosity

(0.195 to 0.200, 0.200 to 0.205, 0.205 to 0.210, 0.210 to 0.215, and above 0.215) are indicated.
198 USDA Forest Service Proceedings RMRS-P-32. 2004

Potenko Genetic Diversity and Mating System of Korean Pine in Russia

Figure 4—Geographical patterns of the distribution of

mean number of alleles per locus. Grey scale gradient

shows the most variable parts of Korean pine natural

range. Four levels of mean number of alleles per locus

(2.00 to 2.05, 2.05 to 2.10, 2.10 to 2.15, and above 2.15)

are indicated.

Table 2—Deviation of genotype frequencies from Hardy-Weinberg

proportions in individual populations (FIS) and the total

population (FIT), degree of genetic differentiation among

populations (FST), and degree of interpopulation gene flow

(Nm).

Locus FIS FIT FST Nm

Aat-3 –0.019 0.004 0.023 6.80

Adh-1 –0.005 0.015 0.020 7.84

Adh-2 –0.009 0.012 0.022 7.11

Gdh 0.052 0.076 0.025 6.24

Lap-1 –0.051 –0.027 0.023 6.80

Lap-2 0.069 0.085 0.017 9.25

Pgm-1 0.025 0.039 0.014 11.27

Pgm-2 0.001 0.008 0.006 26.51

Skdh-1 0.009 0.025 0.016 9.84

Skdh-2 0.051 0.080 0.031 5.00

Mdh-2 –0.015 –0.004 0.011 14.39

Mdh-3 0.008 0.039 0.031 5.00

Mdh-4 0.115 0.133 0.020 7.84

6-Pgd-1 0.050 0.067 0.018 8.73

6-Pgd-2 0.001 0.008 0.006 26.51

Dia-1 0.002 0.022 0.020 7.84

Dia-3 0.018 0.043 0.026 5.99

Fl-Est –0.008 0.007 0.015 10.51

Idh 0.002 0.011 0.009 17.62

Sdh –0.001 0.009 0.009 17.62

Fdh 0.016 0.030 0.014 11.27

Aco –0.002 0.011 0.013 12.15

Gpt –0.013 0.002 0.015 10.51

Mean for 23 loci 0.013 0.030 0.018 10.98

Table 3—Estimations of single locus (ts) and multilocus (tm) outcrossing

rates, fixation index (FIS) and expected inbreeding coefficient

(Fe) based on data from 4 polymorphic loci (standard errors

in parentheses).

Population ts tm FIS Fe

Obluchie 0.885 (0.044) 0.901 (0.041) 0.118 0.052

Galichnoe 1.018 (0.044) 1.001 (0.048) 0.069 0.000

Boicovo 1.042 (0.050) 1.031 (0.049) 0.029 -0.015

Malinovo 0.896 (0.043) 0.906 (0.038) 0.084 0.049

Ustinovka 0.863 (0.053) 0.861 (0.046) -0.031 0.075

Kedrovaya Pad’ 0.958 (0.050) 0.986 (0.043) 0.081 0.007

Petrov‘s Island 0.763 (0.061) 0.751 (0.057) -0.105 0.142

Kievka 0.923 (0.053) 0.912 (0.053) 0.113 0.046

Ternei 0.851 (0.061) 0.852 (0.057) -0.054 0.080

Lesnoi 0.884 (0.046) 0.888 (0.042) 0.009 0.059

levels of gene flow between pine populations than from seed

dispersal by wind.

Unbiased Nei’s genetic distance values between the 25

populations of P. koraiensis were low, averaging 0.003. Low

estimates of Nei’s genetic distances confirm the close genetic

relationship between investigated populations and indicate

a widespread gene flow between populations.

Mating System Analysis

Single locus estimates of outcrossing ranged from 0.763 to

1.042, and multilocus estimates were from 0.751 to 1.031

(table 3). The lowest value tm was found in the Petrov’s

Island population and the highest in the Boicovo population.

Negligible differences were found between single locus and

multilocus estimates of outcrossing in any population.

The mean multi-locus value of outcrossing in this study

(tm=0.909) was lower than that for the three populations

(tm=0.974) studied earlier by Politov and Krutovskii (1994)

but typical for most coniferous forest tree species (Muona

1990; Adams and Birkes 1991; Mitton 1992). The lowest

value tm, on Petrov’s Island, can be attributed to both selfing

and mating among related individuals, supporting the hypothesis that the populating of the island was by a limited

number of migrants. The low estimates of the Ustinovka,

Ternei and Lesnoi populations can be attributed to partial

mating among related individuals due to the founder effect.

Although tm was low in these populations, the estimates of

FIS were either negative or slightly positive, thus indicating
USDA Forest Service Proceedings RMRS-P-32. 2004 199

Genetic Diversity and Mating System of Korean Pine in Russia Potenko

an excess of heterozygotes or practically a Hardy-Weinberg

equilibrium. Any one of these estimates is much lower than

those expected under inbreeding equilibrium, given the

levels of tm (table 3). The relationship between multilocus

estimates of outcrossing (tm) and fixation index (FIS) shown

in figure 5 suggests that the excess of heterozygotes in

Korean pine populations is due to “pseudo-overdominance”

as result of inbreeding depression (Ledig 1986), rather than

selection in favor of heterozygotes (overdominance) as concluded by Politov and Krutovskii (1994) and Krutovskii and

others (1995). These results showed that in Korean pine

populations, the selection against inbred progeny appears

when outcrossing rate is below 0.9 (fig. 5).

It is suggested that a contrary direction of selection occurs

in populations with a deficit of heterozygotes and high

outcrossing rates; that is, selection against heterozygotes.

High outcrossing is probably maintained by a high migration rate between Korean pine populations (Nm=10.98).

However, the validity of the suggested selection against

heterozygotes in Korean pine needs to be field-proven by

making biparental crosses. Selection against hybrid forms of

plants due to outbreeding depression is found in crosses

between distant plants of Delphinium nelsoni (Price and

Waser 1979) and Lotus scoparius (Montalvo and Ellstrand

2001).

Possible microsite differentiation of allele frequencies

that would upwardly bias the fixation index cannot be

excluded as an explanation of a deficit of heterozygotes in

some Korean pine populations. If different subpopulations

sustain different alleles, the allele frequencies will be maintained at a high level in the whole population (Brown 1979).

This phenomenon explains the high level of heterozygosity

in populations with a positive fixation index, although reliable conclusions can only be made after an investigation of

the genetic parameters of subpopulations.

Thus the coastal Korean pine populations we sampled

exhibit different levels of genetic variation and outcrossing.

Results of this study lead to the conclusion that the genetic

structure of the Korean pine populations is under the influence of a complex combination of microevolution factors,

genetic drift, gene flow and natural selection.

Acknowledgments ______________

I thank Andrew Velikov for helpful assistance in the

laboratory analysis, Howard Kriebel for improvement of

English, tables, and figures, and an anonymous reviewer for

helpful comments on the manuscript.

Figure 5—The relationship between multilocus estimates of outcrossing (tm) and

fixation index (FIS).
200 USDA Forest Service Proceedings RMRS-P-32. 2004

Potenko Genetic Diversity and Mating System of Korean Pine in Russia

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USDA Forest Service Proceedings RMRS-P-32. 2004 201

Part IV: White Pine Blister Rust Resistance

Pinus albicaulis (whitebark pine)

Photo credits: Left-J. Barnes. Right (top to bottom): R. Shoal,

B. Danchok, J. Schwandt, J. Barnes
202 USDA Forest Service Proceedings RMRS-P-32. 2004
USDA Forest Service Proceedings RMRS-P-32. 2004 203

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

The authors are with the USDA Forest Service, Dorena Genetic Resource

Center, 34963 Shoreview Road, Cottage Grove, Oregon, 97424, USA. Phone

(541) 767-5716, Fax (541) 767-5709, E-mail: rsniezko@fs.fed.us; A.D. Bower’s

current address: Forest Sciences Department, University of British Columbia, Vancouver, B.C. V6T 1Z4 Canada.

Abstract—In 1995 seed from 13 Pinus monticola (western white

pine) and 12 P. lambertiana (sugar pine) parents previously included in short-term blister rust testing at Dorena Genetic Resource

Center (DGRC), Cottage Grove, OR, were sown to establish field

trials. The parents were chosen to represent a wide array of

resistance responses shown in earlier artificial inoculation trials.

Percentage stem symptoms (cankers and bark reactions) in 1999

and 2000 at the 1996 Happy Camp (HC) trial are reported here.

Sugar pine families have a higher percentage of trees with stem

symptoms (SS percent) than do western white pine families. In

addition to a greater susceptibility to infection overall, the major

gene resistance present in several of these sugar pine families

(conferred by Cr1) is ineffective due to the relatively high frequency

of a virulent (vcr1) strain of rust at this site. Western white pine

families varied from 10.4 to 83.3 SS percent and sugar pine from

73.5 to 91.8 SS percent. The susceptible control lot for western white

pine showed a much greater percentage of trees with stem symptoms (83.3 percent) than any other western white pine seedlot. The

two western white pine families with known major gene resistance

(Cr2) were among the families with lowest infection at this site. The

resistance mechanism of the family with the second lowest level of

stem symptoms is unknown. Moderately strong and significant

positive correlations (r>0.69, p<0.05) existed between SS percent at

HC and SS percent following artificial inoculation at DGRC. The

correlation between needle lesion frequency in DGRC screening and

stem symptoms observed at HC was positive but non-significant

(r>0.5, p>0.1 for sugar pine and western white pine).

Key words: Pinus monticola, Pinus lambertiana, blister rust, Cr1.

Variation in Cronartium ribicola Field

Resistance Among 13 Pinus monticola and

12 P. lambertiana Families: Early Results

from Happy Camp

R.A. Sniezko

A.D. Bower

A.J. Kegley

Introduction ____________________

In Oregon and Washington, the USDA Forest Service

(Region 6) began selecting and screening parent trees of

western white pine (Pinus monticola Dougl. ex D. Don.) and

sugar pine (P. lambertiana Dougl.) for resistance to white

pine blister rust (Cronartium ribicola J.C. Fisch) in the late

1950s. Over 9500 parent trees of these two species have been

selected for rust resistance in natural stands, but variation

in rust hazard by site influences the expected efficacy of field

selection. Progeny of these field selections have been screened

for resistance at Dorena Genetic Resource Center (DGRC)

(Sniezko 1996, Kegley and Sniezko this proceedings). Selections from rust screening have been used to establish seed

orchards in many of the breeding zones, and resistant seed

is now available for some zones.

Operational screening at DGRC generally involves a single

inoculation using two-year old seedlings and the subsequent

assessment of seedlings and assignment into resistance

categories. Primary categories of response in seedlings are

lack of visible stem infections versus presence of visible stem

symptoms. Seedlings screened at DGRC are assigned to

these two categories and are also assessed for other resistance responses (Sniezko 1996, Kegley and Sniezko this

proceedings). Some of the mechanisms preventing stem

infection include: hyper-sensitive reactions (HR) in the

needles conditioned by major genes in sugar pine (Cr1) and

western white pine (Cr2) (Kinloch and Comstock 1981,

Kinloch and others 1999, Kinloch and Dupper 2002), and

two mechanisms hypothesized to be controlled by single

recessive genes: (a) fungicidal reaction in the short shoot

(Hoff and McDonald 1971, McDonald and Hoff 1971), and (b)

premature shed of secondary needles (McDonald and Hoff

1970, McDonald and Hoff 1971). Resistance mechanisms

that may reduce the number of stem infections or the

severity of these infections include: (a) bark reaction (Hoff

1986, Kinloch and Davis 1996), (b) reduced needle lesion

frequency (Hoff and McDonald 1980a, Meagher and Hunt

1996) and (c) tolerance (Hoff and McDonald 1980b). These

mechanisms have been at the core of the Region 6 selection

program for three decades; however, for both sugar pine and

western white pine there has been little or no formal field

validation of the test results from the artificial inoculation

and screening at DGRC or tracking of individual family rust

resistance over time.
204 USDA Forest Service Proceedings RMRS-P-32. 2004

Sniezko, Bower, and Kegley Variation in Cronartium ribicola Field Resistance Among 13 Pinus monticola and 12 P. lambertiana Families:...

Field trials were established in 1996 and 1997 with seedlings from 13 western white pine and 12 sugar pine families

in the first of a series of validation plantings. These 25

seedling families were planted at three test sites, two in

Oregon and one in California. The first and largest was

planted at Happy Camp, California in 1996. This site was

also the first one to show moderate levels of rust infection.

The Happy Camp site on the Klamath National Forest has

been the principal field test site for blister rust resistance

evaluation for the Forest Service’s Region 5 (California)

sugar pine program since 1962 (Kinloch and Byler 1981).

This test site has a high frequency of a strain of blister rust

virulent to the major gene resistance conferred by the Cr1

gene in sugar pine (Kinloch and Comstock 1981). Since Cr1

is neutralized on sugar pine at this site, it is possible to

observe resistance(s) that would be masked by Cr1 at other

sites. The main purpose of these field trials is to validate the

effectiveness and durability of the putative mechanisms of

resistance to blister rust.

This paper examines results five years after planting and

assesses species and family differences in percentage of

trees with stem symptoms (SS percent). We also report on

the correlations between field results (for SS percent) and

results from operational screening of seedlings at DGRC (SS

percent and needle lesion frequency).

Materials and Methods ___________

Twenty-five seedlots (13 western white pine and 12 sugar

pine) were selected from relatively recent blister rust screening trials (“runs”) at Dorena Genetic Resource Center (DGRC).

Two of the western white pine seedlots are full-sib families

from crosses made among resistant parents in DGRC orchards, and one of the western white pine seedlots is a windpollinated collection from a seed orchard at DGRC. The

remaining 22 families are open-pollinated from select trees

in natural stands. The families selected for these field tests

cover a wide range of geographic areas in Oregon and

Washington as well as an array of resistance responses

observed in five years of assessments following artificial

inoculation at DGRC. Families that displayed little or no

resistance in a previous DGRC test were included as low

resistance controls.

Seed was sown in containers in spring 1995, and seedlings

were planted at Happy Camp (HC) in a randomized complete block design in spring 1996 after budbreak. Twelve

blocks were established with generally four trees per family

per block although a few families had fewer trees per block,

and an occasional container held two seedlings that were

then planted together. Ribes sanguineum Pursh, an alternate host to C. ribicola, was inter-planted among the pines

to help ensure uniform exposure to the rust.

Height as well as number and type of stem symptoms (SS)

were assessed in June 1999 and July 2000 at Happy Camp.

For the main analysis in this paper, a tree was recorded as

having SS if there was any sign or symptom of rust infection

on the bole or branches, including small orange discoloration

at the base of infected needles (the initial signs of stem

infection), normal cankers or bark reactions in either 1999 or

2000. Percentage of trees with stem symptoms (SS percent)

was tabulated by family plot and used for analyses. Analyses

of variance were performed using SAS Proc GLM (SAS

Institute 1999) to assess differences between species and

families within species for SS percent.

Pearson’s correlations between family mean SS percent at

Happy Camp and family mean SS percent and Needle

Lesion class (NLclass) at DGRC were calculated using SAS

Proc CORR (SAS Institute 1999). The correlations use results from the 1995 sowing for the 19 families tested in that

year, and a second set of correlations uses the 1995 test data

plus results for the six families tested in other screening

trials (table 1). NLclass is a family and trial-specific value

based upon number of needle lesions or “spots” on all secondary needles on each seedling (Table 2). NLclass values range

from 0 to 4. Generally, the scale is set up to have approximately 25 percent of the seedlings in each needle lesion class

from 1 to 4; seedlings in needle lesion class 0 have no spots.

Results ________________________

Sugar pine had a higher percentage of trees with stem

symptoms (SS percent) than western white pine at the HC

site (fig. 1). Highly significant differences (p<0.0001) for SS

percent existed between species and among western white

pine families (p<0.0001) but not among sugar pine families

(p=0.58). Except for the susceptible western white pine

(control) family, there was no overlap in SS percent among

the 12 sugar pine families and the 13 western white pine

families (fig. 1). Most of the stem symptoms are from infection in autumn 1997.

Overall SS percent (SS observed in 1999 and/or 2000) was

85.4 percent for sugar pine and 43.2 percent for western

white pine (fig. 2). Mean SS percent for western white pine

was 35.2 percent in the 1999 and 30.1 percent in the 2000

(fig. 2). Ten of the 13 western white pine families had lower

SS percent in 2000 than in 1999. SS percent in sugar pine

increased from 70.8 percent in 1999 to 77.4 percent in 2000

(fig. 2). Only one sugar pine family had a lower SS percent in

2000 than in 1999, and two families had the same SS percent

in 1999 and 2000. There was a relatively narrow range in SS

percent among sugar pine families (73.5 to 91.8 percent) but

a very wide range among western white pine families (10.4

to 83.3 percent).

Of two western white pine families notable for their very

low SS percent (table 1 and Fig. 1), Family #22 is known to

have major gene resistance (from Cr2); the resistance mechanism for the other family (#18) is unknown, but it is not Cr2.

The low resistance western white pine control (#16) had the

highest SS percent (table 1).

Strong and significant correlations exist between family

mean SS percent at Happy Camp and those from 1995

artificial screening at DGRC for both sugar pine (r = 0.73,

n=9, p=0.024) and western white pine (r = 0.70, n=10,

p=0.026) (also see fig. 3a and 3b). There were positive but

non-significant correlations between needle lesion frequency

(NLclass) in DGRC screening and SS percent at Happy

Camp for both species (fig. 4a and 4b). Even western white

pine family #25, which was outstanding for NLclass in

several tests at DGRC, is only average for SS percent at HC

(table 1).
USDA Forest Service Proceedings RMRS-P-32. 2004 205

Variation in Cronartium ribicola Field Resistance Among 13 Pinus monticola and 12 P. lambertiana Families:... Sniezko, Bower, and Kegley

Table 1—Summary results for percent stem symptoms, needle lesion class, and major gene resistance from Dorena rust-screening and

Happy Camp, California field planting for 12 sugar pine (SP) and 13 western white pine (WWP) families.

Needle

Field MGR assessment Test lesion % Stem symptoms

ID Female parent Male parent Species resultsb yearc classd Dorena Happy Campf

1a 02176-040 wind SP — 1992 3.25 100.00 89.58

2 10045-689 wind SP — 1995 2.53 100.00 91.83

3 11052-570 wind SP not yet tested 1992 1.75 48.33 87.76

4 11054-370 wind SP — 1992 1.93 100.00 80.56

5 11054-419 wind SP Cr1 (~52%) 1995 1.64 32.20 81.63

6 11054-581 wind SP non-Cr1 1995 2.49 100.00 82.22

7 11054-776 wind SP — 1995 3.30 85.19 88.89

8 11054-903 wind SP Cr1 (~21%) 1995 2.00 43.10 73.47

9 18032-608 wind SP — 1995 1.46 92.59 85.71

10 18033-431 wind SP — 1995 2.30 96.30 87.50

11 18034-404 wind SP — 1995 3.02 94.92 87.50

12 20045-001 wind SP non-Cr1 1995 3.54 96.61 89.80

13 11053-552 wind WWP — 1995 1.48 88.33 38.10

14 03023-509 wind WWP — 1995 2.32 100.00 50.00

15 03024-510 wind WWP non-Cr2 1995 1.75 70.00 32.65

16a 03024-532 wind WWP non-Cr2 1995 3.33 98.33 83.33

17 03024-793 wind WWP — 1995 2.43 91.38 53.19

18 05081-003 wind WWP non-Cr2 1995 1.92 40.00 12.25

19 06023-521 wind WWP not yet tested 1995 1.73 74.51 60.00

20 18034-140 wind WWP non-Cr2 1995 2.82 96.67 40.39

21 1803.5-150 wind WWP non-Cr2 1995 1.95 90.00 57.45

22 15045-816 x 15045-841 wind WWP Cr2 (97%) 1989 2.53 17.02e 10.42

23 15045-823 15045-840 WWP Cr2 (~75%) 1988 2.62 42.50e 33.33

24 21105-052 wind WWP non-Cr2 1995 2.78 89.83 44.44

25 18033-708 18033-703 WWP non-Cr2 1988 0.43 23.33 45.83

aSusceptible control family based on performance in a single artificial inoculation trial at DGRC

bResults from separate inoculation to score hypersensitive reaction (HR) on needles and classify families as to presence or absence of major gene resistance.

Percentage seedlings exhibiting HR indicated in parentheses. Preliminary information indicates the female parent of Family 22 (Orchard Accession # 023220)

may be homozygous dominant for HR. cTest year refers to the year in which the family was sown at Dorena; inoculation occurred the following year.

dFamily mean needle lesion class at DGRC based on the number of lesions on all secondary needles approximately 9 months after artificial inoculation with

C. ribicola. eIn many years the mixture of spores used for inoculation at DGRC contained an unknown frequency of a strain of rust virulent to Cr2 in western white pine. f

Overall percent stem symptoms (present in 1999 and/or 2000) at Happy Camp.

Table 2—Number of needle lesions in each class by test year and trial at Dorena Genetic

Resource Center.

Test Needle lesion classa

year Trial Species 0 1234

1988 4 western white pine 0 1 2-3 4-6 7+

1989 4 western white pine 0 1-3 4-9 10-19 20+

1992 4 sugar pine 0 1-10 11-26 27-50 51+

1995 1 western white pine 0 1-3 4-9 10-20 21+

1995 2 sugar pine 0 1-3 4-9 10-27 28+

aEach seedling is evaluated for number of needle lesions (“spots”) present on secondary needles and

is assigned to a “needle lesion class.” Needle lesion classes are based on counts of number of spots on

seedlings in monitoring plots and are trial specific. Family mean needle lesion class is the average of all

living seedlings in a family.
206 USDA Forest Service Proceedings RMRS-P-32. 2004

Sniezko, Bower, and Kegley Variation in Cronartium ribicola Field Resistance Among 13 Pinus monticola and 12 P. lambertiana Families:...

species

Sugar Pine Western White Pine

% stem symptoms

0

10

20

30

40

50

60

70

80

90

100

species

SP WWP

stem symptoms (%)

0

10

20

30

40

50

60

70

80

90

100

1999 

2000

overall 

Figure 1—Range of means for overall percent stem

symptoms at Happy Camp for 12 sugar pine and 13

western white pine families.

Figure 2—Species means for 1999, 2000, and overall

(present in 1999 or 2000) percent stem symptoms for

12 sugar pine and 13 western white pine families

outplanted at Happy Camp, California.

0 10 20 30 40 50 60 70 80 90 100

0

10

20

30

40

50

60

70

80

90

100

stem symptoms at Dorena (%)

stem symptoms at Dorena (%)

0 10 20 30 40 50 60 70 80 90 100

stem symptoms at Happy Camp (%) stem symptoms at Happy Camp (%)

0

10

20

30

40

50

60

70

80

90

100

Dorena Sow Year 1995

Other Sow Years

(a)

(b)

 Correlations

 SY95 All 

r 0.73 0.52

p 0.02 0.08

n 9 12 

 Correlations

 SY95 All 

r 0.70 0.66

p 0.03 0.01

n 10 13 

Figure 3—Percent stem symptoms at Happy Camp vs.

percent stem symptoms at Dorena for (a) 12 sugar pine

families and (b) 13 western white pine families.

Discussion and Summary ________

The results clearly show that after five years at HC, sugar

pine is significantly more susceptible to blister rust than

western white pine. In a summary of previous studies

involving 16 species of five-needle pines, western white pine

appeared to be slightly less susceptible than sugar pine

(Bingham 1972). In 1999 at HC, sugar pine had 2.7 times as

many stem infections as western white pine (953 vs. 356

total stem infections, Sniezko and others 2000).

Current results from HC demonstrate that SS percent at

this site corresponds fairly well to SS percent at DGRC.

However, at the current level of infection, there appears to

be little or no differentiation between families showing

different levels of needle lesion frequency at DGRC and SS

percent in the field for either western white pine or sugar

pine. Needle lesion frequency may be more associated with

number of stem infections rather than presence or absence

of stem infections. It is still too early to discern the relationships of other resistance traits at DGRC and in the field.

In examining SS percent by individual year, it was noted

that from the 1999 to the 2000 assessment there was a slight

increase (6.7 percent) for sugar pine but a decrease (5.2

percent) for western white pine, and that the total for either

year was more than 8 percent lower than the overall SS

percent (SS present in 1999 or 2000) (fig. 2). Close reexamination of a few trees in spring 2001 showed some

small, fading stem symptoms that could have easily been

missed and will probably not be visible at all within a year

or two.
USDA Forest Service Proceedings RMRS-P-32. 2004 207

Variation in Cronartium ribicola Field Resistance Among 13 Pinus monticola and 12 P. lambertiana Families:... Sniezko, Bower, and Kegley

Although the percentage of sugar pine infected at HC is

already high, there is little mortality as of July 2000. On this

site the sugar pine families with HR show high levels of

infection because of the high frequency of the vcr1 gene

(virulent to Cr1) in the rust population at HC; these two

families would be expected to show much lower infection at

most sites. The lack of significant differences in SS percent

among families in sugar pine may not be a true measure of

field resistance; future assessments will determine whether

differences in bark reaction, mortality or tolerance exist

among families.

One of the biggest expected changes over the next ten

years would be the performance of the two western white

pine families with Cr2 (Families 22 and 23) if a strain of rust

with specific virulence to Cr2 (vcr2) became prominent at

this site. The virulent strain is known to be present in a small

adjacent test established in 1971 (Kinloch, personal communication). Previously, vcr2 had only been documented in a

localized area in Oregon (Kinloch and others 1999, McDonald

and others 1984), but a recent survey has demonstrated its

presence in varying frequency in parts of western Oregon

and at Happy Camp, CA (Sniezko and others 2001).

The performance of the Cr2 families in this test demonstrates that there may be important geographic differences

in blister rust resistance. The resistance conferred by the

Cr2 gene in western white pine appears to be limited geographically to California, Oregon, and southern Washington

(Kinloch and others 2003), but there may also be other types

of resistances that are geographically restricted and not

noted in earlier findings (see discussion below). Elucidation

of geographically restricted resistance mechanisms would

aid breeding efforts and establishing deployment strategies

for resistant seed. At present, operational rust screening at

DGRC and elsewhere only discerns categories of phenotypic

expression, but it is possible that several mechanisms may

have only minor differences in their gross physical expression. For example, only in the mid-1990s has DGRC incorporated an operational screening procedure that separates a

major gene for resistance (hypersensitive reaction in the

needles, see Kinloch and others 1999, Kinloch and Dupper

2002 for details) from other resistance mechanisms in western white pine that also lead to canker-free seedlings after

inoculation such as needle shed or short shoot (Hoff and

McDonald 1971, McDonald and Hoff 1970, Sniezko and

Kegley 2003).

The low level SS percent in Family #18, an open-pollinated, non-Cr2 western white pine family, might be due to

some combination of needle shed or short shoot mechanisms

(both purportedly due to single recessive genes). If this were

true, the frequency of these genes in natural stands would

have to be high and there is no evidence of this in testing at

DGRC; the great majority of families show greater than 90

percent infection in screening trials at DGRC. This could

also be a previously undefined mechanism (mechanism ‘X’),

characterized by low incidence of stem symptoms at DGRC

in testing (35-60 percent SS, relative to 90-100 percent for

most other open-pollinated families) and a negative result

for presence of Cr2 resistance in a separate test. Family #18

at HC fits these parameters. The relatively low SS percent

for this open-pollinated family at DGRC suggests the involvement of a single major gene, but the very low SS percent

at HC suggests a more complicated scenario. Some differences in performance of families in short-term screening and

in the field are not unusual. In a summary of studies in other

plant species Keller and others (2000) note that the resistance phenotype may vary between tests performed under

controlled conditions versus field conditions, or between

seedlings and adult plants. From operational screening of

thousands of western white pine parents at DGRC it appears

that a very low frequency of parents with this type of

resistance (low SS percent and non-Cr2) is present in much

of Oregon and Washington (unpublished data).

Artificial inoculations and short term screening at DGRC

provides a potentially more time- and cost-efficient method

of evaluating progeny of thousands of parent trees for an

array of resistance mechanisms than costly, long-term field

trials. However, field tests are essential for validating effectiveness of the various resistance responses characterized

on young seedlings following artificial inoculation. A wide

array of rust races can be included in artificial screening,

Figure 4—Percent stem symptoms at Happy Camp vs.

needle lesion class (NLclass) at Dorena for (a) 12 sugar pine

families and (b) 13 western white pine families (see table 2

in text for needle lesion classes).

