United States Department of Agriculture

Forest Service Rocky Mountain Research Station Proceedings RMRS-P-76 March 2018

Proceedings of the IUFRO Joint 

Conference: Genetics of Five-Needle 

Pines, Rusts of Forest Trees, and 

Strobusphere

Fort Collins, Colorado June 15–20, 2014
Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint 

conference: Genetics of five-needle pines, rusts of forest trees, and Strobusphere; 2014 

June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. Department of Agriculture, 

Forest Service, Rocky Mountain Research Station. 245 p.

Abstract

Proceedings from the 2014 IUFRO Joint Conference: Genetics of five-needle pines, rusts of forest trees, and 

Strobusphere in Fort Collins, Colorado. The published proceedings include 91 papers pertaining to research 

conducted on the genetics and pathology of five-needle pines and rusts of forest trees. Topic areas are: ecology 

and climate change, common garden genetics, genomic resources, rust resistance, and rust biology, ecology, 

and management.

Keywords: genetic variation, genetic conservation, restoration, Pinus, Populus, rust fungi, disease resistance, 

climate change, Cronartium ribicola

Editor Affiliations

Anna W. Schoettle

USDA Forest Service, Rocky Mountain Research Station

240 West Prospect Road, Fort Collins, CO 80526 USA

Richard A. Sniezko

USDA Forest Service, Dorena Genetic Resource Center

34963 Shoreview Drive, Cottage Grove, OR 97424 USA

John T. Kliejunas

Kliejunas Consulting

27919 193rd Avenue SE, Kent, WA 98042 USA

Cover photo captions: (clockwise from the upper left) Uredinia and telia of Cronartium flaccidum on Paeonia tenuifolia

(J. Kaitera); Wild Populus nigra infected with Melampsora larici-populina in the field (P. Frey); Cronartium ribicola on Pinus 

flexilis in the field (A. Schoettle); Pinus flexilis in Rocky Mountain National Park (C. Holtz); male pollen cones (strobili) on 

Pinus albicaulis seedling at Dorena Genetic Resource Center (A. Schoettle); Mature seed cone of Pinus strobiformis (A. 

Schoettle); Pinus flexilis mortality from Cronartium ribicola on Vedauwoo rock formation during field trip #1 (A. Schoettle); 

field trip #3 in Pinus aristata forest in Mt Goliath Research Natural Area (R. Sniezko).
Pesticide Precautionary Statement 

This publication reports research involving pesticides. It does 

not contain recommendations for their use, nor does it imply 

that the uses discussed here have been registered. All uses 

of pesticides must be registered by appropriate State and/or 

Federal agencies before they can be recommended.

CAUTION: Pesticides can be injurious to humans, domestic 

animals, desirable plants, and fish or other wildlife if they are 

not handled or applied properly. Use all pesticides selectively 

and carefully. Follow recommended practices for the disposal 

of surplus pesticides and pesticide containers.

Papers published in these proceedings were submitted by authors in electronic media. Editing was 

done for readability and to ensure consistent format and style. Authors are responsible for content and 

accuracy of their individual papers and the quality of illustrative materials. Opinions expressed may not 

necessarily reflect the position of the U.S. Department of Agriculture. 

The use of trade or firm names in this publication is for reader information and does not imply 

endorsement by the U.S. Department of Agriculture of any product or service. 
ii

Conference participants at Vedauwoo Recreation Area, Medicine Bow National Forest. Photo by Justin Hof, 

USDA Forest Service.
iii

Proceedings of the IUFRO Joint 

Conference: Genetics of Five-Needle 

Pines, Rusts of Forest Trees, and 

Strobusphere

Fort Collins, Colorado June 15–20, 2014

Technical Editors 

Anna W. Schoettle, USDA Forest Service

Richard A. Sniezko, USDA Forest Service

John T. Kliejunas, Kliejunas Consulting
iv

The Organizers would like to extend a heart-felt thanks to all 

the entities that contributed funds, support, and energy to help 

make this first joint conference and proceedings happen. 

Many thanks to:

USDA Forest Service, Rocky Mountain Research Station 

USDA Forest Service, Pacific Northwest Research Station 

USDA Forest Service, Western Wildland Environmental Threat Assessment Center 

USDA Forest Service, Forest Health Protection, Rocky Mountain Region 

USDA Forest Service, Forest Health Protection, Pacific Northwest Region 

USDA Forest Service, Region 6, Dorena Genetic Resource Center 

USDA Forest Service, Forest Health Protection, Southwestern Region 

USDI Bureau of Land Management, Wyoming Branch 

Whitebark Pine Ecosystem Foundation 

Sierra Pacific Industries 

American Forests
v

Organizing Committees

Co-Chairpersons Anna Schoettle, USDA Forest Service, USA 

 Richard Sniezko, USDA Forest Service, USA

Scientific Committees

IUFRO 2.02.15 Richard Sniezko, USDA Forest Service, USA 

 Anna Schoettle, USDA Forest Service, USA

 Dmitri Politov, Russian Academy of Sciences, Russia

IUFRO 7.02.05 Richard Hamelin, Natural Resources Canada, Canada 

 Pascal Frey, National Institute for Agricultural Research, France 

 Salvatore Morricca, University of Firenze, Italy

Strobusphere David Neale, University of California, Davis, USA

Local Committee Anna Schoettle, USDA Forest Service, USA 

 Kelly Burns, USDA Forest Service, USA

 William Jacobi, Colorado State University, USA

 Diana Tomback, University of Colorado Denver, USA 

 Christina Walters, USDA Agricultural Research Service, USA 

 Chris Richards, USDA Agricultural Research Service, USA 

 Christy Cleaver, Colorado State University, USA 

 Holly Kearns, USDA Forest Service, USA

 Robert Means, USDI Bureau of Land Management, USA

Poster Session Organizers Elizabeth (Libby) Pansing, University of Colorado Denver, USA 

 Marcus Warwell, USDA Forest Service, USA

Field Trip Presenters Dennis Knight, University of Wyoming, USA 

 William Jacobi, Colorado State University, USA

 Kelly Burns, USDA Forest Service, USA

 Christy Cleaver, Colorado State University, USA 

 Sara Alberts, USDA Forest Service, USA 

 Frank Romaro, USDA Forest Service, USA

 Anna Schoettle, USDA Forest Service, USA

 Diana Tomback, University of Colorado Denver, USA 

 Jeff Connor, USDI National Park Service, USA 

 Robert Cain, USDA Forest Service, USA

 Christina Walters, USDA Agricultural Research Service, USA 

 Chris Richards, USDA Agricultural Research Service, USA 

 Patricia Conine, USDA Agricultural Research Service, USA 

 Jeremiah Hyslop, USDA Forest Service, USA

 Mike Monahan, University of Denver, USA

 Smokey Bear, USDA Forest Service, USA
vi

Session Moderators Jill Wegrzyn, University of Connecticut, USA 

 Bruce Moltzan, USDA Forest Service, USA

 Andrew Eckert, Virginia Commonwealth University, USA 

 Holly Kearns, USDA Forest Service, USA

 Bill Libby, University of California, USA

 William Jacobi, Colorado State University, USA

 James Jacobs, USDA Forest Service, USA

 David Neale, University of California, USA 

 Pascal Frey, National Institute for Agricultural Research, France 

 Salvatore Moricca, University of Firenze, Italy 

 Sebastien Duplessis, National Institute for Agricultural Research, France

 Richard Hamelin, University of British Columbia, Canada 

 Diana Tomback, University of Colorado Denver, USA

Logistic Support Katie Renwick, Colorado State University, USA 

 Justin Hof, USDA Forest Service, USA

 John Popp, USDA Forest Service, USA

 Many others!

Registration, Logistics, 

and Conference Service Richard Zabel, Western Forestry and Conservation Association, USA

The Organizers would like to extend a heart-felt thanks to all the entities that contributed funds, support, and 

energy to help make this first joint conference and proceedings happen. 

Many thanks to: 

USDA Forest Service, Rocky Mountain Research Station

USDA Forest Service, Pacific Northwest Research Station

USDA Forest Service, Western Wildland Environmental Threat Assessment Center 

USDA Forest Service, Forest Health Protection, Rocky Mountain Region

USDA Forest Service, Forest Health Protection, Pacific Northwest Region

USDA Forest Service, Region 6, Dorena Genetic Resource Center

USDA Forest Service, Forest Health Protection, Southwestern Region

USDI Bureau of Land Management, Wyoming Branch

Whitebark Pine Ecosystem Foundation 

Sierra Pacific Industries 

American Forests
vii

Preface

This conference was a joint international conference of three groups: IUFRO 2.02.15 (Breeding and 

Genetic Resources of Five-Needle Pines), IUFRO 7.02.05 (Rusts of Forest Trees), and Strobusphere. The 

International Union of Forestry Research Organizations (IUFRO) is a nonprofit, nongovernmental international network of forest scientists that promotes global cooperation in forest-related research and enhances 

the understanding of the ecological, economic, and social aspects of forests and trees. This was the first 

time these three groups have met together to share research in genetics-pathology of five-needle pines and 

rusts of forest trees.

IUFRO Working Group 2.02.15—Breeding and Genetic Resources of Five-Needle Pines

Our Working Party on Breeding and Genetic Resources of Five-Needle Pines is concerned with research 

cooperation and exchange of information on all aspects of genetic research on the five-needle pines. 

This includes provenance and progeny testing, gene conservation, landscape genomics, breeding, species hybridization, clonal propagation and testing, molecular genetics, and the genetics of host-pathogen 

interactions, as well as ecology, evolutionary dynamics, and management of these species. Increasingly, 

though, we are using this knowledge to address issues related to climate change, land use pressure, and 

conservation. We welcome new members interested in these topics. Since 2001, this group has met in the 

United States, Romania, the Republic of South Korea, and Russia and was also a sponsor and organizer of 

the 2012 International Workshop on Disease and Insect Resistance in Forest Trees (IUFRO Working Group 

2.02.15 Website).

IUFRO Working Group 7.02.05—Rusts of Forest Trees

Our Working Party aims to bring together scientists and investigators working on tree rusts. Our goal is to 

foster scientific discussion and exchanges relating to tree rust epidemiology, biology, host-pathogen interactions, resistance, control and management, and genomics. Our working group meets approximately once 

every 4 years in locations in Europe, North America, or Asia. We usually meet in locations that allow us to 

discuss our scientific findings and have field trips in a friendly and relaxed environment that is conducive to 

exchanges and debates. We want to place a strong emphasis on participation of young investigators and 

students, as these meetings provide unique experiences to meet and exchange information with the related 

community (IUFRO Working Group 7.02.05 Website).

Strobusphere

The Strobusphere working group arose as a collaborative effort among researchers that began with a 

multinational White Pine Genomic Resource Workshop held in 2008. The objective of this workshop was to 

discover and identify research objectives, strengths, scope, and resources among the various working agencies. This collaborative effort is designed as a foundation to build wider scientific participation with a scope 

that spans molecular to landscape models, from host to pathogens and alternative hosts (Strobusphere 

Website).
viii
ix

Preface............................................................................................................................................................... vii

Ecology and Climate Change.............................................................................................................................1

Ecology and Genetics of Pinus strobiformis.............................................................................................1

C. Wehenkel, C.Z. Quiñones-Pérez, J.C. Hernández-Díaz, and C.A. López-Sánchez

Climate Change Alters Distribution of Whitebark Pine (Pinus albicaulis) with Respect to 

Land Management Status: Are there Implications for Adaptation?....................................................... 7

Dominique M. Davíd-Chavez, R. Travis Belote, Matthew S. Dietz, and Gregory H. Aplet 

Genetic and Ecological Divergence in Pinus flexilis–Pinus strobiformis–Pinus ayacahuite

Species Complex: Clues to Understanding Speciation in Pines .......................................................... 8

A. Moreno-Letelier, A. Ortiz-Medrano, D. Piñero, and T.G. Barraclough

Southwestern White Pine (Pinus strobiformis) Regeneration Ecology Along Disease 

and Management Gradients in the Southwestern United States ......................................................... 9

B.A. Goodrich and K.M. Waring

Limber Pine Stand Conditions (Pinus flexilis) After White Pine Blister Rust- and 

Mountain Pine Beetle (Dendroctonus ponderosae)-caused Mortality in the Central and 

Southern Rocky Mountains .................................................................................................................10

Christy M. Cleaver, William R. Jacobi, Kelly S. Burns, and Robert E. Means

Present and Possible Past Distribution of Swiss Stone Pine (Pinus cembra) in the 

Austrian Alps—The Ups and Downs of Climate Change? ..................................................................11

B. Heinze and K. Holzer

Forecasting Climate and Disease Impacts on Limber Pine in Rocky Mountain National Park ..............12

W.B. Monahan, T. Cook, F. Melton, J. Connor, B. Bobowski, and F. Krist

Projecting Future Whitebark Pine (Pinus albicaulis) Bioclimatic Envelope Distributions 

Within the Greater Yellowstone Ecosystem Under CMIP5 General Circulation Models 

for the Next Century ............................................................................................................................13

T. Chang, A.J. Hansen, and N. Piekielek

Associations Between Complete Resistance to White Pine Blister Rust and Abiotic 

Stress Tolerances in Limber Pine (Pinus flexilis) ................................................................................14

P.J. Vogan and A.W. Schoettle

Quantifying Tree Species Sustainability from Forest Inventory Data: Current Trends 

in the Pacific Northwestern United States .......................................................................................... 15

H.E. Lintz, A. Gray, A. Yost, R. Sniezko, V. Monleon, C. Woodall, M. Reilly, K. Hutten, 

M. Elliott, and D. Bachelet

Whitebark Pine (Pinus albicaulis) Growth Release Potential Following 

Overstory Disturbance ....................................................................................................................... 16

E. Shanahan, D. Roberts, K.M. Irvine, A. Litt, and K. Legg

Contents
x

White Pine Blister Rust Alters Facilitation Interactions at Treeline: Implications for Treeline 

Communities and Response to Climate Change ............................................................................... 17

D.F. Tomback, L.M. Resler, G.P. Malanson , E.K. Smith-McKenna, S.C. Blakeslee, 

and J.C. Pyatt

Whitebark Pine (Pinus albicaulis) Direct Seeding Trials in the Northern Rocky Mountains: 

the Role of Planting Site and Cache Pilferage by Rodents ................................................................ 18

E.R. Pansing, A.C. Wagner, and D.F. Tomback

White Pine Blister Rust, Mountain Pine Beetles (Dendroctonus ponderosae), and their 

Potential Impact on White Pines: A National Assessment from the National Insect 

and Disease Risk Map ....................................................................................................................... 19

F.J. Krist, A.J. McMahan, and E.L. Smith

Survey of Pinus dalatensis for Gene Conservation in Vietnam ............................................................ 20

P.H. Hai

Common-Garden Genetics.............................................................................................................................. 21

Pinus strobiformis Gene Conservation and Genecology....................................................................... 21

Betsy A. Goodrich, Kristen M. Waring, and Lluvia Flores-Renteria

Genetic Variation in Stomate Densities and Needle Traits in a Rangewide Sampling of 

Whitebark Pine (Pinus albicaulis)....................................................................................................... 30

Patrick Bennett, Jerry Hill, Douglas P. Savin, Angelia Kegley, Anna Schoettle, 

Richard A. Sniezko, Ben Bird, Jeffrey Stone

Genetic Variation in Needle Traits of Whitebark Pine (Pinus albicaulis) Seedling Families: 

Within-Population Variation at Crater Lake National Park .................................................................. 41

Richard A. Sniezko, Jerry Hill, Douglas P. Savin, Rob Mutch, Julianne Sticha, 

Angelia Kegley, and Jen Beck

Adaptive Variation Among Whitebark Pine (Pinus albicaulis) Populations from the 

Interior Northwestern United States in Relation to Climate ................................................................ 54

Marcus V. Warwell

Phenotypic Selection on Growth Rhythm in Whitebark Pine (Pinus albicaulis) in 

Low Elevation Common Gardens....................................................................................................... 59

Marcus V. Warwell

Provenance Variation in Western White Pine (Pinus monticola): The Impact of White Pine 

Blister Rust ......................................................................................................................................... 63

John N. King, Jeff DeBell, Angelia Kegley, Richard A. Sniezko, Geral McDonald, 

and Nicholas Ukrainetz

Whitebark Pine (Pinus albicaulis) Field Screening for Blister Rust Resistance in 

British Columbia: Germination Results............................................................................................... 81

Charlie Cartwright

Monitoring of Blister Rust Resistance, Pathogen Virulence, and Genetic Adaptability of 

Western White Pine (Pinus monticola) at Six Sites in Western Washington, USA ............................ 88

Amy Ramsey, Richard A. Sniezko, Robert Danchok, Dan Omdal, Angelia Kegley, 

Douglas P. Savin, and Sally Long
xi

The Genetic Architecture of Local Adaptation and the Genomic Exploration of Rugged 

Evolutionary Landscapes within Species of Pinus Subgenus Strobus............................................... 91

A.J. Eckert, B.M. Lind, C.J. Friedline, E. Hobson, J.W.R. Zinck, O.P. Rajora, 

D.B. Neale, D. Vogler, and P.E. Maloney

The Efficacy of Ex Situ Conservation of the Mexican and Central American White Pines 

(Pinus ayacahuite and P. chiapensis) in Developing Countries ......................................................... 92

W.S. Dvorak

Forest Health Protection Gene Resource Conservation: A Lasting Legacy .......................................... 93

G. Man and B. Moltzan

A Gene Conservation Program to Capture the Genetic Diversity of Rocky Mountain 

Bristlecone Pine (Pinus aristata) ........................................................................................................ 94

A.W. Schoettle, C.M. Richards, J. Popp, K.S. Burns, B.A. Goodrich, and J.G. Klutsch

Germination of Whitebark Pine (Pinus albicaulis) and Other High Elevation White Pine 

Species Seed After Long-Term Storage: Ex Situ Gene Conservation Potential of 

High Elevation White Pine Species .................................................................................................... 95

Richard A. Sniezko and Angelia Kegley

Genetic Relationship Between Reproductive Morphology and Growth Characteristics 

of Korean Pine (Pinus koraiensis) ...................................................................................................... 96

S.U. Han, H.S. Lee, J.M. Park, and K.S. Kang

Analysis of Genetic Diversity and Spatial Genetic Structure of the Endangered Dwarf Stone 

Pine at Mount Seorak by Using Nuclear Microsatellite Markers ........................................................ 97

H.I. Lim, Y.P. Hong, K.N. Hong, J.G. Han, and K.H. Jang

Population and Evolutionary Genetic Studies of Five-Needle Pines in Russia ..................................... 98

D. Politov, Y. Belokon, M. Belokon, E. Mudrik, T. Poliakova, E. Petrova, 

and S. Goroshkevich

Using Historical Provenance Test Data to Understand Tree Responses to a Changing 

Climate in Sugar Pine (Pinus lambertiana) ........................................................................................ 99

A. Bontemps, J. Wright, R. Sniezko, D. Savin, and J. Schmitt

Genetic Variation in Height and White Pine Blister Rust Resistance in Sugar Pine

(Pinus lambertiana)—15-Year Field Trial Results of Three Six-Parent Half Diallels from 

Three Breeding Zones ..................................................................................................................... 100

Jim Hamlin, Richard A. Sniezko, Fikret Isik, Douglas P. Savin, Angelia Kegley, 

Sally Long, and Robert Danchok

Patterns of Population Structure and Environmental Associations Across the Range 

of Two Ecologically and Economically Important Five-Needle Pine Species ................................... 101

S. Nadeau, N. Isabel, J. Godbout, M. Lamothe, M.-C.Gros-Louis, and K. Ritland

Population Projections and Resistance Gene Frequencies in the Presence of White Pine

Blister Rust: A Population Genetic Model for High Elevation Five-Needle Pines ............................. 102

A.W. Schoettle, J.G. Klutsch, M.F. Antolin, and S. Field
xii

Genomic Resources....................................................................................................................................... 104

Genome Sequencing in Conifers: Implications for Breeding and Gene Resource Management ......... 104

D.B. Neale

Genomic Warfare in the Woods: Scaling Fusiform Rust Resistance from Molecules 

to Landscapes .................................................................................................................................. 105

John M. Davis

Identifying the Genetic Basis of Partial Resistance on Sugar Pine (Pinus lambertiana) Under 

White Pine Blister Rust Infection ...................................................................................................... 106

A. Vázquez-Lobo, C. Vangestel, P.J. Martínez-García, J.L. Wegrzyn, I. Calic, 

J.D. Liechty, and D.B. Neale

Sequencing the Sugar Pine (Pinus lambertiana) Transcriptome......................................................... 107

J.L. Wegrzyn, P.J. Martinez-Garcia, C.A. Loopstra, K. Mockaitis, R. Famula, 

and D.B. Neale

Using Genomics Tools to Assess the Phylogeography of Whitebark Pine (Pinus albicaulis).............. 108

J.A. Gruhn and B.A. Schaal

Targeted Capture Sequencing in Whitebark Pine (Pinus albicaulis) Reveals Rangewide 

Demographic and Adaptive Patterns Despite Challenges of a Large, Repetitive Genome.............. 109

K. Nomi, C. Scelfo-Dalbey, E. Tracy, T. Jennings, R. Cronn, and J. Syring

Genetic Resources for Limber Pine (Pinus flexilis) and a Survey Focusing on Potentially 

Adaptive Single Nucleotide Polymorphisms ..................................................................................... 110

J.B. Mitton, J. Wegrzyn, S.L. Stowell, M. Mitter, L. Kueppers and D. Neale

Cross-Species Transferability of Microsatellites, and Genetic Diversity in Himalayan 

Five-Needle Pine (Pinus wallichaia) and Chir Pine (P. roxburghii) Forests ...................................... 111

H.S. Ginwal and P. Chauhan

Using Transcriptomics to Identify Candidate Genes Associated With Blister Rust Resistance 

in Whitebark Pine (Pinus albicaulis) ................................................................................................. 112

Z. Bair, R. Sniezko, J. Wegrzyn, H. Lintz, and J. Stone

Genomics Study of Western White Pine (Pinus monticola) Genetic Resistance Against 

White Pine Blister Rust .................................................................................................................... 113

J.-J. Liu and R.A. Sniezko

Genetic Markers for Western White Pine (Pinus monticola): Enabling Molecular Breeding for 

Resistance to White Pine Blister Rust ............................................................................................. 114

O.S. Urhan, L. Magalska, M.L. Rust, V. Hipkins, R.A. Sniezko, A.S. Davis, 

M.F. Mahalovich, J.-J. Liu, and G.T. Howe

Using Direct Amplification and Next-Generation Sequencing Technology to Explore Foliar

Endophyte Communities in Experimentally Inoculated Western White Pines (Pinus monticola) ...... 115

Lorinda S. Bullington and Beau G. Larkin

On the Road to Mapping Quantitative Trait Loci of Morphological and Aggressiveness 

Traits in the Poplar Rust Fungus ...................................................................................................... 116

M. Pernaci, S. De Mita, B. Fabre, A. Andrieux, F. Halkett, and P. Frey 
xiii

Genomics, Transcriptomics, and Effectoromics of the Poplar Leaf Rust Fungus 

Melampsora larici-populina .............................................................................................................. 117

B. Petre, S. Hacquard, A. Persoons, N. Saveleva, E. Tisserant, E.Morin, C. Delaruelle, 

J. Petrowski, M. Pernaci, B. Fabre, S. De Mita, F. Halkett, A. Hecker, N. Rouhier, 

P. Frey, and S. Duplessis

Rust Resistance.............................................................................................................................................. 118

Ten-Year Results of a Comandra Blister Rust Screening Trial in the Central Interior 

of British Columbia ........................................................................................................................... 118

Richard W. Reich and Sally John

Patterns of Variation in Blister Rust Resistance in Sugar Pine (Pinus lambertiana) ........................... 124

Bohun B. Kinloch, Jr., Richard A. Sniezko, Douglas P. Savin, Robert Danchok, 

Angelia Kegley, Deems Burton, and Joan Dunlap

Blister Rust Resistance in Whitebark Pine (Pinus albicaulis)—Early Results Following 

Artificial Inoculation of Seedlings from Oregon, Washington, Idaho, Montana, 

California, and British Columbia Seed Sources ............................................................................... 129

Richard A. Sniezko, Angelia Kegley, Robert Danchok, and Sally Long

Blister Rust Inoculation Trials for Whitebark Pine (Pinus albicaulis) in the Canadian 

Kootenay Region .............................................................................................................................. 136

Michael P. Murray and Vicky Berger 

Variation in Blister Rust Resistance and Early Height Growth in Three Populations 

of Southwestern White Pine (Pinus strobiformis) and Implications for Management 

and Conservation ............................................................................................................................. 140

Richard A. Sniezko, Angelia Kegley, James Jacobs, Robert Danchok, Brian Luis, 

Sally Long, and Douglas P. Savin

Persistence of Major Gene Resistance in Western White Pine (Pinus monticola) in 

British Columbia ............................................................................................................................... 149

Richard S. Hunt, Michael Murray, Richard Reich, David Rusch, Alex Woods, 

and Stefan Zeglen

Comparative Look at Rust Infection and Resistance in Limber Pine (Pinus flexilis) 

and Rocky Mountain Bristlecone Pine (P. aristata) Following Artificial Inoculation at 

Three Inoculum Densities ................................................................................................................ 151

William R. Jacobi, Holly S.J. Kearns, Angelia Kegley, Douglas P. Savin, 

Robert Danchok, and Richard A. Sniezko

Southern Rockies Rust Resistance Trial ............................................................................................. 158

Anna W. Schoettle, Kelly S. Burns, William Jacobi, John Popp, Sara Alberts, 

Tim Douville, and Frank Romaro

Sugar Pine (Pinus lambertiana) Partial Resistance Heritability Study ................................................ 162

J. Dunlap, D. Burton, D. Davis, D. Vogler, and B. Westfall

Patterns of Resistance to Cronartium ribicola in Rocky Mountain Bristlecone Pine 

(Pinus aristata) ................................................................................................................................. 163

A.W. Schoettle, R.A. Sniezko, A. Kegley, and K.S. Burns
xiv

Genetic Resistance to Cronartium ribicola in Limber Pine (Pinus flexilis)........................................... 165

A.W. Schoettle, R.A. Sniezko, A. Kegley, and K.S. Burns

Blister Rust Resistance in Whitebark Pine (Pinus albicaulis) at Crater Lake National Park ............... 167

J.S.H. Beck and R.A. Sniezko

The Definitive Guide for White Pine Blister Rust Resistance in White Pine Species

of Western North America (beta 2014.0 version)—A Tree Breeder’s Perspective ........................... 168

Richard A. Sniezko and Angelia Kegley

Performance of Blister Rust Resistant Eastern White Pine (Pinus strobus) Hybrids 

with Himalayan Blue Pine (P. wallichiana) in Field Trials ................................................................. 170

Pengxin Lu

Field-level Resistance of Western White Pine (Pinus monticola) to White Pine 

Blister Rust Assessed in Progeny Trials Planted in Coastal British Columbia ................................. 171

N. Ukrainetz and J.N. King

Genetic Architecture of Quantitative Melampsora larici-populina Leaf Rust Resistance 

in Poplars (Populus spp.) ................................................................................................................. 172

V. Jorge, A. Dowkiw, R. El-Malki, V. Segura, V. Guérin, P. Poursat, P. Faivre-Rampant, 

A. Rae, G. Taylor, and C. Bastien

Rust Biology, Ecology, and Management .................................................................................................... 173

White Pine Blister Rust Hazard Rating for 265 Sites in Southern Oregon, USA ................................ 173

Harv Koester, Douglas P. Savin, Matt Buss, and Richard A. Sniezko 

Genetic Variability in Puccinia psidii as Revealed in Commercial Plantations of 

Eucalyptus Species .......................................................................................................................... 181

Karine Kettener, Júlio César Otto, Juliana Érika de Carvalho Teixeira Yassitepe, 

Edson Luis Furtado, and Celso Luis Marino

Genetic and Genotypic Diversity of Puccinia psidii—The Cause of Guava/Eucalypt/Myrtle Rust

—And Preliminary Predictions of Global Areas at Risk .................................................................... 186

Amy L. Ross-Davis, Rodrigo N. Graça, Acelino C. Alfenas, Tobin L. Peever, 

John W. Hanna, Janice Y. Uchida, Rob D. Hauff, Chris Y. Kadooka, Mee-Sook Kim, 

Phil G. Cannon, Shigetou Namba, Nami Minato, Sofiá Simeto, Carlos A. Pérez, 

Min B. Rayamajhi, Mauricio Morán, D. Jean Lodge, Marcela Arguedas, 

Rosario Medel-Ortiz, M. Armando López-Ramirez, Paula Tennant, Morag Glen, 

Alistair McTaggart, and Ned B. Klopfenstein

Cronartium Rusts Sporulate on a Wide Range of Alternate Hosts in Northern Europe....................... 194

Juha Kaitera, Ritva Hiltunen, Berit Samils, and Jarkko Hantula

White Pine Blister Rust Confirmed on Limber Pine (Pinus flexilis) in Rocky Mountain

National Park .................................................................................................................................... 201

Anna Schoettle, Betsy Goodrich, Jen Klutsch, and Kelly Burns

Efficacy of Early Pruning to Reduce the Incidence of White Pine Blister Rust on 

Sugar Pine (Pinus lambertiana) ....................................................................................................... 205

J. Bronson, J. Petrick, and R. Danchok
xv

Comparative Genomics of the Cronartium Genus .............................................................................. 209

B. Dhillon and R. Hamelin

Colonization History, Host Distribution, and Landscape Features Shape White Pine 

Blister Rust Populations ................................................................................................................... 210

R.C. Hamelin, S. Brar, C.K.M. Tsui, B. Dhillon, M. Bergeron, D.L. Joly, 

and Y.A. El-Kassaby

The World Turned Upside Down: White Pine Blister Rust Pathogen Goes Down to Go Up............... 211

Richard A. Sniezko, Jeff Stone, and Douglas P. Savin

Widening the Landscape: The Role of Ornamental and Wild Trees in the Spread and 

Adaptation of Melampsora larici-populina Leaf Rust ....................................................................... 212

A. Dowkiw, E. Voisin, V. Dievart, T. Servouse, M. Berteloot, V. Guérin, T. Bourgeois, 

A. Andrieux, and P. Frey

Adaptation of Poplar Rust to the Poplar Varietal Landscape .............................................................. 213

K.J. Hayden, C. Xhaard, B. Fabre, F. Halkett, and P. Frey

Impact of White Pine Blister Rust on Resistant and Previously Immune Cultivated 

Ribes and Neighboring Eastern White Pine (Pinus strobus) in New Hampshire, USA .................... 214

I.A. Munck, P. Tanguay, J. Weimer, K. Lombard, S. Villani, and K. Cox

Origin and Distribution in Canada of the Cronartium ribicola vCr Race, Virulent on 

Immune Black Currant (Ribes nigrum) Cultivars Derived from Hunter’s Selections ........................ 215

P. Tanguay

Frequency and Distribution of Virulence in Cronartium ribicola to Two Simply Inherited 

Resistance Genes in Sugar Pine (P. lambertiana) and Western White Pine (P. Monticola) 

in California, USA ............................................................................................................................. 216

D.R. Vogler, A.D. Mix, D. Burton, D. Davis, R.Westfall, and B.B. Kinloch, Jr.

Stem Rust Diseases of Pinus kesiya in Vietnam ................................................................................ 217

P.Q. Thu, S. Kaneko, and M. Imazu

Barcoding the Rust Fungi of Germany ................................................................................................ 218

M. Scholler, L. Beenken, M. Lutz, W. Maier, A. Tahir and M. Thines

Geographic Distribution of Cronartium flaccidum and Peridermium pini in Sweden 

and Finland....................................................................................................................................... 219

B. Samils, P. Barklund, J. Kaitera, and J. Stenlid

Monitoring Trends in White Pine Blister Rust Infection in the Greater Yellowstone Ecosystem.......... 220

E. Shanahan, K.M. Irvine, C. Hollimon, K. Legg, R. Daley, and Greater Yellowstone 

Whitebark Pine Monitoring Working Group

The Proactive Strategy: Preparing the Landscape for Invasion by Accelerating the Evolution 

of Resistance ................................................................................................................................... 221

A.W. Schoettle, K.S. Burns, and R.A. Sniezko
xvi

White Pine Blister Rust Infection Dynamics over 25 Years in Sugar Pine (Pinus lambertiana) 

Progeny Tests on Six Sites Rated as Low, Mid, and High Rust Hazard in Southern 

Oregon, USA .................................................................................................................................... 223

Richard A. Sniezko, Robert Danchok, Douglas P. Savin, Angelia Kegley, 

and Sally Long

White Pine Blister Rust Infection in Whitebark Pine (Pinus albicaulis) Seedlings Over a Wide 

Range of Inoculum Density Levels in an Artificial Inoculation Trial .................................................. 224

Douglas P. Savin, Robert Danchok, Angelia Kegley, and Richard A. Sniezko

Cytological Analyses Reveal Variations in Nuclear Content Along the Urediniosporic Infection 

Cycle of Hemileia vastatrix and Other Rust Fungi ........................................................................... 225

S. Tavares, A.S. Pires, H.G. Azinheira, A.P. Ramos, C. Bispo, C. Andrade, A. Loureiro, 

D. Schmidt, T. Link, R.T. Voegele, R. Gardner, R. Abranches, M.C. Silva, J. Loureiro, 

and P. Talhinhas

Discovery of Mycovirus Community in the White Pine Blister Rust Ecosystems by Rust 

Transcriptome Profiling .................................................................................................................... 227

Jun-Jun Liu, A. Zamany, R.A. Sturrock, R. Hunt, and R.A. Sniezko

Research on the Rust Hyperparasite Coregonladosporium tenuissimum: Status and Prospects ........ 228

S. Moricca, B. Ginetti, A. Ragazzi, A. Lupo, and G. Assante

In Vitro Culture of the Alder (Alnus spp.) Pathogen Melampsoridium hiratsukanum: Practical 

Aspects, Problems and Future Prospects ........................................................................................ 229

B. Ginetti, A. Ragazzi, and S. Moricca

Eucalyptus grandis Anomaly Related to Expression of Defense Genes ............................................. 230

M.C.P. Fuchs-Ferraz, M.L. Acencio, N. Lemke, S. Oda, E.R. Gonzalez, and C.L. Marino

Appendix A—Field Trips ............................................................................................................................... 231

Appendix B—Participant List ....................................................................................................................... 232

Appendix C—Conference Program ............................................................................................................. 240
USDA Forest Service RMRS-P-76. 2018. 1

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

DESCRIPTION

Pinus strobiformis, subgenus Strobus, section 

Quinquefoliae (Gernandt et al. 2005), is commonly 

called Mexican white pine, Arizona white pine, 

Chihuahuan white pine, southwestern white pine, pino 

blanco, pinabete, and pino enano. Extending from 

Alberta (Canada) to the Transverse Volcanic Plateau 

of Mexico, P. strobiformis is part of a species complex 

that includes two geographically adjacent, but nonsympatric, species, namely P. flexilis and P. ayacahuite

(Farjon and Styles 1997; Moreno-Letelier and Piñero 

2009), as well as other species such as P. strobiformis subsp. veitchii, and P. stylesii (Gymnosperm 

Database 2014) (fig. 1). The conservation status of 

Ecology and Genetics of Pinus strobiformis

C. Wehenkel1, *, C.Z. Quiñones-Pérez2, J.C. Hernández-Díaz1, and C.A. López-Sánchez1

1 Universidad Juárez del Estado de Durango, Instituto de Silvicultura e Industria de la Madera and

Programa de Doctorado Institucional en Ciencias Agropecuarias y Forestales, Durango, México

2 Instituto Tecnológico del Valle del Guadiana, Villa Montemorelos, Durango, México

* wehenkel@ujed.mx

P. strobiformis is classified as “least concern and lower 

risk” according to the guidelines of the International 

Union for the Conservation of Nature and Natural 

Resources (IUCN) Red List of Threatened SpeciesTM 

(IUCN 2014). In Mexico, this frequent tree species 

is protected and included in Mexican Regulation 

NOM-059-2010.

Pinus strobiformis is a native, evergreen conifer with 

an open, irregular crown. It can grow to 27 m tall with 

a diameter up to 1 m; the bark is thin, rough, and furrowed. Branches are long and horizontal to pendant. 

Needles are 6 to 10 cm long in fascicles of five. The 

cones are 7 to 25 cm long with reflexed, thick scales; 

Abstract—Extending from the Canadian province of Alberta to the Transverse Volcanic Plateau of Mexico, 

Pinus strobiformis is part of a species complex with two geographically adjacent, but nonsympatric, species, namely P. flexilis and P. ayacahuite. Pinus strobiformis is classified as being of least concern and at 

lower risk according to the guidelines of the International Union for the Conservation of Nature and Natural 

Resources (IUCN) Red List (IUCN 2014). In Mexico, this frequent tree species is protected and included in 

Mexican Regulation NOM-059-2010. Pinus strobiformis appears to have broad environmental tolerances 

and fire resistance when mature. It is often found in mixed-conifer forests that are valuable summer habitat 

for big and small game animals, rodents, and game and nongame birds. At the large scale, analyses of 

spatial genetic structure of P. strobiformis indicated a very weak spatial distance–genetic distance relationship, probably resulting from a strong seed interchange, likely by birds, particularly the Mexican jay 

(Aphelocoma wollweberi), and from a continuous and broad geographic distribution of this tree species. 

The highest genetic diversity was found in the southern populations, indicating the recent colonization of 

the populations at the northern distributional limit. Recently, interest in P. strobiformis has increased due to 

the discovery of white pine blister rust (causal agent: Cronartium ribicola) in the Sacramento Mountains of 

New Mexico (USA).

ECOLOGY AND CLIMATE CHANGE
2 USDA Forest Service RMRS-P-76. 2018.

cones are dehiscent when mature (figs. 2 and 3). Seeds 

are essentially wingless and 10 to 12 mm long (Pavek 

1993; Perry 1991).

DISTRIBUTION AND OCCURRENCE

Pinus strobiformis has a limited distribution. It occurs in the mountains of western Texas, New Mexico, 

Arizona, and southwestern Colorado (USA) (Jones 

1974; Samano and Tomback 2003). It also extends 

along the mountains to central Mexico (Farjon and 

Styles 1997), ranging from northeastern Sonora to 

Western Chihuahua and Durango, northern Zacatecas, 

southern Coahuila, and central Nuevo Leon (U.S. 

Geological Survey 2006). The largest populations 

are found in Mexico (Pavek 1993). This species typically occurs in low densities in southwestern pine, 

mixed-conifer, and spruce-fir (Picea spp.-Abies spp.) 

forests (Stromberg and Patten 1991), which generally 

have been the subject of relatively few studies (Jones 

1974). 

This pine species is associated with several other pine 

species including P. ponderosa, P. arizonica, P. leiophylla, P. engelmannii, and P. cembroides. Pinus strobiformis also is associated with Pseudotsuga menziesii, 

Populus tremuloides, Abies concolor, Abies bifolia, 

Picea pungens, Picea engelmannii, Juniperus deppeana, Cupressus arizonica, Arbutus arizonica, and a 

variety of Quercus species. In Mexico, this tree species 

is associated with a wide variety of endemic Mexican 

species such as Picea chihuahuana, Cupressus lusitanica, Abies durangensis, Abies vejari, and Abies religiosa (Looney and Waring 2013; Simental-Rodríguez 

et al. 2014).

Figure 1—Natural range of Pinus strobiformis (from Little, E.L., Jr., 1971).
USDA Forest Service RMRS-P-76. 2018. 3

Figure 2—Pinus strobiformis trees (a and b) in Durango State, Mexico (photo: C. Wehenkel).

Figure 3—Female flowers (a) and cones (b) of Pinus strobiformis trees in Durango State, Mexico (photo: C. Wehenkel).

a b

a b
4 USDA Forest Service RMRS-P-76. 2018.

ECOLOGY

Pinus strobiformis appears to have broad environmental tolerances (Looney and Waring 2013) and fire resistance when mature (Dieterich 1983). It is widespread 

in mesic sites on ridges, slopes, and canyons of montane zones (Pavek 1993). These zones have a semiarid 

climate, with mild winters and warm summers. The 

average precipitation ranges from 487 to 1,143 mm per 

year. In its U.S. range, P. strobiformis typically grows 

at elevations ranging from 1,900 to 3,000 m above sea 

level (Pavek 1993). In Mexico, it is found at elevations 

from 2,200 to 3,600 m above sea level (AguirreCalderón et al. 2003; Fulé and Covington 1996). 

The range of P. strobiformis is influenced by a variety 

of environmental factors, including elevation (Brady 

and Bonham 1976), moisture availability (Niering and 

Lowe 1984), and temperature (Laughlin et al. 2011). 

The species usually grows on moist, cool sites, where 

it is most likely to be dominant. It is preferentially 

located on dry rocky slopes in high mountains, or as a 

minor component in mixed-conifer forests. The species is found in sites with loamy soil textures ranging 

from shallow, gravelly loams to deep, sandy loams and 

stony silty clay loams (Pavek 1993). Depending on elevation, this species may occur on a variety of aspects 

and topographic settings (Looney and Waring 2013).

GENETICS

Moreno-Letelier and Piñero (2009) presented patterns 

of genetic variation between the taxa in the P. strobiformis complex by chloroplast haplotype (microsatellite) analysis, which supports the concept of significant 

distinctions between each of the taxa named here, and 

between the Sierra Madre Occidental, Sierra Madre 

Oriental, and Jalisco populations of P. strobiformis

(Gymnosperm Database 2014). Two main genetic and 

geographic groups of P. strobiformis were separated by 

the Chihuahuan Desert. Little population differentiation and no isolation by distance within each of the 

two groups were found, suggesting genetic connectivity as a result of population expansions within these 

areas during glacial stages.

Quiñones-Pérez et al. (2014) observed a nonsignificant 

autocorrelation in fine-scale spatial genetic structure 

(SGS) using amplified fragment length polymorphism 

(AFLP), suggesting that the genetic variants of the tree 

species are randomly spatially distributed within sampled plots of 50 m × 50 m. On a larger scale, the SGS 

analyses of P. strobiformis also indicated a very weak 

spatial distance–genetic distance relationship, probably 

resulting from a strong seed interchange, likely by 

birds, particularly the Mexican jay (Aphelocoma wollweberi), and from a continuous and broad geographic 

distribution of this tree species (Looney and Waring 

2013). The overall mean of genetic distances between 

P. strobiformis trees was higher than between Picea 

chihuahuana trees, Populus tremuloides trees, and 

Pseudotsuga menziesii trees in the rare Mexican Picea 

chihuahuana tree community. 

Simental-Rodríguez et al. (2014) found no evidence 

that tree species diversity is related to adaptive AFLP 

structure and mean genetic diversity per AFLP locus 

of P. strobiformis at 12 locations in the Picea chihuahuana tree community on the Mexican Sierra Madre 

Occidental (in contrast to Populus tremuloides, Picea 

chihuahuana, and Pseudotsuga menziesii). As seen in 

Moreno-Letelier and Piñero (2009), the highest genetic diversity was found in the southern populations, 

indicating recent colonization of the populations at the 

northern distributional limit (Petit et al. 2003).

Villalobos-Arámbula et al. (2014) examined crossspecies transferability and usefulness of six nuclear 

microsatellite markers developed in consubgeneric 

eastern white pine (Pinus strobus) with regard to ecologically and commercially important Mexican white 

pine species of conservation genetics concern: Pinus 

chiapensis, P. flexilis, P. strobiformis, P. ayacahuite, 

and P. ayacahuite var. veitchii. Four to six microsatellite loci were found to be polymorphic in different 

species.

MANAGEMENT CONSIDERATIONS

Pinus strobiformis is often found in mixed-conifer forests that are valuable summer habitat for big and small 

game animals, rodents, and game and nongame birds. 

As natural regeneration of clearcut mixed-conifer 

forests on south-facing slopes requires 50 to 100 years, 

these clearcut areas can be a valuable long-term forage 

resource for deer (Odocoileus spp.) and elk (Cervus 

elaphus) (Pavek 1993).
USDA Forest Service RMRS-P-76. 2018. 5

This pine species is the principal host of Apache dwarf 

mistletoe (Arceuthobium apachecum). Infected trees 

have reduced growth and increased susceptibility to 

other infections, insects, and mortality (Hawksworth 

1978; Walters 1978). Pinus strobiformis is infected by 

red ring rot and by root and butt rots (Walters 1978). 

Recently, interest in P. strobiformis has increased 

due to the discovery of white pine blister rust (causal 

agent: Cronartium ribicola) in the Sacramento 

Mountains of New Mexico (Hawksworth 1990). 

Cronartium ribicola is a potentially lethal introduced 

pathogen that threatens white pine species (Conklin 

2004; Schoettle 2004; Zeglen et al. 2010). 

Despite the research to date on white pine blister rust, 

there is insufficient information to predict how this 

disease will affect P. strobiformis in the future (Looney 

and Waring 2013). Other disturbances of P. strobiformis include fire (Danzer et al. 1996; Villanueva-Diaz 

and McPherson 1995), and climate change (Seager et 

al. 2007). A few studies reported effects of insects or 

animals on this species (Hawksworth et al. 2002).

Wood of P. strobiformis is soft, not resinous, and white 

with a slightly darker heartwood. It can be used for 

cabinetry, doors, and window frames. Crooked stems 

and coarse branches make it undesirable for lumber 

(Pavek 1993).

REFERENCES

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Horst; [et al.]. 2003. Análisis estructural de ecosistemas 

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Brady, Ward; Bonham, Charles D. 1976. Vegetation patterns 

on an altitudinal gradient, Huachuca Mountains, Arizona. 

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Danzer, Shelly R.; Baisan, Chris H.; Swetnam, Thomas 

W. 1996. The influence of fire and land-use history 

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mixed-conifer: A case study. Forest Ecology and 

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Botanical Garden. 291 p.

Fulé, Peter Z.; Covington, W.W. 1996. Changing fire 

regimes in Mexican pine forests: Ecological and 

management implications. Journal of Forestry. 94: 33–38.

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Sol; [et al.]. 2005. Phylogeny and classification of Pinus. 

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strobiformis.php. (15 October 2015).

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mistletoe in relation to control. In: Scharpf, Robert F.; 

Parmeter, John R., Jr., tech. coords. Proceedings of the 

symposium on dwarf mistletoe control through forest 

management. Gen Tech. Rep. PSW-31. Berkeley, CA: 

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southern New Mexico. Plant Disease. 74: 938.

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J.C.; Moody, B., tech. coords. Mistletoes of North 

American conifers. Gen. Tech. Rep. RMRS-GTR-98. 

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Service, Rocky Mountain Research Station: 29–56.

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of threatened species. Version 2014.2. http://www.

iucnredlist.org/. (15 October 2015).

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Pap. RM-RP-122. Fort Collins, CO: U.S. Department of 

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[et al.]. 2011. Climatic constraints on trait-based forest 

assembly. Journal of Ecology. 88: 1489–1499. 

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1, conifers and important hardwoods. Misc. Pub. 1146. 

Washington, DC: U.S. Department of Agriculture. 9 p., 

200 maps. 
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Looney, Christopher E.; Waring, Kristen M. 2013. Pinus 

strobiformis (southwestern white pine) stand dynamics, 

regeneration, and disturbance ecology: A review. Forest 

Ecology and Management. 287: 90–102.

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Phylogeographic structure of Pinus strobiformis Engelm. 

across the Chihuahuan Desert filter-barrier. Journal of 

Biogeography. 36: 121–131.

Niering, William A.; Lowe, Charles H. 1984. Vegetation 

of the Santa Catalina Mountains: community types and 

dynamics. Plant Ecology. 58: 3–28.

Pavek, Diane S. 1993. Pinus strobiformis. In: Fire effects 

information system [online]. Missoula, MT: U.S. 

Department of Agriculture, Forest Service, Rocky 

Mountain Research Station, Fire Sciences Laboratory. 

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html. (15 October 2015).

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America. Portland, OR: Timber Press. 231 p.

Petit, Rémy J.; Aguinagalde, Itziar; De Beaulieu, JacquesLouis L.; [et al.]. 2003. Glacial refugia: Hotspots but 

not melting pots of genetic diversity. Science. 300: 

1563–1565.

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Genetica. 63(4): 149–159.

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phenology, seed dispersal, and seed predation in 

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loss. In: Sniezko, R.A.; Samman, S.; Schlarbaum, 

S.E.; [et al.], comps. Breeding and genetic resources 

of five-needle pines: Growth, adaptability and pest 

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the spruce-fir forests on the Pinaleno Mountains, Graham 

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tree species range maps, from Little, E.L., Jr. 1971. 

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hardwoods, vol. 1. Misc. Pub. 1146. Washington, DC: 

U.S. Department of Agriculture. 9 p., 200 maps.

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A.; Rajora, O.P. 2014. Cross-species transferability of 

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markers to five Mexican white pines. Genetics and 

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stand structure in mountains of Sonora, Mexico and New 

Mexico, USA. In: Debano, L.F.; Gottfried, G.J.; Hamre, 

R.H.; [et al.], tech. coords. Biodiversity and management 

of the Madrean Archipelago: The sky islands of 

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USDA Forest Service RMRS-P-76. 2018. 7

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

INTRODUCTION AND METHODS

Climate-based predictions of species distributions provide information for understanding impacts of climate 

change. However, species distribution models do not 

include land management zones as input. Improving 

understanding of geographic shifts will require consideration of management zones where certain activities could facilitate or impede responses to climate 

change. For instance, assisted migration to overcome 

dispersal barriers could be challenged in areas designated for “untrammeled” character (e.g., wilderness). 

Alternatively, large tracts of land with uninterrupted 

disturbance regimes may offer opportunities for 

disturbance-facilitated establishment of species into 

new areas. We focused on current and future distributions of whitebark pine (Pinus albicaulis), a keystone 

species of treeline. Current and future “climate space” 

of whitebark pine were overlaid onto ecoregions and 

land management zones. We also used different thresholds of predicted presence to assess the sensitivity of 

patterns to varying levels of confidence. 

Climate Change Alters Distribution of Whitebark Pine (Pinus 

albicaulis) With Respect to Land Management Status: Are there 

Implications for Adaptation?

D.M. Davíd-Chavez1, *, R.T. Belote2, M.S. Dietz3, and G.H. Aplet4

1 Colorado State University, Human Dimensions of Natural Resources,

Fort Collins, Colorado, USA 

2 The Wilderness Society, Research Department, Bozeman, Montana, USA

3 The Wilderness Society, Research Department, San Francisco, California, USA

4 The Wilderness Society, Research Department, Denver, Colorado, USA

*

 d.mari.david@gmail.com

RESULTS AND CONCLUSIONS

Geographic distribution of viable “climate space” for 

whitebark pine is predicted to contract 86 percent by 

2060 across the western United States based on our 

most inclusive confidence estimate. As climate change 

reduces the total area of viable habitat, distribution of 

whitebark pine in designated wilderness is expected 

to nearly double. The U.S. Department of Agriculture, 

Forest Service climate model predicts that whitebark 

pine range occurring within wilderness boundaries 

will increase from 38 percent in 2010 to 62 percent 

by 2060. Climate adaptation and restoration strategies 

need to consider the influence of land management 

zones as species shift in response to climate change. 

From our analysis, we developed a simple conceptual 

model that managers could use when deciding what 

level of active restoration to perform in protected 

areas set aside to sustain “untrammeled” conditions. 

We discuss our results in the context of ongoing and 

predicted whitebark pine decline, climate change, 

restoration, and wilderness character. 
8 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Genetic and Ecological Divergence in the Pinus flexilis–Pinus 

strobiformis–Pinus ayacahuite Species Complex:

Clues to Understanding Speciation in Pines

A. Moreno-Letelier1, 2, *, A. Ortiz-Medrano1, D. Piñero1,

and T.G. Barraclough2

1 Universidad Nacional Autonoma de México, Departamento de Ecología Evolutiva,

Instituto de Ecologia, México D.F., Mexico 

2 Imperial College London, Division of Biology, Ascot, Berkshire, United Kingdom

*

 aleml@yahoo.com

North America is a secondary center of diversification 

of the genus Pinus. Most of these speciation events are 

fairly recent, producing a pattern of widespread shared 

polymorphism and fuzzy species boundaries. Among 

five-needle pines, the relationships among Pinus 

flexilis, Pinus strobiformis, and Pinus ayacahuite have 

been particularly difficult to determine. To understand 

the speciation process and the phylogenetic relationships in this species complex, we evaluated genetic 

and ecological differentiation using multilocus sequence data and ecological niche model comparisons. 

Our results show Late Pliocene–Early Pleistocene 

divergence and a contrasting pattern of differentiation 

between genes and ecological niche. Cytoplasmic and 

housekeeping nuclear genes show a clear differentiation of P. ayacahuite. However, ecological niche 

differences and candidate genes for drought tolerance 

show a strong differentiation in P. flexilis. Significant 

correlation was found between genetic distance and 

environmental distance in some of these candidate 

genes, as well as evidence of natural selection in 

P. flexilis.

This discrepancy among genes could be evidence of 

a genomic mosaic product of ecological speciation 

in P. flexilis. In adaptive speciation, only those genes 

under natural selection would become differentiated, 

whereas in allopatric or parapatric speciation, differentiation would occur in the entire genome. Additionally, 

the ecological differentiation of P. flexilis could represent a barrier to gene flow with its sister species.

The differentiation of P. flexilis at the ecological and 

candidate gene level suggests that adaptive divergence 

has played a role in speciation. This means that despite 

their long generation times, pines can adapt to new 

environments relatively quickly, and that local adaptation could be an important factor in causing population 

structure and speciation in other five-needle pines. 

Considering the broad latitudinal range of this species 

complex, it is also possible that divergent selection 

could be acting on other genomic regions that were 

not sampled in this study, which in turn would explain 

the differences between the tropical P. ayacahuite and 

subtropical P. strobiformis. Therefore, more work is 

needed to characterize the genetic structure in these 

pines at the genomic level.
USDA Forest Service RMRS-P-76. 2018. 9

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Southwestern White Pine (Pinus strobiformis) Regeneration

Ecology Along Disease and Management Gradients in the 

Southwestern United States

B.A. Goodrich1 and K.M. Waring1, *

1 Northern Arizona University, School of Forestry, Flagstaff, Arizona, USA 

*

 kristen.waring@nau.edu

Southwestern white pine (Pinus strobiformis, SWWP) 

is a component of dry and wet mixed-conifer forests in 

the southwestern United States and provides ecological function and diversity. The species is geographically limited to Arizona, New Mexico, parts of Texas, 

southern Colorado, and northern Mexico, but occurs 

on a wide range of habitats within its distribution. 

Historically, SWWP regeneration has not been well 

studied or monitored in mixed-conifer forests of the 

Southwest, although our understanding of the species’ 

ecology is increasing. Introductions of Cronartium 

ribicola, the pathogen responsible for causing white 

pine blister rust (WPBR), are fairly recent compared 

to other areas in North America, and disease intensity 

is variable in the Southwest. Heavy disease occurs in 

south-central New Mexico, whereas many forests in 

Arizona are not affected yet. However, the disease 

continues to spread west through isolated mountain 

ranges in the Southwest.

Our research seeks to quantify SWWP regeneration 

responses to various silvicultural strategies across 

a range of WPBR intensities, and to determine how 

disease incidence has been affected by recent management in the Southwest. The opportunity to study 

regeneration in stands not yet affected by WPBR allows us to make baseline comparisons for management 

goals and to determine if stand trajectories in areas 

where WPBR has been present for several decades are 

occurring in areas with more recent infestations.

Fifty-five stands were surveyed across the region, including stands with no recent management and stands 

with recent uneven-aged (UEA) treatments (leaving 

two-aged and multi-aged structures). Seedling ages 

(by whorl count) demonstrate germination following 

treatments; survey results indicate that UEA treatments 

successfully regenerate SWWP in areas with and 

without WPBR. Results suggest that SWWP grows 

most successfully in two-aged structures, but models 

interpreting the effects of management, disease, and 

other species’ presence on SWWP densities across the 

region are in progress. Regeneration stem mapping 

will also allow comparisons of species distributions 

and possible competitive interactions between similar 

and different size classes in the understory.

Regeneration success of species at risk of biotic invasions is critical to the sustainability of current and 

future distributions, and SWWP regeneration ecology 

must be better understood in order to make sound 

management decisions in the face of climate change 

and other disturbances. Previous work addressing 

proactive management in other North American fiveneedle pine systems advises that increases in regeneration diversity and densities are critical to maintaining 

populations on the landscape. These results will help 

guide management efforts in sustaining SWWP in the 

presence of an invasive pathogen.
10 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Limber Pine (Pinus flexilis) Stand Conditions After White Pine Blister 

Rust- and Mountain Pine Beetle (Dendroctonus ponderosae)-caused 

Mortality in the Central and Southern Rocky Mountains

C.M. Cleaver1, *, W.R. Jacobi1, K.S. Burns2, and R.E. Means3

1 Colorado State University, Department of Bioagricultural Sciences and Pest Management, Fort Collins, Colorado, USA

2 USDA Forest Service, Forest Health Protection, Golden, Colorado, USA

3 USDI Bureau of Land Management Wyoming, Cheyenne, Wyoming, USA 

* christycleaver@fs.fed.us

Mountain pine beetle (Dendroctonus ponderosae) and 

white pine blister rust (causal agent: Cronartium ribicola) are causing extensive crown dieback and mortality 

in limber pine (Pinus flexilis) in the central and southern 

Rocky Mountains. Ecologically valuable limber pines 

often grow in fragile ecosystems where few other 

trees can grow. The combined effects of mountain 

pine beetle, white pine blister rust, dwarf mistletoe 

(Arceuthobium cyanocarpum), and climate change 

could greatly reduce the biodiversity of these ecosystems. Current status and long-term monitoring of limber 

pine trees and seedlings are needed to advise land managers and to implement restoration. The study objectives 

were to: (1) assess site, stand, and health characteristics 

of seedlings and mature limber pines in Colorado, 

Wyoming, and Montana (USA); (2) determine factors 

that influence the occurrence and incidence of white 

pine blister rust, bark beetles, and dwarf mistletoe; and 

(3) determine factors that affect seedlings, including 

site, stand, and meteorological characteristics, and white 

pine blister rust. In 2011 and 2012, we assessed 22,700 

limber pines on 508 plots in limber pine-dominated 

stands in 25 study areas in Colorado, Wyoming, and 

Montana. Mean density of live limber pine was 311 

stems ha-1. Fifty percent of standing limber pine trees 

were classified as healthy, 26 percent were declining 

or dying, and 24 percent were dead. White pine blister 

rust was the primary damage agent and was widespread, 

occurring in 23 of the 25 study areas, on 73 percent of 

plots with a mean incidence of 26 percent. Bark beetlecaused mortality occurred in all 25 study areas, and 18 

percent of standing limber pines were killed by bark 

beetles. Limber pine dwarf mistletoe occurred within 

20 study areas, on 29 percent of plots, with a mean incidence of 9 percent. Live limber pine seedling density 

averaged 141 stems ha-1. Of all standing live and dead 

limber pine seedlings, 1.5 percent were dying, 4.4 percent were dead, and white pine blister rust occurred on 

5.3 percent of live seedlings. 

We used statistical modeling to determine the meteorological, macrosite, and microsite factors and stand 

factors that influenced the occurrence and incidence of 

white pine blister rust, bark beetles, and dwarf mistletoe 

on limber pines. We also used statistical modeling to 

determine factors that predict density (stems ha-1) of 

limber pine seedlings and proportion of limber pine 

seedlings with white pine blister rust. The widespread 

occurrence of limber pine stands heavily affected by 

mountain pine beetle and white pine blister rust, combined with low regeneration density, suggests that these 

areas should be monitored so land managers can decide 

if restoration efforts are warranted.

ACKNOWLEDGMENTS

This study was funded by the USDA Forest Service, 

Forest Health Monitoring Evaluation Monitoring 

Program; the U.S. Department of the Interior, Bureau 

of Land Management Wyoming; Colorado Agricultural 

Experiment Station; Colorado State Forest Service; and 

Boulder County Open Space. 
USDA Forest Service RMRS-P-76. 2018. 11

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Present and Possible Past Distribution of Swiss Stone Pine

(Pinus cembra) in the Austrian Alps—The Ups and Downs

of Climate Change

B. Heinze1, * and K. Holzer2

1 Federal Research Centre for Forests (BFW), Department of Genetics, Vienna, Austria

2 Klausen-Leopoldsdorf, Austria

*

 berthold.heinze@bfw.gv.at

Abstract—Swiss stone pine (Pinus cembra) is currently restricted to the uppermost subalpine forest belt in 

the central and eastern Alps in Austria, where high mountain national parks are important for this species. 

Climate change threatens this habitat, as global and regional warming tends to enhance the competitiveness of other tree species—European larch (Larix decidua) and Norway spruce (Picea abies)—while an 

upslope migration “escape” is often not possible for orographic reasons. However, records suggest that this 

pattern has recurred throughout post-glacial history, and it is tempting to speculate whether it has resulted 

in the absence of the species from many mountain chains. Human overexploitation has certainly added to 

the species’ current condition. This raises the question as to what approximate minimum number of trees 

is necessary to maintain these tree populations, especially when they are in isolation as a consequence of 

fragmentation of formerly continuous stone pine belts. Any such predictions will also have implications for 

genetic diversity.

We will present results from a survey of literature and data regarding past and present distribution of the 

species in Austria. A striking feature is that many places mentioned in older literature (about 1900) as rare 

outposts often do not seem to have any remaining Swiss stone pine trees, at least in a density relevant 

for forest inventory data. Orographic features (that is, whether the terrain allows the species to escape 

to higher elevations in warmer periods) may have an impact on its current distribution range on the eastern edge of the Alps, as there are dispersal limits that apparently affect the main distribution agent, the 

Eurasian nutcracker bird (Nucifraga caryocatactes). We are starting a genetic investigation in very small 

stands, trying to assess minimum viable population sizes and densities for this pine species. 
12 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Forecasting Climate and Disease Impacts on Limber Pine

(Pinus flexilis) in Rocky Mountain National Park

W.B. Monahan1, 2, *, T. Cook1, 3, F. Melton4, J. Connor5, B. Bobowski5, and F. Krist2

1 U.S. Department of the Interior (DOI), National Park Service, Inventory and Monitoring Division, Fort Collins, Colorado, 

USA

2 USDA Forest Service, Forest Health Technology Enterprise Team, Fort Collins, Colorado, USA

3 USDI, National Park Service, Biological Resource Management Division, Fort Collins, Colorado, USA

4 USDOC, National Aeronautics and Space Administration, Ames Research Center, Cooperative for Research in Earth 

Science and Technology, Moffett Field, California, USA

5 USDI, National Park Service, Rocky Mountain National Park, Estes Park, Colorado, USA

*

 wmonahan@fs.fed.us

Abstract—Five-needle pine management in the face of climate change is challenging because tree 

disease may exacerbate climate impacts in complex ways. Here, using climate-based ecological niche 

models that forecast species’ potential distributions through 2100, plus near-term estimates of tree mortality from the U.S. Department of Agriculture, Forest Service 2013–2027 National Insect and Disease Risk 

Assessment, we quantify the potential impact of climate and disease on limber pine (Pinus flexilis) in 

Rocky Mountain National Park (Colorado, USA). Niche models trained and projected under a wide range 

of conditions and uncertainties all predict the distribution of limber pine in the park to move upslope in 

elevation, resulting in a patchwork of predicted range stability, loss, and colonization under future climate 

change. Disease risk projections further identify areas within the park where limber pine is expected to 

suffer declines in basal area, which, depending on the magnitude, may feed back into the niche models 

to either encourage persistence or accelerate loss. We rank the combined impact of climate and disease 

on limber pine throughout the park and explore how our results may be used to assess vulnerability and 

develop management scenarios.
USDA Forest Service RMRS-P-76. 2018. 13

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Projecting Future Whitebark Pine (Pinus albicaulis) Bioclimatic 

Envelope Distributions Within the Greater Yellowstone Ecosystem 

Under CMIP5 General Circulation Models for the Next Century

T. Chang1, *, A.J. Hansen1, and N. Piekielek1

1 Montana State University, Department of Ecology and Environmental Science, Bozeman, Montana, USA

* tony.chang@msu.montana.edu

Abstract—Projected climate change at a regional level is expected to shift vegetation habitat distributions 

over the next century. For the species whitebark pine (Pinus albicaulis), warming temperatures may result 

in loss of suitable bioclimatic habitat, reducing its distribution within its historical range. This research 

focuses on understanding the patterns of spatiotemporal variability for future projected P. albicaulis suitable 

habitat in the Greater Yellowstone Ecosystem (GYE) through a bioclimatic envelope approach. Because 

intermodal variability from general circulation models (GCMs) leads to differing predictions about the 

magnitude and direction of modeled suitable habitat area, the Coupled Model Intercomparison Project 

Phase 5 (CMIP5) used nine bias-corrected statistically downscaled GCMs to understand the uncertainty 

associated with modeled projections. Modeled P. albicaulis for the 1980–2010 climate showed strong 

presence/absence separations by summer maximum temperatures and springtime snowpack. Patterns of 

projected habitat change by the end of the century suggested a constant decrease in suitable climate area 

from the 2010 baseline for both representative concentration pathways (RCPs) 4.5 and 8.5 climate forcing 

scenarios. Percent suitable climate area estimates ranged from 29 to 2 percent and 10 to 0.04 percent by 

2099 for RCP 4.5 and 8.5, respectively. Habitat projections between GCMs displayed a decrease of variability over the 2010–2099 time period related to consistent warming above the 1910–2010 temperature 

normal after 2070 for all GCMs. A decreasing pattern of projected suitable P. albicaulis habitat area was 

consistent across GCMs, despite strong differences in magnitude. Future ecological research in species 

distribution modeling should consider a full suite of GCM projections in the analysis to better explore range 

contractions/expansions generated by species niche modeling. The results suggest that restoration strategies such as planting of seedlings and controlling competing vegetation may be necessary to maintain 

P. albicaulis in the GYE under the more extreme future climate scenarios.
14 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Associations between Complete Resistance to White Pine Blister 

Rust and Abiotic Stress Tolerances in Limber Pine (Pinus flexilis)

P.J. Vogan1 and A.W. Schoettle1, *

1 USDA Forest Service, Rocky Mountain Research Station, Fort Collins, Colorado, USA

* aschoettle@fs.fed.us

During the naturalization process of white pine blister 

rust (WPBR, caused by Cronartium ribicola), susceptible pine genotypes will be selected against, along with 

any traits that are associated with those genotypes, 

potentially causing a shift in the suite of traits in the 

post-selection population compared to the native population. Consequently, identifying associated traits with 

WPBR resistance is critical for effective restoration 

planning, especially in a changing climate. We sought 

to determine whether complete resistance to WPBR in 

limber pine (Pinus flexilis) is associated with abiotic 

stress tolerances, particularly those related to freezing 

and drought. Previous research has noted that proteins 

involved in partial resistance to C. ribicola also exhibit 

antifreeze capabilities in vitro and very likely enhance 

plant frost tolerance in vivo; these proteins are also expressed in response to cold and, occasionally, drought. 

It is not known if a similar association exists between 

the major gene in limber pine (Cr4) and tolerance of 

abiotic stress. 

We raised 1,020 seedlings of limber pine from 10 

rust-resistant (R) and 10 rust-susceptible (S) openpollinated families (Schoettle et al. 2014), paired as 

1 R and 1 S family from each site of origin to filter 

out potential geographic trait variation. In a series of 

studies, R families exhibited greater frost tolerance 

than S families, which may imply a genetic association 

between Cr4 and cold tolerance gene(s) or a direct 

cryoprotective function of the Cr4 protein product(s) 

itself. Wintertime freezing tolerance of R and S families was not affected by inoculation with C. ribicola

the previous fall. This result suggests that differences 

in frost tolerance between R and S families were not 

induced by pathogen infection or resistance expression 

and instead frost tolerance is a constitutive trait of 

families (Vogan and Schoettle 2015). 

We found a tendency for S family seedlings to retain 

somewhat greater stomatal conductance under modest 

drought than R families and no significant differences under severe (<–2.0 MPa) drought (Vogan and 

Schoettle 2015). Drought during the summer resulted 

in marginally, but not significantly, greater frost tolerance in seedlings the following winter, suggesting that 

mechanisms induced by drought may have enhanced 

cold hardiness in the ensuing months. The correlation 

between cold tolerance and rust resistance may provide an explanation of how Cr4 resistance exists and 

persists in limber pine before the arrival of the corresponding pathogen as cold tolerance traits are usually 

under strong selection in forest trees. 
USDA Forest Service RMRS-P-76. 2018. 15

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Quantifying Tree Species' Sustainability from Forest Inventory Data: 

Current Trends in the Pacific Northwestern United States

H.E. Lintz1, A. Gray2, A. Yost3, *, R. Sniezko4, V. Monleon2, C. Woodall5, M. Reilly6,

K. Hutten7, M. Elliott8, and D. Bachelet9

1 Oregon State University, College of Earth, Ocean, and Atmospheric Science, Oregon Climate Change Research Institute, 

Corvallis, Oregon, USA

2 USDA Forest Service, Pacific Northwest Research Station, Corvallis, Oregon, USA

3 Oregon Department of Forestry, Salem, Oregon, USA

4 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA

5 USDA Forest Service, Northern Research Station, St. Paul, Minnesota, USA

6 Oregon State University, Department of Forest, Ecosystems, and Society, Corvallis, Oregon, USA

7 University of Washington, Department of Forest Ecology, Seattle, Washington, USA

8 Washington State University, Puyallup Research and Extension Center, Puyallup, Washington, USA

9 Oregon State University, Department of Biological and Ecological Engineering,

and Conservation Biology Institute, Corvallis, Oregon, USA

* ayost@odf.state.or.us

Abstract—The task of guiding forests toward a sustainable future is perhaps one of the greatest challenges facing land managers today. Significant shifts in forest location and composition are predicted 

to increase, and trees are long-lived and sessile organisms so management decisions made now must 

confront uncertain environments decades from now. Economically important tree species are predicted 

to decline, disappear, or migrate. How do we make the most-strategic decisions now to ensure long-term 

sustainability of our forests? Characterization of current trends occurring in forest inventories is a key 

ingredient in forest management for an uncertain future. Nature is complex, and prediction from models of 

species distributions have uncertainty. What is occurring now may not necessarily be what models would 

otherwise predict. Here, we present an index that can quantify the sustainability of tree populations and 

meta-populations from forest inventory data to inform scientists, managers, and policy-makers and provide 

corroboration for predictions. We use the index to discover current trends for tree species in the Pacific 

Northwestern United States. We derive the index from data provided by the U.S. Department of Agriculture, 

Forest Service’s Continuous Vegetation Survey and Forest Inventory and Analysis program. We examined 

only fairly commonly occurring species with a minimum of 50 occurrences in the plots. We find that current 

stands of Juniperus occidentalis, Abies procera, Thuja plicata, Tsuga heterophylla, Pinus ponderosa, and 

Pseudotsuga menziesii are on sustainable trajectories. Conversely, we find that Pinus monticola, Arbutus 

menziesii, Pinus albicaulis, Abies lasiocarpa, Taxus brevifolia, Pinus contorta, Abies grandis, Picea englemanii, and Larix occidentalis are not on sustainable trajectories. We quantify these findings by blending 

change in demography and basal area. We argue that our metric is simple, can help track the status of 

our forest resources in a changing climate, and provides information that agrees with what foresters and 

pathologists are noticing on the ground. 
16 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Whitebark Pine (Pinus albicaulis) Growth Release Potential Following 

Overstory Disturbance

E. Shanahan1, *, D. Roberts2, K.M. Irvine3, A. Litt2, and K. Legg1

1 U.S. Department of the Interior (DOI), National Park Service, Greater Yellowstone Monitoring Network,

Bozeman, Montana, USA 

2 Montana State University, Department of Ecology, Bozeman, Montana, USA

3 USDI, U.S. Geological Survey, Northern Rocky Mountain Science Center, Bozeman, Montana, USA 

* erin_shanahan@nps.gov

Much of the information available on successional 

modeling for whitebark pine (Pinus albicaulis) 

communities is based on research conducted in an 

ecosystem influenced by maritime conditions (Pacific 

Northwest). The Greater Yellowstone ecosystem 

(GYE; Wyoming, Montana, and Idaho, USA) is subjected to more continental climatic conditions and as 

a result, the dynamics of whitebark pine populations 

in the GYE may in fact be very different from those 

of maritime populations. Recent survey work in the 

GYE shows establishment is common with variable 

recruitment of young trees in the understory of mature 

forests. Density and age distribution of these individuals vary greatly among stands across the ecosystem. 

By determining historical seedling and sapling growth 

rates and their relation to successional disturbances 

such as fire, bark beetles (Dendroctonus spp.), and 

disease, we will be better equipped to guide restoration 

efforts throughout the GYE and to ensure that management decisions are based on scientific information collected from the ecosystem in which restoration efforts 

are actually occurring. 

Evidence for the occurrence of a growth release 

phenomenon in trees has been documented for several 

shade-tolerant and partially intolerant species following the creation of gaps in the forest canopy due to 

a variety of ecological impacts. By using ring width 

patterns taken from whitebark pine individuals, this 

project examines evidence for growth suppression and 

a hypothetical growth release event in whitebark pine 

understory individuals located in stands where canopy 

composition changed due to the mountain pine beetle 

(D. ponderosae) outbreak of the late 1970s to early 

1980s in the Gallatin Range of the Gallatin National 

Forest (Montana). In some areas, natural regeneration 

of whitebark pine is clearly the optimal form of whitebark pine restoration following widespread mortality. 

To fully evaluate a stand for mechanical restoration, it 

will be important to gain an understanding of historical 

patterns in growth following major canopy disturbance as well as the current demographic structure of 

understory populations and the relationship between 

seedling/sapling age and recruitment potential into the 

canopy. 
USDA Forest Service RMRS-P-76. 2018. 17

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

White Pine Blister Rust Alters Facilitation Interactions at Treeline: 

Implications for Treeline Communities and Response

to Climate Change

D.F. Tomback1, *, L.M. Resler2, G.P. Malanson 3, E.K. Smith-McKenna2,

S.C. Blakeslee1, and J.C. Pyatt1

1 University of Colorado Denver, Department of Integrative Biology, Denver, Colorado, USA

2 Virginia Polytechnic and State University, Department of Geography, Blacksburg, Virginia, USA

3 University of Iowa, Department of Geography, Iowa City, Iowa, USA

* diana.tomback@ucdenver.edu

In some Rocky Mountain treeline ecotones, whitebark 

pine (Pinus albicaulis) serves as a tree island initiator 

more frequently than Engelmann spruce (Picea engelmannii) or subalpine fir (Abies lasiocarpa). Tree island 

initiation begins with a solitary krummholz tree, which 

provides sufficient leeward protection so that another 

tree may become established, resulting in a tree island. 

More trees may establish on the lee side over time. 

Whitebark pine mortality from white pine blister rust 

(pathogen: Cronartium ribicola) reduces opportunities 

for such facilitation. Will declines in whitebark pine 

alter treeline response to climate warming? We have 

surveyed various treeline communities from the Greater 

Yellowstone area (Wyoming, Montana, and Idaho, USA) 

to whitebark pine’s northern limit. 

From 2010 to 2013 at Line Creek, Montana, and 

Divide Mountain, Montana, we examined facilitation 

mechanisms by simulating whitebark pine death from 

blister rust, measuring tree shoot lengths, and evaluating 

performance of common treeline leeward microsites 

with respect to seed germination, seedling survival, and 

11 biophysical variables. In both study areas, plus Parker 

Ridge, Alberta (Canada), we sampled 80 randomized 

15-m × 15-m vegetation plots to characterize community 

composition and structure with respect to local climate, 

hydrology, digital elevation models, and incidence of 

blister rust. Using predictive modeling, we examined 

possible climate and topographic correlates of whitebark 

as tree island initiator both within and across study areas, 

and used agent-based modeling (ABM) to examine response to climate change in the presence of blister rust. 

At Line Creek and Divide Mountain, blister rust incidence was 19.2 and 23.6 percent, respectively. Among 

solitary conifers, whitebark pine was most abundant and 

had the greatest vigor and longest shoot lengths. Death 

of windward whitebark pine reduced shoot length and 

vigor in leeward trees, demonstrating facilitation. 

In planting experiments, new seedlings had higher 

survival on whitebark microsites. Microsites leeward of 

whitebark pine and spruce, relative to rocks or exposed 

sites, had lower photosynthetically active radiation, sky 

exposure, and wind speeds, and more moderate soil and 

air temperatures. Thus, whitebark pine prevalence as a 

tree island initiator results from its hardiness, relative 

abundance, and the protective qualities of its leeward 

microsites. Modeling indicates that growing season 

temperature and slope curvature may predict the incidence of whitebark pine facilitation at local scales. In 

certain treeline ecotones, whitebark pine is abundant, but 

facilitation is less prevalent, very likely because of a more 

moderate climate. The use of ABM combining climate 

warming and blister rust indicated that the latter slowed 

the overall advance of trees while slightly favoring spruce 

and fir in relative abundance. Our combined studies suggest that whitebark pine tolerates the harsh treeline climate better than associated conifers, and losses to blister 

rust will affect treeline response to climate warming.
18 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Whitebark Pine (Pinus albicaulis) Direct Seeding Trials in the 

Northern Rocky Mountains: The Role of Planting Site and Cache 

Pilferage by Rodents

E.R. Pansing1, *, A.C. Wagner1, and D.F. Tomback1

1 University of Colorado Denver, Department of Integrative Biology, Denver, Colorado, USA

* elizabeth.pansing@ucdenver.edu 

Direct seeding is an emerging technique that could 

significantly reduce the labor and costs associated 

with whitebark pine (Pinus albicaulis) restoration, and 

may be compatible with wilderness values. Because 

other restoration methods—including outplanting of 

seedlings—are largely incompatible with maintaining 

wilderness as “untrammeled and natural,” the adoption 

of direct seeding may open an additional 48 percent 

of whitebark pine habitat to restoration efforts in the 

future. 

We conducted a large direct seeding experiment on 

White Calf Mountain, Glacier National Park, Montana 

(USA) and Tibbs Butte, Shoshone National Forest, 

Wyoming (USA) to determine: (1) germination and 

first-year survival rates of simulated caches and how 

rates vary between subalpine forest and the treeline 

ecotone, (2) which planting site characteristics are 

important for germination and survival, and (3) rates 

of cache pilferage by small rodents (Order Rodentiae). 

In 2012, we created simulated Clark’s nutcracker 

(Nucifraga columbiana) caches near three different 

microsite types (trees, rocks, or no object) at randomly 

selected locations using randomly generated cache sizes 

(numbers of seeds per cache, range = 1 to 7). Caches 

were revisited in 2013 and 2014 to assess germination, 

seedling survival, and seed loss. In 2013, 31.9 percent 

of 717 relocated caches germinated (25.6 percent on 

White Calf Mountain, 38.0 percent on Tibbs Butte). 

Odds of germination were 1.78 (95-percent confidence 

interval [C.I.] = 1.29–2.44) times higher on Tibbs Butte 

than on White Calf Mountain. On Tibbs Butte, odds of 

germination were 1.71 (95-percent C.I. = 1.05–2.78) 

times higher at treeline than in the subalpine zone, and 

odds were increased for caches created near rocks or 

no object when compared to caches created near trees. 

About 54 percent of the caches created on both White 

Calf Mountain and Tibbs Butte were pilfered. At both 

study areas in the treeline zone, 62.1 percent of caches 

were pilfered; in the subalpine zone, 46.1 percent were 

pilfered. The estimated odds of pilferage at treeline 

were 3.94 (95 percent C.I. = 1.42–2.57) times the odds 

of pilferage at the subalpine sites. In 2014, 21.3 percent 

and 4.6 percent of the remaining caches germinated on 

Tibbs Butte and White Calf Mountain, respectively, for 

a total of 59.3 percent and 30.2 percent of caches created in 2012 germinating after 2 years. 

Survival rates of seedlings germinated in 2013 were 

66.9 and 35.6 percent on Tibbs Butte and White Calf 

Mountain, respectively. On Tibbs Butte, germination 

seems to be favored near rocks and in the open, and 

further investigation is needed to determine whether 

these findings are consistent throughout the range of 

whitebark pine. Because germination/survival rates exceeding 10 percent are considered successful for direct 

seeding efforts, observed germination rates suggest that 

large-scale direct seeding efforts should be considered 

for restoration. However, determining the regional 

drivers of successful germination and survival (climate, 

soils, seed source, and others) is necessary to ensure a 

full understanding of the processes required for germination and establishment to achieve the greatest success 

of direct seeding efforts. 
USDA Forest Service RMRS-P-76. 2018. 19

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

White Pine Blister Rust, Mountain Pine Beetles

(Dendroctonus ponderosae), and Their Potential Impact on

White Pines: A National Assessment From the National Insect

and Disease Risk Map

F.J. Krist1, A.J. McMahan2, *, and E.L. Smith1

1 USDA Forest Service, Forest Health Technology Enterprise Team, Fort Collins, Colorado, USA

2 Cherokee Nation Technologies, Fort Collins, Colorado, USA

* dmcmahan@fs.fed.us

Abstract—The 2012 National Insect and Disease Risk Map (NIDRM) contains a nationwide strategic 

assessment of the hazard of tree mortality due to insects and diseases, displayed as a series of maps. 

The NIDRM is more than just maps: it is a nationwide, science-based, administrative planning tool resulting from the United States’ forest health community working together to determine the severity and extent 

of potential tree mortality. The NIDRM’s modeling process provides a consistent process through which 

interactive spatial and temporal hazard assessments can be conducted. The NIDRM serves as the U.S. 

Department of Agriculture, Forest Service (USDA FS), Forest Health Protection program’s comprehensive 

analysis of pest hazards, and is referred to in Federal legislation regarding watershed restoration funding to 

the States. Five-needle pines, and their primary disturbance agents—white pine blister rust (causal agent: 

Cronartium ribicola) and mountain pine beetles (Dendroctonus ponderosae)—are among the NIDRM’s 

interactions, which show a potential for catastrophic host impacts over the next 15 years.

This presentation will explain the design and construction of the NIDRM system, and the results for white 

pine ecosystems. These impacts are displayed in map and tabular formats, at various geographic scales 

and for different administrative units, for both the USDA FS and the U.S. Department of the Interior, 

National Park Service. The presentation will also explain the data layers used in the analysis, available for 

downloading, and other website resources where additional information and results can be found (https://

www.fs.fed.us/foresthealth/applied-sciences/mapping-reporting/gis-spatial-analysis/national-risk-maps.

shtml).
20 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Survey of Pinus dalatensis for Gene Conservation in Vietnam

P.H. Hai1, *

1 Vietnamese Academy of Forest Sciences, Hanoi, Vietnam

* phi.hong.hai@vafs.gov.vn

Abstract—Pinus dalatensis is a conifer species of conservation importance in Vietnam. Eighteen 2,500-m2

plots were established in mixed-species forests at different elevations in Vietnam to evaluate the occurrence of P. dalatensis, site conditions, and forest structure; to identify primary associated species; to study 

the morphology of pine samples; and to collect P. dalatensis cones for ex situ gene conservation studies. 

Mature P. dalatensis trees were found at elevations between 1,070 and 1,900 m. The survey also showed 

that the number of P. dalatensis individuals was low, but it was the species in the upper canopy, with 

importance value ranging from 5.6 to 47.4 percent. The principal associated species were Craibiodendron 

syellatum, Podocarpus imbricatus, Dacrydium elatum, Hellicia cochinchinensis, Symplocos poilanei, 

and Quercus langbianensis. The N/D distribution of P. dalatensis forest on most plots was described by 

Mayer or Weibull distributions and suggested that the forests had been selectively thinned. There was no 

regeneration of P. dalatensis in the observed forests. Five-year survival and growth results in ex situ locations where seeds were planted from cones collected in the original survey suggest that P. dalatensis gene 

conservation areas should be established in forest gaps. Plantations of P. dalatensis could be successful, 

but the trees are expected to grow very slowly. Seedbanks could be an alternative method of gene conservation of P. dalatensis.
USDA Forest Service RMRS-P-76. 2018. 21

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

INTRODUCTION

Southwestern white pine (Pinus strobiformis, 

syn. Pinus flexilis var. reflexa) is a high elevation 

five-needle pine. Pinus strobiformis occurs in mixed 

species stands across a range of elevations and forest types in western Texas, southwestern Colorado, 

and throughout New Mexico and Arizona (USA), 

and continues along the mountains south to central 

Mexico (Alexander et al. 1984; Looney and Waring 

2013; Moreno-Letelier and Piñero 2011; Steinhoff and 

Andresen 1971) (fig. 1). This species, like all species 

of North American five-needle pines, is susceptible to 

infection and mortality from the nonnative, invasive 

forest pathogen (Cronartium ribicola) that causes the 

disease white pine blister rust (WPBR) (Kinloch and 

Dupper 2002; Sniezko et al. 2011). White pine blister 

rust was first reported in natural stands of P. strobiformis in 1990 (Hawksworth 1990), and is spreading through the range in New Mexico and Arizona 

(Conklin 2004; Conklin et al. 2009; Fairweather and 

Geils 2011; Looney and Waring 2012). Across the area 

where WPBR was first discovered, about 40,500 ha 

have high WPBR incidence and trees with high severity infections including frequent “topkill” (a condition 

in which needles, branches, and stem tissue above the 

canker location are killed) (Conklin et al. 2009). 

According to available species climate profile predictions, the suitable range of P. strobiformis is expected 

Pinus strobiformis Gene Conservation and Genecology

B.A. Goodrich1, K.M. Waring1, *, and L. Flores-Renteria2

1 Northern Arizona University, School of Forestry, Flagstaff, Arizona, USA

2 San Diego State University, Department of Biology, San Diego, California, USA

* kristen.waring@nau.edu

to decrease under most future climate change scenarios 

(Crookston and Rehfeldt 2008). Predictions of future 

WPBR distributions are not as well understood 

because of difficulty in modeling pathogen/host tree 

interactions (Sturrock et al. 2011), but high hazard 

areas exist in the Southwest (Conklin 2004; Geils et al. 

1999). 

The impacts of WPBR on five-needle pine species 

in northern Rocky Mountain and Pacific Northwest 

ecosystems illustrate the disastrous effects of the 

disease on susceptible hosts on high hazard sites (Fins 

et al. 2002; Tomback and Achuff 2010). Cascading 

negative effects of forest recovery and ecosystem 

services in these areas should be considered a preview 

of what could occur in some southwestern ecosystems 

with no intervention (Schoettle and Sniezko 2007; 

Tomback and Achuff 2010). Negative effects may be 

exacerbated both through reductions of P. strobiformis

genetic diversity with declining suitable habitat and 

through local population extirpations from recent 

large, high-severity fires across the Southwest (fig. 1).

Intervention in this case refers to the use of proactive 

restoration, where silvicultural practices can be used 

to promote natural (stimulated) or artificial (planted) 

regeneration with some resistance to WPBR (Keane 

and Schoettle 2011; Schoettle and Sniezko 2007). Our 

program to sustain P. strobiformis in southwestern 

COMMON-GARDEN GENETICS
22 USDA Forest Service RMRS-P-76. 2018.

mixed-conifer forests includes gene conservation and 

studies of genecology and population genetics. Some 

of our other research directions not addressed here are 

regeneration ecology, silvics, sustainable management, 

and prediction of the future distribution of the species 

using landscape genetics and climate change modeling. Understanding ecophysiological and adaptive 

processes of this species will help inform genetic and 

silvicultural approaches to decrease forest tree vulnerability and increase adaptive capacities in the face 

of disturbances and a changing climate (Bower et al. 

2011; Chmura et al. 2011). 

GENE CONSERVATION

Our seed collections throughout the range of P. strobiformis in the Southwest began in 2012. Although 

our primary goal was to archive seed for the U.S. 

Department of Agriculture, Forest Service (USDA FS) 

Gene Conservation program (Mangold 2011), we have 

collected enough additional seed to support research 

and management projects. Sites were identified using 

the known range of P. strobiformis in Arizona and 

New Mexico in conjunction with information about 

local cone crop production in a given year. At each 

site, we collected cones from up to five trees separated 

by a minimum distance of 200 ft (61 m) and recorded 

major overstory and understory species, presence 

of Ribes shrubs (one of the key secondary hosts for 

WPBR), slope, and aspect (see Schoettle et al. 2011). 

Each collection tree was georeferenced and tagged at 

breast height (1.37 m) for relocation purposes; we also 

recorded total tree height, live crown proportion, diameter at breast height, presence and severity of WPBR 

Figure 1—Pinus strobiformis (southwestern white pine, SWWP) gene conservation seed collection sites across USDA FS 

Region 3; collections from 2012–2014. Recent large fires in collection areas / P. strobiformis habitat included.
USDA Forest Service RMRS-P-76. 2018. 23

infection, and insect infestation levels in cones and 

seeds for each collection tree. In areas where WPBR 

was present, we attempted to collect seeds from 

infection-free trees (putatively resistant), but seeds 

were also collected from trees with visible WPBR 

infections. To date, we have collected cones and seeds 

across much of the range of P. strobiformis in Arizona 

and New Mexico and along local elevational gradients 

where possible (fig. 1). Our seed collections for gene 

conservation represent eight national forests and 

two Indian Reservations (table 1). These collections 

consist of 111 trees (26 populations archived in the 

Gene Conservation program) from 2012 and 2013 and 

59 trees (12 archived populations) from 2014. These 

are in addition to previous collections of 88 families 

and 3 bulk populations of P. strobiformis in USDA FS 

Region 3 (southwestern United States; Sniezko et al. 

2011).

GENECOLOGY RESEARCH

To properly manage P. strobiformis in the face of climate change and WPBR, adaptive traits and responses 

to stressors must be quantified from populations across 

the range; these are considered key objectives in successful restoration efforts in other species of threatened 

five-needle pines (Bower et al. 2011; Hamlin et al. 

2011). There is relatively little information about population differentiation of quantitative traits in P. strobiformis (but see Steinhoff and Andresen 1971). With 

known family and population variation in quantitative 

traits such as phenology, morphology, and drought 

tolerance, we can infer adaptation to geographic or 

climatic gradients, quantify heritability, and reduce 

risk of seedling maladaptation in seed transfer and 

restoration efforts (Bower and Aitken 2008; Bower et 

al. 2011). Because outplanting artificial regeneration 

with natural genetic resistance is a realistic option for 

managers, the risk of seedling maladaptation to new 

sites must be quantified with common garden studies 

(Bower and Aitken 2008; Keane and Schoettle 2011; 

Schoettle and Sniezko 2007).

We began a greenhouse common garden study in 

Flagstaff, Arizona, in spring 2012 with the specific 

objectives to determine family, population, and 

geographic variation in quantitative traits (growth, 

phenology, and the tolerance to low water treatments) 

of P. strobiformis. We used seed from our collections, 

as well as donated seed from previous collections 

(Region 3 Forest Health Protection 1997–2008; 

Conklin et al. 2009; Sniezko et al. 2011), in a design 

Table 1—Pinus strobiformis seed collection sites and year of collection(s) across National Forests in the 

Southwest. Collections from Indian Reservations not shown.

Collection 

year(s) Location District State

Gene 

conservation

sites

2012-2014 Coconino National Forest Peaks AZ 7 

2012 Coconino National Forest Mogollon Rim AZ 2 

2012, 2014 Apache-Sitgreaves National 

Forests 

Black Mesa AZ 3 

2012 Apache-Sitgreaves National 

Forests 

Springerville AZ 1 

2012 Apache-Sitgreaves National 

Forests 

Alpine AZ 1 

2012 Apache-Sitgreaves National 

Forests 

Lakeside AZ 1 

2012 Coronado National Forest Safford AZ 2 

2013 Coronado National Forest Santa Catalina AZ 4 

2014 Coronado National Forest Sierra Vista AZ 1 

2013 Kaibab National Forest Williams AZ 1 

2013 Gila National Forest Reserve NM 1 

2014 Santa Fe National Forest Jemez NM 1 

2013 Lincoln National Forest Sacramento NM 3 

2014 Cibola National Forest Magdalena NM 2 

2012, 2014 Cibola National Forest Mt. Taylor NM 2 
24 USDA Forest Service RMRS-P-76. 2018.

that included 2 to 15 families (half or full-sibling 

offspring from a single mother tree) per population 

across 24 populations of P. strobiformis. Families were 

represented by 18 to 36 seedlings per family dependent 

upon germination rates, and half the seedlings from 

each family were subjected to low water treatments 

over 2 years. Growth, phenology, and physiological 

responses of all families, populations, and watering 

treatments were measured over two growing seasons. 

We hypothesized that (1) growth, phenology, and 

drought tolerance variation would be small within populations but large between populations because of the 

isolated, disjunct nature of P. strobiformis populations; 

(2) population seedling growth and phenology would 

parallel seed source climate (e.g., seedlings from 

source climates with fewer frost-free days would end 

growth earlier; seedlings from warmer source climates 

would grow larger); and (3) growth and survival under 

drought conditions would also parallel seed source 

climate.

Preliminary results indicated that growth trait 

population means such as 6-month height, number 

of branches (potential allocation to growth processes 

other than height), and growth initiation (phases of bud 

and leaf development) were well differentiated and 

appeared to correlate weakly with seed source geographic and climatic clines (fig. 2, R2 = 0.22 and 0.27 

for ambient and low water seedlings, respectively), indicating local adaptation of populations for these traits, 

which will be evaluated with reciprocal transplant 

gardens in the future. Overall, populations from southern sources grew taller than populations from northern 

sources (fig. 2). These patterns continued in the second 

growing season; population means of 14-month-old 

seedling heights were also weakly correlated with seed 

source latitude and southern populations grew larger 

than northern populations (data not shown; R2 = 0.23 

and 0.21 for ambient and low water treatments, respectively). Latitude was a stronger correlate than elevation 

for all growth traits measured in the first season. No 

Figure 2—Relationship between 6-month-old seedling height means (± 1 standard error) and seed 

source latitude across two watering treatments in a greenhouse common garden study in Flagstaff, 

Arizona (ambient and low water). N = 23 populations, 2–15 families per populations, 18–36 seedlings 

per family. 
USDA Forest Service RMRS-P-76. 2018. 25

genotype-by-environment effects were observed in the 

first season for any measured trait and analyses are 

ongoing. Relationships between quantitative traits and 

climate variables are also being investigated.

ONGOING AND FUTURE

RESEARCH DIRECTIONS

We are expanding the initial common garden from the 

greenhouse to the Southwest Experimental Garden 

Array (SEGA), a series of common gardens across an 

elevation gradient in northern Arizona. The first trial 

garden was planted in fall 2014 with 170 families from 

44 populations (fig. 3). Many of these families are also 

included in disease resistance screening trials at the 

USDA FS Dorena Genetic Resource Center (DGRC, 

Cottage Grove, Oregon, USA) in a project funded 

by the USDA FS Forest Health Protection Special 

Technology Development Program (R3-2014-02). 

There are 39 populations of P. strobiformis represented by 154 families growing at DGRC slated to be 

inoculated in fall 2015 for long-term testing of partial 

resistance mechanisms to C. ribicola. A small proportion of these populations were inoculated in fall 2014 

for shorter term major gene resistance tests. We are 

developing two outplanting sites on national forest 

and tribal land in Arizona and New Mexico to test the 

long-term durability of resistant seedlings in the field, 

and families to be included in the outplanting will be 

chosen following early results from resistance trials. 

We are also investigating population genetics across 

the entire range of P. strobiformis, beginning with 70 

populations across the southwestern United States 

and Mexico. In Mexico, foliage was collected from 

19 populations and 169 individuals in the Sierra 

Madre Occidental and Trans Mexican Volcanic Belt in 

May 2014, with additional foliage obtained from the 

botanical collection at the Centro Interdisciplinario 

de Investigación para el Desarrollo Integral Regional 

(CIIDIR, Interdisciplinary Research Center for 

Integrated Regional Development), Durango (fig. 4). 

Figure 3—Initial common garden planting at The Arboretum, Flagstaff, Arizona. Site is part of the Southwest 

Experimental Garden Array. Six-month-old seedlings were planted in early October 2014.
26 USDA Forest Service RMRS-P-76. 2018.

Collections included one population of P. strobiformis var. stylesii from Sierra Madre Oriental and one 

population of P. ayacahuite from Sierra Madre del Sur. 

We also collected foliage from 49 populations across 

Arizona and New Mexico and from one P. flexilis

(limber pine) population in southern Wyoming (fig. 4; 

Wyoming population not included in map). Pinus 

flexilis, P. strobiformis var. stylesii, and P. ayachuite

Figure 4—Pinus strobiformis foliar collection sites across the Southwest United States (Region 3) and 

northern Mexico for use in restriction-site associated DNA sequencing (RADSeq) to identify single 

nucleotide polymorphisms across the genome.
USDA Forest Service RMRS-P-76. 2018. 27

populations were included to distinguish between 

closely related species and any hybridization that may 

occur. 

Restriction-site associated DNA sequencing will be 

used to identify single nucleotide polymorphisms 

across the genome. We are conducting studies to 

understand the dynamics of gene flow, population 

structure, and hybridization to better understand 

the temporal and geographic severity of WPBR and 

climate change on P. strobiformis. Pines are well 

known for their long-distance gene flow and lack of 

incompatibility systems resulting in hybridization. It 

is therefore necessary to quantify the flow of genes 

related to disease resistance, drought tolerance, and 

other potentially adaptive traits across the entire range 

of P. strobiformis and hybrid zones with closely related 

species. 

Future research plans will expand our seed collections across the range of the species (including more 

sites throughout Mexico), add three SEGA common 

gardens to the current experimental design, and add 

families and populations to disease resistance screening trials. Other ongoing and future objectives include 

determining linkages between adaptive traits and 

resistance frequencies, identifying the genetic bases 

for adaptive traits, and providing tools to better predict 

the potential distribution of P. strobiformis under 

future climate change. Our P. strobiformis research 

program combines basic and applied research using 

cross-collaborative efforts to sustain and conserve 

an important tree species in the southwestern United 

States and Mexico. Conserving the genetic diversity 

and defining management strategies to sustain P. strobiformis are critical in a changing climate and with the 

threat of WPBR. We envision a sustainable future for 

P. strobiformis with continued proactive and adaptive 

research and management. 

ACKNOWLEDGMENTS

Funding for these projects has been provided by 

the USDA FS Forest Health Protection (FHP) Gene 

Conservation and Special Technology Development 

Programs, Northern Arizona University’s (NAU’s) 

Technology Research Initiative Fund, McIntire-Stennis 

appropriations to NAU and the State of Arizona, and 

a Genes-to-Environment Fellowship to B. Goodrich. 

Seeds were donated for some projects by Dave 

Conklin (FHP Region 3), Detlev Vogler and Annie 

Mix (Institute of Forest Genetics), Anna Schoettle 

(Rocky Mountain Research Station), and Richard 

Sniezko (Dorena Genetic Resource Center). Amy 

Whipple (NAU Biology, Merriam Powell Center for 

Environmental Research) has been instrumental in 

genomics research and the future research program, 

including SEGA designs. Andrew Eckert (Virginia 

Commonwealth University) has also been instrumental 

in genomics research plans. We thank Brad St. Clair 

(Pacific Northwest Research Station) and James Jacobs 

(FHP Region 3) for reviews on an earlier version of 

this abstract and for their comments, which improved 

the publication. The following helped with lab and 

field work in the United States: Chris Erickson, John 

Benning, Shaughn Dugan, Sean Callahan, Hannah 

Stapleton, Stevia Morawski, Michael Scott, Travis 

Largent, Calvin Rogers, Danny DePinte, Gennaro 

Falco, Eric Vane, Adam Polinko, Connor Meehan, 

Ronnie Kyllo, Kelsey Flathers, Adair Patterson, Tara 

Steadman, Marguerite Rapp, Julian Wilson, Nick 

Dolhyj, and Tanner Laird. In Mexico, foliage collections were facilitated by Socorro González-Elizondo 

(CIIDIR), Francisco Molina-Freaner (Universidad 

Nacional Autonóma de Mexico [UNAM]), Christian 

A. Wehenkel (Universidad Juárez del Estado de 

Durango [UJED]), Aldair Flores (UNAM), Jorge 

Aguilar (Bosques de Árboles de Navidad), Jorge 

Noriega Villa (CIIDIR), Sergio Simental Rodríguez 

(UJED), José F. Martínez (UNAM), Gustavo 

Escobedo (Instituto Nacional de Astrofisica, Óptica y 

Electrónica), Valer Austin (Cuenca de los Ojos), José 

Manuel Pérez Cantú (Cuenca de los Ojos), Mario 

Cirett-Galán (Comision Naciónal de Áreas Naturales 

Protigidas [CONANP]), Isaias Ochoa (CONANP), 

Carlos Cohen (CONANP), Fernando Salazar Jiménez 

(Comisión Nacional Forestal), Ademir Flores, Yolanda 

Rentería, and José Molinar Rodríguez.

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consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Genetic Variation in Stomate Densities and Needle Traits in a 

Rangewide Sampling of Whitebark Pine (Pinus albicaulis)

P. Bennett1, J. Hill2, D.P. Savin3, *, A. Kegley3, A.W. Schoettle4, R.A. Sniezko3, B. Bird4, 5,

and J. Stone1

1 Oregon State University, Department of Botany and Plant Pathology, Corvallis, Oregon, USA

2 USDA Forest Service (retired)

3 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA

4 USDA Forest Service, Rocky Mountain Research Station, Fort Collins, Colorado, USA

5 Colorado State University, Fort Collins, Colorado, USA

* dpsavin@fs.fed.us 

Abstract—Needle dimensions and stomate densities were measured for 300 seedlings representing a 

stratified sampling of 30 families of whitebark pine (Pinus albicaulis) from California, Oregon, Washington, 

Idaho, and Montana (USA), and British Columbia (Canada). The seedlings were selected from a larger 

common garden trial examining white pine blister rust (WPBR) resistance of 225 families from populations 

representing much of the geographic range of whitebark pine. The objective was to examine variation in 

needle characteristics, including needle width, number of stomata, and stomate density, and their relation 

to WPBR infection. Two cohorts of needles were sampled—2008 and 2009—and morphological characteristics on both adaxial (AD) and abaxial (AB) surfaces were measured. Averaged over families and blocks, 

all traits except needle width were larger for the 2009 cohort, and within a cohort, averages were greater 

for variables measured on the AD surface relative to the AB surface. Mean stomate densities (ADEN, 

stomata mm-2) ranged from 29.30 to 40.57 on the AD leaf surface and from 13.75 to 22.52 on the AB leaf 

surface for the 2008 cohort. These values were moderately correlated with stomate densities on the same 

needle surfaces in the 2009 cohort (r = 0.543, p = 0.002 and r = 0.565, p = 0.001 for AD and AB sides, 

respectively). ADEN for the 2009 needle cohort ranged from 38.77 to 52.22 on the AD surface, and from 

18.82 to 29.86 on the AB surface. Family means for needle width (NW) ranged from 0.773 to 1.022 mm 

for the AD surface, and from 0.886 to 1.197 mm for the AB surface across cohorts. Mean numbers of rows 

of stomata, stomate abundance, and stomate density by needle length were significantly correlated with 

NW on the AD surface. Stomate density within rows (RDEN) showed a moderate and significant correlation for the 2009 cohort with number of needle spots resulting from the artificial inoculation with blister rust 

spores (r = 0.543, p = 0.002 and r = 0.438, p = 0.015 for AD and AB, respectively); RDEN was significantly 

correlated with mean temperature in the warmest month. In addition, the number of stomate rows was 

negatively correlated with early canker development (number of cankers) as well as growing season and 

spring precipitation at the seed source. These results suggest that source climate may influence rust symptom development via stomate traits that are under adaptive selection.
USDA Forest Service RMRS-P-76. 2018. 31

INTRODUCTION

Whitebark pine (WBP, Pinus albicaulis) occurs at 

high-elevation sites across much of western North 

America but is currently threatened in large portions 

of its range due to the combined effects of climate 

change, mountain pine beetle (Dendroctonus ponderosae), and white pine blister rust caused by the invasive 

pathogen Cronartium ribicola. This project investigated genetic variation in needle traits of WBP using 

collections from a seedling common garden trial of 

families representing much of the geographic range of 

this forest tree species. Parent tree environmental gradients due to geographic factors such as elevation, latitude, and longitude were considered as possible drivers 

of morphological diversification. A parallel project 

underway examined within-population variation in 

some of the same needle traits in seedling families 

from Crater Lake National Park in southeast Oregon 

(Sniezko et al., this proceedings, Genetic Variation 

in Needle Traits of Whitebark Pine (Pinus albicaulis) 

Seedling Families: Within-Population Variation at 

Crater Lake National Park; hereafter referred to as 

Genetic Variation). 

Stomata mediate the exchange of oxygen, water vapor, and carbon dioxide (CO2) with the atmosphere. 

Control of transpiration allows plants to photosynthesize in a wide range of changing environments, and 

was most likely a driving force behind the ecological 

diversification of plants (Hetherington and Woodward 

2003). Variations in stomate size and density are generally correlated with environmental variables such as 

atmospheric CO2 concentration, as well as geographic 

variables such as elevation (Holland and Richardson 

2009; Schoettle and Rochelle 2000). The size and 

density of stomata influence the rate of conductance 

of carbon to sites of assimilation within the leaf and 

regulate many other ecophysiological functions of leaf 

surfaces (Franks and Beerling 2009). Stomate morphology may also play a significant role in interactions 

with pathogens and pests. Natural openings in leaf 

surfaces such as stomata are important entry points for 

many fungal pathogens, including C. ribicola (Patton 

and Johnson 1970). Consequently, plants have evolved 

to regulate the stomate opening as an important part of 

a general response to microbial invasion (Gudesblat et 

al. 2009; Zeng et al. 2010).

Because many WBP populations face the imminent 

threat of damage by C. ribicola, variations in needle 

morphology in relation to blister rust infection are being studied together with other possible indicators of 

blister rust resistance. Investigation of natural variation 

in needle characteristics such as stomate abundance 

and arrangement may provide some key insights into 

factors influencing susceptibility to the blister rust 

pathogen and its geographic variation.

METHODS

Sample Selection and Inoculations

A subset of 30 families was selected from a common 

garden inoculation study examining resistance to white 

pine blister rust in 225 seedling families from much of 

the range of WBP. Parent trees of the seedlings examined here represent eight seed zones from throughout 

Oregon and Washington as well as individual populations from Idaho, Montana, California, Nevada, and 

British Columbia (fig. 1). The seeds were sown in 

2007 (see Hamlin et al. 2011 for background on seedling culture) in 10-in3 (164-cm3) containers, which 

were then placed into a randomized complete block 

(RCB) design before inoculation with C. ribicola. The 

seedlings were inoculated in 2008 after their second 

growing season. Three blocks were inoculated in early 

September, and the other three blocks in early October; 

different geographic sources of rust were used for each 

inoculation. Infected Ribes leaves were placed on wire 

screens atop the WBP seedlings to allow basidiospore 

release. Basidiospore discharge was monitored on trap 

slides to ensure that all trees received similar inoculum 

exposures. Average spore densities were 3,359 and 

6,013 cm-2 for the first and second inoculations, respectively. Seedlings from all 225 families were transplanted in October 2008 to wooden boxes (0.9 m × 1.2 

m × 0.3 m) in the same RCB design with 12 families 

per box, and 20 boxes per block. Families were in row 

plots (up to 10 seedlings per family row plot). 

In March 2010, needles from both the 2008 and the 

2009 cohorts were collected from 2 seedlings per family per block in all 6 blocks; however, samples from 

only 5 of the 6 blocks were photographed and analyzed 

(300 total seedlings). Assessments of number of needle 

spots, number of stem infections (including zeros), and 
32 USDA Forest Service RMRS-P-76. 2018.

seedling mortality were made in late July–early August 

2009 (inspection 1) and November–December 2009 

(inspection 2) for all seedlings, including the sampled 

ones. Additional assessments of stem infection and 

mortality were made annually 2010–2013 but were not 

used in these analyses.

Needle Measurements and Statistical 

Analyses

Needles were attached to 3-inch × 5-inch (7.6-cm 

× 12.7-cm) index cards with double-sided tape and 

photographed with a Dino-Lite digital USB camera 

(AnMo Electronics Corp., Taipei, Taiwan). Needle 

Figure 1—Map of parent tree locations and seed zone designations used in this study.
USDA Forest Service RMRS-P-76. 2018. 33

dimensions and stomate densities were measured for 

300 sampled seedlings (30 families × 10 seedlings per 

family) for both the 2008 and 2009 needle cohorts. For 

each seedling, four photographs were taken from both 

the abaxial (AB) and adaxial (AD) needle surfaces 

(eight photos total) at a magnification of 200× (fig. 

2a). The abaxial side is the rounded outer surface of a 

needle, and it typically has two resin channels running 

alongside the stomate rows (fig. 2b). An individual 

needle has two adaxial surfaces; these are the inner 

sides of a needle (fig. 2b). Different needles were 

photographed for the AB and AD sides, and two to 

four needles were used for each seedling to obtain the 

photographs. An approximately 2-mm length of needle 

was used for measurements; the 2-mm segments 

were randomly chosen from each needle surface for 

quantification of stomata and rows of stomata (fig. 

2). Needle width was measured in magnified images 

using the line measure tool in the Dino-Lite software 

(fig. 2a). Individual seedling means were calculated 

by averaging these measures for each seedling, and 

analyses of variance (ANOVAs) and linear regressions 

of needle attributes were performed on these means 

(accounting for block differences) using R (R Core 

Team 2016). Family means were calculated from the 

mean of block means, and pairwise correlations were 

performed by using these means and SAS® software 

(version 9.4, SAS Institute Inc., Cary, North Carolina) 

with the CORR procedure.

A sequence of analyses was conducted to explore 

if stomate features mediate the influence of source 

climate of the seed (maternal) parent on early postinfection rust symptom development. Potential significant predictors and groups of intercorrelated predictors 

were explored by using 10-fold cross validation techniques with lasso and elasticnet (Friedman et al. 2008) 

in the glmnet package (Friedman et al. 2010) in R (R 

Core Team 2016). By using these penalized regression 

techniques, three sets of family mean stomate data 

(variables averaged across 2008 and 2009 needles, and 

those data for each year singly) were independently 

analyzed. Each dataset was analyzed in a two-part 

sequence to (1) identify stomate variables that significantly predicted disease development, and (2) identify 

seed source climate variables that explained variation 

in the stomate variables identified in step 1. Each step 

generated linear models to predict disease response 

from stomate traits and predict stomate traits from 

source climatic variables. Normalized (1961–1990) 

means of derived climate variables (table S1) were 

estimated for each source location by thin plate spline 

surfaces (Rehfeldt 2006).

RESULTS

Family (Geographic) Variation in Needle 

Morphologies

Numbers of Stomata, Stomate Density, Numbers 

of Rows of Stomata

Families differed significantly for mean number of 

stomata (nSTOM) on the AD and AB leaf surfaces 

(ANOVA, P < 0.0001 for both sides). Although 

nSTOM for the 2008 and 2009 cohorts differed significantly (ANOVA, P < 0.0001), they were highly 

correlated with each other. Family mean nSTOM 

ranged from 46.75 to 73.73 on the AD surface and 

from 25.25 to 46.58 on the AB surface in the 2008 cohort; means for the 2009 cohort ranged from 70.00 to 

98.65 and 39.88 to 66.01 on the AD and AB surfaces, 

respectively (table 1). The coefficient of variation for 

nSTOM on the AD surface was 10.0 and 8.3 percent 

Figure 2—Magnified images of leaf surfaces: (a) width and 

length measurements shown on photograph of adaxial leaf 

surface; (b) abaxial (top) and adaxial (bottom) leaf surfaces. 

a

b
34 USDA Forest Service RMRS-P-76. 2018.

for the 2008 and 2009 needle cohorts, and 15.4 and 

11.4 percent for AB surfaces in the 2008 and 2009 

cohorts, respectively. The total number of stomata on 

the AD surface of the needles from each of the 2008 

and 2009 cohorts was strongly correlated with needle 

width for that year (table 2), and similar but weaker 

trends were observed for the AB surface. On average, there were fewer stomata per unit length of row 

(RDEN) on the AB surface (10.08 and 11.81 for 2008 

and 2009, respectively) than on the AD surface (10.84 

and 12.33 for 2008 and 2009, respectively) (table 1).

Family mean number of rows of stomata (nROWS) 

varied from 2.31 to 3.48, and from 1.31 to 2.14 on the 

AD and AB surfaces, respectively, in the 2008 cohort; 

family mean nROWS for the 2009 cohort ranged 

from 2.98 to 3.80 and 1.73 to 2.78 for the AD and AB 

surfaces, respectively (table 1); there were 1.60 to 1.69 

times as many nROWS on the AD side relative to the 

AB side for the 2009 and 2008 cohorts, respectively. 

The number of rows on the AD surface for the 2008 

and 2009 cohorts was significantly correlated with 

NW of both the AD and AB surfaces for the respective 

cohorts (table 2). There was also a moderate correlation between the nROWS on the AB surface in the 

2008 cohort and RDEN on the AD surface of the 2008 

cohort (r = 0.374, P = 0.042). nSTOM was generally 

highly correlated with nROWS on the corresponding needle surface within the same needle cohort; for 

example, nSTOM for AD surfaces in the 2009 cohort 

was highly correlated with nROWS for AD surfaces in 

the 2009 cohort (r = 0.879, P < 0.0001).

Stomate density (ADEN) varied from 36.18 stomata 

mm-2 (range of family means: 29.30–40.57) to 44.99 

(38.77–52.22) on the AD surfaces, and from 17.12 

(13.75–22.52) to 22.91 (18.82–29.86) on the AB 

needle surfaces in the 2008 and 2009 needle cohorts, 

respectively (table 1). There was a positive correlation between AD and AB ADEN in the 2009 cohort 

(r = 0.414, P = 0.023) but not in the 2008 cohort 

(r = 0.287, P = 0.12). Values of ADEN were positively 

correlated between the 2008 and 2009 cohorts for both 

needle surfaces (r = 0.543, P = 0.002 and r = 0.565, 

Table S1—Climate variables and their ranges for seed sources used in this study using the model by Rehfeldt (2006).

Climate variable Definition

Range among source locations

max min

mat Mean annual temperature degrees C 5.4 -1.2

map Mean annual precipitation 3320 497

gsp Growing season precipitation, April to September 837 188

mtcm Mean temperature in the coldest month degrees C -1.2 -11.8

mmin Mean minimum temperature in the coldest month degrees C -4.9 -18.1

mtwm Mean temperature in the warmest month degrees C 14.3 8.8

mmax Mean maximum temperature in the warmest month degrees C 23 13.6

sday Julian date of the last freezing date of spring 198 172

fday Julian date of the first freezing date of autumn 260 222

ffp Length of the frost-free period 87 22

dd5 Degree-days >5 degrees C (based on mean monthly temperature) 1142 370

gsdd5 Degree-days >5 degrees C accumulating within the frost-free period 622 118

d100 Julian date the sum of degree-days >5 degrees C reaches 100 190 143

dd0 Degree-days <0 degrees C (based on mean monthly temperature) 1776 394

mmindd0 Degree-days <0 degrees C (based on mean minimum monthly temperature) 3450 1162

smrpb Summer precipitation balance (jul+aug+sep)/(apr+may+jun) 1.03 0.45

smrsprpb Summer/Spring precipitation balance (jul+aug)/(apr+may) 1.2 0.27

sprp Spring precipitation (apr+may) 386 89

smrp Summer precipitation (jul+aug) 168 36

winp Winter precipitation (nov+dec+jan+feb) 1806 205
USDA Forest Service RMRS-P-76. 2018. 35 Table 1—Family means, minimum, and maximum values for needle characteristics and disease assessments. 2008 Needle cohort 2009 Needle cohort Adaxial Abaxial Adaxial Abaxial Trait Mean Min Max P-val Mean Min Max P -val Mean Min Max P -val Mean Min Max P -val nROWS 2.95 2.31 3.48 0.0034 1.75 1.31 2.14 0.0282 3.35 2.98 3.80 0.0195 2.09 1.73 2.78 0.0091 nSTOM 63.34 46.75 73.73 <0.0001 35.01 25.25 46.58 0.0002 82.37 70.00 98.65 0.0009 49.05 39.88 66.01 0.0019 ADEN 36.18 29.30 40.57 0.0094 17.12 13.75 22.52 <0.0001 44.99 38.77 52.22 0.0056 22.91 18.82 29.86 0.0002 RDEN 10.84 9.97 11.66 0.0042 10.08 9.02 11.39 0.001 12.33 11.34 13.22 <0.0001 11.81 11.00 12.87 <0.0001 NW 0.877 0.773 0.977 <0.0001 1.019 0.886 1.136 <0.0001 0.917 0.847 1.022 0.0011 1.073 0.978 1.197 0.0129 HT2 15.88 10.35 21.25 <0.0001 Spots1 106.1 58.8 154.5 0.0053 Cank1 2.28 0.1 6.5 <0.0001 Cank2 8.79 1.1 16.7 <0.0001 nROWS = number of rows of stomata, nSTOM = total number of stomata in 2-mm view, ADEN = stomata mm-2, RDEN = (nSTOM/ 2mm)/ nROWS, LDEN = stomata mm-1 of needle, NW = Needle width (mm), HT2 = seedling height at second inspection (cm), Spots1 = number of needle spots attributed to blister rust infection at first inspection after inoculation, Cank1 = number of blister rust cankers at first inspection after inoculation, Cank2 = number of stem symptoms at second inspection after inoculation = number of normal cankers (NC) + number of partial bark reactions (PBR) + number of bark reactions (BR). Spots1, Cank1, and Cank2 P -values are based on natural log transformed variables.
36 USDA Forest Service RMRS-P-76. 2018.

P = 0.001 for AD and AB sides, respectively). ADEN 

did not correlate significantly with NW but was 

correlated with nROWS on the same needle surface 

within the same cohort. For instance, ADEN on the 

AD needle surface in the 2009 cohort correlated with 

nROWS on the AD needle surface in the 2009 cohort 

(r = 0.674, P < 0.0001), but not the AD or AB surfaces 

in the 2008 cohort. 

Needle Width

The width of AD and AB surfaces of needles (NW) 

from both cohorts varied significantly between families across the range of whitebark pines sampled for 

this analysis (ANOVA): P = 0.0011 (AD 2009), 0.0129 

(AB 2009), <0.0001 (both AD and AB 2008) (table 1). 

The family means for NW on the AD surfaces averaged over the 2008 and 2009 cohorts was 0.897 mm; 

the NW for the AB surfaces averaged over the 2008 

and 2009 cohorts was 1.046 mm. The coefficient of 

variation for NW was similar in magnitude within a 

cohort: 6.4 and 4.6 percent on the AD surfaces and 6.7 

and 4.2 percent on the AB surfaces for 2008 and 2009, 

respectively.

Although the mean NW of the AB side was greater 

than that of the AD sides by a factor of 1.166 (due 

to its curvature), there were fewer stomata, as well 

as fewer rows of stomata on the AB side (1.92 rows 

AB vs. 3.15 rows AD). nSTOM and nROWS were 

correlated with NW on the AD and AB surfaces in the 

corresponding needle cohorts, except for nSTOM and 

nROWS for the AB side in the 2009 needle cohort 

(table 2). 

Disease and Climate Correlations

Stomate traits were correlated with disease symptom 

traits (table 3). Averaged over 2008 and 2009, the 

number of spots present on the entire seedling at the 

first inspection (Spots1) was predicted by RDEN on 

both the AD and AB sides (table 3). Although the 

2009 stomate traits showed similar relationships to the 

yearly averaged data, stomate features of 2008 needles 

lacked the same level of predictive power (P = 0.072 

and P = 0.0019, for RDEN 2008 and AD 2009, respectively). Cank1 and Cank2 were predicted by nROWS 

(AD) (negative correlation) for the yearly averaged 

stomate trait and the 2008 needle cohort, but there 

were no significant correlations when traits for 2009 

needles were analyzed separately. In summary, RDEN 

(AD), RDEN (AB), and nROWS (AD) predicted disease symptoms traits and were used going forward for 

the climate variable analyses.

Elasticnet identified three groups of correlated seed 

source climate variables that correlated with RDEN 

(AD) and RDEN (AB) from the yearly averaged 

stomate data (table 4). The climate variables within 

a group were highly intercorrelated, and univariate 

analyses and LASSO were used to prioritize the 

variable with the greatest predictive power in each 

Table 2—Correlation table for needle morphological characteristics related to needle width.

 NW_AD_08 NW_AD_09 NW_AB_08 NW_AB_09

Characteristic r P r P r P r P 

nROWS_AD_08 0.718 <.0001 0.281 0.133 0.643 0.0001 0.372 0.043

nROWS_AD_09 0.100 0.599 0.568 0.001 0.173 0.361 0.603 0.0004

nROWS_AB_08 0.466 0.009 0.203 0.282 0.423 0.020 0.187 0.324

nROWS_AB_09 -0.251 0.181 0.063 0.740 -0.233 0.216 0.049 0.798

nSTOM_AD_08 0.708 <.0001 0.386 0.035 0.666 <.0001 0.482 0.007

nSTOM_AD_09 0.304 0.102 0.555 0.001 0.347 0.060 0.604 0.0004

nSTOM_AB_08 0.417 0.022 0.207 0.273 0.395 0.031 0.189 0.316

nSTOM_AB_09 -0.100 0.598 0.151 0.424 -0.098 0.608 0.125 0.511

r = Pearson correlation coefficient, P = P-value, nROWS = number of rows of stomata, nSTOM = number of 

stomata, NW = Needle width (mm), AD = adaxial needle surface, AB = abaxial needle surface, 08 = 2008 needle 

cohort, 09 = 2009 needle cohort. 
USDA Forest Service RMRS-P-76. 2018. 37 Table 3—Correlation table for needle morphological characteristics related to disease symptoms. Disease symptom Ave. stomate traits 2008 Stomate traits 2009 Stomate traits Spots1 Stomate traits that predict disease symptom trait RDEN(AD)** RDEN(AB)** RDEN(AD)* RDEN(AD)*** RDEN(AB)** Model Y = (27.35)RDEN(AD)-210.76 Y = (19.78)RDEN(AB)-110.39 Y = (29.76)RDEN(AD)-260.96 Y = (20.30)RDEN(AB)-133.64 P-value 0.010 0.031 0.0019 0.015 Cank1 Stomate traits that predict disease symptom trait nROWS(AD)* nROWS(AD)** -- Model Y = (-2.68)nROWS(AD) +10.19 P -value 0.011 Cank2 Stomate traits that predict disease symptom trait nROWS(AD)** nROWS(AD)* -- Model Y = (-7.82)nROWS(AD)+33.43 P -value 0.044 P-value for univariate analysis: * for <0.10, ** for <0.05, *** for <0.01, **** for <0.001
38 USDA Forest Service RMRS-P-76. 2018.

group. The first group that correlated with the yearly 

averaged RDEN (AD) and RDEN (AB) included 

mean temperature in the warmest month (mtwm), 

degree-days >5 °C (dd5), and degree-days >5 °C accumulating within the frost-free period (gsdd5), each 

of which was a significant predictor of RDEN (AD) 

in univariate analyses, with mtwm providing the best 

fit. The second group included summer precipitation 

balance (smrpb) and summer/spring precipitation 

balance (smrsprpb), which were not significant predictors, but did significantly improve the model fit when 

combined with mtwm (Akaike’s information criterion 

was used as the criterion). The third group of correlated predictors, Julian date of the last freezing date of 

spring (sday) and length of the frost-free period (ffp), 

were not significant singly and did not improve the 

overall fit when modeled with the previous variables. 

RDEN (AB) for 2009 was best predicted by mtwm 

alone with no improvement in fit with additional 

predictors. Elasticnet identified one group of climate 

variables, growing season precipitation (gsp) and 

spring precipitation (sprp), which correlated with the 

yearly averaged nROWS (AD), providing the best fit 

with sprp. nROWS (AD) for 2008 and 2009 singly 

showed mixed results; gsp and sprp were only weakly 

correlated with 2009 nROWS (AD), and sprp provided 

the best univariate model for 2008 nROWS (AD).

DISCUSSION

One of the main objectives of this study was to 

describe the genetic variation in needle morphology 

for seedling families from parent trees representing 

much of the range of Pinus albicaulis. Needle traits 

correlated well between the 2008 and 2009 cohorts, 

and in most cases, some factors such as needle width 

were significantly associated with measures of stomate 

abundance (table 2). Family means for all traits—

stomate counts, stomate densities, and needle dimensions—for the 2009 cohort were larger than those of 

the 2008 cohort (table 1). A study examining withinpopulation genetic variation for the same morphological traits (2009 needle cohort) from this same common 

garden trial showed similar means and ranges for 22 

families from Crater Lake National Park (Sniezko et 

al., this proceedings, Genetic Variation). 

Table 4—Correlation table for needle morphological characteristics related to climate variables. See table S1 for 

climate variable definitions.

Stomatal trait Ave. stomate trait 2008 stomate trait 2009 stomate trait

RDEN(AB) Climatic variables 

that predict 

stomate trait

mtwm*, dd5* -- mtwm**, mmax**

Model Y = (0.19)mtwm + 

9.46

P-value 0.030

RDEN(AD) Climatic variables 

that predict 

stomate trait 

mtwm**, dd5**, gsdd5** mat*, mtwm**, dd5**, 

gsdd5**, d100**

mtwm**, dd5*, gsdd5* 

Model Y = (0.28)mtwm + 

(1.51)smrpb + 7.35

Y = (0.27)mtwm + 

(1.30)smrpb + 6.81

Y = (0.28)mtwm + 

(1.72)smrpb + 7.90

P -value 0.006 0.008 0.012

nROWS(AD) Climatic variables 

that predict 

stomate trait 

sprp**, gsp** gsp*, sprp** sprp* 

Model Y = (0.0012)sprp+2.94 Y = (0.0014)sprp+2.71 

P -value 0.023 0.048

P-value for univariate analysis: * for <0.10, ** for <0.05, *** for <0.01, **** for <0.001
USDA Forest Service RMRS-P-76. 2018. 39

Needle morphology may be of interest when considering factors that influence disease infection. Spores of 

the nonnative blister rust pathogen, C. ribicola, are deposited on needle surfaces and typically enter needles 

through the stomata. Morphological factors, such as 

stomate density or stomate size, might affect the probability of successful pathogen infection and establishment. In the case of western white pine (P. monticola), 

several factors that contributed to reduced needle 

lesion frequency included stomate size and shape, as 

well as needle surface characteristics such as cuticle 

chemistry (Woo et al. 2001). In the current study 

disease incidence and severity were represented by 

number of needle spots and cankers observed in approximately the first year after inoculation. There was 

a significant positive correlation between RDEN and 

the number of needle spots at the first inspection after 

inoculation, suggesting that RDEN may have some 

influence on the successful entry of the pathogen into 

foliage, at least for WBP. In an examination of withinpopulation genetic variation, the relationship between 

RDEN and needle spots was also significant and positive within the Crater Lake National Park population 

(Sniezko et al., this proceedings, Genetic Variation). 

However, RDEN was not significantly associated with 

the number of cankers at either inspection, so RDEN 

does not appear to be related to post-infection disease 

development. 

The number of stomate rows was negatively correlated with the number of cankers that developed on 

seedlings. This result might be counterintuitive, as one 

might expect greater numbers of stem symptoms associated with more rows (and in general, greater numbers) of stomata, providing greater potential frequency 

of infection. However, this study did not include 

measures of stomate size and activity, which may 

also help regulate entry of the fungus into the stem or 

other means of resistance that may be expressed in the 

needles or at the interface between the short shoot and 

the stem. Stomate features may only indirectly affect 

post-infection disease development, and this may be 

mediated through other associated physiological or 

biochemical processes.

Another objective of this study was to examine the 

extent to which geographical or environmental variables affecting the seed (mother) tree in the wild result 

in stomate differences in a common garden. Climatic 

features such as vapor pressure deficit vary along 

latitudinal and elevational gradients and are expected 

to influence needle characteristics such as stomate 

density due to the varying degrees to which trees must 

regulate water loss (Letts et al. 2009). In a study of 

needle morphology in P. flexilis, a significant decrease 

in stomate density was observed with increasing elevation, most likely in response to the relatively xeric 

conditions at higher elevations (Schoettle and Rochelle 

2000). Although variables such as elevation and 

latitude have previously been identified as potential 

drivers of variation in stomate abundance and needle 

morphology in conifers, the variation in the abundance 

and density of stomata across the families in this 

study could not be explained by any combination of 

geographic variables. This is not to imply that climatic 

features do not have a role to play; these same stomate 

traits may be under adaptive selection. Temperature 

in the summer at the seed source predicted stomate 

densities per row, and in some cases this prediction 

was improved with the addition of a summer precipitation variable. Likewise, spring precipitation at the 

seed source predicted the number of stomate rows on 

the adaxial needle surface. This suggests that these 

stomate traits may be adaptations to both summer temperature and spring and summer precipitation patterns. 

Temperature appears to correlate with the number of 

stomata, and spring or summer precipitation with the 

arrangement of stomata on the needle (i.e., number of 

stomate rows).

Although these traits may be adaptive in the climates 

under which they evolved, they may also influence 

susceptibility to WPBR. If increased summer temperatures under climate change select for genotypes with 

increased RDEN (AD) or RDEN (AB), they may also 

increase the infection efficiency of the rust on those 

seed sources. Likewise, if the spring precipitation 

decreases, and plants respond with reduced numbers of 

stomate rows on the adaxial needle surfaces, there may 

be an associated increase in successful canker development following infection. These results suggest that 

stomate traits predict early WPBR disease symptoms 

and that under adaptive selection, seed source climatic 

factors explain some of the variation in those stomate 

traits observed under common garden conditions. 

Consequently, though speculative with this dataset, it 

appears possible that climate change-driven selection 

for stomate traits could affect early rust symptom 
40 USDA Forest Service RMRS-P-76. 2018.

development. In addition, there have most likely been 

blister rust-driven selection pressures on some of the 

populations in this study.

Family variation in needle traits was large across the 

geographic sources included in this study, but it was 

also large in a within-population study (Sniezko et al., 

this proceedings, Genetic Variation). Thus, each population may have adaptive capacity in these traits to 

mitigate some of the impacts of biotic and abiotic influences. Field plantings at Crater Lake National Park 

of some of the families from the within-population 

study may provide a better understanding of the relationship of needle traits to long-term survival under 

a changing climate and the presence of the nonnative 

blister rust. A better understanding of the tolerance 

and resistance traits present in natural populations is 

essential to avoid the collapse of ecologically important five-needle pine ecosystems in western North 

America. For ecologically important, wild-growing 

pine species such as WBP, the introduction and maintenance of quantitative disease resistance (Sniezko et 

al. 2014) in natural ecosystems with plants developed 

through selective breeding will help ensure the longterm survival of these species (Schoettle and Sniezko 

2007). The successful introduction and regeneration of 

native forest stands with resistant planting stock will 

depend on a comprehensive knowledge of tree morphological traits and their adaptive significance, their 

underlying genetic controls, and their roles in influencing pathogen invasion, survival, and proliferation.

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www.fs.usda.gov/treesearch/pubs/21485.

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variation of Pinus flexilis (Pinaceae), a bird-dispersed 

pine, across a range of elevations. American Journal of 

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USDA Forest Service RMRS-P-76. 2018. 41

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

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Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Genetic Variation in Needle Traits of Whitebark Pine (Pinus albicaulis) 

Seedling Families: Within-Population Variation at

Crater Lake National Park

R.A. Sniezko1, *, J. Hill2, D.P. Savin1, R. Mutch3, J. Sticha1, A. Kegley1, and J. Beck4

1 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA 

2 USDA Forest Service (retired) 

3 Rob Mutch Ecosystem Photos, Eugene, Oregon, USA

4 USDI, National Park Service, Crater Lake National Park, Crater Lake, Oregon, USA

* rsniezko@fs.fed.us

Abstract—Genetic variation in needle traits (number of rows of stomata, areal density of stomata, and 

density of stomata in a row, as well as needle length and needle width), seedling height growth, and early 

measures of blister rust (causal agent: Cronartium ribicola) resistance were examined among 22 openpollinated families of whitebark pine (Pinus albicaulis) from Crater Lake National Park (CLNP) in Oregon. 

Needle traits were examined for both the adaxial and abaxial sides. Needle traits were also assessed for 

19 of 22 parent trees from CLNP. The adaxial side of the needle on average had more rows of stomata (3.3 

versus 2.0 rows), a greater density of stomata within a row (12.3 versus 11.2 stomata per mm of row), and 

a much higher areal density of stomata (45.60 versus 22.00 stomata per mm2) than the abaxial side for the 

seedling families. Statistically significant variation among families was noted for many traits, particularly 

needle traits from the adaxial side of the needles as well as for height and rust infection (number of needle 

spots and number of stem symptoms, approximately 8 and 15 months post-inoculation). Individual tree 

heritabilities were low to moderate for needle traits (adaxial side), moderate for height, and very high for 

number of stem infections. Family mean correlations between stomate variables were generally statistically 

significant and moderate to high, with somewhat lower correlations between traits across the two surfaces. 

Needle length was uncorrelated with any of the other traits examined. The density of stomata (adaxial 

side) within a row showed a moderate correlation (0.47) to the number of needle spots; however, neither 

of these measures was correlated with the percentage of seedlings in a family with stem infections. The 

number of needle spots was only moderately correlated with the number of stem infections. Correlations 

between progeny and parental traits were generally significant and moderate to high. The parent–progeny 

mean correlations were highest for some of the traits on the abaxial side. There was evidence of significant 

differences in some traits between the 4 families from the east side (drier) and the 18 families from the 

west side of Crater Lake National Park, with families from the east side tending to have more stomata, 

shorter needles, shorter height, more stem infections, and a much higher percentage of infection 1 year 

after inoculation. The level of within-population variation was similar to that observed among populations 

from much of the range (seedlings grown in the same common garden study). This genetic variation may 

help buffer the Crater Lake population of whitebark pine against some biotic and abiotic factors in the 

future. Recently established genetic field trials at CLNP will provide a means of determining whether the 

characterized genetic variation contributes to maintenance of this population.
42 USDA Forest Service RMRS-P-76. 2018.

INTRODUCTION

Whitebark pine (Pinus albicaulis) is a wide-ranging 

conifer in high elevation forest ecosystems of western 

North America (Tomback and Achuff 2010). It is 

highly susceptible to white pine blister rust, caused by 

the nonnative fungal pathogen Cronartium ribicola. 

Mortality due to blister rust, mountain pine beetle 

(Dendroctonus ponderosae), and a changing climate 

have raised concerns about the future viability of 

many populations of whitebark pine in both the 

United States and Canada (Committee on the Status of 

Endangered Wildlife in Canada 2010; U.S. Fish and 

Wildlife Service 2011). Genetic conservation efforts 

for whitebark pine are underway (Man and Moltzan, 

this proceedings; Sniezko et al. 2011b).

Genetic variation in a species is the cornerstone to its 

potential to evolve in response to biotic and abiotic 

challenges. Genetic variation in a limited number of 

traits has recently been examined in whitebark pine 

(Bower and Aitken 2006, 2008; Hamlin et al. 2011; 

Kegley et al. 2012; Mahalovich et al. 2006; Sniezko et 

al. 2007, 2011a; Warwell, this proceedings, Adaptive 

Variation among Whitebark Pine (Pinus albicaulis) 

Populations from the Interior Northwestern United 

States in Relation to Climate; Warwell, this proceedings, Phenotypic Selection on Growth Rhythm in 

Whitebark Pine (Pinus albicaulis) in Low Elevation 

Common Gardens), but there is little or no information 

to date on variation in needle traits. Needles provide 

the critical photosynthetic surface of whitebark pine, 

and stomata on needles are the entryway to infection 

for the C. ribicola pathogen (Patton and Johnson 

1970). Stomata also regulate the flow of gases such as 

carbon dioxide and water vapor in and out of needles 

and are a key experimental tool to examine plant 

responses to a changing environment (Hetherington 

and Woodward 2003). Genetic variation in needle 

traits may help whitebark pine mitigate the impacts of 

a changing climate, or may aid the understanding of 

some of the genetic resistances to white pine blister 

rust.

Whitebark pine typically has stomata on both the abaxial (AB) and the adaxial (AD) sides of needles (fig. 

1), which can be useful in distinguishing it from other 

five-needle pine species such as western white pine 

(P. monticola) (Hoff 1993). The AB side is the rounded 

outer side of the needle; a whitebark pine needle has 

one AB side and two AD sides (fig. 1). The AB surface 

generally has two resin channels running down the 

Figure 1—Cross section of a whitebark pine fascicle, illustrating how the one abaxial and two adaxial sides of each needle 

fit together to form the five-needle bundles, at approximately 205x magnification (left). The abaxial side at 212x magnification, exhibiting the typical two resin channels on either side of the stomatal rows (family CL04, top right). On the adaxial 

side at 212x magnification, the lack of resin channels can be noted along with an increased amount of stomatal rows (family CL22, bottom right).
USDA Forest Service RMRS-P-76. 2018. 43

needle on both sides of the stomatal rows and generally has only one to three rows of stomata (fig. 1). The 

AD side usually has three or four rows of stomata and 

usually lacks resin channels (fig. 1).

A common garden study of whitebark pine seedling 

families from a wide range of geographic sources 

(populations) offers a unique opportunity to examine 

structural and anatomical aspects of needles within 

Figure 2—Map of Crater Lake National Park, showing the locations of the 25 whitebark pine parent 

trees whose progeny are in the 2007 common garden study. Also shown is the natural density of whitebark pine throughout the Park. Green circles signify the location of the subset of 22 families used for 

the stomate measurements in this study.
44 USDA Forest Service RMRS-P-76. 2018.

and among populations. In this study we assess needle 

length and needle width as well as the frequency and 

distribution of stomata on both the adaxial and abaxial 

sides of needles among a sample of whitebark pine 

seedling progenies from parent trees at Crater Lake 

National Park (CLNP) in southern Oregon (USA) 

(fig. 2). We also examine the relationship between 

these seedling traits and the same traits assessed on 

needles of the parent trees located at CLNP, as well 

as the relationship of the seedling needle traits with 

early height growth and infection with white pine 

blister rust following artificial inoculation. This report 

complements a separate report in which we examine 

genetic differences in these traits among families from 

a wide segment of the geographic range of this species 

(Bennett et al., this proceedings). 

METHODS

Samples collected from two sets of whitebark pine 

materials of CLNP origin were used in this study: (1) 

needles from 22 open-pollinated seedling families that 

were part of a larger common garden study involving 

225 families sown in 2007 at the U.S. Department of 

Agriculture, Forest Service, Dorena Genetic Resource 

Center, Cottage Grove, Oregon (see Hamlin et al. 2010 

for background on the study); and (2) needles from 

a subset of 19 of the 22 parent trees collected in the 

natural population at CLNP (fig. 2). 

The seedlings, in 10-in3 (164 cm3) containers, were 

placed into a randomized complete block (RCB) 

design prior to inoculation with C. ribicola, with six 

blocks. The seedlings were inoculated in 2008, after 

their second growing season. Three blocks were inoculated in early September, and the other three blocks 

in early October; a different geographic source of rust 

was used for each inoculation. Seedlings from all 225 

families were transplanted in October 2008 to wooden 

boxes (0.9 m × 1.2 m × 0.3 m) in the same RCB design with 12 families per box, and 20 boxes per block. 

Families were in row plots (up to 10 seedlings per 

family row plot). 

Two seedlings from each of the 22 CLNP families 

were sampled from each of the 6 blocks (12 seedlings 

total per family) except for 1 seedling family that 

experienced poor germination and growth (9 seedlings 

total collected for this family). Effort was taken to 

collect from the third and eighth seedlings in the family row plot; samples were collected from alternate positions within the row-plot if the seedling had died or 

lacked tissue for sample collection. Needles from each 

seedling were collected during late spring 2010 and 

placed in individual small labeled paper envelopes. 

These envelopes were then bundled into family groups 

with rubber bands, placed into plastic bags, sealed, and 

stored in a freezer (–20 °C) until assessment, which 

occurred between August 2010 and June 2011. 

For assessment, needles from two to four fascicles per 

seedling were separated and affixed to white index 

cards using adhesive tape. Needle lengths (NL) were 

measured with a ruler to the nearest millimeter (mm) 

for four randomly selected needles of each seedling. 

Needles were examined and segments photographed 

under a digital microscope at approximately 200× 

magnification (Dino-Lite model Pro-AM413ZT; 

AnMo Electronics Corp., Taipei, Taiwan). The microscope was calibrated using a 10-mm scale; the 

magnification was input, allowing the software to automatically correct length and width measurements on 

the segment photographs. Segments were about 2 mm 

long, and these lengths were accounted for in density 

calculations.

Needles from 19 parent trees were collected at CLNP 

on September 7, 2009. Three fascicles per tree were 

collected, and an effort was made to collect needles 

from low, middle, and high branches. However, in the 

case of family CL07, all fascicles originated on one 

branch. The parent needles were stored in the same 

location in the freezer as the seedling needles. Needles 

from parent trees were measured after all of the seedling needle measurements were complete and used the 

same protocols as the seedling samples.

For both sets of needle samples, data were collected 

from the 2009 cohort of needles. Data on needle width 

(NW), the number of stomate rows (nROWS), and 

the number of stomata per row (nSR) were collected 

(counts were made from the photographs) for both 

the AD and AB sides. Judgments on which needle 

side was being examined were made on the basis of 

the AB side having two resin channels. Stomate rows 

ending or beginning in the photographed segment were 

commonly observed; in those cases, an estimate to the 

closest tenth of a row was made for nROWS. 
USDA Forest Service RMRS-P-76. 2018. 45

Typically, four AD and four AB measurements were 

made for each seedling sample. There were several 

instances where fewer or more measurements were 

made for a seedling, as a second observer reviewed every photo and changed some AD or AB designations. 

The total number of stomata per approximately 2-mm 

section of the needles was determined (tSTOM). The 

2-mm sections examined were generally toward the 

center section of each needle; the tips and basal sections of needles were not examined. Although efforts 

were made to take each measurement from a different 

needle, due to logistical difficulties (notably twistiness 

of needles), occasionally two measurements were 

made on different regions of the same needle. 

The counts and measurements taken were used to 

calculate several other stomate variables, including 

lineal density (LDEN), areal density (ADEN), and 

stomate frequency within rows (RDEN). LDEN was 

calculated as the number of stomata divided by the 

length of the needle segment in the photo. ADEN was 

calculated as LDEN divided by the needle width in the 

center of the segment photo. RDEN was calculated as 

LDEN divided by the number of rows of stomata in 

the photo. The average of the individual assessments 

for each trait was calculated to create a mean for each 

individual seedling.

For the seedling family means, a mean of means procedure was applied in which the means from the two 

seedlings in a block were averaged to get a family-byblock average, which was then averaged across the six 

blocks to get the overall family average for each trait. 

For the parent trees, there was no replication, so the 

mean is the average of the individual measurements 

for each tree. 

Second-year height (HT2, height through the 2008 

growing season), third-year height (HT3), and data 

on blister rust symptoms were also recorded for the 

seedlings. Number of blister rust needle spots at first 

assessment (summer 2009) (nSPOT1), the number of 

early stem infections present on all trees (nSS2) and 

on trees with stem infections in late fall 2009 (nSS2i), 

and the percentage of seedlings with stem infections at 

second inspection (pSS2) were determined. Only the 

subset of seedlings (maximum 12 seedlings per family) that had needle measurements was used here for 

the mean calculations of rust and growth traits.

Statistical analysis procedures were conducted using 

SAS® 9.4 (SAS Institute Inc., Cary, North Carolina, 

USA) and R 3.0.2 (R Foundation for Statistical 

Computing Vienna, Austria). Family means and parent 

means were used to calculate Pearson correlations 

between all traits by using PROC CORR. Linear 

models were fit by using PROC GLIMMIX. For count 

and proportion data, quasi-Poisson and quasi-binomial 

models were used, respectively. The east side of CLNP 

is drier than the west side (about 8 km apart), and 

separate analyses were set up to examine this contrast. 

Geographic source of rust, families, and side of CLNP 

were treated as fixed effects; blocking variables were 

treated as random effects. Individual tree heritabilities 

(h2

i

) were calculated from variance components in the 

same manner as Hamlin et al. (2011). For heritabilities of count data, square root transformations were 

applied to the individual tree data. Heritability using 

offspring-parent regression was estimated as described 

in Falconer and Mackay (1996). 

RESULTS

Family Variation

Needle Variation: Adaxial Versus Abaxial Side

Although the abaxial side of the needle was wider than 

the adaxial side by a factor of 1.19 for seedlings from 

the 22 families, the AB side averaged fewer rows of 

stomata (2.0 vs. 3.3), a lower density of stomata within 

rows (11.2 vs. 12.3), and a much lower areal stomate 

density (22.0 vs. 45.6) relative to the AD side (table 1). 

Although there was no statistically significant family 

variation in the needle traits for the AB side (although 

NW and ADEN were close to significantly different, 

with P < 0.10), all traits showed significant family 

differences for the AD side (table 1). For the AD side, 

minimum and maximum family means varied by 

greater than 17 percent for NW, 14 percent for RDEN, 

and more than 38 percent for ADEN.

Growth and Rust Infection Traits

There were statistically significant differences among 

families in seedling height (both HT2 [P < 0.0001] 

and HT3 [P < 0.0001]), nSPOT1 (P < 0.0001), nSS2i 

(P < 0.0001), and pSS2 (P = 0.0005). Families showed 

very large variation in these traits. For example, 
46 USDA Forest Service RMRS-P-76. 2018.

family means for HT2 ranged from 6.5 to 15.0 cm, 

and nSPOT1 ranged from 30.5 to 164.1 spots (table 

1). The artificial inoculation of the seedlings was very 

effective, as 100 percent of the seedlings from CLNP 

families had needle spots (no family variation present 

in this trait). Evidence of family differences in NL 

(P = 0.06) was suggested.

Parent Variation (Geographic Variation)

There was large variation in all needle traits for the 

19 parent trees (table 2). Although the abaxial side of 

the needle was wider by a factor of 1.21, as with the 

seedling families, the AB side of parent tree needles 

had fewer rows of stomata (1.9 vs. 3.3), a lower density of stomata within rows (10.6 vs. 11.6) and a much 

lower areal stomata density (18.6 vs. 42.5) than the AD 

side (table 2). There was large variation in means for 

all needle traits (table 2), and the minimum and maximum parental means on the AD side varied by greater 

than 49 percent for NW, 35 percent for RDEN, and 

more than 64 percent for ADEN. Although the needles 

from the parent trees tended to be longer and slightly 

wider than their seedling progeny, the needles from the 

parent trees tended to have fewer stomata than their 

progeny (tables 1 and 2; fig. 3).

Table 1–Overall means and range in family means for needle traits, growth, and blister rust 

infection measures for seedlings of 22 Pinus albicaulis families. Needle traits are shown 

separately for the adaxial (AD) and abaxial (AB) side of the needles except for needle length; 

height and rust means are shown here under the adaxial means. See the text for a description 

of traits.

Adaxial (AD) Abaxial (AB)

 Mean Minimum Maximum p-val Mean Minimum Maximum p-val

nROWS 3.3 2.7 4.0 <.001 2.0 1.6 2.3 0.142

tSTOM 84.0 67.1 98.7 <.001 48.5 38.6 56.5 0.338

LDEN 40.5 32.3 47.5 <.001 23.4 18.6 27.2 0.338

ADEN 45.6 38.8 53.7 <.001 22.0 18.2 26.5 0.069

RDEN 12.3 11.5 13.1 0.017 11.2 10.2 12.4 0.280

NW 0.89 0.81 0.95 0.009 1.06 0.98 1.10 0.066 

NL 39.1 33.9 44.2 0.059 - - - - 

HT2 10.2 6.5 15.0 <.001 - - - - 

HT3 14.6 7.9 19.8 <.001 - - - - 

nSPOT1 89.8 30.5 164.1 <.001 - - - - 

nSS2 6.1 0.4 18.3 <.001 - - - - 

nSS2i 9.4 1.7 19.5 <.001 - - - - 

pSS2 60.5 16.7 100.0 <.001 - - - - 

Table 2–Overall means and range in parental means for needle width and stomate traits for 19 

Pinus albicaulis parent trees at Crater Lake National Park. Needle traits are shown separately 

for the adaxial (AD) and abaxial (AB) side of the needles, except for needle length which is 

shown here under adaxial. See the text for a description of traits.

Adaxial (AD) Abaxial (AB)

 Mean Minimum Maximum Mean Minimum Maximum

nROWS 3.3 2.0 4.3 1.9 0.8 2.7

tSTOM 79.7 55.0 105.8 42.8 17.3 68.0

LDEN 38.4 26.5 50.9 20.6 8.3 32.7

ADEN 42.5 31.3 51.5 18.6 8.0 27.7

RDEN 11.6 10.1 13.6 10.6 6.8 12.5

NW 0.90 0.74 1.10 1.10 0.90 1.20 

NL 50.5 42.0 67.5 - - - 
USDA Forest Service RMRS-P-76. 2018. 47

Correlation Between Traits: Progeny

Family mean correlations (22 families) between the 

stomate variables on the AD and AB sides for the seedling families were generally significant and moderate 

to very high. For example, nROWS on the AD side 

was significantly correlated with NW, tSTOM, LDEN, 

and ADEN of stomata (r = 0.51, 0.93, 0.93, and 0.79, 

respectively) on the AD side. Correlations between 

Figure 3—Relationship between offspring (y-axis) and parents (x-axis) for a variety of needle traits for 19 seedling families 

in a common garden test and the corresponding parent trees at Crater Lake National Park (CLNP). (a) stomatal density in 

rows (RDEN) on the adaxial (AD) side (r = 0.50); (b) number of stomatal rows (nROWS) on the abaxial (AB) side (r = 0.70); 

(c) needle width (NW) on the adaxial (AD) side (r = 0.59); (d) needle length (NL, r = 0.41). Green triangles represent parents 

on the east side of CLNP, while black circles represent the west side. The dotted red line shows the least-squares regression line. Significance at the α = 0.05 and 0.1 levels occurs for correlations with absolute values greater than 0.46 and 0.39, 

respectively. Note the smaller needle length of the three parents and their progenies from the drier east side of Crater Lake 

National Park.
48 USDA Forest Service RMRS-P-76. 2018.

the AD and AB sides of the needles for these variables 

were also generally statistically significant, but somewhat lower (r = 0.33, 0.50, 0.50, and 0.37 for nROWS 

with NW, tSTOM, LDEN, and ADEN, respectively 

(the first and last correlation being nonsignificant at 

the 0.05 level) (table 3). 

Needle width was moderately correlated with tSTOM 

and LDEN on the AD side, but not on the AB side 

(and not with ADEN on either side). Needle width was 

negatively correlated with height on both the AD side 

(r = –0.51, –0.54 with HT2 and HT3) and AB side of 

needles (r = –0.57, –0.59 with HT2 and HT3) but was 

not significantly correlated with measures of blister 

rust infection such as nSPOT1, nSS2i and pSS2, except for a moderate, negative correlation with nSPOT1 

on the AB side (r = –0.43). There was a negative, but 

non-significant, correlation (r = –0.29) on the AD side.

Needle length was uncorrelated with any trait, although the correlation with HT3 was nearly significant 

(r = 0.40, P = 0.06). HT2 and HT3 were significantly 

correlated with nSPOT1 (r = 0.63 and 0.66, respectively) and nSS2i (r = 0.45 and 0.48, respectively), but 

not with nSS2 or pSS2. 

The density of stomata within rows (RDEN) was 

correlated with nSPOT1 (r = 0.47) for the AD side, 

but not for the AB side (r = 0.14) (figs. 4 and 5). The 

number of stem symptoms (both nSS2 and nSS2i) was 

only moderately correlated with nSPOT1 (r = 0.53 and 

0.57, respectively). However, nSS2i was not significantly correlated with pSS2 (r = 0.33). 

Family mean correlations between needle traits between the AD and AB side were significant (or nearly 

significant) for many needle traits (table 3). They were 

generally highest for the same trait on AD versus AB: 

NW (0.89), followed by nROWS (0.59), ADEN (0.58), 

LDEN (0.54), tSTOM (0.54), and RDEN (0.50).

Correlation Between Traits: Parents

Correlations between traits for the 19 parent trees were 

generally significant and moderate to high. For example, nROWS on the AB side was correlated with NW 

(0.67), tSTOM (0.97), ADEN (0.96), and RDEN (0.54) 

on the AB side and also, to a lesser degree, with those 

traits on the AD side (though not correlated with AD 

RDEN). Needle width (both AB and AD) also showed 

moderate-to-high correlation with most measures of 

stomate frequency (except RDEN).

Correlation Between Traits: Parents versus 

Progeny

The parent-progeny correlations were significant for 

the AB but not the AD sides for nROWS (r = 0.70 and 

0.44, respectively) (fig. 4). The correlation between 

parent-offspring for ADEN was also significant for the 

AB side but not the AD side (r = 0.59 and 0.23, respectively). None of the parental needle traits was correlated 

with either seedling height or rust infection traits except 

for ADEN on the AB side with pSS2 (r = 0.48).

East Side Versus West Side

As a group, families from the drier east side (n = 4) 

differed significantly (at P = 0.05) from those on 

the west side (n = 18) for many traits and nearly so 

(P < 0.10) for several other traits (table 4), particularly 

for the AD side of the needles. The difference was 

Table 3–Correlations for stomate traits between abaxial (AB) and adaxial (AD) sides for 

seedlings of 22 Pinus albicaulis families from Crater Lake National Park. Significance at the 

α = 0.05 and 0.1 levels occurs for correlations with absolute values greater than 0.42 and 0.36, 

respectively. See the text for a description of traits.

Adaxial (AD): 

Abaxial (AB): nROWS tSTOM LDEN ADEN RDEN NW

nROWS 0.59 0.49 0.49 0.40 -0.24 0.29

tSTOM 0.50 0.54 0.54 0.54 0.11 0.10

LDEN 0.50 0.54 0.54 0.54 0.11 0.10

ADEN 0.37 0.44 0.44 0.58 0.21 -0.19

RDEN 0.11 0.29 0.30 0.44 0.50 -0.24

NW 0.33 0.20 0.20 -0.19 -0.30 0.89
USDA Forest Service RMRS-P-76. 2018. 49

particularly notable for needle length, areal density, 

and number of stomate rows. The east and west side 

families did not significantly differ for needle width 

(table 4).

Narrow-Sense Individual Tree Heritability

Heritability was calculated by using two techniques, 

one involving variance components and the other 

relying on parent-offspring regression. Individual tree 

heritability (hi

2) calculated from variance components 

was generally higher for the AD side, ranging from 

0.23 to 0.73 (table 5), than for the AB side. Height was 

also very heritable (hi

2 = 0.69 and 0.78 for secondand third-year height, respectively), as was the number 

of stem symptoms at second inspection (hi

2 = 0.92). 