NLclass

NLclass

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

stem symptoms at Happy Camp (%) stem symptoms at Happy Camp (%)

0

10

20

30

40

50

60

70

80

90

100

Dorena Sow Year 1995

Other Sow Years

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

0

10

20

30

40

50

60

70

80

90

100

 Correlations

 SY95 All 

r 0.54 0.53

p 0.14 0.08

n 9 12 

 Correlations

 SY95 All 

r 0.51 0.19

p 0.13 0.52

n 10 13 

(a)

(b)
208 USDA Forest Service Proceedings RMRS-P-32. 2004

Sniezko, Bower, and Kegley Variation in Cronartium ribicola Field Resistance Among 13 Pinus monticola and 12 P. lambertiana Families:...

whereas field testing at any one site would generally rely on

local populations of the pathogen, which may vary from year

to year. However, due to the limitations of a single inoculation on very young seedlings, resistance mechanisms that

manifest themselves more clearly on older, larger trees in

the field may not be identified in operational screening (such

as ontogenetic resistance (Kinloch and Davis 1996); low

canker frequency (Sniezko and others this proceedings)),

thus field plantings serve a complementary function. Correspondence between results from short-term testing at DGRC

and long-term field testing may be dependent upon factors

influenced by the environment, the rust population, and the

nature of the families and resistance mechanisms under

test.

Results from these field tests will allow confirmation of

the field effectiveness of resistance responses observed on

seedlings following artificial inoculation, as well as provide

demonstrations to land managers hoping to use western

white pine or sugar pine in restoration or reforestation

plantings. The plantings will also serve as monitors to

changes in virulence of the rust, and they may help detect

resistance mechanisms or other events not apparent in

short-term screening. For example, from recent observations in fall 2001, some cankers appear inactive but do not fit

the classic pattern of bark reactions.

It may also be very useful to establish some joint field tests

among the blister rust programs in Oregon, Washington,

Canada, Idaho, and California using a small number of

families selected for specific resistance responses in these

different locations. Such plantings may help discern the

presence of geographically limited mechanisms or the influence of environment and local rust populations on host

resistance.

Acknowledgments ______________

The authors would like to thank the Region 5 Genetics

Program for use of the Happy Camp test site; Dean Davis

and Deems Burton for monitoring the site and advice on

assessments; Bro Kinloch and Bob Westfall for early discussions at the trial site; Jude Danielson for help in family

selections from the operational program; Bob Danchok and

Sally Long for leading the assessment efforts; all the staff

and technicians at DGRC whose work and support greatly

facilitated this planting; Paul Zambino, Bro Kinloch, Safiya

Samman, and Rich Hunt for constructive comments on a

earlier version of this manuscript; financial support from

the USDA Forest Service’s Region 6 Forest Health and

Genetics programs in establishing this planting.

References _____________________

Bingham, R.T. 1972. Taxonomy, crossability, and relative blister

rust resistance of 5-needled white pines, p. 271-280. In: R.T.

Bingham, R.J. Hoff, and G.I. McDonald (eds) Biology of rust

resistance in forest trees: Proceedings of a NATO-IUFRO Advanced Study Institute. USDA Forest Service Misc. Publ. 1221.

Hoff, R.J. 1986. Inheritance of Bark Reaction Resistance Mechanism in Pinus monticola by Cronartium ribicola. USDA Forest

Service, Res. Note INT-361. 8pp.

Hoff, R.J. and McDonald, G.I. 1971. Resistance to Cronartium

ribicola in Pinus monticola: short shoot fungicidal reaction. Can.

J. Bot. 49:1235-1239.

Hoff, R.J. and McDonald, G.I. 1980a. Resistance to Cronartium

ribicola in Pinus monticola: reduced needle-spot frequency. Can.

J. Bot. 58:574-577.

Hoff, R.J. and McDonald, G.I. 1980b. Improving rust-resistant

strains of inland western white pine. USDA Forest Service, Res.

Pap. INT-245. 13p.

Keller, Beat, Feuillet, Catherine, and Messmer, Monika. 2000.

Genetics of Disease Resistance: Basic Concepts and Application

in Resistance Breeding. In: A. Slusarenko, R.S.S. Fraser, and

L.C. van Loon, eds. Mechanisms of Resistance to Plant Diseases.

Kluwer Academic Publishers, The Netherlands: 101-160.

Kinloch, Bohun B. and Byler, James W. 1981. Relative effectiveness

and stability of different resistance mechanisms to white pine

blister rust in sugar pine. Phytopathology 71:386-391.

Kinloch, B.B., Jr. 2000. [Personal communication]. U.S. Department of Agriculture, Forest Service, Pacific Southwest Research

Station.

Kinloch, B. B., Jr. and Comstock, M. 1981. Race of Cronartium

ribicolavirulent to major gene resistance in sugar pine. Plant Dis.

65:604-605.

Kinloch, B.B., Jr. and Davis, D. 1996. Mechanisms and inheritance

of resistance to blister rust in sugar pine, p. 125-132. In: B.B.

Kinloch, M. Marosy, and M.E. Huddleston (eds.). Sugar pine:

status, values, and roles in ecosystems: Proceedings of a symposium presented by the California Sugar Pine Management Committee. Univ. Calif. Div. Agr. Res. Publ. 3362.

Kinloch, B.B. and Dupper, G. 2002. Genetic specificity in the white

pine-blister rust pathosystem, Phytopathology 92(3): 278-280.

Kinloch, Bohun B., Sniezko, Richard A., Barnes, Gerald D., and

Greathouse, Tom E. 1999. A major gene for resistance to white

pine blister rust in western white pine from the western Cascade

Range. Phytopathology 89:861-867.

Kinloch, B.B., Jr., Sniezko, R.A., and Dupper, G.E. 2003. Origin and

distribution of Cr2, a gene for resistance to white pine blister rust

in natural populations of western white pine. Phytopathology

93:691-694.

McDonald, G.I. and Hoff, R.J. 1970. Resistance to Cronartium

ribicola in Pinus monticola: early shedding of infected needles.

USDA Forest Service, Res. Note INT 124. 8 pp.

McDonald, G.I. and Hoff, R.J. 1971. Resistance to Cronartium

ribicola in Pinus monticola: genetic control of needle-spots-only

resistance factors. Can. J. For. Res. 1:197-202.

McDonald, G.I., Hansen, E.M., Osterhaus, C.A., and Samman, S.

1984. Initial characterization of a new strain of Cronartium

ribicola from the Cascade Mountains of Oregon. Plant Disease 68:

800-804.

Meagher, M.D. and Hunt, R.S. 1996. Heritability and gain of

reduced spotting vs. blister rust on western white pine in British

Columbia, Canada. Silvae Genet. 45(2-3):75-81.

SAS Institute Inc. 1999. SAS OnlineDoc, Version 8. Cary, NC: SAS

Institute Inc.

Sniezko, R.S. 1996. Developing resistance to white pine blister rust

in sugar pine in Oregon, p. 125-132. In: B.B. Kinloch, M. Marosy,

and M.E. Huddleston (eds.). Sugar pine: Status, values, and roles

in ecosystems: Proceedings of a symposium presented by the

California Sugar Pine Management Committee. Univ. Calif. Div.

Agr. Natural Res. Publ. 3362.

Sniezko, R.A., Bower, A., and Danielson, J. 2000. A comparison of

early field results of white pine blister rust resistance in sugar

pine and western white pine. HortTechnology 10(3): 519-522.

Sniezko, Richard A., Kinloch, Bohun, and Dupper, Gayle. 2001.Geographic distribution of ‘Champion Mine’ strain of white pine

blister rust (Cronartium ribicola) in the Pacific Northwest. 2001

National Forest Health Monitoring meeting, Las Vegas, NV.

Poster presentation. [Online]. Available: http://www.na.fs.fed.us/

spfo/fhm/posters/posters01/geo.pdf [2002, June 22].

Sniezko, Richard A. and Angelia Kegley. 2003. Blister rust resistance of five-needle pines in Oregon and Washington. Proceedings of the Second IUFRO Rusts of Forest Trees Working Party

Conference. 19-23 August 2002. Yangling, China. Forest Research 16 (Suppl.): 101-112.
USDA Forest Service Proceedings RMRS-P-32. 2004 209

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

A.J. Kegley and R.A. Sniezko are with the USDA Forest Service, Dorena

Genetic Resource Center, 34963 Shoreview Road, Cottage Grove, Oregon,

97424, USA. Phone (541) 767-5716; Fax (541) 767-5709; e-mmail:

akegley@fs.fed.us; rsniezko@fs.fed.us.

Abstract—Pinus monticola and P. lambertiana families sown in

1989 and 1994 were inoculated with Cronartium ribicola after two

growing seasons. Development of rust symptoms and mortality

were followed for five years. During this period, 96 to 99 percent of

the seedlings became infected, and 91 to 99 percent of the seedlings

developed stem symptoms in the four screening trials (family means

varied from 30 to 100 percent). Survival of infected seedlings 5 years

after inoculation varied from 1.6 to 13.1 percent for the four trials;

family means varied from 0 to 54.8 percent. The frequency and level

of resistance responses in half-sib progeny of phenotypic selections

from natural stands were generally low, but some families had

survival comparable to the full-sib checklots. In both species the

families with the highest levels of survival generally had higher

than average levels of several resistance responses, including percentage of seedlings without stem symptoms, delayed appearance of

stem symptoms, bark reaction, and survival with stem symptoms.

Levels of bark reaction were generally low in the four trials (3.5 to

12.3 percent of the trees), with half-sib families varying from 0 to

59.9 percent. Many trees with bark reaction also had normal

cankers and died by the end of the assessment period. Selections

from the top individuals within these trials have been grafted into

seed orchards. Breeding efforts are focused on the development of

seedlings with more durable rust resistance for reforestation and

restoration.

Key words: blister rust, resistance responses, screening, pines

Introduction ____________________

The introduced disease white pine blister rust, caused by

the pathogen Cronartium ribicola J.C. Fisch. in Raben., has

devastated populations of five-needle pines in many parts of

western North America. Development of genetic resistance

to this pathogen will be a key to restoration of these species.

Due to land management changes and limited opportunities

Variation in Blister Rust Resistance Among

226 Pinus monticola and 217 P. lambertiana

Seedling Families in the Pacific Northwest

A.J. Kegley

R.A. Sniezko

for planting seedlings on federal lands, maintaining these

species as viable components of the ecosystem will depend

even more on the development of durable resistance.

Evaluation of white pine blister rust resistance and/or

operational screening has occurred for a number of species

in North America, Asia, and Europe (Kim and others 1982;

Blada, this proceedings; Stephan, this proceedings; Daoust

and Beaulieu, this proceedings; McDonald and others, this

proceedings; King and Hunt, this proceedings; Bingham

1983; Zsuffa 1981). In North America, the identification of

some genetically resistant individuals (Bingham 1983;

Kinloch and others 1970; McDonald and others, this proceedings) led to the initiation of several resistance breeding

programs for western white pine (Pinus monticola Dougl. ex

D. Don.) and sugar pine (P. lambertiana Dougl.) (Bingham

1983; King and Hunt, this proceedings; Sniezko 1996; Kinloch

and Davis 1996; Samman and Kitzmiller 1996). In Oregon

and Washington, Region 6 of the USDA Forest Service,

works began on operational screening and breeding of

western white pine (WWP) and sugar pine (SP) for resistance to C. ribicola in the late 1950s, but screening of

whitebark pine (P. albicaulis Engelmann) has only recently

begun. The screening program for all three species is based

at the Dorena Genetic Resource Center (Dorena); the Center

staff work with geneticists and land managers throughout

Oregon and Washington to develop breeding populations

and seed orchards with resistant parent trees. Seedling

common-garden studies (Campbell and Sugano 1987;

Campbell and Sugano 1989) have provided the foundation

for establishing breeding zones in Oregon and Washington

for both SP and WWP. Rust resistance of progeny from

parent trees in natural stands or plantations has been

evaluated for most of these breeding zones; moreover, breeding among resistant progeny has begun for several of the

zones.

Since the 1970s blister rust resistance has been evaluated

using half-sib progeny of phenotypically selected trees from

National Forest lands as well as lands of the USDI Bureau

of Land Management (BLM), and other landowners in Oregon and Washington. The phenotypic selections were made

on sites varying from low to high incidence of blister rust. In

general the selected trees were vigorous and either free of

rust cankers or showed fewer cankers than other WWP or SP

in the local area. Progeny of approximately of 4,900 WWP

and 4500 SP phenotypic selections have now been screened

at Dorena. Based upon screening results, selections among

the progeny of the selected parents have been made and

placed into seed orchards.

The Region 6 program was based on protocols and resistance mechanism studies in WWP by Forest Service re-
210 USDA Forest Service Proceedings RMRS-P-32. 2004

Kegley and Sniezko Variation in Blister Rust Resistance Among 226 Pinus monticola and 217 P. lambertiana Seedling Families in the Pacific Northwest

searchers in Idaho (Bingham 1983; McDonald and others,

this proceedings; Sniezko 1996). Recently, screening of seedlings has been modified to incorporate an assessment for a

major gene conditioning a hypersensitive response (HR) in

needles of sugar pine and western white pine (see Kinloch

and others 1999; Kinloch and Dupper 2002 for details on

screening for major gene resistance); screening for HR complements but does not replace standard operational screening.

Artificial inoculation with blister rust allows seedlings to be

categorized into those with complete resistance (no stem

infection) and those with partial resistance (stem infection

present).

There is little published information on the inherent

levels of blister rust resistance in WWP or SP in Oregon and

Washington (Sniezko 1996). However, a synthesis of results

from the Region 6 program has begun. This paper reports on

the levels of resistance for 226 seedling families of WWP and

217 families of SP, representing a large part of their geographic ranges in Oregon and Washington. The data reveal

the different types of resistance responses and relative

degrees of responses between trials, species, and families,

and compares them to existing checklot families.

Some refinements and modifications to Dorena screening

summaries are incorporated here for the first time. A summary of these four trials along with analyses of other Region

6 studies will improve the advanced-generation breeding

program. Moreover, better comparisons with the resistance

screening and breeding programs in British Columbia, California, and Idaho will be possible.

Materials and Methods ___________

Study Design

Four blister rust screening trials were examined, one

WWP and one SP, from each of two test years, 1989 and 1994.

These trials are denoted as WWP1989, SP1989, WWP1994,

and SP1994. The seedling families in these trials are progeny of parent trees that cover much of the range of WWP in

Oregon and Washington and of SP in Oregon (fig. 1a,b).

In each of the four trials, seed from 120 families were sown

in a randomized complete block design with six blocks.

Within each block, 10 seedlings per family were planted in

row plots. Each trial contained a maximum of 7,200 seedlings (6 blocks x 1,200 seedlings/block). Seedlings were

grown outside in open boxes (0.91 m wide x 1.21 m long x 0.30

m high; 10 boxes per block) for two growing seasons before

artificial inoculation with blister rust.

Families

The majority of families included in each of these trials are

half-sib seedling progeny (“Wild OP” families) of phenotypic

selections from natural forests or plantings (table 1). In the

WWP1994 trial, 42 of 110 Wild OP families were from the

Quinault Indian Nation in western Washington, 57 were

from the Colville National Forest (NF) in eastern Washington, and 11 were from several other National Forests in

Figure 1—Geographic location of parent trees with half-sib progeny included in the four trials.
USDA Forest Service Proceedings RMRS-P-32. 2004 211

Variation in Blister Rust Resistance Among 226 Pinus monticola and 217 P. lambertiana Seedling Families in the Pacific Northwest Kegley and Sniezko

Region 6. In the WWP1989 trial, 45 of 116 Wild OP families

were from the Colville NF, 20 from Mt Hood NF, 28 from the

Umpqua NF, and lesser numbers from other NFs (table 1

and fig. 1). The two SP trials sampled progeny of selected

trees predominantly from the Rogue River NF and Siskiyou

NF in southern Oregon (table 1 and fig.1). Because a few

families had insufficient germination (less than 20 seedlings) for inclusion in analyses, the numbers of Wild OP

families were as follows: 104 in SP1989; 116 in WWP1989;

110 in WWP1994; and 113 in SP1994.

In addition to the 226 WWP and 217 SP Wild OP families,

two to four Dorena full-sib high resistance checklots per

trial, one low resistance seedlot (bulk of three half-sib

families, used only in SP1989), one half-sib bark reaction

checklot (BR checklot) from the Colville NF (only in

WWP1994), and four standard WWP full-sib checklot families from the Forest Service rust resistance program in Idaho

(only in WWP1994; Aram Eramian, personal communication) were included. The Dorena full-sib high resistance

checklots were progeny of crosses among parents that have

been confirmed to have a hypersensitive reaction in the

needles (HR, see Kinloch and Dupper 2002 for discussion of

HR in WWP and SP). Three Dorena full-sib HR checklots

were common between the two WWP trials; two full-sib HR

checklots were common between the two SP trials.

Inoculation

Details of the inoculation procedure have been previously

described by Sniezko (1996) and Samman (1982). Approximately 18 months after sowing, seedlings were moved to a

large room (13.7 m x 10.1 m x 3.0 m) where they were

inoculated with blister rust (table 2). Temperature within

the inoculation chamber was maintained at around 16.7∞C

(62∞F) and relative humidity at 100 percent.

Ribes leaves (the alternate host) infected with C. ribicola

at the telial stage were collected from various forest sites in

Oregon and Washington as well as the Dorena Ribes Garden. These leaves were placed on wire frames above the

seedlings, telial side down. Ribes leaves were randomly

distributed among the six blocks of each trial except in

WWP1994; in that trial, leaves from the Dorena Ribes

Garden were used on Block 5, and leaves from near Champion Mine on the Umpqua NF were used on Block 6. A

virulent pathotype of the rust (with the vcr2 gene) that

neutralizes HR in WWP (and the associated Cr2 gene) had

been noted in both these locations (Kinloch and others 1999).

Blocks 1 through 4 in WWP1994 used Ribes leaves from

areas without known occurrence of vcr2.

Spore fall was monitored until the desired inoculum density was reached for each box (table 2); the Ribes leaves were

Table 1—Geographic location of parent trees of families included in four blister rust screening trials

at Dorena Genetic Resource Center.

Sow Year

Species Breeding Zone National Forest Elevation (ft)c 1989 1994

western 02180 Fremont — 3

white 05010 Mt Baker-Snoqualmie — 1

pine 06020 Mt Hood — 20

09070 Olympicb — 42

15040 Umpqua — 28

16140 Wallowa-Whitman — 3

17110 Wenatchee — 13

18030 Willamette — 9 5

21100 Colville — 45 57

Idahoa — 4

Dorena BR Checka Colville — 1

Dorena HR Checksa Umpqua — 4 4

 Total 120 119

sugar 02176 Fremont All 5 11

pine 10025 Rogue River >4000 17 7

10043 Rogue River <2500 25

10044 Rogue River 2500-4000 25 21

10045 Rogue River >4000 6 42

11053 Siskiyou <2500 3

11054 Siskiyou 2500-4000 27 7

11055 Siskiyou >4000 2

18033 Willamette <3000 19

Dorena HR Checksa Umpqua —d 2 4

Dorena LR Check Umpqua —d 1

 Total 107 117

a The checklots are technically not listed by breeding zone but rather by source (Dorena or Idaho) or by resistance

type HR (hypersensitive reaction), BR (bark reaction), or LR (low resistance). b Although Breeding Zone 09070 corresponds with Olympic NF, the 42 families from breeding zone 09070 in Sow

Year 1994 were from the neighboring Quinault Indian Nation. c

 Unlike sugar pine, elevation is not a criterion for establishing western white pine breeding zones.

dSince these checklots are full-sib crosses between selected parents from different locations, or a mix (LR Check),

elevations are not reported for these families.
212 USDA Forest Service Proceedings RMRS-P-32. 2004

Kegley and Sniezko Variation in Blister Rust Resistance Among 226 Pinus monticola and 217 P. lambertiana Seedling Families in the Pacific Northwest

then removed. After the target inoculum density was reached,

the seedlings were left in the inoculation chamber for approximately 48 hours to ensure spore germination; boxes

were then returned to their previous outdoor location. In

each of the 2 years, the sugar pine and western white pine

trials were inoculated separately.

Assessments of Resistance Traits

Seedlings were assessed for blister rust symptoms six

times over a period of 5 years. The first inspection occurred

approximately 9 months after inoculation. Seedlings were

evaluated for the presence and number of needle lesions

(“spots”). The checklots in each trial were used to monitor the

presence of needle lesions on all secondary needles until the

number of spots reached a maximum. These checklot counts

were then used to establish five needle lesion classes specific

to each trial. The scale was set up to have approximately 25

percent of the seedlings in each needle lesion class from 1 to

4. Table 3 lists the number of spots in each needle lesion class

for each trial.

The second inspection occurred approximately 3 months

after the first inspection (1 year after inoculation). Seedlings

were assessed for the presence of needle lesions, stem symptoms (cankers and bark reactions), and height. Subsequent

inspections of stem symptoms and mortality occurred annually. Bark reaction data was only recorded through the third

year after inoculation (inspection 4).

Based upon data collected from the six inspections, presence or absence of resistance responses (table 4) was determined for each seedling. In general, seedlings were characterized by the presence or absence of needle lesions and/or

stem symptoms, the type of stem symptom, their survival

after 3 and 5 years, and their height after three growing

seasons (1 year after inoculation).

Analyses of variance (Proc GLM, SAS 1999) were performed separately for each of the four trials using family

block means of each trait for two subsets of data: (1) all

families, including checklots and (2) Wild OP families only.

The model included Family and Block effects. Although the

data for many of the traits were not normally distributed, Ftests are fairly robust against this violation (Cochran and

Cox 1967; Zolman 1993).

Table 2—Inoculation summary statistics for four blister rust screening trials at Dorena Genetic

Resource Center.

WWP1989a SP1989 WWP1994 SP1994

Spore Densityb

Target Spore Density 3500 6000 3000 6000

Average Spore Density 3470 5233 3348 7216

Range in Spore Density 3095-3870 3355-6390 2925-3975 6215-8040

% Ribes leaves with vcr2c 85% 65% 33.3% 46.2%

Inoculation Temperature 58-63∞F 60-62∞F 61-64∞F 62-63∞F

Inoculation Humidity 100% 100% 100% 100%

Spore Germination % —d 86.3% 94.7% 95.6%

a

 Where WWP=western white pine and SP=sugar pine and the year indicates year sown for testing.

b The number of basidiospores of Cronartium ribicola per square centimeter. c Percentage of total leaves used that originated from areas where vcr2 pathotype virulent to HR in WWP

have been confirmed. In WWP1989, SP1989, and SP1994, leaves with vcr2 (Ribes Garden and Champion

Creek) were randomly distributed among the blocks. However in WWP1994, potential leaves with vcr2 were

placed on blocks 5 and 6 only. Note: vcr2 does not appear to overcome HR in SP. d

 Basidiospore germination was not assessed in the WWP1989 trial.

Table 3—Number of needle lesions (spots) in each class

for seedlings in four screening trials.

# Spots/Needle Lesion Class

Triala 0 1 234

WWP1989 0 1-2 3-5 6-10 11+

SP1989 0 1 2 3-5 6+

WWP1994 0 1-10 11-25 26-48 49+

SP1994 0 1-2 3-5 6-15 16+

a Where WWP=western white pine and SP=sugar pine and the

year indicates year sown for testing.
USDA Forest Service Proceedings RMRS-P-32. 2004 213

Variation in Blister Rust Resistance Among 226 Pinus monticola and 217 P. lambertiana Seedling Families in the Pacific Northwest Kegley and Sniezko

Results ________________________

Survival and Growth

Survival of Wild OP families 5 years after inoculation

(TSURV5) was low in all trials, averaging about 2 percent to

14 percent (table 5). TSURV5 was higher in the 1989 trials

than in the 1994 trials (table 5). TSURV5 was slightly higher

for SP relative to WWP in 1989, but the reverse was true in

the 1994 trials. Overall survival of rust-infected Wild OP

seedlings (RSURV5) was generally low in all trials, but

varied by trial and species (table 5, fig. 2). Like TSURV5,

RSURV3 and RSURV5 were higher for the 1989 trials than

the 1994 ones (table 5, fig. 2, 3). Fifth-year survival of rustinfected Wild OP seedlings was 9.9 percent and 13.1 percent

for WWP and SP, respectively, in the 1989 trials and 3.2 and

1.6 percent in the 1994 trials (table 5, fig. 3). In the 1989

trials the decline in survival began later, between the third

and fourth inspections (approximately 2 and 3 years after

inoculation), than in the 1994 trials (fig. 2). Despite the very

low overall survival, all four trials had at least one family

with more than 20 percent survival of infected seedlings (see

RSURV5 in table 5 and fig. 3).

RSURV5 of the Idaho full-sib families was moderate, in

the 30 to 40 percent range, slightly lower than the Dorena

BR checklot (56 percent, table 5). For those seedlings with

bark reactions, survival was low but higher than those

with normal stem cankers (BRSURV5 and NCSURV5,

Table 4—Traits and derived variables used in assessing seedlings for blister rust resistance.

Category Trait Description of Trait

General Infected Seedling developed needle lesions and/or stem symptoms

NOINFECT Seedling had no needle lesions present at either first or second inspection and

had no stem symptoms in subsequent inspections.

RSURV3 Infected tree is alive 3 years after inoculation

RSURV5 Infected tree is alive 5 years after inoculation

TSURV5 Seedling (infected or uninfected) is alive 5 years after inoculation

HT3 Total height (cm), including any lammas growth, of seedling after three

growing seasons

Needle Lesions NLC Needle Lesion Class A categorical classification of number of needle lesions (‘spots’) on all

secondary needles on a seedling at first inspection

SPO Spots Only Seedling had needle spots but did not develop stem symptoms through 5

years after inoculation

SPOT% Seedling had needle lesions present at either first (SPOT1%) or second

inspection (SPOT2%)

Stem Symptoms SSFREE Stem symptom free Seedling was free of stem symptoms (initial orange discoloration of the bark,

normal canker or bark reaction) at any of the six inspections following

inoculation

SS Stem Symptoms Seedling exhibited normal canker, bark reaction, or was dead of rust

ESS3 Early Stem Calculated as the ratio of seedlings with stem symptoms (SS) one year after

Symptoms inoculation relative to those with SS three years after inoculation. A lower

value indicates families with relatively slower or delayed appearance of stem

symptomsa

SSAL3 Stem symptom alive Seedling with stem symptoms (SS) and alive (AL) 3 years after inoculationa

SSAL5 Seedling with stem symptoms (SS) and alive (AL) 5 years after inoculationa

NCANK Normal Canker Seedling exhibits initial orange discoloration of the bark or fusiform swelling

with an active orange margin (Kinloch and Davis 1996; Hunt 1997) at any

inspectiona

BR Bark Reaction Seedling exhibits bark reaction, that is, an incompatible interaction with the

fungus (Theisen 1988). BR manifests as a sunken necrotic lesion, often at the

base of a needle fascicle, on stem tissue. When no fungal activity is observed,

the BR is considered ‘complete.’ An ‘incomplete’ or ‘partial’ BR does not

completely halt fungal growth (Kinloch and Davis 1996; Franc 1988)ab

a

 The denominators for the BR%, NCANK%, ESS3%, and SSAL% calculations included only trees with stem symptoms.

b A seedling could be scored as having both a bark reaction and a normal canker due to the presence of (1) multiple stem infections or (2) a transition in status from

normal canker to bark reaction or vice-versa in different inspection years.
214 USDA Forest Service Proceedings RMRS-P-32. 2004

Kegley and Sniezko Variation in Blister Rust Resistance Among 226 Pinus monticola and 217 P. lambertiana Seedling Families in the Pacific Northwest Table 5—Trial means and means of the Dorena and Idaho checklots as well as several Wild OP families with relatively high levels of resistance in four blister rust screening trials. Survival and Growthb Needle Symptomsb Stem Symptomsb no nc br Trial Family Typea infect infect rsurv3 rsurv5 tsurv5 ssal3 ssal5 surv5 surv5 ht NLC spo spot1 spot2 spot ss ess3 br ncank - - - - - - - - - - - - - - - - - - - - - - - - - Percent - - - - - - - - - - - - - - - - - - - - - - - - - - cm - - - - - - - - - - - - - - - - - - - - Percent - - - - - - - - - - - - - - - - - - - - - - WWP 06023-513 Wild OP 97.6 2.4 76.9 52.1 40.0 74.0 36.7 33.9 68.0 31.7 1.6 25.1 88.4 35.3 88.4 72.6 13.9 32.7 61.3 1989 15045-443 Wild OP 98.3 1.7 83.5 54.8 51.7 74.1 32.4 27.1 66.7 29.5 2.7 30.6 98.3 56.8 98.3 67.8 71.8 20.5 64.1 15045-642 Wild OP 100.0 0.0 78.0 42.2 40.0 76.3 37.6 26.5 58.3 29.8 2.6 8.9 100.0 48.7 100.0 91.1 41.3 34.0 80.4 21104-418 Wild OP 100.0 0.0 75.0 35.0 35.0 71.4 28.3 16.1 47.8 37.3 2.2 10.0 100.0 68.3 100.0 90.0 43.2 50.4 78.3 17114-631 Wild OP 100.0 0.0 86.7 54.8 53.3 84.7 44.6 35.0 71.3 32.0 2.4 22.6 100.0 40.4 100.0 77.4 55.3 27.2 67.2 15045-861 x 15045-837 HR check 100.0 0.0 73.0 18.9 18.3 68.7 1.9 1.9 0.0 32.8 2.3 17.2 98.3 37.7 100.0 82.8 51.3 4.2 82.8 15045-861 x 15045-862 HR check 100.0 0.0 88.0 30.6 30.0 83.7 10.0 10.0 0.0 33.5 1.9 22.0 98.3 49.1 98.3 78.0 56.4 2.1 78.0 15045-862 x 18034-392 HR check 90.0 10.0 97.9 65.5 63.3 95.8 39.8 29.3 61.7 36.5 1.7 39.9 90.0 23.1 90.0 50.1 35.0 25.7 43.3 15045-896 x 15045-862 HR check 100.0 0.0 91.7 30.7 30.0 87.2 7.0 2.1 0.0 36.1 2.0 25.7 98.3 27.4 98.3 74.3 56.6 2.4 69.3 Wild OP avg 99.4 0.6 66.6 9.9 9.5 65.3 6.0 4.8 10.8 30.2 2.4 4.6 98.1 59.0 98.4 94.9 65.9 12.3 93.1 SP 18033-317 Wild OP 86.3 4.5 68.8 24.5 21.7 77.8 20.8 14.1 33.3 33.2 1.5 6.5 62.5 32.9 64.6 89.0 31.5 8.9 78.3 1989 18034-112 Wild OP 87.7 11.6 74.6 39.4 36.7 69.8 33.1 6.9 25.0 35.9 1.7 12.2 75.2 31.6 79.5 76.2 40.8 15.4 47.8 10044-212 Wild OP 84.3 15.7 92.6 42.8 46.7 94.3 23.6 14.6 100.0 39.7 1.5 23.1 68.8 27.7 74.8 61.2 46.7 5.7 53.8 10044-230 Wild OP 77.4 22.6 83.7 49.8 36.7 72.2 31.7 9.7 83.3 47.1 1.5 18.8 58.8 25.7 71.3 58.6 34.7 23.6 42.1 11054-877 Wild OP 85.0 15.0 84.8 37.4 45.0 82.1 23.2 16.4 38.9 45.3 1.8 15.4 75.0 33.3 76.7 69.6 45.3 36.9 56.3 B1054-004 x B1054-034 HR check 78.3 22.8 67.4 60.8 68.3 56.7 44.3 17.3 62.5 40.1 1.7 33.6 66.3 17.7 71.3 43.7 80.0 34.4 27.5 B1054-034 x 10044-050 HR check 77.4 22.8 50.3 40.4 51.7 24.5 5.8 6.0 0.0 37.3 1.9 30.0 68.5 33.3 74.1 47.1 67.1 27.3 45.5 Low Resistance Mix LR Check 94.4 5.6 75.6 10.4 15.0 72.6 3.9 2.1 0.0 41.0 2.0 7.0 76.1 43.0 81.7 87.4 52.7 8.1 80.0 Wild OP avg 95.8 3.8 70.0 13.1 14.1 70.6 8.4 4.7 13.5 39.0 2.1 5.5 78.1 52.5 85.1 90.7 58.9 7.2 85.7 WWP 02187-047 Wild OP 98.1 0.0 10.0 6.7 6.7 11.1 5.6 5.6 — 17.1 2.2 4.2 98.1 46.9 98.1 95.8 50.3 0.0 95.8 1994 21105-853 Wild OP 100.0 0.0 41.7 20.0 20.0 31.7 8.9 3.9 10.0 45.5 2.2 11.7 98.3 46.7 100.0 88.3 48.8 31.7 83.3 09070-852 Wild OP 98.3 1.7 30.7 25.2 25.0 9.9 0.0 0.0 0.0 38.0 1.5 25.5 93.1 28.7 93.1 72.8 38.0 7.1 71.2 09070-892 Wild OP 96.7 3.7 39.6 13.9 16.7 26.6 4.0 0.0 5.6 48.7 1.7 10.9 95.0 61.7 95.0 85.4 32.0 32.8 78.3 09070-896 Wild OP 100.0 0.0 20.0 13.3 13.3 11.4 5.4 3.7 16.7 46.4 2.2 8.3 100.0 53.3 100.0 91.7 58.5 25.6 88.3 21104-036 Wild OP 100.0 0.0 34.3 13.7 13.3 32.1 9.6 9.8 17.3 41.1 3.1 5.2 100.0 72.6 100.0 94.8 48.4 38.8 93.1 21105-052 BR check 100.0 0.0 81.5 55.7 55.0 73.3 35.2 29.1 52.0 46.4 2.3 28.7 96.7 52.4 96.7 71.3 35.7 59.9 62.4 17 x 293 Idaho 98.3 1.7 54.4 32.2 33.3 47.3 23.1 17.5 41.7 56.2 2.5 11.7 96.7 50.0 98.3 86.7 38.0 48.2 81.7 221 x 220 Idaho 100.0 0.0 45.0 33.3 33.3 22.4 13.9 2.1 26.7 41.4 3.3 22.8 100.0 53.3 100.0 77.2 43.9 16.5 65.2 208 x 314 Idaho 96.7 3.5 67.8 40.0 41.7 54.0 23.9 20.4 30.7 45.0 2.0 22.0 95.0 31.7 95.0 74.4 22.4 33.5 69.4 222 x 225 Idaho 100.0 0.0 38.3 31.7 31.7 24.2 19.0 17.6 30.0 47.2 3.2 17.2 100.0 43.3 100.0 82.8 49.6 26.3 81.1 15045-861 x 15045-862 HR check 100.0 0.0 40.0 38.3 38.3 2.9 0.0 0.0 0.0 39.8 3.3 43.9 100.0 51.7 100.0 56.1 47.7 2.9 56.1 15045-862 x 15045-837 HR check 100.0 0.0 53.5 51.9 51.7 5.0 0.0 0.0 0.0 48.3 2.9 51.9 100.0 44.8 100.0 48.1 43.3 15.0 48.1 15045-862 x 18034-392 HR check 100.0 0.0 51.7 36.7 36.7 26.5 2.1 2.1 0.0 45.8 2.7 35.6 100.0 50.0 100.0 64.4 54.8 20.9 64.4 15045-896 x 15045-862 HR check 100.0 0.0 40.0 40.0 40.0 0.0 0.0 0.0 0.0 42.4 3.0 40.9 100.0 37.1 100.0 59.1 59.2 2.1 59.1 Wild OP avg 99.5 0.3 6.9 3.2 3.4 4.6 0.7 0.6 1.2 42.3 2.6 2.6 98.4 76.4 98.8 97.1 70.7 7.2 96.8 SP 98-01-018 Wild OP 100.0 0.0 5.0 0.0 0.0 3.7 0.0 0.0 0.0 44.7 2.6 0.0 100.0 75.1 100.0 100.0 79.9 26.1 98.1 1994 B1053-1380 Wild OP 91.7 8.3 53.6 28.7 33.3 2.4 5.2 2.4 33.3 54.2 1.5 34.7 91.7 43.3 91.7 56.9 62.1 11.3 51.8 15043-402 Wild OP 100.0 0.0 20.9 13.7 10.0 16.3 6.9 6.9 — 42.9 2.1 7.9 95.5 60.4 95.5 92.1 66.1 0.0 92.1 B1052-1993 Wild OP 100.0 0.0 5.0 3.3 3.3 5.0 3.3 3.3 0.0 56.7 3.0 0.0 97.9 79.5 100.0 100.0 74.4 4.4 100.0 B2-2400 Wild OP 98.3 1.7 21.1 10.2 11.7 15.1 3.5 3.5 — 53.4 1.7 6.7 91.7 50.7 96.7 91.7 61.4 0.0 91.7 B1054-004 x B1054-034 HR check 79.8 20.2 62.6 60.7 63.3 15.7 15.7 10.0 50.0 49.1 1.9 45.3 79.8 24.3 79.8 34.5 69.3 10.7 31.2 B1054-034 x 10044-050 HR check 94.8 5.4 54.2 50.8 51.7 5.6 5.6 0.0 33.3 51.6 2.3 51.3 91.1 46.3 94.8 43.2 87.8 15.3 40.5 B1054-005 x B1054-034 HR check 92.6 7.4 67.4 46.4 41.7 23.6 18.1 0.0 100.0 60.9 2.0 43.3 90.7 27.0 90.7 49.3 54.2 18.1 42.8 B1054-005 x 10044-049 HR check 93.4 0.0 43.3 37.0 26.7 9.7 9.7 5.6 16.7 58.1 1.6 34.8 85.4 38.5 93.4 65.2 65.3 20.8 62.4 Wild OP avg 99.5 0.2 3.3 1.6 1.6 2.0 0.5 0.4 3.4 47.6 2.9 1.2 97.8 82.9 98.6 98.6 84.5 3.5 98.4

a Where type refers to the classification of the material: Wild OP=half-sib progeny of phenotypic selections, BR check=half-sib family previously screened and having a high level of bark reaction, Idaho=full-sib checklot from the Idaho blister rust program,

HR check=Dorena full-sib checklot with hypersensitive reaction in the needles, LR check=mix of low resistant half-sib material, Wild OP avg=mean of all Wild OP families included in the trial. bTraits are defined in Table 4 except ncsurv5 (survival with normal canker 5 years after inoculation) and brsurv5 (survival with bark reaction 5 years after inoculation).
USDA Forest Service Proceedings RMRS-P-32. 2004 215

Variation in Blister Rust Resistance Among 226 Pinus monticola and 217 P. lambertiana Seedling Families in the Pacific Northwest Kegley and Sniezko

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

% survival

0

10

20

30

40

50

60

70

80

90

100

WWP1989

years after inoculation

SP1989

WWP1994

SP1994

% RSURV5

0

10

20

30

40

50

60

70

80

90

100

% RSURV5

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

% RSURV5

0

10

20

30

40

50

60

70

80

90

100

110

WWP1989

Wild OP mean = 9.9

SP1989

Wild OP mean = 13.1

% RSURV5

0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100

0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100

WWP1994

Wild OP mean = 3.2

SP1994

Wild OP mean = 1.6

Wild OP families

HR full sib checklots

Low resistance checklot

Bark reaction checklot

Idaho full sib checklots

% families

% families

% families

% families

Figure 2—Survival of infected half-sib progeny of western white pine

(WWP) and sugar pine (SP) after inoculation with blister rust. Each

inflection point represents date of assessment.

Figure 3—Distribution of family means for survival of infected seedlings 5 years after inoculation (RSURV5) for four

blister rust screening trials.

respectively, table 5). Survival of the Dorena HR WWP

checklots was about 42 percent in the 1994 trial compared to

36 percent in the 1989 trial. Survival of the two HR SP

checklots common to both test years was slightly higher,

ranging from 40 to 61 percent. RSURV5 of the Low Resistant

SP checklot was lower than the average of the Wild OP

families (10 percent versus 13 percent) (table 5).

Families varied significantly for height in all four trials

(table 6). The checklots were generally intermediate for HT

(fig. 4, table 5). Mean height was greater in the 1994 trials

than the 1989 trials (table 5, fig. 4).

Rust Infection

Infected seedlings in the Wild OP families (infection

percentage) averaged 95.8 to 99.5 percent for the four trials

(table 5). Sugar pine seedlings had more variation in the

frequency of infection than WWP seedlings. Wild OP family

means for SP ranged from 77.4 to 100 percent in the 1989

trial and from 83.3 to 100 percent in the 1994 trial. For WWP
216 USDA Forest Service Proceedings RMRS-P-32. 2004

Kegley and Sniezko Variation in Blister Rust Resistance Among 226 Pinus monticola and 217 P. lambertiana Seedling Families in the Pacific Northwest

Table 6—P-values associated with F-tests for significant differences among families from analyses of variance for each of four blister rust

screening trials at Dorena.

Wild OP families only All families

WWP WWP WWP WWP

Trait 1989 SP 1989 1994 SP 1994 1989 SP 1989 1994 SP 1994

General Infect % 0.2100a <0.0001 0.2430 0.2635 0.0113 <0.0001 0.1656 <0.0003

NOINFECT %b 0.2100 <0.0001 0.0006 0.0183 0.0113 <0.0001 0.0003 <0.0001

RSURV3 % <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

RSURV5 % <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

TSURV5 % <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

HT <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

Needle Lesions NLC <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

SPO % <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

SPOT% 0.1247 <0.0001 0.0349 0.0725 0.0836 <0.0001 0.0152 0.0042

SPOT1 % 0.2309 0.0007 0.0024 0.0197 0.1929 0.0009 0.0009 0.0012

SPOT2 % <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

Stem Symptoms SSFREE % <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

SS % <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

ESS3 % <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

SSAL3 % <0.0001 <0.0001 <0.0001 0.0108 <0.0001 <0.0001 <0.0001 <0.0001

SSAL5 % <0.0001 <0.0001 <0.0001 0.0747 <0.0001 <0.0001 <0.0001 <0.0001

BR % <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

NCANK % <0.0001 <0.0001 0.1453 0.5214 <0.0001 0.0045 0.0017 <0.0001

a

 P-values in bold type are not significant at the a = 0.05 level. b NOINFECT% is not necessarily equal to 100-infect%. To be classified as NOINFECT, a seedling must survive through the end of the assessment period (6th

Inspection) without ever developing needle lesions or stem symptoms.

height (cm)

15 20 25 30 35 40 45 50 55 60 65 70

# families

0

10

20

30

40

50

60

height (cm)

15 20 25 30 35 40 45 50 55 60 65 70

# families

0

10

20

30

40

50

60

height (cm)

15 20 25 30 35 40 45 50 55 60 65 70

# families

0

10

20

30

40

50

60

height (cm)

15 20 25 30 35 40 45 50 55 60 65 70

# families

0

10

20

30

40

50

60

WWP1989

Wild OP mean = 30.2

SP1989

Wild OP mean = 39.0

WWP1994

Wild OP mean = 42.3

SP1994

Wild OP mean = 47.6

Wild OP families

HR full sib checklots

Low resistance checklot

Bark reaction checklot

Idaho full sib checklots

Figure 4—Distribution of family means for height after three growing seasons for the four trials.
USDA Forest Service Proceedings RMRS-P-32. 2004 217

Variation in Blister Rust Resistance Among 226 Pinus monticola and 217 P. lambertiana Seedling Families in the Pacific Northwest Kegley and Sniezko

family means ranged from 96.7 to 100 percent, including the

full-sib families from Dorena and Idaho. Checklots of SP

were not consistent in their infection levels between the two

trial years. In SP1989, the two resistant Dorena checklots

had 77 to 78 percent infection, and the low resistant control

had 94 percent infection, whereas the checklots in SP1994

had 80 to 95 percent infection (table 5). Checklots for WWP

had 90 to 100 percent infection in the two trials. In three of

the four trials, infection percentage was nearly constant

over the course of the evaluation, but in SP1989 infection

levels increased from first to fourth inspection (fig. 5).

There were very few uninfected seedlings (noninfected

seedlings were without any needle lesions and without any

stem symptoms). The mean percentage of uninfected seedlings was less than 1 percent for the Wild OP families in the

WWP1989 trial and the two 1994 trials; the percentage was

about 4 percent for Wild OP families in the SP1989 trial

(table 5).

With a few exceptions, families varied significantly

(p<0.0001) in all traits within each trial, whether or not

checklots were included in the analyses (table 6).

Needle Lesions

The percentage of seedlings with needle lesions (spot

percent) was very high in all trials, averaging 85 to 99

percent among Wild OP families (table 5). At first inspection,

approximately 98 percent of the Wild OP seedlings in

WWP1989, WWP1994, and SP1994 had needle lesions,

while only 78 percent of SP1989 had spots (spot1, table 5). By

the second inspection (approximately 1 year after inoculation),

the percentage of trees with needle spots dropped in all four

trials; Wild OP run averages for spot2 ranged from 59 to 83

percent (table 5, fig. 6). The shedding of needles was most

likely responsible for spot2 values being lower than spot1.

The checklots were among the families with the lowest

percentage of seedlings with needle lesions at second inspection (table 5, fig. 6).

The number of needle lesions per needle lesion class also

varied by trial and species (table 3). In these trials the

minimum number of spots required to enter the highest

needle lesion class (Class 4) ranged from 6 to as high as 49

(table 3). More needle lesions were present in the 1994 trials

relative to the 1989 trials and in western white pine relative

to sugar pine (despite the lower inoculum density in the

WWP trials). Family mean needle lesion class (NLC) ranged

from 1.5 to 4.0, but the distribution of families across classes

shifted depending upon the species and the trial (fig. 7).

Within a trial, the SP HR checklots generally had fewer than

average needle lesions, but the Dorena HR WWP checklots

were not consistent between trials (fig. 7, table 5). There was

a wide range in NLC among the four Idaho full-sib checklots

in WWP1994 (table 5, fig. 7).

The mean percentage of seedlings with needle spots and

no stem symptoms (SPO) was low for Wild OP families in all

trials, ranging from 1.2 to 5.5 percent (table 5). Some Wild

OP families in each trial had more than 10 percent of

seedlings with only needle spots with some families showing

30 to 35 percent seedlings with SPO (table 5, fig. 8). All the

checklot families, except the Low Resistant checklot in

SP1989, showed much higher levels of SPO than the Wild

OP trial mean (table 5, fig. 8).

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

10

20

30

40

50

60

70

80

90

100

% infection

WWP1989

years after inoculation

SP1989

WWP1994

SP1994

Figure 5—Percent infection of half-sib progeny of phenotypic selections of

western white pine (WWP) and sugar pine (SP) in four blister rust screening

trials. Each inflection point represents date of assessment.
218 USDA Forest Service Proceedings RMRS-P-32. 2004

Kegley and Sniezko Variation in Blister Rust Resistance Among 226 Pinus monticola and 217 P. lambertiana Seedling Families in the Pacific Northwest # families

0

10

20

30

40

50

# families

0

10

20

30

40

50

# families

0

10

20

30

40

50

# families

0

10

20

30

40

50

WWP1989

Wild OP mean = 59.0

SP1989

Wild OP mean = 52.5

% SPOT2

0 10 20 30 40 50 60 70 80 90 100

WWP1994

Wild OP mean = 76.4

SP1994

Wild OP mean = 82.9

Wild OP families

HR full sib checklots

Low resistance checklot

Bark reaction checklot

Idaho full sib checklots

% SPOT2

0 10 20 30 40 50 60 70 80 90 100

% SPOT2

0 10 20 30 40 50 60 70 80 90 100

% SPOT2

0 10 20 30 40 50 60 70 80 90 100

NLC

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

# families 0

10

20

30

40

50

60

NLC

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

# families 0

10

20

30

40

50

60

NLC

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

# families 0

10

20

30

40

50

60

NLC

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

# families 0

10

20

30

40

50

60

WWP1989

Wild OP mean = 2.4

SP1989

Wild OP mean = 2.1

WWP1994

Wild OP mean = 2.6

SP1994

Wild OP mean = 2.9

Wild OP families

HR full sib checklots

Low resistance checklot

Bark reaction checklot

Idaho full sib checklots

Figure 6—Distribution of family

means for the percentage of seedlings with needle spots (SPOT2) 1

year after inoculation (second inspection) for the four trials.

Figure 7—Distribution of family

means for needle lesion class (NLC)

for the four trials. NLC is a measure

of the relative frequency of needle

lesions within a test.
USDA Forest Service Proceedings RMRS-P-32. 2004 219

Variation in Blister Rust Resistance Among 226 Pinus monticola and 217 P. lambertiana Seedling Families in the Pacific Northwest Kegley and Sniezko

Stem Symptoms

More than 90 percent of the seedlings in most Wild OP

families developed stem symptoms (SS); the trial average

percentages varied from 91 to 99 percent SS (table 5). Even

so, there were significant differences among families in all

four trials (table 6). The same general pattern of an increase

over time in the percentage seedlings with stem symptoms

developed in the four trials, although there were some

differences in when the SS percent attained a maximum. For

example, in SP1989, SS percent continued to increase slightly

to the end of the evaluation period (sixth inspection, 5 years

after inoculation) whereas SS percent stabilized by the

fourth inspection (3 years after inoculation) in the other

three trials (fig. 9).

Each of the four trials had at least one Wild OP family

with less than 75 percent of the seedlings with stem

symptoms (fig. 10 and table 5); SP1989 had nine Wild OP

families, WWP1989 had five families, and the two 1994

trials each had one family with less than 75 percent of the

seedlings having stem symptoms (table 5, fig. 10). The

Dorena WWP and SP checklots with HR (hypersensitive

reaction in the foliage) generally had among the lowest SS

percent in all trials. The sugar pine checklots with HR # families

0

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120

# families

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# families

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120

# families

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10

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100

110

120

WWP1989

Wild OP mean = 4.6

SP1989

Wild OP mean = 5.5

% SPO

0 10 20 30 40 50 60 70 80 90 100

WWP1994

Wild OP mean = 2.6

SP1994

Wild OP mean = 1.2

Wild OP families

HR full sib checklots

Low resistance checklot

Bark reaction checklot

Idaho full sib checklots

% SPO

0 10 20 30 40 50 60 70 80 90 100

% SPO

0 10 20 30 40 50 60 70 80 90 100

% SPO

0 10 20 30 40 50 60 70 80 90 100

Figure 8—Distribution of family means for percentage of seedlings with needle spots but no

stem symptoms (SPO) in the four trials.

averaged 45 percent and 48 percent SS in the two SP trials,

respectively; the resistant western white pine checklots

averaged 71 percent and 57 percent SS in the WWP trials.

In the WWP1994 trial, the Idaho checklots and the Dorena

BR checklot also showed low SS percent (71 to 87 percent)

relative to the Wild OP families; however, the Dorena HR

checklots were even lower with 48 to 64 percent SS in this

trial (fig. 10 and table 5). The Dorena Low Resistant

checklot in SP1989 had relatively high SS percent (87.4

percent) (table 5, fig. 10).

Normal Cankers—The percentage of Wild OP seedlings

with normal cankers in at least one inspection (NCANK)

varied from 86 to 98 percent for the four trials. The percentage was higher in 1994 trials relative to 1989, but the ranks

between species were not consistent for the 2 years (table 5).

The Wild OP sugar pine families with the lowest NCANK

had values of 42 and 52 percent, whereas the western white

pine families with the lowest NCANK had values of 61 and

71 percent (table 5). Survival of cankered seedlings from

Wild OP families 5 years after inoculation (NCSURV5) was

very low, ranging from 0.4 to 4.8 percent among the four

trials (table 5). Survival of trees with a normal canker was

higher for Wild OP families in the 1989 trials than for the
220 USDA Forest Service Proceedings RMRS-P-32. 2004

Kegley and Sniezko Variation in Blister Rust Resistance Among 226 Pinus monticola and 217 P. lambertiana Seedling Families in the Pacific Northwest

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

% SS

0

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30

40

50

60

70

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100

WWP1989

years after inoculation

SP1989

WWP1994

SP1994

# families

0

10

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60

70

80

90

100

110

# families

0

10

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110

# families

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10

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110

# families

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WWP1989

Wild OP mean = 94.9

SP1989

Wild OP mean = 90.7

% SS

0 10 20 30 40 50 60 70 80 90 100

WWP1994

Wild OP mean = 97.1

SP1994

Wild OP mean = 98.6

Wild OP families

HR full sib checklots

Low resistance checklot

Bark reaction checklot

Idaho full sib checklots

% SS

0 10 20 30 40 50 60 70 80 90 100

% SS

0 10 20 30 40 50 60 70 80 90 100

% SS

0 10 20 30 40 50 60 70 80 90 100

Figure 9—Percentage seedlings with stem symptoms (SS)

of half-sib progeny of western white pine (WWP) and sugar

pine (SP) in four screening trials. Each inflection point

represents date of assessment.

Figure 10—Distribution of family means for seedlings with stem symptoms (SS) in the four trials.

1994 trials. Within a year, survival with a normal canker

was slightly higher for WWP than for SP Wild OP families

(table 5).

The BR checklot in WWP1994 was among the families

with the lowest NCANK percent in that trial (fig. 11, table

5). The Dorena HR checklots had less NCANK percent than

the Wild OP average, and the low resistance checklot approached the mean NCANK percent for SP1989 (table 5).

Bark Reaction (BR)—The incidence of trees with bark

reactions was low in all four trials, ranging from 3.5 to 12.3

percent (table 5, fig. 12). Significant differences existed

among Wild OP families for BR in all four trials (table 6).

WWP showed slightly higher amounts of BR than SP (table

5, fig. 12). In all four trials, the majority of seedling families

had at least one seedling with bark reaction (unpublished

data). In all trials, there was at least one family with more

than 20 percent of the seedlings with bark reaction (fig. 12).

The Wild OP family with the highest BR (50.4 percent) was

from the Colville NF (Family 21104-418) in the WWP1989

trial (table 5). The Dorena SP HR checklots had higher than

average BR in both trials whereas the Dorena WWP HR
USDA Forest Service Proceedings RMRS-P-32. 2004 221

Variation in Blister Rust Resistance Among 226 Pinus monticola and 217 P. lambertiana Seedling Families in the Pacific Northwest Kegley and Sniezko # families

0

10

20

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50

60

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80

90

100

110

120

# families

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10

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100

110

120

# families

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10

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120

# families

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10

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90

100

110

120

% NCANK

0 10 20 30 40 50 60 70 80 90 100

WWP1989

Wild OP mean = 93.1

SP1989

Wild OP mean = 85.7

Wild OP families

HR full sib checklots

Low resistance checklot

Bark reaction checklot

Idaho full sib checklots

WWP1994

Wild OP mean = 96.8

SP1994

Wild OP mean = 98.4

% NCANK

0 10 20 30 40 50 60 70 80 90 100

% NCANK

0 10 20 30 40 50 60 70 80 90 100

% NCANK

0 10 20 30 40 50 60 70 80 90 100

# families

0

10

20

30

40

50

60

70

80

90

0

10

20

30

40

50

60

70

80

90

# families

0

10

20

30

40

50

60

70

80

90

0

10

20

30

40

50

60

70

80

90

% BR

0 10 20 30 40 50 60 70 80 90 100

WWP1989

Wild OP mean = 12.3

SP1989

Wild OP mean = 7.2

Wild OP families

HR full sib checklots

Low resistance checklot

Bark reaction checklot

Idaho full sib checklots

% BR

0 10 20 30 40 50 60 70 80 90 100

WWP1994

Wild OP mean = 7.2

SP1994

Wild OP mean = 3.5

# families # families

% BR

0 10 20 30 40 50 60 70 80 90 100

% BR

0 10 20 30 40 50 60 70 80 90 100

Figure 11—Distribution of family

means for the percentage of seedlings with normal cankers (NCANK)

in the four trials.

Figure 12—Distribution of family

means for the percentage of seedlings with bark reaction (BR) in the

four trials.
222 USDA Forest Service Proceedings RMRS-P-32. 2004

Kegley and Sniezko Variation in Blister Rust Resistance Among 226 Pinus monticola and 217 P. lambertiana Seedling Families in the Pacific Northwest

checklots showed relatively little BR, with a few exceptions

(table 5, fig. 12). The Dorena BR checklot, 21105-052 in

WWP1994, showed the highest BR percent of any family

tested, including the Idaho full-sib checklots (fig. 12, table 5).