The parent-offspring regression resulted in more-equal 

heritabilities between the AD and AB sides. The mean 

of all needle trait heritabilities using this method was 

0.37, compared to 0.29 with the variance component 

method. 

DISCUSSION

There is large within-population variation for needle 

traits, seedling height, and measures of rust infection 

among the 22 seedling families for the CLNP population of whitebark pine. This large genetic variation 

provided an excellent opportunity to examine the 

relationships among these traits.

In general, the relationships between needle traits and 

seedling growth were not significant. An exception 

was a moderate one for needle width (both AD and 

AB) and height; taller families tended to have narrower needles. There was also a moderate negative 

relationship between number of rows of stomata on the 

abaxial side of needles and height. 

In this study of whitebark pine, needle traits were 

generally not significantly correlated with rust infection traits, with the exception of density of stomata 

within rows of the adaxial side of needles and number 

of needle spots (r = 0.47; fig. 5). Bennett et al. (this 

proceedings) reported a significant correlation between 

these two traits for both the AD and AB sides for the 

2008 needle cohort and a nearly significant correlation for the AD side for the 2009 needle cohort. Thus, 

families with more stomata per millimeter within a 

Figure 5—Micrographs of adaxial needles from the families 

with the highest (top) and lowest (bottom) within row density of stomates (RDEN). Family CL25 (top, 212x) had the 

highest stomatal row density as well as high spotting after 

inoculation with blister rust. Family CL02 (bottom, 220x) had 

the lowest stomatal row density as well as the lowest number 

of needle spots. 

Figure 4—Relationship between 22 family means for stomatal density in rows (RDEN) on the adaxial (AD) side of the 

needle (x-axis) and the number of needle spots (nSPOT1, 

y-axis) after inoculation with blister rust spores (r = 0.47, P = 

0.026). Green triangles represent parents on the east side of 

CLNP, while black circles represent the west side. The dotted red line shows the least-squares regression line. 
50 USDA Forest Service RMRS-P-76. 2018.

row tended to have more needle spots, but the number 

of rows or density of stomata over the needle surface 

showed no correlation to the level of needle spotting. 

At the time of sampling in 2010, the 2008 cohort of 

needles (which were present at the time of inoculation) 

was not available for the CLNP population, but there 

were significant and moderate-to-high correlations 

between most needle traits for the 2008 and 2009 

needle cohorts in the related study (Bennett et al., this 

proceedings). There was also a significant negative 

correlation (r = –0.43, P = 0.045) between needle 

width (AB side) and number of needle spots in this 

study, but not in the Bennett et al. (this proceedings) 

study. 

Table 4–East side means (n = 4 families), west side means (n = 18 families), and p-values for the 

significance of side of Crater Lake for seedlings of 22 Pinus albicaulis families from Crater Lake 

National Park. See the text for a description of traits.

 Adaxial (AD) Abaxial (AB)

 East Mean West Mean p-value East Mean West Mean p-value

nROWS 3.45 3.26 0.018 2.14 1.98 0.090

tSTOM 89.9 82.9 0.002 51.8 47.9 0.086

LDEN 43.3 39.9 0.002 25.0 23.0 0.087

ADEN 48.3 45.1 0.003 23.4 21.8 0.084

RDEN 12.6 12.3 0.082 11.0 11.2 0.532

NW 0.90 0.89 0.496 1.06 1.06 0.701

NL 35.7 39.8 0.006 - - - 

HT2 9.3 10.4 0.054 - - - 

HT3 12.9 14.9 0.010 - - - 

nSPOT1 95.8 88.4 0.845 - - - 

nSS2 9.9 5.3 <.001 - - - 

nSS2i 10.9 9.1 0.071 - - - 

pSS2 85.4 55.4 <.001 - - - 

Table 5–Narrow sense individual tree and parent-offspring heritabilities

and standard errors for needle traits, growth, and blister rust infection traits 

for seedlings of 22 Pinus albicaulis families from Crater Lake National 

Park. See the text for a description of traits.

 Variance Components Parent-Offspring Regression

Trait Heritability S.E. Heritability S.E.

NL 0.18 0.15 0.38 0.20

NW (AB) 0.14 0.13 0.23 0.20

NW(AD) 0.26 0.16 0.51 0.17

tSTOM(AB) 0.03 0.09 0.35 0.12

tSTOM(AD) 0.61 0.25 0.35 0.24

LDEN(AB) 0.03 0.09 0.35 0.12

LDEN(AD) 0.61 0.25 0.35 0.24

ADEN(AB) 0.14 0.12 0.48 0.16

ADEN(AD) 0.63 0.27 0.37 0.37

RDEN(AB) 0.04 0.09 -0.07 0.18

RDEN(AD) 0.23 0.17 0.51 0.21

nROWS(AB) 0.09 0.11 0.51 0.13

nROWS(AD) 0.73 0.29 0.45 0.22

HT2 0.69 0.31 - - 

HT3 0.78 0.34 - - 

HT inc 0.37 0.22 - - 

nSPOT1 0.39 0.24 - - 

nSS2 0.92 0.37 - - 
USDA Forest Service RMRS-P-76. 2018. 51

In earlier studies with a different five-needle pine species, western white pine (P. monticola) (Gansel 1956; 

Woo et al. 2001), no significant differences were found 

for stomate frequency traits (or needle width and needle 

length) in pairwise comparisons among susceptible and 

resistance groups, but needles of susceptible families 

had significantly wider and larger stomata (greater area) 

than did those of resistant or seed orchard lots (Woo 

et al. 2001). Size of stomata was not assessed in this 

study; perhaps some measures of both stomate size 

and stomate density would help clarify the relationship 

with the frequency of needle spotting in whitebark pine 

seedlings. 

Number of spots in this study was correlated with the 

number of stem infections but not with the percentage 

of seedlings infected in a family, so other traits play an 

important role in determining which families are most 

resistant (at least for the canker-free trait). The heritability for number of stem infections was much higher 

than that of number of needle spots. Field trials will be 

needed to ascertain the potential merit of reduced needle 

spotting in some seedling families following artificial 

inoculation.

There was a significant correlation between second-year 

seedling height and number of needle spots (r = 0.63), 

with taller families having more needle spots. This 

relationship may be due to several factors including potentially more needles on taller seedlings, more needles 

exposed to rust spores (less clumping), or possibly 

difficulty counting a high number of needle spots on the 

smallest seedlings. In another study (Bennett et al., this 

proceedings) involving families sampled across much 

of the range of whitebark pine, the correlation between 

these traits was low (r = 0.21) and nonsignificant. This 

relationship will be examined in more detail using the 

larger number of families (225) in the common garden 

study and other inoculation trials. It is also notable that 

although the taller families had more needle spots, they 

did not tend to have significantly more stem symptoms 

(r = 0.24 for nSS2). Further investigations and field trials may clarify the underlying relationship.

Nearly all whitebark pine seedlings (99.9 percent) 

developed needle spots in the trial from which the 

CLNP seedlings were sampled, as well as in other trials 

at DGRC (Sniezko et al. 2007; Sniezko, unpublished 

data, on file at DGRC), indicating few escapes at the 

inoculation stage and little capacity of whitebark pine 

for a “no spots” trait. However, families varied greatly 

in the number of needle spots per seedling; some 

seedlings had more than 600 needle spots. There was 

a significant family mean correlation between number 

of needle spots and number of stem symptoms, but 

not with the percentage of seedlings with early stem 

symptoms. In this population, the resistances resulting 

in stem symptom-free seedlings (at 15 months postinoculation) under heavy inoculum pressure appear to 

be unrelated to the number of needle spots. Although 

having fewer needle spots did not result in a lower 

percentage of seedlings exhibiting stem symptoms in 

this study, lower levels of spotting have been postulated 

to play a more significant role in reducing cankering in 

P. monticola in the field (Hoff and McDonald 1980). 

Field studies are needed to confirm the efficacy of this 

trait in both P. monticola and whitebark pine. 

The very large differences among CLNP families in percentage of seedlings showing early stem infection show 

great promise in identifying parents with resistance to 

blister rust, but this trait appears to be unrelated to any 

needle traits examined here. Some families with a low 

percentage of stem symptoms at this early stage also 

showed a marked increase in the percentage of stem 

symptoms over time (R. Sniezko, unpublished data). 

Several restoration trials have been established at CLNP 

since 2009 using many of the seedling families in this 

study. The data from these field sites will allow for 

examination of the relationship of genetic variation in 

these traits in the seedling trial with survival, growth, 

cone production, blister rust resistance, and other traits 

in the field over time.

The statistically significant differences between some of 

the trait means of progeny of east-side versus west-side 

parents were somewhat surprising and may be due to 

selection in the different environments (east side being 

drier) or limitations of the sample set (i.e., sampling of 

four families from the east side vs. 18 families from the 

west side of CRLA). Further examination is needed to 

confirm the trends.

For any additional future efforts to examine needle 

traits, methods for micrographing and analyzing the 

needles may need to be reexamined and standardized. 

Although storage in the –20 °C freezer did not seem 
52 USDA Forest Service RMRS-P-76. 2018.

to affect the clarity of stomata, needle conformation 

could be affected (Wykoff 2002). The twistiness of the 

needles posed some problems. Several micrographs 

appeared to be needles with resin channels on the adaxial side; further investigation suggested that most of 

these needles had a reflective ridge on the needle that 

looked very similar to a resin channel. Others showed 

an unusually high number of stomate rows on the AD 

side. Although this may be an entirely natural event, 

it is also possible that because the angle between the 

two AD sides varies, both of the adaxial sides were 

captured and analyzed in a single micrograph. It is 

also possible that needle curvature caused part of the 

abaxial side to be captured as part of an adaxial photograph. Each photograph was examined by at least two 

people at different times and obvious problems were 

removed from the analysis. However, it is possible 

that some of these unusual observations were naturally 

occurring. In future investigations of needle properties, 

greater care should be taken to ensure only one side of 

the needle is being micrographed. 

An entire needle typically has hundreds to over 1,000 

stomata depending on side. Our subsamples were in 

the center portion of the needle only. Although there 

was genetic variation for the 2-mm subsamples, the 

2-mm subsample data cannot be used to estimate the 

total number of stomata on a needle. 

Whitebark pine showed large heritable variation 

among families from the CLNP population. The large 

genetic variation in whitebark pine needle and white 

pine blister rust infection traits in the CLNP population is encouraging and may help the species to persist 

under pressure of at least some biotic and abiotic 

challenges. 

Future studies should consider including other traits 

such as stomate size. A greater understanding of the 

genetic variation in whitebark pine adaptive traits and 

how it varies within and among populations can help 

guide conservation and restoration strategies.

ACKNOWLEDGMENTS

We acknowledge Crater Lake National Park personnel 

for the seed for this study and data on the parent trees, 

and Elena Thomas for coordinating the collection 

of needles from the parent trees. We thank the U.S. 

Department of Agriculture, Forest Service, Dorena 

Genetic Resource Center personnel for nursery culture 

and other aspects of the trial. We thank Jim Hamlin for 

comments on an earlier version of this paper, and for 

inputs on some of the analyses.

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Adaptive Variation Among Whitebark Pine (Pinus albicaulis) 

Populations from the Interior Northwestern United States

in Relation to Climate 

M.V. Warwell1, *

1 USDA Forest Service, Rocky Mountain Research Station, Moscow, Idaho, USA, and University of Minnesota,

Department of Ecology, Evolution, and Behavior, St. Paul, Minnesota, USA

* mwarwell@fs.fed.us

INTRODUCTION

Many forest tree species are composed of populations that are genetically adapted to only a portion of 

environmental variation that the species occupies as a 

whole. Knowledge from genecology studies on intraspecific patterns of genetic variation in relation to the 

environment is fundamental to gene conservation and 

reforestation (Morgenstern 1996). In particular, knowledge of how genetic variation is related to climate may 

be critical to identify threatened populations and match 

genotypes with suitable climate under rapid ongoing 

climate change (Joyce and Rehfeldt 2013; Rehfeldt 

and Jaquish 2010; Rehfeldt et al. 2014). 

Whitebark pine (Pinus albicaulis) is a keystone and 

foundational species in western U.S. subalpine ecosystems that is threatened by population decline due 

to anthropogenic disturbance (Tomback et al. 2001). 

In recent years, several regional (Hamlin et al. 2008, 

Abstract—Provenance tests of 49 populations of whitebark pine (Pinus albicaulis) revealed moderate 

genetic differentiation for growth potential (growth under conditions approaching physiological optimum). 

Moderate genetic differentiation was also detected for survival after more than 10 years under warm-dry 

climate in the field but not for unconditional expected height, which was considered the best available predictor of fitness. Height was considered unconditional because survival over multiple years was explicitly 

included in the analysis. Genetic divergence for growth potential and survival was found to correspond 

with climatic clines. Population differentiation for growth potential was estimated to require a separation 

of about ±7.4 Julian days for timing of spring warming (D100) between climate of seed sources (≈ ±210 m 

elevation). Differentiation for survival was estimated to require about ±2.66 cm annual spring precipitation 

between climate of population origin. These results inform seed transfer and may provide insight into the 

genetic response of populations under a warming climate, particularly during early growth.

2011; Mahalovich et al. 2006) and rangewide (Bower 

and Aitken 2006, 2008) studies have advanced knowledge of the genecology of whitebark pine genecology. 

Nonetheless, information on genetic variation among 

populations for growth and survival in the field and 

in relation to climate of population origin remains 

limited. Thus, the objectives of the present study were 

to (1) assess genetic variation in growth and survival 

among populations from the interior northwestern 

United States in the field and (2) model this genetic 

variation in relation to climate at population origin. 

This paper reports only preliminary results. 

METHODS

This study used seeds from wind-pollinated cones, 

collected during 1991–1997 from 49 wild whitebark 

populations in the interior northwestern United 

States (fig. 1). Collections were made from 5 to 20 
USDA Forest Service RMRS-P-76. 2018. 55

trees per population. Seeds were sown in 1998, and 

outplanted into two provenance tests in forest openings at Priest River Experimental Forest (lat. 48.3°N, 

long. –116.8°W) in northern Idaho (USA) (fig. 1). 

Test 1 was outplanted in April 24–26, 2000 with 

2-year-old trees representing 46 populations. Test 2 

was outplanted 2.2 km south of test 1 in October 2–5, 

2001 with 4-year-old trees representing 42 populations 

(fig. 2). Thirty-nine populations were common to both 

tests. Soil at the test 2 site was rocky and shallow to 

a hard pan (Page-Dumroese 1993) and appeared to 

be harsher than the test 1 site, where soil was tilled at 

planting, unobstructed, and well drained. Both tests 

were located at a relatively low elevation (671 m) 

where temperatures were warmer (+4.4 to 9.1 °C mean 

annual temperature) and growing seasons were longer 

(+5 to 99 frost-free days) than found among native elevations (1,646 m to 2,774 m) of the study populations.

A randomized complete block design was used in both 

tests. Each seed source was represented by 30 seedlings. Test 1 used five-tree row-plots per population 

within each of six blocks. Test 2 used 10-tree rowplots within each of 3 blocks. Individual trees were 

planted at a spacing of 1 m × 1 m at test 1 and 35.56 

cm × 33.02 cm at test 2. Thinning to prevent competitive interaction among trees was not necessary despite 

close planting in test 2 due to high mortality and the 

species’ characteristic slow growth. Total aboveground 

height measurements on 14-year-old trees were tallied 

in fall 2012 for both tests. Survival and shoot elongation were tallied in fall 2001–2005, 2011, and 2012 in 

test 1, and 2003–2006 and 2010–2012 in test 2. 

The degree of differentiation among populations for 

survival and unconditional expected height was assessed with aster analysis (Geyer et al. 2007; see Shaw 

Figure 1—Location of whitebark pine (Pinus albicaulis) provenances (●) and study site at Priest River 

Experimental Forest (∆). Shaded area indicates predicted contemporary realized climate niche for whitebark 

pine (Warwell et al. 2007). 
56 USDA Forest Service RMRS-P-76. 2018.

et al. 2008) using the reaster function (Geyer 2013) in 

R (R Core Team 2012). Aster analysis permits unified 

analysis of multiple life history stages with appropriate 

statistical distributions. Thus, to assess unconditional height, where survival over multiple years was 

explicitly included in the analysis of height, survival 

was modeled using Bernoulli and height was modeled using normal distributions. For this procedure, 

unconditional expected height was assessed using seed 

source row-tree plot sums for height and mean survival 

to 2012. Differences in growth potential were assessed 

using general linear mixed model analysis using the 

lmer function (Bates et al. 2013) in R (R Core Team 

2012). For this procedure, growth potential was assessed using seed source row-tree plot means for total 

shoot elongation in 2005. 

To model genetic variation with climate, predictions 

for 26 climate variables (Rehfeldt 2006) representing 

the timing of moisture or temperature, or a combination, and their interaction at seed origins were tested 

by comparing the likelihoods of nested models following the statistical framework presented by Leites 

et al. (2012) and using a forward stepwise selection 

approach. Finally, regression analysis was used to describe climate based seed transfer guidelines following 

methods described by Rehfeldt (1983).

RESULTS AND DISCUSSION

After 12 and 11 years of growth in the field, mean 

height for the 14-year-old trees was 155 and 110 cm 

with 39- and 41-percent survival for tests 1 and 2, 

Figure 2—Test 2 site in 2005, 3 years after outplanting. Trees are ~7 years old. Ten-tree row-plots are arranged from the 

foreground to the background edge. 
USDA Forest Service RMRS-P-76. 2018. 57

respectively. Differences between the two test sites 

were statistically significant (P < 0.05) for mean 

height but not survival. In test 1, survival among 

populations varied from 8 to 75 percent; mean apical 

shoot elongation in 2005 varied from 7.3 to 16.5 cm 

and mean height in 2012 varied from 90.4 to 214.4 cm. 

In contrast, populations in test 2 ranged from 19- to 

77-percent survival and 4.6 to 13.7 cm for mean apical 

shoot elongation in 2005, and 18.8 to 124.3 cm for 

mean height in 2012. 

In both tests, no statistically significant (P > 0.05) 

differences among seed sources were detected for 

unconditional expected height, the best available approximation of fitness. In test 2, however, a negative 

trend was apparent for fitness in relation to annual 

spring precipitation (SPRP) at seed source. Survival 

was statistically significantly (P < 0.05) in test 2 but 

not test 1. In test 2, survival was negatively related to 

SPRP at seed source. SPRP accounted for 13.2 percent 

of variation in survival among populations. Variation 

in growth potential was statistically significant 

(P < 0.05) and negatively related to annual dryness and 

winter coldness at the seed source (ADIMMINDD0) 

in test 1 and positively related to timing of spring 

warming (D100) at the seed source in test 2. In test 1, 

ADIMMINDD0 accounted for 9.8 percent of population variation in growth potential. In test 2, D100 

accounted for 21.3 percent of population variation in 

growth potential. Clinal variation for growth potential 

and height generally decreased in a northwest to southeast direction and with decreasing elevation in the 

study region. Conversely, survival and fitness generally increased in a northwest to southeast direction.

Results from the present study indicate that reasonable 

confidence in population differentiation for growth 

potential requires a separation of about ±7.4 Julian 

days for D100 between climates of seed sources. This 

climate range corresponds to an elevation range of 

about ±210 m. These results are in general agreement 

with findings reported by Bower and Aitken (2008). 

In comparison with other western forest species, these 

results suggest that whitebark pine is an intermediate 

between specialist and generalist modes for growth 

potential (Rehfeldt 1994).

Reasonable confidence in population differentiation 

for survival under warm-dry climate requires a separation of about ±2.66 cm SPRP between seed sources. 

Differentiation of survival was assessed using young 

trees grown in a warm-dry climate for more than 10 

years. The warm-dry climate conditions were similar 

to predicted future climate for the contemporary distribution of whitebark pine. Thus, results may provide 

insight into the genetic response of populations under a 

warming climate, particularly during early growth. To 

be sure, populations were tested outside of whitebark 

pine’s native distribution; thus, results may not necessarily indicate performance in whitebark pine’s native 

habitat.

ACKNOWLEDGMENTS

I thank the U.S. Department of Agriculture, Forest 

Service, Rocky Mountain Research Station for funding. In addition, I am grateful to Drs. Dennis Ferguson 

and Ned Klopfenstein for operational support; Donna 

Dekker-Robertson for direction and management of 

seed collections; Paul Leigh, Mose Harris IV, Amanda 

Link, and Debra Eastman for technical field support; 

Charles Geyer, Nicholas Crookston, and Jerry Rehfeldt 

for statistical advisement; and Ruth Shaw and Ned 

Klopfenstein for research advisement. I also thank 

Andrew David for review of an earlier version of this 

paper. 

REFERENCES

Bates, D.; Maechler, M.; Bolker, B. 2013. lme4: Linear 

mixed-effects models using S4 classes. https://cran.rproject.org/web/packages/lme4/index.html. (04 January 

2016).

Bower, A.D.; Aitken, S.N. 2006. Geographic and seasonal 

variation in cold hardiness of whitebark pine. Canadian 

Journal of Forest Research. 36: 1842–1850.

Bower, A.D.; Aitken, S.N. 2008. Ecological genetics and 

seed transfer guidelines for Pinus albicaulis (Pinaceae). 

American Journal of Botany. 95: 66–76.

Geyer, C.J. 2013. Aster: Aster Models. Version: 0.8-27, 

R.p.v. (Ed.). Vienna, Austria: R Foundation for Statistical 

Computing. https://cran.r-project.org/web/packages/aster/

index.html. (04 January 2016).
58 USDA Forest Service RMRS-P-76. 2018.

Geyer, C.J.; Wagenius, S.; Shaw, R.G. 2007. Aster models 

for life history analysis. Biometrika. 94: 415–426.

Hamlin, J.; Kegley, A.; Sniezko, R. 2011. Genetic variation 

of whitebark pine (Pinus albicaulis) provenances and 

families from Oregon and Washington in juvenile height 

growth and needle color. In: Keane, R.E.; Tomback, D.F.; 

Murray, M.P; [et al.], eds. The future of high-elevation, 

five-needle white pines in western North America: 

Proceedings of the high five symposium. Proceedings 

RMRS-P-63. Fort Collins, CO: U.S. Department of 

Agriculture, Forest Service, Rocky Mountain Research 

Station: 133–139.

Hamlin, J.E.; Doede, D.; Kegley, A.; [et al.]. 2008. An 

assessment of genetic variation of Pinus albicaulis

populations from Oregon and Washington in relation 

to height increment, phenology, and form. In: Noshad, 

D.; Woon, N.E.; King, J.; [et al.], eds. Proceedings: 

Breeding and genetic resources of five-needle pines: 

Ecophysiology, disease resistance and developmental 

biology. Seoul: The Director General, Korea Forest 

Research Institute: 24–26.

Joyce, D.G.; Rehfeldt, G.E. 2013. Climatic niche, ecological 

genetics, and impact of climate change on eastern white 

pine (Pinus strobus L.): Guidelines for land managers. 

Forest Ecology and Management. 295: 173–192.

Leites, L.P.; Robinson, A.P.; Rehfeldt, G.E.; [et al.]. 2012. 

Height-growth response to climatic changes differs 

among populations of Douglas-fir: a novel analysis of 

historic data. Ecological Applications. 22: 154–165.

Mahalovich, M.F.; Burr, K.E.; Foushee, D.L. 2006. 

Whitebark pine germination, rust resistance, and 

cold hardiness among seed sources in the Inland 

Northwest: Planting strategies for restoration. In: 

Riley, L.E.; Dumroese, R.K.; Landis, T.D., tech. cords. 

National proceedings: Forest and conservation nursery 

associations—2005. Proceedings RMRS-P-43. Fort 

Collins, CO: U.S. Department of Agriculture, Forest 

Service, Rocky Mountain Research Station: 91–101.

Morgenstern, E.K. 1996. Geographic variation in forest 

trees: Genetic basis and application of knowledge in 

silviculture. Vancouver, BC: UBC Press. 214 p.

Page-Dumroese, D.S. 1993. Susceptibility of volcanic 

ash-influenced soil in northern Idaho to mechanical 

compaction. Res. Note INT-409. Ogden, UT: U.S. 

Department of Agriculture, Forest Service, Intermountain 

Research Station. 5 p.

R Core Team. 2012. R: A language and environment for 

statistical computing. Vienna, Austria: R Foundation for 

Statistical Computing.

Rehfeldt, G.E. 1983. Adaptation of Pinus contorta

populations to heterogeneous environments in northern 

Idaho. Canadian Journal of Forest Research. 13: 

405–411.

Rehfeldt, G.E. 1994. Genetic-structure of western red cedar 

populations in the Interior West. Canadian Journal of 

Forest Research. 24: 670–680.

Rehfeldt, G.E. 2006. A spline climate model for the western 

United States. Gen. Tech. Rep. RMRS-GTR-165. Fort 

Collins, CO: U.S. Department of Agriculture, Forest 

Service, Rocky Mountain Research Station. 21 p.

Rehfeldt, G.E.; Jaquish, B.C. 2010. Ecological impacts and 

management strategies for western larch in the face of 

climate-change. Mitigation and Adaptation Strategies for 

Global Change.15: 283–306.

Rehfeldt, G.E.; Jaquish, B.C.; Sáenz-Romero, C.; [et al.]. 

2014. Comparative genetic responses to climate in the 

varieties of Pinus ponderosa and Pseudotsuga menziesii: 

reforestation. Forest Ecology and Management. 324: 

147–157.

Shaw, R.G.; Geyer, C.J.; Wagenius, S.; [et al.]. 2008. 

Unifying life-history analyses for inference of fitness and 

population growth. American Naturalist. 172: E35–E47.

Tomback, D.F.; Arno, S.F.; Keane, R.E. 2001. The 

compelling case for management intervention. In: 

Tomback, D.F.; Arno, S.F.; Keane, R.E., eds. Whitebark 

pine communities: Ecology and restoration. Washington, 

DC: Island Press: 3–25.

Warwell, M.V.; Rehfeldt, G.E.; Crookston, N.L. 2007. 

Modeling contemporary climate profiles of whitebark 

pine (Pinus albicaulis) and predicting responses to global 

warming. In: Goheen, E.M.; Sniezko, R.A., tech. coords. 

Proceedings of the conference whitebark pine: A Pacific 

Coast perspective. R6-NR-FHP-2007-01. Portland, OR: 

U.S. Department of Agriculture, Forest Service, Pacific 

Northwest Region: 139–142.
USDA Forest Service RMRS-P-76. 2018. 59

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Abstract—Growth rhythm represents the timing of annual plant growth and development in relation to 

the environment. Growth rhythm that is well synchronized with the local climate is understood to confer 

adaptation in plant species. Rapid ongoing climate change threatens to desynchronize growth rhythm for 

many plant populations, yet knowledge of how plant populations undergo selection on growth rhythm and 

how they will respond is limited. Therefore, to evaluate phenotypic selection on growth rhythm, seedlings 

from 49 populations of whitebark pine (Pinus albicaulis), representative of the interior northwestern United 

States, were grown in two common-garden field tests over a 12-year period under climate (+4.4 to 9.1 °C 

mean annual temperature) approximating projected climate change in the present century for the sampled 

species range. In addition, variation in growth rhythm among populations and its relation to the climate 

of the seed source were evaluated to clarify how growth rhythm varies over the study region. Height at 

the end of the study was used as the best available measure of fitness. Survival and the unconditional 

expected value for height were dependent on timing of apical shoot elongation rates within and among 

growing seasons. Comparison of models using survival versus unconditional expected height as the proxy 

for fitness showed that survival strongly influenced selection on elongation rates. The form and magnitude 

of selection on timing and rate of shoot elongation varied between test sites and over time. Analysis of 

the timing of selection detected directional and stabilizing selection on shoot elongation rates in the earliest years whereas only directional selection was detected for selection on elongation rate in later years. 

Differences among populations for growth rhythm were mild and were explained to a moderate extent 

(r2 = 0.08–0.28) by climatic clines. Taken together, these findings suggest that growth rhythm in whitebark 

pine has been mildly selected for in relation to past climate and will undergo phenotypic selection in 

response to ongoing climate change. .

Phenotypic Selection on Growth Rhythm in Whitebark Pine

(Pinus albicaulis) in Low Elevation Common Gardens 

M.V. Warwell1, *

1 USDA Forest Service, Rocky Mountain Research Station, Moscow, Idaho, USA, and University of Minnesota,

Department of Ecology, Evolution, and Behavior, St. Paul, Minnesota, USA

* mwarwell@fs.fed.us

INTRODUCTION

Growth rhythm represents the timing of annual plant 

growth and development in relation to the environment. Growth rhythm that is well synchronized with 

the local climate is understood to confer adaptation in 

woody plant species (Dietrichson 1964; Howe et al. 

2003; Rehfeldt 1992). Rapid ongoing climate change 

threatens to desynchronize growth rhythm for many 

tree populations in the western United States (see, for 

example, Rehfeldt 2004), resulting in maladaptation. Over the long term, the persistence of natural 

populations is determined by the process of evolutionary adaptation. Indeed, evolutionary adaptation is 

expected when the traits under selection vary within 

a given population and are heritable (Darwin 1859; 

Endler 1973; Falconer and Mackay 1996). In keeping 

with this understanding, evolutionary trajectory may 

be estimated when information about selection on the 

phenotype and trait inheritance is available. However, 

selection on phenotype is not well understood for 

many tree species under predicted rapid climate 

change. 
60 USDA Forest Service RMRS-P-76. 2018.

Whitebark pine (Pinus albicaulis) is of particular 

interest given its status as a keystone (Tomback and 

Kendall 2001) and foundational (Ellison et al. 2005) 

species in subalpine forest ecosystems of western 

North America. In addition, due to rapid ongoing 

climate change, whitebark pine’s contemporary 

(1961–1990) realized climate niche is projected to 

be eliminated by mid-century for almost all of its 

distribution in the United States (Warwell et al. 2007). 

Therefore, the primary objectives of the present study 

were to (1) assess variation in growth rhythm among 

populations under warmer and drier climate than the 

climate of seed origin and (2) estimate the direction 

and magnitude of selection on variation in growth 

rhythm. This paper reports only preliminary results. 

METHODS

The present study used whitebark pine provenance 

tests of 46 (test 1) and 42 (test 2) populations, with 39 

populations common to both tests. The provenance 

tests were outplanted in forest openings at Priest River 

Experimental Forest (Idaho, USA) in April 2000 using 2-year-old trees in test 1 and October 2001 using 

4-year-old trees in test 2. These trees were grown over 

a 12-year period under nonnative climate (+4.4 to 

9.1 °C mean annual temperature) that approximated 

projected climate change in the present century for 

whitebark pine’s contemporary distribution. Growing 

conditions were mild at the test 1 site, which was 

characterized by deep, well-drained soil that was tilled 

prior to planting. In contrast, growing conditions were 

harsh at the test 2 site. At that site, air temperature was 

0.25 °C colder over a 17-month period while soil was 

shallow, poorly drained, and rocky. Both provenance 

tests used a randomized complete block design with 

populations planted in 5-tree row-plots in each of 6 

blocks in test 1 and 10-tree row-plots in each of 3 

blocks in test 2. Each provenance was represented by 

30 seedlings. Measurements of annual incremental 

apical shoot elongation growth were recorded approximately weekly to biweekly from late March through 

July in 2001–2005 in test 1 and in 2003–2006 in test 2. 

Because sequential events of annual development tend 

to be intercorrelated (Dietrichson 1964), the timing 

and magnitude of apical shoot elongation were used as 

proxies for growth rhythm. 

To evaluate selection on growth rhythm, the relationship between fitness and timing of intra-annual rate, 

cessation, initiation, and duration of apical shoot 

elongation was assessed with aster models (Geyer and 

Shaw 2008; Geyer et al. 2007) following a general 

multivariate selection approach (Lande and Arnold 

1983). Aster, using the “reaster” function (Geyer 

2013) in R (R Core Team 2012) was used to model 

unconditional expected height, which is an inference of population height that explicitly considers 

population survival. Normal distributions were used 

to model final height in 2012 conditional on survival 

to that year, and Bernoulli was used to model survival 

through each of multiple intervals concluding in 2012. 

Unconditional estimates of final height were used 

as the best available proxy for fitness because they 

provide a more complete measure of population fitness 

than either survival or height measures separately. 

Survival alone was also used as a proxy for fitness. 

Results from selection analyses using unconditional 

height and survival alone were compared to distinguish the influence of height from survival on inferred 

selection. 

RESULTS AND DISCUSSION

Elongation rates at the test 1 site were greater than 

at the test 2 site throughout the study period. Total 

aboveground mean height of survivors in 2012 differed significantly (P < 0.05) between test sites, with 

61.4 cm at the test 1 site and 45.3 cm at the test 2 

site. Survival to 2012 was 39 and 41 percent in tests 

1 and 2, respectively. Survival declined substantially 

in single episodes in the fall following relatively dry 

growing seasons in 2002 at the test 1 site and 2006 at 

the test 2 site.

Timing of initiation, timing of cessation, and duration of elongation were highly correlated. Higher 

elongation rates were correlated with earlier initiation, 

later cessation, and longer growth duration overall. 

Variation in the timing of initiation of shoot elongation 

spanned 2 weeks; timing of cessation of shoot elongation varied by as much as 3 weeks. Finally, total duration of shoot elongation for most individuals ranged 

from about 7 to 10 weeks. 
USDA Forest Service RMRS-P-76. 2018. 61

Survival and the unconditional expected value for 

height were dependent on timing of apical shoot 

elongation rates within and among growing seasons. A 

comparison of models using survival versus unconditional expected height as the proxy for fitness showed 

that survival strongly influenced selection on shoot 

elongation rates. The form and magnitude of selection 

on timing and rate of shoot elongation varied between 

test sites and over time. More complex selection was 

observed in test 1, which had more favorable growing 

conditions than test 2. Analysis of the timing of the 

form of selection detected directional and stabilizing 

selection in the earliest years, whereas only directional 

selection was detected for selection on elongation rate 

in later years (fig. 1). 

A substantial range of variation in timing and rate of 

apical shoot elongation was observed within all populations. Differences among populations for growth 

rhythm were mild and were explained to a moderate 

extent (r2 = 0.08–0.28) by climatic clines. Faster and 

longer growing populations originate from locations 

where winters were milder and precipitation was more 

balanced over the year. This climate tends to occur in 

an increasing southeast to northwest direction in the 

interior northwestern United States. 

Taken together, study findings suggest that growth 

rhythm in whitebark pine has been mildly selected 

for in relation to past climate and is likely to undergo 

further selection in response to ongoing climate 

Figure 1—Fitness surfaces showing observed (open circles) apical shoot elongation rates 

(mm/day) early (Julian dates 108-127) and late (Julian dates 141-155) in the 2004 growing 

season in relation to modeled unconditional expected height (mm) in 2012 at test 1 site.
62 USDA Forest Service RMRS-P-76. 2018.

change. In addition, this study is an initial step toward 

assessing how growth rhythm in pine populations 

may evolve in response to ongoing climate change. 

Continuing research is needed to assess phenotypic selection of growth rhythm and other key adaptive traits 

of forest tree species during seed emergence, early 

establishment, and sexual maturity and under novel 

and contemporary climate. 

ACKNOWLEDGMENTS

I thank the U.S. Department of Agriculture, Forest 

Service, Rocky Mountain Research Station for funding. In addition, I am grateful to Dennis Ferguson 

and Ned Klopfenstein for operational support; Donna 

Dekker-Robertson for direction and management of 

seed collections; Paul Leigh, Mose Harris IV, Amanda 

Link, and Debra Eastman for technical field support; 

Charles Geyer, Nicholas Crookston, and Jerry Rehfeldt 

for statistical advisement; and Ruth Shaw and Ned 

Klopfenstein for research advisement. 

REFERENCES

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natural selection. London: Murray.

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Ellison, A.M.; Bank, M.S.; Clinton, B.D.; [et al.]. 2005. 

Loss of foundation species: Consequences for the 

structure and dynamics of forested ecosystems. Frontiers 

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environmental gradients may be independent of gene 

flow. Science. 179: 243–250.

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University of Minnesota, School of Statistics. 23 p.

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for life history analysis. Biometrika. 94: 415–426.

Geyer, C.J. 2013. Aster: aster models. Version 0.8-27, 

R.p.v. (ed.). Vienna, Austria: R Foundation for Statistical 

Computing. http://CRAN.R-project.org/package=aster. 

(03 January 2016).

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Botany. 81: 1247–1266.

Lande, R.; Arnold, S.J. 1983. The measurement of selection 

on correlated characters. Evolution. 37: 1210–1226.

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Rehfeldt, G.E. 2004. Interspecific and intraspecific variation 

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Tomback, D.F.; Kendall, K.C. 2001. Biodiversity losses: 

The downward spiral. In: Tomback, Diana F.; Arno, 

Stephen F.; Keane, Robert E., eds. Whitebark pine 

communities: Ecology and restoration. Washington, DC: 

Island Press: 243–262. 

Warwell, M.V.; Rehfeldt, G.E.; Crookston, N.L. 2007. 

Modeling contemporary climate profiles of whitebark 

pine (Pinus albicaulis) and predicting responses to 

global warming. In: Goheen, E.M.; Sniezko, R.A., eds. 

Proceedings of the conference whitebark pine: A Pacific 

Coast perspective. R6-NR-FHP-2007-01. Portland, OR: 

U.S. Department of Agriculture, Forest Service, Pacific 

Northwest Region: 139–142.
USDA Forest Service RMRS-P-76. 2018. 63

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Provenance Variation in Western White Pine (Pinus monticola):

The Impact of White Pine Blister Rust

J.N. King1, *, J. DeBell2, A. Kegley3, R.A. Sniezko3, G. McDonald4, and N. Ukrainetz5

1 British Columbia Ministry of Forests (retired)

2 Washington Department of Natural Resources, Olympia, Washington, USA

3 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA

4 USDA Forest Service (retired)

5 British Columbia Ministry of Forests, Surrey, British Columbia, Canada 

* king.forgen@gmail.com

Abstract—The five-needle pines have been an important part of the landscape of North America, but they 

have been severely affected by the introduction of white pine blister rust (causal agent: Cronartium ribicola). However, most of these species have one or more types of some inherent resistance to this exotic 

disease, such as major gene resistance (complete resistance), and some forms of partial resistance. Longestablished provenance trials in western white pine (WWP, Pinus monticola), established with unselected 

populations, allow us to investigate and compare genetic variation in our native populations for this resistance. These trials in British Columbia (Canada) and Washington and Idaho (USA) include seed sources 

that cover all the native range of WWP; the Canadian trials also include some of the selected populations 

from the ongoing genetic improvement effort. Trends shown here for growth (and survival) are similar to 

other reports on WWP—a large rather genetically undifferentiated northern population above latitude 45° 

N and a highly diverse but much less vigorous southern population. But also reported here, after 20 years 

of cumulative rust infection in the trials, is a trend in this northern population, sometimes significant, showing much higher levels of resistance in the Northern Cascades and lower elevation North Coastal sources 

compared to interior sources from Idaho and elsewhere. We describe several factors that could have 

caused these differences, such as natural selection after the early introduction; selection for traits involved 

in general constitutive defenses, which also gives resistance to this nonnative disease; or refugium differences in the primarily undifferentiated northern population. We also note the high effectiveness of the 

selection programs and discuss the inherent resistance defenses to this exotic pathogen that exist in native 

American species of white pine. 

INTRODUCTION

Provenance testing is an important feature in understanding the background genetic variability of forest 

tree species (Morgenstern 1996). In the five-needle 

pines (also known as “white pines” and “soft pines”) 

of North America, this has been especially important 

since the accidental introduction of the exotic white 

pine blister rust (WPBR). The fungal pathogen 

Cronartium ribicola, the causal agent of WPBR, has 

been in North America for about 100 years. Although 

it appears very fragile, with a complex life cycle 

involving five different spore stages, alternating hosts, 

and exacting environmental conditions, it has proved 

remarkably successful in its colonization throughout 

most of the native ranges of the very susceptible North 

American white pines (Geils et al. 2010; Kinloch 

2003). However, unlike other exotic pathogens that 

were also introduced in the early 20th century, in 
64 USDA Forest Service RMRS-P-76. 2018.

Figure 1—Western white pine distribution, sample locations, and trial sites.
USDA Forest Service RMRS-P-76. 2018. 65

particular chestnut blight caused by Cryphonectria 

parasitica on American chestnut (Castanea dentata), 

and Dutch elm disease caused by Ophiostoma ulmi

and O. novo-ulmi on elm species (Ulmus spp.), there 

appears to be considerable native resistance in North 

American white pines to this exotic disease, albeit at 

low frequencies in natural populations (Kegley and 

Sniezko 2004; Kinloch et al. 2003; Sniezko et al. 

2014).

Western white pine (WWP, Pinus monticola), has both 

a wide geographic distribution and one of the most 

comprehensive series of long-term provenance tests 

for the native North American five-needle pines. This 

paper summarizes results from these provenance trials, 

distributed both in the United States and Canada now 

that WPBR has had sufficient time to affect them. It 

draws some conclusions about geographic variability 

in genetic resistance in native WWP to the nonnative 

WPBR, and also about how this important tree species 

is perhaps evolving with this exotic pathogen.

Background to Provenance Research in 

Western White Pine

Two features were earlier noted in studies on the geographic variation and genecology of WWP:

1. There is a marked difference between the widely 

distributed northern populations (Regions I–III) 

and more narrowly distributed southern populations (fig. 1). 

Regions for the southern populations, particularly in the 

Sierra Nevada and Warner Mountains (fig. 1: Region 

VI), are reported as quite distinct both with isozyme 

data (Steinhoff et al. 1983) and with growth and frost 

hardiness data (where it is noted these populations show 

lower growth potential but higher hardiness) compared 

to the northern populations (Rehfeldt et al. 1984). 

In both these studies the populations from Region V 

(Siskiyou) and Region IV, the southern Oregon (USA) 

Cascades, appear transitional between the northern 

population and the Sierra Nevada population.

The observations made from Steinhoff and colleagues’ 

wide-ranging collections are also noted in other collections and studies and many observers have commented 

on the distinct difference in growth and needle characteristics of the Sierra Nevada white pine. In Canadian 

studies, Hunt (1994) and Meagher and Hunt (1998, 

1999) noted southern sources of white pine (Southern 

Cascades, Siskiyou, and Sierra Nevada) are distinct 

from northern sources and are far more susceptible to 

WPBR. An evaluation of early growth and survival in 

a large progeny test of WWP in southwestern Oregon 

by Bower and Sniezko (2004) showed higher growth 

potential from northern sources of WWP. Bower and 

Sniezko (2004) also reported a distinct Californian 

population and a transition zone in the southern 

Oregon Cascades and confirmed Steinhoff’s designation of a broad northern population from northern 

Oregon and eastward into Montana (USA).

2. There is exceptionally wide phenotypic plasticity for the northern populations (fig. 1: Regions 

I–III). 

A commonly observed phenomenon is the wide 

transferability between the interior (Montana, Idaho, 

interior British Columbia [BC]) and coast for growth 

potential (Bower 1987; Steinhoff 1981), but a slightly 

less although still very wide amplitude for cold hardiness (Rehfeldt et al. 1984; Thomas and Lester 1992). 

This is highly unusual as strong genetic differences 

are typically noted between coastal and interior 

populations of most conifers; and strong latitudinal, 

elevational, and other environmental clines are the 

observed norm (Rehfeldt et al. 1984). Steinhoff et al. 

(1983) noted in an isozyme analysis that among-source 

population variability within this northern group 

was very low and even within-source variability was 

generally lower than in other regions. Even though 

populations from the Inland Empire (Idaho, eastern 

Washington, and Montana) and interior BC are now 

separated from the Northern Cascades and Coastal 

populations (fig. 1), the isozyme and the growth and 

adaptive trait data show a lack of strong differentiation 

within this large region, indicating they probably disseminated from a common gene pool after the last ice 

age (Critchfield 1984; Steinhoff et al. 1983). 

Less has been noted on infection and survival differences among WWP provenances to exposure to 

WPBR, but it is interesting to note that the major 

R-gene (major gene resistance; MGR) that confers 

a hypersensitive response resistance (Kinloch et al. 

1970) identified as Cr2 (Kinloch et al. 1999) appears 

absent throughout this northern population and shows 
66 USDA Forest Service RMRS-P-76. 2018.

higher frequencies in the Sierra Nevada (0.005–0.008) 

compared to only 0.001 in the southern Oregon 

Cascades (Kinloch et al. 2003). This observation fits 

well with Critchfield’s hypothesis of a common gene 

pool and bottleneck that did not allow Cr2 to become 

established in the northern population. Another observation on resistance is based on assessment of various 

plantations and trials in BC (Hunt 1994, 2004). Hunt 

(2004) suggests that some of the resistance responses 

selected for in Idaho (e.g., needle shed) can do well in 

the shorter growing season of interior BC, but may not 

perform as well in the longer growing season found in 

coastal environments.

In a study of the genetic architecture of the Steinhoff 

WWP rangewide seed collections using amplified fragmented length polymorphism markers, Kim and others 

(2010) showed with nonmetric multidimensional 

scaling of genetic distance (Kim et al. 2010: fig. 4) a 

potential for a more diverse population structure than 

that described by Steinhoff and others (1983). They 

confirmed the divergence between a northern population of relatively low genetic diversity and the more 

diverse southern population at about 45° N latitude. 

Kim et al. (2010) also found that the transition areas 

between these populations (which they identified as 

clades) east and west of the ridge of the Southern 

Cascades could be quite marked (also Richardson et al. 

2009).

METHODS

Provenance Samples and Field Trial 

Descriptions

Figure 1 shows the distribution of WWP, the provenance source population, and trial locations. Details 

of the U.S. provenance collection information are 

provided by Steinhoff et al. (1983), and the Canadian 

populations and trial sites are described in detail in 

Meagher and Hunt (1998). The Steinhoff collection 

(U.S. trials) had 60 populations in total with 3 to 25 

trees per population. These were considered random 

samples, which means they did not come under the 

intense selection identified for the WPBR screening (King et al. 2010; McDonald et al. 2004). The 

Canadian collection of more than 100 samples (trees or 

mixed seedlots) included random samples encompassing more of the Canadian range of WWP (both Coastal 

and Canadian Rockies). The Canadian collection also 

used the Steinhoff collections for the U.S. Northern 

Rockies Region (Region I-a; fig. 1), and also some 

selected populations. This selected material came from 

various screening programs and included: (1) The 

Porter families—early intensive selections with some 

screening (as described by King and Hunt [2004]); 

(2) The Westar families—selections made in southern 

interior BC using the Idaho protocols (McDonald et al. 

2004) without screening; (3) Dorena Genetic Resource 

Center MGR seedlots from Oregon; and (4) some exotics, of which P. koraiensis is reported here.

Table 1 details the trials we report here with ages, 

final height measurements, and the overall percentage of trees with cankers from blister rust as well 

as blister rust frequency (incidence) for the Idaho 

collection (U.S. Northern Rockies Region I-a) subset 

of seedlots (as this was common to both the U.S. and 

Canadian trials). Rust incidence (presence or absence) 

was recorded as canker frequency in the Results and 

Discussion section (tables 2–7), and canker severity (counted number of cankers, up to 30) was also 

recorded in the U.S. trials. The U.S. trial sites have not 

previously been described, but are randomized complete block (RCB) designs of three 10-tree row blocks 

per seedlot (collection sample) and were established 

in 1983. The Whidbey Island site (Lone Lake) established by Washington State Department of Natural 

Resources (WA DNR) in cooperation with the U.S. 

Department of Agriculture, Forest Service (USDA FS) 

Moscow Forestry Sciences Laboratory (1 in fig. 1) 

and the Lost Valley site (located near McCall, Idaho 

outside the range of WPP on USDA FS land; 4 in fig. 

1) both provided high survival. Another Idaho site 

located at Priest River Experimental Forest (USDA 

FS) lost two full blocks due to early plantation failure 

(5 in fig. 1). One block provided nearly full survival 

and was assessed. The Canadian sites described by 

Meagher and Hunt (1998) are also RCB with 25 replicated blocks of 4-tree row plots and were planted in 

1988; they are described in greater detail later.

DATA ANALYSIS

Analysis of the trial information, which included both 

height and blister rust canker incidence (Canker) and 

severity (Canker Severity), was conducted by using a 

linear mixed-effects model that included the effects of 
USDA Forest Service RMRS-P-76. 2018. 67 Table 1—Site descriptions of provenance trials reported. Site Agency Location Latitude oN Longitude oW Elevation m Mean height and (age) % Blister rust incidence for overall plantation and (Idaho collection) References to sites or materials 1 Whidbey Is. WA DNR Washington, Coastal 48o 03' 122o46' 50 123 –(20) 50% - (54%) a,b 2 Ladysmith Canadian For. Serv. BC Coastal 49o 00' 123o 52' 75 62 – (12) 68% - (79%) c,d,e,f 3 Sechelt Canadian For. Serv. BC Coastal 49o 30' 123o 44' 250 45 – (12) 91% - (96%) c,d,e,f 4 Lost Valley USDA For. Serv. Region 1 Idaho South 44o 59' 116o 30' 1800 35 – (21) 85% - (91%) a,b 5 Priest River USDA For. Serv. Region 1 Idaho North 48o 20' 116o 42' 730 95 – (20) 22% - (24%) a,b 6 Nakusp Canadian For. Serv. BC Interior (South) 50o 15' 117o 46' 680 46 – (12) 35% - (45%) c,d,e 7 Barrière Canadian For. Serv. BC Interior (Central) 51o 19' 119o 54' 725 52 – (12) 16% - (19%) c,d,e 8 Valemount Canadian For. Serv. BC Interior (North) 52o 41' 119o 03' 760 49 – (12) 76% - (81%) c,d,e a Steinhoff et al. (1983). b Rehfeldt et al. (1984). c Hunt (1994). d Meagher and Hunt (1998). e Meagher and Hunt (1999). f King and Hunt (2004). 
68 USDA Forest Service RMRS-P-76. 2018.

interest: Region (R), provenance Population (P), Trial

site (T), and Block (B). Region was based on the original seven regions as designated in Steinhoff et al.’s 

(1983) survey (shown in figure 1), but we also discuss 

the division in terms of northern populations (fig. 1: N, 

Regions I–III), southern Sierra populations (S, Region 

VI), and transitional populations (T, Regions IV and 

V). In the Canadian trials, which had larger sampling 

within the defined northern regions, we also looked 

at regions defined as: Coastal Low elevation (Region 

II-a), Coastal High elevation (or Northern Cascades, 

Region II-b), Idaho (Region I-a), and Interior BC 

(Region I-b) (fig. 1).

In the mixed model the random Population (trees 

sampled) is nested in Region, which is the principal 

fixed effect. The MIXED procedure of SAS® (SAS 

Institute Inc., Cary, North Carolina, USA) was used 

for the normally distributed height data and for canker 

severity; and the GLIMMIX procedure was used for 

rust frequency (incidence), a 1,0 binomial dataset 

(Littell et al. 1996; SAS Institute Inc. 2004). Contrast 

statements allowed testing hypotheses about the fixed 

regional effects or groupings of these fixed effects 

using t-tests. In the analysis of estimates of variance 

of the components associated with random effects of 

Trial, Block, and Population, the significance of each 

of these effects was tested by individually removing it 

from the model and assessing the change in the model 

fit via the observed log likelihood. The reduced model 

lies on the boundary of the parameters' space for the 

variance component; therefore, the P-value associated with these likelihood ratio tests was determined 

by using the technique described in Verbeke and 

Molenberghs (2000: section 6.3.4). All tests have a 

level of significance α = 0.05. In the analysis of the 

Canadian sites, two models were run: one excluded 

the selected populations, and another included these 

populations.

Table 2—Results for canker frequencies (incidence and severity) at the Whidbey Island, Washington DNR Trial Site.

Trait Canker 13 yrs Canker 21 yrs Canker_Severity 21 

Random effect variance S.E. variance S.E. variance S.E.

Block 0 n/a 0.0933 0.1039 0.6646 ns 0.6816

Population (Region) 0.1081 0.1908 0.2358. 0.0866 0.521 *** 0.1251

Fixed effect DF P a DF P a DF P a

Region 5,66 <0.0001 5,57 <0.0001 5,57 0.0002

Region level

Estimated 

canker 

incidence

Confidence 

limits 

Estimated 

canker 

incidence

Confidence 

limits 

Estimated 

canker 

severity

Confidence 

limits 

I N - Interior 0.028 0.017, 0.046 0.537 0.384, 0.682 1.513 -0.268, 3.294

II N - Coastal 0.050 0.028, 0.087 0.302 0.190, 0.444 0.675 -0.955, 2.305

III N - N. Cascades 0.019 0.006, 0.060 0.200 0.111, 0.334 0.574 -0.979, 2.126

IV T - S. Cascades 0.025 0.009, 0.067 0.488 0.327, 0.652 1.884 0.334, 3.433

V T - Siskiyou 0.046 0.011, 0.182 0.680 0.430, 0.857 3.097 1.362, 4.833

VI S – Sierra 0.204 0.136, 0.293 0.712 0.564, 0.826 2.328 0.757, 3.899

Comparison of regions

Difference in 

canker 

incidence

P b Difference in canker 

incidence

P b Difference in severity 

score 

P b

Interior vs. Coastal -0.05 0.1445 0.235 0.0001 0.838 0.0141

Interior vs. N. Cascades 0.009 0.1450 0.337 <0.0001 0.940 0.0242

Coastal vs. S. Cascades 0.025 0.2374 -0.186 0.0187 -1.208 0.0107

Coastal vs. Siskiyou 0.004 0.9359 -0.388 0.0115 -2.422 0.0013

Coastal vs. Sierra -0.154 0.0408 -0.441 0.0007 -1.653 0.0002

  significant <0.0001,** * significant <0.001, ** significant <0.01, n.s. non significant >0.05, n.a. not available a P-value for testing whether all Fixed Region effects are zero is derived from an F distribution. b P-value for testing whether each difference is zero is derived from a t distribution.
USDA Forest Service RMRS-P-76. 2018. 69

RESULTS AND DISCUSSION

The results are discussed in four sections: (1) the 

Washington State DNR trial at Lone Lake on Whidbey 

Island, which was one of the most informative trials 

for survival and completeness of seed sources; (2) the 

coastal sites including analysis of Canadian sites, plus 

in combination with the Whidbey Island (Lone Lake) 

site;(3) the Canadian interior sites analyzed separately; 

and (4) Idaho sites analyzed separately and combined. 

Blister Rust and Height, Lone Lake on 

Whidbey Island, Washington

The Lone Lake trial site on Whidbey Island was the 

most comprehensive of the provenance trials analyzed, 

having the complete Steinhoff collection. White pine 

on this site had good vigorous growth and high overall 

survival but moderate (50 percent) rust incidence measured over several years.

The frequency (incidence) of cankers from blister rust 

was low at age 13, with no region having more than 21 

percent, and most having less than 5 percent (Cankers 

13 years, table 2). This allowed the trees to display 

vigorous growth on this productive site and get ahead 

of later rust development. A significant incidence of 

both branch and stem cankers was recorded only for 

Region VI (the Sierras), where a 20 percent incidence 

of blister rust cankers was significantly different from 

0 percent. The 21-year assessment was quite different 

and by this time the overall incidence (proportions of 

stems infected) of cankering in the trial was 50 percent 

(table 2). Although the Sierra population samples 

again showed the highest frequencies (>70 percent), 

other populations were also higher than 50 percent. 

But the two local populations—the Coastal (Region II) 

and North Cascades (Region III)—had significantly 

lower rust incidence (20–30 percent) than most other 

regions (table 2). Noticeable within the broad region 

Table 3—Results for heights in decimeters at the Whidbey Island Trial (Washington DNR).

Trait Height 4 yrs Height 13 yrs Height 21 yrs

Random effect variance P a variance P a variance P a

Block 0.1600 0.2132 11.81 0.1986 22.12 0.2118

Population (Region) 0.4801 0.0018 24.06 < 0.0004 69.69 < 0.0001

Tree (Population Region) 0.4159 0.0014 146.74 < 0.0001 429.24 < 0.0001

Fixed effect F value P b F value P b F value P b

Region 39.40 <0.0001 37.81 <0.0001 58.41 < 0.0001

Region level

Least 

squares 

mean 

Confidence 

limits 

Least 

squares 

mean 

Confidence 

limits 

Least 

squares 

mean 

Confidence 

limits 

I N - Interior 11.036 10.2, 11.9 70.17 63.2, 77.2 123.64 113.7, 133.6 

II N - Coastal 10.545 9.67, 11.4 64.37 57.3, 71.5 114.19 103.9, 124.5

III N - N. Cascades 10.312 9.35, 11.3 64.56 57.1, 72.0 114.36 103.2, 125.5

IV T - S. Cascades 9.388 8.41, 10.4 54.94 47.4, 62.5 100.12 88.78, 111.5

V T - Siskiyou 7.775 6.34, 9.17 43.30 33.3, 53.3 70.38 54.39, 86.37

VI S – Sierra 5.493 4.60, 6,39 29.95 22.8, 37.1 41.96 31.25, 52.66

Comparison of regions

Difference in 

least 

squares 

P c Difference in least 

squares 

P c Difference in least 

squares 

P c

Interior vs. Coastal 0.492 0.2468 5.804 0.0823 9.45 0.0801

Coastal vs. S. Cascades 1.156 0.0321 9.430 0.0183 14.07 0.0282

Coastal vs. Siskiyou 2.790 0.0004 21.07 0.0002 43.81 <0.0001

Siskiyou vs. Sierra 2.262 0.0038 13.34 0.0131 28.42 0.0017 a P-value for testing whether each variance component is zero is derived from a mixture of χ2 distributions. b P-value for testing whether fixed Regional effects are zero is derived from an F distribution. c P-value for testing whether each difference is zero is derived from a t distribution.
70 USDA Forest Service RMRS-P-76. 2018.

for the northern populations is that interior populations have significantly higher rust incidence than 

the local Coastal populations (Pr > |t| 0.0001) and the 

North Cascades populations (Pr > |t| <0.0001, table 

2). Canker incidence and canker severity (number of 

cankers) tracked each other closely on the U.S. sites 

where this was measured. The Idaho Region populations had twice the severity of the local Coastal or 

Northern Cascades populations (table 2).

Table 3 shows the three height measurements at the 

Whidbey Island site (diameter at age 21 was also 

recorded; it is not reported, but tracked height closely). 

The vigor especially of the Interior populations (>12 m 

on average at age 21) was very marked; they were 

on average 1 m taller than the local populations. The 

southern regions all showed poorer growth compared 

to local sources. The Sierra sources at one-third the 

height of the northern regions suffered poor survival 

caused not only by higher rust incidence but also by 

overgrowth of bigger trees. Very few of the Region VI 

trees were still alive at the 21-year assessment. Figure 

2 shows the increasing height trends of the different 

regions over time.

Blister Rust and Height on Canadian and All 

Coastal Sites

The Ladysmith site, about 200 km north of Whidbey 

Island, is a similar low-elevation productive site with 

average height of more than 6 m at 12 years. Mean 

canker incidence was 68 percent, and showed significant discrimination between the regional groups, as 

did the Whidbey Island site (table 4). Five of the eight 

groups showed greater than 75-percent rust incidence; 

only the North Cascades among the unselected populations had a lower incidence (67 percent). This difference was significant compared to Northern Interior BC 

(Region I-b; Pr > |t| 0.0339). All the selected populations had a significantly lower incidence than the 

unselected, most notably the Dorena MGR population

with only 27 percent versus nearly 70 percent overall. 

Also of interest were the Porter families, which were 

selected quite early in the outbreak and screened 

on rather simple resistance attributes; they showed 

statistically significant higher levels of resistance than 

the local unselected populations (83 percent vs. 46 

percent, Pr > |t| 0.0006, table 4; King and Hunt 2004). 

Figure 2—Height measurements (decimeters) over time at the Whidbey Island site. 
USDA Forest Service RMRS-P-76. 2018. 71 Table 4—Results for canker frequencies (incidence) at the Canadian sites in Coastal BC. Trait Sechelt 1995 Sechelt 2001 Ladysmith Random effect variance S.E. variance S.E. variance S.E. Block 0.4848 0.1825 1.158 0.4874 0.7671 0.2766 Population (Region) 0.1084 0.7112 0.2173 0.2231 0.4005 0.1771 Fixed effect DF P a DF P a DF P a Region (only unselected) 4, 26 0.4343 4, 23 0.3759 4,24 0.0405 Region (with selected) 7, 66 0.0184 7, 52 0.2582 9,88 <.0001 Region level Estimated canker incidence Confidence limits Estimated canker incidence Confidence limits Estimated canker incidence Confidence limits I-a Inland Empire 0.54 0.42, 0.67 0.96 0.91, 0.98 0.80 0.65, 0.89 I-b BC Interior unselected 0.49 0.39, 0.60 0.93 0.95, 0.97 0.86 0.78, 0.92 Westar families 0.39 0.30, 0.50 0.94 0.89, 0.97 0.76 0.68, 0.83 II-a - Coastal (unselected) 0.48 0.36, 0.60 0.95 0.89, 0.98 0.83 0.71, 0.90 Porter families 0.24 0.12, 0.41 0.91 0.74, 0.97 0.46 0.30, 0.62 II-b... high elevation 0.58 0.45, 0.70 0.97 0.91, 0.99 0.92 0.83, 0.96 III N - N. Cascades 0.44 0.30, 0.58 0.92 0.82, 0.97 0.67 0.45, 0.83 IV T - S. Cascades (Dorena) 0.20 0.07, 0.45 0.72 0.33, 0.93 0.27 0.08, 0.62 Comparison of regions Difference in canker incidence P b Difference in canker incidence P b Difference in canker incidence P b Coastal (II-a) vs Interior BC 0.01 0.7318 0.02 0.4187 0.03 0.4579 Coastal (II-a) unselect vs Porter 0.24 0.0328 0.03 0.6647 0.37 0.0006 Interior (BC) vs. Westar families 0.10 0.1379 0.01 0.6911 0.10 0.0160 N. Cascades (III) vs. Interior BC 0.05 0.2286 0.01 0.8123 0.20 0.0339 N. Cascades (III) vs. Idaho (I-a) 0.10 0.1951 0.01 0.1575 0.13 0.2019 Idaho – I-a vs. Dorena 0.34 0.0200 0.24 0.0144 0.53 0.0052 a P-value for testing whether all fixed Regional effects are significantly different is derived from an F distribution. b P-value for testing whether each difference is zero is derived from a t distribution.
72 USDA Forest Service RMRS-P-76. 2018.

Figure 3—Canker frequencies in the coastal trials.
USDA Forest Service RMRS-P-76. 2018. 73

The Sechelt site is another low-elevation coastal site 

on the mainland side of the Salish Sea. This site had 

the highest incidence of rust of all the trials surveyed 

with greater than 90-percent infection at 12 years old, 

a dramatic increase from the 1995 assessment, when 

no group showed more than 58 percent infection. The 

regional differences are nonsignificant (Pr > F 0.3795) 

unlike at Ladysmith or Whidbey Island. Only the 

Dorena MGR seedlot at 72-percent infection (increasing from 20 percent at the 1995 assessment) had a 

significantly lower incidence than the others. Although 

a frequency of 72 percent is higher than what might 

be expected from the segregating Cr2 expression, this 

figure is not in fact significantly different from the 

expected 50 percent (confidence intervals of 33–93 

percent, table 4). Thus, it does not conclusively indicate a breakdown of resistance due to vcr2; further 

monitoring would be needed to verify if in fact vcr2 

was present. This site has not had any other detailed 

assessment, but a walk-through in 2008 indicated a 

still marked proportion of white pines in the stand 

structure (30 percent or greater), many living with 

pronounced cankers.

Results from a combined mixed model run of all the 

coastal sites—Whidbey, Ladysmith, and Sechelt—

based on the last assessment for rust incidence demonstrate the difference between the unselected North 

Cascades (Region III—45 percent cankered) and the 

Interior (Region —64 percent cankered) (fig. 3a; 

Pr > |t| 0.0443). The local Coastal Region II was 54 

percent cankered and was not significantly different 

from either of these. Figure 3b shows the combined 

summary of coastal Canadian sites and includes selected populations. Differences are noted between the 

Interior (Idaho and North BC at 90 percent) and the 

North Cascades at 82 percent. Selected populations 

also showed up well. The Dorena MGR at 48 percent 

had a significantly lower incidence of cankers than 

all other populations except the Porter families at 69 

percent. The exotic P. koriensis had only a 6-percent 

infection rate. The Westar families, representing the 

selected interior families, were not much better than 

the Interior unselected and certainly not as good as the 

North Cascades (Region III). The Westar families representing selections from wild stands in the southern 

interior of BC were not screened.

Heights in the coastal Canadian trials showed the 

same trends as at Whidbey Island with the Idaho 

Region having some of the greatest growth, but unlike 

Whidbey these regional differences were not significant (table 5). However, if the variation in provenance 

samples within regions is investigated more closely, 

some significant trends did emerge. If we split off 

the Northern Rocky Mountain BC samples from the 

Southern Interior (I-b), it shows significantly poorer 

growth than Idaho, Southern Interior BC, and the 

local Coastal population (II-a). These are 18 parent 

trees sampled from areas such as Mount Revelstoke 

and Valemount at the very northern edge of WWP’s 

inland distribution (e.g., trial site 8, fig. 1). It would 

indicate that although this broadly defined northern 

population (Regions I–III) is relatively homogeneous, 

some differentiation is occurring at the species’ edge. 

Another feature on the more detailed redefinition of 

regions is the significant and superior growth of the 

selected Porter families. If this group is taken out of 

the Coastal high elevation group (II-b) and treated 

separately, it shows a marked effect. Individuals in 

the Dorena MGR seedlot (Region IV) as at Whidbey 

Island were significantly smaller at the Ladysmith site; 

but on the heavily rusted Sechelt site they performed 

well, as one of the few healthy populations on this site. 

Blister Rust and Height on the Canadian 

Interior Sites

Among the three Canadian trial sites in Interior BC, 

Nakusp, a moderate productivity site in the Southern 

Interior, had just 35-percent overall infection. Barrière 

in the Thompson River Valley was a more northern 

and severe site but with only 16-percent overall infection. The Valemount site was more than 75 percent 

overall infected and located at the northern edge of 

WWP distribution. The regions (without any of the selected populations) were nonsignificant at all sites, but 

most discriminating at the Valemount site. The same 

trend as the coast was shown: interior populations 

(Region I) had a higher incidence of cankers than the 

low elevation Coastal (Region II-a) or North Cascades 

(Region III) populations (table 6). 

This trend of regional differences is shown graphically 

for the Valemount site in figure 4, with the lower canker 

incidence of North Cascades and low elevation Coastal 
74 USDA Forest Service RMRS-P-76. 2018. Table 5—Results of the mixed-effects model for heights in decimeters for the Coastal Canadian sites. Trait Ladysmith Sechelt Random effect variance P b variance P b Block 7607 0.0005 1760 0.0128 Population(Region) NEa NE Tree (Seedlot Region) 36299 <.0001 32446 <.0001 Fixed effect DF P c DF P c Region 5, 2900 0.2084 5, 1048 0.1583 Region (with selected) 7, 2900 <.0001 6, 1048 0.0077 Region level Least squares mean Confidence limits Least squares mean Confidence limits I-a Inland Empire 63.09 58.82, 67.35 45.42 42.25, 48.60 I-b BC Interior - Westar 62.84 59.05, 66.63 45.74 43.29, 48.18 I-b BC Interior - Northern 58.22 53.54, 62.90 38.03 34.11, 41.95 II-a - Coastal 62.71 NE, NE 44.65 40.54, 48.75 II-b - Coastal high elevation 58.51 NE, NE 45.64 41.29, 49.84 II-a&b Coastal Porter 67.14 64.25, 70.03 47.93 42.28, 53.58 III N - N. Cascades 59.58 55.00, 64.15 41.51 37.52, 45.51 IV Dorena MGR 56.80 51.04, 62.55 44.67 36.28, 53.06 Comparison of regions Difference in least squares P d Difference in least squares P d Local (II-a) vs Idaho 0 0.8341 0.7 0.7323 Local (II-a) vs N Interior 4.5 0.0287 6.6 0.0097 Local vs coastal high (II-b) 2.4 0.0776 0 0.7340 Local vs N. Cascades (III). 2.5 0.1160 3.1 0.2255 Local vs S. Cascades (IV). 3.6 0.0276 0 0.9957 a NE – Not estimable b P-value for testing whether each variance component is zero is derived from a mixture of χ2 distributions. c P-value for testing whether all Fixed Region effects are zero is derived from an F distribution. d P-value for testing whether each difference is zero is derived from a t distribution.
USDA Forest Service RMRS-P-76. 2018. 75

populations (70 percent) and the marked and significant 

effect in the selected populations—particularly the 

greater survival shown by the Region IV Dorena MGR

population (30 percent), the Porter families selections 

(II-Coast select; 45 percent), and even the Westar 

compared to unselected Interior populations (70 vs. 85 

percent).

Regional differences in height were nonsignificant at 

the moderate Nakusp site as they were on the coastal 

sites (table 7). But in the more northern sites the Dorena 

MGR seedlot begins to suffer in performance and at the 

Valemount site even the coastal low elevation sources 

(IIa) begin to suffer (5.1 m vs. 4.3 m (Pr > |t| <0.0001). 

A later walk-through of the Valemount site at 20 years 

showed the Dorena MGR seedlots still alive, but starting 

to be overgrown by the remaining white pine survivors.

Blister Rust and Height on the Lost Valley and 

Priest River, Idaho Sites

The Lost Valley site near McCall, Idaho was similar 

in scope to the Lone Lake site on Whidbey Island; it 

had the complete Steinhoff collection, good growth, 

and high overall survival but a high (85 percent) rust 

incidence measured at 21 years. Although outside the 

normal range of WWP, this area had one of the highest 

incidences of rust among the sites we investigated. The 

Priest River Experimental Forest trial site lost two full 

blocks due to early plantation failure; thus, only the 

one surviving block was assessed. The Priest River site 

was a low rust environment with just over 20 percent 

rust incidence overall.

Overall regional differences in canker frequency were 

nonsignificant in the highly rust-infected Lost Valley 

site (table 8). But contrasts in the Priest River site, 

and the overall analysis, picked up the same results 

as in the Whidbey Island and the Canadian trials. 

Unselected white pines in Idaho (Region I) had overall 

higher susceptibility than the unselected Northern 

Cascades (Region III) (79 percent vs. 49 percent 

cankered) (Pr > |t| 0.0092 in the combined analysis, table 8).

The Lost Valley and Priest River Planting 21-year data 

were also included in a separate analysis (McDonald, 

unpublished data, on file at USDA FS, Moscow 

Table 6—Results of the analysis for canker frequencies (incidence) at the Canadian sites in Interior BC. 

Trait Nakusp Barrière Valemount

Random effect variance S.E. variance S.E. variance S.E.

Block 0.0349 0.0366 0.0793 0.05692 0.2553 0.0916

Population (Region) 0.1190 0.07049 0.1600 0.0646 0.6147 0.1511

Fixed effect DF P a DF P a DF P a

Region (only unselected) 5,25 0.0866 4,31 0.4395 4, 38 0.0852

Region (with selected) 9,92 <.0001 7,63 0.0738 8, 56 0.0007

Region level

Estimated 

canker 

incidence

Confidence 

limits 

Estimated 

canker 

incidence

Confidence 

limits 

Estimated 

canker 

incidence

Confidence 

limits 

I-a Inland Empire 0.456 0.355, 0.561 0.186 0.134, 0.253 0.809 0.661, 0.902

I-b BC Interior 0.444 0.372, 0.520 0.198 0.152, 0.255 0.858 0.782, 0.911

 Westar families 0.39 0.30, 0.50 0.127 0.094, 0.170 0.713 0.593, 0.809

II-a - Coastal 0.425 0.333, 0.522 0.203 0.147, 0.274 0.662 0.549, 0.759

 Porter families 0.181 0.123, 0.258 0.076 0.033, 0.165 0.451 0.252, 0.667

II-b... high elevation 0.333 0.240, 0.440 0.151 0.099, 0.224 0.827 0.715, 0.902

III N. Cascades 0.338 0.229, 0.467 0.137 0.087, 0.210 0.736 0.510, 0.882

IV S. Cascades (Dorena) 0.190 0.080, 0.386 0.049 0.015, 0.152 0.297 0.074, 0.692

Comparison of Regions

Difference in 

canker 

incidence

P b Difference in canker 

incidence

P b Difference in canker 

Incidence 

P b

Interior vs. Westar. Families 0.06 0.0015 0.06 0.0309 0.145 0.0121

Interior vs. Coastal unselect 0.12 0.1271 0.04 0.2215 0.196 0.0043 

Interior vs. Porter families 0.27 <0.0001 0.10 0.0891 0.407 0.0001

Interior vs. Dorena 0.25 0.0275 0.12 0.0115 0.561 0.0019 a P-value for testing whether all Provenance effects are zero is derived from an F distribution. b P-value for testing whether each difference is zero is derived from a t distribution.
76 USDA Forest Service RMRS-P-76. 2018.

Figure 4—Canker frequencies at the Valemount site.

Forestry Sciences Laboratory, Moscow, Idaho). 

Seedlots in this analysis were portioned in a slightly 

different manner based on the separations shown in 

Kim et al. (2010: fig. 4) and data from an unpublished 

nursery and greenhouse experiment conducted at the 

Moscow Forestry Sciences Laboratory. Results suggest some small adjustments to the boundaries shown 

in figure 1 of this paper. These data suggest that the 

Region II and Region III boundary could be shifted 

to the Columbia River along the Oregon-Washington 

boundary. Other subdivisions concurrent with the 

present-day distribution of WWP are also noted: a 

North/South subdivision that follows the Bitteroot 

Divide in the United States and the Columbia River 

(Arrow Lakes) in Canada. Although this still may 

be considered a rather homogenous population, 

differences between the northern populations of 

Regions I–III are starting to emerge as was shown 

in the Canadian Northern Interior sites (Valemount). 

Molecular marker data suggest that a single refugia 

event may be an oversimplification for the northern 

Rocky Mountains.

DISCUSSION OF RESULTS

A result that is consistent but varying in magnitude 

indicates that in the random population selections, as 

in the Steinhoff collection, Region II (North Central 

Cascades) and III (low elevation Coastal) can have as 

much as one-half the level of infections compared to 

the Interior Region I samples or the higher elevation 

Region II (II-b in figure 1). Possible explanations 

include:

1. The effects of natural selection since the WPBR 

introduction to the Port of Vancouver in the 

early 20th century. The spread has been primarily south and west, so it is not surprising to see 

the Northern Cascades and low elevation North 

Coastal areas displaying higher native levels of 

resistance.

2. Details of mortality from WPBR are not shown 

here, but as noted in the Introduction, Hunt 

(2004) has suggested that some of the resistance 

responses valuable in the Interior (e.g., needle 
USDA Forest Service RMRS-P-76. 2018. 77

Table 7—Results for height measurements for the Interior Canadian Sites.

Trait Nakusp Barrière Valemount

Random effect variance P a variance P a variance P a

Block 21.0 0.0006 57.1 0.0005 30.8 0.0008

Seedlot(Region) 20.3 < 0.0001 0 n/a 9.09 0.0033

Tree * Seedlot (Region) 91.05 < 0.0001 156.68 < 0.0001 208.86 < 0.0001

Fixed effect DF P b DF P b DF P b

Region 7,91 <0.0001 6,160 <0.0001 6, 47 <0.0001

Region level

Least 

squares 

mean 

Confidence 

limits 

Least 

squares 

mean 

Confidence 

limits 

Least 

squares 

mean 

Confidence 

limits 

I-a Inland Empire 45.7 41.1, 50.3 52.4 48.9, 55.8 48.6 44.7, 52.5

I-b BC Interior - Westar 48.0 45.5, 50.5 52.9 49.6, 56.2 51.5 48.5, 54.6

I-b BC Interior - Northern 44.9 41.7, 48.0 49.5 45.9, 53.0 50.5 47.2, 53.8

II-a - Coastal 47.5 44.5, 50.4 49.4 46.0, 52.8 42.9 39.8, 46.1

II-b... high elevation 45.6 42.0, 49.3 48.2 44.4, 52.0 47.1 43.1, 51.0

III N - N. Cascades 45.5 39.8, 51.3 49.4 45.7, 53.0 46.6 41.8, 51.3

IV S. Cascades (Dorena) 44.4 34.7, 54.0 46.1 41.7, 50.6 35.9 28.4, 43.5

Comparison of regions

Difference in 

least 

squares 

P c Difference in least 

squares 

P c Difference in least 

squares 

P c

local vs. other interior 3.1 0.0475 3.4 0.0018 1.9 0.1316

local vs. coastal (II-a) 0.5 0.7095 0.1 0.9563 7.5 <0.0001

local vs. coastal high (II-b) 2.4 0.1904 1.3 0.3780 3.4 0.0998

local vs. N. Cascades (III). 2.5 0.3949 0.1 0.9454 3.9 0.1104

local vs. S. Cascades (IV). 3.6 0.4548 3.3 0.0777 14.6 0.0005

a P-value for testing whether each variance component is zero is derived from a mixture of χ2 distributions b P-value for testing whether all Fixed Region effects are zero is derived from an F distribution. c P-value for testing whether each difference is zero is derived from a t distribution.

shed) that do well in the shorter growing season 

of the Interior may do less well in the longer 

growing season of the coast. We found the greatest discrepancy in mortality in the coastal sites.

3. Various constitutive defenses valuable against 

WPBR, but evolved against other unknown pathogens or pests, might have arisen faster in these 

more resistant populations after the postglacial 

northward migration.

4. The evolutionary history of the northern populations appears to be more complex than a single 

introduction, and a great deal more variability 

might exist (see following).