This family had a high BR percent when first tested as a Wild

OP in a 1988 sowing (Sniezko and Kegley 2003). All four of

the Idaho checklots in WWP1994 had higher than average

BR percent (table 5, fig. 12).

Many of the trees with bark reactions also had normal

cankers and died; mean survival 5 years after inoculation of

Wild OP seedlings with BR ranged from 1 to 14 percent in the

four trials (table 5, fig. 13). Families varied greatly for

survival with BR (fig. 13). More of the seedlings with BR in

the 1989 trials survived relative to the 1994 trials (table 5),

and within a year, slightly more SP survived with BR

relative to WWP (table 5).

Early Stem Symptom Percentage (ESS3 Percent)—

The average ESS3 for Wild OP families ranged from 58.9

percent (WWP1989) to 84.5 percent (SP1994) (table 5),

indicating that over half the seedlings in all four trials

showed stem infections approximately 12 months after inoculation. In general the 1989 trials had fewer seedlings # families

0

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50

60

70

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90

100

# families

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# families

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100

# families

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100

% BRSURV5

0 10 20 30 40 50 60 70 80 90 100

Wild OP families

HR full sib checklots

Low resistance checklot

Bark reaction checklot

Idaho full sib checklots

% BRSURV5

0 10 20 30 40 50 60 70 80 90 100

% BRSURV5

0 10 20 30 40 50 60 70 80 90 100

% BRSURV5

0 10 20 30 40 50 60 70 80 90 100

WWP1989

Wild OP mean = 10.8

SP1989

Wild OP mean = 13.5

WWP1989

Wild OP mean = 1.2

SP1989

Wild OP mean = 3.4

Figure 13—Distribution of family means for percentage of seedlings with bark reaction and surviving

5 years after inoculation (BRSURV5) in the four trials.

with early stem symptoms compared with the 1994 trials.

SP1994 showed the highest and least variable family mean

ESS3 percent (fig. 14). The trends between species were not

consistent in the 2 years (table 5). There were significant

differences among Wild OP families in all four trials

(table 6). The WWP checklots had families with more

delayed onset of stem symptoms (lower ESS3 percent) and

included some of the most outstanding families, while the

sugar pine checklots were more variable (fig. 14). All four of

the Idaho full-sib checklots had among the lowest ESS

percent in WWP1994, and the Dorena BR checklot also had

relatively few early stem symptoms (table 5, fig. 14).

Survival of Seedlings with Stem Symptoms—Survival

of seedlings with stem symptoms (SSAL3) was relatively

high (greater than 65 percent) 3 years after inoculation in

the 1989 trials but very low in the 1994 trials (table 5).

However, by the fifth year after inoculation, many of the

seedlings with SS had died; there were few families with

high SSAL5 in any trial (table 5). In the 1989 trials SSAL5

of the best families ranged from 21 to 38 percent, but SSAL5

was less than 10 percent in the best Wild OP families in 1994

(table 5). The Dorena HR full sib checklots were mixed in
USDA Forest Service Proceedings RMRS-P-32. 2004 223

Variation in Blister Rust Resistance Among 226 Pinus monticola and 217 P. lambertiana Seedling Families in the Pacific Northwest Kegley and Sniezko

% ESS

# families

0

10

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30

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60

# families

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60

# families

0

10

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60

0

10

20

30

40

50

60

WWP1989

Wild OP mean = 65.9

SP1989

Wild OP mean = 58.9

0 10 20 30 40 50 60 70 80 90 100

% ESS

0 10 20 30 40 50 60 70 80 90 100

% ESS

0 10 20 30 40 50 60 70 80 90 100

% ESS

0 10 20 30 40 50 60 70 80 90 100

# families

WWP1994

Wild OP mean = 70.7

SP1994

Wild OP mean = 84.5

Wild OP families

HR full sib checklots

Low resistance checklot

Bark reaction checklot

Idaho full sib checklots

Figure 14—Distribution of family means for the percentage of seedlings with early stem symptoms

(ESS3) in the four trials.

performance but generally had low fifth-year survival of

seedlings with stem symptoms (SSAL5 percent) (table 5).

Two of the full sib HR checklots, SP Family B1054-004 x

B1054-034 and WWP Family 15045-862 x 18034-392 were

outstanding for SSAL5 in the 1989 trials, and B1054-004 x

B1054-034 was also well above the Wild OP mean for SSAL5

in 1994 (table 5). The Dorena BR checklot was well above the

Wild OP mean with 35.2 percent SSAL5 in WWP1994 (table

5). Only 3.9 percent of the seedlings in the low resistant

sugar pine checklot survived with SS in SP1989 (table 5).

Checklot Performance—More seedlings from Dorena

WWP full-sib HR checklots developed stem symptoms (SS

percent) in 1989 relative to 1994 (table 5), even though

survival of infected seedlings was fairly comparable between

the 2 years. In spite of a higher percentage of seedlings with

stem symptoms in 1989, those with normal cankers had

higher survival than those in 1994 (NCSURV5) (table 5).

Mean needle lesion class was higher for the Dorena WWP

full sib checklots in 1994 relative to 1989. On average, the

Dorena WWP full sib checklots had fewer needle lesions

than the Wild OP mean in 1989, while the reverse was true

in 1994 (table 5).

For WWP1994, the five Idaho full-sib families generally

had higher survival, bark reaction, and survival of seedlings with stem symptoms than the Wild OP families. The

Idaho families also had fewer seedlings with stem symptoms and fewer with early stem symptoms than the Wild

OP families. The Dorena BR checklot generally had higher

levels of resistance responses than the Idaho checklots, and

only one of the Idaho families had fewer early stem symptoms (table 5).

Of interest in the WWP1994 trial was whether or not the

Idaho full-sib checklots and Wild OP families exhibited a

differential reaction when inoculated with a pathotype of

rust known to be virulent to HR in WWP. Survival of the

infected seedlings from the Dorena and Idaho checklots in

WWP1994 was relatively consistent across the first four

blocks (in which the pathotype of rust virulent to HR in

WWP was excluded), with the Dorena checklots showing

higher survival (fig. 15). However, in block 5 and 6 where a

pathotype of rust virulent to HR in WWP was present,

survival of the four Dorena checkots was dramatically lower,

approaching zero in block 6. The Dorena checklot with

known bark reaction, however, had higher survival in blocks
224 USDA Forest Service Proceedings RMRS-P-32. 2004

Kegley and Sniezko Variation in Blister Rust Resistance Among 226 Pinus monticola and 217 P. lambertiana Seedling Families in the Pacific Northwest

Figure 15—Survival of infected seedlings of several sources

of western white pine, by block, in the 1994 trial, with and

without a pathotype of rust virulent to HR in western white

pine.

1234 5 6

% survival

0

10

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30

40

50

60

70

80

90

100

wild, open-pollinated families

Dorena HR full sib checklots

bark reaction check

Idaho full sib checklots 

virulent pathotype

absent

virulent pathotype

present

block

5 and 6. There was essentially little or no change in the

average survival of the Wild OP families or the Idaho

checklots (fig. 15).

SP Checklots—Survival of infected seedlings from the

Dorena full-sib checklots was relatively high (37 percent to

68 percent) in both 1989 and 1994 (table 5). The two full-sib

checklots common to 1989 and 1994 had similar levels of

RSURV5 and SS percent in both trials (table 5). A relatively

high proportion (20 percent) of seedlings in the full-sib

checklots showed no infection in 1989, and Family B1054-

004 x B1054-034 also had a high proportion of uninfected

seedlings in 1994 (noinfect, table 5). This family also had a

relatively high proportion of seedlings alive with stem symptoms five years after inoculation in both trials (table 5). Bark

reaction was higher for the full-sib checklots in 1989 relative

to 1994 (table 5, fig. 12). In 1989 mean NLC of the full-sib

checklots and the low resistance checklot were close to the

run mean of Wild OP families, whereas in 1994, the checklots

had fewer than average needle lesions (table 5). In both

years, all checklots were below the mean for presence of

needle lesions 1 year after inoculation (table 5, fig. 6).

Discussion _____________________

Artificial inoculation was successful in infecting more

than 95 percent of the 2-year-old seedlings in each trial. This

level of infection is probably similar to a high hazard field

site but reflects only a single inoculation event. It is unknown whether the use of inoculum in these tests from a

wide geographic area increased the infection and mortality

levels beyond that of using inoculum from a single geographic source.

SP1989 had 13 percent fewer trees with spots than the

other three trials. This is likely attributable to the much

lower and more variable inoculum density relative to SP1994

(5,200 spores/cm2 versus 7,200 spores/cm2, respectively,

table 2). It has been suggested that prevention of needle

infection (corresponds to ‘no infect’ in this paper) is a threshold trait, dependent upon inoculation intensity (Hoff and

McDonald 1980a). Results from experiments using different

inoculum densities of fusiform rust (C. quercuum f. sp.

fusiforme) on loblolly (P. taeda L.) and slash pine (P. elliottii

Engelmann) indicated that intermediate levels of resistance

were more distinguishable at lower inoculum densities (Laird

and others 1974). Even when the inoculum density is within

the targeted range for WWP or SP, the number of needle

lesions per seedling can vary remarkably by trial. Worth

noting is that the two SP HR checklots common to these

trials had slightly higher percentages of seedlings with stem

symptoms in SP1989 even though the mean for the Wild OP

families was nearly 8 percent less than in SP1994. Recent

data for SP1989 show stem symptoms on trees previously

stem symptom free, presumably from natural infections in

1995 and 1997. This had been noted in western white pine;

Hunt (1990) reported the presence of stem symptoms on 84

percent of unspotted seedlings in a 1987 inoculation trial.

Furthermore, many of those trees without spots had latent

canker development (Hunt 1990).

In general, survival provides the ultimate guideline of

utility of resistance for immediate use. Five years after

inoculation, the mortality in all four trials was high. Only a

few families examined here (including checklots) had moderate levels of survival. Both SP and WWP are very susceptible. However, results are dependent on the trial—families,

inoculum source, and environmental conditions—and the

traits examined. The low survival of progeny from phenotypic selection described here is similar to other reports;

Zsuffa (1981) observed very low survival of progeny of

resistant eastern white pine (P. strobus L.) selections in

artificial screening trials (2 percent, family means ranging

from 0 to 11.3 percent). Hoff (1984) reported that nearly 90

percent of the cankered western white pine seedlings are

dead by the fourth year after inoculation.

One of the major differences between the 1994 and 1989

tests was the lower final survival (total and/or rust-infected

survival) in the 1994 tests. The lower survival may have

been influenced by the 1994 nursery regime that resulted in

the presence of late season lammas growth on both species,

and/or a higher number of needle lesions on the trees. Rust

infection on lammas growth may circumvent putative resistance mechanisms that prevent stem infection (McDonald

and Hoff 1971).

While sugar pine had higher percentages of seedlings

without stem symptoms and higher survival relative to

western white pine in the 1989 trials, the reverse was true

in the 1994 trials. A previous summary of relative blister

rust resistance of five-needle pines ranked sugar pine as

slightly more susceptible than western white pine (Bingham

1972) as did a field study (Sniezko and others 2000).

The length of the evaluation period after inoculation can

have dramatic results on the interpretation of the level of

resistance in families (for example, SSAL3 versus SSAL5

for WWP1989 and SP1989). Much of the mortality at

Dorena occurs after the third year following inoculations.
USDA Forest Service Proceedings RMRS-P-32. 2004 225

Variation in Blister Rust Resistance Among 226 Pinus monticola and 217 P. lambertiana Seedling Families in the Pacific Northwest Kegley and Sniezko

The operational program in Idaho terminates formal assessments following the third year after inoculation (Franc

1988). The availability of longer-term data gives the Region

6 program the option to make selections using either the

third year or fifth year data.

Trees with normal cankers surviving 5 years after inoculation (a subgroup of SSAL5) are relatively rare for both

species. On the surviving trees, some of these cankers are

noted to be inactive during the final inspection. Further

tracking of the surviving but cankered trees over time would

be informative, as large trees with old basal cankers have

been observed in the field for both WWP and SP (Hoff 1984;

Sniezko and others, this proceedings; Dean Davis, personal

comm.) as well as eastern white pine (Hirt 1948).

Bark reaction was present in low frequency in many

families and moderate frequency in a few families. Many of

the individuals with bark reaction died by the end of the 5-

year test period. Survival of seedlings with bark reaction

ranged from 1.2 to 13.5 percent in the four trials, but some

families had more than 50 percent. Generally trees with

complete bark reactions would be expected to live (unless the

seedling was girdled or the seedling also had a normal

canker). All of the SP resistant checklots had bark reaction

levels higher than the Wild OP mean and fewer trees free of

stem symptoms than expected. Incompatible or aborted

bark reactions have been reported on SP seedlings with HR;

these symptoms were associated with infected primary

needles (Kinloch and Littlefield 1977; Kinloch and Comstock

1980). It is possible that some of the SP HR seedlings with

these atypical symptoms were classified as having stem

symptoms.

In examining the Wild OP families for a given trial, there

were a few families that approached the levels of survival,

bark reaction, or stem-symptom-free of the Dorena or Idaho

full-sib checklots. In the 1989 tests, several families had

survival levels as high or higher than some of the HR

checklots, but in the two 1994 tests, the survival of the Wild

OP families was generally much lower than the checklots. In

the 1994 tests, SSAL5 levels were very low in the Wild OP

families; not many infected trees survived 5 years after

inoculation. There may be opportunities for within-family

selection for families with a very low incidence for some of

the resistant responses.

The Dorena full-sib checklots used in these trials are

known to segregate for HR, and they provide linkages

between tests. The presence of a pathotype virulent to the

HR in WWP (vcr2) notably reduced the expected survival of

the Dorena full-sib checklots in WWP1989, as well as in the

two blocks of WWP1994 in which the Ribes sources with this

pathotype were used. In comparison the Idaho full-sib families, the bark reaction checklot, and many Wild OP families

in WWP1994 did not show increased levels of stem symptoms when challenged with the virulent pathotype.

Although there was wide variation in family mean needle

lesion class (NLC) within each of the four trials, most trees

developed stem infections. Individuals with reduced needle

lesion frequency (fewer spots) were hypothesized to have

fewer stem infections and higher survival (Hoff and McDonald

1980b). However, number of needle lesions in artificial

screening has been found to be a poor predictor of cankering

in the field (Hunt 2002). In a paired test of high and low

spotting individuals within a family planted in the field, the

low spotting individuals were as likely to develop stem

infections as their highly spotted siblings (Hunt 1990).

However, in our trials, there was a trend for the families

with higher mean NLC to have a higher percentage early

stem symptoms (unpublished data). This has been previously noted with WWP; seedlings with fewer spots generally

had fewer cankers 16 to 18 months post-inoculation (Hunt

2002; Meagher and Hunt 1996). Similarly, Hunt (1990)

observed that many spot-free WWP seedlings had latent

stem symptom development. The implication is that selection for fewer needle lesions may be an indirect selection for

delayed stem symptom development.

Several of the resistance responses evaluated here (bark

reactions, needle lesion class, latent development of stem

symptoms (low ESS3), and stem symptom alive) may be

forms of partial resistance. These types of resistances may

need several generations of breeding to increase utility for

field use. Control crosses among parents with partial resistance traits and testing of their progeny are underway in the

Region 6 program. Additionally, field validation of families

with complete or partial resistances is in progress (Sniezko

and others, this proceedings). Questions remain about

whether additional breeding would increase the survival of

trees with bark reactions and which of these resistance

responses may prove to be more effective in the field as the

trees get larger. Many of the families with relatively higher

levels of survival expressed more than one resistance response. It is unknown whether these resistances are under

the control of genes in tightly linked loci or whether they

represent a continuum of response, delaying the expression

of disease symptoms or reducing the severity of those

symptoms.

At least four mechanisms that lead to complete resistance

(lack of stem symptoms) in western white pine have been

previously described (Hoff and McDonald 1980a, Hoff and

McDonald 1971, McDonald and Hoff 1970, Kinloch and

others 1999). There is tentative evidence of a fifth one

(unpublished data). One mechanism for complete resistance

has been described in sugar pine (Kinloch and others 1970).

The hypersensitive reactions in SP and WWP are known to

be under the control of separate, single major genes, and

premature needle shed and fungicidal reaction in the short

shoot are hypothesized to be under the control of separate

single recessive genes. However, there may be more than

one resistance mechanism or gene underlying a particular

phenotypic expression. Differentiating similar phenotypes

controlled by different genes will be difficult without virulent strains specific to each, or without the aid of molecular

techniques.

The Region 6 program has evaluated blister rust resistance of progeny of thousands of sugar pine and western

white pine selections. Results from the trials examined here

indicate that progeny of most of these parents are very

susceptible. Given the high susceptibility of very young

seedlings, natural regeneration may be very unlikely on

many sites, exacerbating the decline of these species. Stabilizing and reversing the decline of WWP and SP will be

dependent on the development and deployment of resistant

material coupled with use of silvicultural tools such as

appropriate site selection and pruning. Selections of progeny showing one or more resistance responses have been

made and grafts have been established in breeding orchards
226 USDA Forest Service Proceedings RMRS-P-32. 2004

Kegley and Sniezko Variation in Blister Rust Resistance Among 226 Pinus monticola and 217 P. lambertiana Seedling Families in the Pacific Northwest

and seed orchards. Resistant seed is available for some

breeding zones for both western white pine and sugar pine.

Advance-generation breeding has started for WWP. Major

gene resistance in WWP and SP are likely to play an

important role for immediate restoration and reforestation

efforts, but partial resistance traits will likely be more

important in the future development of durable resistance.

Acknowledgments ______________

The Region 6 Area Geneticists and the many tree improvement workers who made all the phenotypic selections and

organized the cone collections. Bob Danchok, the late Delbert

Albin, and all members of the Dorena rust screening team

for the extensive assessments. Jude Danielson for help in

organizing the operational elements of the program. The

USFS Region 6 Genetics and Forest Health programs for

funding and logistical support, and the Bureau of Land

Management and Quinault Indian Nation provided seed

from their field selections. Joan Dunlap and Scott Kolpak for

their reviews of an earlier draft of this paper.

References _____________________

Bingham, R.T. 1972. Taxonomy, crossability, and relative blister

rust resistance of 5-needled white pines, p. 271-278. In: R.T.

Bingham, R.J. Hoff, and G.I. McDonald (eds.) Biology of Rust

Resistance in Forest Trees: Proceedings of a NATO-IUFRO Advanced Study Institute. USDA Forest Service Misc. Publ. 1221.

Bingham, R.T. 1983. Blister rust resistant western white pine for

the Inland Empire: the story of the first 25 years of the research

and development program. USDA Forest Service General Technical Report INT-146. 45 p.

Campbell, R.K. and Sugano, A.I. 1987. Seed zones and breeding

zones for sugar pine in southwestern Oregon. USDA Forest

Service Research Paper PNW-RP-383. 18 p.

Campbell, R.K. and Sugano, A.I. 1989. Seed zones and breeding

zones for white pine in the Cascade Range of Washington and

Oregon. USDA Forest Service Research Paper PNW-RP-407.

Cochran, W.G. and Cox, G.M. 1967. Experimental designs, 2nd

edition. John Wiley and Sons, Inc. New York. 611 p.

Franc, G.C. 1988. The white pine program in the northern region, p.

21-26. In: R.S. Hunt, ed. Proc. of a western white pine management symposium. Nakusp, BC. Pacific Forestry Centre, Victoria,

BC.

Hirt, R.R. 1948. Evidence of resistance to blister rust by eastern

white pine growing in the northeast. J. For. 46:911-913.

Hoff, R.J. 1984. Resistance to Cronartium ribicolain Pinus monticola:

Higher Survival of Infected Trees. USDA Forest Service Research Note INT-343, 6p.

Hoff, R.J. and McDonald, G.I. 1980a. Improving rust-resistant

strains of inland western white pine. USDA Forest Service

Research Paper INT-245. 13 p.

Hoff, R.J. and McDonald, G.I. 1980b. Resistance to Cronartium

ribicola in Pinus monticola: reduced needle-spot frequency.

Can. J. Bot. 58:574-577.

Hoff, R.J. and McDonald, G.I. 1971. Resistance to Cronartium

ribicola in Pinus monticola: short shoot fungicidal reaction. Can.

J. Bot. 49:1235-1239.

Hunt, R.S. 1990. Blister rust in inoculated and plantation-tested

western white pine in British Columbia. Can. J. For. Res.

12:279-282.

Hunt, R.S. 1997. Relative value of slow-canker growth and bark

reactions as resistance responses to white pine blister rust.

Can. J. Plant Pathol. 19:352-357.

Hunt, R.S. 2002. Relationship between early family-selection traits

and natural blister rust cankering in western white pine. Can. J.

Plant Path. 24:200-204.

Kim, H.J., Yi, C.K., and La, Y.J. 1982. Present status of Korean pine

(Pinus koraiensis) blister rust and relative resistance of three

species of five-needled pines to Cronartium ribicola. Research

Report of Forest Research Institute, Seoul, Korea. 29:239-252.

Kinloch, B.B., Jr. and Comstock, M. 1980. Cotyledon test for major

gene resistance to white pine blister rust in sugar pine. Can. J.

Bot. 58: 1912-1914.

Kinloch, B.B. and Davis, D. 1996. Mechanisms and inheritance of

resistance to blister rust in sugar pine, p. 125-132. In: B.B.

Kinloch, M. Marosy, and M.E. Huddleston (eds.). Sugar pine:

status, values, and roles in ecosystems: Proceedings of a symposium presented by the California Sugar Pine Management Committee. Univ. Calif. Div. Agr. Res. Publ. 3362.

Kinloch, B.B., Jr. and Dupper, G.E. 2002. Genetic specificity in the

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Kinloch, B.B., Sniezko, R.A., Barnes, G.D., and Greathouse, T. E.

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western white pine from the western Cascade Range. Phytopathology 89:861-867.

Laird, P.P., Knighten, J.L., and Wolfe, R.L. 1974. Reduced inoculum

density enhances sensitivity of resistance detection in fusiform

rust resistance testing. USDA Forest Service Southeastern Area,

State and Private Forestry, Division of Forest Pest Management

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(Suppl.): 101-112.

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of sugar and western white pines. USDA Forest Service Region 6

Tree Improvement paper 13. 24 p.

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181-183.
USDA Forest Service Proceedings RMRS-P-32. 2004 227

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

The authors are with the Natural Resources Canada, Canadian Forest

Service, Pacific Forestry Centre.

Abstract—To demonstrate the existence of the dominant Cr2

blister rust resistance gene in Dorena derived stock already producing seed in British Columbia, about 50 seedlings/parent tree were

inoculated and examined for hypersensitive needle spots. Seedlings

from 33 of 42 canker-free parents produced hypersensitive spots,

confirming the presence of Cr2 in the parent trees. To determine if

an existing pathotype might have already overcome the Cr2 gene in

British Columbia, seedlings from three suspect trees (because they

were canker-free for 10 years, but recently became cankered) were

inoculated. The Cr2 gene was absent in these three trees. Additionally, seedlings from two seedlots known to possess the Cr2 gene were

subjected to two consecutive annual inoculations. This double inoculation was repeated on five different occasions with a composite

inoculum. Canker-free seedlings following the first inoculation

remained canker-free after a second inoculation, consistent with the

uncompromised expression of Cr2.

Key words: Cr2, blister rust, resistance genes

Introduction ____________________

As early as the 1960s a single dominant gene for resistance

to white pine blister rust (Cronartium ribicola J.C. Fisch.)

was suspected in western white pine (Pinus monticola D.

Don) from the Champion mine region of Oregon. Cankerfree trees from this source were incorporated into the USDA

Forest Service seed orchard at the Dorena Genetic Resource

Center, near Cottage Grover, Oregon. By 1984 this resistance had failed (McDonald and others), which was later

attributed to the failure of a single dominant gene, called

Cr2 (Kinloch and others). We obtained two seed collections

from the Dorena seed orchard - one bulked collection of 32

Champion Mine-derived parents (Champion), and one

bulked from six Washington State parents (WA). We out

planted the resulting seedlings in plantations across southConfirmation of Dominant Gene Resistance

(Cr2) in U.S. White Pine Selections to White

Pine Blister Rust Growing in British

Columbia

R.S. Hunt

G.D. Jensen

A.K. M. Ekramoddoullah

ern British Columbia (BC) in 1986 and 1987 (Hunt 1987).

Unaware that these lots could possess the dominant Cr2

gene, we reported that these two lots had greater resistance

than other lots in two coastal and two interior plantations

(Hunt 1994). Since then, we have tried to (a) confirm that

these healthy trees possess the Cr2 gene; (b) determine if a

virulent pathotype might exist in BC which overcomes the

Cr2 gene; and (c) determine whether the resistant phenotype would be maintained under repeated inoculation.

Materials and Methods ___________

The seven plantations with the Champion and WA seedlots

have been monitored for rust incidence annually, or biennially (Hunt 1994). Some of the surviving trees from these two

Dorena seedlots have produced seed, particularly in the two

most rusted coastal plantations. We collected seed from 42

of these parents and three trees that were canker-free for 10

years but then developed rust. In 2000 50 seeds/parent were

stratified and 1-0 seedlings bearing only primary needles

were inoculated in a ribes (disease) garden with a composite

of six BC rust sources initially collected from throughout the

range of western white pine on Vancouver Island. The

seedlings were examined for hypersensitive spots, the manifestation of the Cr2 gene (Kinloch and others 1999), in

February 2001.

To determine if repeated inoculation would nullify the

dominant resistance, or whether it would be maintained,

two seedlots known to possess the Cr2 gene respectively

were obtained from Dorena (Kinloch and others 1999; registration # 119-15045-845 X OP (our seedlot #1) and #119-

15045-845X15045-841 X OP (our seedlot #2)), sown and

grown in 1994, 1996, and 1997. Some were first inoculated

at age 4-months (only primary needles present) others at 16-

months (secondary needles present) in the ribes (disease)

garden. Seedling survival was attributed to the Cr2 gene. All

survivors were re-inoculated the following year. From the

1994 inoculation, 40 grafted ramets were produced from

seven seedlings, and these were inoculated.

Results ________________________

The most severely damaged plantation had 49 percent of

the Dorena derived trees canker-free compared to only 0.2

percent for the other provenances after 15 years (fig. 1);

comparable numbers for the other plantation were 66 and 13
228 USDA Forest Service Proceedings RMRS-P-32. 2004

Hunt, Jensen, and Ekramoddoullah Confirmation of Dominant Gene Resistance (Cr2) in U.S. White Pine Selections to White Pine Blister Rust ...

percent. Seventeen of 18 canker-free plantation trees from

the Champion seedlot produced seedlings with hypersensitive spots (fig. 2) typical for Cr2 (family mean percent Cr2 49;

range 21 to 100). None of the seedlings produced by the

parent lacking Cr2 possessed Cr2. From the WA seedlot 16

of 24 plantation trees also produced hypersensitive spots

(family mean percent Cr2 41; range 21 to 60). The seven WA

plantation trees lacking Cr2 processed few seedlings with

hypersensitive spots (family mean percent 1; range 0 to 5).

The percentage of Cr2 in the offspring of the three recently

cankered parents was 0, 0, and 5.

Many of the seedlings from the two registered Dorena

seedlots possessing the Cr2 gene died from fusarium root rot

making survival rates variable. Nevertheless, the over-all

mean survival was greater than 50 percent for each seedlot.

None of the canker-free survivors from the first inoculation

developed rust cankers from a second inoculation (table 1).

In addition, none of the 40 ramets became infected, but in six

the rootstock became infected. In these, the rust failed to

cross the graft union over 4 years (fig. 3).

Row 1 Row 2 Row 3 Row 4 Row 5 Row 6 Row 7 Row 8 Row 9 Row 10 Row 11 Row 12 Row 13 Row 14

Group Seedling

12 4 Dor

3 Dor

2 Dor

1 Dor

11 4

3

2

1

10 4 WA WA

3 WA WA

2 WA WA

1 WA WA

9 4 Dor WA

3 Dor WA

2 Dor WA

1 Dor WA

8 4 WA Dor WA WA

3 WA Dor WA WA

2 WA Dor WA WA

1 WA Dor WA WA

7 4 Dor

3 Dor

2 Dor

1 Dor

6 4

3

2

1

5 4 Dor WA Dor

3 Dor WA Dor

2 Dor WA Dor

1 Dor WA Dor

4 4

3

2

1

3 4 Dor Dor

3 Dor Dor

2 Dor Dor

1 Dor Dor

2 4 WA WA Dor

3 WA WA Dor

2 WA WA Dor

1 WA WA Dor

1 4 Dor

3 Dor

2 Dor

1 Dor

Robert's Creek Plantation

Figure 1—A plantation depicting

14 provenances of western white

planted in four-tree row plots, with

each provenance replicated 10

times. White denotes canker-free

pine, black cankered pine, grey

other conifers and missing trees.

Trees labelled Dor = a bulked

Dorena seed orchard collection,

and WA = a collection from Washington State trees growing in the

Dorena seed orchard.

Figure 2—Blister rust infection spots in pine needles. The top

two needles show normal spotting; the bottom two have

necrotic halos typical of hypersensitive spots in western white

pine as a result of the Cr2 gene.
USDA Forest Service Proceedings RMRS-P-32. 2004 229

Confirmation of Dominant Gene Resistance (Cr2) in U.S. White Pine Selections to White Pine Blister Rust ... Hunt, Jensen, and Ekramoddoullah

Table 1—Percentage Cr2 gene in two western white pine families

based on survival after an initial inoculation with Cronartium

ribicola and canker-free survivors after a second

inoculation. No survivors from the first inoculation became

cankered from a second inoculation, but numbers were

reduced by fusarium root rot.

Seedlot Seedlings Inoculation Canker-free Cr2

(no.) (year) (no.) (%)

1 44a 1995&6 10 29

1 24a 1996&7 12 54

1 20b 1997&8 10 50

1 446a 1997&8 214 55

1 498b 1998&9 324 74

2 76a 1995&6 28 74

2 40a 1996&7 28 77

2 37b 1997&8 22 59

2 336a 1997&8 182 73

2 448b 1998&9 305 80

a

 First inoculated at 4 months.

b

 First inoculated at 16 months.

Figure 3—Failure of Cronartium ribicola to infect a western white

pine scion possessing the Cr2 gene from an infected root stock.

Discussion _____________________

No apparent failure of the dominant resistance gene has

been observed in seven BC plantations over 15 years. Inoculation of seedlings from 33 healthy parents derived from

these plantations produced hypersensitive spots, confirming they possessed the Cr2 gene. Additionally, inoculation of

seedlings possessing the Cr2 gene with a composite inoculum resulted in high survivor ratios as would be expected

from a heterozygous parent pollinated with orchard pollen.

Re-inoculated survivors remained canker-free, confirming

the stability of the Cr2 gene. Even scions possessing the Cr2

gene failed to become infected from C. ribicola infected rootstock after 4 years. In contrast, seedlings from three recently

cankered parents derived from the same Dorena stocks

produced normal spots and thus lack the Cr2 gene. We

conclude that we have failed to demonstrate the existence of

a virulent pathotype of Cronartium ribicola to the Cr2 gene

in BC and so far the Cr2 gene appears stable in BC even

when nonselected stocks are devastated (fig. 1).

Most blister rust cankers in BC occur within 3 m of the

ground (Hunt 1991); that is, when the plantations are young

(usually less than 15 years old). It appears that the Cr2 gene

could be deployed successfully in isolated areas in BC, and

if the resistance failed, the trees likely will have out-grown

their most susceptible age before a virulent pathotype could

have an opportunity to build-up damaging levels of inoculum. However, if a series of age classes were planted in

contiguous areas, a new virulent pathotype could be limiting

to the younger plantations.

Acknowledgments ______________

We thank Drs. Ray Steinhoff and Richard Sniezko for

supplying seedlots from the Dorena Genetic Resource Center.

References _____________________

Hunt, R.S. 1987. Is there a biological risk of western white pine

provenances to root diseases? In G.A. DeNitto (complier) West.

Int. For. Dis. Wk. Conf. 35: 29-34. USDA For. Serv., San Franciso

CA.

Hunt, R.S. 1991. Operational control of white pine blister rust by

removal of lower branches. For. Chron. 67: 284-287.

Hunt, R.S. 1994. Transferability of western white pine within and

to British Coumbia – blister rust resistance. Can. J. Plant Pathol.

16:273-278.

Kinloch, B.B., R.A. Sniezko, G.D. Barnes, and T.E. Greathouse.

1999. A major gene for resistance to white pine blister rust in

western white pine from the western Cascade Range. Phytopathology 89: 861-867.

McDonald, G.I., E.M. Hansen, C.A. Osterhaus, and S. Samman.

1984. Initial characterization of a new strain of Cronartium

ribicolafrom the Cascade Mountains of Oregon. Plant Disease 68:

800-804.
230 USDA Forest Service Proceedings RMRS-P-32. 2004

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

The authors are Forest Geneticists, Forest Research Institute, Sos.

Stefanesti 128, Post Office 11, Bucharest, Romania. E-mail:

ioan_blada@yahoo.com and flaviu popescu@toptech.ro, respectively.

Abstract—This paper reports information about genetic variation

in blister rust resistance (BRR), survival (TS) and total height

growth (H) over 20 years in a Pinus strobus x P. peuce hybrid

population. Highly significant (p<0.001) differences among hybrid

families were found for the three traits. With minor exceptions,

female and male x female effects were significant (p<0.05) or highly

significant (p<0.01; p<0.001) over the whole testing period for BRR,

TS and H. However, male effects were significant (p<0.05) only for

H. This suggested that the traits were controlled by additive and

non-additive genes. Over 20 years, the additive variances ranged

between 26 and 39 percent for BRR, between 17 and 62 percent for

TS and between 31 and 44 percent for H. Non-additive variances

ranged between 37 and 60 percent for BRR, between 34 and 78

percent for TS and between 31 and 54 percent for H. Therefore, both

variances were important for the traits involved. Narrow-sense

heritability estimates at the family level ranged between 0.290 and

0.430 for BRR, between 0.177 and 0.633 for TS and between 0.382

and 0.531 for H. Heritabilities at the individual level ranged

between 0.024 and 0.067 for BRR and between 0.064 and 0.167 for

H. Parents of good general combining ability were found for both

BRR and H. Strong age-age genetic correlations were found between

BRR and TS while correlations between BRR and TS on one hand

and H on the other were low. The high-parent heterosis was

negative while the mid-parent one was positive, accounting for 21.3

percent for BRR, 35.4 percent for TS and 10.4 percent for H. A

variable genetic gain for the three traits could be expected, suggesting that hybrid planting could be profitable.

Key words: Pinus strobus, P. peuce, factorial cross, hybrid,

additive variance, heritability, combining ability,

genetic correlation

Age Trends in Genetic Parameters of Blister

Rust Resistance and Height Growth in a

Pinus strobus x P. peuce F1

 hybrid

population

Ioan Blada

Flaviu Popescu

Introduction ____________________

It is well known that eastern white pine (Pinus strobus L.)

has wide genetic variability and high productivity in plantations in North America, Europe and the Far East (Kriebel

1983).

The blister rust (Cronartium ribicolaJ.C. Fisch. in Rabenh.)

migrated from its Siberian gene center to Europe (Leppik

1967) and by 1900 to North America where it caused losses

in P. strobus, P. monticola Dougl. and P. lambertiana Dougl.

(Bingham and Gremmen 1971). The inefficiency of conventional control methods of the pathogen has stimulated interest in genetic improvement of resistance. The eastern white

pine improvement program was based on both intraspecific

crosses (Riker and others 1943; Heimburger 1972a; Zsuffa

1981) and interspecific ones (Patton 1966; Heimburger

1972; Zsuffa 1979).

Interspecies breeding with white pines was tried in attempts to introduce resistance factors into eastern white

pine from related more resistant species, like Balkan pine

(Pinus peuceGriseb.) and Himalayan pine (Pinus wallichiana

Jacks.). According to Heimburger (1972a), introduction of

resistance genes in eastern white pine may be the only way

to form a realistic program with this species. In a previous

investigation, an F1hybrid population from reciprocal crosses

between eastern white pine and Balkan pine supported

evidence that extranuclear genes controlling blister-rust

resistance and growth traits could be found in eastern white

pine (Blada 1992). In addition, more recent investigations

(Blada 2000a) suggested that parents with a good general

combining ability were found in eastern white pine, not only

for growth traits but also for blister-rust resistance.

The P. strobus x P. peuce F1 hybrids have demonstrated

good blister rust resistance (Patton 1966; Heimburger 1972a;

Blada 1989; 2000a) and growth traits (Leandru 1982;

Blada 2000a; 2000b).

Introduction to Romania of eastern white pine took place

at the end of the 19th century, but it was intensified after

1960 when about 300,000 ha were planted with this species;

with its 20.2 m3/year/ha it proved to be a fast growing species

(Radu 1974). At the same time, blackcurrant (Ribes nigrum

L.), one of the alternate hosts of the blister rust pathogen,

was planted on large scale to produce fruits. Very soon,

coincident with blackcurrant spreading, heavy attacks of

blister rust occurred all over the country (Blada 1987; 1990).

Because the blister rust cannot be controlled by conventional

methods (Bingham and others 1953), a genetic improvement
USDA Forest Service Proceedings RMRS-P-32. 2004 231

Age Trends in Genetic Parameters of Blister Rust Resistance and Height Growth in a Pinus strobus x P. peuce F1 hybrid population Blada and Popescu

program was launched in Romania in 1977 (Blada 1990).

The program included both intra- and inter-specific crossing. The practical objective of the program was to establish

hybrid seed orchards consisting of parents with high general

combining ability for blister rust resistance (Blada 1982).

Because of financial reasons, this objective has been only

partially fulfilled. Several progress reports on this subject

were previously published or presented in symposia (Blada

1987; 1989; 1990; 1992; 1994; 2000a; 2000b).

The objective of this paper is to present the age trends in

genetic parameters for blister rust resistance, tree survival and

total height growth over 20 years of testing in a P. strobus x

P. peuce F1 hybrid population.

Materials and Methods ___________

Parents, Mating Design, and Progenies

The initial material consisted of five eastern white pine

female and five Balkan pine male parents selected in nonimproved planted populations free of blister rust and of

unknown origin, The selections could be considered random

samples with respect to any trait, except reproductive fertility. In 1979, a 5 x 5 factorial mating design (Comstock and

Robinson, 1952) was completed. The seed samples were

stratified according to Kriebel’s (1973) recommendations

and then sown (spring 1981) in individual polyethylene pots

(22x18 cm) in a potting mixture consisting of 70 percent

spruce humus and 30 percent sand. The progenies grew in

pots throughout the first six years and they were the subjects

of the nursery test reported earlier (Blada 1987).

Inoculation and Experimental Design

The 25 full-sib hybrid families and two open pollinated

progenies were artificially inoculated three times with blister rust, in late August 1982, 1983 and 1984, when they were

two, three and four years old, respectively. During each

inoculation period, the potted trees were introduced into a

polyethylene tent and arranged in randomized complete

block design with 14-seedling row-plots in each of the three

blocks. The two open pollinated progenies, representing the

means of the two parent species, were included as controls.

Inoculum consisted of heavily infected leaves of Ribes nigrum

L. collected from a single plantation. Other details concerning inoculation and inoculation tents were similar to those

described by Bingham (1972).

At age six, the hybrids and controls were planted out at

3x3 m spacing, in the Caransebes-Valisor Forest District, at

about 45∞27’ N latitude and 22∞07’ E longitude and 310 m

altitude. As expected, owing to the heavy controlled artificial

inoculation, a variable number of seedlings per family were

killed during the nursery stage, so that only about 10

seedlings per plot could be used for planting in the field test.

Hence, a randomized complete block design with 10-seedling row-plots in each of the three blocks was used. No

thinning was carried out by age 20.

Measurements

Several traits were assessed when the hybrids were 5-, 9-,

11-, 13-, 17- and 20-years-old. However, only three traits for

each age were presented in this report. (table 1). The blister

rust resistance was scored using an index that took into

account both the number and severity of the lesions. Its

numerical values assigned were: 1 = dead tree or total

susceptibility (all trees killed by rust in previous years were

included in this cumulative category); 2 = four or more

serious stem lesions; 3 = three severe stem lesions; 4 = three

more or less severe stem lesions; 5 = two severe stem lesions;

6 = two more or less severe stem lesions; 7 = one severe stem

lesion; 8 = one more or less severe stem lesion; 9 = branch or

very light stem lesions; 10 = free of lesions or total resistance.

Percentages of the trees surviving were calculated based on

blister rust resistance index data, i.e. all trees with a score

2 to 10 were considered tree survivors. All percentages were

transformed to the arc sin square root for analysis.

Plot-mean data were subject to randomized block and

factorial analysis of variance (Hallauer and Miranda 1981)

and within-plot variance was calculated by a separate analysis (Becker 1984) where only six (out of 10) planted trees per

plot were taken into account. It should be stressed that a few

families had only six surviving trees / block; this is the

reason why only six trees per block, taken at random, were

included in the analysis.

Statistical Analyses

In order to estimate the genetic components of variance

the following statistical model, applied to plot means, was

assumed:

xijkh = m + Mi + Fj + (MF)ij + Bk + eijkh (1)

where: xijkh = the observation of the h-th full-sib family from

the cross of the i-th male and j-th female in the k-th block;

m = general mean; Mi = the effect of the i-th male (I =

1,2,...I); Fj = the effect of the j-th female (j= 1,2,...J); (MF)ij =

the effect of the interaction of the i-th male and j-th female;

Bk = the effect of the k-th block (k = 1,2,...K) and eijkh = the

random error.

Table 1—Traits measured for 25 full-sib hybrid families and two open pollinated families.

Traits Measured at ages.... Units Symbols

Blister-rust resistance 5, 9, 11, 13, 17, 20 Scale 1...10 BRR. 5 ... BRR. 20

Survival 5, 9, 11, 13, 17, 20 % S. 5 ... S. 20

Total height growth 5, 9, 11, 13, 17, 20 dm H. 5 ... H. 20
232 USDA Forest Service Proceedings RMRS-P-32. 2004

Blada and Popescu Age Trends in Genetic Parameters of Blister Rust Resistance and Height Growth in a Pinus strobus x P. peuce F1 hybrid population

Since the parents were assumed to be random samples

from a random mating population, and the hybrid families

were planted in a complete randomized block design, a

random model for statistical analysis (table 2) was used

(Comstock and Robinson 1952).

Standard errors (SE) of variance components were computed by the formula given by Anderson and Bancroft

(1952).

To estimate effectiveness of selection for early traits, three

types of heritabilities were calculated.

The first heritability is the one commonly used for estimating the ratio of genetic (additive and non-additive) to

total phenotytic variance which is appropriate for estimating gain from selection among hybrid families when they are

vegetatively propagated. This is broad-sense heritability

(h2

1) and was estimated by Grafius and Wiebe’s (1959)

formula:

h2

1 = s2G /s2Ph.1 = (s2M + s2F + s2MF) /

(s2

M + s2F + s2MF + s2e / k) (2)

where k = number of blocks.

Mass selection genetic gain (Falconer 1981) was estimated by:

DG1 = i1 h2

1 sPh.1 (3)

where: i1= the selection intensity taken from Becker (1984);

sPh.1 is the phenotypic standard deviation of the family

mean.

The second heritability is appropriate for estimating gain

from selection among half-sib hybrid families when they are

sexually propagated. This is narrow-sense heritability (h2

2)

at the family level and was estimated by Grafius and Wiebe’s

(1959) formula:

h2

2 = s2A / s2Ph.1 = (s2M + s2F) /

(s2

M + s2F + s2MF + s2e / k) (4)

and gain from half-sib family selection was estimated by:

DG2= i1 h2

2 sPh.1 (5)

The third heritability is the one commonly used for estimating genetic gain from mass selection among randomly

placed seedlings. This is individual tree narrow-sense heritability (h2

3) and was estimated by:

h2

3 = s2A / s2Ph.2 = (s2M + s2F) /

(s2

M + s2F + s2MF + s2p + s2W) (6)

where: s2

W = within plot variance; s2p = plot error = s2e –

s2

W/n; n = seedlings per plot.

The mass selection gain was estimated by:

DG3= i1 h2

3 sPh.2 (7)

where s2

Ph2 is the phenotypic standard deviation and it

refers to individual tree values.

If the best general combining ability parents (gca) are to

be selected and intermated, then the genetic gain was

calculated as twice the average of gca’s or the average of the

breeding values of the three selected parents for the next

breeding works (table 10).

The heterosis was calculated according to Hallauer and

Miranda’s (1981) formula:

He1 = [(Hy - HP) / HP] ·100 (8)

He2 = [(Hy - MP) / MP] ·100 (9)

where: Hy, HP and MP are the hybrid mean, the highparent mean and the mid-parent mean, respectively. As

shown above, two estimates of heterosis were computed: one

that compared to the best parent (He1) and the other that

compared to the mean of the parents from open pollinated

controls (He2). According to the broad, modern concept, there

exists positive or negative heterosis, luxuriant, adaptive,

selective or reproductive heterosis and labile or fixed heterosis (Mac Key 1976). Only positive and negative heterosis was

estimated in this experiment.

Genetic coefficient of variation (GCV) was calculated by

formula:

CGV =( ̃ s2

G / X) 100 (10)

where: s2

G and X are the genetic variance and trait mean,

respectively.

General combining ability (gca) effects of each parental

tree were calculated, using Griffing’s (1956) method 4,

adapted to a factorial mating design. The statistical model

was:

Xij. = X... + gi + gj + sij + eijk (11)

where: Xij. is the mean of the i-th female tree crossed to the

j-th male tree over k replications; X... is the general mean;

gi is the gcai effect associated with the i-th female tree; g.j is

the gcaj effect associated with j-th male tree; sij is the sca

effect associated with the cross between the i-th female tree

and j-th male tree; eijk is the residual effect.

The computational formulae were as follows:

gcai = xi. - X... (12)

gcaj = x.j - X... (13)

Table 2—Analysis of variance of factorial meting design random effects model, in

a random complete block in one environment.

Source of variation Df MS E (MS)

Total KIJ-1

Blocks (B) K-1 MSB

Hybrids (Hy) IJ-1 MSHy

 -Females (F) J-1 MSF s2

W + s2p + Ks2MF + KIs2F

 -Males (M) I-1 MSM s2

W + s2p + Ks2MF + KJs2M

 -M x F (J-1)(I-1) MSMF s2

W + s2p + Ks2MF

Pooled error (IJ-1)(K-1) MSE s2

W + s2p

Within plot KIJ(n-1) MSW s2

W
USDA Forest Service Proceedings RMRS-P-32. 2004 233

Age Trends in Genetic Parameters of Blister Rust Resistance and Height Growth in a Pinus strobus x P. peuce F1 hybrid population Blada and Popescu

where: xi. = the mean of the F1 resulting from crossing the ith female tree with each of the male parent; x.j = the mean

of the F1 resulting from crossing the j-th male parent with

each of the female tree;

To examine relationships among traits, genetic correlations coefficients were estimated using Falconer’s (1981)

formula.

Results and Discussions _________

Blister Rust Attack Evolution

Table 3 summarizes survival at ages 6 and 20. At the end

of the nursery test, survival in the F1 hybrid population was

74.3 percent, while in open pollinated control populations

of P. strobus and P. peuce,the survival was 22.7 percent and

98.2 percent, respectively (table 3, column 3). The field test

showed that, at age 20, survival in hybrids, P. strobus and

P. peuce was 70.7 percent, 15.5 percent and 88.9 percent,

respectively (table 3, column 5). Blister rust susceptibility

was high in eastern white pine, very low in Balkan pine and

low in hybrids. The mortality was due mostly to blister rust

and only a few to other causes (see table 3, columns 7 and 8).

It should be emphasized that the field test had been laid out

in a site free of any Ribes species and blister rust, as well.

Consequently, no new infections took place, so that the trees

were killed only due to the infections that resulted from the

control inoculation. Also, it was noticed that during the

nursery test, both stem and branch cankers were evident,

but later only stem basal cankers occurred. This suggests

the absence of the local secondary infections.

Concerning the tree growth, the following phenomenon

was observed:

• when the canker was marginal to the stem, the infected

tree grew normally up to about 12-15 years of age;

• when the canker spread and reached the half stem

diameter, the growth was slow and the tree was dead

after a few years;

• when the canker had spread over half the stem diameter, the tree was dead in the same or in the next season

of vegetation.

Genetic Variation

The analysis of variance indicated highly significant (p <

0.001) differences among hybrid family means for blister

rust resistance, survival and total height growth at all ages

(table 4, row 2). At age 20, blister rust resistance varied from

6.3 to 9.1, survival between 49.9 percent and 91.3 percent

and total height from 88.3 dm to 110.0 dm. Hence, selection

at the family level within the hybrid population could be

carried out for the three economically important traits.

There was a large genetic variation among parents within

each sex (species) for the three traits examined over years.

An important finding of this experiment was that the effects

of eastern white pine female parents were significant (p <

0.05) for blister rust resistance and highly significant (p <

0.01; p < 0.001) for survival at most ages. Significant differences among eastern white pine female parents for total

height growth were found by the end of the testing period, i.

e. at ages 17 and 20 (table 4, row 3).

Balkan pine as male parents had significant (p < 0.05)

effects on height growth through age 17 but had no significant effects on blister rust resistance and survival at any age

(table 4, row 4). The results suggested that: (i) an additive

genetic control in these three traits occurred; (ii) high gca

parents could be selected within the eastern white pine

parental population. Similar results were found in other

experiments with P. strobus xP. peuce hybrids (Blada 1989).

Male x female interaction effects were highly significant

(p < 0.01; p < 0.001) for the three traits at all ages (table 4,

row 5), suggesting non-additive gene action.

The genetic coefficient of variation at the family level

(table 5) was, in general, moderate ranging between 8.0 and

12.3 percent for blister rust resistance, between 7.6 and 18.3

percent for survival and between 5.0 and 10.8 percent for

total height growth.

Variance Components

Variance component estimates, standard errors and dominance ratios were listed in table 5.

The contribution of the GCA variance to the total phenotypic variance ranged over years from 26 to 39 percent for

blister rust resistance, 17 to 62 percent for survival and 31

to 44 percent for total height growth. The contribution of

SCA variance for the same traits ranged from 37 to 60

percent, 34 to 78 percent and 31 to 54 percent, respectively.

The ratio of dominance to additive variance for blister rust

resistance was greater than 1 at all ages indicating that

dominance variance was of higher importance. However, the

additive variance was only slightly lesser than dominance

variance, suggesting that both variances could be used in a

Table 3—Tree survival and evolution of blister rust attack.

Nursery test at age 6 Field test at age 20

Inoculated Planted Total killed Killed trees Killed by

trees at age 2 Survival trees Survival trees by rust other causes

Genotype No. No. (%) No. (%) No.(%)** No. (%)** No.(%) *** No.(%)***

1 2345678

P. strobus 150 34 (22.7) 34 23 (15.5) 127 (84.5) 124 (97.6) 3 (2.4)

Hybrids 1050 780 (74.3) 780* 742 (70.7) 308 (29.3) 299 (97.1) 9 (2.9)

P. peuce 109 107 (98.2) 107 97 (88.9) 12 (11.1) 4 (33.3) 8 (66.7)

* Some families had more and some other less than 10 trees / block; ** calculated as against inoculated trees (column 2).

*** Calculated as against total killed trees (column 6).
234 USDA Forest Service Proceedings RMRS-P-32. 2004

Blada and Popescu Age Trends in Genetic Parameters of Blister Rust Resistance and Height Growth in a Pinus strobus x P. peuce F1 hybrid population

Table 4—Mean squares and F-tests for the P. strobus x P. peuce F1 hybrid factorial analyses.

Source of Traitsa

variation DF BRR.5 BRR. 9 BRR. 11 BRR. 13 BRR. 17 BRR. 20 S. 5 S. 9 S. 11

Replications 2 0.054 0.035 0.036 0.054 0.171 0.146 2.675 1.418 2.13

Hybrids 24 2.520*** 1.503*** 2.775*** 2.520*** 2.537*** 2.590*** 112.328*** 295.800*** 332.71***

 -Females (F) (4) 5.527* 2.692 6.702* 5.527* 7.261* 7.512* 184.804 904.224** 1259.58***

 -Males (M) (4) 2.398 2.248 1.121 2.398 1.526 1.671 108.036 229.358 191.20

 -M x F (16) 1.799*** 1.020** 2.207*** 1.799*** 1.609*** 1.589*** 95.282*** 160.304*** 136.37***

Error 48 0.142 0.246 0.154 0.142 0.130 0.120 1.953 2.241 5.80

Within plot b 540 3.480 6.407 8.348 8.684 14.980 14.950 - - -

Source of Traits

variation DF S. 13 S. 17 S. 20 H. 5 H. 9 H. 11 H. 13 H. 17 H. 20

Replications 2 7.842 3.03 3.26 0.075 0.242 0.314 0.792 5.796 3.647

Hybrids 24 287.044*** 312.92*** 262.08*** 0.491*** 2.243*** 7.730*** 14.793*** 50.217*** 113.538***

 -Females (F) (4) 832.042** 1145.85** 901.62** 0.123 3.408 6.967 19.628 105.040* 243.275*

 -Males (M) (4) 249.610 95.72 101.45 1.765** 4.410* 19.029* 35.662* 87.649* 149.623

 -M x F (16) 160.153*** 158.99*** 142.35*** 0.265** 1.410*** 5.096*** 8.366*** 27.153*** 72.082***

Error 48 4.240 5.27 2.88 0.056 0.263 1.013 0.862 5.215 2.765

Within plot b 540 - - - 2.484 3.006 10.568 14.124 38.801 75.871

a See Table 1 for list of traits.

b The within plot variance was calculated by a separate analysis; *p = 5 percent; **p = 1 percent;***p = 0.1 percent.

breeding program. The ratio of dominance to additive variance for both survival and total height growth did not show

a clear trend over the testing period. But each of these

variances was sufficient for their practical use for the improvement of blister rust resistance, survival and total

height growth. The trend in the contribution of the error

variance to the phenotypic variance for blister rust resistance and height growth declined significantly with age,

that is, from 35 percent at age nine to 12 percent at age 20

and from 29 percent at age 11 to 7 percent at age 20,

respectively. In contrast, for survival, the error variance

displayed a slight continuous decline, ranging from 5 percent at age five to 3 percent at age 20. This decline supports

the expectation that genetic estimates of the three traits

become more accurate with age.

The additive variance component associated with female

parent (P. strobus) effects ranged from 16 to 39 percent for

blister rust resistance, from 15 to 59 percent for survival and

from 0 to 27 percent for total height growth of the phenotypic

variance. The contribution of male parent effects to the same

traits ranged from 0 to 12 percent, 0 to 6 percent and 12 to

44 percent, respectively. Thus, it is evident that the magnitude of the variance component associated with female

parent effects was far greater than that associated with

male parent effects over the whole testing period. These

results were consistent with those reported elsewhere (Blada

1989) for another experiment with P. strobus x P. peuce

hybrids.

The female additive variance components were associated

with standard errors smaller than the estimates themselves

in all but four cases thus making heritability estimates

fairly reliable. However, the standard errors of the male

variances for blister rust and survival were higher in six

cases than the estimates themselves, at most ages, and

suggesting non-accurate estimates.

Heritability

The broad-sense (h2

1) and narrow-sense (h22) heritabilities at the family level, as well as individual-tree narrowsense (h2

3) heritabilities calculated over testing period are

represented in table 6.

The magnitude of heritability estimates for blister rust

resistance, survival and total height growth indicated that

these traits may be under moderate to high genetic control,

but there is an apparent age dependency. For blister rust

resistance, the estimated narrow-sense heritability at the

family level was 0.325 at age 5 and increased to 0.430 at age

20. Similarly, for survival the heritability was lowest at age

five (h2

2 = 0.177), but increased to 0.516 at age 20. The family

narrow-sense heritability for total height growth ranged

between 0.382 and 0.531. According to table 6, the trend in

the three types of heritability for blister rust resistance and

height growth was in general consistent over the 20 years

testing period. The change in heritability in long rotation

crops such as trees is not surprising since genes involved in

height growth control may change with age (Namkoong and

others 1988) and these changes may be related to different

growth phases (Franklin 1979). Perhaps the accumulative

nature of blister-rust resistance and tree survival was responsible for increasing heritability with age and the ageage genetic correlations.