Besides the regional difference within the northern 

population for rust resistance, we also note that the 

northern population did not perform uniformly well 

for growth and survival across all sites. Samples from 

Southern Interior BC and Northern U.S. Rockies 

(Idaho I-a in tables) performed particularly well for 

growth across all sites—at the very favorable sites of 

Whidbey Island and Ladysmith (VI)—nearly 10 percent higher than the local populations. However, the 

Northern Canadian Rockies samples (I-b) (from the 

far north of its range) do poorly for growth on coastal 

sites, and Coastal populations have poor growth in the 

Northern Canadian Rockies. Kim et al. (2010) suggest 

that the glacial refugia in the Rockies and subsequent 

recolonizations might be more complex than a simple 

founder effect and that genotypic diversity is more 

likely to arise at the fringes of the range for a species 

(Kim et al. 2010; Petit et al. 2003) and McDonald (unpublished data, on file at USDA FS, Moscow Forestry 

Sciences Laboratory, Moscow, Idaho). 
78 USDA Forest Service RMRS-P-76. 2018. Table 8—Results of the GLIMMIX analyses for canker frequencies (incidence) at the Idaho sites. Trait Lost Valley Priest River Combined Idaho Random effect variance S.E. variance S.E. variance S.E. Population (Region) 1.3839 0.4914 0.4442 0.3457 0.7646 0.2715 Block * Region 0 . Fixed effect DF P a DF P a DF P a Region 5,52 0.6577 5,56 0.3457 5,53 0.1226 Region level Estimated canker incidence Confidence limits Estimated canker incidence Confidence limits Estimated canker incidence Confidence limits I N - Interior 0.91 0.78, 0.97 0.24 0.16, 0.35 0.79 0.21, 0.98 II N - Coastal 0.90 0.73, 0.97 0.21 0.085, 0.43 0.78 0.21, 0.98 III N - N. Cascades 0.76 0.44, 0.93 0.03 0.004, 0.21 0.49 0.069, 0.92 IV T - S. Cascades 0.84 0.56, 0.96 0.22 0.094, 0.43 0.71 0.16, 0.97 V T - Siskiyou 0.92 0.39, 0.99 0.31 0.053, 0.79 0.83 0.24, 0.99 VI S – Sierra 0.89 0.69, 0.97 0.40 0.18, 0.68 0.83 0.28, 0.98 Comparison of regions Difference in canker incidence P b Difference in canker incidence P b Difference in canker incidence P b Interior vs. Coastal 0.01 0.8902 0.03 0.7795 0.10 0.8247 Interior vs. N. Cascades 0.14 0.1079 0.20 0.0378 0.30 0.0092 a P-value for testing whether all Provenance effects are zero is derived from an F distribution. b P-value for testing whether each difference is zero is derived from a t distribution.
USDA Forest Service RMRS-P-76. 2018. 79

The results also show that early first generation 

phenotypic selection programs have been very effective against WPBR. The Porter families selected and 

screened in the 1950s (King and Hunt 2004) proved 

particularly effective as a group. This adds to the 

evidence from the unselected populations that low frequency, probably constitutive defenses, exist for resistance to this disease beyond just the MGR expression 

of Cr2. The value of ongoing selective breeding to take 

advantage of the inherent resistance found in the North 

American white pine and accelerate the natural selection process is well documented (King et al. 2010; 

Sniezko et al. 2014). In the most heavily infected sites 

(Sechelt at 90 percent and Valemount at 80 percent) 

the MGR seedlot from the Dorena program appeared 

the only effective resistant lots although many nonMGR trees still survived in later surveys of these 

severe sites with both active and non-active cankers. 

More recent trials that include seedlots with known 

partial resistance have shown good field resistance in 

very severely infected sites and in artificial inoculation 

trials (Sniezko et al. 2014; Ukrainetz and King, poster 

presented at IUFRO meeting, March 2016, Rotorua, 

New Zealand). In the long term this approach is likely 

to be more successful for stable resistance.

The existence of this genetic variation to WPBR is the 

foundation for the breeding work with western white 

pine. This paper shows that at least low-to-moderate 

differences exist in the level of resistance between 

geographic populations of WWP, with perhaps the 

highest incidence of resistance (but still relatively low) 

in the North Cascades populations. Previous work has 

shown geographic variation in one type of resistance, 

MGR (Kinloch et al. 2003). It also suggests that the 

efficacy of resistance might vary by site conditions 

(coastal or interior, high or low elevation) (Hunt 2004), 

and effectiveness under a changing climate will need 

to be monitored. Resistance programs seek to capture 

this resistance, and they are well underway in much of 

the range of WWP (King and Hunt 2004; McDonald 

et al. 2004; Sniezko et al. 2014). A new series of trials 

involving many of the best resistant seedlots known 

(from BC, Idaho, Oregon, and Washington) is being 

established in Oregon, Washington, and BC in 2014 

and 2015 to further examine the efficacy of resistance 

from different geographic sources. 

ACKNOWLEDGMENTS

Thanks to Ray Hoff in the early 1990s for encouraging Richard Sniezko to consider assessment of the 

Whidbey Island trial and to Boyd Wilson for organizing a visit by Sniezko in the early 1990s that led to 

setting up further assessments of this trial. The USDA 

Forest Service and Washington Department of Natural 

Resources employees who helped with assessments 

are gratefully acknowledged. Very constructive reviews were provided by Jim Hamlin, Rich Hunt, Alvin 

Yanchuk, and Anna Schoettle.

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Whitebark Pine (Pinus albicaulis) Field Screening for Blister Rust 

Resistance in British Columbia: Germination Results

C. Cartwright1, *

1 British Columbia Ministry of Forests, Lands and Natural Resource Operations, Cowichan Lake Research Station,

Tree Improvement Branch, Mesachie Lake, British Columbia, Canada

* charlie.cartwright@gov.bc.ca

Abstract—Five hundred whitebark pine (Pinus albicaulis) parent trees from 80 populations were collected 

from throughout the range of whitebark pine, and are to be deployed in long-term field tests to screen for 

resistance to blister rust. The objective is to identify individuals to provide resistant seed for restoration of 

the species now undergoing catastrophic population declines throughout most of British Columbia (BC), 

Canada. Two series of four test sites, each with half of the open-pollinated families, are to be deployed 

across the range in BC with the first series to be established in 2015 and the second 2 years later. Survival 

and effects of rust will be assessed periodically for seedlings in the field, and total height will be measured. 

Along with resistance, it is anticipated that facets of genetic architecture, safe seed transfer distances, and 

likely impacts of climate change will be determined. To date, the first series has been sown and germination data collected. Stratification and nursery methods are discussed in light of the figures, in particular 

variation in germination based on seed source, consequences of storage and scarification, and efficacy of 

x-raying seed to determine viability. 

INTRODUCTION

Although work with whitebark pine (Pinus albicaulis) 

has been underway for decades in British Columbia 

(BC), activities have increased in light of its classification as endangered in 2012 under the Government of 

Canada Species at Risk Act (SARA). This is in response to the report by the Committee on the Status of 

Endangered Wildlife in Canada (2010), in which it was 

concluded that the species would be reduced to a quarter of its range if remedial action were not taken. Prior 

to that there had been reports by forest pathologists 

(Campbell and Antos 2000; Zeglen 2002) documenting the results of province-wide surveys attributing 

catastrophic population declines largely to white pine 

blister rust (caused by the fungal pathogen Cronartium 

ribicola) and to a lesser extent mountain pine beetle 

(Dendroctonus ponderosae). From forest geneticists 

there had been studies suggesting how to ameliorate 

these challenges for whitebark (Bower and Aitken 

2008; Krakowski and Kolotelo 2009) given success 

with related species (King 2010). Seed collections 

had been initiated building an ex situ reserve at the 

BC Ministry of Forests and Range Tree Seed Centre, 

and a limited number of individual tree seed lots had 

been sent for rust resistance screening at the U.S. 

Department of Agriculture, Forest Service (USDA FS), 

Dorena Genetic Resource Center in Cottage Grove, 

Oregon (USA).

As a consequence of the listing of whitebark pine in 

Canada, a recovery plan must be filed for Federal 

lands. Though much of the habitat in Canada is under 

provincial jurisdiction, language within SARA specifies that the Federal action must be supported. As a 

result, activities have become more organized and 

energized, and goals have been set. One of the goals is 
82 USDA Forest Service RMRS-P-76. 2018.

to identify blister rust-resistant trees and seed sources 

for use in restoration. To this end a proposal for establishment of two series of provenance trials with family 

structure by the provincial forest service was approved 

for funding. The first set of field tests, which will also 

elucidate genetic architecture, inform seed transfer 

guidelines, and ascertain long-term durability of the 

genotypes, was sown in early 2014. A second rust 

resistance screening proposal with controlled inoculation of 120 different families to be held in nursery beds 

was subsequently also approved (Murray and Berger, 

Blister Rust Inoculation Trials for Whitebark Pine 

(Pinus albicaulis) in the Canadian Kootenay Region, 

this proceedings). A description of the field testing 

project follows, along with some details of methods 

used and germination results.

Producing propagules effectively is of considerable 

consequence to the testing process. As noted by Leslie 

and Wilson (2011), sound whitebark pine seed is difficult to obtain because of erratic cone production, 

predation, and unpredictable seed development. It has 

been suggested that delayed seed germination as a result of underdeveloped embryos at the time of dispersal may be an ecological strategy that is a product of 

selection (Tomback et al. 2001). Complex dormancy 

is also an issue, and low germination rates are noted 

often (Arno and Hoff 1990; McCaughey and Schmidt 

1990). These obstacles have led to varied and involved 

seed stratification methods, though seed are generally 

brought out of cold storage, x-rayed to determine their 

stage of development, dipped in fungicidal baths, 

soaked for several days in running water (Mahalovich 

et al. 2006), stratified for several months often at different temperatures (Burr et al. 2001), and then scarified (Leslie and Wilson 2011; Pitel and Wang 1990) 

before sowing into planting containers or germination 

trays. 

Stratification of the whitebark seed for the field trials at the BC Ministry of Forests, Lands and Natural 

Resources Operations Kalamalka Forestry Centre 

nursery in Vernon follows a method for Siberian pine 

(P. sibirica) that had been in circulation at the facility for more than a decade, but is of unknown origin. 

Our investigations into refining protocols for growing 

whitebark pine started in 2012 with comparison of 

germination of scarified and unscarified seed of five 

trees (table 1). The practice of seed nicking prescribed 

by our approach was evaluated by staff (V. Berger, 

2013, unpublished data, on file at Kalamalka Forestry 

Centre, Vernon, BC) and it was found that nicking 

improved germination by about 20 percent versus 

un-nicked seed. In another facet of the nursery trials, 

it was found that if care was taken to remove empty 

or rotten seeds when nicking, the germinants relative 

to the number of sown seed had a germination percent 

as much as 13 percent higher than for germinants relative to all seed put into stratification (V. Berger, 2013, 

unpublished data on file at Kalamalka Forestry Centre, 

Vernon, BC). This cull of bad seeds reduced nursery 

costs. The present study provides germination results 

for the stratification process used at Vernon.

METHODS

Seed Sources

Five hundred families will be screened across two 

series of field trials, the first of which is described 

here. A total of 257 wind-pollinated families from 46 

populations went into stratification in mid-November 

2013, of which 156 trees were from BC; 12 from 

across USDA FS Region 1; 8 from USDA FS Region 

4; and 81 from USDA FS Region 6. Seed x-ray and 

germination data for the Region 6 families from earlier 

work were made available (R. Sniezko, 2012, unpublished data on file at the Dorena Genetic Resource 

Center, Cottage Grove, Oregon). Of the BC trees, 50 

were from the Chilcotin, 44 from the Kootenays, 28 

from the Coast-Interior Transition, 22 from the Interior 

North, and 12 from the Rocky Mountains. Survival, 

height, and health of the trees will be recorded in the 

nursery and then in the field, but only germination data 

are described here.

Stratification

Seed was stored at below-freezing temperatures for as 

long as 10 years, then x-rayed before use to estimate 

the numbers needed to produce the required seedlings. 

The seed was soaked in a 3 percent solution of hydrogen peroxide for no longer than an hour, though as 

little as 15 minutes. Seed were rinsed in running water 

to remove residue of the previous treatment and put 

into mesh bags. These were then placed in a bucket 

with running water allowed to gently flow over them. 

Aeration was enhanced with an aquarium pump (fish 
USDA Forest Service RMRS-P-76. 2018. 83 Table 1—Comparison of percent germination of scarified and un-scarified seed. Tree Nicked seed Discarded nicked seed Seed not nicked Germinated nicked seed Germinated not nicked seed Germinated all nicked seed (%) Germinated of sound nicked seed (%) Germinated not nicked seed (%) 1401 Moyie 165 13 185 103 88 62 68 48 1413 Baldy 169 5 179 129 94 76 79 53 1420 Baldy 179 10 171 99 47 55 59 27 1304 Panorama 191 6 164 140 112 73 76 68 1404 Puddingburn 172 15 167 128 96 74 82 57 Average 68 73 51
84 USDA Forest Service RMRS-P-76. 2018.

tank oxygenator) and the bath continued for about 48 

hours (fig. 1). Mesh bags were then placed as flat as 

possible on sterilized sand in plastic totes in a manner 

intended to maximize contact with the sand.

Sand is sterilized before use by adding enough water 

to make it moist, but not runny, covered with aluminum foil, then baked in an oven preheated to 94 °C 

until the center of the sand reaches 82 °C. Baking was 

continued for 30 minutes, with care not to overheat the 

sand. The first layer of bags was covered with more 

sand and a second layer was then laid out. Up to four 

layers can be placed in a 20-cm-deep tote (fig. 2). A lid 

was put on the tote at an angle such that there is air exchange, but moisture will not escape quickly. The sand 

was periodically checked so that it remained moist, but 

water did not sit in the bottom. The totes were kept at 

room temperature for a month at 15 to 20 °C, and then 

at 2 to 4 °C for 3 months. 

Seed that germinated early are removed and placed in 

the growing containers; otherwise the mesh bags are 

removed and the seed scarified after cool stratification. 

Scarification entails cutting 1 to 3 mm from the radicle 

end of the seed with a razor or scalpel such that a bit of 

white is showing. The opening allows the opportunity 

to detect empty or rotten seed and discard them. 

The seed for each open-pollinated family was sown 

into two 412A (short 77) StyroblocksTM (Beaver 

Plastics, Acheson, Alberta, Canada), which have a 

volume of 125 ml per cavity, with the intent of raising them in the nursery for two growing seasons. 

The blocks were single or double sown based on the 

proportion of good seed as determined by X rays. The 

growing medium was exclusively long-strand peat 

(premium grade) to assure ample air space to encourage root development. The seed was placed in the 

cavity and covered with forestry grit. The Styroblocks 

were misted and watered so that seeds were in constant 

contact with moisture. Upon germination, if there 

were two plants per cavity, one was transplanted to 

an empty cell. This was done before secondary roots 

appeared, at which point the operation is easy and 

successful. It would be possible to upgrade seed to 

remove more empty ones before sowing, or germinate 

them in a tray and then transplant to each cell of a 

Styroblock, but the approach described had financial 

advantages.

Data Collection and Analysis

Seeds were counted by tree (open-pollinated family) 

before stratification, and numbers discarded during 

scarification were recorded. The good seeds were sown 

and germinants counted on the 16th, 30th, and 49th 

day following. Seed was considered germinated when 

the radicle had achieved a length equivalent to the 

length of the seed. Seeds that did not germinate were 

sampled to determine if they were defective in order 

Figure 1—Whitebark seeds soaking in running water with 

extra aeration.

Figure 2—Mesh bags of seed to be buried in sterilized sand.
USDA Forest Service RMRS-P-76. 2018. 85

to help account for germination rates. Descriptive 

statistics and Pearson correlation coefficients were 

determined by using SAS® software (Ver. 9.3, 2010; 

SAS Institute Inc., Cary, North Carolina, USA).

RESULTS AND DISCUSSION

Germination is described here as the number of germinants relative to the number of seed sown rather than 

compared to the number of seed before nicking. The 

average germination across all families was 49 percent, 

but 47 percent if it is calculated relative to the number 

of seed put into stratification. In previous sowings, the 

difference between these rates has been as much as 13 

percent (V. Berger, 2013, unpublished data on file at 

Kalamalka Forestry Centre, Vernon, BC), but was accomplished with experienced technical staff. Due to the 

large quantity of seed (>60,000) to process, they were 

nicked by a rotating crew of six to eight nursery workers over 5 days. Nicking requires precision and practice 

to avoid damage and should be done in a warm and 

unhurried environment by competent staff. 

Germination percent can be expected to vary by family, seed source, program, geographic descriptors, 

climate, and date of collection among other factors. 

Regionally there were very large differences, with 

the greatest being between Canadian (35 percent 

germination) and American (78 percent germination) 

sources. Differences among the United States regions 

were less consequential, ranging from 73 percent to 81 

percent. Provenance-level variation was particularly 

pronounced for the Canadian populations and ranged 

from several sources with few germinants to some 

with more than 90 percent germination. Regionally 

the Kootenays had the best average germination at 43 

percent, with the South Coast–Interior Transition sites 

the worst at 23 percent. 

Family-by-family comparison of the same lots grown 

in this study and earlier in Oregon revealed a considerable differential. The current sowing averaged 79 percent germinations across the 81 Region 6 families used 

in common, but the mean for those grown previously 

in Oregon was 51 percent (fig. 3). It is likely that some 

features of the current study stratification were more 

effective. Both approaches used x-rays of the seed, 

sterilized them, and had stratification length that was 

similar and at similar temperatures. Features that are 

not common to both protocols included extra aeration 

in seed soaking, use of sand versus a naked stratification, and the nicking process.

Variable germination rates for BC regions are difficult 

to explain due to effects confounded by collection in 

different years, by different agencies with different 

storage. The large regional differences suggest that 

Figure 3—Percent germination of representative families (from Region 6) tested at nurseries in both Oregon and 

British Columbia.
86 USDA Forest Service RMRS-P-76. 2018.

climate was important, but mean annual temperature 

and mean annual precipitation explained little of the 

variation in germination (R < 0.1) as was the case 

for Pearson correlations with latitude, longitude, and 

elevation. The strongest influence (R > 0.7) was the 

length of storage, which agrees with the findings of 

McCaughey and Schmidt (1990), though not with 

Bower et al. (2011) or Sniezko and Kegley (this proceedings). Seed most likely deteriorated in storage, 

though lower germination rates may be due also to 

excessive seed moisture in some lots, which was not 

consistently monitored in the BC collections in the 

past.

Random sampling of 300 ungerminated seed after the 

last assessment of germination revealed 23 percent 

were morphologically normal and sound but failed to 

germinate. A further 18 percent of good seed were rotten. Fifteen percent were empty, but were not detected 

during the nicking process; the same number had a 

full-length corrosion cavity but no embryo. Fourteen 

percent had no cavity, and another 3 percent had a 

short cavity. The remaining seeds were only partially 

filled. Though the sound seed might eventually have 

germinated, the rotten well-developed seed is consistent with deterioration in storage. 

X-ray images of seed from families that germinated 

poorly often appeared more promising than the results, 

which would be consistent with undetected damage 

from storage or delayed germination. Data for x rays 

done at three different facilities were available. Those 

for BC seed originating from the ex situ reserve at 

the BC Tree Seed Centre showed a good correlation 

between germination percentage and sound seed as 

detected by x-ray (R = 0.8). For the seed from Oregon 

the information was clearly useful, but the images obtained at Kalamalka Forestry Centre were misleading, 

perhaps due to negative effects of storage. 

Results suggest that fresher seed be used when possible and that monitoring seed moisture content should 

be undertaken before long-term storage. X-raying seed 

is beneficial, as may be the case for extra aeration 

during the soaking of seed and stratification in sand. 

Nicking appears to be effective in increasing germination rates and eliminates some unviable seeds, thereby 

reducing sowing costs. The process was expensive, 

accounting for over half of the first-year nursery 

expense. A more recent study (Robb 2014) suggests 

that with longer stratification, particularly at room 

temperature, scarification is not necessary for good 

germination.

ACKNOWLEDGMENTS

The work described was funded by the Gene 

Conservation Technical Advisory Committee of the 

Forest Genetics Council of British Columbia. The 

methods described were developed over time by technical staff of the Kalamalka Forestry Centre in Vernon, 

British Columbia, specifically Vicky Berger, Giselle 

Phillips, and Randy Armitage. The seed for 81 families from the U.S. Department of Agriculture, Forest 

Service, Pacific Northwest Region (Region 6) along 

with germination and x-ray data for them from earlier 

work, was provided by Richard Sniezko of the Dorena 

Genetic Resources Center, Cottage Grove, Oregon.

REFERENCES

Arno, Stephen F.; Hoff, Raymond J. 1990. Pinus albicaulis

Engelm. whitebark pine. In: Burns, Russell M.; Honkala, 

Barbara H., tech. coords. Silvics of North America. 

Volume 1. Conifers. Agric. Handb. 654. Washington, 

DC: U.S. Department of Agriculture, Forest Service: 

268–279.

Bower, Andrew D.; Aitken, Sally A. 2008. Ecological 

genetics and seed transfer guidelines for Pinus albicaulis

(Pinaceae). American Journal of Botany. 95(1): 66–76. 

Bower, Andrew D.; Kolotelo, Dave; Aitken, Sally A. 2011. 

Effect of storage and stratification on germination 

of whitebark pine seeds. Western Journal of Applied 

Forestry. 26(1): 24–29.

Burr, Karen E.; Eramian, Aram; Eggleston, Kent. 2001. 

Growing whitebark pine seedlings for restoration. In: 

Tomback D.F.; Arno, S.F.; Keane, R.E., eds. Whitebark 

pine communities: Ecology and restoration. Washington, 

DC: Island Press: 325–345. 

Campbell, Elizabeth M.; Antos, Joseph A. 2000. 

Distribution and severity of white pine blister rust and 

mountain pine beetle in British Columbia. Canadian 

Journal of Forest Research. 30: 1051–1059.

Committee on the Status of Endangered Wildlife in Canada. 

2010. COSEWIC assessment and status report on the 

whitebark pine Pinus albicaulis in Canada. Ottawa: 

Committee on the Status of Endangered Wildlife in 

Canada. 44 p.
USDA Forest Service RMRS-P-76. 2018. 87

King, John N. 2010. Management of white pine: Assessing 

the effectiveness of our selections and breeding programs 

by measuring “field resistance.” In: Cleary, M., ed. 

Proceedings of the 3rd western white pine management 

conference. Kamloops, BC: BC Ministry of Forest and 

Range: 76–82.

Krakowski, Jodie; Kolotelo, Dave. 2009. Genetic 

conservation strategy for whitebark pine in British 

Columbia. Victoria, BC: Ministry of Forests and Range, 

Gene Conservation Technical Advisory Committee. 20 p. 

Leslie, Adrian; Wilson, Brendan. 2011. No free lunch: 

Observations on seed predation, cone collection, and 

controlled germination of whitebark pine from the 

Canadian Rockies. In: Keane, Robert E.; Tomback, 

Diana F.; Murray, Michael P.; [et al.], eds. The future 

of high-elevation, five-needle white pines in western 

North America: Proceedings of the high five symposium. 

Proceedings RMRS-P-63. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky 

Mountain Research Station: 348–354.

Mahalovich, Mary F.; Burr, Karen E.; Fouchee, David 

L. 2006. Whitebark pine germination, rust resistance 

and cold hardiness among seed sources in the Inland 

Northwest: Planting strategies for restoration. In: Riley, 

L.E.; Dumroese, R.K.; Landis, T.D., tech. coords. 

National proceedings: Forest and conservation nursery 

associations—2005. Proceedings RMRS-P-43. Fort 

Collins, CO: U.S. Department of Agriculture, Forest 

Service, Rocky Mountain Research Station: 91–101. 

McCaughey, Ward W.; Schmidt, Wyman C. 1990. 

Autecology of whitebark pine (Pinus albicaulis Engelm.) 

In: Schmidt, W.C.; MacDonald, K.J., eds. Proceedings of 

the symposium on whitebark pine ecosystems: Ecology 

and management of a high mountain resource. Gen. 

Tech. Rep. INT-270. Fort Collins, CO. U.S. Department 

of Agriculture, Forest Service, Intermountain Forest and 

Range Experiment Station: 85–96.

Pitel, J.A.; Wang, B.S.P. 1990. Physical and chemical 

treatments to improve germination of whitebark 

pine seed. In: Schmidt, W.C.; MacDonald, K.J., eds. 

Proceedings of the symposium on whitebark pine 

ecosystems: Ecology and management of a high 

mountain resource. Gen. Tech. Rep. INT-270. Fort 

Collins, CO: U.S. Department of Agriculture, Forest 

Service, Intermountain Forest and Range Experiment 

Station: 130–133.

Robb, Lindsay, 2014. Whitebark pine (Pinus albicaulis

Engelm.) germination method. (Poster), Alberta Tree 

Improvement and Seed Centre, Box 750, Smokey Lake, 

Alberta. 

Tomback, Diana F.; Anderies, Angela J.; Carsey, Katherine 

S.; [et al.]. 2001. Delayed seed germination in whitebark 

pine and regeneration patterns following the Yellowstone 

fires. Ecology. 82(9): 2587–2600.

Zeglen, Stephan 2002. Whitebark pine and white pine blister 

rust in British Columbia, Canada. Canadian Journal of 

Forest Research. 32(3): 1265–1274.
88 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Monitoring of Blister Rust Resistance, Pathogen Virulence, and 

Genetic Adaptability of Western White Pine (Pinus monticola)

at Six Sites in Western Washington, USA

A. Ramsey1, *, R.A. Sniezko2, R. Danchok2, D. Omdal1, A. Kegley2, D.P. Savin2, and S. Long2

1 Washington Department of Natural Resources, Resource Protection Division, Olympia, Washington, USA 

2 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA 

* amy.ramsey@dnr.wa.gov

INTRODUCTION

Western white pine (WWP, Pinus monticola) is a wideranging, long-lived native conifer species in western 

North America. It is highly susceptible to the introduced fungal pathogen Cronartium ribicola, causative 

agent of white pine blister rust (WPBR). The impacts 

of the blister rust have reduced the incidence of WWP 

in natural ecosystems and have led to reluctance in 

using the species in both restoration and reforestation. 

Research and operational programs to develop genetic 

resistance to WPBR have been ongoing for more 

than five decades, with regional programs based in 

Oregon (USA), Idaho (USA), and British Columbia 

(Canada). Progeny of thousands of parent trees have 

been screened for rust resistance in short-term artificial 

inoculation trials, and several types of resistance have 

been uncovered (Sniezko et al. 2014). Seed orchards 

have been established using the products of the earlier 

resistance work, and breeding to increase the level 

of genetic resistance continues. However, relatively 

few field trials, and notably multisite field trials with 

a diverse array of geographic sources and types of 

resistance, have been established to examine field 

resistance and durability of resistance (Sniezko et al. 

2012).

This series of field trials examines WWP families and 

orchard seedlots from a wide array of geographic origins (British Columbia, Idaho, Oregon, Washington) 

at six diverse sites in western Washington (USA). 

The group of families and orchard seedlots includes 

materials that should encompass most (if not all) of the 

known types of resistance to white pine blister rust in 

WWP, and represents among the best resistant seedlots 

currently available from the three operational screening programs working to develop resistance to WPBR. 

In addition to testing the efficacy and durability of the 

different types of resistance in the field, these sites can 

help increase our understanding of the dynamics of 

rust infection over time, refine rust hazard guidelines, 

and determine whether seedlots from other breeding 

zones are adapted to sites in western Washington.

OBJECTIVES

The current and future objectives for this study 

include: 

1. Assessing the current rust incidence and impacts 

(mortality). Establishing baseline for long-term 

monitoring. 

2. Examining growth, vigor, and reproductive status 

of western white pine. Establishing baseline for 

long-term monitoring.

3. Assessing impacts of abiotic and biotic agents 

on WWP seed sources from diverse geographic 

origins.
USDA Forest Service RMRS-P-76. 2018. 89

4. Assessing the level and durability of partial resistance over an array of environments.

5. Monitoring durability of major gene resistance 

(MGR).

6. Providing updates to landowners and aerial 

surveyors in western Washington on the levels 

of rust incidence and field resistance currently 

available.

METHODS

Six 3-ac (1.21-ha) trials were established in 2006 

across western Washington. Thirty-six seedlots were 

planted across the sites (two sites had 30), with 1,269 

to 1,365 individual seedlings planted at each site. A 

randomized complete block design was used, with 

seven blocks per site and three to seven seedlings per 

seedlot per block. The seedlots originated from many 

areas across the region, including the Gifford Pinchot 

National Forest (NF), Mount Baker-Snoqualmie 

NF, Olympic NF, Wenatchee NF, and Colville NF 

in Washington; Mount Hood NF, Umpqua NF, and 

Confederated Tribes of Warm Springs in Oregon; 

British Columbia; and the Bingham F2 seed orchard in 

Idaho. 

Incidence and severity assessments of WPBR were 

made in 2010, 2011, and 2013. During the assessments 

tree damage, tree vigor, and rust severity, including 

the number of WPBR cankers, were measured. During 

the 2013 assessments, tree height and reproductive 

status were also measured. HOBO® (Onset®, Bourne, 

Massachusetts, USA) weather stations were installed 

at each site in 2013 to measure temperature and relative humidity.

RESULTS AND DISCUSSION

In 2013, overall survival—taking into account all 

mortality agents, including root disease and animal 

damage—was 90 percent or greater at all sites, even 

in the known WPBR-susceptible seedlots (fig. 1a). 

WPBR mortality was generally less than 1 percent 

across all sites, with the greatest amount of mortality from rust at the Cayuse and Rocky Raccoon sites 

(fig. 1b). As expected, WPBR infection levels were 

higher than the mortality, with most seedlots having 

less than 25 percent of the trees infected, except at 

Shuwah Jigsaw (fig. 1c). At the Shuwah Jigsaw site, 

47 percent of the trees were infected and there was 

an average of 4.3 infections per tree (fig. 1d). The 

susceptible control had the highest overall infection 

(45 percent) across the six sites and was the family 

with the highest percentage stem infection on four 

of the six sites, including 98 percent at the Shuwah 

Jigsaw site (where it averaged more than nine cankers 

per tree). The low-to-moderate level of rust infection 

is encouraging, but it is likely due to a combination of 

genetic resistance and initial low rust hazard. A few 

trees putatively originating from a parent homozygous 

for major gene (Cr2) resistance now show cankers, potentially indicating the presence of a low frequency of 

virulent genotypes (vcr2), or environmental conditions 

such that the resistance is not 100 percent effective. 

Further investigations are pending. As with other sites, 

the level of rust infection and mortality are expected 

to increase over time (Sniezko et al. 2012), hence the 

importance of a network of “permanent” field trials 

such as this to monitor rust infection over time in a 

changing environment.

Less than 10 percent of the trees on most sites had pollen or cones, but the trees on Shuwah Jigsaw stand out 

again, as 50 percent of some seedlots had reproductive 

features (fig. 1e). The height data show that generally the fast growing seedlots at one site are also fast 

growing at other sites; trees were notably shorter at the 

Caveman site (fig. 1f). Six similar sites will be established in eastern Washington in 2015 (and one site in 

western Oregon in December 2014). Future analyses will 

look at more details of the geographic origin of the parent trees and their performance in western Washington.

ACKNOWLEDGMENTS

We appreciate funding from a U.S. Department of 

Agriculture, Forest Service Region 6 Forest Health 

Monitoring Evaluation Monitoring grant and extensive 

assistance with trial assessments from the Dorena 

Genetic Resource Center technicians.

REFERENCES

Sniezko, R.A.; Smith, J.; Liu, J-J.; [et al.]. 2014. Genetic 

resistance to fusiform rust in southern pines and white 

pine blister rust in white pines—A contrasting tale of two 

rust pathosystems—Current status and future prospects. 

Forests. 5(9): 2050–2083. doi:10.3390/f5092050.
90 USDA Forest Service RMRS-P-76. 2018.

Figure 1—Growth, survival, white pine blister rust (WPBR) impacts and cone and/or pollen reproduction in six western white pine 

(P. monticola) genetic trials through 2013 (sites planted in 2006): (a) percent tree survival by site, (b) percent mortality from WPBR, 

(c) percent trees with WPBR stem symptoms, (d) mean number of cankers per infected tree, (e) percent trees with pollen or cones, 

and (f) mean 2013 height (cm). (Note: X-axis is site location/name: 1 = Caveman, 2 = Cayuse, 3 = High Chaparral, 4 = Rocky 

Raccoon, 5 = Shuwah Jigsaw, 6 = Stevens Left, 7 = Mean performance over all six sites. Each bar spans the range of the seedlot 

means at each site (30 or 36 seedlots, depending on the site) so that the minimum and maximum for each trait can be compared. 

The black line is the overall site average. The green line is the seedlot mean for a seedlot known to have Major Gene Resistance. 

The red line is the seedlot mean for a known susceptible seedlot. Green bars represent the 2 sites with lowest percentage of trees 

with stem symptoms, red bars for the highest 2 sites and yellow bars for the 2 intermediate sites.)

Sniezko, R.A.; Danchok, R.; Hamlin, J.; [et al.]. 2012. 

White pine blister rust resistance of 12 western white 

pine families at three field sites in the Pacific Northwest. 

In: Sniezo, R.A.; Yanchuk, A.D.; Kliejunas, J.T.; [et al.], 

tech. coords. Proceedings of the fourth international 

workshop on the genetics of host-parasite interactions 

in forestry: Disease and insect resistance in forest trees. 

Gen. Tech. Rep. PSW-GTR-240. Albany, CA: U.S. 

Department of Agriculture, Forest Service, Pacific 

Southwest Research Station: 356–367. https://www.

fs.fed.us/psw/publications/documents/psw_gtr240/psw_

gtr240_356.pdf. (07 March 2015).
USDA Forest Service RMRS-P-76. 2018. 91

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Naturally occurring genetic diversity is the material 

upon which much of the biodiversity we attribute 

to forested ecosystems is based. The structuring of 

genetic diversity within natural populations of forest 

trees results from the interplay of numerous evolutionary forces, such as genetic drift, migration, and natural 

selection. Landscape genomics is concerned with testing hypotheses about how these interacting evolutionary forces change over geographical space and time 

to shape naturally occurring patterns of this diversity. 

Here, I will use a landscape genomic approach to illustrate how genetic drift, migration, and natural selection 

interact to determine patterns of genetic diversity, including the genetic diversity underlying fitness-related 

phenotypes, at multiple spatial scales within natural 

populations of forest trees.

I will accomplish this task by arguing that populations 

of sugar pine (Pinus lambertiana), western white pine 

(P. monticola), and whitebark pine (P. albicaulis) 

are adapted to local site conditions within the Lake 

Tahoe Basin of California and Nevada, USA. More 

The Genetic Architecture of Local Adaptation and the Genomic 

Exploration of Rugged Evolutionary Landscapes Within Species of 

Pinus Subgenus Strobus

A.J. Eckert1, *, B.M. Lind2, C.J. Friedline1, E. Hobson1, J.W.R. Zinck3, O.P. Rajora4,

D.B. Neale5, D. Vogler6, and P.E. Maloney7

1 Virginia Commonwealth University, Department of Biology, Richmond, Virginia, USA

2 Virginia Commonwealth University, Graduate Program in Integrative Life Sciences, Richmond, Virginia, USA

3 Public Health Agency of Canada, Sir Frederick G. Banting Research Centre, Ottawa, Ontario, Canada

4 University of New Brunswick, Faculty of Forestry and Environmental Management,

Fredericton,New Brunswick, Canada

5 University of California, Davis, Department of Plant Sciences, Davis, California, USA

6 USDA Forest Service, Pacific Southwest Research Station, Institute of Forest Genetics, Placerville, California, USA

7 University of California, Davis, Department of Plant Pathology, Davis, California, USA

* aeckert@vcu.edu

specifically, I will test the hypothesis that local adaptation is apparent at local spatial scales (~600 km2), 

despite large amounts of pollen-mediated gene flow 

among local populations. I will also highlight, however, several instances where clearly adaptive patterns 

of genetic diversity have been influenced by local 

demographic events. Building upon these results, I will 

then illustrate how the observed patterns of molecular 

genetic diversity for a varied set of functional genes 

are organized relative to the phenotypic variation 

used to highlight local adaptation. The drivers of 

these patterns are very likely the multilocus aspects of 

molecular genetic diversity as it contributes to local 

adaptation at fine spatial scales. Last, I will draw upon 

emerging examples from eastern white pine (P. strobus) and foxtail pine (P. balfouriana) to illustrate the 

promising role that species of Pinus subgenus Strobus

will play in our understanding of the genetic architecture of local adaptation and the ability of this architecture to rapidly respond to environmental change within 

natural populations of forest trees.
92 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

The Efficacy of Ex Situ Conservation of the Mexican and Central 

American White Pines (Pinus ayacahuite and P. chiapensis)

in Developing Countries 

W.S. Dvorak1, *

1 North Carolina State University, Department of Forestry and Environmental Resources,

Camcore, Raleigh, North Carolina, USA

* dvorak@ncsu.edu

Mexican white pine (Pinus ayacahuite) and Central 

American white pine (P. chiapensis) are two of the 

largest tree species in the highlands of Mexico and 

Central America. Pinus ayacahuite occurs from 

northern Mexico to central Honduras as three varieties: 

brachyptera, veitchii, and ayacahuite. Its southernmost 

variety, P. ayacahuite var. ayacahuite, is the most 

threatened of the three in its native environment. Pinus 

chiapensis has a smaller geographic range than P. ayacahuite, from central Mexico to western Guatemala. 

Many of the remnant populations of P. chiapensis are 

endangered throughout its geographic range. Locally, 

these white pines are used for rustic furniture and 

woodcarvings.

Over the last three decades, the International Tree 

Breeding and Conservation Program (Camcore) at 

North Carolina State University has made small 

research seed collections of 9,250 mother trees in 380 

populations of 25 different pine species in Mexico and 

Central America for the establishment of field tests and 

ex situ conservation plantings in Latin America and 

southern Africa. In its genetic testing and ex situ conservation work of the white pines, Camcore sampled 

16 populations and 428 mother trees of P. ayacahuite

and 16 provenance and 380 trees of P. chiapensis in 

Mexico and Central America. Genetic material was 

subsequently established in Brazil, Colombia, and 

South Africa. 

The two white pines are some of the most difficult for 

which to successfully develop ex situ conservation 

programs because of their strict site requirements and 

slow initial growth that requires long-term weed control and trial maintenance. However, most detrimental 

to conservation success is that no markets for white 

pine wood have been developed in the southern hemisphere to interest the private sector or the small grower 

to establish commercial plantings of the species, even 

though Camcore members have seed stands of P. chiapensis and second-generation progeny trials available 

to continue low-level breeding and improvement work. 

The places where a species like P. chiapensis thrives 

as an exotic are also the same locations best suited for 

short-rotation species, such as Eucalyptus grandis and 

its hybrids.

This presentation broadly addresses ex situ conservation efforts of the Central American and Mexican pines 

and highlights the challenges of protecting these species. A case study is provided on how the accidental 

introduction of a disease (pitch canker, caused by 

Fusarium circinatum) into South Africa has changed 

the private sector’s view of the importance of pine 

ex situ conservation, the need for having access to 

alternate species, and the benefits of developing pine 

hybrids as a defense against future potential threats to 

exotic plantation forests.
USDA Forest Service RMRS-P-76. 2018. 93

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Forest Health Protection Gene Resource Conservation:

A Lasting Legacy

G. Man1 and B. Moltzan1, *

1 USDA Forest Service, Forest Health Protection, Washington, DC, USA

* bmoltzan@fs.fed.us

Abstract—The U.S. Department of Agriculture, Forest Service (USDA FS) has a strong tradition of improving the resilience of forested landscapes. In 2007, a national workshop on genetic conservation classified 

nine native white pine species as important points for genetic conservation. Added support in 2008 and 

2009 from Forest Health Protection and in-kind contributions from other branches of the USDA FS and 

its partners have facilitated and accelerated several gene conservation actions, including individual tree 

seed collections of Pinus albicaulis, P. aristata, P. balfouriana, P. flexilis, P. longaeva, and P. strobiformis. 

Forest Health Protection began a formal strategic approach to gene conservation in 2010. The USDA FS 

established a Gene Conservation Steering Committee and a charter, which was endorsed in December 

2011. The program seeks to conserve tree species at risk from the negative impacts of mountain pine 

beetle (Dendroctonus ponderosae), white pine blister rust (caused by the pathogen Cronartium ribicola), 

and climate change, as well as other disturbance factors. Through this effort, more than 2,200 collections 

have been stored for conservation, many of which focus on pine. To help assess and prioritize other at-risk 

forest tree species, the USDA FS has just launched Project CAPTURE (Conservation Assessment and 

Prioritization of Forest Trees Under Risk of Extirpation). This 3-year effort will assess the vulnerabilities of 

major forest trees in the United States to help guide not just gene conservation, but also restoration and 

climate change mitigation actions.
94 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

A Gene Conservation Program to Capture the Genetic Diversity of 

Rocky Mountain Bristlecone Pine (Pinus aristata)

A.W. Schoettle1, *, C.M. Richards2, J. Popp1, K.S. Burns3, B.A. Goodrich1, and J.G. Klutsch1

1 USDA Forest Service, Rocky Mountain Research Station, Fort Collins, Colorado, USA 

2 USDA Agricultural Research Service, National Center for Genetic Resources Preservation, Fort Collins, Colorado, USA

3 USDA Forest Service, Forest Health Protection, Golden, Colorado, USA

* aschoettle@fs.fed.us

Abstract—Rocky Mountain bristlecone pine (Pinus aristata) has a narrow core geographic and elevational 

distribution and is threatened by rapid climate change, white pine blister rust (caused by Cronartium 

ribicola), and bark beetles (Dendroctonus spp.). The core distribution of P. aristata is in central and 

southern Colorado and extends into northern New Mexico with a disjunct population on the San Francisco 

Peaks in northern Arizona (USA). The first genetic diversity study of P. aristata revealed low species- and 

population-level genetic diversity (He = 0.070 and 0.062, respectively) and moderate among-population 

differentiation (FST = 0.131) (Schoettle et al. 2012). Genetic diversity correlated with longitude, latitude, 

and elevation, and a strong mountain island effect may contribute to substructuring and isolation. The 

combination of low genetic diversity, moderate population isolation, and a protracted regeneration dynamic 

puts populations at risk for extirpation by novel stresses (Schoettle et al. 2012), highlighting the need for ex 

situ gene conservation. 

Using the knowledge of the variation and distribution of genetic diversity, we developed and executed 

an efficient gene conservation sampling design and collection. Ten populations within each of 6 collection areas corresponding to the observed genetic substructuring were identified; 10 individual tree seed 

collections and a bulk collection (20 seed trees equally represented) were targeted from each of the 60 

populations. Seed trees are georeferenced and sampled for seeds and needle tissue; each collection is 

stored for gene conservation at –18 °C at the U.S. Department of Agriculture (USDA) Agricultural Research 

Service, National Center for Genetic Resources Preservation lab (Fort Collins, Colorado) with a working collection maintained by the USDA Forest Service, Rocky Mountain Research Station (Fort Collins, 

Colorado). Ecological data on each population were also recorded (site data including physical characteristics, stand density, species composition, and disturbance history) to complement the genetic collection.
USDA Forest Service RMRS-P-76. 2018. 95

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Germination of Whitebark Pine (Pinus albicaulis) and Other High 

Elevation White Pine Species Seed After Long-Term Storage:

Ex Situ Gene Conservation Potential of High Elevation

White Pine Species 

R.A. Sniezko1, * and A. Kegley1

1 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA

* rsniezko@fs.fed.us

Abstract—Genetic variation in a plant species is a key to its ability to survive and evolve in the face of 

changing environmental pressures. Due to insect and disease impacts, changes in fire regimes, and a 

changing climate, many populations of high elevation white pine species continue to experience high 

mortality levels and potentially worrisome decreases in genetic variation. In recent years, some trees rated 

highly for resistance to the nonnative white pine blister rust (causal agent: Cronartium ribicola) have been 

killed by fire or mountain pine beetle (Dendroctonus ponderosae). Ex situ gene conservation offers the 

possibility to conserve the genetic variation within a species before much of it is lost. For many conifer 

species, freezer storage of seed offers a relatively inexpensive, long-term method of storing germplasm 

for potential future use. However, seed of some conifers does not store well for long time periods under 

currently used freezer technology, and few data are available for many species such as the high elevation 

white pine species of western North America.

The U.S. Department of Agriculture, Forest Service (USDA FS) and cooperators have embarked on a 

program to collect and conserve seed of these high elevation white pine species. Most of these seedlots 

have been collected since 2008. Knowledge of seed viability after one or more decades of storage would 

be useful for assessing the utility of the freezer storage for these species. A test was begun in 2010 for 

whitebark pine (Pinus albicaulis), limber pine (P. flexilis), southwestern white pine (P. strobiformis), Rocky 

Mountain bristlecone pine (P. aristata), and foxtail pine (P. balfouriana). Some organizations in the United 

States and Canada had seedlots available that had been in cold storage or freezer storage for 7 to 32 

years and made seed available for a germination test at the USDA FS Dorena Genetic Resource Center 

(Cottage Grove, Oregon, USA). We report here on the results of germination testing of these oldest known 

seedlots of these five species and the implications for ex situ gene conservation efforts and future testing. 

Results indicate that at least some seedlots of species such as whitebark pine and foxtail pine can be 

stored for several decades and show very high germination percentage in subsequent testing.
96 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Genetic Relationship between Reproductive Morphology and Growth 

Characteristics of Korean Pine (Pinus koraiensis)

S.U. Han1, H.S. Lee1, J.M. Park2, and K.S. Kang2, *

1 Korea Forest Research Institute, Suwon, Republic of Korea

2 Seoul National University, Seoul, Republic of Korea 

* kangks84@snu.ac.kr

Korean pine (Pinus koraiensis) is an important species 

for timber as well as pine nut production in Korea. 

The selection of “plus” trees began in 1959 and 

seed orchards have been established with grafts and 

seedlings propagated from these selected individuals. 

There are 300 “plus” trees and 100 ha seed orchards 

of P. koraiensis in Korea. Further, 244 open-pollinated 

families are being tested at four 18.5-ha sites. The 

main purposes of this study were: (1) to investigate 

the correlation among cone, seed, and growth traits of 

P. koraiensis; (2) to examine the possibility that such 

traits could be useful indicators for progeny testing and 

further selection; and (3) to provide information on 

genetic parameters for the establishment of advancedgeneration seed orchards.

Open-pollinated cones of P. koraiensis plus trees were 

collected in 1981, and the morphological characteristics of cones and seeds were examined. The seeds 

were sown in a nursery in 1982 and grown for 4 years. 

In 1986, the seedlings were planted in three sites containing 20 trees in each of 6 replications with 1.8-m 

× 1.8-m spacing as a progeny test plantation. The 

correlations between morphological traits of cones and 

seeds from each plus tree and growth characteristics 

(height, diameter at breast height [d.b.h., diameter at 

1.37 m] and volume) in plantations were investigated. 

Variance components and heritabilities for growth 

characteristics were estimated in the plantation at ages 

10, 20, and 30 years.

Seedling height at the nursery bed showed a strong 

correlation with most cone and seed characteristics. 

Numbers of cones per tree and seeds per cone were 

negatively correlated with seedling height and statistically significant at the nursery bed. Cone diameter 

and cone weight showed a positive correlation with 

seedling height. Seed length, width, thickness, and 

weight were all positively correlated with seedling 

height at the nursery and in the plantation at age 5. 

From age 10, tree height, d.b.h., and volume were 

compared with cone and seed traits. None of the cone 

traits was correlated with growth characteristics from 

age 10. Some seed traits such as seed length, thickness, 

and weight were correlated with growth until age 10. 

After that age, the correlations between seed traits and 

growth characteristics were also nonsignificant, except 

between seed length and d.b.h. at age 20. Analyses 

suggest that the correlation between reproductive morphology and growth characteristics becomes weaker 

as trees grow older. It seems likely that cone and seed 

traits of P. koraiensis, which bears middle-sized seeds, 

are related until trees enter the mid-term growth phase. 

Heritability estimation showed that growth characteristics were under genetic control. Family heritability 

was much higher than individual heritability, implying 

that family selection would be a better option for the 

establishment of advanced-generation seed orchards of 

P. koraiensis.
USDA Forest Service RMRS-P-76. 2018. 97

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Analysis of Genetic Diversity and Spatial Genetic Structure of the 

Endangered Dwarf Stone Pine at Mount Seorak by using Nuclear 

Microsatellite Markers

H.I. Lim1, Y.P. Hong1, K.N. Hong1, J.G. Han1, *, and K.H. Jang1

1 Korea Forest Research Institute, Forest Genetic Resources Division, Suwon, Republic of Korea

* han6430@gmail.com

Abstract—Dwarf stone pine (Pinus pumila), which occurs in northeastern Asia, is found only in a limited 

area at Daecheongbong (1,550–1,700 m above sea level) on Mount Seorak in South Korea. Dwarf stone 

pines are distributed over about 10 ha in this location. To establish strategies for adequate conservation 

and sustainable management of this endangered species, 57 individuals were selected at 12-m intervals 

in the study site (3.75 ha). Thirty-three nuclear microsatellite (nSSR) primers, developed from P. koraiensis

and P. strobus, were tested and 10 of them revealed successful amplification products. However, only 3 of 

the 10 primer pairs turned out to be polymorphic. Fourteen alleles were observed in three polymorphic loci. 

The observed number of alleles per locus ranged from four to five. The observed heterozygosity (0.294) 

was very low compared to the expected heterozygosity (0.489), which resulted in a high value for the 

fixation index (0.4). This may have resulted from reproduction of progenies by crossing among genetically 

closely related individuals. Dwarf stone pines have been maintained as a single isolated relic population 

for such a long time (at least since the last ice age) that inbreeding among kinship individuals could not 

be avoided in the Daecheongbong area. This postulation could be supported by the high proportion of 

monomorphic loci (i.e., 7 of 10 nSSR loci). However, any apparent genetic patches were not observed in a 

previous study using I–SSR markers for spatial autocorrelation analysis of individuals 12 m apart. Failure 

to observe any spatial genetic structures at the study site may have resulted from either an inadequate 

sampling strategy developed for the earlier study or the limited number of nSSR loci analyzed in this study. 

Further work will be performed at a finer sampling scale (e.g., sampling individuals at 4-m intervals) and 

with additional polymorphic loci.
98 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Population and Evolutionary Genetic Studies of

Five-Needle Pines in Russia

D. Politov1, *, Y. Belokon1, M. Belokon1, E. Mudrik1, T. Poliakova1, E. Petrova2,

and S. Goroshkevich2

1 Vavilov Institute of General Genetics, RAS, Moscow, Russia

2 Institute of Climatic and Ecological Systems, Siberian Branch RAS, Tomsk, Russia

* dvp@vigg.ru

Three species of stone pines are native to Russia: 

Siberian stone pine (Pinus sibirica), dwarf Siberian 

pine (P. pumila), and Korean pine (Pinus koraiensis). 

Genetic differentiation by allozyme loci placed P. 

sibirica in one clade with European stone pine (P. 

cembra), whereas the Far Eastern species P. pumila

and P. koraiensis represent another lineage more 

closely associated with the Asiatic white pines that 

have winged seeds: P. armandii, P. parviflora, and P. 

wallichiana. Molecular data on the large divergence 

of sibirica–cembra and pumila–koraiensis clades are 

supported by analysis of needle anatomy. The taxonomic position of the North American stone pine (P. 

albicaulis) is intermediate between the Asiatic stone 

pines and the North American white pines P. flexilis, 

P. monticola, and others.

In the central parts of their ranges, stone pine gene 

pools are subjected to integrating factors: extensive 

gene flow and balancing selection in similar environmental conditions. Populations at the margins of 

their range experience differentiating forces: genetic 

drift, disruptive selection, and gene exchange with 

related species. When analyzing natural populations 

and clonal archives of P. sibirica ecotypes, we observed significant correlations between frequencies 

of particular alleles at allozyme loci and geographic 

coordinates as well as climatic conditions. Analyses 

of multilocus genotypes showed good correspondence with recognized forest seed zoning, in many 

cases refining borders of seed zones, regions, and 

subregions. The sympatry zone between P. sibirica and 

P. pumila is characterized by interspecific gene exchange through hybridization with bidirectional gene 

introgression of nuclear and mitochondrial markers. 

More genetically similar P. pumila and P. koraiensis

do not form a hybrid zone. Formation of stone pine 

lineages and species putatively included multiple 

events of reticulate evolution. Analysis of allozyme 

and simple sequence repeat loci has proven that multitrunk forms in P. sibirica and clumps in P. pumila are 

different genotypes (but often related as half-sibs) and 

therefore represent the result of germination of several 

seeds from the caches of nutcrackers (Nucifraga spp.). 

A substantial level of heterozygote deficiency was 

recorded in several P. pumila stands that were characterized by a low ratio of plant height to the square of 

a clump. This indicates inbreeding, reflecting a high 

proportion of selfing and consanguineous mating due 

to the high probability of sedimentation of the pollen 

cloud within a clump of genetically related individuals. 

In response to global climate change, it is evident 

that treelines for major stone pine species are shifting 

to the north on the plain and upslope in subalpine 

mountain belts. Descriptions of local populations and 

inventories of genetic resources of stone pines are 

urgently needed to ensure conservation of amongand within-species genetic diversity. Formation of 

genetic collections and seed banks should be based on 

knowledge of the genetic structure of populations at 

different types of markers (nuclear, cpDNA, mtDNA) 

and candidate genes responsible for adaptation and 

economically important traits.
USDA Forest Service RMRS-P-76. 2018. 99

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Using Historical Provenance Test Data to Understand Tree 

Responses to a Changing Climate in Sugar Pine (Pinus lambertiana)

A. Bontemps1, J. Wright2, *, R. Sniezko3, D. Savin3, and J. Schmitt1

1 University of California, Davis, Davis, California, USA 

2 USDA Forest Service, Pacific Southwest Research Station, Davis, California, USA

3 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA

* jessicawwright@fs.fed.us 

Climate change is a major concern for forest managers 

involved in ecological restoration, in part because they 

are responsible for planting trees that will be components of a healthy and resilient ecosystem for the 

next 100 to 200 years. Anticipated changes in climate 

include changes in temperature, precipitation, and 

length of the growing season. Trees locally adapted to 

their native climate may not be able to survive or grow 

in novel climates. Current afforestation strategies may 

thus require the use of seeds from populations already 

adapted to future climates. 

Researchers can help to identify these populations 

thanks to provenance tests. Provenance tests are 

studies where genotypes from different sources (or 

provenances), coming from the entire range of the species or only a subset, are transplanted onto a common 

site. In fact, individuals are moved from one climate, 

are planted in a new one, and can be monitored for survival and growth. Therefore, this experimental design 

is ideal for understanding how trees respond to novel 

climates.

In this study we will focus on two sets of sugar pine 

(Pinus lambertiana) provenance tests. The first involves four test sites, two in California (USA) and two 

in Oregon (USA). Sixty-nine sources, spanning the 

entire range of this species from Mexico to Oregon, 

were planted in each of the four sites starting in 1984. 

The second test, Harrel, has only one planting site; 124 

sources of sugar pine were planted on that site in 1992. 

Using information on where the seeds were collected, 

we can generate data for a set of climate variables 

(monthly average, minimum, and maximum temperatures; and monthly total precipitation) using available 

models. 

The aim of our study will be to assess whether the 

growth and survival of the trees in the transplanted 

site is influenced by the climate at the home site. Our 

hypothesis is that trees are better adapted to the climate at the home site and thus, the growth and survival 

of transplanted trees will be negatively affected by a 

strong difference in climate between the home and the 

transplanted sites.
100 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Genetic Variation in Height and White Pine Blister Rust Resistance in 

Sugar Pine (Pinus lambertiana)—15-Year Field Trial Results of Three 

Six-Parent Half Diallels From Three Breeding Zones

J. Hamlin1, R.A. Sniezko2, *, F. Isik3, D.P. Savin2, A. Kegley2, S. Long2, and R. Danchok2

1 USDA Forest Service, Umpqua National Forest, Roseburg, Oregon, USA

2 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA

3 North Carolina State University, Department of Forestry and Environmental Resources, Raleigh, North Carolina, USA 

* rsniezko@fs.fed.us

Sugar pine (Pinus lambertiana) is a long-lived conifer 

species found in forest ecosystems from southern 

California to northern Oregon (USA). It is highly 

susceptible to white pine blister rust (WPBR), caused 

by the fungal pathogen Cronartium ribicola. Two 

long-term operational programs to develop populations 

of sugar pine with genetic resistance to WPBR, one in 

Oregon and one in California, have been ongoing for 

more than four decades. Most of the resistance work 

involves artificial inoculation of young seedlings. A 

small percentage of sugar pine parents have complete 

resistance (a hypersensitive-like response in the 

needles in the absence of a virulent strain of the rust) 

under the control of a single major dominant gene. 

In addition, there is a low frequency of partial resistance in some sugar pine parents. Partial resistance 

is thought to be under the control of multiple genes 

and more durable than the hypersensitive-like needle 

resistance. Both types of resistance could potentially 

be combined to increase survival at most field sites. 

However, detailed field trials to examine partial resistance in sugar pine are relatively rare. 

In 1998, a large field trial was established in Happy 

Camp, California, to examine partial resistance, its 

inheritance, and its durability. The trial was established 

with 39 full-sib families, produced from crosses 

among three six-parent half diallel mating designs 

(18 parents). The parents of the three diallels, all field 

selections from the early years of the Oregon program, 

represented three different breeding zones for sugar 

pine in southern Oregon. Prior rust resistance screening of seedling progeny showed only low levels of 

partial resistance (at least for 2-year-old seedlings) 

in these full-sib families. Post-planting survival was 

excellent, and height averaged 88, 312, and 537 cm 

in 2001, 2007, and 2012, respectively. Rust infection 

(stem cankers) was extremely low until 2007. In 2007, 

the site averaged 73-percent infection, with full-sib 

family means ranging from 50 to 96 percent; in 2012, 

the site averaged 89-percent infection, and family 

means ranged from 66 to 100 percent. The number of 

stem infections per tree averaged 4.7, and full-sib family means ranged from 1.9 to 14.3 in 2007; number of 

stem infections increased to 8.0 in 2012, with family 

means ranging from 3.2 to 19.1. Mortality from rust 

was relatively low through 2012, with full-sib family 

means ranging from 3 to 47 percent. The level of partial resistance exhibited by some sugar pine families is 

encouraging. The results will be used to help examine 

genetic differences in growth and rust resistance 

among the breeding zones, examine inheritance of 

some components of partial resistance, and provide 

possible selections for sugar pine seed orchards and for 

future breeding to increase rust resistance.
USDA Forest Service RMRS-P-76. 2018. 101

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Patterns of Population Structure and Environmental Associations 

Across the Range of Two Ecologically and Economically Important 

Five-Needle Pine Species

S. Nadeau1, *, N. Isabel2, J. Godbout2, M. Lamothe2, M.-C. Gros-Louis2, and K. Ritland1

1 University of British Columbia, Department of Forest and Conservation Sciences, Faculty of Forestry,

Vancouver, British Columbia, Canada 

2 Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Sainte-Foy, Québec, Canada 

* simon.nadeau.ubc@gmail.com

Abstract—Under rapid global warming, it is critical for us to better understand the capacity of forest trees 

to adapt to a changing climate, especially for species that are more at risk such as five-needle pines. 

Western white pine (Pinus monticola) and eastern white pine (Pinus strobus) have fragmented ranges and 

have suffered declines due to harvesting, fire suppression, and the white pine blister rust (causal agent: 

Cronartium ribicola). We identified and compared patterns of genetic diversity and adaptation to climate in 

these two species by using a set of 267 orthologous genes, including candidate genes for growth, bud phenology, and resistance to biotic and abiotic stresses. Genotyping resulted in 158 and 153 successful single 

nucleotide polymorphisms (SNPs) for P. monticola and P. strobus, respectively. Each set of SNPs was 

genotyped on rangewide samples of 362 P. monticola individuals (61 populations) and 840 P. strobus individuals (133 populations). Analyses were conducted separately in each of the two species. STRUCTURE 

analyses identified two genetics clusters in each species, corresponding to north-south genetic discontinuities, as well as significant hierarchical substructure within each of those clusters. The use of FST outlier 

analyses revealed that about 6 percent and about 9 percent of SNPs were under selection in P. monticola

and P. strobus, respectively. The use of different methods of analysis to detect environmental associations 

revealed that about 16 to 22 percent of P. monticola SNPs and about 15 to 30 percent of P. strobus SNPs 

were significantly associated with one or more climate variables. We looked for strong candidates by combining the results from all FST outlier and environmental association analyses. About 4 percent and about 

5 percent of the SNPs in P. monticola and P. strobus, respectively, were detected by at least two different 

methods, highlighting candidate SNPs for future adaptation studies in western and eastern white pine.
102 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Population Projections and Resistance Gene Frequencies in the 

Presence of White Pine Blister Rust: A Population Genetic Model

for High Elevation Five-Needle Pines

A.W. Schoettle1, *, J.G. Klutsch2, M.F. Antolin3, and S. Field3

1 USDA Forest Service, Rocky Mountain Research Station, Fort Collins, Colorado, USA

2 Colorado State University, Department of Agricultural and Resource Economics; and USDA Forest Service,

Rocky Mountain Research Station, Fort Collins, Colorado, USA

3 Colorado State University, Department of Biology, Fort Collins, Colorado, USA

* aschoettle@fs.fed.us

The slow growth and long generation time of the fiveneedle pines have historically enabled these trees to 

persist on the landscape for centuries, but without sufficient regeneration opportunities these same traits hinder the species’ ability to adapt to novel stresses such 

as the exotic disease white pine blister rust (WPBR). 

Increasing the frequency of resistance to WPBR is 

the foundation for options to sustain five-needle pine 

species in the presence of the pathogen Cronartium 

ribicola. The objective of management intervention 

in the high elevation ecosystems is to promote pine 

population resilience for many generations (Schoettle 

and Sniezko 2007; Schoettle et al. 2012). However, 

greater understanding of the regeneration cycle and the 

potential for increasing the frequency of resistance are 

needed. This is especially critical for the WPBR pathosystem as WPBR kills trees of all ages and therefore 

affects multiple stages of the regeneration cycle. 

We developed a population genetic model, parameterized for high elevation five-needle pines, to improve 

our understanding of pine population dynamics in the 

presence of WPBR and identify key processes that 

affect resistance gene frequencies over time (Field et 

al. 2012). This model allows us to address questions 

such as: (1) What frequency of resistance to WPBR 

is enough to sustain a population under different 

initial conditions? (2) What regeneration densities are 

sufficient to promote increased frequency of resistance 

over time? (3) What is the effect of competition on 

population sustainability and frequency of resistance 

over time? (4) During what state of infestation and 

impact by WPBR is intervention most effective? The 

matrix model includes six age classes (cohorts): seeds, 

primary seedlings (1–4 yrs old), secondary seedlings 

(5–20 yrs), saplings (21–40 yrs), young adults (41–90 

yrs), and mature adults (>90 yrs). The model includes 

nonlinear functions for the effects of competition 

(leaf area index) on germination and cone production. 

Population size, incidence of infection by WPBR, and 

frequency of a simply inherited dominant WPBR resistance gene by age class over time are all included in 

the model output. This presentation will demonstrate 

that regeneration is a stabilizing factor in populations 

and that how and when it is managed, in coordination 

with gene frequencies for resistance, will play an important role in the establishment of a new equilibrium 

for a host population and its ecosystems in the presence of a nonnative pest.

REFERENCES

Field, S.G.; Schoettle, A.W.; Klutsch, J.G.; [et al.]. 2012. 

Demographic projection of high-elevation white pines 

infected with white pine blister rust: a nonlinear disease 

model. Ecological Applications. 22: 166–183.
USDA Forest Service RMRS-P-76. 2018. 103

Schoettle, A.W.; Klutsch, J.G.; Sniezko, R.A. 2012. 

Integrating regeneration, genetic resistance and timing 

of intervention for the long-term sustainability of 

ecosystems challenged by non-native pests—A novel 

proactive approach. In: Sniezko, R.A.; Yanchuk, A.D.; 

Kliejunas, J.T.; [et al.], tech. coords. Proceedings of 

the fourth international workshop on the genetics of 

host-parasite interactions in forestry: Disease and 

insect resistance in forest trees. Gen. Tech. Rep. PSWGTR-240. Albany, CA: U.S. Department of Agriculture, 

Forest Service, Pacific Southwest Research Station: 

112–123. 

Schoettle, A.W.; Sniezko, R.A. 2007. Proactive intervention 

to sustain high elevation pine ecosystems threatened by 

white pine blister rust. Journal of Forest Research. 12: 

327–336.
104 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Genome Sequencing in Conifers: Implications for Breeding and

Gene Resource Management

D.B. Neale1, *

1 University of California, Davis, Department of Plant Sciences, Davis, California, USA

* dbneale@ucdavis.edu

Abstract—The genomes of conifers were only recently sequenced due to their extremely large size and 

complexity. With the advent of next-generation sequencing technologies and improved sequence assembly 

methods, the genomes of Norway spruce (Picea abies), white spruce (Picea glauca), and loblolly pine 

(Pinus taeda) were reported in the last year. These genome sequences begin to reveal the size and complexity of conifer genomes and the path of genome evolution these ancient species have followed. Of more 

practical value, the genome sequences now provide the necessary “roadmap” to apply modern genomic 

technologies for future breeding and gene resource applications. In this presentation, I will begin with a 

description of how the PineRefSeq team generated the genome sequence for loblolly pine, what we have 

learned about conifer genomes, and how this knowledge is being applied to sequencing of the sugar pine 

(Pinus lambertiana) genome, the first white pine genome to be sequenced.

GENOMIC RESOURCES
USDA Forest Service RMRS-P-76. 2018. 105

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Genomic Warfare in the Woods: Scaling Fusiform Rust Resistance 

From Molecules to Landscapes

J.M. Davis1, *

1 University of Florida, School of Forest Resources and Conservation, Gainesville, Florida, USA

 * jmdavis@ufl.edu

Abstract—Heteroecious rust fungi coevolve with two or more plant host species. The fusiform rust pathogen Cronartium quercuum f. sp. fusiforme (Cqf) incites rust symptoms on leaves of oak species (Quercus

spp., the telial host) and gall symptoms on stems of pine species (Pinus spp., the aecial host). Genome 

sequencing of Cqf and loblolly pine (P. taeda) enabled us to identify candidate genes for corresponding avirulence and resistance loci, respectively. Because allelic variation at these interacting loci enables 

prediction of disease incidence in the field, markers should be useful for understanding co-evolution of 

pathogen and host species. We also envision the future development of a molecular toolkit for directly 

assessing pathogen allelic diversity at avirulence loci in Cqf spore collections. This information could be 

used to guide breeding and deployment of corresponding resistance loci in pine plantings.
106 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Identifying the Genetic Basis of Partial Resistance on Sugar Pine 

(Pinus lambertiana) Under White Pine Blister Rust Infection 

A. Vázquez-Lobo1, 2, *, C. Vangestel1, 3, P.J. Martínez-García1, 2,

J.L. Wegrzyn1, 4, I. Calic1, J.D. Liechty1, and D.B. Neale1

1 University of California, Davis, Plant Sciences Department, Davis, California, USA

2 Universidad Nacional Autónoma de México, Instituto de Ecología,

Departamento de Ecología Evolutiva, Distrito Federal, México

3 Ghent University, Department of Biology, St. Pietersnieuwstraat, Gent, Belgium

4 University of Connecticut, Department of Ecology and Evolutionary Biology, Storrs, Connecticut, USA

* eva@ecologia.unam.mx

Abstract—Since its arrival in North America in the early 1900s, white pine blister rust (WPBR; causal 

agent: Cromartium ribicola) has infected and threatened natural populations of at least five white pine 

species. An important step toward learning how to control the rise of diseases is the identification of 

natural defense mechanisms of hosts. In genetic terms, plant resistance is classified as one of two types: 

the complete resistance or major gene resistance (monogenic) and the incomplete or partial resistance 

(polygenic). Both mechanisms have been documented in sugar pine (Pinus lambertiana) under WPBR 

infection, although their genetic basis remains unknown. To identify genes involved in pine partial resistance, a progeny of 1,000 individuals from a controlled pollination cross was exposed to inoculation of 

WPBR at Happy Camp Outplant Site (Klamath National Forest, California, USA). Phenotypes of young 

trees under infection were quantified by considering five different symptoms: normal active cankers (N), 

normal active blights (NBL), normal bark reaction (NBR), blights (BL), and bark reactions (BR). More than 

50 percent of trees showed no sign of infection, and the most frequent symptom (in about 30 percent of 

trees) was the presence of normal active cankers overlapping with other symptoms. There is no statistically 

significant correlation between occurrences of symptoms, indicating that each symptom could be related 

with different genetic responses. The 2 parents and 93 individuals from progeny, including healthy and 

highly infected trees, were selected for genotyping using a 384-single nucleotide polymorphism (SNP) chip 

(Golden Gate Assay, Illumina®, San Diego, California), covering more than 260 genes of general functions, 

stress response, and candidates for disease resistance. Deviation from the expected segregation ratio 

revealed five genes associated with survivorship in early developmental stages. Associations between 

genetic variation and the five symptoms were detected through distribution of the minor allele frequency 

in affected cases and the use of full model association tests, to recognize association of genotypes with 

symptoms. Twenty-seven SNPs in 23 genes were associated with different symptoms. Annotations of 

these genes indicate similitude to genes previously associated with disease response in angiosperms. 

Most associations were related with allele frequency, although evidence for heterozygous advantage was 

found for some SNPs. Associated genes indicate that different responses to infection are related with proteins involved in general defense mechanisms such as signal transduction (protein kinases), inhibition of 

pathogen growth (proteases), and synthesis of plant antibiotics such as phytoalexins (chalcone synthases). 

These results provide evidence of the cellular responses of pines to infections and may be evaluated for 

use in breeding programs based on gene-assisted selection. 
USDA Forest Service RMRS-P-76. 2018. 107

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Sequencing the Sugar Pine (Pinus lambertiana) Transcriptome

J.L. Wegrzyn1, *, P.J. Martinez-Garcia2, C.A. Loopstra3, K. Mockaitis4,

R. Famula2, and D.B. Neale2

1 University of Connecticut, Department of Ecology and Evolutionary Biology, Storrs, Connecticut, USA

2 University of California, Davis, Department of Plant Sciences, Davis, California, USA

3 Texas A&M University, Department of Ecosystem Science and Management, College Station, Texas, USA

4 Indiana University, Department of Biology, Bloomington, Indiana, USA

*

 jill.wegrzyn@uconn.edu

Abstract—The PineRefSeq project is responsible for genome and transcriptome sequencing of three conifer species. Sugar pine (Pinus lambertiana) will be the second pine and largest genome sequenced, with 

an estimated size of 34 Gb. Few genomic resources currently exist to assist in the assembly and annotation of this large, complex genome. To generate these resources and define the gene space, samples were 

obtained for 14 different tissue types including those categorized as vegetative, reproductive, and early 

development. Vegetative organs (needle, stem, and roots) from two individuals were subjected to a variety 

of abiotic stressors, including temperature stress (both extreme heat and cold), water stress (drought 

and flooding), and salinity. Additionally, a biotic stress in the form of white pine blister rust (WPBR; causal 

agent: Cronartium ribicola) inoculations was used to generate sequencing libraries from vegetative organs. 

Illumina® (San Diego, California) deep sequencing was conducted on all libraries in the form of HiSeq (150 

bp PE reads) and MiSeq (300 bp PE reads). To combat existing challenges in transcriptome assembly from 

short reads, a new strategy known as “Iso-Seq” was applied to six libraries. This methodology uses single 

molecule technology from PacBio® (Pacific Biosciences of California, Inc., San Mateo, CA) to generate 

full-length transcripts that can reach 6 Kb in length. De novo assembly of all libraries (Illumina and PacBio) 

will generate the full reference transcriptome. Error correction provided by the short reads will improve 

the accuracy of the long transcripts obtained with the Iso-Seq approach. This will be the first attempt to 

generate full transcripts with next-generation sequencing techniques in conifers. The improved reference 

will facilitate expression analysis as well as the ability to compare against needle transcriptomes generated 

in several other white pine species, including whitebark pine (P. albicaulis), limber pine (P. flexilis), and 

western white pine (P. monticola).
108 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Using Genomics Tools to Assess the Phylogeography of Whitebark 

Pine (Pinus albicaulis)

J.A. Gruhn1, * and B.A. Schaal1

1 Washington University in St. Louis, Department of Biology, St. Louis, Missouri, USA

*

 jenniferagruhn@gmail.com

Abstract—The particularly heterogeneous climate, geology, and ecology of northwestern North America 

have created complex evolutionary histories for species, generally explained by patterns of ancient vicariance or dispersal in combination with more recent colonization from glacial refugia. Studies aiming to 

resolve the phylogeography of species in this region have been hampered by the limitations of species 

sampling as well as crude or biased analyses based on few molecular markers. Here, we are resolving 

these issues by using genomics tools that capture thousands of informative genetic markers, to analyze 

the geographic and evolutionary history of whitebark pine (Pinus albicaulis). Whitebark pine has a relatively 

unique geographic range that spans the western parts of the United States and Canada, providing the 

potential to test, within a single and consistent analysis, several alternative phylogeographic hypotheses 

posed for the region. Using the high-throughput restriction digest-based sequencing method genotypeby-sequencing (GBS), we are assessing the genetic structure across ≥35 whitebark pine populations 

representing the range of the species. This analysis will aid in the identification of those populations, 

across the species’ geographic distribution, that are genetically unique and yet currently of conservation 

concern. Implementing species distribution modeling (ecological niche modeling) as an independent 

phylogeographic assessment, we are also constructing the historical geographic distribution of the species, 

during several points in time, to infer the influence of previous events on the species’ present-day genetic 

structure. Last, we are combining these phylogeographic data with predictions about the future range of 

whitebark pine, as it is affected by climate change and white pine blister rust (causal agent: Cronartium 

ribicola), to further identify the most threatened and yet genetically distinct populations across the species’ 

range.
USDA Forest Service RMRS-P-76. 2018. 109

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Targeted Capture Sequencing in Whitebark Pine (Pinus albicaulis) 

Reveals Rangewide Demographic and Adaptive Patterns Despite 

Challenges of a Large, Repetitive Genome

K. Nomi1, C. Scelfo-Dalbey1, E. Tracy1, T. Jennings2, R. Cronn2, and J. Syring1, *

1 Linfield College, McMinnville, Oregon, USA

2 USDA Forest Service, Pacific Northwest Research Station, Corvallis, Oregon, USA

*

 jsyring@linfield.edu

Abstract—Whitebark pine (Pinus albicaulis) covers an expansive range in western North America, 

and is considered a keystone species of subalpine environments. Whitebark is susceptible to multiple 

threats—climate change, white pine blister rust (causal agent: Cronartium ribicola), mountain pine beetle 

(Dendroctonus ponderosae), and fire exclusion—and is suffering significant mortality rangewide, prompting 

the tree to be listed as “endangered” both by the International Union for Conservation of Nature (IUCN) and 

by the Canadian Government. Conservation collections (in situ and ex situ) are being initiated to preserve 

the genetic legacy of the species. Reliable, transferrable, and highly variable genetic markers are essential 

for quantifying the genetic profiles of seed collections relative to natural stands, and ensuring the completeness of conservation collections. 

We evaluated the use of hybridization-based capture to enrich targeted genomic regions from the 30+ GB 

genome of whitebark, and to evaluate genetic variation across loci, trees, and geography. Probes were 

designed to capture 7,849 distinct genes, and screening was performed on 48 trees. Despite the inclusion 

of repetitive elements in the probe pool, the resulting dataset provided information on 4,452 genes and 32 

percent of targeted positions (528,873 sites), and we were able to identify 12,390 segregating sites from 47 

trees. Variation at single nucleotide polymorphism sites reveals strong geographic trends in heterozygosity and allelic richness, with trees from the southern Cascade and Sierra Nevada Ranges showing the 

greatest distinctiveness and differentiation. Our results show that targeted capture produces high-quality, 

codominant genotypes from the large genome of whitebark, and the resulting data can be readily integrated into management and gene conservation activities.
110 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Genetic Resources for Limber Pine (Pinus flexilis) and a Survey 

Focusing on Potentially Adaptive Single Nucleotide Polymorphisms

J.B. Mitton1, *, J. Wegrzyn2, S.L. Stowell1, M. Mitter1, L. Kueppers3, and D. Neale2

1 University of Colorado, Department of Ecology and Evolutionary Biology, Boulder, Colorado, USA

2 University of California, Davis, Department of Plant Sciences, Davis, California, USA

3 University of California, Merced, Sierra Nevada Research Institute, Merced, California, USA

*

 mitton@colorado.edu

Abstract—RNA-sequencing studies of limber pine (Pinus flexilis) and Engelmann spruce (Picea 

engelmannii) were conducted to identify single nucleotide polymorphisms (SNPs) in the Front Range of 

Colorado (USA). Each study was based on nine trees, five above treeline (3,353 m above sea level) on 

Niwot Ridge, and four trees near the lower limit of the local distribution (2,347 m above sea level), in the 

hopes of finding genetic markers with elevational clines in frequencies. Each study yielded more than 

10,000 highly reliable SNPs. Annotations produced identifications for about 70 percent of the contigs. 

Comparisons with other conifer species revealed that unidentified sequences had been reported for other 

species, so although the genes have not been identified, they appear to be functional. 

One of our objectives is to identify genes that play a role in site-specific adaptation, especially with respect 

to elevation. To increase the probability of choosing potentially adaptive genes, we have been screening for 

SNPs that change both the amino acid and its functional group (polar, nonpolar, acidic, basic); these sorts 

of changes are most likely to change the kinetic and physiological performance of the protein. Preliminary 

data will be presented on SNPs that change frequency along an elevational gradient in the Front Range of 

Colorado.
USDA Forest Service RMRS-P-76. 2018. 111

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Cross-Species Transferability of Microsatellites, and Genetic 

Diversity in Himalayan Five-Needle Pine (Pinus wallichaia)

and Chir Pine (P. roxburghii) Forests

H.S. Ginwal1, * and P. Chauhan1

1 Forest Research Institute – India, Division of Genetics and Tree Propagation, Uttaranchal, India

*

 ginwalhs@icfre.org; ginwalhs@rdiffmail.com

Abstract—Pine forests in India are found in inland forests at elevations ranging between 400 and 2,500 

m above sea level. Five species indigenous to India are chir pine (Pinus roxburghii), blue pine (P. wallichiana), Khasi pine (P. kesiya), Chilgoza pine (P. gerardiana), and Teriasserian pine (P. merkussi). Of these, 

P. roxburghii, P. wallichiana, and P. gerardiana are found in the Himalayan Mountains, whereas P. kesiya

and P. merkussi are indigenous to Assam and Myanmar. As part of our studies on population genetic 

analysis of P. wallichiana and P. roxburghii, we report here the results of detecting and testing microsatellites and genetic diversity in these species. Eighty simple sequence repeat (SSR) primers taken up from 

P. thunbergii, P. sylvestris, P. resinosa, P. taeda, P. merkussi, and P. densiflora were used for trans-specific 

amplification in P. wallichiana and P. roxburghii, which included 33 chloroplast and 47 nuclear microsatellite 

loci. In P. wallichiana, 26 primer loci showed positive amplification, revealing a transfer rate of 32.5 percent. 

Primers from P. thunbergii and P. sylvestris showed almost identical rates of transfer of 75.0 percent and 

76.9 percent, respectively. A transfer rate of just 3.1 percent was observed from P. taeda whereas no 

transfer was seen from P. resinosa, P. merkussi, and P. densiflora. In P. roxburghii, very high transfer rates 

of primer pairs were observed from P. thunbergii (95 percent), P. sylvestris (100 percent), and P. merkussi

(80 percent). 

By using 12 chloroplast SSR (cpSSR) markers, 18 populations of P. wallichiana from the states of 

Uttarakhand, Himachal Pradesh, and Jamu and Kashmir, which together represent the natural range 

of distribution, were evaluated for genetic diversity. A maximum of 6 cpSSR alleles and 61 cpSSR size 

variants were found among 18 populations. The total gene diversity ranged from 0.45 to 0.68, which is 

moderate in comparison to other gymnosperms. The highest level of gene diversity was concentrated in 

populations from Himachal Pradesh. Similarly, 55 populations of P. roxburghii along the distribution range 

in the Himalayas were studied with 14 cpSSR markers. A maximum of 9 cpSSR alleles and 81 cpSSR size 

variants were found among 55 populations. The highest level of haplotypic diversity was concentrated in a 

population from Kiarala, Himachal Pradesh. The cpSSRs suggest that P. roxburghii maintains a high level 

of diversity (mean h = 0.65). Estimated gene flow between the populations was moderate in P. wallichiana

(Nm = 0.43) and P. roxburghii (Nm = 0.58). Most of the variation resided within populations in both the 

species. Results are interpreted in the context of conservation of genetic resources of P. wallichiana and 

P. roxburghii in the Himalayas.
112 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Using Transcriptomics to Identify Candidate Genes Associated with 

Blister Rust Resistance in Whitebark Pine (Pinus albicaulis)

Z. Bair1, *, R. Sniezko2, J. Wegrzyn3, H. Lintz4, and J. Stone1

1 Oregon State University, Department of Botany and Plant Pathology, Corvallis, Oregon, USA 

2 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA

3 University of Connecticut, Department of Ecology and Evolutionary Biology, Storrs, Connecticut, USA 

4 Oregon State University, Oregon Climate Change Research Institute, Corvallis, Oregon, USA

*

 bairz@science.oregonstate.edu

Abstract—Whitebark pine (Pinus albicaulis) is an iconic high elevation tree species currently threatened 

by climate change, mountain pine beetle (Dendroctonus ponderosae), and white pine blister rust, a 

lethal disease caused by the invasive fungal pathogen Cronartium ribicola that afflicts all North American 

white pines. A major goal in managing pine populations vulnerable to blister rust is identifying sources of 

heritable resistance that can be used in operational breeding programs. U.S. Department of Agriculture, 

Forest Service (USDA FS) breeding programs have identified major resistance genes in four other Pinus

species, but no such R gene is evident in whitebark pine. However, genetic variation in susceptibility to 

blister rust has been observed in naturally occurring populations. Although resistance phenotypes of whitebark pine differ from those in other pine species, certain seed parents are known to produce offspring that 

consistently develop fewer rust cankers compared to other seed parents under the same disease pressure, 

suggesting a genetic basis for partial resistance. 

In collaboration with the USDA FS Dorena Genetic Resource Center (Cottage Grove, Oregon, USA), germplasm was collected from more than 50 mother trees, representing whitebark pine populations throughout 

the Pacific Northwest. In particular, seedling families yielded from both Mount Rainier (Washington, USA) 

and Mount Hood (Oregon) appear especially promising and will be the primary focus of bioinformatic 

investigation. Three days after inoculation with blister rust, needle tissue was sampled and flash-frozen 

to preserve each individual's RNA expression profile. Half of the seedlings from each family served as 

a control group and were inoculated only after sampling. This sampling strategy permits comparisons 

between both experimental conditions, while still revealing the resistance phenotype of each individual 

tree seedling. By using transcriptomics to analyze differential expression between these resistant and susceptible individuals, candidate genes associated with blister rust resistance can be detected. Additionally, 

comparisons between control and inoculated groups can reveal whether the resistance is constitutive or 

induced. Furthermore, co-expression analyses can be used to characterize gene networks and infer potential mechanisms of resistance. Once candidate resistance genes are identified, markers can be developed 

to assist breeders in screening whitebark pine individuals for resistance.
USDA Forest Service RMRS-P-76. 2018. 113

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Genomics Study of Western White Pine (Pinus monticola)

Genetic Resistance Against White Pine Blister Rust

J.-J. Liu1, * and R.A. Sniezko2

1 Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, Victoria, British Columbia, Canada 

2 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA

*

 Jun-jun.liu@nrcan.gc.ca

Western white pine (WWP, Pinus monticola) is a 

long-lived conifer with an extensive geographic range 

in western North America. It is of high interest in ecological studies and forest breeding because of its high 

susceptibility to the invasive disease white pine blister 

rust (WPBR, caused by the fungus Cronartium ribicola). However, P. monticola lacks genomic resources 

and is evolutionarily far away from plants with available draft genome sequences. Use of high-throughput 

RNA-sequencing (RNA-seq) technology is a costeffective strategy to generate substantial transcriptome 

data for global transcript profiling and DNA marker 

discovery. We report here the RNA-seq analysis results 

using Illumina® (San Diego, California, USA) deep 

sequencing of P. monticola infected with C. ribicola. 

De novo gene assembly was used to generate the first 

P. monticola consensus transcriptome of primary needles, which contained 39,439 unique transcripts with 

an average length of 1,303 bp and a total length of 51.4 

Mb. About 23,000 Pinus orthologous genes (POGs) 

and 200 disease resistance gene analogs (RGAs) encoding NBS-LRR proteins were identified by BLAST 

search against the Pinus gene index database and plant 

R gene families, respectively. Comparison of transcriptomes from WPBR-susceptible and -resistant white 

pine genotypes revealed about 1,000 differentially 

expressed genes (DEGs) with statistical significance 

during early stages of the compatible and incompatible 

P. monticola–C. ribicola interactions. 

In silico single nucleotide polymorphism (SNP) detection identified more than 100,000 high quality SNPs 

in the above three groups of candidate genes (POGs, 

RGAs, and DEGs) by a bulked segregation-based 

RNA-seq analysis. To estimate efficiency of in silico 

SNP discovery, 432 SNPs were selected to develop genotyping assays using Sequenom® iPlex (Sequenom, 

San Diego, California) technology. About 70 percent 

of in silico SNPs were successfully genotyped and 53 

percent of the total SNPs were detected with predicted 

nucleotide variations in a collection of resistance 

germplasm. SNP clustering analyses consistently 

identified distinct populations, each composed of multiple full-sib seed families by parentage assignment. 

Linkage disequilibrium analysis identified 21 genes 

in significant association with major gene (Cr2) resistance. Genotyping verification by TaqManTM (Thermo 

Fisher Scientific, Waltham, Massachusetts, USA) 

qPCR indicated that at least one SNP locus provided 

an excellent marker for Cr2 selection among populations across western North America (Oregon and 

Washington [USA] and British Columbia [Canada]).

The validated SNP markers can be used as novel 

genomic resources for genetic, evolutionary, and 

ecological studies in P. monticola and related fiveneedle pines. Our results demonstrate that integration 

of RNA-seq-based transcriptome analysis and highthroughput genotyping is an effective approach for discovery of a large number of nucleotide variations and 

for identification of functional gene variants associated 

with adaptive traits in a non-model species.
114 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Genetic Markers for Western White Pine (Pinus monticola):

Enabling Molecular Breeding for Resistance to

White Pine Blister Rust

O.S. Urhan1, *, L. Magalska1, M.L. Rust2, V. Hipkins3, R.A. Sniezko4, A.S. Davis2,

M.F. Mahalovich5, J.-J. Liu6, and G.T. Howe1

1 Oregon State University, Department of Forest Ecosystems and Society, Corvallis, Oregon, USA

2 University of Idaho, Department of Forest, Rangeland and Fire Sciences, Moscow, Idaho, USA

3 USDA Forest Service, National Forest Genetics Laboratory, Placerville, California, USA

4 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA

5 USDA Forest Service, Northern, Rocky Mountain, Southwestern, and Intermountain Regions,

Forest Sciences Laboratory, Moscow, Idaho, USA

6 Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, Victoria, British Columbia, Canada

*

 oguz.urhan@oregonstate.edu

Abstract—Western white pine (Pinus monticola) is an economically and ecologically important conifer 

that has been severely affected by blister rust, a disease caused by the introduced fungal pathogen 

Cronartium ribicola. Because of this disease, the use of western white pine for reforestation is limited. 

The U.S. Department of Agriculture, Forest Service and others have resistance breeding programs to (1) 

identify the amount and type of genetic resistance in natural populations of western white pine and (2) 

develop durable disease resistance in breeding populations. These breeding programs can be enhanced 

by adopting advanced genomic technologies and new marker-assisted breeding approaches such as 

“breeding-without-breeding” (BWB) and “genomic selection.” The BWB approach allows breeders to 

pursue advanced-generation breeding without crossing—that is, using wind-pollinated mating designs that 

avoid time-consuming activities and associated costs of controlled crosses. Furthermore, it may be possible to identify genetically resistant trees by determining the parentage of trees growing in field plantations 

that have been established with seed orchard seed. By using single nucleotide polymorphic (SNP) genetic 

markers to infer parentage, breeders may be able to select resistant genotypes from extensive operational 

plantations, field experiments, and nursery trials established using wind-pollinated seedlots from rustresistant seed orchards. The success of BWB will depend on the characteristics of the (1) SNP markers 

(e.g., allele frequencies and genotyping success rate), (2) seed orchards (e.g., number and relatedness 

of the genotypes in the orchard), and (3) field and nursery tests (e.g., heritability of rust resistance traits). 

Our objectives are to (1) develop SNP genetic markers for western white pine, (2) use the SNP markers 

to conduct a proof-of-concept trial of BWB, and (3) transfer marker-assisted breeding technologies to tree 

breeders to accelerate resistance breeding and lay the foundation for genomic selection.
USDA Forest Service RMRS-P-76. 2018. 115

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Using Direct Amplification and Next-Generation Sequencing 

Technology to Explore Foliar Endophyte Communities in 

Experimentally Inoculated Western White Pines (Pinus monticola)

L.S. Bullington1, * and B.G. Larkin1

1 MPG Operations, LLC, Missoula, Montana, USA

*

 LBullington@MPGRanch.com

Abstract—Fungal endophytes can influence disease severity and survival of infected trees. Here we characterized the endophyte community in western white pine (Pinus monticola), an important species in the 

northwestern United States that has largely been decimated by pathogenic fungi. We also assessed the 

ability to successfully inoculate seedlings with desirable endophytes, with the long-term goal of providing a 

protective microbiome and added defense against pathogens. Pinus monticola seedlings were inoculated 

in the field with potential pathogen antagonists and fungi isolated from healthy mature trees. After inoculations, direct amplification and next-generation sequencing were used to characterize fungal endophyte 

communities, and explore interspecific competition, diversity, and co-occurrence patterns in needle tissues. 

Negative co-occurrence patterns between inoculated fungi and potential pathogens, as well as many other 

species, suggest early competitive interactions. Our study explores early endophyte community assemblage and shows that fungal inoculations may influence tree growth. 
116 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

On the Road to Mapping Quantitative Trait Loci of Morphological and 

Aggressiveness Traits in the Poplar Rust Fungus

M. Pernaci1, *, S. De Mita1, B. Fabre1, A. Andrieux1, F. Halkett1, and P. Frey1

1 Institut National de la Recherche Agronomique, Université de Lorraine,

Interactions Arbres–Microorganismes, Champenoux, France

*

 michael.pernaci@nancy.inra.fr

Abstract—Poplar (Populus deltoides) is an important tree species in Europe, both for the wood industry 

and for its increasing contribution to energy production systems. The main disease limiting the growth 

of poplar is poplar rust, caused by the basidiomycete Melampsora larici-populina (Mlp). The selection of 

poplar cultivars with qualitative resistance has led to repeated failures since 1980, due to the breakdown 

of each resistance type by the rise of new virulence factors. The search for more-durable resistance has 

led breeders to target quantitative resistance. Meanwhile, pathologists have focused on the potential for 

rapid evolution of several fungal quantitative life history traits (LHTs) related to aggressiveness or dispersal 

potential, or a combination thereof (Lannou 2012). We used a quantitative genetics approach to determine 

whether these traits are heritable and directly affect the fitness of the pathogen as these characteristics can lead to rapid changes in pathogen populations in response to selection pressures exerted by 

the host. Mapping the traits of interest requires the construction of a high-resolution genetic map of the 

fungus and the measurement of segregating traits in progeny. Using techniques developed by our group 

to allow completion of the sexual cycle of Mlp in the laboratory, we adopted a strategy of genetic mapping 

by the resequencing (Huang et al. 2009), based on about 145,000 single nucleotide polymorphisms, of 

96 offspring derived from selfing of the 98AG31 strain (reference genome, 462 scaffolds; Duplessis et al. 

2011). Our preliminary analyses have already brought together 81 scaffolds in 23 linkage groups. A single 

virulence trait against P. deltoides, 'L270 -3,' segregates in the progeny, making it a potentially mappable 

qualitative LHT. Concerning the segregation of quantitative LHTs, we observed a clear segregation of both 

morphological traits of urediniospores (length, width, volume and length/width ratio) and aggressiveness 

traits (latent period, infection efficiency, sporulation capacity, lesion size), which is encouraging for future 

mapping of quantitative trait loci. 
USDA Forest Service RMRS-P-76. 2018. 117

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Genomics, Transcriptomics, and Effectoromics of the Poplar Leaf 

Rust Fungus Melampsora larici-populina

B. Petre1, 2, 3, S. Hacquard1, 2, †, A. Persoons1, 2, N. Saveleva1, 2, E. Tisserant1, 2,

E. Morin1, 2, C. Delaruelle1, 2, J. Petrowski1, 2, M. Pernaci1, 2, B. Fabre1, 2, S. De Mita1, 2,

F. Halkett1, 2, A. Hecker1, 2, N. Rouhier1, 2, P. Frey1, 2, and S. Duplessis1, 2, *

1 Institut National de la Recherche Agronomique/Université de Lorraine Interactions

Arbres/Micro-organismes, Centre INRA Nancy Lorraine, Champenoux, France

2 Université de Lorraine/Université de Lorraine Interactions Arbres/Micro-organismes,

Faculté des Sciences et des Technologies, Vandoeuvre-lès-Nancy, France

3 Norwich Research Park, the Sainsbury Laboratory, Norwich, United Kingdom

† Current address: Max Planck Institute for Plant Breeding Research,

Department of Plant-Microbe Interactions,Cologne, Germany

*

 sebastien.duplessis@inra.fr

Melampsora larici-populina is causing leaf rust disease 

in poplar (Populus spp.) worldwide and poses serious 

problems to poplar breeders in France and in northern 

Europe. As with other rust fungi, its obligate biotroph 

status hinders laboratory investigations. Understanding 

the biology of rust fungi and deciphering the molecular 

mechanisms underlying biotrophy have long been a problem. Most of the knowledge gained was largely descriptive and limited to a few rust species; only recently has 

substantial progress been made in the characterization of 

avirulence determinants of the flax rust Melampsora lini. 

The pioneer sequencing of the genomes of the poplar 

rust fungus M. larici-populina and of the wheat stem 

rust fungus Puccinia graminis f. sp. tritici in 2011 has 

opened great perspectives for the molecular study of 

this group of fungi. Comparative analyses with recent 

reports have confirmed the typical profile of rust fungi 

genomes. These genomes are rather large (>89 Mb) 

and contain many repetitive and transposable elements 

(~50 percent of the genome) and a very large number of 

predicted genes (>16,000), including many large gene 

families of unknown function with no homology outside 

the Pucciniales. Within the genomes are several hundreds 

of genes encoding candidate effectors that are likely to 

be involved in the fungal virulence. These effectors are 

small secreted proteins specifically expressed in planta.

In the present talk, we will briefly introduce some basics 

about the poplar rust genome. We will provide an overview of our current knowledge gained by genome-wide 

oligoarrays and RNA-Seq on M. larici-populina gene 

expression at different stages of the complex lifecycle 

of this heteroic and macrocyclic rust, particularly during 

successful infection of the telial host (poplar) and of the 

aecial host (larch, Larix spp.). We will present our current 

strategy to characterize virulence effectors and illustrate a 

few candidates under investigation. Finally, we will provide an overview of ongoing genomics projects including 

the refinement of the genome assembly through the 

construction of a genetic map and a population genomics 

approach to identify the gene(s) responsible for a breakdown in the major gene resistance in poplar plantations.

ACKNOWLEDGMENTS

We acknowledge the support of public grants overseen 

by the French National Research Agency (ANR) as part 

of the “Investissements d’Avenir” program (ANR-11-

LABX-0002-01, Lab of Excellence ARBRE) and the 

Young Scientist Grant POPRUST to Sébastien Duplessis 

(ANR-2010-JCJC-1709-01) and by the Région Lorraine 

(Researcher Award to Sébastien Duplessis). We also 

acknowledge the support of poplar rust genomics by 

the U.S. Department of Energy Joint Genome Institute 

through the 2014 Community Science Program.
118 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Abstract—Improved resistance to comandra blister rust (causal agent: Cronartium comandrae) can 

improve lodgepole pine (Pinus contorta var. latifolia) survival and growth, but we lack information about 

resistance structure in this pathosystem. Incidence of comandra and other diseases was monitored annually in a 130-seedlot trial designed to both characterize the resistance structure in lodgepole pine and 

identify resistant families for inclusion in the tree improvement program in British Columbia, Canada. By 

10 years of age, incidence of new comandra infection had largely stabilized on all three trial sites, having 

reached 79, 70, and 22 percent, respectively. However, close proximity to the alternate host plant, bastard 

toadflax (Geocaulon lividum), resulted in an increased risk of infection by comandra, and relatively high 

family-level variation between sites. Although most families appeared to be highly susceptible, a few 

families exhibited a moderate level of resistance. Based on these results, it is recommended that future 

additional screening of a much larger number of families be conducted to identify a sufficient number of 

families for either augmenting existing orchards or establishing a comandra-resistant orchard.

Ten-Year Results of a Comandra Blister Rust Screening Trial in the 

Central Interior of British Columbia

R.W. Reich1, * and S. John2

1 British Columbia Ministry of Forests, Lands and Natural Resource Operations, Forest Health Program,

Omineca Region, Prince George, British Columbia, Canada

2 Isabella Point Forestry, Salt Spring Island, British Columbia, Canada

* reichr1@cnc.bc.ca

INTRODUCTION

Comandra blister rust (caused by Cronartium comandrae) is one of the most lethal diseases of young 

lodgepole pine (Pinus contorta var. latifolia) (van der 

Kamp and Spence 1987; Woods et al. 2000). It occurs 

throughout the Canadian province of British Columbia 

(BC), but is most common in the Sub Boreal Spruce 

(SBS) biogeoclimatic zone, and alternates between 

bastard toadflax (Geocaulon lividum) and lodgepole 

pine (Ziller 1974). Management options include 

increased composition of nonhost species in reforestation, but some of the colder, drier, or more nutrientpoor ecosystems may not be well suited for these other 

species (Reich and van der Kamp 1993). Increased 

planting density of lodgepole pine (proportional to 

risk) is practiced in some areas, but this can be costly 

where risk to comandra is very high (British Columbia 

Ministry of Forests 1996). Hazard rating for comandra 

(Jacobi et al. 1993) identified several key variables 

including proximity to alternate host. Numerous other 

authors previously reported that infection is typically 

found in close proximity to alternate hosts (Andrews 

and Harrison 1959; Applegate and Seay 1971; Cordell 

et al. 1969). Although resistance to comandra has not 

been thoroughly investigated in the past, a formal disease assessment of a BC Ministry of Forests progeny 

field test in 2000 showed significant and large differences in family infection rates. Based on this result, a 

field trial was designed specifically to test comandra 

resistance of genotypes established in the Bulkley 

Valley low-elevation seed orchard (Orchard 219).

RUST RESISTANCE
USDA Forest Service RMRS-P-76. 2018. 119

METHODS

Three sites within established pine plantations in 

the central interior of BC were selected based on 

high incidence of comandra, and abundant cover of 

Geocaulon lividum, an alternate host of comandra. 

Each 2-ha site (about 100 m × 200 m) was prepared 

in fall 2003 by mechanically mulching the existing 

trees. In June 2004, seedlings from 130 seedlots were 

planted on a 1.5-m-square grid, in single tree plots, 

with 50 blocks per site (Alpha design). Most seedlots 

(hereafter referred to as “families”) consisted of 50 

seedlings, typically from open-pollinated families, 

though several controlled-cross families were included 

to evaluate specific putative resistant families. A total 

of 7,440 seedlings, including internal buffer seedlings, 

were monitored.

Two sites, Endako and Thompson, were assessed 

starting in 2006, 24 months after planting, and the 

third site, Holy Cross, was assessed starting in 2007, 

36 months after planting. Detailed annual assessments 

were continued through 2014, representing 10 full 

growing seasons in the field. Disease assessments in 

2006 through 2008 consisted of recording tree condition, and counting the number of individual infections 

(severity) by comandra, as well as by western gall 

rust (causal agent: Endocronartium harknessii), and 

stalactiform blister rust (causal agent: Cronartium 

coleosporioides). From 2009 to 2014, the tree condition and only the occurrence (not the count of infections) of a new rust species on a tree were recorded. 

Starting in 2012, an additional form or severity was 

also recorded to facilitate rust impact modeling. For all 

rusts, either the estimated percent stem encirclement 

(for stem-infected trees), or the distance of branch 

infections from the stem (for branch-only infected 

trees), was recorded for the most lethal-appearing stem 

or branch infection per rust per tree. In 2007, a count 

of the number of G. lividum stems was recorded within 

each 1.5-m × 1.5-m tree cell to evaluate the source of 

spread for comandra. A geographic information system 

(GIS) exercise was later used to calculate the distance, 

in 1-m intervals, from each tree to the closest G. lividum plant for determining the risk of infection based 

on proximity to the alternate host plant. For evaluating 

the source of spread of stalactiform, the presence 

of common red paintbrush (Castilleja miniata) was 

also assessed within the 1.5-m grid on all three sites 

in 2007. For stalactiform, the presence and general 

abundance of both yellow rattle (Rhinanthus minor) 

and cow wheat (Melampyrum lineare) were also noted 

at the site level.

RESULTS

Comandra Occurrence by Site

Although the three sites were selected to maximize 

risk to comandra infection, only two of the three sites 

were infested with a sufficiently high incidence to allow reliable screening for comandra resistance (fig. 1). 

The two sites with the highest infection, Endako 

(79-percent incidence of infection) and Thompson 

(70-percent incidence), were located about 12 km 

apart. They apparently had very similar weather conditions, based on the similarity of the timing and incidence of comandra on an annual basis. The third site, 

Holy Cross, which was more than 100 km southeast of 

the other two sites, recorded significantly less comandra (22-percent incidence), and its timing was very different from that of the other two sites. By 2006, both 

of the western sites had about 35-percent comandra, 

with close to 0 percent noted at Holy Cross. By 2007, 

both of the western sites had about 50-percent cumulative comandra incidence, compared to only 2 percent 

at the Holy Cross site. Given that it takes 2 to 3 years 

for infections to become noticeable, this difference 

probably indicates very different weather, both in 2004 

and 2005, between these two geographic regions. The 

incidence of comandra at all three sites had apparently 

stabilized by age 10, with very little new infection occurring. The vast majority of infections by comandra 

on lodgepole pine in BC are typically restricted to a 

zone less than 0.5 m above ground, so it is reasonable 

to assume that the lack of new infections is related to 

the development of age-based resistance. 

Ranking Resistance to Comandra

The primary interest in this trial is the structure of resistance in the lodgepole pine population, and whether 

there are any resistant families that can be used to 

increase the resistance of the improved tree orchards 

for BC. Sorting families by increasing comandra 

incidence at the Thompson site allows a systematic 

evaluation of the family variation due to site (fig. 2). 

After 10 growing seasons, the vast majority of the 
120 USDA Forest Service RMRS-P-76. 2018.

0 

0.1 

0.2 

0.3 

0.4 

0.5 

0.6 

0.7 

0.8 

0.9 

1 

Comandra incidence (%) 

Seedlot 

2014 Thompson 2014 Endako 

Figure 2—Cumulative mean family comandra incidence at Thompson and Endako, sorted by Thompson 

comandra incidence.

0 

10 

20 

30 

40 

50 

60 

70 

80 

90 

100 

2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 

Comandra bister rust incidence (%) 

Thompson 

Endako 

Holy Cross 

Figure 1—Cumulative comandra incidence at all three sites monitored annually.
USDA Forest Service RMRS-P-76. 2018. 121

families appear to be highly susceptible, with only a 

few showing somewhat lower comandra incidence. 

Especially given the large number of families in this 

trial, this result may suggest that there exists very low 

natural resistance in the lodgepole pine population as a 

whole. This would be reasonable to expect, especially 

if most of the pine population is not normally exposed 

to challenge by comandra on a regular basis.

The difference between the moderately resistant families and the highly susceptible ones is more distinct 

when comparing the severity of infection (count of 

infections per tree) rather than the mean incidence at 

the family level (Feng et al. 2013). The most susceptible families may be almost twice as susceptible as the 

least susceptible when basing the comparison on incidence, whereas the most susceptible families are close 

to six times more susceptible when using severity as 

the basis for comparison. 

Although variability in family-level comandra incidence between sites should have been minimized 

through the robust trial design (50 open-pollinated 

seedlings planted in single-tree plots within 50 blocks 

per site), the family rankings varied considerably 

between sites. A correlation R2 calculated annually 

for the two most highly infested sites never rose much 

higher than about 0.50. This would generally indicate 

that only about 50 percent of the variability was accounted for by genotype, which is considerably lower 

than expected for this type of study.

Spatial Correspondence of Comandra and its 

Alternate Host

At an early stage in the monitoring of this trial it 

became apparent that the highly clustered distribution 

of comandra incidence may be related to the highly 

clustered spatial distribution of the alternate host, and 

that this factor may affect the outcome of ranking 

families for resistance. In particular, ranking families 

using the average family incidence of all sites may 

introduce error, as trees within the same site may be 

exposed to dramatically different levels of challenge. 

Combining trees that are challenged at a low level with 

trees challenged at a high level is unlikely to yield reliable rankings, unless their proportions are similar. 

Figure 3—Cumulative comandra incidence by proximity to alternate host for all three sites, monitored annually.

0% 

10% 

20% 

30% 

40% 

50% 

60% 

70% 

80% 

90% 

100% 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 

Comandra incidence (%) 

Distance from tree to the nearest Geocaulon lividum plant (m) 

Thompson 2014 

Thompson 2013 

Thompson 2012 

Thompson 2011 

Thompson 2010 

Thompson 2009 

Thompson 2008 

Thompson 2007 

Thompson 2006 

Endako 2014 

Endako 2013 

Endako 2012 

Endako 2011 

Endako 2010 

Endako 2009 

Endako 2008 

Endako 2007 

Endako 2006 

Holy Cross 2014 

Holy Cross 2013 

Holy Cross 2012 

Holy Cross 2011 

Holy Cross 2010 

Holy Cross 2009 

Holy Cross 2008 

Holy Cross 2007 
122 USDA Forest Service RMRS-P-76. 2018.

A visual inspection of the mapped distribution and 

intensity of comandra incidence, in relation to the 

distribution and clustering of the alternate host, exhibited a distinct spatial correspondence on all three 

sites. Results of using the GIS technique to display the 

cumulative risk of infection by comandra in relation 

to the proximity to the nearest alternate host plant 

at each annual measurement show a sharp decrease 

in risk over a very short distance from the alternate 

host (fig. 3). The shape of this function appears to be 

relatively independent of the incidence or the site. This 

proximity relationship is explored in greater detail 

through the development of a flexible semi-parametric 

generalized additive model to reduce ranking error 

(Feng et al. 2013). This approach was used in an 

expanded, but unpublished, study by the same authors 

to more reliably rank families by modeling the effect 

of proximity to alternate host, abundance of alternate 

host, and other factors such as site topographic effects 

(e.g., slope, elevation, and aspect).

Other Rusts and Forest Health Factors

Several observations were made regarding the other 

two rusts. Western gall rust occurred at a lower than 

expected frequency and incidence on all three sites. 

Stalactiform blister rust occurred at a much higher 

than expected incidence at only one of the three sites. 

Stalactiform incidence at the Endako site appeared 

to follow a rapid and widespread invasion in 2004 or 

2005 by R. minor, a known transient annual (Westbury 

2004). Rhinanthus minor was not observed until 

2010 at the Holy Cross site and not until 2012 at the 

Thompson site. Although C. miniata was highly clustered and somewhat abundant on all three sites, it did 

not appear to result in distinctly clustered stalactiform 

infection. It may not be as effective an alternate host 

for stalactiform as R. minor, though the trial was not 

designed to evaluate this factor. Melampyrum lineare

was present only on the Holy Cross site, and was 

very abundant and widespread. It did not appear to 

cause widespread stalactiform on pine, though the 

weather may not have been suitable for infection by 

stalactiform.

Trial Design, Assessment Schedule, and Tree 

Condition

The results of this trial demonstrated the importance 

of careful site selection, the need for replication of 

test sites, and the dependence on suitable weather for 

successful screening using field trials. This trial also 

demonstrated the need for detailed annual monitoring 

given both the rapidly changing annual rate of new infection, and the rapid rate of mortality of trees infected 

at a very young age. Further, it demonstrated the value 

of mapping the alternate host distribution in climates 

where spatial infection gradients may develop over 

very short distances from the alternate host.

CONCLUSIONS

Detailed annual monitoring and subsequent analysis 

have shown that after 10 growing seasons, the comandra incidence has both stabilized and reached an 

adequate threshold for reliable screening on two of 

three sites. Although very few families demonstrated 

resistance in relation to the large number of families 

tested, this result may possibly indicate that the 

lodgepole pine population as a whole may contain a 

relatively low level of resistance to an endemic pathogen. Screening of additional lodgepole pine families 

to further bolster the number of comandra-resistant 

families is highly recommended.

REFERENCES

British Columbia Ministry of Forests. 1996. Pine stem rust 

management guidebook. Forest Practices Code of British 

Columbia. Victoria, BC. https://www.for.gov.bc.ca/tasb/

legsregs/fpc/fpcguide/pinestem/pine-toc.htm [Accessed 

March 13, 2017].

Andrews, E.A.; Harrison, M.D. 1959. Cronartium 

comandrae in Wyoming. Plant Disease Reporter. 43: 

418–419.

Applegate, H.W.; Seay, T. 1971. Two new hosts for 

Comandra blister rust. Plant Disease Reporter. 55: 697.

Cordell, C.E.; Applegate, H.W.; Huffman, P.J. 1969: Pond 

pine, a new host for comandra blister rust in eastern 

Tennessee. Plant Disease Reporter. 53: 694.
USDA Forest Service RMRS-P-76. 2018. 123

Feng, C.X.; Dean, C.B.; Reich, R. 2013. Impact of 

misspecifying spatial exposures in a generalized additive 

modelling framework with application to the study of the 

dynamics of comandra blister rust in British Columbia. 

Environmetrics. 24(2): 63–80.

Jacobi, W.R.; Geils, B.W.; Taylor, J.E.; [et al.]. 1993. 

Predicting the incidence of comandra blister rust on 

lodgepole pine: Site, stand and alternate host influences. 

Phytopathology. 83:630–637.

Reich, R.W.; van der Kamp, B.J. 1993. Frost, canker, and 

dieback of Douglas-fir in the central interior of British 

Columbia. Canadian Journal of Forest Research. 23(3): 

373–379.

van der Kamp, B.J.; Spence, M. 1987. Stem diseases of 

lodgepole pine in the British Columbia interior following 

juvenile spacing. The Forestry Chronicle. 64: 334–339. 

Westbury, D.B. 2004. Rhinanthus minor L. Journal of 

Ecology. 92: 906–927. 

Woods, A.J.; Nussbaum, A.; Golding, B. 2000. Predicted 

impact of hard pine stem rusts of lodgepole pine 

dominated stands in central British Columbia. Canadian 

Journal of Forest Research. 30: 476–481.

Ziller, W.G. 1974. The tree rusts of western Canada. Publ. 

1392. Victoria, BC: Environment Canada, Forestry 

Service, Pacific Forest Research Centre. 272 p.
124 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

White pines exhibit both qualitative and quantitative 

resistance to white pine blister rust, which is caused 

by the fungal pathogen Cronartium ribicola (Sniezko 

et al. 2014). Species from several western U.S. States 

have a major gene at low frequencies that confers 

virtual immunity to the disease; and most also show 

heritable quantitative variation in infection frequency, 

as well as the ability to abort or heal infections that do 

occur (Kinloch et al. 2012). Here, we summarize extensive data from different tests, some previously published, showing the magnitude and pattern of variation 

in both mechanisms throughout the geographic range 

of sugar pine (Pinus lambertiana). We then discuss 

how this variation could be developed and deployed 

to effect durable resistance in synthetic populations of 

this ecologically and commercially important species.

The frequency of Cr1, the major gene for resistance 

in sugar pine, was assayed over many years following 

established protocols (Kinloch 1992). Open-pollinated 

seedlings from more than 15,000 seed parents in 50 

seed zones were analyzed, covering most of the range 

of the species across more than 14 degrees of latitude 

from northern Oregon to Baja California (USA). 

Seedlings identified with Cr1 in these assays were 

outplanted at a test site near Happy Camp in northern 

Patterns of Variation in Blister Rust Resistance in

Sugar Pine (Pinus lambertiana)

B.B. Kinloch, Jr.1, *, R.A. Sniezko2, D.P. Savin2, R. Danchok2, A. Kegley2,

D. Burton3, and J. Dunlap4

1 USDA Forest Service, Pacific Southwest Research Station (retired) 

2 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA

3 USDA Forest Service, Pacific Southwest Region, Placerville Genetic Resources Unit (retired)

4 USDA Forest Service, Pacific Southwest Region, Placerville Genetic Resources Unit, Placerville, California, USA

* bkinloch@earthlink.net

California for a secondary screen for partial resistance 

(also called slow rusting resistance) (Kinloch et al. 

2012). Development of different kinds of resistance 

was needed as defense against the possibility of 

virulence to Cr1 becoming widespread. At this site, a 

mutant virulent allele of the rust (vcr1, cognate to Cr1) 

had already arisen and become predominant as a result 

of selection by Cr1 host genotypes concentrated on the 

site. With Cr1 neutralized, different forms of partial 

resistance could become expressed and assessed, thus 

identifying individual trees with both Cr1 and partial 

resistance. 

Field resistance was examined separately on an extensive collection of 64 seed sources, covering approximately the same range as the Cr1 assay, planted at 

mid-elevation in the central Sierra Nevada (Kitzmiller 

2004; fig. 1). Major subregions represented included 

western Oregon/Cascades, Klamath Mountains, 

eastern Cascades, North Coast, northern and southern 

Sierra Nevada, South Coast, Transverse and Peninsula 

Ranges, and the Sierra San Pedro Martir. Three other 

sites planted with most of the same sources were also 

examined. Traits recorded were number of infections 

per tree, and whether the infections were normal 
USDA Forest Service RMRS-P-76. 2018. 125

or reactive; reactive responses show bark or blight 

responses to infection, leading to partial or complete 

remission of disease symptoms (Kinloch et al. 2012). 

Mean frequency of Cr1 was 0.0208 (range: 

0.0000–0.0886) (Kinloch 1992; J. Dunlap, unpublished data on file at USDA Forest Service, Pacific 

Southwest Region, Placerville Genetic Resources 

Unit, Placerville, California). Very low frequencies 

in northern latitudes increased almost clinally to the 

south, peaking around 0.06 in the southern Sierra, 

before dropping again in the Transverse and Peninsula 

Ranges and becoming undetectable at the southern 

extremity of sugar pine in the Sierra San Pedro Martir 

in Baja, Mexico (fig. 2). Cr1 frequency was negatively 

correlated (–0.62) with latitude. Several exceptions to 

this pattern were evident, including an anomalous high 

value (0.0886) in an outlier population on the south 

coast of California. 

Challenge with blister rust was severe on three of the 

four field test sites, ranging from 84 to 92 percent 

of trees infected. Correlations of seed sources with 

infection among the sites were positive and generally 

high, but due to low survival and maintenance problems on other sites, only the site with the most seed 

sources, good survival, and highest overall infection 

(Fitch Rantz) (Kitzmiller 2004) is considered further. 

Percentage of trees infected among seed sources on 

this site ranged from 72 to 98, and was the reverse 

pattern of Cr1, with a highly significant (0.74) correlation with latitude. A much more sensitive index of 

susceptibility was infection frequency (counts of separate infections per tree), which ranged from 0 to over 

100 for individual trees, and averaged from 3 to 29 

among seed sources (fig. 3). No clear pattern emerged, 

but when seed sources were grouped over subregions, 

extreme northern and southern sources tended to have 

fewer, and Sierra Nevada sources more, infections 

(fig. 4). 

A derivative parameter of individual infection counts 

is the proportion of trees with no active infections 

(NAI), that is, trees with complete remission of symptoms (resulting from bark, blight, or other reactions), 

or those that were never infected. This parameter is 

perhaps the clearest representation of partial resistance, as it integrates the several different components 

into one index (Kinloch et al. 2012). Mean NAI was 

almost 5 percent (range: 1.5–13 percent; fig. 5). Again, 

there was no obvious geographic pattern over seed 

zones or broader subregions, but all seed sources have 

abundant trees available for selection for this trait. 

The generally wide distribution and strong inheritance 

of both major gene and quantitative resistances provide 

a clear and compelling strategy for developing durable resistance. Cr1 is nearly totipotent, and protects 

against all ambient variability in the rust population 

except genotypes carrying the vcr1 allele. Although 

Cr1 is specifically (and completely) vulnerable to this 

allele, partial resistance is not; it is no more vulnerable to vcr1 than wild type inoculum. Consequently, 

Figure 1—Locations of seed sources and plantations for 

sugar pine provenance trials.
126 USDA Forest Service RMRS-P-76. 2018.

Figure 2—Frequency of Cr1 in different seed zones of sugar pine. (No data for zones colored red.)

Figure 3—Average number of blister rust infections per tree in different seed zones of sugar pine. (No data 

for zones colored red.)
USDA Forest Service RMRS-P-76. 2018. 127

Figure 4—Average number (horizontal lines) and range (solid bars) of blister rust infections per tree in broad 

sub-regions of sugar pine, arrayed by latitude (WOC, western Oregon and Cascades; EC, eastern Cascades; KM, 

Klamath Mountains; NSN, northern Sierra Nevada; NC, north coastal; SSN, southern Sierra Nevada; SC, south 

coastal; T-P, Transverse and Peninsula Ranges; SSPM, Sierra San Pedro Martir.

Figure 5—Selection ratio: average number of trees with no active infections (NAI), by seed zones of sugar pine. 

(No data for zones colored red.)
128 USDA Forest Service RMRS-P-76. 2018.

tandem selection of Cr1 with partial resistance can effectively restrict both rate and quantity of infection by 

all inocula. This would greatly lower the probability 

of infection by vcr1 (already rare in natural populations). The two resistances are mutually reinforcing, 

and developed together in synthetic populations could 

conceivably reduce the amount of disease to endemic 

levels in a few generations of selection and breeding.

ACKNOWLEDGMENTS

A U.S. Department of Agriculture, Forest Service 

(USDA FS) Forest Health Monitoring grant (WCF-09-01) provided partial financial support for the 

latest (2009) assessment of the four field trials. The 

assistance of USDA FS personnel from Regions 5 and 

6 and from the Pacific Southwest Research Station in 

assessing the various trials is gratefully acknowledged. 

REFERENCES

Kinloch, B.B., Jr. 1992. Distribution and frequency of a 

gene for resistance to white pine blister rust in natural 

populations of sugar pine. Canadian Journal of Botany. 

70: 1319–1323.

Kinloch, Bohun B., Jr.; Burton, Deems; Davis, Dean A.; 

[et al.]. 2012. Strong partial resistance to white pine 

blister rust in sugar pine. In: Sniezko, R.A.; Yanchuck, 

A.; Kliejunas, J.; [et al.], tech. coords. Proceedings 

of the fourth international workshop on the genetics 

of host-parasite interactions in forestry: Disease and 

insect resistance in forest trees. Gen. Tech. Rep. PSWGTR-240. Albany, CA: U.S. Department of Agriculture, 

Forest Service, Pacific Southwest Research Station: 

80–91.

Kitzmiller, J. 2004. Adaptive variation in sugar pine. In: 

Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott 

E.; [et al.], eds. 2004. Breeding and genetic resources 

of five-needle pines: Growth, adaptability and pest 

resistance. IUFRO Working Party 2.02.15. Proceedings 

RMRS-P-32. Fort Collins, CO: U.S. Department of 

Agriculture, Forest Service, Rocky Mountain Research 

Station: 103–123.

Sniezko, R.A.; Smith, J.; Liu, Jun-Jun; [et al.]. 2014. 

Genetic resistance to fusiform rust in southern pines and 

white pine blister rust in white pines—A contrasting 

tale of two rust pathosystems—Current status and future 

prospects. Forests. 5(9): 2050–2083; doi:10.3390/

f5092050.
USDA Forest Service RMRS-P-76. 2018. 129

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Blister Rust Resistance in Whitebark Pine (Pinus albicaulus)—Early 

Results Following Artificial Inoculation of Seedlings from Oregon, 

Washington, Idaho, Montana, California, and British Columbia Seed 

Sources

R.A. Sniezko1, *, A. Kegley1, R. Danchok1, and S. Long1

1 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA

* rsniezko@fs.fed.us

Abstract—Whitebark pine (Pinus albicaulis) is very susceptible to white pine blister rust, caused by the 

fungal pathogen Cronartium ribicola. Operational programs to develop resistant populations of whitebark 

pine are underway. These programs rely on artificial inoculation of young seedlings and can evaluate 

hundreds of parent trees using seedling progeny in a relatively short time. The trial reported here was 

undertaken at Dorena Genetic Resource Center (Cottage Grove, Oregon, USA). Inoculation was very 

effective and produced a high incidence and severity of disease in the seedlings with 99.9 percent of 

seedlings exhibiting needle spots and averaging more than 125 needle spots per seedling 9 months postinoculation (mpi), and more than 11 stem symptoms per seedling and over 77 percent of seedlings with 

stem symptoms approximately 15 mpi. Overall, 47 of the 225 families had 100 percent of seedlings with 

stem symptoms 15 mpi. In this summary, we examine the variation among seed sources in one measure 

of rust resistance, the percentage of seedlings with early stem infections. The results showed large differences among populations from Oregon, Washington, Idaho, Montana, and California (USA), and British 

Columbia (Canada). The highest levels of resistance were observed in some seed sources from Oregon 

and Washington, with some sources showing less than 60 percent stem infection. The two Idaho and 

Montana seed lots that had been rated moderately rust-resistant in previous testing elsewhere were among 

the most susceptible in this trial, with 92.4 percent of seedlings with stem symptoms approximately 15 mpi. 

This was very likely due to lower effective inoculation in the prior test.

Caution is warranted in extrapolating results to the field, especially when the effective inoculation level is 

relatively low and suboptimal, but the best candidates, to date, are likely to be those from trials such as 

this one with a very high effective level of inoculation. However, repeated testing of a subset of seed lots 

is warranted to improve confidence. Long-term field testing, with a susceptible control, is also essential to 

establish the relationship between the seedling screening assays as well as to examine durability of resistance. Field trials, with susceptible controls, have been established in Oregon and are just starting to show 

rust infection. Land managers have been notified of the highest ranking parents, and seed from these can 

be used in restoration efforts or to establish seed orchards. A synthesis of results underway from the full 

5-year post-inoculation data from this trial and several other trials at Dorena Genetic Resource Center will 

provide further insights on genetic resistance to white pine blister rust.
130 USDA Forest Service RMRS-P-76. 2018.

INTRODUCTION

Whitebark pine (Pinus albicaulis), a long-lived conifer 

in high elevation ecosystems of the western United 

States and Canada, is highly susceptible to white pine 

blister rust, caused by the nonnative fungal pathogen 

Cronartium ribicola. Developing populations of trees 

with genetic resistance to this pathogen has been an 

essential strategy for reforestation in species such as 

western white pine (P. monticola) and sugar pine (P. 

lambertiana) (King et al. 2010; Kinloch et al. 2012; 

McDonald et al. 2004; Sniezko 2006; Sniezko et al. 

2014). In many areas, restoration of whitebark pine 

is being done or is being contemplated (Beck and 

Sniezko, this proceedings), and the use of rust-resistant 

whitebark pine is seen as the key to success in many 

areas. Several programs to develop resistant populations of whitebark pine in the United States and British 

Columbia (Canada) are underway, and similar plans 

are being contemplated in Alberta (Canada). Until relatively recently, little was known about the components 

of genetic resistance and their frequency in whitebark 

pine, but some detailed reports of results in the first 

few years post-inoculation are now available (Kegley 

et al. 2012; Sniezko et al. 2007). Many aspects of 

blister rust resistance in whitebark pine are similar to 

those observed in western white pine (see review in 

Sniezko et al. 2014).

Tens of thousands of whitebark pine seedlings 

have been evaluated for rust resistance at the U.S. 

Department of Agriculture, Forest Service, Dorena 

Genetic Resource Center (DGRC; Cottage Grove, 

Oregon, USA; 45.78°N, 122.96°W, 244.1 m above sea 

level). These resistance screening trials have provided 

insights into the levels of the natural genetic resistance, 

some of its components (Sniezko et al. 2011), and an 

indication of the geographic distribution of resistance, 

as well as some caveats on extrapolation of results. 

Each study is monitored for 5 years post-inoculation; 

however, in this overview we will focus on the first 

15 months post-inoculation (mpi) for one of the large 

recent studies and present results on differences among 

seed sources in the percentage of seedlings with stem 

symptoms (normal cankers and bark reactions).

METHODS

This report provides a partial summary of early results 

from a seedling trial (SY2007R4) involving 225 halfsib families in which the level of effective inoculation 

was extremely high (99.9 percent needle spotting). The 

artificial inoculation used standard DGRC protocols, 

which have been used with success for greater than 20 

years (see Sniezko et al. 2011 for some details). The 

trial included 14 half-sib seedling families previously 

rated as “highly susceptible” or “highly resistant” 

to serve as controls and to examine repeatability of 

results over years.

The trial was sown in 2007 and inoculated in 

September 2008, as 2-year-old seedlings. See Hamlin 

et al. (2011) for some background on the trial, including geographic sources for the 215 Oregon and 

Washington families; the additional families from 

Idaho and Montana (2), California (3), and British 

Columbia (5) were not included in the height and foliage color summary by Hamlin et al. (2011). The 18 

Oregon and Washington populations are represented 

by families from different national forests, national 

parks, and Confederated Tribes of Warm Springs (1 to 

42 families per population).

RESULTS AND DISCUSSION

The spore density used in artificial inoculation can 

elicit varying results, depending on the pre- and postinoculation environment of seedlings and other factors. 

The effectiveness of the inoculation can be discerned 

by examining key factors such as the number of needle 

spots on a seedling, the percentage of seedlings with 

needle spots, and the number of stem infections on 

each seedling (most notably on susceptible controls). 

In this trial, the four susceptible control families had 

96.7 to 100 percent of seedlings with stem symptoms 

and 9.3 to 18.6 stem infections per seedling. Figures 

1, 2 and 3 show the high level of needle spots and 

cankering in this trial. At DGRC, over decades of 

trials, we have generally observed that more than 95 

percent of seedlings develop needle spots for the various white pine species evaluated. In this whitebark 
USDA Forest Service RMRS-P-76. 2018. 131

Figure 1—Whitebark pine seedling with hundreds of white pine blister rust needle spots, ~9 months 

post-inoculation.

Figure 2—Heavily cankered whitebark pine seedlings ~12 months post-inoculation. 
132 USDA Forest Service RMRS-P-76. 2018.

pine trial the artificial inoculation was extremely 

effective, with means greater than 125 needle spots 

per seedling and 99.9 percent of seedlings displaying 

needle spots approximately 9 months mpi. The level 

of needle spotting per seedling (the first symptom of 

blister rust infection noted after inoculation) varied 

dramatically among families (25.8 to 247.4) and populations (50.3 to 165.1) (Sniezko, unpublished data on 

file at DGRC). The trial averaged 11.3 stem symptoms 

per seedling and 77.5 percent of seedlings with stem 

symptoms ~15 mpi. Forty-seven of the 225 families 

reached 100 percent of seedlings ~15 mpi. This level 

of infection might correspond to a field site with very 

high levels of infection (many cankers per tree and a 

very high percentage of infected trees).

Families that show resistance in a trial such as the 

one reported here should be the best candidates for 

establishing whitebark pine plantings that will show 

the highest levels of field survival with exposure to 

the rust pathogen over the hundreds of years’ lifespans 

of these ecologically important trees. However, field 

testing on sites of high rust hazard will be needed to 

confirm the relationship of the seedling rust-resistance 

testing and field resistance. 

The 21 geographic sources in this trial varied greatly 

in the level of stem infections (fig. 4). There was also 

large variation among families within populations 

(Sniezko, unpublished data). Many of the sources 

with the highest level of resistance (based on low 

early stem infection percentage) appear to be from 

the Cascade Mountains of Oregon and Washington; 

those from eastern Oregon, the Interior West (Idaho 

and Montana), California, and British Columbia show 

much lower levels of rust resistance. Results from 

other studies have also suggested that the highest 

levels of resistance may be in the Pacific Northwest 

Figure 3—Heavily cankered whitebark pine seedling (left) and canker-free seedling (right), from half-sib family 

from seed collected from a Wenatchee National Forest parent tree, ~13 months after artificial inoculation.
USDA Forest Service RMRS-P-76. 2018. 133

populations (Kegley et al. 2012; Mahalovich et al. 

2006; Sniezko et al. 2007). Hundreds of additional 

seedling families are now under test, and the results 

will help better delineate geographic patterns. 

The high level of stem symptom-free percentage for 

some families and populations at the 15-month assessment suggests that the resistance could be similar to 

the major gene resistance found in other white pine 

species such as P. monticola, P. lambertiana, P. flexilis, 

and P. strobiformis (reviewed in Sniezko et al. 2014). 

However, other tests at DGRC indicate that the whitebark pine seedlings in the highest canker-free families 

do not exhibit the hypersensitive-like reaction in the 

needles observed in those four species (Sniezko et al. 

2008; Sniezko, unpublished data). The frequency and 

the level of resistance in these whitebark pine families 

is higher than generally seen in much more extensive 

resistance testing of half-sib families of P. monticola

and P. lambertiana (Kegley and Sniezko 2004; 

Sniezko, unpublished data); at least one half-sib family of P. monticola from the Cascades of Washington 

shows a relatively low percentage of seedlings with 

stem symptoms and is confirmed not to be from major 

gene resistance (see Sow #75 in table 1 in Sniezko et 

al. 2014). The high level of resistance shown in some 

of these open-pollinated whitebark pine families is 

comparable to or higher than that seen at DGRC in the 

Bingham F2 orchard seedlot of P. monticola (second 

generation of improved stock) (see Sow #71 in table 

1 in Sniezko et al. 2014). This is very encouraging 

considering that whitebark pine was initially considered to be the most susceptible of the North American 

five-needle pine species (Hoff et al. 1980). Further 

summary and analyses of this whitebark pine trial 

and other trials of this species underway or recently 

completed at DGRC will provide greater elucidation 

of the type and frequency of blister rust resistance in 

whitebark pine.

Fourteen families represented in earlier trials at DGRC 

were included as controls in this trial. Of these 14, the 

4 families with the highest percentages of seedlings 

with early stem infection in earlier trials also exhibited 

very high percentages of seedlings with early stem 

infection in this trial (100 percent for three of the 

Figure 4—Genetic variation among seed sources in percentage of seedlings with early blister rust stem symptoms 

~15 months post-inoculation. The number of half-sib families represented in each seed source mean is indicated 

above the bar (varies from 1 to 42). The 18 Oregon and Washington sources refer to individual National Forest, 

National Park and Confederated Tribes of Warm Springs boundaries from which the seedlots were collected.
134 USDA Forest Service RMRS-P-76. 2018.

families). In contrast, the 10 families showing a lowto-moderate level of early stem infection in a previous 

trial also showed a low-to-moderate percentage of 

early stem infections in this trial (40 to 65.3 percent).

The two families from the Interior West (Idaho and 

Montana) had previously been rated as moderately 

to highly resistant in an artificial inoculation trial 

elsewhere (Mahalovich et al. 2006), but appear to 

have been subjected to a much lower level of effective inoculation. These two families were among the 

most susceptible families in this trial (fig. 4) (Sniezko 

and Kegley 2015; Sniezko et al. 2011), with not only 

a very high early stem infection percentage (92.4 

percent) and number of stem infections per seedlings 

(10.3) but also 98.1 percent mortality 3 years after 

inoculation. In a second trial (also inoculated in 2008), 

with a different geographic source of rust inoculum, 

the two families from the Interior West source again 

rated poorly for resistance (97.9 percent of seedlings 

with stem infections ~15 mpi). Ratings of families 

or seed sources for relative resistance levels may be 

influenced by the effective level of inoculation. A 

less effective inoculation may give some indication 

of relative resistance among sources, but may not be 

indicative of how these sources will survive on higher 

hazard field sites. It is more likely that a less effective 

inoculation would lead to designating seedling families 

as resistant when they in fact escaped inoculation due 

to poor spore coverage or physiological status of the 

seedling. 

The presence of families and populations with relatively moderate levels of stem symptoms in this trial 

is encouraging given the highly effective inoculation. 

Land managers have been notified of the highest 

ranking parents, and seed from these can be used in 

restoration efforts or to establish seed orchards. A 

more comprehensive summary of all resistance traits 

compiled over the 5-year post-inoculation period for 

this trial and other trials at DGRC is underway.

Additional half-sib families from British Columbia are 

now being tested at DGRC (Murray and Berger, this 

proceedings), and some of these show promise in the 

early stages of the test. Hundreds of other selections 

are now in testing in both the Pacific Northwest and 

Interior West rust-resistance testing programs, and 

updates on trends and levels of rust resistance will be 

presented over time.

Extrapolating seedling testing results to field resistance 

over the life of long-lived whitebark pine needs to be 

done with caution. Results from trials with low effective inoculation should be used with caution or the trial 

repeated with a higher effective inoculation. At least 

a subset of seedling families should be included in 

multiple tests in different years to demonstrate repeatability of performance as well as to provide linkages 

between different trials. Long-term field trials with 

susceptible controls should be established to provide 

validation of the seedling screening results. In the 

Pacific Northwest region (Oregon and Washington) 

and British Columbia, field trials with known susceptible controls have been established in several areas to 

examine field resistance and durability of resistance 

(e.g., Beck and Sniezko, this proceedings; Murray and 

Berger, this proceedings).

ACKNOWLEDGMENTS

We thank all those who contributed seed and the 

technicians who completed many of the assessments. 

Funding for this project was through the USDA Forest 

Service Genetic Resource and Forest Health Protection 

programs in Region 6 as well as Crater Lake National 

Park (CRLA) and Mount Rainier National Park 

(MORA). We thank Regina Rochefort (MORA), and 

Laura Hudson and Jen Beck (CRLA) for their assistance. We thank Michael Murray, Jodie Krakowski, 

Everett Hansen, Bohun Kinloch, Jr., Bruce Moltzan, 

Jun-Jun Liu, and James Jacobs for their reviews of an 

earlier version of this paper. 

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Hamlin, J.; Kegley, A.; Sniezko, R. 2011. Genetic variation 

of whitebark pine (Pinus albicaulis) provenances and 

families from Oregon and Washington in juvenile 

height growth and needle color. In: Keane, Robert E.; 

Tomback, Diana F.; Murray, Michael P.; [et al.], eds. 

The future of high-elevation, five-needle white pines in 

western North America: Proceedings of the high five 

symposium. Proceedings RMRS-P-63. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky 

Mountain Research Station: 133–139.
USDA Forest Service RMRS-P-76. 2018. 135

Hoff, R.; Bingham, R.T.; McDonald, G.I. 1980. Relative 

blister rust resistance of white pines. European Journal of 

Forest Pathology. 10: 307–316.

Kegley, A.J.; Sniezko, R.A. 2004: Variation in blister 

rust resistance among 226 Pinus monticola and P. 

lambertiana seedling families in the Pacific Northwest. 

In: Sniezko, R.A.; Samman, S.; Schlarbaum, S.E.; [et 

al.], eds. Breeding and genetic resources of five-needle 

pines: Growth, adaptability and pest resistance, IUFRO 

Working Party 2.02.15. Proceedings RMRS-P-32. Fort 

Collins, CO: U.S. Department of Agriculture, Forest 

Service, Rocky Mountain Research Station: 209–226. 

https://www.fs.fed.us/rm/pubs/rmrs_p032/rmrs_

p032_209_226.pdf. (04 October 2015).

Kegley, A.; Sniezko, R.A.; Danchok, R.; [et al.]. 2012. 

Blister rust resistance among 19 families of whitebark 

pine, Pinus albicaulis, from Oregon and Washington—

Early results from an artificial inoculation trial. In: 

Sniezko, R.A.; Yanchuk, A.D.; Kliejunas, J.T.; [et al.], 

tech. coords. Proceedings of the fourth international 

workshop on the genetics of host-parasite interactions 

in forestry: Disease and insect resistance in forest trees. 

Gen. Tech. Rep. PSW-GTR-240. Albany, CA: U.S. 

Department of Agriculture, Forest Service, Pacific 

Southwest Research Station: 311–315. https://www.

fs.fed.us/psw/publications/documents/psw_gtr240/psw_

gtr240_311.pdf. (04 October 2015).

King, J.N.; Noshad, D.A.; Smith, J. 2010. A review of 

genetic approaches to the management of blister rust in 

white pines. Forest Pathology. 40: 292–313.

Kinloch, B.B., Jr.; Burton, D.; Davis, D.A.; [et al.]. 2012. 

Strong partial resistance to white pine blister rust in 

sugar pine. In: Sniezko, R.A.; Yanchuk, A.D.; Kliejunas, 

J.T.; [et al.], tech. coords. Proceedings of the fourth 

international workshop on the genetics of host-parasite 

interactions in forestry: Disease and insect resistance in 

forest trees. Gen. Tech. Rep. PSW-GTR-240. Albany, 

CA: U.S. Department of Agriculture, Forest Service, 

Pacific Southwest Research Station: 80–91. https://www.

fs.fed.us/psw/publications/documents/psw_gtr240/psw_

gtr240_080.pdf. (04 Oct 2015).

Mahalovich, M.F.; Burr, K.E.; Foushee, D.L. 2006. 

Whitebark pine germination, rust resistance and cold 

hardiness among seed sources in the Inland Northwest: 

Planting strategies for restoration. In: Riley, L.E.; 

Dumroese, R.K.; Landis, T.D., tech. coords. National 

proceedings: Forest and conservation nursery association. 

Proceedings RMRS-P-43. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky 

Mountain Research Station: 91-101.

McDonald, G.I.; Zambino, P.J.; Sniezko, R.A. 2004. 

Breeding rust-resistant five-needled pines in the western 

United States: Lessons from the past and a look to the 

future. In: Sniezko, R.A.; Samman, S.; Schlarbaum, 

S.E; [et al.], eds. Breeding and genetic resources 

of five-needle pines: Growth, adaptability and pest 

resistance, proceedings of IUFRO working party 2.02.15 

conference. Proceedings RMRS-P-32. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky 

Mountain Research Station: 28–50. https://www.fs.fed.

us/rm/pubs/rmrs_p032/rmrs_p032_028_050.pdf. (04 

October 2015).

Sniezko, R.A. 2006. Resistance breeding against nonnative 

pathogens in forest trees—Current successes in North 

America. Canadian Journal of Plant Pathology. 28: 

S270–S279.

Sniezko, R.A.; Kegley, A.J.; Danchok, R. 2008. White pine 

blister rust resistance in North American, Asian, and 

European species—Results from artificial inoculation 

trials in Oregon. Annals of Forest Research 51: 53–66

Sniezko, R.A.; Kegley, A. 2015. Report on results of Interior 

West (Region 1) whitebark pine seedlots evaluated 

in two screening trials at Dorena Genetic Resource 

Center. Rep. R6-DGRC-2015-01. Cottage Grove, OR: 

U.S. Department of Agriculture, Forest Service, Pacific 

Northwest Region. 

Sniezko, R.A.; Kegley, A.J.; Danchok, R.S.; [et al.]. 2007. 

Variation in resistance to white pine blister rust among 43 

whitebark pine families from Oregon and Washington—

Early results and implications for conservation. In: 

Goheen, E.M.; Sniezko, R.A., tech. coords. Proceedings 

of the conference whitebark pine: A Pacific Coast 

perspective. R6-NR-FHP-2007-01. Portland, OR: U.S. 

Department of Agriculture, Forest Service, Pacific 

Northwest Region: 82–97. https://www.fs.usda.gov/

Internet/FSE_DOCUMENTS/fsbdev2_025894.pdf. (04 

October 2015).

Sniezko, R.A.; Mahalovich, M.F.; Schoettle, A.W.; [et 

al.]. 2011. Past and current investigations of the genetic 

resistance to Cronartium ribicola in high-elevation 

five-needle pines. In: Keane, R.E.; Tomback, D.F; 

Murray, M.P.; [et al.], eds. The future of high-elevation, 

five-needle white pines in western North America: 

Proceedings of the high five symposium. Proceedings 

RMRS-P-63. Fort Collins, CO: U.S. Department of 

Agriculture, Forest Service, Rocky Mountain Research 

Station: 246–264. https://www.fs.fed.us/rm/pubs/rmrs_

p063/rmrs_p063_246_264.pdf. (04 October 2015).

Sniezko, R.A.; Smith, J.; Liu, J-J.; [et al.]. 2014. Genetic 

resistance to fusiform rust in southern pines and white 

pine blister rust in white pines—A contrasting tale of two 

rust pathosystems—Current status and future prospects. 

Forests. 5(9): 2050–2083. doi:10.3390/f5092050.
136 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Blister Rust Inoculation Trials for Whitebark Pine (Pinus albicaulis) in 

the Canadian Kootenay Region

M.P. Murray1, * and V. Berger2

1 British Columbia Ministry of Forests, Lands and Natural Resource Operations, Nelson, British Columbia, Canada

2 British Columbia Ministry of Forests, Lands and Natural Resource Operations, Kalamalka Forestry Centre,

Vernon, British Columbia, Canada

* michael.murray@gov.bc.ca

Abstract—Controlled inoculations of whitebark pine (Pinus albicaulis) families in British Columbia (BC), 

Canada, took place in 2013 and 2014 at the Kalamalka Forestry Centre in Vernon. This effort has focused 

on 40 families from the Kootenay region with results (identification of disease-resistant families) expected 

over the next 5 years. Meanwhile, demand continues for further screening of whitebark pine from other 

regions in Canada. New funding from the BC Forest Genetics Council will permit work to continue. Our 

plan is to inoculate 40 additional families per year (2016–2018). Seedlings will be collected by multiple 

partners such as national and provincial parks and possibly mining companies.

INTRODUCTION

Whitebark pine (Pinus albicaulis) is a widespread and 

valuable tree species in western Canada that continues 

to suffer widespread mortality due to the introduced 

blister rust disease (agent: Cronartium ribicola). 

The decline appears to have increased during the 

last 20 years. In 2000, Campbell and Antos (2000) 

reported on 54 stands across the range in British 

Columbia (BC) and remarked that on average about 

50 percent of whitebark pine was killed or infected 

by white pine blister rust. They noted the worst rates 

of rust infection were in the northwest (Smithers) and 

southeast (Fernie). A more recent survey of the Rocky 

Mountains in Canada and Montana (USA) (Smith 

et al. 2008) found mortality had increased from 26 

percent (1996) to 61 percent (2004). Due to steep 

declines, the Canadian government declared whitebark 

pine as endangered under the Species at Risk Act in 

June 2012.

As many denuded populations have few surviving 

trees, their future is jeopardized. Several authors 

predict that whitebark pine will be locally extirpated 

where it is less favored and rust thrives (Krakowski 

et al. 2003; Smith et al. 2008; Tomback and Achuff 

2010). Screening selected families for rust resistance 

is a critical component for maintaining whitebark pine 

(Environment Canada 2013; Schoettle and Sniezko 

2007). To do this, artificially inoculating seedlings 

with Cronartium ribicola followed by post-inoculation 

assessments of seedling health and survival are useful 

for identifying disease-resistant families (Sniezko et al. 

2011). 

LAUNCH (2013)

During August 2013 an inoculation trial was performed 

at the Kalamalka Forestry Centre (KFC), Vernon, BC, 

operated by the BC Ministry of Forests, Lands and 

Natural Resource Operations. Seedlings representing 

10 whitebark pine families from BC’s Kootenay region 

were used. These same families were also submitted 

to the U.S. Department of Agriculture, Forest Service 

(USDA FS) facility, Dorena Genetic Resource Center 
USDA Forest Service RMRS-P-76. 2018. 137

(DGRC), for tandem inoculations to provide a comparison of results. Each family consisted of 50 individuals 

per inoculation run (10 families × 50 seedlings × 2 runs 

= 1,000 seedlings). Our methods were adapted from the 

protocol in use by DGRC (Danchok et al. 2004). This 

included randomized blocks of seedlings and achieving 

a basidiospore load of 3,000 cm-2. 

The first run took place within a greenhouse where a 

plastic frame (polyvinyl chloride piping) formed an 

inoculation chamber. The second run was conducted 

in a growth chamber room. Temperatures were maintained near 20 °C and relative humidity was kept in the 

high 90s (percent). We achieved a target spore loading 

of about 3,000 spores cm-2 in our greenhouse run. 

However, there were no basidiospores produced in the 

growth chamber run; this was due to overmature telia 

on the Ribes leaves. Thus, these uninoculated seedlings 

were allocated to a field trial in Glacier National Park 

(BC).

During the following spring (2014) separate surveys 

were conducted by DGRC and Kalamalka personnel for needle spots and cankers associated with 

the inoculated seedlings. In comparing spot counts 

between KFC and DGRC, there is notable incongruence (fig. 1). A subsequent survey for cankers was 

conducted in November 2014 (14 months post-inoculation). Again, surveys indicated very low numbers of 

cankers on seedlings inoculated at Kalamalka (fig. 2). 

Although germination of spores was verified, we cannot explain the low incidence of symptoms. A variety 

of factors may have intervened, including additional 

biological agents, seedling physiological traits, and 

low virulence. 

2014 AND BEYOND

Seedlings from an additional 30 Kootenay families 

were inoculated at KFC in August 2014. All inoculations were conducted in the chamber room with spore 

Figure 1—Spots counts observed on needles of inoculated seedlings at 9–10 months post-inoculation.
138 USDA Forest Service RMRS-P-76. 2018.

loads averaging 4,400 cm-2. In September 2014, 

three field screening trials were established to screen 

non-inoculated seedlings from the same 40 families 

in natural (uncontrolled) environments. About 50 

seedlings from each family were planted at each site 

(fig. 3). During the same week in September, a fourth 

screening trial was established in Glacier National 

Park near Golden, BC. This trial used only 10 families 

(from the failed inoculation run of 2013).

Demand continues for screening additional families. 

New funding from the BC Forest Genetics Council 

will permit work to continue. In December, cones and 

seeds from 26 new families were submitted by Parks 

Canada. These seedlings will be ready for inoculating 

in 2016. Our plan is to perform controlled inoculations 

on up to 40 additional families per year until 2018. 

The number of families is likely to vary each year 

based on cone production and capabilities of cone 

collecting partners. These partners include national 

and provincial parks and possibly First Nations 

groups. In addition, coal and gold mining operations 

are expanding in whitebark pine habitat. The industry 

is expressing an interest in using whitebark pine for 

rehabilitation (Moody and Clason 2013).

ACKNOWLEDGMENTS

The inoculation team consisted of Ministry of Forests, 

Lands and Natural Resource Operations (FLNRO) 

staff based at the Kalamalka Forestry Centre: Randy 

Armitage, Vicky Berger, Ward Strong, and Nick 

Ukrainetz. Many thanks for useful advice from 

Joan Dunlap, John Gleason, and Det Vogler (U.S. 

Department of Agriculture, Forest Service [USDA FS], 

Institute of Forest Genetics, Placerville, California), 

Angelia Kegley and Richard Sniezko (USDA FS 

Dorena Genetics Resource Center, Dorena, Oregon), 

Rich Hunt, Canadian Forest Service (retired), Jodie 

Krakowski (FLNRO), and Paul Zambino (USDA FS). 

Figure 2—Canker counts observed on inoculated seedlings at 14 months post-inoculation..
USDA Forest Service RMRS-P-76. 2018. 139

REFERENCES

Campbell, E.M.; Antos, J.A. 2000. Distribution and severity 

of white pine blister rust and mountain pine beetle in 

British Columbia. Canadian Journal of Forest Research. 

30: 1051–1059.

Danchok, R.; Sharpe, J.; Bates, K.; [et al.]. 2004. 

Operational manual for white pine blister rust inoculation 

at Dorena Genetic Resource Center. Unpublished report 

on file at: U.S. Department of Agriculture, Forest 

Service, Dorena Genetic Resource Center, Cottage 

Grove, OR. 31 p.

Environment Canada. 2013. Draft recovery strategy for 

whitebark pine recovery in Canada. Recovery strategy 

series. 62 p.

Krakowski, J.; Aitken, S.N.; El-Kassaby, Y.A. 2003. 

Inbreeding and conservation genetics in whitebark pine. 

Conservation Genetics. 4(5): 581–593.

Moody, R.; Clason, A.J. 2013. Whitebark restoration and the 

mining industry: Potential for collaboration? Nutcracker 

Notes. 23: 17–18.

Schoettle, A.W.; Sniezko, R.A. 2007. Proactive intervention 

to sustain high-elevation pine ecosystems threatened by 

white pine blister rust. Journal of Forest Research. 12(5): 

327–336.

Smith, C.M.; Wilson, B.; Rasheed, S.; [et al.]. 2008. 

Whitebark pine and white pine blister rust in the Rocky 

Mountains of Canada and Northern Montana. Canadian 

Journal of Forest Research. 38: 982–995.

Sniezko, R.A.; Mahalovich, M.F.; Schoettle, A.W.; [et 

al.]. 2011. Past and current investigations of the genetic 

resistance to Cronartium ribicola in high-elevation 

five-needle pines. In: Keane, Robert E.; Tomback, 

Diana F.; Murray, Michael P.; [et al.], eds. 2011. The 

future of high-elevation, five-needle white pines in 

western North America: Proceedings of the high five 

symposium. Proceedings RMRS-P-63. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky 

Mountain Research Station: 246–264. https://www.

fs.usda.gov/treesearch/pubs/38234. (13 October 2015).

Tomback, D.F.; Achuff, P.2010. Blister rust and western 

forest biodiversity: Ecology, values and outlook for white 

pines. Forestry Pathology. 40: 186–225.

Figure 3—Planting a rust screening trial at Idaho Peak, above Slocan Lake, BC (September 2014).
140 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Variation in Blister Rust Resistance and Early Height Growth in

Three Populations of Southwestern White Pine (Pinus strobiformis) 

and Implications for Management and Conservation

R.A. Sniezko1, *, A. Kegley1, J. Jacobs2, R. Danchok1, B. Luis1, S. Long1, and D.P. Savin1

1 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA 

2 USDA Forest Service, Region 3, Forest Health Protection, New Mexico Zone, Albuquerque, New Mexico, USA 

* rsniezko@fs.fed.us

Abstract—Southwestern white pine (SWWP, Pinus strobiformis), a long-lived conifer in Mexico and the 

southwestern United States, is very susceptible to the nonnative pathogen Cronartium ribicola, the cause 

of white pine blister rust. Due to the recent introduction of C. ribicola to the Southwest, the future viability 

of many populations of SWWP is in jeopardy. Fortunately, some early studies showed that there is at least 

a low frequency of genetic resistance in SWWP. In partnership with the U.S. Department of Agriculture, 

Forest Service (USDA FS), Southwestern Region Forest Health Protection and colleagues at Northern 

Arizona University, several trials to evaluate genetic resistance to the rust are now underway at the USDA 

FS Dorena Genetic Resource Center (Cottage Grove, Oregon, USA). We summarize here early results 

from a seedling inoculation trial involving 40 half-sib families from three populations in New Mexico. 

Sources of the three populations were the Lincoln, Cibola, and Santa Fe National Forests. Seventeen of 

the 20 selections from the Lincoln National Forest were from putatively canker-free trees present within a 

heavily infested stand; the three others were cankered trees and serve as susceptible controls. There is 

currently a low incidence of rust infection on the Cibola and Santa Fe National Forests, and the 20 parents 

selected there were considered random selections. Data collected in this experiment show differences 

between the tested SWWP populations in early height growth as well as resistance to C. ribicola. The 

Lincoln National Forest population was taller and more resistant to C. ribicola than the randomly collected 

populations from the Cibola and Santa Fe National Forests.

INTRODUCTION

Populations of southwestern white pine (SWWP, 

Pinus stobiformis), a long-lived conifer native to the 

American Southwest and Mexico, are increasingly 

threatened by white pine blister rust (WPBR) disease, 

caused by the nonnative fungal pathogen Cronartium 

ribicola (fig. 1). This disease was first reported causing damage to SWWP in the Sacramento Mountains 

of southern New Mexico in 1990 (Hawksworth 1990). 

Since then, the fungus has spread throughout central 

and southern New Mexico and the White Mountains 

of Arizona (Conklin et al. 2009; Fairweather and Geils 

2011). On the Lincoln National Forest in southern 

New Mexico, incidence of infection has exceeded 

80 percent in some areas (Conklin 1994, 2004), with 

many infected trees in advanced stages of decline. 

Some large mature trees had hundreds of cankers 

(fig. 2) and topkill (Conklin 2004).

Both major gene resistance and partial resistance have 

been previously documented in this species (Hoff et 

al. 1980; Kinloch and Dupper 2002; Sniezko et al. 

2008), but relatively few families have been evaluated 

for partial resistance. In this common garden study 
USDA Forest Service RMRS-P-76. 2018. 141

we examine variation in early height growth and 

resistance to WPBR among half-sib seedling families 

of SWWP originating from three populations in New 

Mexico: Cibola (CNF), Santa Fe (SFNF), and Lincoln 

(LNF) National Forests. The study is ongoing, and 

we report here only the early trends among the three 

forest populations in seedling growth, effectiveness of 

the artificial inoculation, and some elements of early 

resistance.

METHODS

Seed from 40 families of SWWP from the CNF, 

SFNF, and LNF were sown in March 2009 at the U.S. 

Department of Agriculture, Forest Service (USDA FS) 

Dorena Genetic Resource Center (DGRC; Cottage 

Grove, Oregon, USA). The seedlots were individual 

tree collections from 10 parent trees each from the 

CNF and the SFNF and from 20 parent trees on the 

LNF. Phenotypically canker-free SWWP surrounded 

by heavily infected individuals had been previously 

documented in the Bradford Canyon area of the LNF 

(Conklin et al. 2009). The parent trees from the LNF 

included 17 putatively resistant trees that did not 

exhibit observable cankering and 3 trees that had 

moderate-to-heavy cankering (to serve here as susceptible controls). The parent trees from the CNF and 

SFNF populations were in areas with little or no rust at 

the time of selection and represent random selections 

from those populations.