Individual narrow-sense heritabilities differed over years.

Their estimates were low ranging from 0.024 to 0.067 for

blister rust resistance and low to moderate ranging between

0.064 and 0.167 for height growth.

As expected, the broad-sense heritability estimates were

much greater than the narrow-sense estimates. The narrowsense heritabilities are used in conventional breeding while

broad-sense ones are also important as vegetative propagation methods and economical methods of producing specific
USDA Forest Service Proceedings RMRS-P-32. 2004 235

Age Trends in Genetic Parameters of Blister Rust Resistance and Height Growth in a Pinus strobus x P. peuce F1 hybrid population Blada and Popescu

Table 5—Variance components (percents in brackets), standard errors (SE), dominance ratios, genetic coefficient of variation (GCV) and trait

means (X).

Traitsa

Parameters BRR. 5 BRR. 9 BRR. 11 BRR. 13 BRR. 17 BRR. 20 S. 5 S. 9 S. 11

s2

GCA-F ± SE 0.248 (25) 0.111 (16) 0.300 (26) 0.249 (25) 0.377 (38) 0.395 (39) 5.968 (15) 49.594 (46) 74.881 (59)

±0.216 ±0.106 ±0.263 ±0.216 ±0.282 ±0.291 ±7.421 ±34.985 ±48.576

s2

GCA-M ± SE 0.040 (4) 0.082 (12) –0.072 (0) 0.040 (4) –0.006 (0) 0.005 (0) 0.850 (2) 4.604 (4) 3.655 (3)

±0.100 ±0.089 ±0.065 ±0.101 ±0.069 ±0.073 ±4.666 ±9.520 ±7.959

Total s2

GCA 0.288 0.193 0.300 0.289 0.377 0.400 6.818 54.198 78.536

s2

SCA± SE 0.592 (56) 0.258 (37) 0.648 (60) 0.552 (56) 0.493 (49) 0.490 (49) 31.110 (78) 52.688 (48) 43.523 (34)

±0.200 ±0.114 ±0.245 ±0.200 ±0.179 ±0.177 ±10.588 ±17.812 ±15.157

Total s2

G 0.840 0.451 0.984 0.841 0.870 0.890 37.928 106.886 122.059

s2

e± SE 0.142 (15) 0.246 (35) 0.154 (14) 0.142 (15) 0.130 (13) 0.120 (12) 1.953 (5) 2.241 (2) 5.803 (4)

±0.028 ±0.049 ±0.031 ±0.028 ±0.026 ±0.024 ±0.390 ±0.448 ±1.160

s2

Ph 0.982 0.697 1.138 0.983 1.000 1.010 39.881 109.127 127.861

s2

W 3.480 6.407 8.348 8.684 14.980 14.950 — — —

s2

p –0.438 –0.822 –1.237 –1.305 –2.367 –2.372 — — —

s2

SCA : s2GCA 1.9 : 1.0 1.3 : 1.0 2.3 : 1.0 1.9 : 1.0 1.3 : 1.0 1.2 : 1.0 4.6 : 1.0 1.0 : 1.0 0.6 : 1.0

s2

GCA-F : s2GCA-M 6.2 : 1.0 1.3 :1.0 1.0 : 0.0 6.2 : 1.0 1.0 : 0.0 1.0 : 0.0 7.0 : 1.0 10.8 : 1.0 20.5 : 1.0

GCV (%) 11.5 8.0 12.1 11.5 12.1 12.3 7.6 14.9 16.7

Mean 8.0 8.4 8.2 7.9 7.7 7.7 81.4 Arc 69.3 Arc 66.0 Arc

97.8 % 87.4 % 83.5 %

Traits

Parameters S. 13 S. 17 S. 20 H. 5 H. 9 H. 11 H. 13 H. 17 H. 20

s2

GCA-F ± SE 44.793 (42) 65.791 (54) 50.618 (51) –0.009 (0) 0.133 (14) 0.125 (4) 0.751 (13) 5.192 (24) 11.413 (27)

±32.222 ±44.245 ±34.847 ±0.008 ±0.135 ±0.291 ±0.778 ±4.088 ±9.500

s2

GCA-M ± SE 5.964 (6) –4.218 (0) –2.727 (0) 0.100 (44) 0.200 (20) 0.929 (27) 1.820 (31) 4.023 (18) 5.169 (12)

±10.245 ±5.105 ±5.025 ±0.068 ±0.173 ±0.741 ±1.385 ±3.427 ±5.978

Total s2

GCA 50.757 65.791 50.618 0.100 0.333 1.054 2.571 9.225 16.582

s2

SCA± SE 51.971 (48) 51.237 (42) 46.492 (46) 0.070 (31) 0.382 (39) 1.361 (40) 2.502 (42) 7.313 (34) 23.106 (54)

±17.797 ±17.669 ±15.818 ±0.030 ±0.158 ±0.325 ±0.931 ±3.037 ±8.011

Total s2

G 102.728 117.028 97.110 0.170 0.715 2.415 5.073 16.538 39.688

s2

e± SE 4.240 (4) 5.276 (4) 2.877 (3) 0.056 (25) 0.263 (27) 1.013 (29) 0.862 (14) 5.215 (24) 2.765 (7)

±0.848 ±1.055 ±0.575 ±0.111 ±0.053 ±0.203 ±0.172 ±1.043 ±0.553

s2

Ph 106.978 122.304 99.987 0.225 0.978 3.428 5.935 21.753 42.453

s2

W — — — 2.484 3.006 10.568 14.124 38.801 75.871

s2

p — — — –0.192 –0.238 –0.748 –1.492 –1.252 –9.880

s2

SCA : s2GCA 1.0 : 1.0 0.8 : 1.0 0.9 : 1.0 0.7 : 1.0 1.1 : 1.0 1.3 : 1.0 1.0 : 1.0 0.8 : 1.0 1.4 : 1.0

s2

GCA-F : s2GCA-M 7.5 : 1.0 1.0 : 0.0 1.0 : 0.0 0.0 : 1.0 0.7 : 1.0 0.1 : 1.0 0.4 : 1.0 1.3 : 1.0 2.2 : 1.0

GCV (%) 15.8 18.3 17.2 10.8 5.9 5.6 5.0 5.5 6.6

Mean 64.1 Arc 59.0 Arc 57.2 Arc 3.8 14.2 27.8 44.7 73.6 96.1

80.8 % 73.5 % 70.7 %

a See Table 1 for list of traits.

s2

GCA-F and s2GCA-M = additive variance due female and male parent trees, respectively.
236 USDA Forest Service Proceedings RMRS-P-32. 2004

Blada and Popescu Age Trends in Genetic Parameters of Blister Rust Resistance and Height Growth in a Pinus strobus x P. peuce F1 hybrid population

Table 6—Estimates of phenotypic variance (s2

Ph.1, s2Ph.2), phenotypic standard deviations (sPh.1, sPh.2), family broad sense and narrow sense

heritabilities (H2

1, h22) and individual narrow sense heritabilities (h23).

Traitsa

Parameters BRR. 5 BRR. 9 BRR. 11 BRR. 13 BRR. 17 BRR. 20 S. 5 S. 9 S. 11

s2

Ph.1 0.888 0.533 1.035 0.888 0.913 0.930 38.579 107.633 123.993

s2

Ph.2 4.320 6.858 9.332 9.525 15.850 15.840 — — —

sPh.1 0.942 0.730 1.017 0.942 0.955 0.964 6.211 10.375 11.135

sPh.1 2.078 2.619 3.055 3.086 3.981 3.980 — — —

H2

1=h2bs 0.947 0.847 0.951 0.947 0.953 0.957 0.983 0.993 0.984

h2

2=h2ns 0.325 0.363 0.290 0.325 0.413 0.430 0.177 0.503 0.633

h2

3=h2W 0.067 0.028 0.032 0.030 0.024 0.025 — — —

Traits

Parameters S. 13 S. 17 S. 20 H. 5 H. 9 H. 11 H. 13 H. 17 H. 20

s2

Ph.1 104.141 118.786 98.089 0.188 0.803 2.753 5.359 18.276 40.610

s2

Ph.2 — — — 2.654 3.721 12.983 19.197 55.339 115.559

sPh.1 10.205 10.899 9.904 0.434 0.896 1.659 2.315 4.275 6.373

sPh.1 — — — 1.629 1.929 3.603 4.381 7.439 10.750

H2

1=h2bs 0.966 0.985 0.990 0.901 0.891 0.877 0.946 0.905 0.977

h2

2=h2ns 0.487 0.554 0.516 0.531 0.415 0.382 0.480 0.505 0.408

h2

3=h2W — — — 0.064 0.089 0.081 0.134 0.167 0.143

a See Table 1 for list of traits.

H2

1=h2bs = s2G /s2Ph.1; h22=h2ns = s2GCA / s2Ph.1; h23=h2W = s2GCA /s2Ph.2; s2G = s2M+ s2F + s2MF; s2GCA = s2M + s2F;

s2

Ph.1 = s2M+ s2F+ s2MF+ s2e/R; s2Ph.2 = s2M+ s2F+ s2MF+ s2p + s2W; s2p = plot error = s2e - s2W/n; n = 6; s2e = variance error.

crosses, such as supplemental mass pollination, become

available (Zobel and Talbert 1984). However, heritability

estimates were high enough to ensure genetic progress in

improving blister rust resistance, survival and height growth

using P. strobus x P. peuce F1 hybrids.

Combining Abilities

The general combining ability (gca) effects estimated for 10

parents and 18 traits over years were presented in table 7.

Both positive and negative gca effects which differed from

the test mean were found for both male and female parents

for most traits. The range of estimated gca effects among

parents suggested that it may be possible to select parents

with superior breeding values for blister rust resistance,

tree survival and height growth.

At age 20, (fig. 1), the eastern white pine female Parent 7

had the largest positive gca effects for both blister rust

resistance (gca = 0.827 points) and survival (gca = 8.070 arc

sin) whereas the Parent 1 was the second highest for blister

rust resistance but the fourth for survival. At the same age,

among Balkan pine parents, the male Parent 20 had the

largest positive gca effects for both blister rust resistance

(gca = 0.420 points) and total height growth (gca = 5.185 dm).

On the other hand, the female Parent 2 and Parent 8 were

the worst because of their negative effects for all traits at age

20. With one exception, the Balkan male parents had low

effects on both blister rust resistance and height growth. A

primary objective of tree breeding involves choosing the best

parents for mating, especially when the trait to be improved

is quantitatively inherited. Hence the parents 7 and 1 should

be selected as good gca parents for blister rust resistance

and, on the other hand, parents 3 and 20 should be selected

as good parents for height growth. Taking into account that

blister rust resistance trait has the first priority in improvement, the parents 7, 1, and 20 should be used for blister-rust

resistance breeding, as they have the ability of transmitting

to their offspring a good level of resistance.

Genetic Correlations

Genetic correlations for traits involved in 5-, 9-, 11-, 13-,

17- and 20-year-old hybrids are presented in table 8.

Within trait age-age genetic correlations ranged from

0.406 to 1.00 for blister rust resistance, 0.106 to 0.963 for

survival and 0.010 to 0.926 for total height growth. Correlations between blister rust resistance and survival at the

same age, except age five, were moderate to high ranging

between 0.425 and 0.897. They appear to have a significant

predictive value for early selection purposes. Based on these

data, one may expect that selection for high blister rust

resistance at age five or survival at age nine should result in

high blister rust resistance and a high survival at age 20.

Trait-trait genetic correlations between blister rust resistance or survival, on one hand, and height growth on the

other, were low and very low. These results suggested that
USDA Forest Service Proceedings RMRS-P-32. 2004 237

Age Trends in Genetic Parameters of Blister Rust Resistance and Height Growth in a Pinus strobus x P. peuce F1 hybrid population Blada and Popescu

Table 7—General combining ability (gca ) effects of 10 parents for tested traits.

Traitsa

Parents BRR. 5 BRR. 9 BRR. 11 BRR. 13 BRR. 17 BRR. 20 S. 5 S. 9 S. 11 S. 13 S. 17 S. 20 H. 5 H. 9 H. 11 H. 13 H. 17 H. 20

gca - females

1 0.338 –0.014 0.660 0.338 0.490 0.480 –3.875 0.678 3.358 0.917 6.148 2.964 –0.017 –0.277 –0.388 –1.392 –1.731 –0.068

2 –0.876 –0.508 –0.860 –0.876 –0.730 –0.726 –1.695 –7.335 –9.228 –6.443 –9.112 –8.216 0.094 0.463 0.112 –0.192 –1.511 –2.928

3 0.138 0.092 0.600 0.138 0.156 0.154 4.072 3.025 3.872 0.370 2.435 5.484 –0.146 –0.444 0.992 2.288 4.529 6.252

7 0.698 0.639 0.093 0.698 0.796 0.827 3.365 11.065 11.545 11.690 9.715 8.070 0.036 –0.310 –0.834 –0.645 0.189 0.812

8 –0.296 –0.208 –0.493 –0.296 –0.710 –0.733 –1.869 –7.435 –9.548 –6.536 –9.188 –8.303 0.032 0.570 0.119 –0.059 –1.477 –4.068

gca - males

13 –0.289 –0.561 –0.340 –0.289 –0.137 –0.153 1.178 –2.215 –1.135 –1.656 1.008 –0.636 0.464 –0.250 0.232 0.015 –1.697 –1.528

14 0.031 –0.054 0.060 0.031 0.010 –0.013 1.152 –3.455 –2.082 –3.090 –3.138 –1.870 0.168 –0.290 –1.281 –1.539 –0.131 0.478

15 –0.509 0.019 –0.207 –0.509 –0.450 –0.453 2.125 –1.642 –2.568 –3.996 –1.405 –1.350 0.022 –0.564 –0.728 0.832 –0.411 –1.095

18 0.371 0.072 0.340 0.371 0.196 0.200 –4.642 0.925 –0.428 4.144 3.562 4.564 –0.402 0.330 –0.092 –0.279 –1.864 –3.042

20 0.398 0.526 0.147 0.398 0.383 0.420 0.185 6.385 6.212 4.597 –0.028 –0.710 –0.254 0.776 1.686 2.635 4.103 5.185

a See Table 1 for list of traits.
238 USDA Forest Service Proceedings RMRS-P-32. 2004

Blada and Popescu Age Trends in Genetic Parameters of Blister Rust Resistance and Height Growth in a Pinus strobus x P. peuce F1 hybrid population

10.7

6.2

5.4

2.6 2

-0.2

-2

-5.9

-9.2 -9.5

15

10

5

0

-5

-10

-15

7 20 18 3 14 13 15 2 8 Parents

x

gca effects (%) for blister rust resistance

Blister rust resistance

11.4

7.8

6.4

4.2

-0.9 -1 -1.9 -2.6

-11.6 -11.7

15

10

5

0

-5

-10

-15

7 18 1 13 20 2 8 Parents

x

gca effects (%) for survivors

Survival

6.5

5.4

0.8 0.5

-0.1

-1.1

-1.6

-3 -3.2

-4.2

8

6

4

2

0

-2

-4

-6

3 7 14 1 15 2 18 8 Parents

gca effects (%) for total height growth

Total height growth

1

3 15 14

20 13

Figure 1—General combining ability (gca) effects for blister-rust resistance, survival

and total height growth at age 20.
USDA Forest Service Proceedings RMRS-P-32. 2004 239

Age Trends in Genetic Parameters of Blister Rust Resistance and Height Growth in a Pinus strobus x P. peuce F1 hybrid population Blada and Popescu

Table 8—Genetic correlations among traits.

Traitsa BRR. 9 BRR. 11 BRR. 13 BRR. 17 BRR. 20 S. 5 S. 9 S. 11 S. 13 S. 17 S. 20 H. 5 H. 9 H. 11 H. 13 H. 17 H. 20

BRR. 5 0.809 0.676 1.000 0.926 0.918 0.281 0.816 0.803 0.874 0.751 0.754 –0.405 –0.178 0.019 0.078 0.227 0.232

BRR. 9 — 0.406 0.809 0.759 0.759 0.312 0.868 0.774 0.783 0.568 0.661 –0.561 –0.187 –0.108 0.031 0.236 0.148

BRR. 11 — 0.676 0.644 0.626 0.106 0.474 0.506 0.425 0.559 0.487 –0.397 –0.285 0.037 0.135 0.328 0.352

BRR. 13 — 0.926 0.918 0.281 0.816 0.803 0.874 0.751 0.754 –0.405 –0.178 0.019 0.078 0.227 0.232

BRR. 17 — 0.998 0.342 0.869 0.871 0.891 0.790 0.778 –0.306 –0.265 0.053 0.098 0.303 0.344

BRR. 20 — 0.348 0.877 0.880 0.897 0.792 0.775 –0.316 –0.253 0.070 0.114 0.325 0.361

S. 5 — 0.398 0.414 0.379 0.324 0.390 0.229 –0.383 0.110 0.232 0.395 0.363

S. 9 — 0.963 0.906 0.762 0.817 –0.378 –0.278 0.061 0.174 0.351 0.309

S. 11 — 0.917 0.841 0.851 –0.247 –0.290 0.087 0.175 0.365 0.408

S. 13 — 0.845 0.839 –0.323 –0.208 0.054 0.086 0.210 0.237

S. 17 — 0.921 –0.232 –0.452 –0.038 –0.032 0.148 0.304

S. 20

— –0.287 –0.430 –0.045 –0.001 0.143 0.231

H. 5 — 0.043 0.073 0.010 0.093 0.093

H. 9 — 0.567 0.454 0.065 0.016

H. 11 — 0.926 0.660 0.564

H. 13 — 0.829 0.702

H. 17 — 0.876

a See Table 1 for list of traits.
240 USDA Forest Service Proceedings RMRS-P-32. 2004

Blada and Popescu Age Trends in Genetic Parameters of Blister Rust Resistance and Height Growth in a Pinus strobus x P. peuce F1 hybrid population

100

90

80

70

60

50

40

30

20

10

0

10

9

8

7

6

5

4

3

2

1

0

(%)

110

90

100

80

70

60

50

40

30

20

10

0

(dm)

106.5

96.1

67.6

88.9

70.7

15.5

3.5

7.7

9.2

Resistance

(scale 1...10)

Trait BRR. 20 SV. 20 H. 20

Genotype

HPH (%) –16.3 –20.4 –9.8

MPH (%) +21.3 +35.4 +10.4

P. s. H. P.p. P. s. H. P.p. P. s. H. P.p.

P. strobus

Hybrid

P. peuce

the two categories of traits were inherited independently,

and hence, tandem selection cannot be applied.

Contradictory age-age within trait correlation estimates

was found in total height growth. Thus, correlations involving height growth at ages five and nine were very low with

two exceptions. In contrast, correlations involving ages

between 11 and 20 were high and very high, ranging between 0.564 and 0.926. Consequently, early selection at age

11 could result in height growth improvement at age 20, and

presumably at rotation age, too.

Heterosis

Parent and hybrid performances and the two types of

heterosis exhibited at age 20 are illustrated in figure 2.

It should be pointed out that the eastern white pine is the

best parent species for growth whereas the Balkan pine is

the best parent species for blister rust resistance.

At age 20, the estimates of the high-parent heterosis were

negative for the three traits, that is, the hybrid performances

were lower than those of the best parent for each trait. For

example, for height growth, the hybrid mean was 9.8 percent

lower than the mean of the white pine but greater than

Balkan pine. Similarly, the mean blister rust resistance and

tree survival were 16.3 and 20.4 percent, respectively, lower

than the mean of Balkan pine bulk lot.

Mid-parent heterosis was positive for the three involved

traits. The heterosis estimates accounted for 21.3 percent for

blister rust resistance, 35.4 percent for survival and 10.4

percent for height growth.

The hybrids inherited a high blister rust resistance and

were fast growing. Thus, the eastern white pine measured an

average of 3.5 points in blister rust resistance and 15.5

percent in tree survival while the hybrid measured 7.7 points

and 70.7 percent, respectively; that is, 120 percent and 356

percent more. Also, the hybrid mean exceeded the mean of the

Balkan pine in height growth. The Balkan pine measured

67.6 dm in height while the hybrid measured 96.1 dm, or 42

percent more. Therefore, the hybrids inherited high blister

rust resistance and faster growth from their parents.

Figure 2—Pinus strobus, P. peuce and P. strobus x P. peuce F1 hybrid performance at age 20,

high-parent heterosis (HPH) and mid-parent heterosis (MPH).
USDA Forest Service Proceedings RMRS-P-32. 2004 241

Age Trends in Genetic Parameters of Blister Rust Resistance and Height Growth in a Pinus strobus x P. peuce F1 hybrid population Blada and Popescu

Selection and Genetic Gain

Based on these results, selection could be made at both the

family and individual level. The genetic gain that could be

achieved in all traits and all ages are presented in table 9.

If the best 5, 8, 11, or 14 out of 25 hybrid families were

selected at age 20, a genetic gain of 7.2,, 5.8, 4.7,and 3.7

percent in blister rust resistance, 12, 9.7, 7.7 and 6.1 percent

in survival and 3.6, 2.9, 2.3 , and 1.8 percent in height growth

could be expected.

Selection at individual level could make an additional

gain. So, if at age 20 the best 5, 10, 15, and 20 percent

individuals within the best hybrid families were selected, a

genetic again of 2.7, 2.3, 2.0, and 1.8 percent in blister rust

resistance and 3.3, 2.8, 2.5, and 2.2 percent in total height

growth could be achieved.

Genetic gain was calculated as twice the average of the

gca,

s, at age 20. The average breeding value of the best

parents was presented in table 9.

The best three parents for blister rust resistance were

female trees 7 and 1 and male tree 20. Their average

breeding value was 1.151 points, which represent a genetic

gain of 15 percent in the overall mean (7.7 points), for blister

rust resistance (table 10, column 4). Similarly, for total

height growth, the best three parents were 3, 7, and 20, and

their average breeding value was 8.165 dm, which represent

a genetic gain of 8.5 percent in the overall mean (96.1 dm) for

height growth

The estimated genetic gains indicated that a program

aimed at improving blister rust resistance and height growth

through interspecific hybridisation could be successfully

achieved.

Table 9—Expected genetic gain (DG) according to the intensity of selection and hybrid age.

DG (%) selecting the best

DG (%) selecting the best 5, 8, 5, 10, 15 or 20%

11 or 14 hybrid families of 25 individuals within the best

tested hybrid families

Traitsa 5 8 11 14 5 10 15 20

BRR. 5 5.1 4.1 3.3 2.6 3.6 3.0 2.7 2.4

BRR. 9 4.2 3.4 2.7 2.2 1.8 1.5 1.4 1.2

BRR. 11 4.8 3.9 3.1 2.5 2.5 2.1 1.8 1.7

BRR. 13 5.2 4.2 3.4 2.6 2.4 2.1 1.8 1.6

BRR. 17 6.9 5.5 4.4 3.5 2.6 2.2 1.9 1.7

BRR. 20 7.2 5.8 4.7 3.7 2.7 2.3 2.0 1.8

S. 5 1.8 1.5 1.2 0.9 — — —

S. 9 10.1 8.1 6.5 5.1 ————

S. 11 14.4 11.5 9.3 7.3 ————

S. 13 10.4 8.4 6.7 5.3 ————

S. 17 13.8 11.1 8.9 7.0 — — —

S. 20 12.0 9.7 7.7 6.1 ————

H. 5 8.2 6.6 5.3 4.1 5.7 4.8 4.3 3.8

H. 9 3.5 2.8 2.3 1.8 2.5 2.1 1.8 1.7

H. 11 3.1 2.5 2.0 1.6 2.2 1.8 1.6 1.5

H. 13 3.3 2.7 2.2 1.7 2.7 2.3 2.0 1.8

H. 17 3.9 3.2 2.5 2.0 3.5 3.0 2.6 2.4

H. 20 3.6 2.9 2.3 1.8 3.3 2.8 2.5 2.2

a See Table 1 for list of traits.

Table 10—General combining ability (gca) estimates, breeding values (BV) and genetic gains (DG) if selected the best three

parents for blister-rust resistance, tree survivors and total height growth.

Blister-rust resistance Survival Total height growth

Select Select Select

parents gca BV DGa parents gca BV DGa parents gca BV DGa

- - - Points - - - % - - - - - - - -% - - - - - - - - - - - - dm - - - - - %

7 0.827 1.654 21.5 7 11.4 22.4 31.7 3 6.252 12.50 13.0

1 0.480 0.960 12.5 3 7.8 15.6 22.1 20 5.185 10.37 10.8

20 0.420 0.840 10.9 18 6.4 12.8 18.1 7 0.812 1.624 1.7

Mean 0.577 1.151 15.0 8.5 17.1 24.0 4.083 8.165 8.5

a

 Calculated against test mean, that is, 7.7 for blister-rust resistance, 70.7 percent for survival and 96.1 dm for total height growth.
242 USDA Forest Service Proceedings RMRS-P-32. 2004

Blada and Popescu Age Trends in Genetic Parameters of Blister Rust Resistance and Height Growth in a Pinus strobus x P. peuce F1 hybrid population

Conclusions____________________

At age 20, the P. strobus x P. peuce F1 hybrids exhibited a

mid-parent heterosis in blister rust resistance, survival and

total height growth.

Highly significant genetic variation over 20 years was

detected in the hybrid population to warrant improvement

for the three traits involved, using additive as well as nonadditive genetic variances.

Additive variance for height growth was found within both

P. strobus and P. peuce parent populations, whereas the

additive variance associated with blister rust resistance and

survival was found within female parent (P. strobus) population, only. Non-additive variance was also consistently

involved in the three tested traits.

The magnitude of variation in gca effects suggested that

in both initial populations it is possible to detect parents

with high breeding values for the traits under study. An

important finding was that good gca parents were found

within eastern white pine, not only for growth but for blister

rust resistance, as well.

Narrow sense heritability estimates at the hybrid family

level suggested that the tested traits were under moderate

to high genetic control over all ages.

The within trait age-age genetic correlations suggested

that selection for blister rust resistance and height growth

at an early age should result in high improvement of the

respective traits, at age 20.

Because of the weak trait-trait correlations, at all ages,

between growth and blister rust resistance, no tandem

selection can be applied.

The estimated genetic gains indicated that planting P.

strobus x P. peuce F1 hybrids in operational planting programs seems to be promising

Acknowledgements _____________

The authors wish to acknowledge the substantial technical assistance of S. Tanasie, A. Dragila and C. Dinu. Also, we

express our gratitude to Professor Scott Schlarbaum from

the University of Tennessee and to Dr. Richard Sniezko from

the Dorena Genetic Resource Center, Oregon, who reviewed

an early draft of this paper and made useful suggestions.

Also, the authors acknowledge the thorough comments of

the two, unknown for us, reviewers.

References _____________________

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USDA Forest Service Proceedings RMRS-P-32. 2004 243

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

Richard A. Sniezko, Robert S. Danchok, Joseph M. Linn, and Angelia

J. Kegley are with the USDA Forest Service, Dorena Genetic Resource

Center, 34963 Shoreview Road, Cottage Grove, Oregon, 97424, U.S.A.,

Phone (541) 767-5700, Fax (541) 767-5709, Email: rsniezko@fs.fed.us.

Bohun B. Kinloch Jr. is with the USDA Forest Service, Pacific Southwest

Research Station, Box 245, Berkeley, California, 94701, U.S.A. Email:

bkinloch@fs.fed.us. Andrew D. Bower is with the University of British

Columbia, Forest Sciences Department, Vancouver, B.C. V6T 1Z4

CANADA Email: adbower@interchange.ubc.ca.

Abstract—Two field sites were established between 1968 and 1974

using canker-free western white pine seedlings from full-sib families previously inoculated with white pine blister rust (Cronartium

ribicola) at Dorena Genetic Resource Center. Many individuals

planted on these sites had been identified as the resistant segregants for a major gene for resistance (Cr2). However, a strain of

rust with specific virulence (vcr2) to this gene has been found at high

frequency at and near these sites. In 1997, 27 and 92 families had

surviving individuals at Blodgett Creek (BC) and Grass Creek (GC),

respectively. Most of the trees on both sites were infected (99.1

percent at BC; 92.5 percent at GC). Despite heavy incidence of

infection, there was striking variation in its intensity. Individual

trees ranged from 0 to more than 200 cankers, and families also

varied dramatically. Many of the trees at both sites continue to grow

well, despite heavy infection. Wide variation in infection frequency

and survival among and within families on these sites demonstrates

that even the earliest selections from the program possess mechanisms of resistance other than Cr2.