Seedlings were grown for two growing years, 1 year 

in supercell Cone-tainersTM (Ray Leach, Canby, 

Oregon) (164 cm3) in family blocks in the greenhouse, 

Figure 1—Southwestern white pine (a) sapling with several branch cankers, (b) bole canker with aecia, and (c) large 

tree with many cankers and top kill from rust (photo credits: Mary Lou Fairweather (a & b); Betsy Goodrich (c)).
142 USDA Forest Service RMRS-P-76. 2018.

Figure 2— “Discovery Tree”—the first infected Pinus strobiformis tree found in the 

Southwest, as it appeared in 1990. Branch 

‘flagging’ from hundreds of blister rust cankers is from 5–6 year-old infections. The 

tree is located 2 miles NE of Cloudcroft, on 

the Lincoln National Forest (photo: Dave 

Conklin).

followed by transplanting to 0.9 m × 1.2 m × 0.3 m 

boxes outside for the second growing season (fig. 3). 

Twelve to 60 seedlings were available for each family 

(mean = 47), and seedlings were transplanted into family row plots in a randomized complete block design 

with 6 blocks and up to 10 seedlings per family per 

block. Seedlings were inoculated in September 2010 

with basidiospores of C. ribicola (fig. 4) (see Kegley 

and Sniezko 2004 and Sniezko et al. 2008, 2011 for 

general protocols used in inoculations at DGRC). 

Mean inoculum density was 4,527 spores/cm2; basidiospore germination was 98.7 percent. Both primary 

and secondary needles were present at the time of 

inoculation. 

Seedlings were assessed in spring 2011 for secondyear height (HT2), number of needle spots on secondary needles and primary needles, and number of stem 

symptoms (cankers or bark reactions). Additionally, 

the percentage of secondary needle necrosis caused 

by needle blight fungi and environmental factors was 

recorded for each seedling. Subsequent annual assessments were done in late fall and early winter of 

2011, 2012, 2013, and 2014; presence and number of 

stem symptoms by type (normal canker or bark reaction), severity of stem symptoms, and mortality were 

recorded. Third-year height (HT3) was recorded in fall 

2011; this was the new growth in the year following 

inoculation with rust, including any late-season lammas growth. The severity score quantified the cumulative impact of all the stem symptoms for a seedling 

at each assessment. It ranged from 0 to 9 (where 0 = 

no stem symptom; 1 to 4 = very small or nongirdling 

infection(s); 5 = at least one stem symptom encircling 

the diameter of the seedling, but little vertical extension; 6 to 8 = extensive cankering encompassing much 

of the seedling radially and vertically; 9 = seedling 

dead from rust). Here, we report on some assessments 

through winter 2011, using the four groups (populations) of families: (1) CNF, (2) SFNF, (3) LNF-R 

(from canker-free parents), and (4) LNF-S (three 

susceptible controls).

Statistical analyses were conducted by using SAS® 

9.4 (SAS Institute Inc., Cary, North Carolina, USA). 

The analyses of variance (ANOVAs) for heights were 

based on family-plot means using PROC MIXED. 

Forests and families nested within forests were considered fixed effects; block effects were considered 

random. For HT3, the severity of rust was added to 

the model as an explanatory variable. For analyses 

on binomial responses such as mortality, a logistic 

regression model with logit link was fit using PROC 

GLIMMIX, with forests treated as fixed effects and 

random block effects.

RESULTS

There were statistically significant differences among 

the four populations and among families within populations for both second-year height (pre-inoculation, 

P < 0.0001 for both) and third-year height (1 year 

post-inoculation, P < 0.0001 for both). Seedlings from 

the LNF-R were on average taller (20.1 cm for the 17 

resistant parents) than those from CNF (19.2 cm) or 

SFNF (18.2 cm) or those from the three susceptible 

parents on the LNF (18.5 cm) for HT2. Some of the 

height differences between families within a population were also visibly striking (fig. 5). In this trial, 
USDA Forest Service RMRS-P-76. 2018. 143

Figure 3—Southwestern white pine (Pinus strobiformis) trial shortly after artificial inoculation with 

Cronartium ribicola.

Figure 4—Southwestern white pine (Pinus strobiformis) in Dorena Genetic Resource Center ‘fog’ 

chamber just prior to artificial inoculation with Cronartium ribicola.
144 USDA Forest Service RMRS-P-76. 2018.

there was a significant negative correlation between 

third-year height and the severity of rust infection at 

the time of inspection (P = 0.002). Seedlings with a 

one-unit higher canker severity score were on average 

0.9 cm shorter (95-percent confidence interval from 

0.3 to 1.4 cm shorter). 

The inoculation of the seedlings with C. ribicola was 

very effective in this experiment, with 97.3 and 99.6 

percent of seedlings exhibiting characteristic needle 

spots on the primary and secondary needles, respectively (fig. 6). The mean number of needle spots per 

seedling on secondary needles was 40.9 at 9 months 

post-inoculation (mpi).

Stem symptoms, cankers, and bark reactions appeared 

relatively quickly; 56.9 percent of seedlings had stem 

symptoms 9 mpi and 87.0 percent had stem symptoms 

at 15 mpi (fig. 7). At second assessment (~15 mpi), the 

seedling progenies from the LNF-R averaged 75.7 percent with stem infections versus 94.1, 98.0, and 90.7 

percent with stem infections for those from the CNF, 

SFNF, and LNF-S, respectively; the mean number of 

stem infections per family was 11.2 (ranging from 2.9 

to 20.7 for the 40 half-sib families). Mean severity of 

rust ~15 mpi varied from 3.9 for LNF-R to >6.0 for the 

other three populations in this study. 

The percent secondary needle necrosis present in early 

spring 2001 in this study varied significantly for the 

four groups of seedling families studied (P < 0.0001), 

with 54, 67, 59, and 68 percent of seedlings affected 

on the LNF-R, LNF-S, CNF, and SFNF, respectively. 

The mean percentage of secondary tissue affected 

overall was 8.2 percent. Only 10.4 percent of seedlings 

had more than 20 percent of secondary tissue affected. 

Figure 5—Large within-population variation in height growth after second growing season for two families from 

Cibola National Forest. Family ‘49’ is taller (row plot on left, mean 2nd year height = 23.5cm) and the much shorter 

Family ’45’ (row plot on right, mean height = 17.7cm).
USDA Forest Service RMRS-P-76. 2018. 145

After accounting for forest differences, there was no 

evidence of a relationship between either the probability of a seedling exhibiting needle necrosis or the 

percentage of secondary tissue affected and the percentage of seedlings cankered (P = 0.54 and P = 0.90, 

respectively).

Eight percent (150 of the 1,872) of the trees in the trial 

were killed by C. ribicola over the 15-mpi monitoring 

period reported here. This mortality varied significantly by population (P < 0.0001), from 2.5 percent 

for LNF-R, to 7.5 percent for LNR-S and 12.5 percent 

for both CNF and SFNF. One family from CNF had 36 

percent rust-related mortality at this early stage. 

Figure 6—Southwestern white pine (Pinus strobiformis) seedling from Santa Fe National Forest (Sow #59) with 

hundreds of needle spots after blister rust inoculation; this family had 41 needle spots per seedling and 94.4 percent of seedlings with stem symptoms (and 13 stem symptoms per seedling) at 9 and 15 months post-inoculation, 

respectively.

DISCUSSION

Differences in early height growth were found between the populations and may suggest a need for 

establishing breeding zones within this species. Other 

trials are now underway to examine in more detail 

the differences in some adaptive traits in this species 

(Goodrich et al., this proceedings). The rapid development of cankers during the first growing season after 

inoculation affected normal season growth, the lammas 

growth at the end of the growing season, or both; seedlings with the highest severity ratings for rust showed 

the most impact to growth in the 15 months following 

inoculation.
146 USDA Forest Service RMRS-P-76. 2018.

The overall frequency of canker-free seedlings was 

low (<6 percent) at 15 mpi in the seedling families 

from the parental selections from the SFNF and CNF, 

populations which originated from stands with little 

or no blister rust present. A low frequency of cankerfree trees was also found in several earlier seedling 

inoculation studies of SWWP resistance to WPBR in 

the United States (Hoff 1980) and Germany (Stephan 

1986). These data and the high infection (>80 percent) 

in some natural stands (Conklin 2004) confirm the 

high susceptibility of SWWP and the relatively low 

Figure 7—Southwestern white pine seedling from Cibola National Forest family (Sow #50) with numerous stem cankers 14 months after inoculation; this 

family had 58 needle spots per seedling (9 months post-inoculation) and 94.8 

percent of trees with stem symptoms and 17 stem infections per seedling, at 

~15 months post-inoculation.

incidence of genetic resistance to WPBR in this species. Seedling families from the canker-free parent 

trees on the LNF had the lowest levels of early stem 

infection (mean = 75.7 percent) following artificial 

inoculation, but several families from this population had more than 90 percent of seedlings with stem 

symptoms. The parent trees from LNF are in an area 

of very high incidence and severity of rust infections, 

and 17 of the 20 selections tested were canker-free at 

the time of seed collection. Efficacy of selection was 

expected to be higher in this population than that from 
USDA Forest Service RMRS-P-76. 2018. 147

the other two forests given the presence of C. ribicola

within the LNF for decades before selection, and 

this holds true for the early component of resistance 

reported here. A smaller, earlier trial of progeny from 

other parent trees from LNF also indicated a relatively 

high frequency of resistance, including a relatively 

low percentage of trees with stem symptoms (42.5 

percent) 39 mpi (Sniezko et al. 2008). The lower level 

of stem symptoms in the earlier trial may be due to the 

different families utilized, a higher effective inoculation in the trial reported in this paper, or a combination 

thereof. In previous seedling testing at the USDA FS 

Institute of Forest Genetics (Placerville, California), 

progeny of the 17 canker-free LNF parents showed no 

evidence of 1:1 segregation for major gene resistance 

(D.A. Conklin, personal communication, 2008). The 

primary needles (which are more susceptible than 

secondary needles) present on the seedlings in this 

test may have led to somewhat higher stem infection 

than if only secondary needles had been present. 

Regardless, selections from the canker-free LNF 

parents were more resistant to WPBR in this trial than 

those from the other two geographic areas. Field trials 

to confirm the actual level and durability of resistance 

are needed before extrapolating any seedling assay 

such as this one to long-term field results.

This trial is still ongoing, but large differences among 

families and populations in resistance to WPBR are 

apparent. Analyses are currently underway, and in 

later summaries we will discuss the family and seed 

source variation in additional rust-resistance traits over 

the 5-year post-inoculation assessment period. In this 

study we observed necrosis on the secondary needles 

for each of the seedling families. The data indicated 

that although a moderate frequency of needle necrosis 

caused by an unknown incitant was present in each of 

the seedling families, it affected only a small percentage of the secondary needle tissue on most seedlings. 

The incitant probably had very little impact on cankering incidence, as we did not observe any association 

between percent necrosis and percent cankered. An 

additional 158 families from throughout the U.S. 

portion of the range of SWWP were sown in 2014 for 

blister rust inoculation in 2015, and this will provide a 

broader estimate of the frequency and distribution of 

resistance to C. ribicola in this species. 

Field trials to validate resistance observed in seedling 

inoculation trials and to examine its durability are 

planned. Seed collections for gene conservation have 

begun, and studies of genetic variation in adaptive 

traits are underway (Goodrich et al., this proceedings). 

Other management options for mitigating the effect 

of WPBR in natural stands have been discussed elsewhere (Conklin et al. 2009). Some of the high-hazard 

sites in the Southwest may necessitate planting of 

resistant seedlings, but much can also be done by favoring white pine in silvicultural treatments. Although 

thinning of stands can decrease the likelihood of standreplacing fire and thereby conserve possible resistant/

tolerant white pines, it also increases the understory 

sunlight and in some areas, especially when combined with underburning, may lead to an increase in 

populations of Ribes species, the alternate host for C. 

ribicola. Fortunately, there are some forested areas in 

the Southwest with little to no presence of susceptible 

Ribes species (M.L. Fairweather email to Sniezko, 

Feb. 12, 2015; Geils et al. 1999; Looney 2012; van 

Arsdel et al. 1998).

Southwestern white pine is very susceptible to white 

pine blister rust, and the frequency of naturally occurring resistance is low. Overall forest management 

goals, as well as an understanding of the natural resistance present in SWWP, will have to be considered if 

the region’s white pine resources in many areas are to 

remain viable. Given the relatively recent introduction 

of WPBR to the Southwest, it is imperative that we 

identify and quantify resistance in this species, including the frequency of occurrence over the landscape. 

Based on permanent plot monitoring, it is clear that 

retaining SWWP on high-hazard sites will be difficult 

without resistant planting stock. The first rust-resistant 

parents have now been identified, and seed can be 

collected from them to use for reforestation and restoration. These selections should also be grafted into 

orchards or clone banks to ensure retention of these 

valuable resistant parents.

ACKNOWLEDGMENTS

Thanks to the U.S. Department of Agriculture, Forest 

Service (USDA FS) Dorena Genetic Resource Center 

personnel for their contributions; Dave Conklin 
148 USDA Forest Service RMRS-P-76. 2018.

(USDA FS Region 3 Forest Health Protection, retired) 

for project initiation and funding; and timber staff on 

the Lincoln National Forest, Cibola National Forest, 

and Santa Fe National Forest in National Forest 

System Region 3 for organizing seed collections 

and for partial funding. We also thank Mary Lou 

Fairweather, Betsy Goodrich, and Kristen Waring for 

their comments on an earlier version of this paper. 

Photo credits: Richard Sniezko, unless indicated 

otherwise.

REFERENCES

Conklin, D.A. 1994. White pine blister rust outbreak on the 

Lincoln National Forest and Mescalero – Apache Indian 

Reservation, New Mexico. R-3-94-2. Albuquerque, 

NM: U.S. Department of Agriculture, Forest Service, 

Southwestern Region, Forest Health Protection. 12 p.

Conklin, D.A. 2004. Development of the white pine blister 

rust outbreak in New Mexico. R3-04-01. Albuquerque, 

NM: U.S. Department of Agriculture, Forest Service, 

Southwestern Region, Forest Health Protection. 17 p. 

https://www.fs.usda.gov/Internet/FSE_DOCUMENTS/

stelprdb5238472.pdf. (15 October 2015).

Conklin, D.A. 2008. Phone discussion with Dr. Richard 

Sniezko. Discussions on the nature of resistance of the 

seedlots he requested to test for blister rust resistance at 

Dorena Genetic Resource Center in the 2009 trial. 

Conklin, D.A.; Fairweather, M.L.; Ryerson, D.E.; [et al.]. 

2009. White pines, blister rust, and management in 

the Southwest. R3-FH-09-01. Albuquerque, NM: U.S. 

Department of Agriculture, Forest Service, Southwestern 

Region, Forest Health Protection. 16 p. https://www.

fs.usda.gov/treesearch/pubs/33753. (23 January 2018).

Fairweather, M.; Geils, B. 2011. First report of the white 

pine blister rust pathogen, Cronartium ribicola, in 

Arizona. Plant Disease. 95: 494.

Geils, B.W.; Conklin, D.A.; van Arsdel, E.P. 1999. A 

preliminary hazard model of white pine blister rust 

for the Sacramento Ranger District, Lincoln National 

Forest. Res. Note RMRS-RN-6. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky 

Mountain Research Station. 6 p.

Hawksworth, F.G. 1990. White pine blister rust in southern 

New Mexico. Plant Disease. 74: 938.

Hoff, R.; Bingham, R.T.; McDonald, G.I. 1980. Relative 

blister rust resistance of white pines. European Journal of 

Forest Pathology. 10: 307–316.

Kegley, A.J.; Sniezko, R.A., 2004. Variation in blister 

rust resistance among 226 Pinus monticola and P. 

lambertiana seedling families in the Pacific Northwest. 

In: Sniezko, R.A.; Samman, S.; Schlarbaum, S.E.; [et 

al.], eds. Breeding and genetic resources of five-needle 

pines: Growth, adaptability and pest resistance, IUFRO 

Working Party 2.02.15. Proceedings RMRS-P-32. Fort 

Collins, CO: U.S. Department of Agriculture, Forest 

Service, Rocky Mountain Research Station: 209–226. 

https://www.fs.fed.us/rm/pubs/rmrs_p032/rmrs_

p032_209_226.pdf . (15 October 2015).

Kinloch, B.B., Jr.; Dupper, G.E. 2002. Genetic specificity in 

the white pine-blister rust pathosystem. Phytopathology. 

92: 278–280.

Looney, C. 2012. Cronartium ribicola effects on white pine 

growth, vigor and survival in Arizona and New Mexico. 

Flagstaff, AZ: Northern Arizona University. Master of 

Science thesis. 138 p. 

Sniezko, R.A.; Kegley, A.J.; Danchok, R. 2008: White pine 

blister rust resistance in North American, Asian, and 

European species—Results from artificial inoculation 

trials in Oregon. Annals of Forest Research. 51: 53–66. 

http://www.afrjournal.org/index.php/afr/article/view/145. 

(15 October 2015).

Sniezko, R.A.; Mahalovich, M.F.; Schoettle, A.W.; [et 

al.]. 2011. Past and current investigations of the genetic 

resistance to Cronartium ribicola in high-elevation 

five-needle pines. In: Keane, R.E.; Tomback, D.F.; 

Murray, M.P.; [et al.], eds. The future of high-elevation, 

five-needle white pines in western North America: 

Proceedings of the high five symposium. Proceedings 

RMRS-P-63. Fort Collins, CO: U.S. Department of 

Agriculture, Forest Service, Rocky Mountain Research 

Station: 246–264. http://www.fs.fed.us/rm/pubs/rmrs_

p063/rmrs_p063_246_264.pdf. (15 October 2015). 

Stephan, B.R. 1986. The IUFRO experiment on resistance 

of white pines to blister rust (Cronartium ribicola) in 

northern Germany. Proceedings of the 18th IUFRO 

World Congress; Ljubljana; September 1986. Division II. 

Vol. I: 80–89.

van Arsdel, E.P.; Conklin, D.A.; Popp, J.B.; [et al.]. 

1998. The distribution of white pine blister rust in the 

Sacramento Mountains of New Mexico. Finnish Forest 

Research Institute Research Papers. 712: 275–283. 
USDA Forest Service RMRS-P-76. 2018. 149

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Persistence of Major Gene Resistance in Western White Pine

(Pinus monticola) in British Columbia

R.S. Hunt1, *, M. Murray2, R. Reich3, D. Rusch4, A. Woods5, and S. Zeglen6

1 Victoria, British Columbia, Canada (retired) 

2 British Columbia Ministry of Forests, Lands and Natural Resource Operations (BC MFLNRO),

Nelson, British Columbia, Canada

3 BC MFLNRO, Prince George, British Columbia, Canada

4 BC MFLNRO, Williams Lake, British Columbia, Canada

5 BC MFLNRO, Smithers, British Columbia, Canada

6 BC MFLNRO, Nanaimo, British Columbia, Canada

* ribicola@gmail.com

White pine blister rust, a devastating disease caused 

by the rust pathogen Cronartium ribicola, has been 

attacking and killing western white pines (Pinus monticola) for more than 100 years since its introduction 

to the Canadian province of British Columbia (BC) 

(Hunt 2009). There is a single dominant (major) gene 

for resistance (MGR) that produces a hypersensitive 

infection spot similar to that observed in resistant 

reactions in other plants attacked by rust pathogens. 

It was first found in a limited geographic area of 

southern Oregon, USA, known as the Champion 

Mines (Kinloch et al. 1999). Resistant Champion 

Mines clones were established at Dorena, Oregon, 

in the mid-1960s; since then, however, the pathogen 

has circumvented this resistance at Dorena and other 

areas in southern Oregon (Kinloch et al. 2004). This 

MGR resistance was imported to BC in seed collected 

from Dorena. Resistance came from seedlots from two 

geographic areas: (1) open-pollinated (OP) Champion 

Mines clones, and (2) OP Washington State, USA 

clones. Seedlings were planted into nine root disease 

sites in 1986 and 1987 throughout BC. Details of the 

plantation design using these 2 pine sources and 12 

other provenances can be found in Hunt (1987) and 

Meagher and Hunt (1998). The first seedlot was also 

planted into three coastal and three interior provenance 

trials in 1988 (see Meagher and Hunt 1998) and the 

second seedlot into three demonstration plantations in 

1994. These trees have been examined for growth and 

blister rust periodically over the years. At the previous 

IUFRO rust meeting held in OR in 2001, we (Hunt et 

al. 2004) reported that this resistance appeared to be 

persistent. In this report we provide a 2014 update. 

The growth of the Washington trees was consistently 

superior to that of the Champion Mines-derived trees. 

At age 10 to 12, it was apparent in several of the 

plantations that about 50 percent of the trees in each 

of these two seedlots were rust resistant. Seed was 

collected from a sample of these canker-free trees. By 

using the protocol of inoculating 50 seedlings/parent 

as recommended by Bro Kinloch, it was determined 

that 50 percent, or all, of their offspring produced 

hypersensitive needle reactions indicative of heterozygous and homozygous MGR resistance, respectively. 

The MGR resistance within the Washington trees was 

attributed to pollination at Dorena from Champion 

Mines clones, because none of these trees was homozygous resistant. Rust surveys and observations in 

2013 and 2014 of six root disease sites continued to

demonstrate that there was no apparent failure of MGR 

resistance (table 1; trees missing or those killed by 

other agents were omitted from the dataset, and MGR 

and neighboring geographic sources were pooled). 

The six provenance sites have not been formally 

inspected since 2001. The demonstration plantation 
150 USDA Forest Service RMRS-P-76. 2018.

at Woss (northern Vancouver Island) was inspected in 

2007. Washington sources at the other two plantations 

(Texada Island and southern Vancouver Island) were 

inspected in 2011 for rust status prior to scion collection. In each of these three plantations, the Washington 

source was least cankered at about 50 percent.

In BC, most cankers were located close to the ground 

(Hunt 1982, 1998) so the three demonstration plantations have largely grown through that young age when 

they were most susceptible to attack. To continue 

monitoring for the persistence of MGR resistance in 

BC, younger plantations should be inspected. More 

recently, the BC Forest Service has imported three 

seedlots from Dorena for operational use. We have periodically monitored one of these younger plantations 

and confirmed hypersensitive needle spots by inoculating offspring from canker-free parents. A 2013 survey 

of a 2002 trial using these three lots on three coastal 

sites, averaged 64, 66, and 70 percent canker-free trees 

per lot compared to 27, 29, and 30 percent canker-free 

trees for non-MGR selections. To date there is no evidence of MGR failure in BC.

REFERENCES

Hunt, R.S. 1982. Blister rust in British Columbia I. The 

possibilities of control by branch removal. The Forestry 

Chronicle. 58: 136–138.

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

white pine provenances to root disease? In: DeNitto, 

G.A., comp. Proceedings of the 35th annual Western 

International Forest Disease Work Conference. Portland, 

OR: U.S. Department of Agriculture, Forest Service: 

29–34. 

Hunt, R.S. 1998. Pruning western white pine in British 

Columbia to reduce white pine blister rust losses: 10-year 

results. Western Journal of Applied Forestry. 13: 60–63. 

Hunt, R.S. 2009. History of western white pine and blister 

rust in British Columbia. The Forestry Chronicle 85: 

516–520.

Hunt, R.S.; Jensen, G.D.; Ekramoddoullah, A.K. 2004. 

Confirmation of dominant gene resistance (Cr2) in 

U.S. white pine selections to white pine blister rust 

growing in British Columbia. 2001 Proceedings, IUFRO 

2.02.15. Breeding and genetics resources of five-needle 

pines. Proceedings RMRS-P-32. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky 

Mountain Research Station: 227–229.

Kinloch, B.B., Jr.; Sniezko, R.A.; Barnes, G.D.; [et al.]. 

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.; Dupper, G.E. 2004. 

Virulence gene distribution and dynamics of the white 

pine blister rust pathogen in western North America. 

Phytopathology. 94: 751–758.

Meagher, M.D.; Hunt, R.S. 1998. Early height growth of 

western white pine provenances in British Columbia 

plantations. Western Journal of Applied Forestry. 13: 

47–53.

Table 1—Blister rust infection on pooled sources of western white pine across seven plantation sites. 

Canker-free (%) white pine from four pooled sourcesa

Site Pool #1 Pool #2 Pool #3 Pool #4

Marl, 51°31 W117°25 87 89 88 53

Tisdall, 52014 W120054 85 84 62 73

Beaver FR, 54°3 W122°10 75b 75b 11b 75b

Busk, 49°38 W117°9 63 24 7 0 

Sharpe, 55°07 W127°21 56 12 0 4b

Alberni, 49°14 W124°44 52 12 6 0 

Smallwood, 49030 W117o26 40c 31 4 0 a Pooled sources: #1 MGR, #2 BC coast, #3 BC interior/Idaho/Montana, and #4 California/Warner Mts. b Small sample size. c After blister rust killed many young trees, drought appeared to preferentially kill the large survivors. In the 

MGR group this meant loss of canker-free trees and a reduction in the sample size to 30.
USDA Forest Service RMRS-P-76. 2018. 151

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Comparative Look at Rust Infection and Resistance in Limber Pine 

(Pinus flexilis) and Rocky Mountain Bristlecone Pine (P. aristata) 

Following Artificial Inoculation at Three Inoculum Densities

W.R. Jacobi1, *, H.S.J. Kearns2, A. Kegley3, D.P. Savin3, R. Danchok3, and R.A. Sniezko3

1 Colorado State University, Fort Collins, Colorado, USA 

2 USDA Forest Service, Forest Health Protection, Sandy, Oregon, USA

3 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA

* William.jacobi@colostate.edu

INTRODUCTION

Limber pine (Pinus flexilis) and Rocky Mountain 

bristlecone pine (Pinus aristata) are important components of forest ecosystems in the southern Rocky 

Mountains, United States. Both species are susceptible to white pine blister rust caused by the fungal 

pathogen Cronartium ribicola. However, very little is 

known about the relative resistance of the two species. 

In this study, we used artificial inoculation of seedlings 

to compare the relative susceptibilities of these two 

species. The objective of the inoculation test was 

to compare the performance of seedlings from bulk 

seedlots of limber pine and bristlecone pine exposed 

to three inoculum densities (low, medium, and high). 

This information will provide guidelines for inoculum 

density levels for future trials of these species and 

a first look at the extent of resistance present in the 

general population and its efficacy under exposure to 

increasing levels of inoculum.

METHODS

The seedlings were grown by the Colorado State 

Forest Service Nursery in Fort Collins, Colorado, USA 

from bulk seedlots collected from parent trees in the 

Rollinsville, Colorado, area. Limber pine seedlings 

were 3 years old and bristlecone pine 2 years old when 

transported by covered truck on August 14, 2003, to 

the U.S. Department of Agriculture, Forest Service, 

Dorena Genetic Resource Center (DGRC; Cottage 

Grove, Oregon, USA). Seedlings were transported and 

inoculated in 30-cell StyroblocksTM (Beaver Plastics, 

Acheson, Alberta, Canada). A randomized complete 

block design with three replications of three treatments for the two species was used for inoculation and 

outplanting. Thirty limber pine seedlings were used in 

each block-by-treatment combination for a total of 90 

seedlings per treatment. Twenty-four bristlecone pine 

seedlings were used per treatment block for a total 

of 72 seedlings per treatment. The limber pine seedlings were grown in removable containers placed in 

Styroblocks, and individual seedlings were randomly 

assigned to the treatment blocks. Seedling numbers 

were equalized (30 seedlings per treatment block) 

among the blocks. Limber pine seedlings were also 

spaced out (15 seedlings per Styroblock) to increase 

the likelihood of infection. The bristlecone seedlings, 

however, were directly planted into the Styroblocks, so 

an entire Styroblock was randomly assigned to a treatment block. 

Trees were inoculated on September 18, 2003, with 

naturally infected Ribes leaves placed above the 

trees (Ribes species are the main alternate host). The 
152 USDA Forest Service RMRS-P-76. 2018.

infected Ribes leaves were collected from forest stands 

in Oregon and Washington (USA) and represent nonvcr2 sources. Treatments consisted of three target 

inoculum densities: low (3,000 basidiospores cm-2), 

medium (6,000 basidiospores cm-2), and high (9,000 

to 10,000 basidiospores cm-2). At DGRC, 3,000 spores 

cm-2 is the density used in operational inoculations 

of western white pine (P. monticola); 6,000 spores 

cm-2 is the density used in inoculation trials of sugar 

pine (P. lambertiana). When the targeted inoculum 

Figure 1—Limber pine seedlings with stem symptoms and mortality following inoculation by 

C. ribicola at three inoculum spore densities.

Figure 2—Bristlecone pine seedlings with stem symptoms and mortality following 

inoculation by C. ribicola at three inoculum spore densities.
USDA Forest Service RMRS-P-76. 2018. 153

density was reached, the Ribes leaves were removed 

from that treatment. The actual inoculation densities 

achieved were somewhat higher than the target (3,260, 

8,227, and 12,053 spores cm-2) (table 1). Basidiospore 

germination was monitored at inoculation and was at 

least 90 percent for all treatments except the rep one 

high density, which was 52 percent. Seedlings were 

transplanted in October 2003 to three standard DGRC 

outdoor planting boxes (0.9 m wide × 1.2 m long × 0.3 

m high). One box was used per species replication, and 

there were three contiguous rows of 10 limber pine 

and 8 to 9 bristlecone pine seedlings per treatment. 

Seedlings were assessed for needle lesions (spots) 

twice during the first year and for stem symptoms 

yearly for 5 years and then at 10 years. 

RESULTS

At the first-year assessments, we noted that inoculum 

spore density did not affect measures of infection on 

limber pine as much as Rocky Mountain bristlecone 

pine (table 2). The effect of inoculum density could be 

noted in bristlecone pine after 2 years, but limber pine 

was so susceptible that the low spore density caused as 

much infection as the high concentration (figs. 1 and 

2). We also found that averaged over all spore densities at 1 year post-inoculation, limber pine exhibited 

a much higher percentage (61 percent) of stems with 

symptoms than bristlecone pine (26 percent). By 10 

years post-inoculation, 83 percent of limber pine and 

76 percent of bristlecone pine seedlings exhibited stem 

symptoms, with little change from the 5-year assessment (figs. 1, 2, 3–5; table 2). There was little or no 

change in percentage of stems infected after year 2, so 

we assume there was little to no natural infection from 

spores in the DGRC area between year 1 and 10. Aecia 

production occurred by year 3 on many seedlings (figs. 

5 and 6). On average, 83 percent of limber pine and 

74 percent of bristlecone pine seedlings were dead 10 

years post-inoculation, and the temporal dynamics of 

mortality were similar (figs. 1 and 2). 

At the first assessment, 10 months post-inoculation, 

limber pine showed a much higher incidence of needle 

spots per tree (15 to 23; fig. 6) compared to the 1 to 2 

spots per tree on Rocky Mountain bristlecone pine at 

the three inoculum densities (table 2). The percentage 

of seedlings with spots on secondary needles was also 

much higher in limber pine (mean of 92 percent), with 

89 percent of seedlings exhibiting needle spots at the 

low inoculum density, 98 percent at medium density, 

and 90 percent at high density. In Rocky Mountain 

bristlecone pine the mean was 35 percent of seedlings 

with needle spots, with 23 percent at low inoculum 

density, 42 percent at medium density, and 45 percent 

at high density. Rocky Mountain bristlecone pine seedlings had a fairly high percentage of primary needles 

with needle spots (34 to 69 percent over the three 

densities), whereas limber pine had only 3 to 7 percent 

of seedlings with primary needle spots. This difference 

is most likely because the limber pine seedlings were 

3 years old and had relatively few primary needles 

present compared to the 2-year-old bristlecone pine 

seedlings. 

SUMMARY AND DISCUSSION

The bulk seedlots from the Rollinsville, Colorado, 

area produced Rocky Mountain bristlecone and limber 

pine seedlings that were very susceptible to white pine 

blister rust. The levels of inoculum density utilized 

influenced infection levels in bristlecone but not limber pine. The lowest inoculum density level used here 

(~3,260 spores cm-2) may be adequate in future trials 

for limber pine, but Rocky Mountain bristlecone pine 

is likely to require higher inoculum densities. 

Over all inoculum densities, limber pine had a higher 

percentage (61 percent) of stems infected than bristlecone (26 percent) 1 year after inoculation, but by year 

5 this was narrowed to 83 and 75 percent, respectively, 

indicating that latent canker development (a possible 

partial resistance trait) was more common in bristlecone pine (table 2). Both species had latent infections 

developing after those present 1 year post-inoculation. 

The 26-percent survival by bristlecone and 17-percent 

survival of limber pine at year 10 may indicate a 

source of resistance in the source population as seen 

by subsequent tests (Schoettle et al. 2011, 2014). 

However, only a few inoculated seedlings with stem 

infections survived the duration of the trial, indicating 

limited occurrence of bark resistance. 

The canker-free seedlings in limber pine may be predominantly due to major gene resistance (Schoettle et 

al. 2014). Major gene resistance has been noted in limber pine (Schoettle et al. 2014) and would not generally be influenced by inoculum density, but this type of 
154 USDA Forest Service RMRS-P-76. 2018. Table 2—Average number of spots and stem symptoms per tree and percentages of pine seedlings with stem symptoms and killed by white pine blister rust infections. Inoculum density Year one needle spots/tree Year one stem symptoms/tree % seedlings with stem symptoms / % mortality year one year five year ten limber bristlecone limber bristlecone limber bristlecone limber bristlecone limber bristlecone low 15 1 3.3 0.2 55/ 0 11/ 0 84/ 78 62/ 53 84/ 83 64/ 58 Medium 23 1 7.2 0.6 72/ 0 31/ 0 89/ 84 77/ 73 89/ 88 78/ 77 High 19 2 4.2 0.9 55/ 0 36/ 0 77/ 76 87/ 84 77/ 77 87/ 87 Table 1—Mean inoculation time, inoculum density, and basidiospore germination percentage (+ 1 standard error) for three treatments of bristlecone and limber pine. Treatment Duration (h) Mean inoculum density % basidiospore germination Low 7.1 + 0.3 3260 + 155 94.7 + 2.6 Medium 11.4 + 1.4 8227 + 590 95.0 + 1.5 High 11.3 + 0.1 12053 + 670 81.0 + 14.7 
USDA Forest Service RMRS-P-76. 2018. 155

Figure 3—Limber pine seedling 1 year after inoculation with needle spots.

Figure 4—Limber pine seedling 1 year after inoculation with stem symptoms and 

needle spots.
156 USDA Forest Service RMRS-P-76. 2018.

Figure 5—Limber pine with aecia 3 years after inoculation. Figure 6—Bristlecone pine 3 years after inoculation with 

aecia on the stem.

resistance has not been confirmed in bristlecone pine. 

The increasing level of infection in bristlecone pine 

with increasing inoculum density may suggest that partial resistance is present and can be at least somewhat 

eroded at very high inoculum densities. Future trials 

should investigate even higher levels of inoculum 

density to ascertain whether some level of resistance 

is maintained in Rocky Mountain bristlecone pine 

under extreme pathogen pressure. The inoculum levels 

used at DGRC may need adjustment depending on 

the seedling culture (seedling physiological state) and 

local environment pre- and post-inoculation. Primary 

needles are more susceptible than secondary needles. 

Nursery protocols that provide young seedlings with 

predominantly secondary needles, which are likely 

to give the best correspondence with field resistance, 

should be developed. 

Artificial inoculation trials to examine blister rust 

resistance using seed from individual parent tree collections (half-sib seedlots) has begun for seedlings 

of both limber pine and Rocky Mountain bristlecone 

pine (Schoettle et al. 2011, 2014; Sniezko et al. 2011, 

2016). Such trials will help delineate the level and 

type of partial resistance, as has been noted in other 

species (Sniezko et al. 2014). Field trials of these two 

species to validate resistance from seedling trials, like 

those established for P. monticola, P. lambertiana, and 

whitebark pine (P. albicaulis), are needed. Several 

small trials have recently been established (see 

Schoettle et al., this proceedings, Southern Rockies 

Rust Resistance Trial). If confirmed in other tests, the 

level of resistance found in the bulk seedlots used in 

this study is encouraging, and the information can be 

used by land managers for restoration efforts. 
USDA Forest Service RMRS-P-76. 2018. 157

REFERENCES

Schoettle, Anna W.; Sniezko, Richard A.; Kegley, Angelia; 

[et al.]. 2011. Preliminary overview of the first extensive 

rust resistance screening tests of Pinus flexilis and Pinus 

aristata. In: Keane, R.E.; Tomback, D.F.; Murray, M.P.; 

[et al.], eds. The future of high-elevation, five-needle 

white pines in western North America: Proceedings of 

the high five symposium. Proceedings RMRS-P-63. Fort 

Collins, CO: U.S. Department of Agriculture, Forest 

Service, Rocky Mountain Research Station: 265–269.

Schoettle, Anna W.; Sniezko, Richard A.; Kegley, Angelia; 

[et al.]. 2014. White pine blister rust resistance in limber 

pine: Evidence for a major gene. Phytopathology. 

104(2):163–173.

Sniezko, Richard A.; Danchok, Robert; Savin, Douglas 

P.; [et al.]. 2016. Genetic resistance to white pine 

blister rust in limber pine (Pinus flexilis): Major gene 

resistance in a northern population. Canadian Journal 

of Forest Research. 46 (9): 1173–1178. doi:10.1139/

cjfr-2016-0128.

Sniezko, Richard A.; Mahalovich, Mary Francis; Schoettle, 

Anna W.; [et al.]. 2011. Past and current investigations 

of the genetic resistance to Cronartium ribicola in 

high-elevation five-needle pines. In: Keane, R.E.; 

Tomback, D.F.; Murray, M.P.; [et al.], eds. The future 

of high-elevation, five-needle white pines in western 

North America: Proceedings of the high five symposium. 

Proceedings RMRS-P-63. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky 

Mountain Research Station: 246–264.
158 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Southern Rockies Rust Resistance Trial

A.W. Schoettle1, *, K.S. Burns2, W. Jacobi3, J. Popp1, S. Alberts4, T. Douville4, and F. Romaro4

1 USDA Forest Service, Rocky Mountain Research Station, Fort Collins, Colorado USA

2 USDA Forest Service, Rocky Mountain Region, Forest Health Protection, Golden, Colorado, USA 

3 Colorado State University, Fort Collins, Colorado, USA 

4 Medicine Bow National Forest, Laramie, Wyoming, USA 

* aschoettle@fs.fed.us

The nonnative pathogen Cronartium ribicola, the 

cause of the lethal white pine blister rust (WPBR) disease, is spreading through limber pine (Pinus flexilis) 

and Rocky Mountain bristlecone pine (P. aristata) 

forests of the southern Rocky Mountains (USA). An 

integrated regional program—the Proactive Strategy—

is characterizing the infestation and gaining ecological 

and genetic knowledge of these less well studied 

ecosystems. This knowledge will provide the science 

foundation for early interventions to mitigate the development of ecological impacts to the high mountain 

headwater ecosystems (Burns et al. 2008; Schoettle 

and Sniezko 2007; Schoettle et al., this proceedings, 

The Proactive Strategy: Preparing the Landscape for 

Invasion by Accelerating the Evolution of Resistance). 

Identifying and developing planting material with genetic resistance to WPBR, and understanding its field 

performance, is essential for managers to sustain these 

forests into the future. 

The Southern Rockies Rust Resistance Trial (SRRRT) 

was begun in 2013 to field verify genetic resistance 

to WPBR identified during seedling inoculation 

screening tests of limber and Rocky Mountain 

bristlecone pines conducted in collaboration with 

the U.S. Department of Agriculture, Forest Service 

(USDA FS) Dorena Genetic Resource Center (DGRC; 

Cottage Grove, Oregon, USA) and the Institute of 

Forest Genetics (IFG; Placerville, California, USA) 

(Schoettle et al. 2011, 2014). This field test will assess 

if the resistance frequencies and disease symptom 

phenotypes that develop under natural growing and 

inoculation conditions at SRRRT are similar to those 

observed for the same families under the controlled 

inoculation tests of young seedlings in a different 

environment and with a different geographic source 

of the pathogen. Growing conditions, rust hazard, and 

inoculum sources can affect the expression of disease 

resistance and susceptibility, so field verification 

increases restoration deployment guidance and the 

potential for restoration success. 

An existing administrative site on the Pole Mountain 

Unit of Laramie Ranger District, Medicine Bow 

National Forest that was used as a USDA FS nursery 

in the past was revitalized for this project. Seed from 

previously tested resistant and susceptible individualtree collections (i.e., families) was sown March 

22–23, 2012, and seedlings grown at the Colorado 

State Forest Service Nursery (Fort Collins, Colorado). 

Thirteen limber pine families and 11 Rocky Mountain 

bristlecone pine families are included in the study and 

represent seed sources from throughout the southern 

Rocky Mountains (fig. 1). Administrative approval 

and site preparation were completed in 2012 and 2013. 

More than 700 seedlings were outplanted August 

26–28, 2013, and another 700 seedlings planted May 

27–28, 2014 (figs. 2 and 3). White pine blister rust is 

common in the limber pine forest in and around the 

SRRRT site, providing a natural source of inoculum to 
USDA Forest Service RMRS-P-76. 2018. 159

the seedlings; ponderosa pine (P. ponderosa) is also a 

common forest species in the area. The seedlings will 

be periodically assessed for signs and symptoms of 

WPBR over the next 10 years. This project was funded 

largely by the USDA Forest Service Rocky Mountain 

Region, Regional Office and is a cooperative effort 

between the Medicine Bow National Forest, Forest 

Health Protection, Rocky Mountain Research Station, 

and Colorado State University. Wyoming Conservation 

Corps provided a volunteer field crew that was instrumental in completing site preparation, and citizen 

and University of Wyoming volunteers assisted with 

seedling plantings.

Additional field trials of southern Rockies seed sources 

have been established at Happy Camp (California) 

with previously tested families of both species: Rocky 

Mountain bristlecone pine planting in 2006 and limber 

pine in 2012. The Happy Camp planting site is a component of the Pacific Southwest Region’s Sugar Pine 

Resistance Program and offers exposure to the virulent 

strain of the C. ribicola (vcr1) that can overcome Cr1 

complete resistance in sugar pine (P. lambertiana). 

Additional previously tested P. flexilis seedlings from 

the southern Rocky Mountains have also been planted 

in 2008 in an area of the Deschutes National Forest 

(Oregon). 

All of these plantings, and those at SRRRT, include 

families that showed resistance in seedling inoculation 

testing at DGRC or IFG as well as families shown to 

be highly susceptible to C. ribicola. The susceptible 

families are monitored to detect exposure of the seedlings to rust spores to confirm that both resistant and 

Figure 1—Source locations of the WPBR-resistant and susceptible families in the southern Rocky Mountains 

and the location of the SRRRT site. Yellow and red symbols denote source locations for bristlecone and limber 

pine families, respectively.
160 USDA Forest Service RMRS-P-76. 2018.

Figure 2—A. Planting seedling on the site. B. Overview of planting area.

A.

B.
USDA Forest Service RMRS-P-76. 2018. 161

susceptible seedlings are being challenged over time. 

The resistant limber pine families have shown predominantly major gene resistance (Schoettle et al. 2011, 

2014). Resistance in Rocky Mountain bristlecone pine 

is less well characterized at this point (Jacobi et al., 

this proceedings; Schoettle et al., this proceedings, 

Patterns of Resistance to White Pine Blister Rust in 

Rocky Mountain Bristlecone Pine (Pinus aristata)). 

REFERENCES

Burns, K.S.; Schoettle, A.W.; Jacobi, W.R.; Mahalovich, 

M.F. 2008. Options for the management of white 

pine blister rust in the Rocky Mountain Region. Gen. 

Tech. Rep. RMRS-GTR-206. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky 

Mountain Research Station. 26 p. https://www.fs.usda.

gov/treesearch/pubs/29450. (02 January 2016).

Schoettle, A.W.; Burns, K.S.; Cleaver, C.M.; Connor, J.J. 

[in press]. Proactive limber pine conservation strategy 

for the greater Rocky Mountain National Park Area. 

Gen. Tech. Rep. RMRS-GTR-xxx. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky 

Mountain Research Station.

Schoettle, A.W.; Sniezko, R.A. 2007. Proactive 

intervention to sustain high elevation pine ecosystems 

threatened by white pine blister rust. Journal of Forest 

Research. 12: 327–336. http://www.springerlink.com/

content/9v91t44278w74430/fulltext.pdf. (02 January 

2016).

Schoettle, A.W.; Sniezko, R.A.; Kegley, A.; Burns, K.S. 

2014. White pine blister rust resistance in limber pine: 

evidence for a major gene. Phytopathology. 104: 163–

173. https://apsjournals.apsnet.org/doi/pdfplus/10.1094/

PHYTO-04-13-0092-R. (02 January 2016).

Schoettle, A.W.; Sniezko, R.A.; Kegley, A.; Burns, K.S. 

2011. Preliminary overview of the first extensive rust 

resistance screening tests of Pinus flexilis and Pinus 

aristata. In: Keane, R.E.; Tomback, D.F.; Murray, 

M.P.; Smith, C.M., eds. The future of high-elevation, 

five-needle white pines in western North America: 

proceedings of the high five symposium. Proceedings 

RMRS-P-63. Fort Collins, CO: U.S. Department of 

Agriculture, Forest Service, Rocky Mountain Research 

Station: 265–269. https://www.fs.fed.us/rm/pubs/rmrs_

p063/rmrs_p063_265_269.pdf. (02 January 2016).

Figure 3—Overview of the SRRRT site.
162 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Sugar Pine (Pinus lambertiana) Partial Resistance Heritability Study

J. Dunlap1, *, D. Burton2, D. Davis2, D. Vogler3, and B. Westfall4

1 USDA Forest Service, Region 5 Genetic Resources Program, Placerville Nursery, Camino, California, USA 

2 USDA Forest Service, Region 5 Genetic Resources Program (retired)

3 USDA Forest Service, Pacific Southwest Research Station, Institute of Forest Genetics, Placerville, California, USA

4 USDA Forest Service, Pacific Southwest Research Station, Albany, California, USA

* jdunlap@fs.fed.us

The health and regeneration of sugar pine (Pinus 

lambertiana), a major species in the mountain forests 

of California (USA), has been substantially affected by 

an exotic pathogen, Cronartium ribicola, which causes 

white pine blister rust. Seedlings from trees with major 

gene resistance (MGR) are being used to increase field 

survival in plantations. Partial resistance (PR) due to 

multiple genes is considered to be more durable than 

MGR, providing protection to sugar pines exposed to 

a virulent form of blister rust. In 2006, a field study 

was established on the Klamath National Forest to 

examine PR traits and their inheritance in progeny from 

128 families whose parents were known to produce 

rust-susceptible (FR) or PR progeny as well as parents 

having PR traits but without information about their 

progeny. The crossing scheme was a mixed-factorial 

diallel; the field design was randomized incomplete 

blocks with noncontiguous single-tree plots. Trees 

were planted at close spacing, irrigated, and naturally 

exposed to blister rust over multiple years from onsite 

Ribes species, the alternate hosts. Data on mortality 

from rust, number of rust cankers, and canker types 

were collected from 2010 to 2013. Progeny from PR 

parent(s) had a substantially lower percentage of trees 

that died from blister rust (8 to 53 percent with three to 

one PR trees in a family) when compared to progeny 

from FR parents (64 to 99 percent with one to two FR 

parents in a family). Progeny of PR parents also had 

a higher percentage of trees that were clean of rust (6 

to 23 percent) and with no active infections (17 to 58 

percent) compared to progeny from FR parents (0 to 

2 percent clean of rust; 0 to 10 percent with no active 

infection). Progeny from other selected parents, based 

on their PR phenotypes, performed more like progeny 

from one PR parent or intermediate between one and 

two PR parents, depending on the trait examined. 

Quantitative analyses from 2-year data revealed that 58 

and 78 percent of the variance in PR inheritance was 

additive (compare with Kinloch et al. 2012). Further 

analyses are needed on more parents and to examine 

components of heritability. Seed orchards with trees 

having MGR, PR, or both traits have been established 

for the western Sierra Nevada and are in development 

for northern California forests. 

REFERENCES

Kinloch, Bohun B., Jr.; Burton, Deems; Davis, Dean A.; [et 

al.]. 2012. Strong partial resistance to white pine blister 

rust in sugar pine. In: Sniezko, Richard A.; Yanchuk, 

Alvin D.; Kliejunas, John T.; [et al.], tech coords. 

Proceedings of the fourth international workshop on 

the genetics of host-parasite interactions in forestry: 

Disease and insect resistance in forest trees. Gen. Tech. 

Rep. PSW-GTR-240. Albany, CA: U.S. Department of 

Agriculture, Forest Service, Pacific Southwest Research 

Station: 81–90.
USDA Forest Service RMRS-P-76. 2018. 163

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Patterns of Resistance to Cronartium ribicola in Rocky Mountain 

Bristlecone Pine (Pinus aristata)

A.W. Schoettle1, *, R.A. Sniezko2, A. Kegley2, and K.S. Burns3

1 USDA Forest Service, Rocky Mountain Research Station, Fort Collins, Colorado, USA

2 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA 

3 USDA Forest Service, Forest Health Protection, Golden, Colorado, USA

* aschoettle@fs.fed.us

The core distribution of Rocky Mountain bristlecone 

pine (Pinus aristata) extends from central Colorado 

into northern New Mexico with a disjunct population 

in northern Arizona (USA). Populations are primarily 

at high elevations and often define the alpine treeline, 

but the species is also found in open mixed-conifer 

stands with ponderosa (P. ponderosa) or pinyon (P. 

edulis) pines. On dry, exposed sites the stands are open 

and bristlecone is commonly the only species present. 

The combination of the pine’s adaptive traits with 

infrequent disturbance has enabled trees on these sites 

to attain ages of more than 2,500 years. These same 

traits and conditions will inevitably hinder the ability 

of bristlecone pine to adapt to novel anthropogenic 

stresses such as climate change and infection by the 

nonnative pathogen (Cronartium ribicola) that causes 

the lethal disease white pine blister rust (WPBR). 

Infection of bristlecone pine by C. ribicola was first 

documented in the field in 2003 in south-central 

Colorado (Blodgett and Sullivan 2004). These threats 

and the species’ unique aesthetic and ecological roles 

make bristlecone pine a species of conservation interest, and we have begun implementing the Proactive 

Strategy (Schoettle 2004). 

The frequency of genetic resistance to WPBR will 

affect the trajectory of bristlecone pine populations 

in the future. Here we report results from two of our 

studies of rust resistance for bristlecone pine families 

(Schoettle et al. 2011). Seeds collected in 2001 

from 184 individual trees across 11 sites throughout 

Colorado were sown in 2002. The 3-year-old seedling 

families were inoculated with C. ribicola at Dorena 

Genetic Resource Center (Cottage Grove, Oregon, 

USA) in 2005. Seeds for an additional smaller test 

were sown in April 2009 and seedlings inoculated 

with C. ribicola in September 2009. Needle infection 

lesions (spots) were easily identified on needles of the 

seedlings inoculated at a young age (2009 trial), but 

were less obvious on needles of the seedlings inoculated when older (2005 trial). However, WPBR stem 

symptoms developed on many of the older seedlings 

in the 2005 trial for which infection spots were not 

observed, suggesting that needle lesions are not a good 

early measure of infection in older bristlecone pine 

seedlings. Bristlecone pine expresses multiple WPBRresistant phenotypes, including canker-free seedlings, 

seedlings with partial bark reactions, seedlings developing stem infections later or showing longer survival 

with stem infections, and seedlings developing stem 

infections with infections becoming inactive over time; 

the frequency of each varied geographically. 

REFERENCES

Blodgett, J.T.; Sullivan, K.F. 2004. First report of white pine 

blister rust on Rocky Mountain bristlecone pine. Plant 

Disease. 88: 311.
164 USDA Forest Service RMRS-P-76. 2018.

Schoettle, A.W. 2004. Ecological roles of five-needle pines 

in Colorado: Potential consequences of their loss. In: 

Sniezko, Richard A.; Samman, Safiya; Schlarbaum, 

Scott E.; [et al.], eds. Breeding and genetic resources 

of five-needle pines: Growth, adaptability and pest 

resistance. IUFRO Working Party 2.02.15. Proceedings 

RMRS-P-32. Fort Collins, CO: U.S. Department of 

Agriculture, Forest Service, Rocky Mountain Research 

Station: 124–135.

Schoettle, A.W.; Sniezko, R.A.; Kegley, A.; Burns, K.S. 

2011. Preliminary overview of the first extensive rust 

resistance screening tests of Pinus flexilis and Pinus 

aristata. In: Keane, R.E.; Tomback, D.F.; Murray, 

M.P.; Smith, C.M., eds. The future of high-elevation, 

five-needle white pines in western North America: 

Proceedings of the high five symposium. Proceedings 

RMRS-P-63. Fort Collins, CO: U.S. Department of 

Agriculture, Forest Service, Rocky Mountain Research 

Station: 265–269.
USDA Forest Service RMRS-P-76. 2018. 165

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Genetic Resistance to Cronartium ribicola in Limber Pine (Pinus flexilis)

A.W. Schoettle1, *, R.A. Sniezko2, A. Kegley2, and K.S. Burns3

1 USDA Forest Service, Rocky Mountain Research Station, Fort Collins, Colorado, USA 

2 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA

3 USDA Forest Service, Forest Health Protection, Golden, Colorado, USA

* aschoettle@fs.fed.us

Limber pine (Pinus flexilis) is being threatened by the 

lethal disease white pine blister rust (WPBR) caused 

by the nonnative pathogen Cronartium ribicola. The 

types and frequencies of genetic resistance to the rust 

in native populations before invasion are likely to 

determine the potential success of restoration or proactive measures. 

The first extensive inoculation trials using individual 

tree seed collections from more than 100 limber 

pine trees confirm that genetic segregation of a stem 

symptom-free trait to blister rust is consistent with 

inheritance by a single dominant resistance gene (Cr4) 

and the resistance allele appears to be distinct from 

the Cr2 allele in western white pine (P. monticola) 

(Schoettle et al. 2014). The frequency of the Cr4 allele 

across healthy and recently invaded populations in the 

southern Rocky Mountains was unexpectedly high (5.0 

percent, ranging from 0 to 13.9 percent), and northern 

Colorado (USA) appears to be a hotspot. Other tests 

by us and others suggest that the Cr4 allele may be 

absent in populations in the U.S. northern Rockies and 

California. An ongoing inoculation trial of 74 limber 

pine families confirms the low frequency of bark 

reaction and resistance mechanisms for slow canker 

development.

Genetic resistance is present in limber pine populations and occurs at frequencies that are detectable even 

in populations that have not yet been invaded by the 

pathogen (i.e., sampling without the benefit of putative 

resistance phenotypic selection in the field). The 

limber pine seed trees that our trials reveal to have the 

Cr4 allele will serve as monitors for the evolution of 

the vcr4 virulent allele in C. ribicola (Schoettle et al., 

in press). To date, the Cr4 trees in populations sampled 

here and exposed to WPBR for more than 20 years remain disease-free. The combination of low stand densities and open canopies of southern Rocky Mountain 

limber pine stands and the lack of a strong relationship 

between proximity to Ribes and disease incidence 

in limber pine suggest long-distance transport of C. 

ribicola spores in these ecosystems. The atmospheric 

mixing of spores from multiple C. ribicola populations over extensive landscapes may reduce localized 

selection pressure on C. ribicola and, therefore, slow 

the proliferation of virulence. However, relying on 

Cr4 alone to sustain limber pine populations into the 

future in the presence of increasing WPBR pressure is 

not recommended and investigations to identify other 

more durable types of resistance to WPBR in limber 

pine are underway. The identification of Cr4 and other 

types of resistance in native populations of limber pine 

early in the invasion process in this region provides 

useful information for predicting near-term impacts 

and structuring long-term management strategies 

(Schoettle and Sniezko 2007). 

REFERENCES

Schoettle, A.W.; Burns, K.S.; Cleaver, C.M.; Connor, J.J. 

[in press]. Proactive limber pine conservation strategy 

for the greater Rocky Mountain National Park Area. 
166 USDA Forest Service RMRS-P-76. 2018.

Gen. Tech. Rep. RMRS-GTR-xxx. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky 

Mountain Research Station.

Schoettle, A.W.; Sniezko, R.A. 2007. Proactive 

intervention to sustain high elevation pine ecosystems 

threatened by white pine blister rust. Journal of Forest 

Research. 12: 327–336. http://www.springerlink.com/

content/9v91t44278w74430/fulltext.pdf. (05 January 

2016).

Schoettle, A.W.; Sniezko, R.A.; Kegley, A.; [et al.]. 

2014. White pine blister rust resistance in limber pine: 

Evidence for a major gene. Phytopathology. 104:163–

173. https://apsjournals.apsnet.org/doi/pdfplus/10.1094/

PHYTO-04-13-0092-R. (05 January 2016).
USDA Forest Service RMRS-P-76. 2018. 167

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Blister Rust Resistance in Whitebark Pine (Pinus albicaulis) at Crater 

Lake National Park

J.S.H. Beck1 and R.A. Sniezko2, *

1 USDI, National Park Service, Crater Lake National Park, Crater Lake, Oregon, USA 

2 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA 

* rsniezko@fs.fed.us

Abstract—Whitebark pine (WBP, Pinus albicaulis) has been declining at Crater Lake National Park 

(CLNP) in southern Oregon, USA for several decades due to infection with white pine blister rust, caused 

by the pathogen Cronartium ribicola. This decline has been exacerbated in the past decade by significant 

levels of additional mortality caused by the mountain pine beetle (MPB, Dendroctonus ponderosae). In 

2003, CLNP initiated a partnership with the Dorena Genetic Resource Center (DGRC) to collect WBP 

cones from phenotypically rust-resistant trees within the park and test progeny of these parent trees for 

genetic resistance to blister rust. As part of this effort: (1) seed is collected from candidate trees and seedlings are infected via artificial inoculation to assess their resistance and rate the parents’ rust resistance; 

(2) trees identified as rust resistant are protected from MPB attack by application of verbenone; (3) extra 

seed is available for use in (i) genetic trials to gather further information on adaptive genetic variation in 

progeny of CLNP parent trees, (ii) ex situ gene conservation, and (iii) restoration plantings at CLNP; and 

(4) information is made available to the public on the status of WBP at CLNP. Through fall 2013, seed 

collections have been made from 101 parent trees, and 63 of these are currently in rust resistance trials 

at DGRC. Half of the seedling families show low-to-moderate levels of resistance. CLNP implemented 

a collection tree monitoring program in 2012 that tracks the fate of trees with progeny in rust resistance 

screening trials on an annual basis; results from monitoring can be directly tied to those from resistance 

screening trials and restoration outplantings. Thirty-one percent of trees with progeny showing the highest 

level of rust resistance are now dead; all were killed by MPB attack.

It is imperative to keep efforts going to identify new rust-resistant trees, protect trees with known or potential rust resistance, and preserve their genetic resources before they are lost. Seedlings from trees with 

known resistance to blister rust have been planted in restoration outplantings at CLNP. Family identity 

has been retained for the seedlings in these plantings to permit more information to be learned about 

the genetic variation present in the population of WBP trees at CLNP and its influences on traits such as 

survival, growth, and rust resistance. Small quantities of seed from most of the resistant parent trees are 

currently being stored for gene conservation and potential restoration use.
168 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

The Definitive Guide for White Pine Blister Rust Resistance in

White Pine Species of Western North America (beta 2014.0 

version)—a Tree Breeder’s Perspective

R.A. Sniezko1, * and A. Kegley1

1 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA

* rsniezko@fs.fed.us 

The nine native species of white pines in the United 

States (four of which also occur in Canada) are all 

highly susceptible to white pine blister rust caused by 

the fungal pathogen Cronartium ribicola. Research 

and applied programs in the United States and Canada 

to develop resistant populations of white pine species 

have been underway for more than 50 years. The 

regional program based at the U.S. Department of 

Agriculture Forest Service’s Dorena Genetic Resource 

Center (DGRC; Cottage Grove, Oregon, USA) has the 

most experience and continuity and has been active for 

over 50 years. The DGRC has evaluated rust resistance 

in families of eight of the nine species of white pines 

that are native to the United States, for an array of 

resistance responses in seedling inoculation trials (and 

seedlings for the remaining species, foxtail pine [Pinus 

balfournia], are slated for inoculation in fall 2015). 

Inoculations of seedlings of more than 10,000 families 

have been completed over this time. A subset of these 

families has been tested several times in different 

years or with different geographic sources of rust and 

differing inoculum densities, or a combination thereof. 

Another subset of these families—western white 

pine (P. monticola), sugar pine (P. lambertiana), and 

whitebark pine (P. albicaulis)—has been used to established field trials to validate the results of seedling 

screening and to monitor the durability of the different 

types of resistance (Sniezko et al. 2012). The extensive 

experience gained through this large series of trials 

has helped refine the understanding of the types of 

resistance and their frequencies (Kegley and Sniezko 

2004; Sniezko et al. 2008, 2011, 2012, 2014). These 

screening trials have focused on both major gene 

resistance (present in four of the species; see Schoettle 

et al. 2014) and other types of resistance apparent in 

the 5 years of observations following each artificial 

inoculation (Kegley and Sniezko 2004; Sniezko et al. 

2011, 2012, 2014). 

In this talk, we will present some results from both 

inoculation trials and field tests to provide insights into 

the current state of knowledge of frequency, levels, 

and types of rust resistance present (using western 

white pine, whitebark pine, southwestern white pine 

[P. strobiformis], and sugar pine as examples) in our 

North America species. Results from the latest control 

crosses of western white pine will be used to help 

assess the potential level of resistance that may be 

possible in the next generation of improvement. A detailed overview of the genetic resistance present, using 

western white pine as an example, has recently been 

published (Sniezko et al. 2014).

REFERENCES

Kegley, A.; Sniezko, R.A. 2004. Variation in blister rust 

resistance among 226 Pinus monticola and 217 P. 

lambertiana seedling families in the Pacific Northwest. 

In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, 

Scott E.; [et al.], eds. Breeding and genetic resources 

of five-needle pines: Growth, adaptability, and pest 
USDA Forest Service RMRS-P-76. 2018. 169

resistance. Proceedings RMRS-P-32. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky 

Mountain Research Station: 209–226.

Schoettle, A.W.; Sniezko, R.A.; Kegley, A.; [et al.]. 

2014. White pine blister rust resistance in limber pine: 

Evidence for a major gene. Phytopathology. 104(2): 

163–173.

Sniezko, R.A.; Danchok, R.; Hamlin, J.; [et al.]. 2012. 

White pine blister rust resistance of 12 western white 

pine families at three field sites in the Pacific Northwest. 

In: Sniezko, R.A.; Yanchuk, A.D.; Kliejunas, J.T.; [et 

al.], tech. coords. Proceedings of the fourth international 

workshop on the genetics of host-parasite interactions 

in forestry: Disease and insect resistance in forest trees. 

Gen. Tech. Rep. PSW-GTR-240. Albany, CA: U.S. 

Department of Agriculture, Forest Service, Pacific 

Southwest Research Station: 356–367.

Sniezko, R.A.; Kegley, A.J.; Danchok, R. 2008. White pine 

blister rust resistance in North American, Asian, and 

European species—Results from artificial inoculation 

trials in Oregon. Annals of Forest Research. 51: 53–66.

Sniezko, R.A.; Mahalovich, M.F.; Schoettle, A.W.; [et 

al.]. 2011. Past and current investigations of the genetic 

resistance to Cronartium ribicola in high-elevation 

five-needle pines. In: Keane, R.E.; Tomback, D.F.; 

Murray, M.P.; [et al.], eds. The future of high-elevation, 

five-needle white pines in western North America: 

Proceedings of the high five symposium. Proceedings 

RMRS-P-63. Fort Collins, CO: U.S. Department of 

Agriculture, Forest Service, Rocky Mountain Research 

Station: 246–264.

Sniezko, R.A.; Smith, J.; Liu, J-J.; [et al.]. 2014. Genetic 

resistance to fusiform rust in southern pines and white 

pine blister rust in white pines—A contrasting tale of two 

rust pathosystems—Current status and future prospects. 

Forests. 5(9): 2050–2083. doi:10.3390/f5092050.
170 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Performance of Blister Rust-Resistant Eastern White Pine

(Pinus strobus) Hybrids With Himalayan Blue Pine

(P. wallichiana) in Field Trials

P. Lu1, * 

1 Ontario Ministry of Natural Resources, Ontario Forest Research Institute, Sault Ste. Marie, Ontario, Canada

* pengxin.lu@ontario.ca

Abstract—Introducing white pine blister rust (caused by Cronartium ribicola) resistance genes to eastern 

white pine (Pinus strobus) from Eurasian five-needle pine species is a breeding strategy used in Ontario, 

Canada, to help regenerate eastern white pine at sites with high hazard ratings. Among Eurasian fiveneedle pine species, Himalayan blue pine (P. wallichiana) was identified as a desirable resistance gene 

donor because some of its hybrids with P. strobus demonstrated heritable and strong blister rust resistance 

as well as improved adaptation and growth after backcrossing to P. strobus. Data from field trials provided 

evidence of the effectiveness of this breeding approach and a resistance screening protocol that evaluates 

resistance by using the post-inoculation survival rate of 6-month-old seedlings.

At a field site near Sault Ste. Marie, Ontario, where greater than 90 percent of previously planted eastern 

white pine trees died of blister rust or had rust cankers at a plantation age of 10 years, 3.7 percent of 

replanted hybrid progeny from eight top-ranking open-pollinated families (7.0 percent for all hybrids) 

developed stem or branch cankers, compared with 13.3 percent of commercial eastern white pine controls 

4 years post-trial establishment. Furthermore, none of the 263 trees propagated (by using rooted cuttings) 

from seedlings that survived blister rust infection had developed stem or branch cankers in the same trial. 

In another field trial, hybrid progeny of P. strobus with P. wallichiana, which had about a 75-percent P. 

strobus pedigree, demonstrated a growth rate and resistance to weevil (superfamily Curculionoidea) attack 

that were similar to those of pure eastern white pine at a trial age of 9 years. Because somatic embryogenesis (SE) is operationally applicable for vegetative propagation of eastern white pine and its hybrids, clonal 

deployment of SE emblings from elite hybrid backcrosses carrying up to a 94 percent P. strobus pedigree 

may be an appealing strategy to forest products companies and others for regenerating white pine at sites, 

such as those in northern Ontario, where eastern white pine is often decimated by blister rust.
USDA Forest Service RMRS-P-76. 2018. 171

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Field-Level Resistance of Western White Pine (Pinus monticola)

to White Pine Blister Rust Assessed in Progeny Trials Planted in 

Coastal British Columbia

N. Ukrainetz1 and J.N. King2, *

1 British Columbia Ministry of Forests, Lands and Natural Resource Operations, Tree Improvement Branch,

Victoria, British Columbia, Canada

2 British Columbia Ministry of Forests, Lands and Natural Resource Operations, Tree Improvement Branch (retired)

* king.forgen@gmail.com

Abstract—Partial resistance of western white pine (Pinus monticola) to the invasive pathogen Cronartium 

ribicola (white pine blister rust) was detected in controlled inoculation experiments, which found variation 

in resistance among half-sib families of parents selected for resistance in natural stands. Information from 

these experiments was used to select parent trees for breeding. Breeding was conducted on some of 

the parent trees in natural stands, but mainly on grafts in seed orchards. The resulting F1 crosses were 

deployed in four series of field progeny tests (16 test sites) in coastal British Columbia, Canada between 

2001 and 2006. Approximately 100 full-sib cross families were established per series. In addition to providing a population for recurrent selection, the F1 full-sib design will allow some genetic analysis. Included 

in these field trials were a series of diallels created at the U.S. Department of Agriculture, Forest Service 

Dorena Genetic Resource Center (Cottage Grove, Oregon, USA), which will help this genetic analysis.

The field tests have been assessed for blister rust infection and height growth. Some preliminary genetic 

analysis results will be presented. One finding is that selfed families had higher levels of resistance, 

but a lower growth rate, suggesting the possibility that recessive genes may be involved in partial resistance. Although not explicitly part of the F1 program, major gene resistance has also been deployed 

in British Columbia. Families with major gene resistance do well against blister rust on coastal sites in 

British Columbia although certain families appear more susceptible to other fungal pathogens such as 

Dothistroma pini (cause of Dothistroma needle blight). We present these results and discuss some of the 

implications for the breeding and orchard programs in British Columbia.
172 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Genetic Architecture of Quantitative Melampsora larici-populina

Leaf Rust Resistance in Poplars (Populus spp.)

V. Jorge1, *, A. Dowkiw1, R. El-Malki1, V. Segura1, V. Guérin1, P. Poursat3,

P. Faivre-Rampant2, A. Rae4, G. Taylor5, and C. Bastien1

1 Institut National de la Recherche Agronomique, Unité Amélioration, Génétique et Physiologie Forestières, Orléans, France

2 Institut National de la Recherche Agronomique, Unité de Recherches en Génomique Végétale, Evry, France

3 Institut National de la Recherche Agronomique, Génétique et Biomasse Forestières Orléans, France

4 Cherry Valley Farms Limited, United Kingdom

5 University of Southampton, Plants & Environment Lab, United Kingdom

* veronique.jorge@inra.fr

Poplar (Populus spp.) leaf rusts caused by Melampsora

species are considered among the most crucial sanitary 

problems threatening poplar stands worldwide. Several 

major resistance factors specific to poplar rusts have 

been identified, each of them discovered by using a 

single Melampsora species. Most of them are inherited 

from Populus species that did not co-evolve with the 

pathogen. The origin and function of such “exapted” 

resistance are thus called into question.

The purpose of this presentation is to compare the genetics of exapted and co-adapted resistance to M. medusae f. sp. deltoidae (Mmd) and to M. larici-populina

(Mlp), respectively. To achieve this, resistance quantitative trait locus (QTL) against Mlp was studied in two 

pedigrees. The first, an F2 P. trichocarpa × P. deltoides

family, was previously evaluated for resistance to 

Mmd, and a major gene for resistance, Mmd1, inherited from the P. trichocarpa grandparent, was mapped. 

The second pedigree was an F1 P. nigra family.

In the F2 pedigree, 17 QTLs were detected that are 

involved in the 3 components of quantitative resistance 

to the 5 Mlp strains inoculated in laboratory. These 

QTLs explain between 3 and 30 percent of the phenotypic variance. None of these QTLs has a broad-range 

effect. No major resistance factor against Mlp was 

identified in this pedigree, showing that Mmd1 is not 

functional against Mlp strains tested.

In the P. nigra family, 11 QTLs explaining between 2 

and 60 percent of the phenotypic variation observed 

after inoculation with the two Mlp strains were 

detected. All except one of these QTLs were traitor strain-specific. A major QTL inherited from the 

most resistant parent and located at the end of LG I 

explained more than 60 percent of uredinia size variation after inoculation with one Mlp strain and showed 

a moderate effect after inoculation with the other 

strain. Genomic mining of the QTL region revealed a 

cluster of 25 NB-LRR genes. Those results confirm in 

P. nigra the gene-for-gene model suggesting specific 

interactions between QTLs for quantitative resistance 

and Mlp strains. 

The results presented will be compared and discussed together with results of previous studies on 

Melampsora/Salicaceae pathosystems.
USDA Forest Service RMRS-P-76. 2018. 173

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

White Pine Blister Rust Hazard Rating for 265 Sites in

Southern Oregon, USA

H. Koester1, D.P. Savin2, M. Buss2, and R.A. Sniezko2, *

1 USDI Bureau of Land Management (retired), Medford, Oregon, USA

2 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA

* rsniezko@fs.fed.us

Abstract—Sugar pine (Pinus lambertiana) and western white pine (Pinus monticola) are two important 

long-lived conifer species in forests of southwestern Oregon (USA) and elsewhere. Both of these white 

pine species have been in decline for decades due largely to the presence of the nonnative white pine 

blister rust, caused by the fungal pathogen Cronartium ribicola. Many land managers are interested in 

keeping these white pine species present in forest ecosystems or in managed forests, but the very high 

susceptibility of these species to the blister rust and the lack of information rating the landscape for rust 

hazard have prevented their full use. This study provides an overview of the rust infection levels at 265 

sites in southwestern Oregon during the 1980s, including the frequency of cankered trees at each site and 

maps showing the geographic distribution patterns of the differing levels of infection. This information will 

assist land managers in plans for deploying seed from the blister rust resistance programs. All except one 

site had infected white pine species, and the mean percentage of trees with cankers over the 265 sites 

was 43.9 percent, with the average number of cankers per tree ranging from 0 to >11 per site. The mapping of the levels of infection showed some areas of notable low and high infection. Cankers tended to be 

within 2.4 m of the ground, suggesting that early branch pruning of planted trees might increase survival. 

This extensive one-time survey of sites is valuable in helping delineate areas of lowest and highest infection levels, but other data suggest that such surveys may underestimate the level of infection and should 

be complemented by a subset of permanent plots. With the widespread presence and impact of the rust, 

the continued development and use of genetically resistant sugar pine and western white pine seed will be 

vital in retaining or restoring these species to desirable levels in southwestern Oregon.

INTRODUCTION

Sugar pine (SP, Pinus lambertiana) and western white 

pine (WWP, Pinus monticola) are long-lived conifers 

that are important components of forest ecosystems 

in southwestern Oregon (USA). Both of these pines 

are very susceptible to the nonnative fungal pathogen 

Cronartium ribicola, cause of white pine blister rust 

(WPBR). Detailed knowledge of the geographic distribution of the rust and its intensity can be an important 

aid to managers developing restoration or reforestation 

prescriptions with these species. 

The Medford District of the U.S. Department of the 

Interior, Bureau of Land Management (BLM) contains 

nearly 405,000 ha of forest land in southwestern 

Oregon, including many areas with sugar pine, and to 

a lesser extent, western white pine. In the mid-1980s, 

BLM personnel completed a WPBR hazard survey 

of 265 sites in southwestern Oregon. The objective 

of the survey was to document how common the rust 

was in this area and to provide information on relative 

levels of site hazard for rust over this area. This is the 

largest and most intensive WPBR rust hazard survey 

RUST BIOLOGY, ECOLOGY, AND MANAGEMENT
174 USDA Forest Service RMRS-P-76. 2018.

of an area for sugar pine, and perhaps any five-needle 

pine species. The information provided here will serve 

as a valuable tool for land managers in southwestern 

Oregon for use in determining where five-needle pine 

species may be used for reforestation and restoration, 

and where genetically tested resistant trees should be 

used for forest development projects.

In this paper, we quantify rust hazard over the 

landscape for the 265 locations surveyed. Hazard is 

quantified by hazard rating, as discussed by Geils et al. 

(1999) and Burns et al. (2008):

Forest disease outbreaks are described in the 

epidemiological terms of rates of spread and 

intensification, incidence, severity, damage, 

and ecological impacts. Hazard rating provides a means for combining these attributes 

into a relative index or classification for comparing disease potential among sites. Rust 

epidemiology is strongly influenced by genetics, abundance, and proximity of hosts and by 

microclimate... (Geils et al. 1999: 2).

Hazard can be quantified several different 

ways, such as the proportion of infected trees, 

percent mortality, or the # and severity of cankers. By delineating rust hazard zones, managers can focus on white pine management in 

those areas where successful management is 

more likely to be heavily impacted (Burns et al. 

2008: 9).

We present hazard rating data in separate maps using 

proportion of trees affected, number of cankers per 

tree, and rust index.

METHODS

The areas examined in this project were portions of 

southwestern Oregon, primarily BLM’s Medford 

District forest lands, and some adjacent U.S. 

Department of Agriculture, Forest Service (USDA FS) 

and private timber company lands (fig. 1). All BLM 

sites selected were from the Medford District forest 

inventory database of young stands with a component 

of either SP or WWP. Generally, sites were included 

only if they had a minimum of 50 trees and ranged in 

size from 8 to 10 ac (3 to 4 ha), up to 100 ac (40.5 ha). 

Due to various factors, 71 of the 265 sites had fewer 

than 50 trees sampled (lowest number was 25), and 6 

sites had more than 50 trees sampled (1 site had 100 

trees sampled).

Sites were evaluated over a several-year period in 

the mid-1980s. Of the 265 sites, 244 sites contained 

a component of sugar pine, and 21 had western white 

pine present. The scatter of sites in this study provides 

a fairly systematic and representative coverage of 

sites in this area with white pine species. Wild/natural 

regeneration trees occurred on 255 sites, and another 

10 sites had been previously planted (in the mid-to-late 

1960s) with seedlings from putatively rust-resistant 

(RR) parent trees (RRSP and RRWWP). Between 67 

and 2,780 trees were planted at the RR sites.

For the 255 naturally regenerated stands, field crews 

generally inspected 40 to 50 trees per site using a random path method (i.e., walk 50 paces and look left and 

then assess the nearest tree). However, in some sites 

where white pines were at very low frequency, most 

trees in sight were selected to achieve the objective of 

a 40- to 50-tree count. Dead trees were sampled if they 

fell into the random scheme (same as for live trees) 

provided that mortality was recent, and the number 

of cankers could be assessed. The 10 RRSP/RRWWP 

sites were sampled in a slightly modified manner to 

predominantly include only trees (marked or tagged) 

of the RR plot, but some control or natural trees were 

included in some sites.

For individual sample trees, measurements included 

age, height, number of cankers (main stem and 

branches), presence/absence of cankers on the main 

stem, and height of the highest canker. Tree age was 

approximated by counting whorls. For each site, aspect 

and elevation were noted, and township, range, and 

section were used to obtain latitude and longitude at 

the center of each section for mapping purposes.

Basic site criteria were that the trees should be at least 

10 years old and generally less than 6 m in height to 

allow them to be potentially exposed to rust infection 

for a number of years, and to allow for proper identification and counting of cankers. Sites with steep slope 

allowed for taller trees to be assessed. Sites where 

pines were being suppressed by overstory or brush 

were not chosen for assessment.

The collected individual tree data for each site were 

used to calculate percentage of infected trees (PI). 

Rust index (RI), where RI = number cankers per 1,000 

needles per year, was calculated by using a program 
USDA Forest Service RMRS-P-76. 2018. 175

Figure 1—Geographic distribution of lowest to highest rust hazard sites, classified by (a) rust index, (b) average number of 

cankers per tree, and (c) percentage of trees infected. Sugar pine (Pinus lambertiana, 235 sites represented) and western 

white pine (Pinus monticola, 20 sites represented) are shown with filled circles and triangles, respectively. The map area is 

shown in the inset. Sites with improved stock were excluded. Datum used was North American Datum 1983; projection used 

was Albers Equal Area Conic. Maps were generated by Jennifer Christie.

a. b.

c.
176 USDA Forest Service RMRS-P-76. 2018.

developed earlier for western white pine (Hagle et al. 

1989). Only the 255 naturally regenerated sites were 

used to create maps. For mapping rust hazard, rust 

index was divided into three groups, as follows: 

Rust Hazard:

High: Rust index ≥0.009 (~>70 percent of 

trees with cankers)

Medium: Rust index 0.001 to 0.009 (~50 percent of trees with cankers)

Low: Rust index up to 0.001 (~25 percent of 

trees with cankers)

Rust index is a measurement of a tree's or site's rust 

infection level and therefore susceptibility to additional blister rust infections. According to the USDA 

FS Forest Health Technology Enterprise Team [n.d.], 

“The blister rust index, a measure of white pine blister 

rust infection level or potential for a site, area, or 

stand, drives the model. The initial value of the blister 

rust index can be provided by the user, calculated 

from current infections provided with inventory data, 

or calculated based on Ribes populations (white pine 

blister rust-Cronartium ribicola-is heteroecious and its 

alternate host is Ribes).” 

For comparison, we also show maps of the relative rust 

hazard based on number of cankers per tree (using five 

categories) and percentage of trees with cankers (five 

Figure 2—Relationship between tree height and age for 265 

stands surveyed for rust hazard on BLM’s Medford District.

Figure 3—Distribution of percent infected with white pine 

blister rust for (a) sugar pine stands, (b) western white pine 

stands, and (c) sites planted with seedlings from putatively 

resistant parents.
USDA Forest Service RMRS-P-76. 2018. 177

Table 1—Summary information for 255 natural (n.s.) and 10 planted (RR) stands of sugar pine (Pinus 

lambertiana) and western white pine (Pinus monticola) assessed for blister rust in southwest Oregon.

Type of 

site

Number 

of sites Measure Elevation 

(m)

Average 

age

Average 

height (m)

Percent 

infected

Average 

number 

of 

cankers

SP n.s. 235 Ave 930 29.9 3.8 43.6 2.06

Min 340 13 1.8 0 0.00

Max 1,890 65 10.4 94 11.50

SP RR 9 Ave 1,000 19.2 3.3 37.2 1.70

Min 680 17 2.1 6 0.16

Max 1,250 23 4.3 60 3.41

WWP 

n.s.

20 Ave 1,470 24.9 3.5 51.8 2.20

Min 640 15 1.8 4 0.06

Max 1,740 57 7.3 86 5.04

WWP RR 1 1,100 20 4.3 16 0.46

categories). Mean plot data and calculated variables 

(such as rust index) generated after the survey were 

used in creating the map and graphs for this summary.

Statistical analyses were conducted using R version 

3.0.2 (R Foundation for Statistical Computing, Vienna, 

Austria). Natural log transformations were applied to 

the average number of cankers per site and the rust 

index values, and a logit transformation was applied to 

the percent infected data. Very low levels of infection 

(0 to ~5 percent) were rounded to 0.0000 on the rust 

index scale, so the seven sites with these values were 

dropped from linear regression analyses involving rust 

measures. The 10 sites with improved stock were also 

removed from all linear regression analyses because 

inference is desired for naturally regenerated stands.

RESULTS

Most trees surveyed here were less than 7 m tall, but 

the age of trees varied from about 12 years to more 

than 50 years old (fig. 2). There is strong evidence 

of an association between the average height and the 

average age of trees in the naturally regenerated stands 

(Pearson’s correlation = 0.225, P = 0.0003). It is estimated that the mean height at a site is 0.97 m greater 

for each 10-year increase in average age (95-percent 

confidence interval from 0.44 m to 1.49 m or greater). 

Of the 265 sites surveyed, 264 had one or more trees 

with blister rust cankers; the mean across sites was 

43.6 percent infection and 2.1 cankers per tree for 

the 235 SP natural stand sites (table 1; figs. 3, 4). The 

number of cankers per tree per site ranged from near 0 

to >11 (fig. 4).

Percent infection and number of cankers per tree are 

strongly related to rust index (fig. 4). Rust index (as 

well as infection percentage and number of cankers 

per tree) varied widely among sites (figs. 1, 3, 4). The 

mean height of the highest canker was less than 1.8 m 

(6 ft) for most sites (fig. 5).

The natural logarithm of rust index is strongly associated with both the logit of percent infection (Pearson’s 

correlation = 0.890, P <0.0001, fig. 6a) and the natural 

logarithm of the average number of cankers per tree 

(Pearson’s correlation = 0.930, P < 0.0001; fig. 6b). 

The mean rust index changes from low hazard to medium hazard and from medium hazard to high hazard 

at 28.5 and 78.0 percent infected trees, respectively. 

Similarly, the mean rust index changes from low 

hazard to medium hazard and from medium hazard to 

high hazard at an average of 0.58 and 4.85 cankers per 

tree, respectively.

There is strong evidence of an association between 

the average height of the highest canker in naturally 

regenerated stands with nonzero rust index and the average age of the stand (Pearson’s correlation = 0.639, 

P < 0.0001, fig. 7). For each 10-year increase in the 
178 USDA Forest Service RMRS-P-76. 2018.

Figure 4—Relationship between (a) average number of cankers/tree and rust index (RI), (b) percent infected and RI, and 

(c) percent infected and average number of cankers/tree.

average age, the mean height of the highest canker is 

estimated to be 0.42 m higher (95-percent confidence 

interval from 0.36 m to 0.49 m higher). There is also 

strong evidence of an association between the percentage of trees infected at a site and the average age of 

the site (P = 0.008). Younger stands in general had a 

greater percentage of trees infected; the odds of infection were on average 52 percent greater at a site for 

each 10 years younger (95-percent confidence interval 

from 30 percent to 78 percent greater).

DISCUSSION AND SUMMARY

The data summarized here and elsewhere (Goheen 

and Goheen 2014; Sniezko et al. 2012, 2013) indicate 

that the risk of blister rust is ubiquitous in the portion 

of southern Oregon in this study. However, the site 

rust hazard varies from low to relatively high (fig. 

3). Some geographic clumping of low and mid-tohigh hazard areas occurs (fig. 3). Land managers can 

use this information to guide the use of genetically 

rust-resistant stock, branch pruning, and restoration 

options. 

Based on past observations, most of the trees with 

lower bole infections are expected to die. The impact 

of blister rust reported here is likely to be an underestimate due to the one-time survey of the stands. The 

survey excludes most trees that have previously died 

from rust, and obviously does not account for future 

infections. Other studies (Sniezko et al. 2012, 2013) 

suggest the impacts could be much greater over time. 

Further, the criteria for stand selection may have 

excluded areas of highest rust hazard due to low frequency of surviving WWP or SP in those areas.

On many sites, rust-resistant stock or pruning of 

branches, or both, will be needed to help maximize 

survival and keep these species as viable components 

in forest ecosystems or managed plantings. Sugar pine 

and western white pine will continue to decline in 

southwestern Oregon without the utilization of seedlots with genetic resistance to blister rust. The BLM 

and USDA FS manage seed orchards for sugar pine 

and western white pine and are involved in breeding 

and resistance testing to further increase the level of 

resistance (Sniezko et al. 2014; Waring and Goodrich 

2012). Most of the cankers noted are generally within 

2.4 m of the ground (fig. 5), suggesting that branch 
USDA Forest Service RMRS-P-76. 2018. 179

Figure 5—Frequency histogram denoting the average 

height of the highest canker at each of the 264 sites with 

a canker present. The vertical black line shows the overall 

average.

Figure 6—Fitted regression line and 95% prediction intervals 

for estimating the natural logarithm of rust index from (a) 

the logit of percent infected and (b) the natural logarithm of 

the average number of cankers per tree. The dashed line is 

the boundary between low and medium rust hazards, while 

the dotted line is the boundary between medium and high 

hazards.

pruning (and removing needles attached directly to the 

main bole) could potentially be effective in any reforestation or restoration plantings in these areas. Several 

studies to examine efficacy of pruning are now underway. Note that this would help survival for current 

trees on the landscape, but not the ensuing generations. 

Only an increase in durable resistance will provide that 

ongoing benefit.

The maps and data summarized here provide a planning tool to land managers wishing to retain these species in forest ecosystems, restore these species, or use 

them in managed forests. The value of such maps has 

been touted elsewhere: 

Hazard maps provide a tool for management 

planning at forest and project scales, a basis 

for stratifying landscapes for damage and 

impact surveys, and a foundation for additional 

epidemiological and ecological studies (Geils 

et al. 1999: 2). 

From a practical standpoint, the number of cankers 

per tree or percentage of trees with cankers provides a 

useful rating of site rust hazard when rust index is not 

calculated.

The effect of a changing climate on the incidence and 

intensity of blister rust infection is unknown. The data 

for this study were collected decades ago. However, 

recent studies suggest that blister rust continues to be 

very active in southwestern Oregon (Sniezko et al. 

2012, 2013). Permanent plots would provide a better 

tracking of the impacts of blister rust dynamics, and 

would complement a large-scale one-time survey such 

as this.

ACKNOWLEDGMENTS

We are grateful for the multiyear persistence of the 

many employees of the BLM Medford District to set 

up and complete the study. These employees were Jim 

Langhoff, Glenn Calascibetta, Paul Sweeney, Carrie 
180 USDA Forest Service RMRS-P-76. 2018.

Figure 7—Relationship between the average height of the 

highest canker and the average age of the stand for natural 

stands with nonzero rust indices. The linear least squares 

regression line is shown with a dotted line.

Sweeney, Gina Rogers, Alex Pancheco, and Larry 

Hutchinson. The project was partially funded by the 

U.S. Department of Agriculture, Forest Service Forest 

(USDA FS) Health Protection (FHP). We thank Bob 

Harvey of the FHP group for his guidance and assistance with the hazard-rating field data collection process, the program analysis process, and the adaptation 

of western white pine program inputs to sugar pine 

inputs. This gratitude also extends to Geral McDonald, 

USDA FS Intermountain Research Station, for the 

use of his basic Idaho western white pine rust index 

program for our Oregon sugar pine project. We thank 

Jim Hamlin and Angelia Kegley for their reviews of 

early versions of the materials for this paper, Terry 

Fairbanks for providing copies of some of the files for 

this recent summary, and Jennifer Christie for development of the maps.

REFERENCES

Burns, Kelly S.; Schoettle, Anna W.; Jacobi, William R.; 

[et al.]. 2008. Options for the management of white 

pine blister rust in the Rocky Mountain region. Gen. 

Tech. Rep. RMRS-GTR-206. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky 

Mountain Research Station. 26 p.

Geils, Brian W.; Conklin, David A.; van Arsdel, Eugene P. 

1999. A preliminary hazard model of white pine blister 

rust for the Sacramento Ranger District, Lincoln National 

Forest. Res. Note RMRS-RN-6. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky 

Mountain Research Station. 6 p.

Goheen, E.M.; Goheen, D.J. 2014. Status of sugar and 

western white pines on Federal forest lands in southwest 

Oregon: Inventory query and natural stand survey results. 

SWOFIDSC-14-01. Jacksonville, OR: U.S. Department 

of Agriculture, Forest Service, Forest Health Protection, 

Southwest Oregon Forest Insect and Disease Service 

Center. 71 p.

Hagle, Susan K.; McDonald, Geral I.; Norby, Eugene 

A. 1989. White pine blister rust in northern Idaho 

and western Montana: alternatives for integrated 

management. Gen. Tech. Rep. INT-261. Ogden, UT: U.S. 

Department of Agriculture, Forest Service, Intermountain 

Research Station. 35 p. https://www.fs.fed.us/rm/pubs_

int/int_gtr261.pdf. (13 October 2015).

Sniezko, R.A.; Danchok, R.; Long, S.; [et al.]. 2012. Longterm monitoring of white pine blister rust infection and 

survival at 10 sugar pine evaluation sites: II. Growth and 

survival of sugar pine through age 25 in six progeny tests 

of low to high blister rust hazard in southwestern Oregon. 

USDA Forest Service 2012 Forest Health Monitoring 

National Meeting. Poster. https://www.fhm.fs.fed.us/

posters/posters12/Sniezko_Long_term_Monitoring_

Poster.pdf. (13 October 2015).

Sniezko, R.A.; Danchok, R.; Long, S.; [et al.]. 2013. 

Growth and survival of sugar pine through age 25 in six 

progeny tests on sites of low to high blister rust hazard in 

southwest Oregon. In: Browning, J., comp. Proceedings 

of the 60th annual western international forest disease 

work conference. [Place of publication and Publisher 

unknown]:167–170. 

Sniezko, R.A.; Smith, J.; Liu, J.-J.; [et al.]. 2014. Genetic 

resistance to fusiform rust in southern pines and white 

pine blister rust in white pines—A contrasting tale of two 

rust pathosystems—Current status and future prospects. 

Forests. 5(9): 2050–2083. doi:10.3390/f5092050.

USDA Forest Service, Forest Health Technology Enterprise 

Team. [n.d.]. White pine blister rust model. Washington, 

DC. https://www.fs.fed.us/foresthealth/applied-sciences/

fvs-models/fvs-whitepineblisterrust.shtml [Accessed 

March 2, 2017].

Waring, K.M.; Goodrich, B. 2012. Artificial regeneration of 

five-needled pines of western North America: A survey 

of current practices and future needs. Tree Planters’ 

Notes. 55 (2): 55–71.
USDA Forest Service RMRS-P-76. 2018. 181

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Genetic Variability in Puccinia psidii as Revealed in Commercial 

Plantations of Eucalyptus Species

K. Kettener1, J.C. Otto1, J.É. Yassitepe2, E.L. Furtado3, and C.L. Marino1, *

1 Universidade Estadual Paulista (UNESP), Institute of Biosciences, Department of Genetics, Botucatu, São Paulo, Brazil

2 Fibria Celulose, Jacareí, São Paulo, Brazil

3 UNESP, Faculdade de Ciências Agronômicas, Plant Protection Department, Botucatu, São Paulo, Brazil 

*

clmarino@ibb.unesp.br

Abstract—The climates of central and southern regions of São Paulo state (Brazil) are suitable for pathogens such as Puccinia psidii, which causes a common and serious disease in eucalyptus plantations less 

than 2 years old. We studied the genetic variability of P. psidii in commercial plantations of Eucalyptus species by random amplified polymorphic DNA molecular markers. All 56 isolates of P. psidii analyzed showed 

high polymorphism and there was no evidence of preferential infection in different plant tissues. In the 

analysis of pooled pustules according to the clone of origin, we observed the formation of two groups, one 

with isolates from susceptible clones and the second group only with isolates from resistant clones, indicating that isolates infecting resistant clones are more genetically similar than isolates infecting susceptible 

clones.

INTRODUCTION

Puccinia psidii was originally described from common 

guava (Psidium guajava) in eastern Brazil in 1884. 

It became notorious for its host jumps to nonnative 

Eucalyptus, first observed in Brazil in the 1940s. It 

caused substantial economic damage to large Brazilian 

plantations in the 1970s (Coutinho et al. 1998). The 

wide host range of the rust fungus and its potential 

threats to many native or introduced species in 

Myrtaceae has become a major concern to many countries, including New Zealand, Australia, South Africa, 

and Brazil, where Myrtaceae species are the major 

components of the forests and ecosystem (Coutinho et 

al. 1998; Glen et al. 2007; Tommerup et al. 2003).

The host jump in Brazil is currently cited as a prime 

example in the literature illustrating the potential 

danger for host jumps following anthropocentric 

movement of potential hosts (Slippers et al. 2005; 

Wingfield et al. 2001). The probability of a new race is 

particularly high when a particular resistance gene is 

used extensively in breeding (Keiper et al. 2003). 

Breeding programs are underway to improve resistance of eucalypts to P. psidii. In E. grandis, resistance 

is controlled by a locus of main effect, which makes 

this species useful in breeding programs (Junghans 

et al. 2003). However, based on the pattern of inheritance, slight changes in the pathogen’s genetic structure can overcome this host resistance gene and render 

it ineffective (Graça et al. 2011).

The lack of information about the origin and genetic 

variation of the pathogen population has hindered 

choosing the best form of management aimed at reducing disease.
182 USDA Forest Service RMRS-P-76. 2018.

Our aim is to determine the genetic relationships 

among P. psidii isolates from Eucalyptus species in 

Brazil with random amplified polymorphic DNA 

(RAPD) markers, allowing us to investigate whether 

the pathogen has undergone a genetic change that 

could enable it to break the barrier of plant resistance. 

Results of this research are expected to assist in developing effective control measures.

METHODS

Fungal Isolates

Single uredinial isolates of P. psidii were collected 

from six different Eucalyptus clones, totaling 56 pustules (table 1). These clones are located in the city of 

Jacarei, São Paulo (Brazil) and were provided by the 

Fibria SA breeding program. 

DNA Extraction

Genomic DNA was extracted by using Chelex® 

(Sigma-Aldrich Co., St. Louis, Missouri, USA) 

resin. The protocol is to add to each sample 100 μl 

of 5 percent Chelex, then to soak the spores until the 

entire contents are homogenized, and to add 80 mg 

of proteinase K. Samples were placed in a water bath 

overnight at 65 °C. The next day, the samples were 

exposed to a temperature of 95 °C water bath for 15 

minutes, removed, and centrifuged for 1 minute at 

14,000 rpm. The supernatant was transferred to new 

tubes.

RAPD Genotyping

We genotyped 56 isolates with the primers OPAD-01, 

OPM-16, OPM-10, OPX-06 by Operon Technologies 

(Olive Branch, Mississippi, USA). Each 13-μL polymerase chain reaction (PCR) contained 10 ng total 

genomic DNA, 0.8 mM of dNTP, 1923 mM of MgCl2, 

0.36 percent of bovine serum albumin (BSA), 1 unit 

of Taq DNA polymerase (Invitrogen®; Thermo Fisher 

Scientific, Waltham, Massachusetts, USA), 0.3 mM of 

primer, and autoclaved deionized water. The PCR reaction was performed in a PT 100TM thermocycler (MJ 

Research, Waltham, Massachusetts) with one cycle at 

95 °C for 3 minutes, followed by 40 cycles at 92 °C 

for 1 minute, 35 °C for 1 minute, and 72 °C for 2 minutes; a final elongation step of 5 minutes at 72 °C; and 

finally a holding step at 10 °C. The PCR products were 

separated by using a 2.5 percent agarose gel.

Data Analysis

A binary matrix recording specific RAPD fragments as 

present (1) or absent (0) was generated for each genotype, and the data obtained plotted on a Microsoft® 

Excel® spreadsheet.

Analysis of diversity and genetic distance was 

performed with the software PopGene 1.32. A dendrogram was generated based on UPGMA analysis of 

array-based genetic distance of Nei (1978).

Figure 1—Cluster diagram based on genetic distance 

calculated from the presence/ absence of polymorphic 

markers across the pustules grouped by clone. Pop 1, 

2, 3, 4, 5 and 6: name of each group of pustules in the 

clones. 
USDA Forest Service RMRS-P-76. 2018. 183

Table 1—Plant location of Puccinia psidii pustules collected from six Eucalyptus sp. clones obtained from 

crosses between susceptible and resistant trees of E. grandis located in Jacarei-SP, Brazil, from Fibria SA 

breeding program. 

Sample Number Description

1 Clone A, Plant 1, Leaf 1, Tree top

2 Clone A, Plant 1 ,Leaf 2 , Pock 1 ,Tree top 

3 Clone A, Plant 1, Leaf 2 ,Pock 2 ,Tree top 

4 Clone A ,Plant 1, Leaf 3 ,Tree bottom

5 Clone A, Plant 1, Leaf 4 , Tree bottom

6 Clone A, Plant 2, Leaf 1, Tree top

7 Clone A, Plant 2, Leaf 2, Tree bottom 

8 Clone A, Plant 2, Leaf 3, Pock 1, Tree bottom 

9 Clone A, Plant 2, Leaf 3, Pock 2, Tree bottom 

10 Clone A, Plant 2, Leaf 4, Tree top

11 Clone B, Plant 1, Leaf 1, Pock 1, Tree top

12 Clone B, Plant 1, Leaf 1, Pock 2, Tree top 

13 Clone B, Plant 1, Leaf 1, Pock 3, Tree top 

14 Clone B, Plant 1, Leaf 1, Pock 4, Tree top 

15 Clone B, Plant 1, Leaf 2, Pock 1, Tree top 

16 Clone B, Plant 1, Leaf 2, Pock 2, Tree top 

17 Clone B, Plant 1, Leaf 2, Pock 3, Tree top 

18 Clone B, Plant 1, Leaf 2, Pock 4, Tree top 

19 Clone B, Plant 1, Leaf 2, Pock 5, Tree top 

20 Clone B, Plant 1, Leaf 3, Pock in the stem, Tree top 

21 Clone B, Plant 2 ,Leaf 1, Pock in the petiole, Tree top

22 Clone B, Plant 2, Pock in the stem, Tree top 

23 Clone B, Plant 3, Leaf 1, Pock 1, Tree top

24 Clone B, Plant 3, Leaf 1, Pock 2, Tree top

25 Clone C, Plant 1, Leaf 1, Tree top 

26 Clone C, Plant 1, Leaf 2, Tree top

27 Clone C, Plant 1, Leaf 3, Pock 1, Tree top

28 Clone C, Plant 1, Leaf 3, Pock 2, Tree top 

29 Clone C, Plant 1, Leaf 3, Pock 3, Tree top 

30 Clone C, Plant 2, Leaf 1, Tree top 

31 Clone C, Plant 2, Leaf 2, Pock in veins, Tree top 

32 Clone C, Plant 4, Leaf 1, Pock in veins, Tree top 

33 Clone D, Plant 1, Leaf 1, Pock 1, Tree top

34 Clone D, Plant 1, Leaf 1, Pock 2, Tree top

35 Clone D, Plant 1, Leaf 2, Pock 1, Tree top

36 Clone D, Plant 1, Leaf 2, Pock 2, Tree top

37 Clone D, Plant 2, Leaf 1, Pock in the petiole, Tree top

38 Clone D, Plant 2, Leaf 2, Tree top

39 Clone D, Plant 2, Leaf 3, Pock in the petiole, Tree top

40 Clone D, Plant 2, Leaf 4, Pock in the petiole, Tree top

41 Clone D, Plant 3, Leaf 1, Tree top

42 Clone D, Plant 3, Leaf 2, Tree top

43 Clone E, Plant 1, Leaf 1, Tree top

44 Clone E, Plant 1, Leaf 2, Tree top

45 Clone E, Plant 1, Leaf 3, Pock 1, Tree top

46 Clone E, Plant 1, Leaf 3, Pock 2, Tree top

47 Clone E, Plant 1, Leaf 3, Pock 3, Tree top

48 Clone E, Plant 2, Leaf 1, Tree top

49 Clone E, Plant 2, Leaf 3, Pock 1, Tree top

50 Clone E, Plant 2, Leaf 3, Pock 2, Tree top

51 Clone E, Plant 2, Leaf 4, Tree top

52 Clone F, Plant 1, Leaf 1, Tree top

53 Clone F, Plant 1, Leaf 2, Tree top 

54 Clone F, Plant 1, Leaf 3, Tree top 

55 Clone F, Plant 1, Leaf 4, Pock 1, Tree top 

56 Clone F, Plant 1, Leaf 4, Pock 2, Tree top 
184 USDA Forest Service RMRS-P-76. 2018.

RESULTS AND DISCUSSION

Two dendrograms were built, one with the pustules 

grouped by clone and another with the pustules 

separated without regard to the origin clone. In the 

dendrogram with pustules grouped by clone (fig. 1), 

we observed two main groups. Group I with isolates 

from pop1, pop2, and pop3 was subdivided into a 

cluster formed by pop1 and pop2 with a similarity coefficient of 0.1. Group II is an isolate of pop3. The two 

groups were correlated, with a similarity coefficient of 

0.2. In group II, formed by pop4, pop5, and pop6, two 

clusters were observed with a few genetic polymorphisms (fig. 1). Note that the first group contains only 

Figure 2—Cluster diagram based on genetic distance calculated from the presence or absence of polymorphic markers across the pustules not grouped by clone. Pop 1 until 56: name of each pustule. 
USDA Forest Service RMRS-P-76. 2018. 185

susceptible clones and the second group, only resistant 

clones, which means that Puccinia isolates from resistant clones are genetically more similar than isolates 

from susceptible clones.

The analysis of pustules separately (unrelated to the 

clone of origin) showed high genetic diversity. We 

observed the formation of many groups with no apparent relationship between the pustules taken from the 

same clone forest (fig. 2). There was no relationship 

between the pustule positions along the tree, showing 

no fungus preference to infect a specific tree tissue. 

This high diversity was expected because the fungus is 

native to Brazil. 

These results open a favorable perspective to the 

development of specific fungus markers for each 

Eucalyptus clone. These and similar results can provide a basis for choosing new strategies for improving 

plant resistance.

ACKNOWLEDGMENTS

We thank the National Council for Scientific and 

Technological Development (CNPq) for funding this 

project, and Fibria SA for providing the P. psidii populations (from Eucalyptus) and other facilities. 

Competing Interests: The study presented in the manuscript is a collaboration between the University of São 

Paulo State and Fibria Pulp and Paper. The collaboration did not involve any financial agreement between 

the two parties. Juliana Érika de Carvalho Teixeira 

Yassitepe was employed by Fibria at the time of the 

study, but is no longer with the company. This does not 

alter the policies on sharing data and materials. 

REFERENCES 

Coutinho, T.A.; Wingfield, M.J.; Alfenas, A.C.; [et al.]. 

1998. Eucalyptus rust—A disease with the potential for 

serious international implications. Plant Disease. 82: 

819–825.

Glen, M.; Alfenas, A.C.; Zauza, E.A.V.; [et al.]. 2007. 

Puccinia psidii: a threat to the Australian environment 

and economy—a review. Australasian Plant Pathology 

36: 1–16.

Graça, R.N.; Aun, C.P.; Guimarães, L.M.S.; [et al.]. 

2011. A new race of Puccinia psidii defeats the Ppr-1 

resistance gene in Eucalyptus grandis. Australasian Plant 

Pathology. 40: 442–447.

Junghans, D.T.; Alfenas, A.C.; Brommonshenckel, S.H.; 

[et al.]. 2003. Resistance to rust (Puccinia psidii Winter) 

in Eucalyptus: Mode of inheritance and mapping of a 

major gene with RAPD markers. Theoretical and Applied 

Genetics. 108: 175–180.

Keiper, F.J.; Hayden, M.J.; Park, R.F; [et al.]. 2003. 

Molecular genetic variability of Australian isolates of 

five cereal rust pathogens. Mycological Research. 107: 

545–556.

Nei, M. 1978. Estimation of average heterozygosity and 

genetic distance from a small number of individuals. 

Genetics. 76: 379–390.

Slippers, B.; Stenlid, J.; Wingfield, M.J. 2005. Emerging 

pathogens—Fungal host jumps following anthropogenic 

introduction. Trends in Ecology and Evolution. 20: 

420–421.

Tommerup, I.C.; Alfenas, A.C.; Old, K.M. 2003. Guava rust 

in Brazil—A threat to eucalyptus and other Myrtaceae. 

New Zealand Journal of Forestry Science. 33: 420–428.

Wingfield, M.J.; Slippers, B.; Roux, J.; [et al.]. 2001. 

Worldwide movement of exotic forest fungi, especially 

in the tropics and the Southern Hemisphere. BioScience. 

51: 134–140..
186 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Genetic and Genotypic Diversity of Puccinia psidii—The Cause of 

Guava/Eucalypt/Myrtle Rust—And Preliminary Predictions of Global 

Areas at Risk

A.L. Ross-Davis1, R.N. Graça2, A.C. Alfenas2, T.L. Peever3, J.W. Hanna1, J.Y. Uchida4,

R.D. Hauff5, C.Y. Kadooka4, M.-S. Kim6, P.G. Cannon7, S. Namba8, N. Minato8, S. Simeto9,

C.A. Pérez10, M.B. Rayamajhi11, M. Morán12, D.J. Lodge13, M. Arguedas14, R. Medel-Ortiz15,

M.A. López-Ramirez15, P. Tennant16, M. Glen17, A. McTaggart18, and N.B. Klopfenstein1, *

1 USDA Forest Service, Rocky Mountain Research Station, Moscow Forestry Sciences Laboratory, Moscow, Idaho, USA

2 Universidade Federal de Viçosa, Departamento de Fitopatologia, Brazil

3 Washington State University, Department of Plant Pathology, Pullman, Washington, USA

4 University of Hawaii at Manoa, Department of Plant and Environmental Protection Sciences, Honolulu, Hawaii, USA

5 Department of Lands and Natural Resources, Division of Forestry and Wildlife, Honolulu, Hawaii, USA

6 Kookmin University, Department of Forestry, Environment and Systems, Kookmin University, South Korea

7 USDA Forest Service, Forest Health Protection, Vallejo, California, USA

8 University of Tokyo, Department of Agricultural and Environmental Biology, Japan

9 Instituto Nacional de Investigación Agraria Tacuarembó, Programa Forestal, Uruguay

10 Universidad de la República, Facultad de Agronomía, Departamento de Protección Vegetal, Uruguay

11 USDA Agricultural Research Station, Invasive Plant Research Laboratory, Fort Lauderdale, Florida, USA

12 Desarrollos Madereros S.A., Paraguay

13 USDA Forest Service, Forest Products Laboratory, Puerto Rico

14 Instituto Tecnológico de Costa Rica, Escuela de Ingeniería Forestal, Costa Rica

15 Universidad Veracruzana, Instituto de Investigaciones Forestales, Mexico

16 University of the West Indies, The Biotechnology Centre, Jamaica

17 University of Tasmania, Tasmanian Institute of Agriculture, Australia

18 University of Queensland, Queensland Alliance for Agriculture and Food Innovation, Australia

*

nklopfenstein@fs.fed.us

INTRODUCTION

Puccinia psidii (Basidiomycota, Uredinales) is a 

biotrophic rust fungus that was first reported in 

Brazil from guava (Psidium guajava) in 1884 (Winter 

1884) and later from nonnative eucalypt (Eucalyptus 

capitelata and Corymbia citriodora, syn. Eucalyptus 

citriodora; Joffily 1944). Considered to be of 

Neotropical origin, the rust has also been reported to 

infect diverse myrtaceous hosts elsewhere in South 

America, Central America, the Caribbean, Mexico, the 

United States (California, Florida, and Hawaii), Japan, 

Australia, China, and most recently South Africa and 

New Caledonia (Carnegie et al. 2010; Giblin 2013; 

Kawanishi et al. 2009; MacLachlan 1938; Marlatt and 

Kimbrough 1979; Mellano 2006; Pérez et al. 2011; 

Roux et al. 2013; Uchida et al. 2006; Zambino and 
USDA Forest Service RMRS-P-76. 2018. 187

Nolan 2011; Zhuang and Wei 2011). Given the rate 

at which the pathogen is spreading and its wide host 

range, the objectives of this study are to determine 

how much genetic diversity exists within populations 

across the species’ putative native range as well as 

areas of recent introduction, evaluate possible pathways of spread, and predict the geographic area that is 

climatically suitable for the species to determine areas 

at risk of invasion. 

METHODS

Six microsatellite loci were scored for P. psidii

samples derived from diverse hosts and geographic 

locations (table 1). Samples were assigned to genetic 

clusters using a Bayesian genetic clustering algorithm 

implemented in STRUCTURE v2.3.4 (Pritchard 

et al. 2000). Posterior probabilities were estimated 

for K = 1 to K = 12 assuming an admixture model. 

Table 1—Geographic origin and host of confirmed Puccinia psidii genotypes.

Origin Host Clustera

Australia Pilidiostigma glabrum Pandemic

Backhousia oligantha Pandemic

Rhodomyrtus psidioides Pandemic

Brazil Eucalyptus spp. Eucalypt

Eugenia uniflora Guava

Myrciaria cauliflora Guava

Psidium guajava Guava

Psidium guineenese Guava

Syzygium cumini Guava

Syzygium jambos Eucalypt

Costa Rica Callistemon lanceolatus Pandemic

Syzygium jambos Pandemic

Jamaica Pimenta dioica Allspice

Syzygium jambos Pandemic

Mexico Syzygium jambos Pandemic

Paraguay Eucalyptus grandis Eucalypt

Puerto Rico Syzygium jambos Pandemic

Uruguay Psidium guajava Guava

Eucalyptus grandis Eucalypt

Eucalyptus globulus Eucalypt

Myrrhinium atropurpurea Guava

United States Eugenia koolauensis Pandemic

Melaleuca quinquenervia Pandemic

Metrosideros excelsa Pandemic

Metrosideros polymorpha Pandemic

Myrcianthes fragrans Pandemic

Myrtus communis Pandemic

Rhodomyrtus tomentosa Pandemic

Syzygium jambos Pandemic

a (1) Allspice = collections from Pimenta dioica from Jamaica; (2) Pandemic = collections from diverse hosts 

from Australia, the Caribbean, Mexico, and the United States; (3) Eucalypt = collections from Eucalyptus spp. 

and Syzygium jambos from Brazil, Paraguay, and Uruguay; (4) Guava = collections from Psidium guajava, 

Psidium guineense, Syzygium cumini, Myrciaria cauliflora, Eugenia uniflora, and Myrrhinium atropurpureum var 

octandrum from Brazil and Uruguay.
188 USDA Forest Service RMRS-P-76. 2018.

STRUCTURE analyses were performed with clonecorrected data (unique multilocus genotypes, MGs). 

Fifty thousand burn-in generations were employed for 

each of 10 replicate runs of 1,000,000 generations of 

the Markov chain Monte Carlo (MCMC) sampler for 

each K. The optimal value of K was inferred using the 

method of Evanno et al. (2005). Population genetic 

analyses of all samples grouped by genetic cluster 

were performed using GenAlEx v6.501 (Peakall 

and Smouse 2006). To examine relationships among 

MGs, a minimum spanning network was estimated 

using the genetic distance measure of Bruvo et al. 

(2004). Bioclimatic modeling was performed using 

all documented rust occurrences and reduced sets of 

the eucalypt, pandemic, and guava biotypes coupled 

with 19 bioclimatic variables in MaxEnt (Maximum 

Entropy Species Distribution Modeling) v3.3.3K. 

Cross-validation was used to verify results among 10 

replicate runs. Quantum GIS was used to create the 

final output in a cumulative format according to prediction probability. 

RESULTS AND DISCUSSION

Among the P. psidii samples collected from Australia, 

Brazil, Costa Rica, Jamaica, Mexico, Paraguay, Puerto 

Rico, Uruguay, and the United States, 26 unique MGs 

were found. A Bayesian clustering analysis and a minimum spanning network revealed four major genetic 

clusters among the MGs: (1) Allspice = collections 

from Pimenta dioica from Jamaica; (2) Pandemic 

= collections from diverse hosts from Australia, 

the Caribbean, Mexico, and the United States; (3) 

Eucalypt = collections from Eucalyptus species and 

Syzygium jambos from Brazil, Paraguay, and Uruguay; 

and (4) Guava = collections from Psidium guajava, 

Psidium guineense, Syzygium cumini, Myrciaria cauliflora, Eugenia uniflora, and Myrrhinium atropurpureum var. octandrum from Brazil and Uruguay (table 

1; figs. 1 and 2). 

All loci were polymorphic, with 5 to 11 alleles detected per locus. Genetic diversity was highest within 

the guava cluster, followed by the pandemic, the 

allspice, and finally the eucalypt clusters (table 2). 

Similar to previous attempts to assess the geographic 

invasive potential of the species (Booth and Jovanovic 

2012; Booth et al. 2000; Elith et al. 2013; Glen et al. 

2007; Kriticos et al. 2013; Magarey et al. 2007), the 

model using all records of occurrence projects many 

areas throughout the world as having some degree of 

probability of suitable climate for P. psidii (fig. 3). 

Models using reduced sets of the eucalypt, pandemic, 

and guava biotypes show predictions of different 

distributions (figs. 3 and 4). Inoculation tests can also 

help to assess threats posed by different pathogen 

biotypes. For example, Silva et al. (2014) showed that 

the eucalypt biotype was highly virulent on Hawaiian 

’ōhi’a lehua (Metrosideros polymorpha) plants (fig. 5), 

and Zauza et al. (2010) showed that diverse myrtaceous species from Australia varied greatly in their 

resistance/susceptibility to one race of the eucalypt 

biotype. 

When bioclimatic modeling is coupled with inoculation tests, it suggests that different biotypes pose 

different risks to various global regions, should they 

be introduced. This work builds on previous research 

that shows that diversity must be considered when 

assessing the invasive threat posed by this pathogen 

Table 2—Summary of genetic variation among clusters of Puccinia psidii. 

Allspicea Pandemic Eucalypt Guava

Alleles per locus 2.50 (0.43) 3.50 (0.67) 2.33 (0.21) 4.83 (0.60)

Effective alleles per locus 2.26 (0.35) 2.91 (0.45) 1.97 (0.25) 3.40 (0.39)

Private alleles 4 9 1 11

Shannon’s index 0.78 (0.18) 1.07 (0.16) 0.70 (0.12) 1.35 (0.12)

Number of MGs 5 9 6 6 

Observed heterozygosity 0.77 (0.16) 1.00 (0.00) 0.69 (0.19) 0.61 (0.13)

Expected heterozygosity 0.49 (0.10) 0.62 (0.05) 0.44 (0.08) 0.69 (0.04)

Fixation index -0.62 (0.16) -0.67 (0.13) -0.39 (0.31) 0.14 (0.16)

a

Clusters of Allspice, Pandemic, Eucalypt, and Guava are described in Table 1.
USDA Forest Service RMRS-P-76. 2018. 189

Figure 1—A Population structure inferred using a Bayesian clustering algorithm implemented in STRUCTURE at K = 4 

(estimated ln probability = -418.9).

Figure 2—Minimum spanning network of multilocus genotypes represented by red for collections from Pimenta 

dioica (Jamaica), blue for collections from Eucalyptus spp. and Syzygium jambos (Brazil, Paraguay, and Uruguay), 

green from the pandemic cluster (which includes collections from diverse hosts from the Australia, the Caribbean, 

Mexico, and United States), and yellow from other hosts (collections from Psidium guajava, Psidium guineense, 

Syzygium cumini, Myrciaria cauliflora, Eugenia uniflora, and Myrrhinium atropurpureum var octandrum). Sizes of 

circles are proportional to MG frequency and connections are labeled with Bruvo genetic distances if different from 

0.06, which corresponds to one mutational step at one locus. Loops in the network indicate multiple tied minimum 

spanning trees.
190 USDA Forest Service RMRS-P-76. 2018.

Figure 3—Maxent models of suitable climate space (potential distribution) for 

Puccinia psidii based on 19 global bioclimatic variables derived from the WorldClim 

(worldclim.org) database for A all biotypes (245 occurrence points); B eucalypt 

biotype (88 occurrence points); C pandemic biotype (89 occurrence points); and D 

guava biotype (65 occurrence points).
USDA Forest Service RMRS-P-76. 2018. 191

Figure 4—Assessment of Brazilian strain virulence to Hawaiian ’Ōhi’a (Metrosideros polymorpha) (Silva et al. 

2014). The Brazilian Puccinia psidii eucalypt biotype was highly virulent on Hawaiian ’ōhi’a plants.

Figure 5—Maxent models of suitable climate space (potential distribution) for Puccinia psidii in Hawaii 

based on 19 global bioclimatic variables derived from the WorldClim (worldclim.org) database for A all 

biotypes; B eucalypt biotype; C pandemic biotype; and D guava biotype.
192 USDA Forest Service RMRS-P-76. 2018.

to myrtaceous hosts worldwide (Graça et al. 2013). 

Future studies that include P. psidii genotypes from 

additional geographic areas will allow a better prediction of global geographic areas that are climatically 

suitable for P. psidii and for subspecies-level genetic 

clusters, which will provide a more robust assessment 

for pathways of spread and areas at risk of invasion.

ACKNOWLEDGMENTS

Eric Pitman was instrumental in the bioclimatic modeling efforts. 

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assessment of high risk areas for Puccinia psidii

(Eucalyptus rust) in the Neotropics and Australia. 

Agriculture Ecosystems and Environment. 82: 295–301.

Booth, T.H.; Jovanovic, T. 2012. Assessing vulnerable 

areas for Puccinia psidii (eucalyptus rust) in Australia. 

Australasian Plant Pathology. 41: 425–429. 

Bruvo, R.; Michiels, N.K.; D’Souza, T.G.; [et al.]. 2004. 

A simple method for the calculation of microsatellite 

genotype distances irrespective of ploidy level. 

Molecular Ecology. 13: 2101–2106. 

Carnegie, A.J.; Lidbetter, J.R.; Walker, J.; [et al.]. 2010. 

Uredo rangelii, a taxon in the guava rust complex, newly 

recorded on Myrtaceae in Australia. Australasian Plant 

Pathology. 39: 463–466. 

Elith, J.; Simpson, J.; Hirsch, M.; [et al.]. 2013. Taxonomic 

uncertainty and decision making for biosecurity: 

Spatial models for myrtle/guava rust. Australasian Plant 

Pathology. 42: 43–51. 

Evanno, G.; Regnaut, S.; Goudet, J. 2005. Detecting the 

number of clusters of individuals using the software 

STRUCTURE: A simulation study. Molecular Ecology. 

14: 2611–2620. 

Giblin, F. 2013. Myrtle rust report: New Caledonia. 

Maroochydore, Queensland: University of the Sunshine 

Coast. 18 p. 

Glen, M.; Alfenas, A.C.; Zauza, E.A.V.; [et al.]. 2007. 

Puccinia psidii: A threat to the Australian environment 

and economy—A review. Australasian Plant Pathology. 

36: 1–16. 

Graça, R.N.; Ross-Davis, A.L.; Klopfenstein, N.B.; [et al.]. 

2013. Rust disease of eucalypts, caused by Puccinia 

psidii, did not originate via host jump from guava in 

Brazil. Molecular Ecology. 22: 6033–6047. 

Joffily, J. 1944. Ferrugem do eucalipto. Bragantia. 4: 

475–487. 

Kawanishi, T.; Uemastu, S.; Kakishima, M.; [et al.]. 2009. 

First report of rust disease on ohia and the causal fungus, 

Puccinia psidii, in Japan. Journal of Genetic Plant 

Pathology. 75: 428–431. 

Kriticos, D.J.; Morin, L.; Leriche, A.; [et al.]. 2013. 

Combining a climatic niche model of an invasive 

fungus with its host species distributions to identify 

risks to natural assets: Puccinia psidii sensu lato in 

Australia. PLoS ONE. 8(5): e64479. doi:10.1371/journal.

pone.0064479. 

MacLachlan, J.D. 1938. A rust of the pimento tree in 

Jamaica, BWI. Phytopathology. 28: 157–170.

Magarey R.D.; Fowler. G.A.; Borchert, D.M.; [et al.]. 2007. 

NAPPFAST: An internet system for the weather-based 

mapping of plant pathogens. Plant Disease. 91: 336–345.

Marlatt, R.B.; Kimbrough, J.W. 1979. Puccinia psidii on 

Pimenta dioica in south Florida. Plant Disease Reporter. 

63: 510–512. 

Mellano, V. 2006. Rust on myrtle found in San Diego 

County. Healthy Garden-Healthy Home, University 

of California Cooperative Extension Retail Nursery 

Newsletter. 1: 3. 

Peakall, R.; Smouse, P.E. 2006. GENALEX 6: Genetic 

analysis in Excel. Population genetic software for 

teaching and research. Molecular Ecology Notes. 6: 

288–295. 

Pérez, C.A.; Wingfield, M.J.; Altier, N.A.; [et al.]. 2011. 

Puccinia psidii infecting cultivated Eucalyptus and 

native myrtaceae in Uruguay. Mycological Progress. 10: 

273–282. 

Pritchard, J.K.; Stephens, P.; Donnelly, P. 2000. Inference 

of population structure using multilocus genotype data. 

Genetics. 155: 945–959. 

Roux, J.; Greyling, I.; Coutinho, T.A.; [et al.]. 2013. The 

Myrtle rust pathogen, Puccinia psidii, discovered in 

Africa. IMA Fungus. 4: 155–159. 

Silva, A.C.; Andrade, P.M.T.; Alfenas, A.C.; [et al.]. 2014. 

Virulence and impact of Brazilian strains of Puccinia 

psidii on Hawaiian 'ohi'a (Metrosideros polymorpha). 

Pacific Science. 68: 47–56. 
USDA Forest Service RMRS-P-76. 2018. 193

Uchida, J.; Zhong, S.; Kilgore, E. 2006. First report of a rust 

disease on 'ōhi'a caused by Puccinia psidii in Hawaii. 

Plant Disease. 90: 524.

Winter, G. 1884. Repertorium. Rabenhorstii fungi europaei 

et extraeuropaei, Centuria XXXI et XXXII. Hedwigia. 

23: 164–172. 

Zambino P.J.; Nolan, P.A. 2011. First report of rust 

caused by Puccinia psidii on paperbark, Melaleuca 

quinquenervia, in California. Plant Disease. 95: 

1314–1315. 

Zauza, E.A.V.; Alfenas, A.C.; Old, K.; [et al.]. 2010. 

Myrtaceae species resistance to rust caused by Puccinia 

psidii. Australasian Plant Pathology. 39: 406–411

Zhuang, J.-Y.; Wei, S.-X. 2011. Additional materials for the 

rust flora of Hainan Province, China. Mycosystema. 30: 

853–860.
194 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Cronartium Rusts Sporulate on a Wide Range of Alternate Hosts

in Northern Europe

J. Kaitera1, *, R. Hiltunen2, B. Samils3, and J. Hantula4

1 Natural Resources Institute Finland, Oulu, Finland

2 University of Oulu, Botanical Gardens, Oulu, Finland

3 Swedish University of Agricultural Sciences, Department of Forest Mycology and Plant Pathology,

Uppsala, Sweden

4 Natural Resources Institute Finland, Vantaa, Finland

* juha.kaitera@luke.fi

INTRODUCTION

Cronartium flaccidum is a serious pathogen of twoneedle pines, especially of Pinus sylvestris, in northern 

Fennoscandia (Samils et al. 2010). The rust spreads 

via a number of alternate hosts in natural forests and 

gardens, among which species in genera Melampyrum, 

Pedicularis, Vincetoxicum, and Paeonia are the most 

important known ones (Gäumann 1959; Kaitera et 

al. 1999). In northern Europe, the most important 

species is Melampyrum sylvaticum (Kaitera et al. 

2005), while in southern Europe the respective species is Vincetoxicum hirundinaria (Gäumann 1959). 

Cronartium ribicola is a common pathogen on fiveneedle pines in arboretums, botanical gardens, and 

parks in northern Europe (Gäumann 1959; Kaitera et 

al. 2013). The rust spreads mainly via Ribes (Kaitera 

and Nuorteva 2006).

METHODS

To test the susceptibility of known and potential 

alternate hosts to C. flaccidum and C. ribicola, 109 

plant species in 18 families were inoculated using various Fennoscandian spore sources in 2008–2013. The 

inoculations were performed in the greenhouse and in 

the laboratory, and plants were also exposed to natural 

inoculum in the field (e.g., Kaitera et al. 2015). 

RESULTS AND DISCUSSION

Over the years, 45 plant species in 13 families supported uredinial or telial sporulation of C. flaccidum

on tested leaves, while C. ribicola sporulated on 16 

species or cultivars in 5 families (Kaitera and Hiltunen 

2011, 2012; Kaitera et al. 2012, 2015). Among the 

tested species, hemiparasites were highly susceptible to both rusts. All of the tested genera of family 

Orobanchaceae included species that were susceptible 

to C. flaccidum in the greenhouse and in the laboratory. Some of these species were also infected when 

exposed to natural inoculum in the field (Kaitera et al. 

2015). 

Cronartium ribicola sporulated on species in genera 

such as Bartsia (fig. 1), Castilleja (fig. 2), Pedicularis

(fig. 3), Mentzelia (fig. 4), Loasa (fig. 5), Tropaeolum

(fig. 6) and Ribes (figs. 7–9). Cronartium flaccidum

sporulated on species in genera such as Melampyrum

(figs. 10–13), Pedicularis (figs. 14–17), Rhinanthus

(figs. 18–19), Euphrasia (figs. 20–23), Loasa

(figs. 24–25), Bartsia (fig. 26), Impatiens (figs. 

27–28), Vincetoxicum (fig. 29), Asclepias (fig. 30), 

Apocynum (fig. 31), Nicotiana (fig. 32), Swertia (fig. 

33), Veronica (figs. 34–38), Nemesia (figs. 39–40), 

Paeonia (figs. 41–45), Tropaeolum (fig. 46), Verbena

(figs. 47–48), Saxifraga (figs. 49–50), Castilleja (fig. 

51) and Myrica (fig. 52).
USDA Forest Service RMRS-P-76. 2018. 195

In conclusion, a high number of previously unknown 

alternate hosts can support rust sporulation under 

laboratory and greenhouse conditions, as well as in the 

field, and therefore, may also spread rust epidemics 

under favorable conditions in natural forests. 

REFERENCES

Gäumann, E. 1959. Die Rostpilze Mitteleuropas. Beiträge zur 

Kryptogamenflora der Schweiz. 12: 85–93.

Kaitera, J.; Hiltunen, R. 2011. Susceptibility of Pedicularis

spp. to Cronartium ribicola and C. flaccidum in Finland. 

Forest Pathology. 41: 237–242.

Kaitera, J.; Hiltunen, R. 2012. New alternate hosts for the 

rusts Cronartium ribicola and Cronartium flaccidum

in Finland. Canadian Journal of Forest Research. 42: 

1661–1668.

Kaitera, J.; Hiltunen, R.; Hantula, J. 2015. Cronartium rusts 

sporulation on hemiparasitic plants. Plant Pathology. 

64(3): 738–747.

Kaitera, J.; Hiltunen, R.; Samils, B. 2012. Alternate host 

ranges of Cronartium flaccidum and Cronartium ribicola

in northern Europe. Botany. 90: 694–703.

Kaitera, J.; Kauppila, T.; Hiltunen, R. 2013. Incidence of 

Cronartium ribicola in Finnish botanical gardens and fiveneedle pine plantations in northern Finland. Scandinavian 

Journal of Forest Research. 28: 746–751.

Kaitera, J.; Nuorteva H. 2006. Susceptibility of Ribes spp. to 

pine stem rusts in Finland. Forest Pathology. 36: 225–246.

Kaitera, J.; Nuorteva, H.; Hantula, J. 2005. Distribution and 

frequency of Cronartium flaccidum on Melampyrum

spp. in Finland. Canadian Journal of Forest Research. 35: 

229–234.

Kaitera, J.; Seitamäki, L.; Hantula, J.; [et al.]. 1999. 

Inoculation of known and potential alternate hosts with 

Peridermium pini and Cronartium flaccidum aeciospores. 

Mycological Research. 103: 235–241.

Samils, B.; Ihrmark, K.; Kaitera, J.; [et al.]. 2010. Genetic 

structure of Scots pine blister rust (Cronartium flaccidum

and Peridermium pini). Phytopathologia Mediterranea. 49: 

428.

Figure 1—Uredinia of Cronartium ribicola on Bartsia alpina. 

Figure 2—Uredinia and telia of C. ribicola on Castilleja sulphurea. 

Figure 3—Uredinia and telia of C. ribicola on Pedicularis palustris ssp. palustris. 

Figure 1–52—(1) Uredinia (white arrow heads) and telia (white arrows) of Cronartium ribicola (nos. 1-9) and C. flaccidum

(nos. 10–52) on abaxial leaf surface of the plants.

Figure 4—Uredinia of C. ribicola on Mentzelia lindleyi. 

Figure 5—Uredinia of C. ribicola on Loasa triphylla. 

Figure 6—Uredinia of C. ribicola on Tropaeolum majus. 
196 USDA Forest Service RMRS-P-76. 2018.

Figure 7—Uredinia of C. ribicola on Ribes nigrum ‘Mortti’. 

Figure 8—Uredinia of C. ribicola on Ribes spicatum subsp. spicatum.

Figure 9—Uredinia of C. ribicola on Ribes laxiflorum. 

Figure 10—Telia of Cronartium flaccidum on Melampyrum sylvaticum. 

Figure 11—Telia of C. flaccidum on Melampyrum pratense. 

Figure 12—Telia of C. flaccidum on Melampyrum nemorosum. 

Figure 13—Telia of C. flaccidum on Melampyrum arvense. 

Figure 14—Uredinia and telia of C. flaccidum on Pedicularis palustris ssp. palustris. 

Figure 15—Telia of C. flaccidum on Pedicularis sceptrum-carolinum. 

Figure 16—Telia of C. flaccidum on Pedicularis groenlandica. 
USDA Forest Service RMRS-P-76. 2018. 197

Figure 20—Uredinia and telia of C. flaccidum on Euphrasia minima. 

Figure 21—Telia of C. flaccidum on Euphrasia brevipila. 

Figure 22—Uredinia and telia of C. flaccidum on Euphrasia stricta. 

Figure 23—Uredinia and telia of C. flaccidum on Euphrasia officinalis. 

Figure 24—Uredinia and telia of C. flaccidum on Loasa tricolor. 

Figure 25—Uredinia and telia of C. flaccidum on Loasa triphylla. 

Figure 17—Uredinia of C. flaccidum on Pedicularis dolichorrhiza. 

Figure 18—Uredinia and telia of C. flaccidum on Rhinanthus minor. 

Figure 19—Telia of C. flaccidum on Rhinanthus aestivalis. 
198 USDA Forest Service RMRS-P-76. 2018.

Figure 29—Uredinia and telia of C. flaccidum on Vincetoxicum hirundinaria. 

Figure 30—Uredinia of C. flaccidum on Asclepias incarnata. 

Figure 31—Uredinia of C. flaccidum on Apocynum cannabinum. 

Figure 32—Telium of C. flaccidum on Nicotiana rustica.

Figure 33—Uredinia and telia of C. flaccidum on Swertia fedtschenkoana. 

Figure 34—Uredinia and telia of C. flaccidum on Veronica longifolia. 

Figure 35—Uredinia of C. flaccidum on Veronica incana. 

Figure 36—Uredinia and telia of C. flaccidum on Veronica krylovii. 

Figure 26—Uredinia and telia of C. flaccidum on Bartsia alpina. 

Figure 27—Uredinia and telia of C. flaccidum on Impatiens balsamina. 

Figure 28—Uredinia of C. flaccidum on Impatiens glandulifera. 
USDA Forest Service RMRS-P-76. 2018. 199

Figure 41—Uredinia and telia of C. flaccidum on Paeonia tenuifolia. 

Figure 42—Uredinia of C. flaccidum on Paeonia officinalis. 

Figure 43—Uredinia and telia of C. flaccidum on Paeonia lactiflora. 

Figure 44—Uredinia of C. flaccidum on Paeonia anomala. 

Figure 45—Uredinia of C. flaccidum on Paeonia obovata. 

Figure 46—Uredinia of C. flaccidum on Tropaeolum majus. 

Figure 37—Telia of C. flaccidum on Veronica daurica. 

Figure 38—Uredinia and telia of C. flaccidum on Veronica grandis. 

Figure 39—Uredinia of C. flaccidum on Nemesia versicolor. 

Figure 40—Uredinium of C. flaccidum on Nemesia strumosa. 
200 USDA Forest Service RMRS-P-76. 2018.

Figure 50—Telia of C. flaccidum on Saxifraga hostii. 

Figure 51—Uredinia and telia of C. flaccidum on Castilleja miniata. 

Figure 52—Telia of C. flaccidum on Myrica gale. 

Figure 47—Uredinia and telia of C. flaccidum on Verbena canadensis. 

Figure 48—Uredinia of C. flaccidum on Verbena x hybrida. 

Figure 49—Uredinia of C. flaccidum on Saxifraga exarata. 
USDA Forest Service RMRS-P-76. 2018. 201

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

White Pine Blister Rust Confirmed on Limber Pine (Pinus flexilis)

in Rocky Mountain National Park

A.W. Schoettle1, *, B. Goodrich1, J. Klutsch1, and K.S. Burns2

1 USDA Forest Service, Rocky Mountain Research Station, Fort Collins, Colorado, USA 

2 USDA Forest Service, Forest Health Protection, Rocky Mountain Region, Golden, Colorado, USA

* aschoettle@fs.fed.us

On August 24, 2009, while working in Rocky 

Mountain National Park (Estes Park, Colorado, USA) 

on the Conserving Limber Pine Genetic Diversity 

for Possible Future Restoration at Rocky Mountain 

National Park project (see Schoettle et al. 2011, 

2013, in press), a crew from the U.S. Department of 

Agriculture, Forest Service, Rocky Mountain Research 

Station (RMRS) noticed a small limber pine (Pinus 

flexilis) with a dead top along Highway 36 near the 

Many Parks Curve (fig. 1). The tree was about 2 m 

tall and 7 to 10 cm in diameter at breast height (d.b.h., 

1.37 m) and occurred at about 3,000 m in elevation. 

The UTM coordinates for the tree are E445370, 

N4471762 (WGS84, Z13N). The signs on the main 

stem were consistent with symptoms caused by the 

canker fungus Cronartium ribicola, which causes 

white pine blister rust (WPBR). Mature trees and 

seedlings in the area around the infected tree were 

surveyed on September 11, 2009. One branch in the 

upper crown of a mature tree at 3,020 m in elevation 

resembled WPBR because of the extensive chewing 

by squirrels (Family Sciuridae). The tree was flagged 

and its UTM coordinates are E445230, N4471698 

(WGS84, Z13N). No other suspected cankers were 

found. Similar surveys of mature and young trees 

were conducted at each of 17 research sites within the 

eastern side of the park and no other suspected WPBR 

infections were observed.

Ribes inerme, a susceptible alternate host for C. 

ribicola, is present along Hidden Valley Creek in the 

Beaver Ponds area, suggesting that this area could 

serve as a source of basidiospores to infect limber 

pine up slope if the rust were present in the area. No 

evidence of rust infection of the Ribes plants in the 

Beaver Ponds area was seen during a 2009 inspection.

These observations provided strong evidence for the 

presence of WPBR in Rocky Mountain National Park. 

Plans were made to revisit the area in early summer 

2010 in an attempt to observe the bright orange aecia 

that offer a phenotypic identification of the disease. 

On June 22, 2010 RMRS and Rocky Mountain 

National Park employees surveyed the Many Parks 

Curve and Beaver Ponds areas of the park. Both areas 

were walked with global positioning system devices 

and an attempt was made to survey all limber pine 

within the area for signs or symptoms of WPBR by 

using binoculars or inspecting smaller trees closely. 

At Many Parks Curve, two crews of two people each 

walked parallel to the road for about 1.3 km and surveyed more than 289 limber pine trees and 95 limber 

pine seedlings (<1.34 m in height) over 2.6 ha (fig. 

1). No WPBR infections were found. The limber pine 

tree above the road in the Many Parks Curve area 

found in 2009 with a possible canker (fig. 1) had no 
202 USDA Forest Service RMRS-P-76. 2018.

sporulation, suggesting that the infection was successfully removed the previous summer. The second tree 

found in 2009 with the squirrel chewing also had no 

evidence of sporulation.

At Beaver Ponds, one crew of three people walked 

about 1.2 km and surveyed 78 mature limber pine 

trees and 25 limber pine seedlings (<1.34 m in height) 

between the creek and the ridgeline over 1.8 ha (fig. 

1). One mature limber pine tree was found with a C. 

ribicola sporulating canker (figs. 1 and 2). The tree 

was tagged with a round metal tag (#4149); the UTM 

coordinates for the tree are E 445914, N 4472182 

(WGS84, Z13N), at an elevation of 2,786 m. The tree 

is within 200 m of the stream in a riparian area with 

substantial canopy cover by limber and lodgepole (P. 

contorta) pines as well as some Engelmann spruce 

(Picea engelmannii). The d.b.h. was 28 cm. The single 

canker was 25 cm long on a branch about 1.25 m 

above the ground (fig. 3). Approximately 9 cm of the 

branch had aecial blisters at both the proximal and 

distal ends of the canker; the proximal sporulating 

region was within 20 cm of the main tree bole. The 

aecial blisters had already ruptured (fig. 4). A small 

dead twig was present in the center of the canker and 

a needle on that twig, when alive, was most likely the 

tissue that was initially infected. With further dissection of the cankered branch, our best estimate is that 

infection may have occurred 4 to 6 years before 2010. 

No other cankers were detected in the immediate area 

or on the infected tree. This is the first confirmed case 

of WPBR in Rocky Mountain National Park. 

Figure 1—White pine blister rust survey areas of Many Parks Curve and Beaver Ponds in Rocky Mountain National Park. 

A limber pine possibly infected with Cronartium ribicola was found above Trail Ridge Road in Many Parks area in July 2009 

and a confirmed white pine blister rust canker found in the Beaver Ponds area in 2010 and another up the road in 2017.
USDA Forest Service RMRS-P-76. 2018. 203

Figure 2—The limber pine tree found infected with white 

pine blister rust in the Beaver Ponds area of the Rocky 

Mountain National Park. The cankered branch is the lowest 

branch on the left (yellow arrow). 

The cankered branch was removed. Annual inspections 

in early summer have been made in this area and no 

additional infections have been identified (C. Cleaver, 

Colorado State University, and A. Schoettle, personal 

communication, 2015). Cronartium ribicola was detected, however, on R. inerme in the area in fall 2015 

(K. Burns, personal communication, 2015). Because 

WPBR is difficult to identify in the early stages of an 

outbreak, it is possible there are more infected trees in 

the area.

REFERENCES

Schoettle, A.W.; Burns, K.S.; Cleaver, C.M.; Connor, J.J. 

[in press]. Proactive limber pine conservation strategy 

for the greater Rocky Mountain National Park Area. 

Gen. Tech. Rep. RMRS-GTR-xxx. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky 

Mountain Research Station.

Schoettle, A.W.; Connor, J.; Mack, J.; [et al.]. 2013. 

Establishing the science foundation to sustain highelevation five-needle pine forests threatened by novel 

interacting stresses in four western National Parks. 

The George Wright Forum. 30: 302–312. http://www.

georgewright.org/303schoettle.pdf. (05 October 2015).

Schoettle, A.W.; Goodrich, B.A.; Klutsch, J.G.; [et al.]. 

2011. The proactive strategy for sustaining five-needle 

pine populations: An example of its implementation 

in the southern Rocky Mountains. In: Keane, R.E.; 

Tomback, D.F.; Murray, M.P.; [et al.], eds. The future 

of high-elevation, five-needle white pines in western 

North America: Proceedings of the high five symposium. 

Proceedings RMRS-P-63. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky 

Mountain Research Station: 323–334. https://www.fs.fed.

us/rm/pubs/rmrs_p063/rmrs_p063_323_334.pdf. (05 

October 2015).
204 USDA Forest Service RMRS-P-76. 2018.

Figure 3—The distal end of the canker with the aecia is on the left and the proximal end of the canker, also with aecia, 

is on the right. Overall canker length is 25 cm. The tree bole is to the right.

Figure 4—Proximal sporulating end of the canker (ruler included for 

scale). Note that the aecial blisters have already ruptured and begun 

releasing the orange aeciospores.
USDA Forest Service RMRS-P-76. 2018. 205

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Efficacy of Early Pruning to Reduce the Incidence of White Pine 

Blister Rust on Sugar Pine (Pinus lambertiana)

J. Bronson1, *, J. Petrick2, and R. Danchok3

1 USDA Forest Service, Pacific Northwest Region, Forest Health Protection, Central Point, Oregon, USA

2 USDA Forest Service, Dorena Genetic Resource Center (retired)

3 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA

* jjbronson@fs.fed.us

INTRODUCTION

Sugar pine (Pinus lambertiana) is a five-needle 

pine native to the mixed-conifer forests of the Sierra 

Nevada and Cascade Ranges of western North 

America. Sugar pine has great aesthetic, ecological, 

and economic value and is among the largest of all 

pines. Like other five-needle pines, sugar pine is susceptible to white pine blister rust (WPBR), caused by 

the nonnative pathogen Cronartium ribicola. Although 

tree-breeding efforts have been the most successful 

strategy against WPBR, artificial pruning by removing 

the lowest branches (where infections are most common) can potentially enhance survival of five-needle 

pines exposed to WPBR. Artificial pruning has been 

used successfully in WPBR management programs for 

western white pine (Pinus monticola), although most 

pruning is not done until trees are 10- to 15-year-old 

saplings. Earlier pruning against WPBR cankers and 

pruning on sugar pine have received less attention. 

As part of a larger restoration effort involving several 

Federal agencies, personnel from the U.S. Department 

of Agriculture, Forest Service, Dorena Genetic 

Resource Center (DGRC; Cottage Grove, Oregon, 

USA) established monitoring plots to measure the efficacy of early pruning on the incidence and impact of 

WPBR on planted sugar pine seedlings.

METHODS

Ten planting sites with moderate-to-high WPBR pressure were selected in the southern Cascade Range of 

Oregon, near the Little River on the Umpqua National 

Forest. For each site, sixteen 202-m2 plots were 

established, cleared of woody vegetation, and planted 

with improved and unimproved sugar pine seedlings. 

Twenty-four families of containerized seedlings were 

grown at the DGRC and outplanted in fall 2000. 

These families comprised eight resistant families 

(SB orchard), eight F1 orchard families, and eight 

demonstrated nonresistant families (Wild). Each plot 

was planted with the same mix of 24 seedlings, one 

representative per family per plot. 

Using a paired plot design, half of the plots at each site 

were randomly selected for the pruning treatment, and 

the other half remained untreated as controls. All trees 

in the treated plots were pruned following the fourth 

growing season (2003, when they were 1.2 to 1.5 m in 

height) and every 4 years thereafter. Whole branches 

were removed, starting at the ground, leaving about 

50 percent canopy remaining. Periodic pruning will 

continue until the trees are pruned to a height of 3 m. 

Surveys began in the fall of the fourth year and continued every 2 to 4 years through 2012. Data collected 
206 USDA Forest Service RMRS-P-76. 2018.

0 

100 

200 

300 

400 

500 

600 

700 

2003 2008 2010 2012 

Total Number of Bole Cankers 

Year 

Pruned 

Not Pruned 

94 96 134 107 82 70 33 45 95 113 60 123 113 70 67 39 49 110 98 61

0 

0.2 

0.4 

0.6 

0.8 

1 

1.2 

1 2 3 4 5 6 7 8 9 10 

Mean No. Bole Cankers per Tree 

Site Number 

Pruned 

Not Pruned 

Figure 1—The total number of bole cankers on live trees for each year recorded.

Figure 2—The mean number of bole cankers on live trees per site in 2012. Error bars indicate ± standard error 

of the mean. Numbers inside bars indicate the total number of live trees on the site indicated.
USDA Forest Service RMRS-P-76. 2018. 207

during the surveys include: mortality (and cause if 

evident), number and location of branch cankers, 

number and location of bole cankers, percentage of 

the bole circumference girdled by cankers, and other 

comments. The number of branch cankers on trees in 

treatment plots was recorded prior to pruning.

RESULTS AND DISCUSSION

Cumulative percent mortality on all sites was surprisingly similar between pruned and unpruned plots 

for the years recorded; in fact, cumulative mortality 

in 2012 was identical, 55 percent, for pruned and 

unpruned plots. Similarly, the percentage of trees that 

were killed by WPBR differed little between pruned 

and unpruned plots; 57 and 59 percent of mortality, 

respectively, was attributed to WPBR. WPBR was the 

primary mortality agent although all of the plots contained trees that suffered from animal and insect damage as well. The average height of trees in 2012 was 3.1 

m on pruned plots, and 3.4 m on unpruned plots. Height 

growth of trees was highly variable within and between 

plots, but appeared to be minimally affected by early 

pruning. Between 2004 and 2012, the average increase 

in height of trees was 1.9 m (±0.9 m SD) on the pruned 

plots and 2.3 m (±1.1 m SD) on unpruned plots. 

Overall, more total cankers were observed on untreated, control plot trees than on treated, pruned plot 

trees. As of 2012, trees in unpruned plots contained almost three times as many branch cankers as the pruned 

plots (2,147 and 785, respectively). This result is confounded by the pruning treatment that removes lower 

branches and reduces potential infection area. Analysis 

accounting for this reduction in lower branches and 

resistance capacity may reveal more subtle differences 

between treatment, family resistance, or both. 

The most promising result thus far has been the reduction of bole (stem) cankers on trees in pruned plots. 

As of 2012, there were 292 bole cankers on trees on 

the pruned plots, compared to 562 bole cankers on 

unpruned plots (fig. 1). Trees on the pruned plots 

have had lower numbers of bole cankers consistently 

throughout the study (table 1). The average number 

of bole cankers recorded per tree has also been substantially less on pruned plots (fig. 2). In addition, 

pruning appears to reduce the percentage of trees with 

bole cankers regardless of seedling type (fig. 3). As of 

2012, 16 percent of the F1 orchard families on pruned 

plots had bole cankers, compared to 31 percent on 

unpruned plots. Although early pruning has not yet 

resulted in a significant reduction in WPBR-caused 

mortality, the lower number of bole cankers on these 

trees is very promising. It is likely that we will not 

see the full benefits of pruning for several more years. 

Surveys are expected to continue through 2020.

Table 1—Total number of bole cankers in each site according to treatmenta and year.

 2003 2008 2010 2012

Site 

No. P NP P NP P NP P NP

1 14 12 54 66 67 99 36 81

2 12 8 17 44 27 73 24 59

3 6 8 42 94 33 98 47 90

4 21 38 35 40 23 37 27 65

5 31 62 33 62 40 63 35 45

6 152 142 48 49 36 64 16 17

7 60 53 59 78 76 74 14 31

8 3 6 5 22 7 32 17 44

9 7 14 53 60 108 92 59 102

10 31 13 31 28 33 24 18 28

Mean 34 36 38 54 45 66 29 56 a P - Pruned (treated) plots. NP – Unpruned (control) plots
208 USDA Forest Service RMRS-P-76. 2018.

0% 

10% 

20% 

30% 

40% 

50% 

60% 

F1 SB Wild 

Percent of trees with bole canker 

Seedling Type 

Pruned 

Not Pruned 

Figure 3—The percent of live trees with at least one bole canker by seedling type. F1 

– F1 DGRC orchard families. SB – OP resistant families. Wild – screened susceptible 

families. All families are open-pollinated progeny of phenotypic selections.
USDA Forest Service RMRS-P-76. 2018. 209

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Comparative Genomics of the Cronartium Genus

B. Dhillon1, * and R. Hamelin1

1 University of British Columbia, Forest Sciences Center, Department of Forest and Conservation Sciences,

Vancouver, British Columbia, Canada

* bddhillo@uark.edu

Abstract—The obligate basidiomycete Cronartium ribicola, causal agent of white pine blister rust, has 

been responsible for the decline of five-needle pines across the North American landscape. This nonnative 

pathogen, originally from Asia, was first introduced into eastern North America from Europe. Multiple introductions of infected plant material from Europe have been recorded on the Eastern seaboard, whereas a 

single introduction has been described in the Pacific Northwest region. By decimating the natural stands of 

western white pine (Pinus monticola), C. ribicola has played a significant role in shifting the dominant tree 

species in the northwestern United States. We have sequenced the C. ribicola genome and two other rust 

pathogens using next-generation sequencing technology. Cronartium comandrae, a related rust species 

native to North America, is found on two- and three-needle pines and causes the comandra blister rust 

disease. Both of these rust pathogens are heteroecious, requiring two different plant hosts to complete 

their life cycle. In contrast, Peridermium harknessii, responsible for the western gall rust disease on twoand three-needle pines, is autoecious, requiring only one host for its survival. Through sequence analyses 

of C. ribicola and comparative studies with the related rust pathogens, we aim to explore and understand 

the underlying factors that determine the differences among these three rust species at the genomic level. 

This will help answer some fundamental questions about the biology, host preference, and etiology of each 

pathogen and its evolutionary significance.
210 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Colonization History, Host Distribution, and Landscape Features 

Shape White Pine Blister Rust Populations

R.C. Hamelin1, *, S. Brar1, C.K.M. Tsui1, B. Dhillon1, M. Bergeron1, D.L. Joly2,

and Y.A. El-Kassaby1

1 University of British Columbia, Department of Forest and Conservation Sciences, Vancouver, British Columbia, Canada 

2 Natural Resources Canada, Laurentian Forestry Centre, Québec City, Québec, Canada 

* rhamelin@nrcan.gc.ca

Abstract—White pine blister rust, caused by Cronartium ribicola (Basidiomycota, Pucciniales), was introduced into North America at the beginning of the 20th century on pine seedlings imported from Europe and 

has had serious economic and ecological impacts on white pines. We applied a population and landscape 

genetics approach to understand the patterns of introduction and colonization as well as population 

structure and migration of C. ribicola. We characterized 1,341 samples of C. ribicola from 76 geographic 

locations in North America using 31 single nucleotide polymorphisms and evaluated the effect of landscape 

features, host distribution, and colonization history on the population structure. We identified eastern and 

western genetic populations in North America that are strongly differentiated. Eastern populations have 

genetic diversity two to five times higher than western ones, which can be explained by the repeated accidental introductions of the pathogen in northeastern North America as opposed to the single documented 

introduction in western North America. These distinct genetic populations are maintained by a barrier to 

gene flow that corresponds to a region where host connectivity is interrupted. Furthermore, additional cryptic spatial differentiation was identified in western North America that corresponds to landscape features, 

such as mountain ranges. These results highlight the importance of monitoring this invasive alien tree 

disease to prevent admixture of the eastern and western populations where different pathogen races occur.
USDA Forest Service RMRS-P-76. 2018. 211

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

The World Turned Upside Down: White Pine Blister Rust Pathogen 

Goes Down to Go Up

R.A. Sniezko1, *, J. Stone2, and D.P. Savin1

1 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA

2 Oregon State University, Department of Botany and Plant Pathology, Corvallis, Oregon, USA

* rsniezko@fs.fed.us

Abstract—Cronartium ribicola, the fungal pathogen that causes white pine blister rust, infects pines 

through the needles and progresses to the stem, where cankers typically develop and seedlings or trees 

are killed after the canker girdles the tree. An alternate host, usually species of Ribes in North America, 

harbors the spore stage (basidiospores) that infects pines; there is thought to be no pine-to-pine infection 

via spores. However, in this report we present evidence of pine-to-pine transmission of white pine blister 

rust via root grafting. At Dorena Genetic Resource Center (DGRC; Cottage Grove, Oregon, USA), 2-yearold seedlings of North American white pine species are inoculated with C. ribicola to evaluate genetic 

resistance. After inoculation, the seedlings are assessed for up to 5 years for the different components of 

resistance. Most stem infections appear within 2 to 3 years following artificial inoculation, and mortality 

often occurs within a few years after appearance of stem symptoms (cankers). Some trees remain cankerfree after artificial inoculation, due either to resistance or occasionally being an “escape.” Canker-free trees 

in some of the trials have been followed casually for up to 15 years.

In several of these trials (notably in western white pine, Pinus monticola), we have noted new cankers 

developing at the base of several trees that were previously canker-free, more than 8 years following 

inoculation. Such cankers could have resulted from new infections from ambient local rust basidiospores. 

New infections attributed to local inoculum have been observed sporadically, occurring on upper branches 

in surviving trees. In trees with new basal cankers, however, there was no foliage at the base of the tree to 

permit normal infection through needles. Alternatively, the cankers could be latent or slow-developing infections on resistant trees. A third explanation to consider is that the new cankers result from transmission of 

the rust fungus from the root system of one seedling to an adjacent tree via root grafting and manifest as 

a canker at the base of the previously non-cankered tree. The frequency of cankers resulting from putative root-grafting was 3.0 percent (46/1,539) and 1.5 percent (33/2,214) in the two trials summarized to 

date. The seedlings at DGRC are planted at close spacing, permitting a near-optimum scenario in which 

to observe this phenomenon. The excavations of roots of several surviving newly cankered trees have 

revealed root grafting between the live trees and adjacent rust-killed individuals. Histology work is now 

underway to examine the roots for presence of white pine blister rust. At least in these resistance trials, if 

root grafting occurs, it could potentially bypass some resistances in previously canker-free trees.
212 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Widening the Landscape: The Role of Ornamental and Wild Trees in 

the Spread and Adaptation of Melampsora larici-populina Leaf Rust

A. Dowkiw1, *, E. Voisin1, V. Dievart2, T. Servouse2, M. Berteloot1, V. Guérin1,

T. Bourgeois1, A. Andrieux3, and P. Frey3

1 Institut National de la Recherche Agronomique, Amélioration Génétique et Physiologie Forestières, Orléans, France

2 Institut National de la Recherche Agronomique, Génétique et Biomasse Forestières, Orléans, France

3 Institut National de la Recherche Agronomique, Interactions Arbres-Microorganismes, Champenoux, France

* arnaud.dowkiw@inra.fr

Abstract—Poplar rust, caused by Melampsora larici-populina, has become the primary disease affecting 

poplars (Populus spp.) in northern Europe after several cultivars selected for complete resistance became 

susceptible following pathogen adaptation. Although understanding pathogen adaptation requires working 

at the genetic and molecular levels both in the cultivated host and in the pathogen, landscape-level study 

of all other partners of the interaction is also needed to understand the process.