Key words: white pine blister rust, field resistance, western white

pine, virulence

Introduction ____________________

Since its introduction to western North America near

Vancouver, B.C. in 1910 (Mielke 1943), white pine blister

rust (Cronartium ribicola J. C. Fisch.) has caused widespread damage and mortality to western white pine (Pinus

monticola Dougl. ex D. Donn) and other five-needle pines. In

Field Resistance to Cronartium ribicola in

Full-Sib Families of Pinus monticola in

Oregon

Richard A. Sniezko

Bohun B. Kinloch Jr.

Andrew D. Bower

Robert S. Danchok

Joseph M. Linn

Angelia J. Kegley

the mid-1950s, the USDA Forest Service began an operational breeding program for blister rust resistance in Oregon

and Washington (USDA Forest Service Region 6) to produce

seedlings of western white pine for reforestation. The development of resistant populations of western white pine through

breeding was seen as the best avenue for re-establishing this

species. The early program included tree selection in the

field, controlled pollination of selections, and artificial inoculation of the progeny.

Many of the early field selections for which progeny tests

showed the most dramatic genetic resistance came from

candidate trees in two areas in Oregon. The area with the

highest frequency of canker-free trees was a natural secondgrowth stand dating from approximately 1920 in the Champion Mine area on the Cottage Grove Ranger District of the

Umpqua National Forest in the Western Cascade Range.

The second source of highly resistant parent trees was a

plantation established with seedlings of unknown origin

between 1916 and 1935 in the Bear Pass area on the Sweet

Home Ranger District of the neighboring Willamette National Forest.

Survival of the parent trees at Champion Mine and Bear

Pass after repeated natural epidemics was due primarily to

a single dominant gene (Cr2) for resistance (Kinloch and

others 1999). However, a new strain of rust appeared in the

Champion Mine area around 1970 (McDonald and others

1984). Trees formerly free of infection became heavily infected. By 1994, all resistant parent trees in the Champion

Mine area were dead from rust. Many of the resistant trees

in the Bear Pass area still show no infection even though a

low frequency of the virulent strain (vcr2) has recently been

detected in this area (unpublished data).

Few long-term plantings (25 years or more) have tracked

occurrence of blister rust in individual resistant families of

western white pine. The plantings at Blodgett Creek (BC)

and Grass Creek (GC) were primarily established with

canker-free survivors of full-sib families after artificial inoculations (geographic origins are indicated in fig. 1). Soon

after planting BC and GC, budgetary constraints and personnel departures resulted in their virtual abandonment. In

1996 and 1997 BC and GC were remonumented and assessed for survival, growth, and incidence of blister rust.

This paper reports on the status of these two plantings

following 23 to 29 years of exposure to blister rust.
244 USDA Forest Service Proceedings RMRS-P-32. 2004

Sniezko, Kinloch Jr., Bower, Danchok, Linn, and Kegley Field Resistance to Cronartium ribicola in Full-Sib Families of Pinus monticola in Oregon

Material and Methods ____________

The BC and GC plantings were established between 1968

and 1974 with family row plots or blocks consisting primarily of healthy, canker-free seedlings from families that had

been artificially inoculated and assessed for blister rust

infection in the Region 6 program (table 1). The seedling

families had been inoculated two to three times and assessed

for stem symptoms. The seedlings planted at BC were from

two different sowings: 1968 and 1969. Seedlings from both

sowings were artificially inoculated two times with C. ribicola

by suspending Ribes sp. leaves bearing mature telia over

them in a moist enclosure at ambient temperatures. Seedlings planted at GC from sowings in 1959, 1962, 1963, and

1964 were artificially inoculated three times, and the seedlings at GC from the 1965 and 1966 sowings were inoculated

twice. Seedlings were held for several years after the last

inoculation, and age at planting varied. The number of trees

planted per family varied depending upon results from the

artificial inoculation. A few noninoculated families were

also included.

The Blodgett Creek site is on a low elevation, relatively

flat bench area and appears to have a higher exposure to rust

than the Grass Creek site, which is on a steep, high elevation, south-facing slope. Both sites are within 10 miles of the

Champion Mine area, where many of the parents originated,

and where a strain of rust virulent to Cr2 was first identified

(McDonald and others 1984, Kinloch and others 1999).

A total of 62 families (1,726 seedlings total) from 65

parents with one to 90 trees per family were planted at BC

in late 1973 and early 1974. A total of 120 families (3,751

seedlings total) from 82 parents with one to 180 trees per

family were represented in three plantings (1968, 1970, and

1972) at GC (table 1). The majority of families at both sites

were full-sibs from controlled crosses made among 120

ortets in natural stands. The parents represented at BC

came from four National Forests: Umpqua, Willamette, Mt.

Hood, and Mt. Baker-Snoqualmie (fig. 1); parents at GC

were predominantly from the Umpqua, Willamette, Mt.

Hood, and Mt. Baker-Snoqualmie, with a few selections from

BLM lands and an unknown source from Idaho.

Data on survival after first growing season were available

for all plantings at the BC site and for the first (1968)

planting at GC (table 2). After extensive remonumentation

of each site in 1996, every identifiable tree (living and dead)

was assessed for survival, diameter, and frequency and

location (bole versus branch) of rust infection. When possible, the presence of cankers was determined for dead trees.

Frequency of infections was assessed using a scale of 0 to 6

that was geometric, rather than arithmetic, in which each

 

 

 

Grass Creek Parentals

120° W

100 200

Blodgett Parentals

Plantations Sites

0

National Parks

Portland

45° N

National Forests

Tree Locations

Oregon

Seattle

Washington

Kilometers

Blodgett

Grass Creek

Figure 1—Geographic distribution of western white pine parent trees

represented at experimental plantings at Blodgett Creek and Grass

Creek. Insert represents plantations in Champion Mine area.

Table 1—Establishment and background information for two plantings of western white pine in Oregon.

Blodgett Creek Grass Creek

Latitude 43.678∞ N 43.601∞ N

Longitude 122.718∞ W 122.580∞ W

Elevation 2250 ft (690 m) 3800 ft (1160 m)

Aspect Southwest South

Topography Mostly flat with some areas sloped 5-35% Slopes 5-45% with several flat bench areas

Distance from Champion Mine (km) ~8 miles (12.8 km) NW ~3 miles (~4.8 km) ENE

Year(s) Established 1973, 1974 1968, 1970, 1972

Number of Families Planted 62 120

Total Number of Trees Planted 1726 3751

Families Surviving in 1997 27 92

Trees Remaining in 1997 404 1579
USDA Forest Service Proceedings RMRS-P-32. 2004 245

Field Resistance to Cronartium ribicola in Full-Sib Families of Pinus monticola in Oregon Sniezko, Kinloch Jr., Bower, Danchok, Linn, and Kegley

succeeding class interval was approximately double that of

the preceding class interval. Trees in canker class (CCL) 0

had no cankers; class 1 was (arbitrarily) set at 1 to 3 cankers;

class 2, 4 to 9; class 3, 10 to 21; class 4, 22 to 50; class 5, 51

to 100; class 6, greater than 100. Because of the long

intervals between establishment and assessment, lack of a

replicated design, and nonscalar measurement of cankers/

tree, no formal statistical analyses were possible.

Results ________________________

The 1997 survey of the two sites indicated that 99.1 and

92.5 percent of the living trees have blister rust infections at

BC and at GC, respectively. The mean CCL per living tree

was 4.33 at BC (approximately 62 cankers) and 3.60 at GC

(approximately 42 cankers) (table 2). Family mean CCL

ranged from 1.67 to 5.60 at BC and from 0 to 6 at GC (fig. 2),

but very few families were in CCL 2 or lower (less than 10

cankers). The number of canker-free trees was greater at GC

(73) than at BC (3) (table 2).

Mean diameter (at 1.3 m) of surviving trees was 19.9 cm

and 16.3 cm at BC and GC, respectively (table 2). Overall

survival (including mortality within the first year following

planting) was 19.2 percent at BC and 26 percent at GC.

Survival varied by family (fig. 3). Some of the families with

the highest survival (greater than 50 percent) differed dramatically in numbers of cankers per tree; this is also true for

trees within families (fig. 4a – d for examples). When firstyear mortality is excluded, survival was 46.7 percent at BC

and 41.1 percent for the 1968 planting at GC (table 2).

Table 2—Summary of mean survival, growth and rust status of two plantings of western white pine in Oregon.

Blodgett Creek Grass Creek

First year survival 41.2% n/a

Total survival in 1997 332 trees (19.2%) 974 trees (26.0%)

Survival in 1997 as % of first year survival 46.7% 41.1% (1968 planting)

n/a (1970 and 1972 plantings)

Identifiable trees in 1997 404 total 1579 total

313 healthy (77.5%) 755 healthy (47.8%)

19 sick or dying (4.7%) 219 sick or dying (13.9%)

33 dead <5 years (8.2%) 190 dead <5 years (12.0%)

39 dead >5 years (9.7%) 395 dead >5 years (25.0%)

20 dead, rust status unknown (1.3%)

Mean diameter 19.9 cm 16.3 cm (overall)

17.9 cm (1968 planting)

18.3 cm (1970 planting)

15.2 cm (1972 planting)

Mean canker class per treea 4.33 3.60 (overall)

4.70 (1968 planting)

3.09 (1970 planting)

3.30 (1972 planting)

Range of family mean canker class 1.67-5.60 0-6

Canker-free trees 3 (0.4%) 73 (6.3%)

a Frequency of infection was assessed for each tree using a scale of 0 to 6. Trees in class 0 had 0 cankers; class 1, 1-3; class 2, 4-9; class

3, 10-21; class 4, 22-50; class 5, 51-100; class 6, >100.

Figure 2—Distribution of family mean canker class (based

on numbers/tree of individual trees) of 1997 survivors at

(a) Blodgett and (b) Grass Creek.
246 USDA Forest Service Proceedings RMRS-P-32. 2004

Sniezko, Kinloch Jr., Bower, Danchok, Linn, and Kegley Field Resistance to Cronartium ribicola in Full-Sib Families of Pinus monticola in Oregon

Figure 3—Distribution of family means for percent survival

from establishment at (a) Blodgett and (b) Grass Creek

Recent mortality was greater at GC (38.3 percent) than at

BC (17.9 percent) (table 2).

Atropellis canker (caused by Atropellis sp.) was present

on trees at both sites (14 trees at BC and 189 trees at GC);

some families at GC had more than 50 percent of living trees

with Atropellis. Care was taken to distinguish this disease

from white pine blister rust.

Blodgett Creek

In 1997, 332 of the 404 identifiable trees (82 percent) were

still alive, with 27 of the original 62 families still represented

(with one to 31 surviving trees per family). Survival from

time of planting for these 27 families was low to moderate

(fig. 3a) but higher if first-year mortality was excluded.

Excluding first-year mortality, survival varied from 18.8 to

85.7 percent, with an overall mean of 46.7 percent. Survival

in the past 10 to 15 years was generally high for all families.

Of the 72 dead trees, 33 appeared to have died within the

previous 5 years (table 2).

The majority of living trees had more than 22 cankers

(CCL greater than 3), and more than 20 percent of the trees

had over 100 cankers (fig. 5a). On a family mean basis, the

number of cankers per tree varied from five to more than 100

(fig. 2). Only three trees were canker-free on this site. Of the

living trees, 73 percent had both bole cankers and branch

cankers, and no trees had only bole cankers. A small percentage (5.1) of trees had only branch cankers that were relatively new (less than 5 years old), while most trees (82.5

percent) had both recent and old branch cankers. Only six

trees had all cankers dead or inactive, and these were also

the six trees with the fewest cankers. The two families

largest in diameter had among the fewest cankers (fig. 6a).

Grass Creek

In 1997, 974 of 1579 remonumented trees were still alive,

and 98 of the 120 families were still represented by identifiable living and dead trees (with one to 101 trees per family

still alive). Survival from time of planting was only 26.0

percent, but some families had 100 percent survival (fig. 3b);

survival in the past 10 to 15 years varied widely by family

and somewhat by planting year but was high for many

families. Nearly one-third (190 of the 605 trees) of the

mortality recorded in 1997 appears to have occurred within

the previous 5 years (table 2).

Trees in the 1968 planting averaged more than twice the

number of cankers as the 1970 and 1972 plantings (table 2

and fig. 5b-d). Over 50 percent of trees in the 1968 planting

had more than 50 cankers (CCL 5 and 6), while less than 15

percent of the trees in any of the three plantings were

canker-free (fig. 5b-5d). Most families averaged CCL 3 or

higher (minimum of 10 cankers/tree) (fig. 2b). Of the living

trees, 63 percent had both bole and branch cankers, while

only two trees had bole cankers only. Few trees (64) had only

branch cankers that were relatively recent; most had old and

recent branch cankers (82.2 percent). All cankers appeared

to be dead or inactive on 15 trees. Overall, there was no

strong relationship between diameter and number of cankers (fig. 6b).

A total of 73 trees, coming from 21 different families, at

this site were canker-free, with most of these being in the

1970 and 1972 plantings (fig. 5b-d). These families had from

one to 30 trees canker-free, and where more than 15 trees

had been planted, the family mean percentage of canker-free

trees was generally low (less than 10 percent). Family 103

(18034-374 x 18034-391), which originally had only three

trees planted, still had 100 percent survival, and no cankers

were apparent in 1997. Of six trees planted in Family 117

(06020-501 x 06020-511), the two survivors were cankerfree. Family 43 (18034-395 x 18035-386) had 33 of the

original 35 (94.3 percent) trees planted surviving in 1997,

and 12 of these (36.3 percent) were canker-free (fig. 4c).

Follow-up visits (after 1997) detected a canker in one of the

trees in Family 103, and branch cankers are also present on

all of the formerly canker-free trees in Family 43.

One parent, 15040-836, was involved in six of the eight

crosses with highest survival; overall it was used in 18

crosses at GC. GC Family 16 (15045-835 x 15045-836) had

high survival despite the presence of many cankers (fig. 4d).

Another interesting parent is 06020-511 from Mt. Hood

National Forest. At BC, 06020-511 was the female parent in
USDA Forest Service Proceedings RMRS-P-32. 2004 247

Field Resistance to Cronartium ribicola in Full-Sib Families of Pinus monticola in Oregon Sniezko, Kinloch Jr., Bower, Danchok, Linn, and Kegley

Figure 4—Canker class distribution (1997 assessment) of living trees for two relatively resistant (a, c) and two susceptible (b, d) families at

Grass Creek.

Figure 5—Canker class distribution of trees

alive in 1997 at (a) Blodgett plantings established in 1973-1974 and Grass Creek plantings

established in (b) 1968, (c) 1970 and (d) 1972.
248 USDA Forest Service Proceedings RMRS-P-32. 2004

Sniezko, Kinloch Jr., Bower, Danchok, Linn, and Kegley Field Resistance to Cronartium ribicola in Full-Sib Families of Pinus monticola in Oregon

two crosses and the male parent in two crosses. At GC,

06020-511 was represented in three families, twice as the

male parent and once as an apparent self (Family 119). GC

Family 119 had 180 seedlings planted; in the 1997 inventory, 101 of these trees were still alive, of which 30 (29.7

percent) were canker-free (fig. 4a). Many other trees in this

family appeared vigorous despite having large, old bole

cankers. Although only one of the two families at BC with

06020-511 as the female parent had surviving trees in 1997,

this family had the lowest mean canker class (1.67) and the

second largest mean DBH (24.6 cm) (fig. 6a).

Discussion _____________________

The high level of rust infection at these sites was somewhat unexpected since most of the seedlings were cankerfree after heavy artificial inoculation. However, recent investigations make it apparent that the main cause of the

high level of infection is the presence of a virulent strain of

rust (Kinloch and others 1999, Kinloch and Dupper 2002).

Many of the parents represented in the planting are now

known to carry a specific gene for resistance (Cr2) that is

neutralized by a corresponding gene for virulence (vcr2) in

the pathogen in a gene-for-gene relationship (Kinloch and

Figure 6—Family mean canker class per living tree

versus diameter (dbh) at (a) Blodgett and (b) Grass

Creek.

others 1999, Kinloch and Dupper 2002). High inoculum

loads and intense selection pressure caused a sudden and

dramatic increase in vcr2 in these plantations.

The families planted on these sites were some of the first

selections made in the Region 6 program (Kinloch and others

1999). Despite a relatively narrow genetic base among these

early families, wide variation in infection frequency and

survival of trees on these sites indicates that non-Cr2 resistance is also present. This became apparent only after Cr2

was neutralized by vcr2, thereby unmasking independent,

unrelated mechanisms. These mechanisms are forms of

partial resistance that reduce infection rate or allow the tree

to survive after infection. Similar results have been reported

for progenies of sugar pine (P. lambertiana Dougl.) with a

different major gene for resistance to blister rust (Cr1). After

many years exposure to a strain of rust (vcr1) with specific

virulence to Cr1, most of the sugar pine trees were killed, but

a significant number exhibited mechanisms of partial resistance unrelated to Cr1 that enabled them to survive and in

many cases heal (Kinloch and Davis 1996). One of these

mechanisms included infection frequency differences of a

similar magnitude to those observed at BC and GC.

Wave years of infection occurred frequently at both sites

after plantation establishment. Many trees have dozens or

more cankers but are still showing vigorous growth. Some

individuals have large bole cankers that have been present

for over two decades (an indication of tolerance), for example, family ‘119’ at GC. One reason many of these trees

are still alive is that most of the cankers at these two sites are

branch cankers more than 0.5 m from the main stem and so

are unlikely to reach the bole. These two plantings will

continue to be monitored to see if the cumulative blister rust

impacts over time lead to mortality directly (bole girdling) or

indirectly (crown thinning or predisposition to other agents

such as bark beetles), and whether the resistance is durable.

From a silvicultural point of view, it is encouraging to see

trees that still thrive after nearly 30 years of intense white

pine blister rust exposure.

Due to the gap in data collection between trial establishment and the 1997 assessment as well as the substantial

first-year postplanting mortality, it is not possible to clearly

delineate all of the resistance mechanisms that might be

present. Although the physiological basis and inheritance of

additional mechanisms is unknown, at least several phenotypes have been documented: canker-free, low infection

frequency (fewer than average cankers present), tolerance

(vigorous tree with large bole cankers), and bark reaction

(healed or inactive cankers present). The gap in data limits

some of the specific information that could have been garnered for each family, such as for small, ephemeral bark

reactions.

These two sites represent some of the earliest field plantings

of the first resistant trees produced by the Region 6 western

white pine blister rust resistance program. Although only

canker-free seedlings were deployed, survival of these trees

over nearly 30 years of exposure to a virulent strain of rust

can be attributed to partial resistance mechanisms. These

mechanisms may provide the foundation for establishing

durable resistance in future generations of western white

pine. Collections of wind-pollinated lots from several trees at

BC and GC have been made, and these seedlots have been

included in recent rust-screening trials.
USDA Forest Service Proceedings RMRS-P-32. 2004 249

Field Resistance to Cronartium ribicola in Full-Sib Families of Pinus monticola in Oregon Sniezko, Kinloch Jr., Bower, Danchok, Linn, and Kegley

Since these two sites were established, progeny of thousands of parent trees have been screened for a more diverse

set of putative resistance mechanisms (Sniezko 1996), and

replicated field validation tests have been established

(Sniezko and others this proceedings, Sniezko and others

2000). Modifications to the operational screening program

continue as new information on resistance mechanisms

becomes available. Resistant seed from the breeding program will be used to help restore and maintain western

white pine as a valuable component of the forest ecosystems

in Oregon and Washington.

Acknowledgments ______________

The authors acknowledge Gerry Barnes (Dorena Center

Manager from 1966-1977) whose first-hand knowledge of

the history of early screening and establishment of these

stands was invaluable, and Paul Zambino for his review of

an earlier version of this paper. We thank those involved in

establishing these plantings, including Cliff Woody, Ted

Degerstrom, Ron Myers, Mack St. Clair, Gero Mitchelen,

Roland Fergason, Ernie Degerstrom, and Marge Janisch as

well as the USDA Forest Service’s forest health protection

group for funding key portions of this work.

References _____________________

Kinloch, Bohun B., Jr. and Davis, Dean. 1996. Mechanisms and

inheritance of resistance to blister rust in sugar pine, p. 125-132.

In: B.B. Kinloch, M. Marosy, and M.E. Huddleston (eds.). Sugar

pine: status, values, and roles in ecosystems: Proceedings of a

symposium presented by the California Sugar Pine Management

Committee. Univ. Calif. Div. Agr. Res. Publ. 3362.

Kinloch, Bohun B. and Dupper, Gayle E. 2002. Genetic specificity

in the white pine-blister rust pathosystem. Phytopathology

92:278-280.

Kinloch, B.B., Jr., Sniezko, R.A., Barnes, G.D., and Greathouse,

T.E. 1999. A major gene for resistance to white pine blister rust

in western white pine from the Western Cascade Range. Phytopathology 89:861-867.

McDonald, G.I., Hansen, E.M., Osterhaus, C.A., and Samman, S.

1984. Initial characterization of a new strain of Cronartium

ribicola from the Cascade Mountains of Oregon. Plant Disease

68: 800-804.

Mielke, J.L. 1943. White pine blister rust in North America. Yale

University School of Forestry. Bulletin 52. 155 pp.

Sniezko, R.S. 1996. Developing resistance to white pine blister rust

in sugar pine in Oregon, p. 125-132. In: B.B. Kinloch, M. Marosy,

and M.E. Huddleston (eds.). Sugar pine: Status, values, and roles

in ecosystems: Proceedings of a symposium presented by the

California Sugar Pine Management Committee. Univ. Calif. Div.

Agr. Natural Res. Publ. 3362.

Sniezko, R.A., Bower, A., Danielson, J. 2000. A comparison of early

field results of white pine blister rust resistance in sugar pine and

western white pine. HortTechnology 10(3): 519-522.
250 USDA Forest Service Proceedings RMRS-P-32. 2004

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel,

Howard B., eds. 2004. Breeding and genetic resources of five-needle pines:

growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR,

USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins,

CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station.

Kwan-Soo Woo is with the Tree Breeding Division, Forest Research

Institute, Korea Forest Service // Suwon, Omokchundong 44-3, Kyonggido,

441-350, South Korea. Telephone: 82-(0)31-290-1106; Fax: 82-(0)31-292-

4458, e-mail: woo9431@yahoo.co.kr. Geral I. McDonald is with the USDA

Forest Service, Rocky Mountain Research Station, 1221 South Main Street,

Moscow, ID 83843, U.S.A. Telephone: (208) 883-2343; Fax: (208) 883-2318, email: gimcdonald@fs.fed.us. Lauren Fins is with the Department of Forest

Resources, University of Idaho, Moscow, ID 83844-1133, U.S.A. Telephone:

(208) 885-7920; Fax: (208) 885-6226, e-mail: lfins@uidaho.edu.

Abstract—Growth conditions for nursery-grown western white

pine seedlings have been shown to affect levels of blister rust

infection (from Cronartium ribicola). In an experiment initially

designed to test the influence of environmental conditions at two

nurseries in northern Idaho on the blister rust pathosystem, western white pine seedlings of a single resistant seedlot were unintentionally held in cold storage for 6 months longer at one nursery than

at the other. Inoculation of these long-stored seedlings with blister

rust spores occurred at 1 month after growth resumed under

nursery conditions, versus 7 months for those with shorter storage.

Infection percent was nearly double and infection efficiency (infections per unit area of stomata) was 70 times greater on the seedlings

with only 1 month of growth than on the seedlings with the more

mature foliage. Since the seedlings had originated from the same

genetic source, the overwhelming difference suggests that phenology and/or nursery regimes can strongly influence infectability of

seedling needles in western white pine using artificial inoculations.

If phenology is the key factor, it may help explain why infection

levels have been relatively high on northern Idaho resistant selections when grown at milder locations. Furthermore, if resistance

genes can be selectively activated by manipulating phenology,

molecular tools that examine gene expression might be employed to

enhance our understanding of environmental regulation of genes

for blister rust resistance.

Key words: Cronartium ribicola, blister rust pathosystem, infection efficiency, phenology.

Influence of Seedling Physiology on

Expression of Blister Rust Resistance

in Needles of Western White Pine

Kwan-Soo Woo

Geral I. McDonald

Lauren Fins

Introduction ____________________

Western white pine’s (Pinus monticola Dougl.) susceptibility to the blister rust fungus (Cronartium ribicola J.C.

Fisch. in Rabenh.) has been shown to vary with tree age,

type, and age of needles, and age of the shoot (Lachmund

1933; Pierson and Buchanan 1938; Bingham 1972; Hunt

1991). But the environment in which seedlings are reared

may also influence their susceptibility to infection. This

possibility became apparent when, at three years postinoculation with C. ribicola spores, 47.6 percent of the seedlings

grown in one nursery in northern Idaho were dead, compared to only 29.8 percent mortality of the seedlings grown

in a second Idaho nursery. The result was the reverse of

expectations because the seedlings grown in the second

nursery were from a previously untested seed orchard,

which was later determined to have low rust resistance

compared to those grown in the first nursery. It appeared

that, in addition to genetic variation, factors associated

with nursery location and/or growing regimes had influenced “susceptibility,” or perhaps “infectability” of the

seedlings to C. ribicola (Eramian and Foushee, personal

communication).

If rust resistance levels do vary as a function of growth

environment, then estimates of resistance levels of the same

genetic stock may vary widely depending on the nursery

regime in which the stock is grown, or between stocks placed

in field environments and their nursery-grown counterparts. Such variation in estimated resistance would imply a

need to refine nursery rearing and testing protocols such

that they produce comparable test results that are reliable

predictors of long-term resistance levels under a variety of

field conditions.

The original objective of the study reported here was to

address this issue and test the hypothesis that blister rust

infection levels in western white pine seedlings are indeed

influenced by differences in nursery growing environment.

We began by growing seedlings of the same genetic stock in

two nurseries in northern Idaho, with the intent of following their routine protocols and exposing the seedlings to

blister rust spores at the end of their second growing season

after germination. However, an unexpected physiological

difference was induced between the stocks when the seedlings at one of the nurseries were accidentally kept in cold

storage six months longer than their counterparts at the

second nursery. As environmental conditions during inoculation and/or disease development can influence the expression of blister rust-resistance genes in white pines, we
USDA Forest Service Proceedings RMRS-P-32. 2004 251

Influence of Seedling Physiology on Expression of Blister Rust Resistance in Needles of Western White Pine Woo, McDonald, and Fins

recognized the possibility that the physiological differences induced by the accidental extension of cold storage

might also have important and potentially profound effects

on expression of blister rust resistance in the seedlings

(Yokata 1983; Bower 1987; Hunt and Meagher 1989;

McDonald and others, this volume). As a measure of the

relative infectability and early expression of resistance in

the two stocks by C. ribicola, we compared their levels of

needle infection at 5 months postinoculation. Subsequent

mortality from rust was not evaluated.

Materials and Methods ___________

Plant Material and Artificial Inoculation

Western white pine seedlings from a single genetic source

(blister rust-resistant F2 from the R.T. Bingham White Pine

Seed Orchard in Moscow, ID, as described in Hoff and others

1973) were grown for two growing seasons at two nurseries

in northern Idaho: Potlatch Corporation’s Nursery in

Lewiston, and the University of Idaho Forest Research

Nursery in Moscow, hereafter referred to as the Lewiston

and Moscow nurseries. The seedlings from both nurseries

were inoculated with spores of C. ribicola at the USDA

Forest Service Nursery in Coeur d’Alene, ID, in September

1999 using the routine procedures described in Mahalovich

and Eramian (1995). The rust inoculum was collected from

an established Ribes garden that had been inoculated with

aeciospores collected from blister rust cankers in locations in

Idaho, western Montana, and eastern Washington. Spore

cast was monitored on slides that were placed at regular

intervals among the seedlings.

At the time they were inoculated, the seedlings from the

Moscow Nursery were about 36 cm tall and had calipers of

0.6 to 0.8 cm. Seedlings from the Lewiston Nursery, which

had inadvertently been left in cold storage from December

1998 to early August 1999, were physiologically immature

and substantially smaller, measuring about 22 cm in height,

with a caliper of 0.3 to 0.5 cm, and had short (but expanding)

secondary needles. We entered 200 seedlings into this study

(25 per nursery in each of four replications). Nursery groups

were randomized within replications, with one plot per

nursery per replication.

In the months following inoculation, the seedlings were

irrigated but no nutrients were supplied. At 5 months

postinoculation, the seedlings were inspected for needle

spots as a measure of successful penetration by basidiospore

germ tubes. Overall seedling survival was 98.5 percent (197/

200.) Needle spots were counted on all needles. Infection

efficiency (IE) was calculated as:

[1] IE = spots per seedling / infective target area per

seedling / spore cast

Infection efficiency was determined for each seedling and

averaged for each nursery (McDonald and others 1981,

1991). The number used for spore cast in this equation was

determined the by spore count on a monitoring slide that was

closest to the seedlings during the inoculation process.

Infective target area (ITA) (cm2/seedling) was estimated as:

[2] ITA = SR x N x L (cm) x 0.01 (cm)

where SR= average number of stomatal rows on two adaxial

sides per needle, N= number of needles per seedling, and L=

average length of stomatal row (estimated by needle length)

(McDonald and others 1981). Average number of stomatal

rows and needle length were based on samples of six needles

per seedling (one from each of six fascicles, two each from the

top, middle, and bottom of each seedling). Lesion frequency

was calculated as the number of needle spots/infective target

area. Subsequent mortality as a function of blister rust

infection was not evaluated.

Statistical Analyses

Data were analyzed using the SAS-PROC GLM (general

linear model) statistical package and Type III Sums of

Squares (SAS Institute Inc. 1989). Data were transformed

by arcsine when the exploratory PROC UNIVARIATE test

showed they were not normally distributed.

Results ________________________

Seedlings from the Moscow Nursery averaged 3.25 spots

per seedling (range: 0 to 50) whereas seedlings from the

Lewiston Nursery averaged 87.83 (range: 0 to 710) (table 1).

Infection efficiency was significantly higher on seedlings

Table 1—Infection efficiency of Cronartium ribicola on western white pine seedlings from two nurseries in northern Idahoa.

Infective target Spore

Number of area concentration Germination Infection

Nurseries Percent infection spots/seedling (cm2/seedling) (spores/cm2) (%) Lesion freq. efficiency

Lewiston 95 87.83 94.15 5521.20 44.77 1.050 2.1x10–4

(11.04) (4.30) (232.04) (1.42) (0.138) (2.9x10–5)

Moscow 51 3.25 322.36 4465.75 61.39 0.011 3x10–6

(0.73) (11.31) (116.58) (1.08) (0.002) (6x10–7)

aValues are means with SE given in parentheses. P-values (shown below) are based on comparisons between samples of seedlings from the Lewiston versus the

Moscow nurseries when P<0.05. Needle spots were counted on all needles per seedling (P=0.0009); Infective target area was estimated by average number of stomatal

rows on two adaxial sides per needle x number of needles per seedling x average length of stomatal row (estimated by needle length) x 0.01cm (P=0.0037); Spore

concentration = total number of spores in ten fields per slide x 60.2 (P=0.6574); Germination % (P=0.003); Lesion frequency = # spots/infective target area (P=0.0025);

Infection efficiency = spots per seedling/infective target area per seedling/spore cast (P=0.0105).
252 USDA Forest Service Proceedings RMRS-P-32. 2004

Woo, McDonald, and Fins Influence of Seedling Physiology on Expression of Blister Rust Resistance in Needles of Western White Pine

from the Lewiston Nursery (mean=2.1x10–4) compared to

those grown in the Moscow Nursery (mean=3x10–6)

(P=0.0105). Also statistically significant were differences in

the number of spots per seedling (P=0.0009), infective target

area (P=0.0037), spore germination percentage (P=0.003),

and lesion frequency (P=0.0025). Only five of 98 seedlings

from the Lewiston Nursery had no spots, compared to 49 of

99 for the Moscow seedlings. No statistical difference was

found between nurseries in spore concentration (P=0.6574).

Compared to the seedlings grown in the Moscow Nursery,

seedlings grown in the Lewiston Nursery had a relatively

small infective target area (94 versus 322 cm2/seedling) and,

although spore germination percentage was lower on nearby

monitoring slides (45 vs 61 percent), the Lewiston Nursery

seedlings developed more rust spots per seedling than did

those from the Moscow Nursery.

The mean spore concentration (spores/cm2) for the two

stocks at the end of the inoculation period was 4,994 spores/

cm2, ranging from 2,047 to 11,920 spores/cm2. Mean spore

germination was 53 percent (range 25 to 82 percent).

Discussion _____________________

Compared to the Moscow Nursery seedlings, the Lewiston

Nursery seedlings had 27 times the number of spots and 70

times the infection efficiency (table 1). As the seedlings from

the two nurseries had originated from the same genetic

source (open pollinated seed from the same seed orchard),

and the seedlings were inoculated at the same time and

under the same inoculation conditions, observed differences

in infection were not likely to be a function of genetic

differences or differences in inoculation conditions. The

most likely explanation of the observed differences in infection is either nursery cultural practices and/or a difference

in developmental and physiological state of the seedlings at

the time of inoculation.

As previous attempts to infect white pine seedlings grown

in the Lewiston Nursery had resulted in relatively low

infection percentages (Foushee, personal communication),

the standard nursery practice used at the Lewiston Nursery

is not a likely explanation for the observed, relatively high

infection levels in this study. However, the Lewiston Nursery seedlings were kept in cold storage for six months longer

than the seedlings from the Moscow Nursery and were

removed from cold storage only one month prior to inoculation with rust. The seedlings had small needles (that were

probably still expanding) and succulent tissues when they

were inoculated. These observations suggest that some

needle resistance mechanisms may not be fully operational

in needles that have not reached their full development

within a current growing season.

In addition, the very low frequency of needle spotting on

the seedlings from the Moscow Nursery (compared to target

levels of greater than 90 percent for routine rust screenings)

may indicate a nursery regime that, at least temporarily,

protects seedlings from infection. If so, rust screening that

includes only artificial inoculations of their seedlings will

not reflect actual long-term rust resistance levels under field

conditions.

Data on needle spots were not recorded by needle type or

location on the seedlings. However, it was clear that most of

the observed rust spots appeared on current-year needles

that were at the tops of the western white pine seedlings

from both nurseries. Although it is possible that this reflects

a purely spatial phenomenon, with uppermost younger

needles having greater spore deposition than those lower on

the stem, our results with P. monticola are consistent with

previous findings for P. monticola and P. strobus that current needles are more susceptible to blister rust than older

needles (Snell 1936; Van Arsdel 1968; Hunt 1991). Alternatively, physio-mechanical attributes may explain the differences, since stomata in older needles are less active than

those of the current-year needles (Hirt 1938).

Bingham (1973) reported that seedling height was significantly related to the frequency of needle spots on nonresistant western white pine seedlings, with taller seedlings

more highly cankered than shorter ones. However, this

relationship did not hold true for three types of resistant

stocks, one of which consisted of a bulk lot of F2 seedlings

similar to those used in our study. In either case, our result

differs from Bingham’s in that the relatively small seedlings

from Lewiston Nursery had, by a large margin, more needle

spots than the seedlings grown in the Moscow Nursery.

The average spore germination for our study was 53 percent,

considerably lower than the overall average of 73 percent for

the 1999 routine inoculations at the Coeur d’Alene Nursery

(Eramian, personal communication; data on file at the

USDA Forest Service Coeur d’Alene Nursery). Also, spore

germination percentages on the slides near the Lewiston

seedlings were consistently lower than those near the Moscow seedlings (45 percent versus 61 percent respectively).

The reasons for the overall lower spore germination and the

differences between samples near the two stock types are not

apparent but may be related to variation in microsite associated with seedling size and foliage density.

Target spore deposition at the Coeur d’Alene Nursery is

6,000 to 7,000 spores per square centimeter and 95 percent

or higher infection percent (Eramian, personal communication). Thus the mean spore deposition of 5,258 spores per

square centimeter was lower than desired, but the 95 percent infection of the Lewiston Nursery seedlings indicates

the deposition of spores and the environmental conditions in

the inoculation tent were sufficient to achieve a high level of

infection in at least one of the groups. The reason for the low

infection percentage for the Moscow nursery seedlings is not

known.

We found no relationship between needle spot development and either spore concentration or percent spore germination on slide traps (table 1), suggesting that at relatively

high spore concentrations, seedling physiology may have

more influence on infection efficiency than either spore

concentration or percent germination.

An investigation of actual physiological differences between the groups was beyond the scope of this study.

However, in other studies, a western white pine protein,

Pin m III, was found to increase both during winter months

and in tissue infected with blister rust (Ekramoddoullah and

others 1995; Ekramoddoullah and others 1998). The normal

winter increase in the protein was suppressed in rustinfected trees with the slow canker growth resistance mechanism, further suggesting a relationship between Pin m III

and rust resistance. If the abundance of Pin m III is related

to rust resistance, and if it is relatively easy to manipulate
USDA Forest Service Proceedings RMRS-P-32. 2004 253

Influence of Seedling Physiology on Expression of Blister Rust Resistance in Needles of Western White Pine Woo, McDonald, and Fins

by subjecting trees to differing lengths of cold storage, the

protein may be a useful indicator of desirable or undesirable

genotypes for selection in tree breeding programs. This

hypothesis is easily tested by subjecting groups of resistant

and susceptible seedlings to long versus short cold storage

treatments. If there is a relationship only the resistant

stocks exposed to normal cold storage period are predicted to

show low levels of the protein.

Our results are also consistent with the infection of

Japanese stone pine shoots by Endocronartium sahoanum

(Kaneko and Harada 1995) after cold storage synchronization. Increased susceptibility in these related situations

argues for the existence of a physiological cause associated

with cold-storage treatment and/or immature tissue.

In our study, seedlings established under different nursery environments displayed different infection levels, but

the likely causal factors were confounded and could not be

isolated to explain the differences. Physiological and developmental conditions, growing regimes and nursery environment may all have influenced infectability of the study

seedlings.

Needle infection was higher on current-year needles than

on older needles (magnitude not quantified in this study) but

older needles may have been sheltered from inoculum by the

newer foliage, or they may have been less physiologically

active. The highest infection levels occurred on physiologically immature needles. It appears that some needle resistance mechanisms may not be fully developed in actively

growing, nonmature, succulent needles. If true, movement

of stock to other climatic conditions, or differences in annual

weather patterns under field conditions may influence the

effectiveness of some resistance mechanisms, and may, at

least in part, account for suspected “wave year” phenomena.

However, it is also possible that, in this study, the effect of

needle maturation state was confounded by physiological

changes associated with the prolonged period of cold storage

and/or the nursery growing regime. The “needle immaturity” hypothesis could be tested by inoculating second year

seedlings of the same genetic stock at monthly intervals

from June through September, or by inoculating only in

September using groups of seedlings (of the same genetic

stock) that had been subjected to different periods of cold

storage.

Our results suggest that expression of genes related to

rust resistance in northern Idaho white pine seedlings is

sensitive to physiological state as related to phenology and/

or to growing regimes that alter needle infectability in the

nursery. If phenology is a critical factor, new molecular tools,

such as cDNA PCR detection assays and micro arrays may

facilitate experiments designed to explore environmental

regulation of resistance genes and their association with

phenologic traits. If variation in growing regimes is found to

critically affect the infectability of seedling needles, then

either protocols for evaluating resistance must be fine-tuned

to address this variation, or growth regimes and testing

protocols should be standardized for test seedlings across

nurseries.

Any program that relies on artificial inoculation for evaluating seedlings for rust resistance must also include longterm field tests under a variety of conditions to fully assess

resistance and validate the predictions of early screenings.

If studies determine that rust infection under field conditions generally tends to be substantially higher than is

indicated by current rust screening procedures for tree

improvement programs, it may be useful to alter testing

protocols to better mimic field conditions, such as including

multiple exposures to rust spores, and exposing seedlings to

spores earlier, potentially beginning when teliospores are

first produced on the Ribes leaves. Further study is needed

to test these hypotheses and to refine rust screening procedures (and perhaps nursery rearing procedures) that, together, will provide accurate long-term predictions of rust

resistance.

Acknowledgments ______________

The authors thank Paul Zambino and Ned Klopfenstein

for their thoughtful reviews of the manuscript, Aram Eramian

for providing critical baseline information on routine rust

screenings at the Coeur d’Alene Nursery, and the Inland

Empire Tree Improvement Cooperative for its support of the

original study on which the manuscript is based.

References _____________________

Bingham, R.T. 1972. Artificial inoculation of large number of Pinus

monticola seedlings with Cronartium ribicola. In Biology of rust

resistance in forest trees: proceedings of a NATO-IUFRO advance study institute. R.T. Bingham, R.J. Hoff, and G.I. McDonald,

eds. USDA Misc. Publ. 1221, p. 357-372.

Bingham, R.T., Hoff, R.J., and McDonald, G.I. 1973. Breeding

blister rust resistant western white pine. VI. First results from

field testing of resistant planting stock. USDA Forest Service

Research Note INT-179. USDA Forest Service, Intermountain

Forest and Range Experiment Station, Ogden, Utah 84401. 12 p.

Bower, R.C. 1987. Early comparison of an Idaho and a coastal source

of western white pine on Vancouver Island. West. J. App. For.

2:20-21.

Ekramoddoullah, A.K.M., Davidson, J.J., and Taylor, D.W. 1998. A

protein associated with frost hardiness of western white pine is

up-regulated by infection in the white pine blister rust

pathosystem. Can. J. For. Res. 28:412-417.

Ekramoddoullah, A.K.M., Taylor, D.W., and Hawkins, B.J. 1995.

Characterization of a fall protein of sugar pine and detection of its

homologue associated with frost hardiness of western white pine

needles. Can. J. For. Res. 25:1137-1147.

Eramian, A.D. 1997, 2003. Personal communication. USDA Forest

service, Coeur d’Alene Nursery, Coeur d’Alene, Idaho.

Foushee, D. 1997. Personal communication. USDA Forest service,

Coeur d’Alene Nursery, Coeur d’Alene, Idaho.

Hirt, R.R. 1938. Relation of stomata to infection of Pinus strobus by

Cronartium ribicola. Phytopathology 28:180-190.

Hoff, R.J., McDonald, G.I., and Bingham, R.T. 1973. Resistance to

Cronartium ribicola in Pinus monticola: structure and gain of

resistance in the second generation. USDA For. Serv. Res. Note

INT-178, Intermountain Forest and Range Experiment Station,

Ogden, Utah. 8 p.

Hunt, R.S. 1991. The effect of age on the susceptibility to blister rust

of western white pine seedlings. In 3rd International IUFRO Rust

of Pine Working Party Conference. Ed. By Hiratsuka, Y., J. K.

Samoil, P. V. Blenis, P. E. Crane, and B. L. Laishley. Sep. 18-22,

1989. Banff, Alberta, Canada, pp. 287-290.

Hunt, R.S., and Meagher, M.D. 1989. Incidence of blister rust on

“resistant” white pine (Pinus monticola and P. strobus) in coastal

British Columbia plantations. Can. J. Plant Pathol. 11 (4):419-423.

Kaneko, S., and Harada, Y. 1995. Life cycle of Endocronartium

sahoanum and its nuclear condition in axenic culture. Pp 95-100.

In: Proceedings of the 4th IUFRO “Rust of Pines” Working Party

Conference ed by Kaneko, S., K. Katsuya, M. Kakishima, and

Y. Ono. Tsukubo, Japan.
254 USDA Forest Service Proceedings RMRS-P-32. 2004

Woo, McDonald, and Fins Influence of Seedling Physiology on Expression of Blister Rust Resistance in Needles of Western White Pine

Lachmund, H.G. 1933. Resistance of the current season’s shoots of

Pinus monticola to infection by Cronartium ribicola. Phytopathology 23:917-922.

Mahalovich, M.F., and Eramian, A. 1995. Breeding and seed orchard plan for the development of blister rust resistant white

pine for the Northern Rockies. Draft tree improvement plan.

USDA Forest Service Northern Region. 59 p.

McDonald, G.I., Hoff, R.J., and Samman, S. 1991. Epidemiologic

function of blister rust resistance: A system for integrated management. In 3rd International IUFRO Rust of Pine Working Party

Conference. Ed. by Hiratsuka, Y., J. K. Samoil, P. V. Blenis, P. E.

Crane, and B. L. Laishley. Sep. 18-22, 1989. Banff, Alberta,

Canada, pp. 235-255.

McDonald, G.I., Hoff, R.J., and Wykoff, W.R. 1981. Computer

simulation of white pine blister rust epidemics. I. Model formulation. USDA For. Serv. Res. Pap. INT-258, Intermountain Forest and Range Experiment Station, Ogden, Utah. 136 p.

McDonald, G.I., Zambino, P. and Sniezko, R. In press. Breeding

rust-resistant pines in the western United States: Lessons from

the past and a look to the future. This Volume.

Pierson, R.K. and Buchanan, T.S. 1938. Susceptibility of needles

of different ages on Pinus monticola seedlings to Cronartium

ribicola infection. Phytopathology. 29:833-839.

SAS Institute Inc. 1989. SAS/STAT user’s guide, version 6.4th ed.

SAS Institute Inc., Cary, N.C.

Snell, W.H. 1936. The relation of the age of needles of Pinus

strobus to infection by Cronartium ribicola. Phytopathology

26:1074-1080.

Van Arsdel, E.P. 1968. Stem and needle inoculations of eastern

white pine with the blister rust fungus. Phytopathology 58:512-514.

Yokota, S-I. 1983. Resistance of improved Pinus monticola and

some other white pines to the blister rust fungus Cronartium

ribicola, of Hokkaido, Japan. European Journal of Forest Pathology

13:389-402.
USDA Forest Service Proceedings RMRS-P-32. 2004 255

Part V: Conference Attendees

Conference attendees at the BLM’s Sprague Seed Orchard

Photo courtesy of T. Tuttle
256 USDA Forest Service Proceedings RMRS-P-32. 2004
USDA Forest Service Proceedings RMRS-P-32. 2004 257

Chang-Young Ahn

Korea Forest Research Institute

Omokchun-dong 44-3

Suwon 441-350 Republic of Korea

Alexander H. Alexandrov

Forest Research Institute

132 St. Kliment Ohridski Blvd.

1756 Sofia, Bulgaria

forestin@bulnet.bg

Nabil Atalla

2310 Winslow Park Dr.

Medford, OR 97504

Bruce Barr

Sierra Pacific Industries

P.O. Box 39

Stirling City, CA 95978 USA

Phone: (530)873-9192

Fax: (530)873-2463

bbarr@spi-ind.com

Jerome Beatty

USDA Forest Service

Forest Health Protection

1601 North Kent Street

RPC, 7th Floor (FHP)

Arlington, VA 22209

Phone: (703)605-5332

Fax: (703)605-5353

jbeatty@fs.fed.us

Jordan Bennett

University of Victoria

P.O. Box 3020

Victoria, BC V8W 3N5 Canada

Phone: (250)721-7113

Fax: (250)721-6611

muchachoverde@hotmail.com

Ioan Blada

Forest Research Institute

SOS Stefaresti, NR 128

Bucharest 11 Romania

ioan_blada@yahoo.com

Andrew Bower

University of British Columbia

Forest Sciences Department

Vancouver, B.C. V6T 1Z4 Canada

adbower@interchange.ubc.ca

Conference Attendees _____________________________________________

Patti Brown

Canadian Forest Products LTD

4858 Skylark Rd

Sechelt, BC V0N 3A2 Canada

Phone: (604)885-5905

Fax: (604)885-7614

pabrown@mail.canfor.ca

Todd Burnes

University of Minnesota

495 Borlaug Hall

St. Paul, MN 55108 USA

Phone: (612)625-6231

Fax: (612)625-9728

burne002@tc.umn.edu

Deems Burton

USDA Forest Service

P.O. Box 377

Happy Camp, CA 96039 USA

Phone: (530)493-2243

Fax: (530)493-1796

dburton@fs.fed.us

Mo-Mei Chen

University of California, Berkeley

1001 Valley Life Sciences Bldg

Berkeley, CA 94720 USA

Phone: (510)643-0633

Fax: (510)643-5390

mmchen@nature.berkeley.edu

Wan-Yong Choi

Korea Forest Research Institute

Omokchun-dong 44-3

Suwon 441-350 Republic of Korea

wychoi@foa.go.kr

Russell Cox

Tennessee Division of Forestry

2416 Fletcher Luck Lane

Knoxville, TN 37916 USA

Phone: (865)594-6432

Fax: (865)594-5430

racox@state.tn.us

Tim Crowder

Timberwest

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USDA Forest Service

P.O. Box 377

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Natural Resources Canada

Canadian Forest Service

Laurentian Forestry Centre

1055 du P.E.P.S.

P.O. Box 3800

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Fax: (418) 648-5849

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Abul K. Ekramoddoullah

Canadian Forest Service

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Fax: (250)363-0775

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SUNY Environ. Science and Forestry

1 Forestry Drive

Syracuse, NY 13210 USA

Phone: (315)470-5746

Fax: (315)470-6934

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Lauren Fins

University of Idaho

Department of Forest Resources

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Fax: (208)885-6226

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John Hawkins

Sierra Pacific Industries

P.O. Box 39

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Phone: (530)873-9192

Fax: (530)2463

jhawkins@spi-ind.com
258 USDA Forest Service Proceedings RMRS-P-32. 2004

Conference Attendees

Liang Hsin

USDI Bureau of Land Management

P.O. Box 2695

Portland, OR 97208 USA

Phone: (503)808-6079

Fax: (503)808-6021

lhsin@or.blm.gov

Richard Hunt

Pacific Forestry Centre

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Phone: (250)363-0640

rhunt@pfc.forestry.ca

Anatoly Iroshnikov

Research Institute of Forest

Genetics and Breeding

105 Lomonosov Str

394087 Voronezh Russia

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Fax: +(0732)53-94-36

ilgis@lesgen.vrn.ru

mail@lesgen.vrn.ru

Angelia Kegley

Dorena Genetic Resource Center

USDA Forest Service

34963 Shoreview Rd

Cottage Grove, OR 97424 USA

Phone: (541)767-5703

Fax: (541)767-5709

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Hayatabad, Peshawar Pakistan

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John King

Ministry of Forests

P.O. Box 9519 STN Prov Govt

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Bohun Kinloch

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bkinloch@fs.fed.us

Jay Kitzmiller

USDA Forest Service

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Chico, CA 95928 USA

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Harvey Koester

USDI Bureau of Land Management

3040 Biddle Rd

Medford, OR 97504 USA

Phone: (541)618-2401

Fax: (541)618-2400

hkoester@or.blm.gov

Jodie Krakowski

Forest Sciences Dept.

University of British Columbia

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Vancouver, British Columbia V6T 1Z4

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Howard B. Kriebel

Ohio State University

625 Medford Leas

Medford, NJ 08055 USA

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Fax: 609-654-3625

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Umpqua National Forest

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jlinn @fs.fed.us

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Sierra Pacific Industries

P.O. Box 39

Stirling City, CA 95978 USA

Phone: (530)873-9192

Fax: (530)873-2463

glunak@spi-ind.com

Gordon Lyford

USDI Bureau of Land Management

Sprague Seed Orchard

Phone: (541)476-4432

Gordon_Lyford@or.blm.gov

Mary Frances Mahalovich

USDA Forest Service

1221 S Main Street

Moscow, ID 83843 USA

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Sheila Martinson

USDA Forest Service

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Portland, OR 97208 USA

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Geral McDonald

USDA Forest Service

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Fax: (208)883-2318

gimcdonald@fs.fed.us

Mike Meagher

MDM FORGENE

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Victoria, BC Canada

Phone: (250)727-7675

Fax: (250)727-7609

mmeagher@pfc.forestry.ca

Robert Ohrn

USDI Bureau of Land Management

1717 Fabry Rd SE

Salem, OR 97306 USA

Phone: (503)589-6883

Fax: (503)375-5715

bob_ohrn@blm.gov

David Oline

Southern Oregon University

1250 Siskiyou Blvd

Ashland, OR 97520 USA

Phone: (541)552-6799

OlineD@sou.edu

Dmitri Politov

Vavilov Institute of General

Genetics

Russian Academy of Sciences

3 Gubkin Str GSP-1

Moscow 119991 Russia

Phone: (095) 135-5067

Fax: (095) 132-8962

dvp@vigg.ru

Christine Redmond

WA Department of Natural Resources

P.O. Box 47017

Olympia, WA 98504 USA

Phone: (360)407-7576

Fax: (360)459-6872

christine.redmond@wadnr.go

Marc Rust

IETIC

UI College of Natural Resources

Moscow, ID 83844 USA

Phone: (208)885-7109

Fax: (208)885-6226

mrust@uidaho.edu
USDA Forest Service Proceedings RMRS-P-32. 2004 259

Conference Attendees

Safiya Samman

USDA Forest Service

Forest Health Protection

1601 North Kent Street

RPC, 7th Floor (FHP)

Arlington, VA 22209

Phone: (703)605-5341

Fax: (703)605-5353

ssamman@fs.fed.us

Scott Schlarbaum

University of Tennessee

Dept. of Forestry, Wildlife & Fisheries

Knoxville, TN 37901 USA

Phone: (865)974-7993

Fax: (865)974-4733

tenntip@utk.edu

Anna Schoettle

USDA Forest Service

240 W Prospect Rd

Fort Collins, OR 80526 USA

Phone: (970)498-1333

Fax: (970)498-1212

aschoettle@fs.fed.us

Richard Sniezko

USDA Forest Service

Dorena Genetic Resource Center

34963 Shoreview Rd

Cottage Grove, OR 97424 USA

Phone: (541)767-5716

Fax: (541)767-5709

rsniezko@fs.fed.us

Richard Stephan

Institute for Forest Genetics

Siekerlandstrasse 2

D-22927 Grosshansdorf Germany

Tel/Fax: +49 - (0)4102 - 63555

r-c.stephan@t-online.de

Paul Stover

USDA Forest Service

2375 Fruitridge Rd

Camino, CA 95709 USA

Phone: (530)642-5030

Fax: (530)642-5099

pstover@fs.fed.us

Terry Tuttle

USDI Bureau of Land Management

1884 Bristol Drive

Medford, OR 97504 USA

Phone: (541)779-3396

Fax: (541)846-9119

terry_tuttle@blm.gov

Nick Vagle

USDA Forest Service

P.O. Box 440

Grants Pass, OR 97528 USA

Phone: (541)471-6810

Fax: (541)471-6514

nvagle@fs.fed.us

Huo-Ran Wang

Division of Exotic Forestry

Research Institute of Forestry

Chinese Academy of Forestry

Beijing 100091 China

Phone: (086)010 62889683

Fax: (086)010 62884927; 62872015

wanghr@rif.forestry.ac.cn

Kwan-Soo Woo

Tree Breeding Division

Forest Research Institute

Korea Forest Service // Suwon,

Omokchundong 44-3,

Kyonggido, 441-350, South Korea

Telephone: 82-(0)31-290-1106

Fax: 82-(0)31-292-4458,

woo9431@yahoo.co.kr
260 USDA Forest Service Proceedings RMRS-P-32. 2004

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Flagstaff, Arizona Reno, Nevada

Fort Collins, Colorado* Albuquerque, New Mexico

Boise, Idaho Rapid City, South Dakota

Moscow, Idaho Logan, Utah

Bozeman, Montana Ogden, Utah

Missoula, Montana Provo, Utah

Lincoln, Nebraska Laramie, Wyoming

*Station Headquarters, Natural Resources Research Center,

2150 Centre Avenue, Building A, Fort Collins, CO 80526

AB C D

E FG

H I J

From upper left corner of back cover:

A. Pinus sibirica (D. Politov)

B. P. strobus (G. Daoust)

C. P. sibirica (D. Politov)

D. P. cembra (B.R. Stephan)

E. P. koraiensis (A. Boyarinov)

F. P. wallichiana x P. strobus (G. Daoust)

G. P. armandii var. amamiana (T. Sugaya)

H. P. balfouriana (D. Oline)

I. P. cembra (B.R. Stephan)

J. P. koraiensis (A. Boyarinov)

K. P. albicaulis (R. Sniezko)

L. P. monticola (R. Sniezko)

M. P. parviflora (B.R. Stephan)

N. P. lambertiana (R. Sniezko)

O. P. pumila (V. Potenko)

P. P. peuce (B.R. Stephan)

K L M

NOP

Back Cover Design: Ryan Berdeen