We will present the results from a 3-year project aimed at elucidating the role of wild poplars in the adaptive potential of the pathogen to an exotic and noncommercially deployed resistance gene. Melampsora 

larici-populina isolates were sampled in different environments with different selective pressures. We found 

that up to 13 percent of the isolates collected on wild Lombardy poplar (P. nigra) trees in environments 

where the resistance gene is absent were able to defeat the gene. This finding demonstrates that wild host 

populations act as a reservoir for pathogen adaptation.

We will also present initial results from an ongoing project where ornamental poplar and larch (Larix spp.) 

(aecial host) trees have been recorded, mapped, and characterized in urban and rural areas outside the 

natural range of sympatry between alternate hosts. This project aims at elucidating how scattered trees 

outside the forest influence the timing and rate of rust epidemics.
USDA Forest Service RMRS-P-76. 2018. 213

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Adaptation of Poplar Rust to the Poplar Varietal Landscape

K.J. Hayden1, 2, C. Xhaard1, 2, B. Fabre1, 2, F. Halkett1, 2, and P. Frey1, 2, *

1 Institut National de la Recherche Agronomique, Interactions Arbres–Microorganismes, Champenoux, France

2 Université de Lorraine, Interactions Arbres–Microorganismes, Vandoeuvre-les-Nancy, France

*

 pascal.frey@inra.fr

The breakdown of host resistance to plant pathogens 

is of critical concern in agriculture, forestry, and the 

management of natural systems. Evolution of the fungal poplar rust pathogen Melampsora larici-populina

has been shown to have been dramatically influenced 

by the deployment of resistance genes in commercial 

poplar (Populus spp.) plantations, with pathogen populations inundated by virulent genotypes (Xhaard et al. 

2011). The deployment and subsequent breakdown of 

resistance genes in poplar plantations provide an experimental system for understanding the dynamics of 

pathogen evolution in response to resistance breeding, 

which will be critical to formulating effective management strategies in the future.

We describe a combined retrospective and prospective 

approach, integrating population genomics, landscape 

epidemiology, and evolution of life history traits of the 

poplar rust fungus. First, the records of poplar genotypes deployed across France over the last 17 years, 

along with genotypes of M. larici-populina collected 

across France, have been used to make overlaying 

maps of host and pathogen resistance and virulence 

genotypes. We demonstrate that the virulence 

landscape continues to be dominated by the sweep 

described by Xhaard et al. (2011); regions heavily 

planted with poplar resistance type R7 are still dominated by the corresponding virulence type, along with 

virulence types that “hitchhiked” across the landscape 

during the original sweep. These genotypes persist, despite a reduction in planting of R7, and a near-absence 

of resistance types corresponding to the hitchhikers. In 

addition, the beginnings of a new sweep are emerging 

in regions in which a recently overcome resistance 

type (R8) has been more widely planted. We also 

describe plans to forecast the ability of the pathogen to 

evolve resistance to the future deployment of quantitative resistance types. To accomplish this objective, we 

will use the information learned from objective 1, and 

experimental tests of tradeoffs in pathogen aggressiveness, reproduction, and spread in response to quantitative resistance (resistance measured by degree). These 

forecasts will provide a framework for the future 

management of poplar and other forest plantations, and 

will also provide testable hypotheses with which to 

continue to improve understanding of host–pathogen 

co-evolution.

REFERENCES

Xhaard, C.; Fabre, B.; Andrieux, A.; [et al.]. 2011. The 

genetic structure of the plant pathogenic fungus 

Melampsora larici-populina on its wild host is 

extensively impacted by host domestication. Molecular 

Ecology. 20: 2739–2755.
214 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Impact of White Pine Blister Rust on Resistant and Previously 

Immune Cultivated Ribes and Neighboring Eastern White Pine

(Pinus strobus) in New Hampshire, USA

I.A. Munck1, *, P. Tanguay2, J. Weimer3, K. Lombard3, S. Villani4, and K. Cox4

1 USDA Forest Service, State and Private Forestry, Forest Health Protection, Durham, New Hampshire, USA

2 Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Sainte-Foy, Québec, Canada 

3 New Hampshire Division of Forests and Lands, Concord, New Hampshire, USA

4 Cornell University, Department of Plant Pathology and Plant-Microbe Biology, Geneva, New York, USA

* imunck@fs.fed.us

White pine blister rust (WPBR; causal agent: 

Cronartium ribicola) has been a threat to both forest resources and agricultural commodities since its introduction to North America in the early 1900s. Eastern white 

pine (Pinus strobus) is the most economically important 

timber species in New Hampshire (USA). From 1917 to 

1970 Ribes throughout the State was eradicated to protect the timber industry. After the lifting of the Federal 

Quarantine Act (USDA Bureau of Entomology and 

Plant Quarantine Federal Domestic Plant Quarantine 

number 63 [1938]) restricting Ribes and the more recent 

availability of immune and resistant Ribes cultivars, 

several States in the Northeast amended their restrictions to allow planting of selected Ribes cultivars. In 

New Hampshire, 19 WPBR-resistant or immune Ribes

cultivars were available for planting. In 2011, WPBR 

was reported for the first time on immune black currant 

(Ribes nigrum cv. Titania) in Connecticut. The objectives of this study were to evaluate the impact of WPBR 

on cultivated resistant and immune Ribes cultivars and 

neighboring white pines.

To accomplish our objectives, 42 sites with cultivated 

Ribes were surveyed in late July through September 

2013. At each site, incidence and severity were assessed 

on up to five plants per Ribes cultivar. Heavily infected 

leaves of each cultivar were collected for polymerase 

chain reaction (PCR) analyses conducted at Cornell 

University. The same methods were used to assess 

WPBR incidence and severity of wild Ribes found 

in the vicinity. Samples from immune black currants 

infected with WPBR from New Hampshire were used 

to inoculate immune Ribes cultivars from the Canadian 

Clonal Genebank in Harrow, Ontario. In addition, the 

following data were collected for the 12 nearest eastern 

white pines within 300 m of cultivated Ribes: WPBR 

incidence, diameter at breast height (1.37 m above the 

ground), distance, and cardinal direction from cultivated 

Ribes. 

Two hundred and fifty-five plants of 19 Ribes cultivars 

and 445 white pines were evaluated. ‘Titania’ was the 

most frequently planted Ribes cultivar. Incidence of 

WPBR ranged from 0 to 60 percent and from 0 to 100 

percent for immune and resistant Ribes cultivars, respectively. As expected, mean WPBR severity on resistant Ribes was limited to less than 6 percent of leaf area 

whereas WPBR severity on previously immune Ribes

was greater, 14 percent of leaf area. All infected Ribes

cultivars were PCR-positive for C. ribicola. Samples 

from New Hampshire successfully infected immune 

Ribes cultivars from the Canadian Clonal Genebank. 

It was more likely to find infected pines neighboring 

infected immune black currants (probability = 0.18) 

than neighboring infected resistant Ribes (0.09), or 

WPBR-free Ribes (0.02). Results from this study led 

to the removal of the previously immune black currant 

cultivars ‘Consort,’ ‘Coronet,’ ‘Crusader,’ and ‘Titania’ 

from the New Hampshire approved list for planting. The 

breakdown of WPBR immunity poses a threat to the 

white pine resource and to cultivated Ribes production.
USDA Forest Service RMRS-P-76. 2018. 215

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Origin and Distribution in Canada of the Cronartium ribicola vCr

Race, Virulent on Immune Black Currant (Ribes nigrum) Cultivars 

Derived from Hunter’s Selections

P. Tanguay1, *

1 Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Québec City, Québec, Canada 

* philippe.tanguay@nrcan.gc.ca

Abstract—Black currant (Ribes nigrum) breeding programs were undertaken by different countries as a 

mean to control pests and pathogens, including Cronartium ribicola, the agent responsible for white pine 

blister rust (WPBR). This rust pathogen requires currants as its alternate telial host to complete its life cycle 

and infect five-needle pines, often causing mortality. The R. ussuriense Cr dominant resistance gene was 

introgressed into R. nigrum to generate the first immune black currant cultivars, called Hunter’s selections. 

Three of these cultivars (‘Coronet,’ ‘Crusader,’ and ‘Consort’) were commercially released in the 1950s, 

and further bred to generate the more recent C. ribicola-immune cultivars such as ‘Titania,’ ‘Tisel,’ and 

‘Blackcomb.’ Breakdown of the Ribes Cr resistance gene was reported in Connecticut in 2011. The same 

year, the vCr race was observed on immune Ribes cultivars in Quebec. Aecidiospores carrying the vCr

gene were sampled on eastern white pine (Pinus strobus) cankers in spring 2012 and 2013. In late summer 2013, vCr stains were present in fields of black currant producers in Quebec, New Brunswick, Nova 

Scotia, and Prince Edward Island, but absent in southern Ontario (Canada). Genetic analyses suggest the 

vCr race is not from a new introduction, but rather resulted from either a novel mutation or DNA recombination in a North American strain.
216 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Frequency and Distribution of Virulence in Cronartium ribicola to Two 

Simply Inherited Resistance Genes in Sugar Pine (Pinus lambertiana) 

and Western White Pine (P. monticola) in California, USA

D.R. Vogler1, *, A.D. Mix2, D. Burton3, D. Davis3, R. Westfall4, and B.B. Kinloch, Jr.1

1 USDA Forest Service, Pacific Southwest Research Station, Institute of Forest Genetics (retired) 

2 USDA Forest Service, Pacific Southwest Research Station, Institute of Forest Genetics, Placerville, California, USA

3 USDA Forest Service Pacific Southwest Region (retired) 

4 USDA Forest Service, Pacific Southwest Research Station, Albany, California, USA

* dvogler @fs.fed.us

Abstract—For the last two decades, we have been documenting in California (USA) the frequency of 

virulence in the blister rust pathogen Cronartium ribicola to simply inherited genes for complete resistance 

in sugar pine [Pinus lambertiana (Cr1)] and western white pine [P. monticola (Cr2)]. At the Happy Camp 

Outplant Site in the north, seedlings of both species have been planted extensively for silvicultural and 

research purposes. At Mountain Home Demonstration State Forest in the southern Sierra Nevada, sugar 

pine is a major component of the giant sequoia- (Sequoiadendron giganteum) dominated forests. At both 

sites, blister rust infection occurs at high frequency because of near-optimal climatic conditions and an 

abundance of Pinus and Ribes hosts. Our test protocol requires suspending telia-bearing Ribes leaves, 

collected in early fall from several sites at each field location, over resistant (R_) and susceptible (rr) 

juvenile seedlings of each pine host. We inoculate pines for 72 hours in the dark in dew chambers under 

optimal conditions for infection at the U.S. Department of Agriculture, Forest Service, Institute of Forest 

Genetics in Placerville. As infection appears, we monitor resistant and susceptible needle-spot reactions, 

observe their development over 6 months, and confirm onset of definitive stem infection. We report here 

results from the mid-1990s through spring 2014. At both sites, virulence in C. ribicola has increased in 

frequency and become more widespread.
USDA Forest Service RMRS-P-76. 2018. 217

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Stem Rust Diseases of Pinus kesiya in Vietnam

P.Q. Thu1, *, S. Kaneko2, and M. Imazu3

1 Vietnamese Academy of Forest Sciences, Forest Protection Research Center, Hanoi, Vietnam 

2 National Institute of Agrobiological Sciences, Ibaraki, Japan

3 Shinshu University, Nagano, Japan

* phamquangthu@vafs.gov.vn

Surveys of two stem rust diseases of Pinus kesiya

were implemented in Da Lat, Lam Dong province, in 

the Central Highlands of Vietnam. Symptoms of the 

first disease, a gall rust, are very similar to those of 

Cronartium quercuum in North America. Our surveys 

found many Quercus cambodiensis trees severely infected by the uredinial and telial states of a Cronartium

species, later identified by microscopic characteristics 

as Cronartium orientale, which is native to eastern 

Asia. The aecial state of C. orientale occurs on various 

two-needle pines. We tentatively identified the gall 

rust on P. kesiya as C. orientale. We conducted inoculation experiments to demonstrate host alternation by 

C. orientale. 

The gall rust of P. kesiya is very common in both 

forest plantations and natural stands in Lam Dong 

province. Disease incidence and severity vary among 

survey areas. In cases of high disease severity, almost 

all branches of a pine tree have galls, and exhibit poor 

form due to the death of many branches and stems. 

Disease incidence and severity are lowest in young 

plantations, reach the greatest levels in 5- to 10-yearold plantations, and are lower again in 15-year-old 

plantations. Main stems that have galls are very brittle.

The second rust found during surveys was a blister 

rust, with symptoms much like those produced by C. 

flaccidum and Endocronartium species, although these 

species have not previously been recorded in Vietnam. 

The blister rust attacks stems and branches of the pine. 

The period of aeciospore production is longer than 3 

months. The germination type of aeciospores differed 

from that of Endocronartium, which has a pine-to-pine 

life cycle. The microscopic characteristics of the aecial 

states are also identical to those of C. flaccidum. By 

comparisons with C. flaccidum from Japan and Russia 

based on nucleotide sequences of the D1/D2 region 

of 28S rDNA, the blister rust on P. kesiya was identified as C. flaccidum. This species is a host-alternating 

species, but no suspected alternate hosts have been 

found in Da Lat. A pine-to-pine biological form of this 

species is known in Europe. To clarify the life cycle 

in Da Lat, an artificial inoculation experiment with 

aeciospores onto seedlings of P. kesiya was conducted 

in Hanoi. However, the seedlings remained uninfected 

for 2 years after the inoculation. Further studies on the 

life cycle are needed. 

The damage by the two rusts to young pine plantations 

is very severe. Infected trees were very susceptible 

to attack by bark beetles (Dendroctonus spp.) and the 

longhorned beetle Monochamus alternatus, a vector of 

the nematode Bursaphelenchus species, which causes 

pine wilt disease. Measures for minimizing disease 

impacts based on integrated pest management methods 

are discussed.
218 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Barcoding the Rust Fungi of Germany

M. Scholler1, *, L. Beenken2, M. Lutz3, W. Maier4, A. Tahir5, and M. Thines5

1 Staatliches Museum für Naturkunde Karlsruhe, Karlsruhe, Germany 

2 Swiss Federal Institute of Technology Zurich, Institute of Integrative Biology, Zurich, Switzerland

3 Universität Tübingen, Institut für Evolution und Ökologie, Evolutionäre Ökologie der Pflanzen, Tuebingen, Germany

4 Bundesforschungsinstitut für Kulturpflanzen, Institut für Epidemiologie und Pathogendiagnostik,

Julius Kühn-Institut, Braunschweig, Germany

5 Biodiversity and Climate Research Centre, Frankfurt, Germany

* scholler@naturkundeka-bw.de

The German Barcode of Life (GBOL) project is a 

large-scale DNA barcoding initiative to assess the 

biodiversity of animals, fungi, and plants of Germany. 

Here we introduce the subproject focusing on rust 

fungi (Pucciniales, Basidiomycota). This is the only 

group of fungi represented in the initial 3.5-year phase 

of the project. 

The goal of the rust project is a complete DNAbarcode sequence library for the roughly 500 rust 

species reported for Germany. The DNA barcode 

database will link the extensive information available 

on morphology, ecology, and host range studies with 

barcoding data linked to individual specimens. Rust 

fungi are exceptionally well studied in Central Europe. 

A significant number of cryptic species may be recovered; alternatively, several species described from 

different hosts may need to be merged. Further, the full 

life cycles and host relationships, particularly of some 

host-alternating species, await elucidation. These data 

will allow identification of rust specimens in museum 

collections, which to date could not be identified because of missing discriminatory spore states. 

Most DNA samples are extracted from herbarium 

specimens of the fungus collections preserved at the 

Natural History Museum Karlsruhe in Karlsruhe 

Germany, but targeted collecting of additional specimens will also be necessary. In the first phase of the 

project, primer selection and optimization were found 

to be essential for successful generation of DNA 

barcodes. The internal transcribed spacer (ITS) rDNA 

region and the large subunit of the rDNA are suitable 

common fungal barcode markers. To facilitate highthroughput barcoding, methods have been developed 

for semiautomatic DNA extraction from herbarium 

specimens and polymerase chain reactions in 96 well 

plates.

Currently the following groups are extensively DNAbarcoded: Uromyces pisi s.l., an especially complicated complex of species, and the genera Melampsora

and Coleosporium. An example of an as-yet unidentified Coleosporium species on invasive Solidago (goldenrod) is presented. 
USDA Forest Service RMRS-P-76. 2018. 219

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Geographic Distribution of Cronartium flaccidum and Peridermium 

pini in Sweden and Finland

B. Samils1, *, P. Barklund1, J. Kaitera2, and J. Stenlid1

1 Swedish University of Agricultural Sciences, Department of Forest Mycology and Plant Pathology, Uppsala, Sweden

2 Natural Resources Institute Finland, Oulu, Finland

* berit.samils@slu.se

Abstract—Extensive attacks of blister rust on Scots pine (Pinus sylvestris) are widespread across northern Sweden, especially in Norrbotten and Västerbotten Counties. The first reports of damage came in 

2002, and an inventory in 2008 showed that 130,000 ha, or 34 percent of young pine forests in this region, 

were infested. A follow-up inventory in 2012 did not indicate any decrease in rust attacks. What induced 

the sudden epidemic is not known. The blister rust on Scots pine is caused by two forms of the fungus: 

the macrocyclic host-alternating Cronartium flaccidum, and Peridermium pini (syn. Endocronartium pini), 

which spreads clonally from pine to pine. Melampyrum sylvaticum is considered to be the most important 

alternate host of C. flaccidum in the northern parts of Sweden and Finland, but several other plants from 

various families are also suitable alternate hosts. The two fungi were recently suggested to belong to the 

same species, as molecular marker data show little overall genetic differentiation between the fungi. It is 

possible to separate the two forms by using molecular markers because P. pini is characterized by totally 

homozygotic aeciospores and C. flaccidum aeciospores contain heterozygous loci.

To study the population genetic structure and geographic distribution of C. flaccidum and P. pini, aeciospore samples were collected from eight different locations in Sweden and five locations in Finland in 2011. 

In total, 220 samples were analyzed by using seven microsatellite markers. Our results showed that C. 

flaccidum dominates the fungal populations in the epidemic areas in northern Sweden. We could not detect 

any distinct geographic separation between C. flaccidum and P. pini, although specific locations or areas 

were dominated by one or the other form. Populations of C. flaccidum in the epidemic areas in northern 

Sweden were genetically similar to the neighboring populations in northern Finland. 
220 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Monitoring Trends in White Pine Blister Rust Infection in the

Greater Yellowstone Ecosystem

E. Shanahan1, *, K.M. Irvine2, C. Hollimon3, K. Legg1, R. Daley1, and Greater Yellowstone 

Whitebark Pine Monitoring Working Group4, 5, 6

1 USDI National Park Service, Greater Yellowstone Monitoring Network, Bozeman, Montana, US

2 USDI U.S. Geological Survey, Northern Rocky Mountain Science Center, Bozeman, Montana, USA

3 Montana State University, Department of Mathematics, Bozeman, Montana, USA

4 USDA Forest Service, Forest Health Protection

5 USDI National Park Service, Grand Teton National Park, Moose, Wyoming, USA

6 USDI National Park Service, Yellowstone National Park, Wyoming, USA 

* erin_shanahan@nps.gov

Abstract—Due to the significant role of whitebark pine (Pinus albicaulis) in the Greater Yellowstone 

ecosystem (Wyoming, Montana, and Idaho, USA) and recent declines of mature, cone-bearing trees, 

monitoring trends in the health and status of whitebark pine populations is an informative component for 

current and future management of the species. Since 2004, the Interagency Whitebark Pine Monitoring 

Program has been documenting whitebark pine health across this ecosystem with data compiled from 176 

permanent transects in 150 randomly selected pure and mixed whitebark pine stands. Specific long-term 

monitoring objectives for this program include estimating (1) the rate at which white pine blister rust (causal 

agent: Cronartium ribicola) infection is changing over time, (2) severity of infection and the transition of 

infection from what is regarded as less harmful to infections considered more lethal, (3) trends in and 

influences on mortality, and (4) recruitment of understory constituents. From 2004 to 2011, each of the 

176 transects was surveyed twice for the presence of blister rust and three times for mortality. Using these 

cumulative data, we have completed the first statistical analysis examining step-trends in blister rust infection, transition of blister rust severity, and overall mortality. This monitoring affords a better understanding 

of the overall condition of the ecosystem and allows managers to gain knowledge, to make informed 

decisions, and to work more effectively with other agencies and individuals for the benefit of parks, other 

Federal lands, and privately held resources. 
USDA Forest Service RMRS-P-76. 2018. 221

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

The Proactive Strategy: Preparing the Landscape for Invasion

by Accelerating the Evolution of Resistance

A.W. Schoettle1, *, K.S. Burns2, and R.A. Sniezko3

1 USDA Forest Service, Rocky Mountain Research Station, Fort Collins, Colorado, USA 

2 USDA Forest Service, Forest Health Protection, Golden, Colorado, USA

3 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA

* aschoettle@fs.fed.us

Restoration of forests devastated by nonnative invaders often dominates the attention of forest managers 

and their actions. However, taking a broader view 

of the invasion beyond the crisis areas reveals opportunities where proactive management can alter the 

outcome of the invasion in threatened areas. Proactive 

management moves past the idea of protecting the 

hosts from exposure to the established nonnative 

invader and shifts toward facilitating naturalization by 

preparing the landscape to sustain critical ecosystem 

function into the future in the presence of the invader 

(Schoettle and Sniezko 2007).

Increasing the frequencies of durable resistance or 

tolerance traits within tree populations is accepted as 

a promising avenue for the coexistence of native tree 

species and nonnative pathogens (King et al. 2010). 

Therefore the objective of proactive intervention is 

to position the ecosystem to facilitate the evolution 

of pathogen resistance in the tree populations upon 

invasion. Diversifying the age class structure by 

stimulating regeneration in the healthy forest will 

provide a larger population size for resistance selection upon invasion and simultaneous selection in both 

the younger and older cohorts (Schoettle and Sniezko 

2007). Accelerating the generation time and natural 

selection process through silvicultural treatments will 

reduce the ecological consequences of mortality in any 

one cohort and increases the potential for development 

of durable resistance within the population while 

maintaining broad genetic diversity. Planting seedlings 

or seed from seed trees that have heritable resistance 

before pathogen invasion will provide additional time 

for these individuals to mature and contribute to gene 

flow before the overstory is heavily affected upon 

invasion. Screening for resistance and planting early, 

rather than after the stand is heavily infested, reduces 

the window of time when forest regeneration may be 

limited by seed availability. 

Early attention to threatened but not yet infected 

species or stands also provides opportunity for gene 

conservation. Seed, pollen, or tissue collections can be 

made from the full genetic diversity of the population 

before the potential bottleneck caused by high pathogen-induced mortality. In the case of Pinus aristata, 

rangewide seed and tissue samples have now been 

archived before population impacts by Cronartium 

ribicola, and working collections are available for 

research collaborations.

The Proactive Strategy has been adopted for P. 

flexilis, P. aristata, P. longevea, and P. strobiformis

in the southern Rocky Mountains, the Southwest, 

and portions of the Great Basin (which covers parts 

of six western States) for the white pine blister 

rust pathosystem (Goodrich et al., Pinus strobiformis Gene Conservation and Genecology, this 
222 USDA Forest Service RMRS-P-76. 2018.

proceedings; Schoettle et al. 2011, 2013) and for 

healthy Chamaecyparis lawsoniana in stands at risk 

for impacts from Phytophthora lateralis. It has been 

incorporated into the National Strategic Framework 

for Invasive Species Management (2013) (http://www.

fs.fed.us/foresthealth/publications/Framework_for_

Invasive_Species_FS-1017.pdf).

REFERENCES

King, J.N.; David, A.; Noshad, D.; [et al.]. 2010. A review 

of genetic approaches to the management of blister rust 

in white pines. Forest Pathology. 40: 292–313.

Schoettle, A.W.; Connor, J.; Mack, J.; [et al.]. 2013. 

Establishing the science foundation to sustain highelevation five-needle pine forests threatened by novel 

interacting stresses in four western National Parks. 

The George Wright Forum. 30: 302–312. http://www.

georgewright.org/303schoettle.pdf. 

Schoettle, A.W.; Goodrich, B.A.; Klutsch, J.G.; [et al.]. 

2011. The proactive strategy for sustaining five-needle 

pine populations: An example of its implementation 

in the southern Rocky Mountains. In: Keane, R.E.; 

Tomback, D.F.; Murray, M.P.; [et al.], eds. The future 

of high-elevation, five-needle white pines in western 

North America: Proceedings of the high five symposium. 

Proceedings RMRS-P-63. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky 

Mountain Research Station: 323–334. 

Schoettle, A.W.; Sniezko, R.A. 2007. Proactive 

intervention to sustain high elevation pine ecosystems 

threatened by white pine blister rust. Journal of Forest 

Research. 12: 327–336. http://www.springerlink.com/

content/9v91t44278w74430/fulltext.pdf.
USDA Forest Service RMRS-P-76. 2018. 223

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

White Pine Blister Rust Infection Dynamics Over 25 Years in Sugar 

Pine (Pinus lambertiana) Progeny Tests on Six Sites Rated as Low, 

Mid, and High Rust Hazard in Southern Oregon, USA

R.A. Sniezko1, *, R. Danchok1, D.P. Savin1, A. Kegley1, and S. Long1

1 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA 

* rsniezko@fs.fed.us

Abstract—Sugar pine (SP, Pinus lambertiana), western white pine (WWP, P. monticola), whitebark pine 

(P. albicaulis), and limber pine (P. flexilis)—the four white pine species native to Oregon (USA)—are very 

susceptible to white pine blister rust (WPBR), caused by the fungal pathogen Cronartium ribicola. High 

infection and mortality have been documented on some sites, but few or no long-term evaluations of the 

dynamics of rust infection over time have been made. We report here on rust infection over approximately 

25 years for six SP trials established in southwestern Oregon. The sites had been rated for rust hazard by 

using protocols developed for WWP, and the hazard for the sites ranged from low to high. Open-pollinated 

seedling progeny of phenotypic parent tree selections were established at the sites (36 to 49 families per 

site), with 31 families in common. The families included some highly susceptible, some with low levels of 

partial resistance, and some with major gene resistance (MGR).

Performance at 5 and 10 years after planting was consistent with the hazard ratings; all sites showed rust 

infection, and the sites rated as high hazard showed the highest incidence of stem infection (cankers). 

Little mortality was observed at age 5, but the high hazard sites experienced moderate to high levels of 

mortality by age 10. By age 15, infection levels on the low-to-moderate hazard sites reached high levels; 

but mortality levels were still relatively low, whereas mortality on the two high hazard sites was high. At 

the latest assessment, 23 to 27 years post-planting, all sites showed high levels of infection (81.4 to 94.8 

percent) and mortality (61.9 to 93.4 percent), with the low-to-moderate hazard sites lagging only slightly 

behind the high hazard sites. Excluding nonrust mortality and averaged over all 6 sites, only 8.9 percent of 

the 8,340 trees were canker-free and only 22.4 percent were alive. Approximately 60 percent of the surviving trees were cankered (with the percentage varying by site, from 21.2 to 77.0 percent), and mortality is 

expected to increase. A notable number of cankered trees seemed to be restricting the radial growth of the 

canker, suggesting that some form of partial resistance may be operating. The families with MGR had the 

lowest infection at all sites. Most of the cankers were relatively low in the tree, which suggests that branch 

pruning could be an effective strategy to enhance survival at least on some sites. Hazard rating for WPBR 

in southern Oregon has value, but these data indicate that it may not be a static value; perhaps “wave” 

years or changes in rust hazard (for example, due to adjacent stand management or fire, which potentially 

increases the number of alternate hosts) can affect the long-term predictive ability of current rust hazard 

rating protocols.
224 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

White Pine Blister Rust Infection in Whitebark Pine (Pinus albicaulis) 

Seedlings Over a Wide Range of Inoculum Density Levels in an 

Artificial Inoculation Trial 

D.P. Savin1, R. Danchok1, A. Kegley1, and R.A. Sniezko1, *

1 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA

* rsniezko@fs.fed.us

Abstract—Whitebark pine (Pinus albicaulis) is a wide-ranging high elevation conifer of forest ecosystems 

in the western United States and Canada. It is highly susceptible to white pine blister rust (WPBR), caused 

by the nonnative fungal pathogen Cronartium ribicola. Whitebark pine has been in decline in many forest 

ecosystems for decades, with WPBR being a major causative agent. In Canada, it has been listed as 

an endangered species, and in the United States it has been proposed for listing under the Endangered 

Species Act. Gene conservation efforts have begun and programs to identify parent trees of whitebark pine 

genetically resistant to WPBR are underway. In WPBR testing, seedling progeny of candidate parent trees 

are artificially inoculated with C. ribicola basidiospores to determine the level of genetic resistance conveyed by each parent. Progeny of hundreds of parent trees have now been evaluated for resistance. Thus 

far, no complete resistance (a hypersensitive-like response in the needles that precludes stem infection in 

the absence of a virulent strain of rust) such as that which has been found in four other species of white 

pines native to the western United States has been found in whitebark pine. However, in rust resistance 

screening, the whitebark pine seedling families show a range of partial resistance from very highly susceptible to moderately resistant. In most inoculation trials, the seedling progeny are usually inoculated with 

a single spore density (usually ~3,000 spores cm-2, at Dorena Genetic Resource Center, Cottage Grove, 

Oregon, USA) to help determine the relative rankings of the families. Little is known about how varying 

spore density levels might affect the level of resistance shown in this species. We report on results from a 

trial where 2-year-old seedlings from 7 families (2 susceptible, 5 resistant) were inoculated (in September 

2011) with 12 spore densities ranging from 50 to 15,400 spores cm-2. Inoculation was very successful, with 

99.1 percent of seedlings showing needle spots. Averaged over all inoculum levels, 80.8 percent of seedlings had cankers; the two susceptible families averaged 97.9 percent of seedlings with cankers, whereas 

73.9 percent of the seedlings in the five resistant families were cankered. In the most resistant family, only 

59.6 percent of the seedlings had cankers. Through December 2013, overall mortality was 59.3 percent. 

Higher spore densities applied during artificial inoculation resulted in increased levels of needle spots, 

severity of stem symptoms, percentage of trees with stem symptoms, and mortality percentage.
USDA Forest Service RMRS-P-76. 2018. 225

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Cytological Analyses Reveal Variations in Nuclear Content Along

the Urediniosporic Infection Cycle of Hemileia vastatrix

and other Rust Fungi

S. Tavares1, 2, A.S. Pires1, 2, H.G. Azinheira1, A.P. Ramos3, C. Bispo4, C. Andrade4,

A. Loureiro1, D. Schmidt5, T. Link5, R.T. Voegele5, R. Gardner4, R. Abranches2,

M.C. Silva1, J. Loureiro6, and P. Talhinhas1, 2, *

1 Instituto de Investigação Científica Tropical, BioTrop, Centro de Investigação das Ferrugens do Cafeeiro,

Quinta do Marquês, Oeiras, Portugal

2 Universidade Nova de Lisboa, Instituto de Tecnologia Química e Biológica, Plant Cell Biology Laboratory, Oeiras, Portugal

3 Universidade de Lisboa, Instituto Superior de Agronomia, CEER–Biosystems Engineering, Lisboa, Portugal

4 Instituto Gulbenkian de Ciência, Oeiras, Portugal

5 Universität Hohenheim, Institut für Phytomedizin, Stuttgart, Germany

6 University of Coimbra, CFE–Centre for Functional Ecology, Department of Life Sciences, Coimbra, Portugal

* ptalhinhas@iict.pt

The recent completion of genome sequencing for some 

rust fungi has contributed to suggest a link between 

biotrophic specialization and genome size expansion. 

The measurement of genome sizes for a selection of 

rust fungi has revealed some of the largest genomes 

among fungi, with nine rust species with haploid 

genomes between 300 and 780 Mbp. In particular, 

Uromyces appendiculatus, Phakopsora pachyrhizi, U. 

transversalis, and Hemileia vastatrix genomes were 

measured as 652, 716, 746, and 772 Mbp, respectively. 

Moreover, flow cytometric analysis of H. vastatrixinfected coffee (Coffea spp.) leaf samples consistently 

yielded profiles compatible with the occurrence of 

three different nuclear contents (n, 2n, and 4n). The 

n and 2n populations contained relatively similar 

numbers of nuclei, whereas the 4n population clearly 

represented a minority. Pre-sporulating and sporulating (urediniosporic) coffee leaf infections presented 

these three nuclei populations. The n population was 

absent from both resting urediniospores and germinating urediniospores but reappeared in appressoria. 

These findings are compatible with the occurrence of 

karyogamy and meiosis prior to sporulation, although 

parasexuality phenomena cannot be ruled out. In fact, 

although several orthologs of meiosis-specific genes 

are present in the H. vastatrix genome, only one of 

these was identified in H. vastatrix expressed sequence 

tag (EST) libraries obtained from germinating urediniospores, appressoria, and pre-sporulating infected 

leaves. Some karyogamy-related genes were identified 

both in the genome and in the EST libraries. Moreover, 

the occurrence of three different nuclear contents was 

also verified in infected host samples (at the urediniosporic stage, just before sporulation) for at least 

Phakopsora pachyrhizi, Puccinia pelargonii-zonalis, 

U. appendiculatus, and U. fabae, in contrast with the 

absence of the n population in resting and germinating 

urediniospores. This finding suggests that this phenomenon may be common across the Pucciniales.

ACKNOWLEDGMENTS

This work is being funded by Portuguese National 

Funds through Fundação para a Ciência e a Tecnologia 
226 USDA Forest Service RMRS-P-76. 2018.

(project PTDC/AGR-GPL/114949/2009 and grants 

SFRH/BPD/47008/2008, SFRH/BPD/65965/2009, 

and SFRH/BPD/88994/2012), and by the Deutscher 

Akademischer Austauschdienst (DAAD, Germany) in 

the scope of the Portuguese-German bilateral collaboration project “Functional and cytological characterisation of Hemileia vastatrix genes expressed during the 

infection process of coffee leaves.”
USDA Forest Service RMRS-P-76. 2018. 227

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Discovery of Mycovirus Community in White Pine Blister Rust 

Ecosystems by Rust Transcriptome Profiling

J.-J. Liu1, *, A. Zamany1, R.A. Sturrock1, R. Hunt1, and R.A. Sniezko2

1 Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, Victoria, British Columbia, Canada 

2 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA

* Jun-jun.liu@nrcan.gc.ca

In North America, the nonnative rust fungus 

Cronartium ribicola kills immature native five-needle 

pine trees and indirectly predisposes maturing trees 

to secondary attack by insects and other fungi, which 

may result in severe mortality. Since the early 1900s 

when C. ribicola arrived, environmental factors, individual trees, the rust fungus, and insects (for example, 

mountain pine beetle [Dendroctonus ponderosae]) 

have all interacted to dramatically change forested ecosystems across North America. To date our white pine 

blister rust (WPBR) research has generated genomics 

data from the WPBR ecosystem, including transcriptomes and single nucleotide polymorphisms (SNPs) 

from healthy western white pines (WWPs, Pinus 

monticola), whitebark pines (WBPs, P. albicaulis), and 

infected white pines (down to 60 percent of transcripts 

from tree tissues, up to 40 percent of transcripts from 

C. ribicola), and from C. ribicola strains at different 

stages of its life cycle by RNA-sequencing technology. Transcriptome analyses have identified many 

expressed genes derived from mycoviruses. Even 

though the genomic information from organisms other 

than white pine and C. ribicola accounted for about 1 

percent of the total sequence data, this eco-genomics 

strategy showed complex interactions of multiple 

organisms in the WPBR ecosystem. Bioinformatics 

analysis indicates that there are multiple mycoviral 

double-stranded RNAs (ds-RNAs) in C. ribicola, 

the presence of which was further supported by dsRNA extraction and gel electrophoresis analysis. We 

observed variation in mycoviral sequences (including 

presence and absence) across C. ribicola strains collected from British Columbia (Canada) and Oregon 

(USA). Kinloch et al. (2004) provided evidence and 

suggested cytoplasmic inheritance of C. ribicola

virulence (both vcr1 and vcr2), so our discovery of 

novel mycoviral ds-RNAs as cytoplasmic genetic factors in C. ribicola may open new avenues for WPBR 

management. We are investigating rust mycoviruses 

to develop cytoplasmic markers for epidemiological 

diagnosis and study of biodiversity. Progress on this 

and our related work will be further discussed.

REFERENCES

Kinloch, Bohun B., Jr.; Sniezko, Richard A.; Dupper, Gayle 

E. 2004. Virulence gene distribution and dynamics of 

the white pine blister rust pathogen in western North 

America. Phytopathology. 94(7): 751–758.
228 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Research on the Rust Hyperparasite Cladosporium tenuissimum: 

Status and Prospects

S. Moricca1, *, B. Ginetti1, A. Ragazzi1, A. Lupo2, and G. Assante3

1 Università di Firenze, Dipartimento di Scienze delle Produzioni Agroalimentari e dell’Ambiente, Florence, Italy 

2 Università del Sannio, Dipartimento di Scienze Biologiche ed Ambientali, Facoltà di Scienze, Benevento, Italy

3 Università di Milano, Dipartimento di Patologia Vegetale, Milan, Italy

* salvatore.moricca@unifi.it

The use of biological agents to control plant diseases is 

an attractive approach, especially at a time when managers and other stakeholders seek to limit the impact of 

synthetic pesticides. Further, chemical pesticides cannot be used in natural habitats because of their expense 

and potential negative effects on ecosystems. 

Mycoparasites are viewed as effective and environmentally friendly tools for the management and 

control of many important rust pathogens. Several 

species of the genus Cladosporium are mycoparasites 

of rust fungi. Of these, C. tenuissimum strongly inhibits important rust agents in the genera Melampsora, 

Cronartium, Peridermium, Puccinia, and Uromyces. 

It is also effective against nonrust fungal pathogens 

and oomycota. It suppresses disease by various means 

such as mechanical action, enzymatic action, and the 

production of metabolites that have antifungal properties. It reduces the number of rust propagules and their 

longevity, and hence curtails the diseases these rusts 

cause in a controlled environment (greenhouse).

Despite all these positive indications, several factors 

have so far made it difficult to use C. tenuissimum as a 

conventional biofungicide. Among these complicating 

factors are the complexity and variability of the habitat 

in which the mycoparasite occurs, the response of the 

other partners of the three-way interaction (the host 

and the parasite), the mode of application of the mycoparasite (its formulation and its combination with other 

control measures), and variations in mycoparasite virulence. In field tests the effectiveness of the fungus did 

not reach the level required to be commercially used 

for crop protection.

The situation might be completely different, however, 

in the prevention and control of human diseases. Here 

there are high hopes that a way will be found to use 

this mycoparasite, or the metabolites it produces, to 

protect human beings against disease. Cladosporols 

A–E, the family of metabolites that the fungus produces in vitro, strongly inhibited in vitro the growth 

of several Candida species, and it has recently been 

demonstrated that some of these metabolites are also 

effective antitumor agents, inhibiting the proliferation 

of cancer cells. The possible use of these molecules 

as therapeutic tools opens unexpected and promising 

avenues of research.
USDA Forest Service RMRS-P-76. 2018. 229

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

In Vitro Culture of the Alder (Alnus spp.) Pathogen Melampsoridium 

hiratsukanum: Practical Aspects, Problems, and Future Prospects

B. Ginetti1, A. Ragazzi1, and S. Moricca1, *

1 Università di Firenze, Dipartimento di Scienze delle Produzioni Agroalimentari e dell’Ambiente, Florence, Italy 

* salvatore.moricca@unifi.it

Abstract—The exotic alder (Alnus spp.) rust fungus Melampsoridium hiratsukanum has recently spread 

throughout several European countries. In Italy, it has been found heavily parasitizing alder leaves at the 

end of summer in several alpine valleys. Here the fungus is very aggressive on gray alder (A. incana) 

growing in riparian formations, and in thickets at the upper elevations of the forest, where decline and 

mortality of trees as a result of the strong parasitic action of this fungus could seriously compromise the 

stability of slopes and river banks. A better understanding of this micro-organism, and particularly a way 

to cultivate it axenically, would be a great step forward, as it would enable new and hitherto unexplored 

aspects of this still largely unknown rust agent to be elucidated. The in vitro axenic culture would be essential to advance cellular, developmental, and molecular research on the fungus, as well as to explore in vitro 

its nutrition, physiology, and interaction with its host(s).

Here we report on the growth trials that have successfully obtained the growth of saprophytic mycelium 

of M. hiratsukanum on some synthetic media in the laboratory. Urediniospores were used as an inoculum 

source for mass spore seeding. Axenic cultures were obtained on several media having a variety of 

organic and inorganic constituents, demonstrating that the nutritional requirements of the fungus are relatively non-specific. We report on the main obstacles in obtaining axenic growth of the rust fungus and on 

practical ways to eliminate unwanted microbial contaminants and to obviate other drawbacks. We also give 

some useful hints for obtaining stable and sustained growth. Implications for future research may include 

new characters (for example, morphology-based and molecular-based) to improve fungus taxonomy and to 

elucidate in vitro its host range in a simplified, controlled system.
230 USDA Forest Service RMRS-P-76. 2018.

In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds. 2018. Proceedings of the IUFRO joint conference: Genetics of 

five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15–20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p.

Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure 

consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. 

Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture.

Eucalyptus grandis Anomaly Related to Expression of Defense Genes

M.C.P. Fuchs-Ferraz1, M.L. Acencio2, N. Lemke2, S. Oda3, E.R. Gonzalez3, and C.L. Marino1, 4 *

1 Universidade Estadual Paulista, Instituto de Biociências/Departamento de Genética, Botucatu, São Paulo, Brazil

2 Universidade Estadual Paulista, Instituto de Biociências/Departamento de Física e Biofísica, Botucatu, São Paulo, Brazil

3 Empresa Suzano Papel e Celulose SA, Itapetininga, São Paulo, Brazil 

4 Universidade Estadual Paulista, Instituti de Biotecnologia, Botucatu, São Paulo, Brazil

* clmarino@ibb.unesp.br

In collaboration with the Suzano Papel and Celulose 

SA company, we identified an anomaly derived from 

a full-sib population in a controlled cross between two 

Eucalyptus grandis individuals. Abnormal seedlings 

died in a few months and showed significant phenotypic differences (P < 0.05), including seedling height, 

seedling stem-base diameter, and individual leaf area, 

compared with normal seedlings. Previous genetic 

analysis of the full-sib progeny showed a Mendelian 

segregation of 3 (normal): 1 (abnormal) (χ2 = 2.32, 

P > 0.1), suggesting that the lethality phenotype is 

controlled by a recessive allele (Tambarussi 2006). 

To identify the genes associated with the anomaly 

detected, we compared the transcriptional profiles of 

leaf, stem, and root tissue of normal and abnormal 

plants by RNA-seq technology. RNA-seq analysis 

showed the most differentially expressed genes were 

upregulated in the anomalous plants in all tissues 

analyzed. Most of these upregulated genes belong to 

functional groups of defense response and metabolism. 

Functional and metabolic analysis also demonstrated 

gene ontology and pathways involved in defense response upregulated in the abnormal plants. 

Our results suggest that this lethality is associated 

with inappropriate activation of the immune system, 

similar to the phenomenon of hybrid necrosis. Hybrid 

necrosis is a deleterious genetic incompatibility 

often associated with negative epistatic interactions 

(Bateson-Dobzhansky-Muller model) or single-locus 

incompatibility. Due to some characteristics such as 

rapid nucleotide sequence evolution and high copy 

number, response genes have a predisposition to cause 

genetic incompatibility. Therefore, three gene families 

differentially expressed (thaumatin, Bet v1, and chitinase class I) were considered as candidates for causing 

the negative interactions. Research is underway to 

identify the locus by using high-resolution mapping 

with the molecular markers derived from the candidate 

families of which the genes are differentially expressed 

between normal and lethality plants.

REFERENCE

Tambarussi, E.V. 2006. Associação de marcador RAPD 

e desenvolvimento de marcador SCAR para um tipo 

de anomalia de viveiro em Eucalyptus grandis [course 

conclusion paper] Botucatu: Faculdade de Ciências 

Agronômicas, Universidade Estadual Paulista “Júlio de 

Mesquita Filho”.
USDA Forest Service RMRS-P-76. 2018. 231

Appendix A—Field Trips

Trip 1—Tuesday, June 17, 

all day: Southern Rocky 

Mountains Forested 

Ecosystems

Native forested landscapes of 

southern Wyoming challenged 

by rusts, bark beetles, and other 

disturbances. Tour several Pinus 

flexilis research installations. 

Presenters include: Dennis

Knight, William Jacobi, Kelly 

Burns, Christy Cleaver, Sara 

Alberts, Anna Schoettle, and

Frank Romaro.

Trip 2—Thursday, June 19, 

all day: Rocky Mountain 

National Park

All-day field trip to Rocky 

Mountain National Park to visit 

a treeline Pinus flexilis stand and 

other locations with spectacular 

mountain views. Presenters 

include: Jeff Connor, Diana 

Tomback, Bob Cain, and Anna 

Schoettle.

Trip 3—Friday, June 20, all day: Mount Goliath Research Natural Area and Mount Evans 

Ecosystems

Optional all-day field trip to Mount Evans (a “14er”) west of Denver with a stop at the Mount Goliath Research 

Natural Area and a short walk on the Walter Pesman Alpine trail through ancient Pinus aristata at treeline. 

Presenters include: Jeremiah Hyslop, Anna Schoettle, and Diana Tomback.

Trip 4—Wednesday, June 18, mid-day: USDA Agricultural Research Service, National Center for 

Genetic Resources Preservation

Genetic resources (plants and animals) are preserved using state-of-the-art technology that often involves cryogenics. Presenters include: Christina Walters, Patricia Conine, and Chris Richards.

APPENDICES
232 USDA Forest Service RMRS-P-76. 2018.

Appendix B—Event Participant List (updated March 2018): Genetics of 

Five-Needle Pines and Rusts in Mountain Landscapes. June 15 – 20, 2014

1. Peter Achuff

Park Canada (retired)

Box 70 

Mountain View, Alberta T0K 1N0 Canada

403-653-4966

plachuff@gmail.com 

2. Thobayet Alshahrani

King Saud University

P.O. Box 2460

Riyadh, Saudi Arabia 11451

thobayet@yahoo.com

3. Zolton Bair

Oregon State University

Cordley 2074

Corvallis, OR 97331

541-740-2973

bairz@science.oregonstate.edu

4. Patrick Bennett

Oregon State University

Cordley 2082

Corvallis, OR 97331

541-737-5260

bennetpa@science.oregonstate.edu

5. James Blodgett

USDA Forest Service

8221 S Highway 16

Rapid City, SD 57702

605-716-2783

jblodgett@fs.fed.us

6. Aurore Bontemps

UC Davis

5346 Storer Hall

Davis, CA 95618

aurore.bontemps@yahoo.fr

7. Andrew Bower

USDA Forest Service

1835 Black Lake Blvd. SW

Olympia, WA 98512

360-956-2405

abower@fs.fed.us

8. Joshua Joseph Bronson

USDA Forest Service

2606 Old Stage Rd.

Central Point, OR 97502

541-858-6124

jjbronson@fs.fed.us

9. Lorinda Bullington

MPG Ranch

1001 S. Higgins Suite A3

Missoula, MT 59801

406-547-4413

lhunt@mpgranch.com

10. Kelly Burns

USDA Forest Service

1617 Cole Blvd., Bldg. 17

Lakewood, CO 80401-3305

303-236-8006

ksburns@fs.fed.us

11. Charlie Cartwright

BC Ministry of Forests

P.O. Box 335

Mesachie Lake, BC V0R 2N0 Canada

250-749-6811

charlie.cartwright@gov.bc.ca

12. Kristen Chadwick

USDA Forest Service

16400 Champion Way

Sandy, OR 97055

503-668-1474

klchadwick@fs.fed.us

13. Tony Chang

Montana State University

310 Lewis Hall

Bozeman, MT 59717

406-944-2670

tony.chang@msu.montana.edu
USDA Forest Service RMRS-P-76. 2018. 233

14. Christy Cleaver

USDA Forest Service

3815 N. Schreiber Way

Coeur d’Alene, ID 83815

208-765-7343

christycleaver@fs.fed.us

15. Dominique David-Chavez

Colorado State University

20 Glacier Court

Bozeman, MT 59715

406-548-8743

d.mari.david@gmail.com

16. John Davis

University of Florida

Box 110410

Gainesville, FL 32611

352-262-4363

jmdavis@ufl.edu 

17. Braham Dhillon

Department of Plant Pathology,

Plant Sciences Bldg, Rm 217

495 N Campus Dr

Fayetteville, AR 72701

bddhillo@uark.edu 

18. Arnaud Dowkiw

French Nat. Inst. for Ag. Research

2163 Avenue da la Pomme de Pin

CS 40001 - Ardon

Champenoux, France 45075

33-23-841-2012

arnaud.dowkiw@inra.fr

19. Joan Dunlap

USDA Forest Service (retired)

2375 Fruitridge Rd.

Camino, CA 95709

530-642-5031

jdunlap@fs.fed.us

20. Sebastien Duplessis

French Nat. Inst. for Ag. Research

Tree-Microbe Interactions Department

INRA Centre Grand Est Nancy

Champenoux, France 54280

33-383-39-40-13

Sebastien.duplessis@inra.fr

21. William Dvorak

NC State University

Campus Box 8008

Raleigh, NC 27695

919-515-6424

dvorak@ncsu.edu

22. Richard Hamelin

University of British Columbia

3rd Floor, Forest Sciences Centre

#3032-2424 Main Mall

Vancouver, BC V6T 1Z4 Canada

604-827-4441

richard.hamelin@ubc.ca

23. Andrew Eckert

Virginia Commonwealth University

1000 W Cary St. Room 126

Richmond, VA 23284

804-828-0800

aeckert2@vcu.edu

24. Sam Foster

USDA Forest Service (retired)

240 W Prospect Road

Fort Collins, CO 80526

25. Pascal Frey

French Nat. Inst. for Ag. Research

Tree - Microbe Interactions Department

INRA Centre Grand Est Nancy UMR 1136

Champenoux, France 54280

33-383-394-056

Pascal.frey@inra.fr

26. Nancy Gillette

USDA PSW Res. Station (retired)

PO Box 245

Berkeley, CA 94701

27. Harish Singh Ginwal

Forest Research Institute – India

Uttaranchal, India

91 135-275-5473

ginwalhs@icfre.org
234 USDA Forest Service RMRS-P-76. 2018.

28. Jennifer Gruhn

Washington University - St. Louis

1 Brookings Drive

Campus Box 1137

St. Louis, MO 63130

704-776-8160

jenniferagruhn@gmail.com

29. Phi Hong Hai

Vietnamese Academy of Forest Sciences

46 Co Nhue, Dong Ngac

Hanoi, Vietnam 0844

84 913-309-746

phi.hong.hai@vafs.gov.vn

30. Matt Horning

USDA Forest Service

63095 Deschutes Market Rd

Bend, OR 97701

541-408-1711

mhorning@fs.fed.us

31. Jingyu Han

Korea Forest Research Inst.

39 Onjeong-ro, Gwonseon-gu

Suwon, Korea 441-350

82-31-290-1157

han6430@gmail.com

32. JeriLyn Harris

USDA Forest Service

740 Simms St.

Golden, CO 80401

303-275-5155

jerilynharris@fs.fed.us

33. John Hart

1390 Curt Gowdy Dr.

Cheyenne, WY 82009

307-630-5202

34. Berthold Heinze

Federal Research Centre for Forests

Seckendorff-Gudent Weg 8

Vienna, Austria 1130

43-187-838-2219

berthold.heinze@bfw.gv.at

35. Valerie Hipkins

USDA Forest Service

2480 Carson Rd.

Placerville, CA 95667

530-622-1609

vhipkins@fs.fed.us

36. Justin Hof

USDA Forest Service

16400 Champion Way

Sandy, OR 97055

503-668-1646 

justinhof@fs.fed.us

37. Richard S. Hunt

4043 Hopesmore Place

Victoria BC V8N 5T1 Canada

250 5952117

ribicola@gmail.com

38. William Jacobi

Colorado State University (retired)

Department BSPM

Fort Collins, CO 80523

william.jacobi@colostate.edu

39. James Jacobs

USDA Forest Service

1992 Folwell Ave.

St. Paul, MN 55108

651-649-5266 

jamesjjacobs@fs.fed.us

40. Veronique Jorge

French Nat. Inst. for Ag. Research

2163 Avenue de la Pomme de Pin

CS 40001 - Ardon

Champenoux, France 45075

33-23-841-7828

veronique.jorge@inra.fr

41. Juha Kaitera

Natural Resources Institute Finland 

Natural Resources and Bioproduction

Paavo Havaksen tie 3

P.O. Box 413

FI-90014 University of Oulu, Finland

+358-40-801-5553

Juha.kaitera@luke.fi
42. Kyu-Suk Kang

Seoul National University

1 Gwanak-ro, Gwanak-gu

Seoul, Republic of Korea 151-921

kangks84@snu.ac.kr

43. Holly Kearns

USDA Forest Service

16400 Champion Way

Sandy, OR 97055

503-668-1475

hkearns@fs.fed.us

44. Chris Keefe

BLM

5353 Yellowstone Rd.

Cheyenne, WY 82009

307-775-6101

ckeefe@blm.gov

45. Angelia Kegley

USDA Forest Service

34963 Shoreview Dr.

Cottage Grove, OR 97424

541-767-5711

akegley@fs.fed.us

46. John King

BC Ministry of Forests (retired)

2838 Murray Drive

Victoria, BC V9A 2S5 Canada

250-721-3814

king.forgen@gmail.com

47. Bohun Kinloch

1440 Le Roy Avenue

Berkeley, CA 94708

510-704-7745

bkinloch@earthlink.net

48. Bill Libby

University of California

28 Valencia Rd.

Orinda, CA 94563

925-376-5896

william.libby0@gmail.com

49. Eleonore Lickl

Fed. College & Res. Inst. for Chem. Industry

Rosensteingasse 79

Vienna, Austria A1170

43-664-732-10-696

eleonore.lickl@schule.at

50. Jun-Jun Liu

Canadian Forest Service

506 West Burnside Road

Victoria, BC V8Z 1M5 Canada

250-298-2378

jun-jun.liu@nrcan.gc.ca

51. Pengxin Lu

Ontario Forest Research Institute

1235 Queen Street East

Sault Ste Marie, Ontario P6A 2E5 Canada

705-946-7415

pengxin.lu@ontario.ca

52. Glenn Lunak

Sierra Pacific Ind. (retired)

2849 Northgate Dr.

Chico, CA 95973

53. Helen Maffei

USDA Forest Service (retired)

60395 Deschutes Market Rd.

Bend, OR 97701

54. Mary Frances Mahalovich

USDA Forest Service

1221 S. Main St.

Moscow, ID 83843

208-883-2350

mmahalovich@fs.fed.us

55. John Major

Canadian Forest Service

1350 Regent St.

Fredericton, NB E3B 5P7 Canada

506-452-3262

jmajor@nrcan.gc.ca
236 USDA Forest Service RMRS-P-76. 2018.

56. Celso Luis Marino

Institute of Biosciences

UNESP - State Public University

Departamento de Genetica

Botucatu, Brazil 18618970

55-14-388-00363

clmarino@ibb.unesp.br

57. Andrew McMahan

USDA Forest Service

2150 A Centre Ave.

Fort Collins, CO 80526

58. Robert Means 

BLM (deceased) 

5353 Yellowstone Road

Cheyenne, WY 82009

59. Jeffry Mitton

University of Colorado

Campus Box 334

Boulder, CO 80309

303-817-1175

jeffry.mitton@colorado.edu

60. Annette Mix

USDA Forest Service (retired)

2480 Carson Rd.

Placerville, CA 95667

61. Bruce Moltzan

USDA Forest Service

201 14th St. SW

Washington, D.C. 20250

703-605-5336

bmoltzan@fs.fed.us

62. Mike Monahan

University of Denver

Denver, CO 80208

303-871-3540

mmonahan@du.edu

63. William Monahan

USDA Forest Service

2150 A Centre Ave

Fort Collins, CO 80526

wmonahan@fs.fed.us 

64. Alejandra Moreno-Letelier

Nat. Autonomous University of Mexico

Insurgentes Sur 3493

UH Villa Olimpica

Mexico D.F., Mexico 14020

555-622-9001

aleml@yahoo.com

65. Salvatore Moricca

University of Florence

Piazzale delle Cascine 28

Firenze, Italy 50144

39-055-328-8349

salvatore.moricca@unifi.it

66. Isabel Munck

USDA Forest Service

271 Mast Road

Durham, NH 03824

603-833-5575

imunck@fs.fed.us

67. Michael Murray

BC Ministry of Forests

401-333 Victoria Street

Nelson, BC V1L 4K3 Canada

250-354-6852

michael.murray@gov.bc.ca

68. Simon Nadeau

University of British Columbia

102-2280 W. 6th Avenue

Vancouver, BC V6K 1V4 Canada

604-349-5196

simon.nadeau.ubc@gmail.com

69. David Neale

University of CA-Davis

Mail Stop 4

262 C Robbins hall

Davis, CA 95616

530-754-8431

dbneale@ucdavis.edu

70. Amy Nicholas

US Fish & Wildlife

5353 Yellowstone Rd. #308A

Cheyenne, WY 82009

amy_nicholas@fws.gov
USDA Forest Service RMRS-P-76. 2018. 237

71. Brent Oblinger

USDA Forest Service

63095 Deschutes Market Rd.

Bend, OR 97701

530-902-7134

boblinger@fs.fed.us

72. Elizabeth Pansing

University of Colorado

Dept. of Integrative Biology

Campus Box 171

Denver, CO 80217

303-912-5668

elizabeth.pansing@ucdenver.edu

73. Tom Parchman

Univ. of Nevada-Reno

Dept. of Biology

Reno, NV 89557

775-784-4484

tparchman@unr.edu 

74. Dana Perkins

BLM (retired)

1387 S. Vinnell Way

Boise, ID 83709

75. Michael Pernaci

French Nat. Inst. for Ag. Research

Tree - Microbe Interactions Department

UMR 1136

Champenoux, France 54280

76. Dmitry Politov

Russian Academy of Sciences

3 Gubkin St. GSP-1

Moscow, Russia 119991

7-916-574-5580

dmitri_p@inbox.ru

77. Kevin Potter

NC State University

3041 Cornwallis Rd.

Research Triangle Park, NC 27709

919-549-4071

kpotter@ncsu.edu

78. Zulema Quinones Perez

Univ. Juárez del Estado de Durango

Durango, Mexico 34120

zule_qp@hotmail.com

79. Richard Reich

College of New Caledonia 

The University of British Columbia 

British Columbia, Canada

Reichr1@cnc.bc.ca

80. Daniel Reinhart

National Park Service

P.O. Box 168

Yellowstone National Park, WY 82190

Dan_Reinhart@nps.gov

81. Amy Ross-Davis

USDA Forest Service

1221 South Main Street

Moscow, ID 83843

82. Berit Samils

Swedish University of Ag. Sciences

Dept. of Forest Mycology/Plant Path.

Box 7060

Uppsala, Sweden SE-75755

46-73-030-6944

berit.samils@slu.se

83. Doug Savin

USDA Forest Service

34963 Shoreview Dr.

Cottage Grove, OR 97424

541-767-5703

dpsavin@fs.fed.us

84. Anna Schoettle

USDA Forest Service

240 West Prospect Road

Fort Collins, CO 80526

970-498-1333

aschoettle@fs.fed.us

85. Markus Scholler

Museum of Natural History – Germany

Abteilung Biowissenschaften Erbprinzenstr

Baden-Wurttemberg, Germany 76133

49-0721-175-2810

scholler@naturkundeka-bw.de

86. Erin Shanahan

National Park Service

2327 University Way, Suite 2

Bozeman, MT 59715

406-581-0398

erin_shanahan@nps.gov
238 USDA Forest Service RMRS-P-76. 2018.

87. Eric Smith

USDA Forest Service (retired)

2150 Centre. Ave., Bldg. A Ste. 331

Fort Collins, CO 80526

88. Richard Sniezko

USDA Forest Service

34963 Shoreview Dr.

Cottage Grove, OR 97424

541-767-5716

rsniezko@fs.fed.us

89. John Syring

Linfield College

900 SE Baker St.

McMinnville, OR 97128

503-883-2466

jsyring@linfield.edu

90. Pedro Manuel Talhinhas

Instituto de Investigacao Cientifica Topical

Quinta do Marques

Oeiras, Portugal 2780-505

91. Philippe Tanguay

Canadian Forest Service

1055 rue du PEPS

C.P, 10380 succ. Ste-Foy

Québec, Canada G1V 4C7

418-648-7556

philippe.tanguay@nrcan.gc.ca

92. Pham Quang Thu

Vietnamese Academy of Forest Sciences

Hanoi, Vietnam

84 043-836-2376

phamquangthu@vafs.gov.vn

93. Diana Tomback

Whitebark Pine Ecosystem Foundation

PO Box 17943

Missoula, MT 59808

303-556-2657

diana.tomback@ucdenver.edu

94. Oguz Serif Urhan

Oregon State University

321 Richardson Hall

Corvallis, OR 97331

541-602-7653

oguz.urhan@oregonstate.edu

95. Alejandra Vazquez Lobo Yuren

Centro de Investigación en Biodiversidad y

Conservación, Universidad Autónoma del 

Estado de Morelos, Avenida Universidad 

1001, 62209 Cuernavaca, Morelos, Mexico 

ale.lobo17@gmail.com

96. Brian Verhulst

National Park Service

1000 US Hwy 36

Estes Park, CO 80517

970-586-1444

brian_verhulst@nps.gov

97. Detlev Vogler

USDA Forest Service (retired)

2480 Carson Rd.

Placerville, CA 95667

530-621-6881

dvogler@fs.fed.us

98. Aaron Wagner

University of Colorado

12395 Madison Court

Thornton, CO 80241

aaron.wagner@ucdenver.edu

99. Kristen Waring

Northern Arizona University

P.O. Box 15018

Flagstaff, AZ 86011

928-523-4920

kristen.waring@nau.edu

100. Marcus Warwell

USDA Forest Service

1221 South Main Street

Moscow, ID 83843

208-883-2305

mwarwell@fs.fed.us

101. Jill Wegrzyn

University of Connecticut

75 N Eagleville Road, Unit 3043

Storrs, CT 06269

619-316-8548

jill.wegrzyn@uconn.edu
USDA Forest Service RMRS-P-76. 2018. 239

102. Christian Wehenkel

Univ. Juárez del Estado de Durango

Aptdo. Postal 741 Zona Centro

Durango, Mexico 34000

wehenkel@ujed.mx

103. Xiaoxin Wei

Chinese Academy of Sciences

20 Nanxincun, Xiangshan

Beijing, China 100093

weixx@ibcas.ac.cn

104. Jami Westerhold

American Forests

1220 L Street NW; Ste. 750

Washington, DC 20005

202-370-4516

jwesterhold@americanforests.org

105. Jessica Wright

USDA Forest Service

1731 Research Park Dr.

Davis, CA 95618

530-759-1742

jessicawwright@fs.fed.us

106. David Yih

Trinity College

43 Dean St.

Hartford, CT 06114

860-246-2077

yyih@wesleyan.edu

107. Richard Zabel

Western Forestry & Consv. Assoc.

4033 SW Canyon Rd.

Portland, OR 97221

503-226-4562

richard@westernforestry.org

________________________

Participant Count: 107
240 USDA Forest Service RMRS-P-76. 2018.

Appendix C—IUFRO Joint Conference - Genetics of 5-Needle Pines, 

Rusts of Forest Trees & Strobusphere Program

Sunday June 15 At the Hilton

1600‐1900 Registration in Atrium

Set up posters

1700‐2030 Welcome Social and Poster Session

Monday June 16 At the Hilton ‐ One Session

0700‐0800

duration

0745‐0820

Introduction

&

Welcome 35

0820‐0845 1 Keynote D. Neale Genome Sequencing in Conifers: Implications for Breeding and Gene Resource Management 25

0845‐0910 2 Keynote

R.C. Hamelin, S. Brar, C.K.M. Tsui, B Dhillon, M.

Bergeron, D.L. Joly and Y.A. El‐Kassaby

Colonization History, Host Distribution and Landscape Features Shape White Pine

Blister Rust Populations. 25

0910‐0935 3 Keynote J. Davis

Genomic Warfare in the Woods: Scaling Fusiform Rust Resistance from Molecules to

Landscapes 25

0935‐0945 Q&A 100945‐1000 4

B. Petre, S. Hacquard, A. Persoons, N. Saveleva, E.

Tisserant, E.Morin, C. Delaruelle, J. Petrowski, M.Pernaci, B. Fabre, S. De Mita, F. Halkett, A. Hecker, N.Rouhier, P. Frey, and S. Duplessis

Genomics, Transcriptomics and Effectoromics of the Poplar Leaf Rust Fungus

Melampsora larici‐populina 15

1000‐1015 5

Zhen‐Zhen Hao, Yan‐Yan Liu, Liu Liu, Xiao‐Xin Wei

and Xiao‐Quan Wang Evolution and Biogeography of section Quinquefoliae (subgenus Strobus, Pinus) 15

1015‐1025 Q&A 101025‐1040 Break View Posters 151040‐1055 6

V. Jorge, A. Dowkiw, R. El‐Malki, V. Segura, V. Guérin, P.

Poursat, P. Faivre‐Rampant, A. Rae, G. Taylor, and C.

Bastien

Genetic Architecture of Quantitative Melampsora larici‐populina Leaf Rust Resistance

in Poplars 15

1055‐1110 7 R. Reich and S. John Ten Year Results of a Comandra Blister Rust Screening Trial in the Central Interior of British Columbia 15

1110‐1125 8 R.A. Sniezko and A. Kegley The Definitive Guide for White Pine Blister Rust Resistance in White Pine Species of Western North America (beta 2014.0 version) – A Tree Breeders Perspective 15

1125‐1140 9

R.A. Sniezko, B. Kinloch, D.P. Savin, R. Danchok, and A.

Kegley Provenance Variation in Blister Rust Resistance in Sugar Pine 15

1140‐1155 Q&A 151155‐1300 Lunch Included 65

IUFRO Joint Conference ‐ Genetics of

5‐Needle Pines, Rusts of Forest Trees

& Strobusphere (6/13/14)

PROGRAM

Moderator 1: Jill Wegrzyn

DiscussionDiscussion

TOPIC: Genetic Resistance ‐ all species

Moderator 2: Bruce Moltzan

Discussion

REGISTRATION

Joint White Pine

& Rust Group Session (all day)

Foster/Schoettle/Hamelin/Neale/Sniezko

TOPIC: Genomics
USDA Forest Service RMRS-P-76. 2018. 241 1300‐1325 10 B. Heinze and K. Holzer Present and Possible Past Distribution of Pinus cembra in the Austrian Alps – The Ups and Downs of Climate Change? 25 1325‐1340 11 K. J. Hayden, C. Xhaard, B. Fabre, F. Halkett, and P. Frey Adaptation of Poplar Rust to the Poplar varietal landscape 15 1340‐1355 12 P.J. Vogan and A.W. Schoettle Associations Between Complete Resistance to White Pine Blister Rust and Abiotic Stress Tolerances in Limber Pine (Pinus flexilis James) 15 1400‐1410 Q&A 10 1410‐1425 13 M.V. Warwell Genecology and Phenotypic Evolution of Whitebark Pine under Warm‐Dry Climate: joint analysis of 12‐year survival and growth of 49 populations in common gardens. 15 1425‐1440 14 A. Bontemps, J. Wright, R. Sniezko, D. Savin, and J. Schmitt Using Historical Provenance Test Data to Understand Tree Responses to a Changing Climate in Sugar Pine, Pinus lambertiana 15 1440‐1455 15 D.F. Tomback, L.M. Resler, G.P. Malanson, E.K. Smith‐ McKenna, S.C. Blakeslee, and J.C. Pyatt White Pine Blister Rust Alters Facilitation Interactions at Treeline: Implications for Treeline Communities and Response to Climate Change 15 1455‐1505 Q&A 10 1505‐1520 Break 15 1520‐1530 16 Holly Kearns Session Introduction 10 1530‐1545 17 F.J. Krist, A.J. McMahan, E.L. Smith White Pine Blister Rust, Mountain Pine Beetles, and their Potential Impact on White Pines: A National Assessment from the National Insect and Disease Risk Map 15 1545‐1600 18 A. J. McMahan and E.L. Smith Spatial Modeling of Past and Future Losses of White Pines of the Western U.S. 15 1600‐1615 19 C. Cleaver, W. Jacobi, K. Burns, and R. Means Limber Pine Stand Conditions after White Pine Blister Rust and Mountain Pine Beetle Caused Mortality in the Central and Southern Rocky Mountains 15 1615‐1730 20 Bob Means (BLM); Dan Reinhart (NPS); Peter Achuff (Canada); Amy Nicholas (FWS); Sara Alberts (USFS) Panelist Presentations and Discussion with Audience Participation 75 1800‐2030 POSTER SESSION 150 TOPIC: Western Wildland Environmental Threat Assessment Center Sponsored Session: Patterns of WPBR disease, impacts and risk across the landscape – Implications on decision making for conservation of whitebark pine and other 5 needle pines Moderator 4: Holly Kearns ATRIUM ‐‐‐ Cash bar, light appetizers, posters (Dinner on your own) TOPIC: Adaptations & Landscape Variation Moderator 3: Andrew Eckert Discussion Discussion View Posters
242 USDA Forest Service RMRS-P-76. 2018. Tuesday June 17 Field Trip 0730‐2000 FIELDTRIP to Southern Wyoming; dinner at Avogadro's Number in Fort Collins (included); Group Photo Wednesday June 18 At the Hilton ‐ CONCURRENT SESSIONS PINES duration 0745‐0800 0830‐0845 22 P. H. Hai Study of Occurences of Pinus dalantensis De Ferré for Gene Conservaiton in Vietnam 15 0845‐0855 Q&A 10 0855‐0910 23 A. Moreno‐Letelier, A. Ortiz‐Medrano, D. Piñero, and T.G. Barraclough Genetic andEecological Divergence in Pinus flexilis‐Pinus strobiformis ‐Pinus ayacahuite Species Complex: Clues to Understanding Speciation in Pines 15 0910‐0925 24 C. Wehenkel, C.Z. Quiñones‐Pérez, S.L. Simental‐ Rodríguez, C.A. López Sanchez Genetic Structure in Pinus strobiformis Engelm. on the Sierra Madre Occidental, Mexico 15 0925‐0940 25 K.M. Waring and B.A. Goodrich Pinus strobiformis Gene Conservation, Adaptive Traits and Climate Change 15 0940‐1055 Q&A 15 1055‐1010 Break 15 1010‐1025 26 D. Politov, Y. Belokon, M. Belokon, E. Mudrik, T. Poliakova, E. Petrova, and S. Goroshkevich Population and Evolutionary Genetic Studies of 5‐Needle Pines in Russia 15 1025‐1040 27 M. F. Mahalovich and D.L. Foushee Pinus albicaulis Engelm. Genetic Restoration Program for the Inland West (USA): a first generation of improvement 15 1040‐1055 28 G. Man and B. Moltzan Forest Health Protection Gene Resource Conservation a Lasting Legacy 15 1055‐1105 Q&A Discussion 10 Instructions and head over to NCGRP 30 NCGRP tours and Lunch 105 Return to Hilton 20 1345‐1400 29 P. Lu Performance of Blister Rust Resistant Eastern White Pine Hybrids with Himalayan Blue Pine in Field Trials 15 1400‐1415 30 C. Cartwright, N. Ukrainetz, and M. Murray Whitebark Pine Field Screening for Blister Rust Resistance: Early results. 15 1415‐1430 31 J. N. King Field Level Resistance of Western White Pine to White Pine Blister Rust Assessed in Progeny Trials Planted in Coastal British Columbia 15 1430‐1445 32 M. P. Murray Blister Rust Inoculation Trials for Canadian Whitebark Pine 15 1445‐1500 33 S.U. Han, H.S.Lee, J.M. Park and K.S. Kang Genetic Relationship between Reproductive Morphology and Growth Characteristics of Pinus koraiensis 15 Announcements 1110‐1345 FIELDTRIP CONCURRENT SESSIONS (a) 5‐needle pines, (b) Rusts TOPIC: Gene Conservation & Restoration Moderator 5: Bill Libby TOPIC: Gene Conservation & Restoration, continued Moderator 6: Bill Jacobi TOPIC: Rust Resistance Trials, Field Performance & Genetic Variation Moderator 7: James Jacobs 0800‐0830 21 Keynote W.S. Dvorak The Efficacy of Ex Situ Conservation of the Mexican and Central American White Pines, Pinus ayacahuite and Pinus chiapensis, in Developing Countries 30 Discussion Discussion View Posters
USDA Forest Service RMRS-P-76. 2018. 243 1500‐1515 Q&A 15 1515‐1535 Break 20 1535‐1550 34 A.J. Eckert, B.M. Lind, C.J. Friedline, E. Hobson, J.W.R. Zinck, O.P. Rajora, D.B. Neale, D. Vogler, and P.E. Maloney The genetic architecture of local adaptation and the genomic exploration of rugged evolutionary landscapes within species of Pinus subgenus Strobus 15 1550‐1605 35 J.L. Wegrzyn, P.J. Martinez‐Garcia, C.A. Loopstra, K. Mockaitis, R. Famula, and D.B. Neale Sequencing the sugar pine transcriptome 15 1605‐1620 36 J.B. Mitton, J. Wegrzyn, S. L. Stowell, M. Mitter, L. Kueppers and D. Neale Genetic Resources for Limber Pine and a Survey Focusing on Potentially Adaptive SNPs 15 1620‐1635 37 J.A. Gruhn, B.A. Schaal Using Genomics Tools to Assess the Phylogeography of Whitebark Pine 15 1635‐1645 Q&A 10 1645‐1700 38 A. Vázquez‐Lobo, C. Vangestel, P.J. Martínez‐García, J.L. Wegrzyn, I. Calic, J.D. Liechty, and D.B. Neale Identifying the Genetic Basis of Partial Resistance on Sugar Pine (Pinus lambertiana ) Under White Pine Blister Rust Infection (WPBR). 15 1700‐1715 39 S. Nadeau, N. Isabel, J. Godbout, M. Lamothe, M.‐ C.Gros‐Louis, and K. Ritland Patterns of Population Structure and Environmental Associations Across the Range of Two Ecologically and Economically Important Five‐Needle Pine Species 15 1715‐1730 40 J.‐J. Liu and R.A. Sniezko Genomics Study of Western White Pine Genetic Resistance Against White Pine Blister Rust 15 1730‐1745 41 H. S. Ginwal and P. Chauhan Cross Species Transferability of Microsatellites, and Genetic Diversity in Himalayan Five Needle Pine (Pinus wallichaia A.B. Jacks) and Chir pine (P. roxburghii Sarg.) Forests 15 1745‐1800 Q&A 15 1800‐1830 1830‐1900 Business meetings Dinner on your own take posters down Overall Strobusphere discussion Discussion Poster viewing TOPIC: Strobusphere Special Session Moderator 8: David Neale Discussion Discussion
244 USDA Forest Service RMRS-P-76. 2018. RUSTS 0745‐0800 0800‐0820 42 A. Dowkiw, E. Voisin, V.Dievart, T. Servouse, M. Berteloot, V. Guérin, T. Bourgeois, A. Andrieux, and P. Frey Widening the Landscape: The Role of Ornamental and Wild Trees in the Spread and Adaptation of Melampsora larici‐populina Leaf Rust 15+5 0820‐0840 43 P. Tanguay Origin and Distribution in Canada of the Cronartium ribicola vCr Race, Virulent on Immune Blackcurrant Cultivars Derived from Hunter’s Selection 15+5 0840‐0900 44 I.A. Munck, P. Tanguay, J. Weimer, K. Lombard, S. Villani, K. Cox Impact of White Pine Blister Rust on Resistant and Previously Immune Cultivated Ribes and Neighboring Eastern white pine in New Hampshire 15+5 0900‐0920 45 B. Samils, P. Barklund, J. Kaitera, and J. Stenlid Geographic Distribution of C. flaccidum and P. pini in Sweden and Finland 15+5 0920‐0940 46 A. Ross‐Davis, N. Klopfenstein, and 24 others Genetic and Genotypic Diversity of Puccinia psidii  ‐ The Cause of Guava/Eucalypt/Myrtle Rust ‐ and Preliminary Predictions of Global Areas at Risk 15+5 0940‐1000 47 J. Kaitera, R. Hiltunen, B. Samils, and J. Hantula Cronartium Rusts Sporulate on a Wide Range of Alternate Hosts in Northern Europe 15+5 1000‐1020 Break View Posters 20 1020‐1040 48 D.R. Vogler, A.D. Mix, D. Burton, D. Davis, R. Westfall, and B.B. Kinloch Jr. Frequency and Distribution of Virulence in Cronartium ribicola to Two Simply‐ Inherited Resistance Genes in Sugar Pine and Western White Pine, Respectively, at Locations in Northern Siskiyou County and in the South Central Sierra Nevada of California 15+5 1040‐1100 49 R.S. Hunt, M. Murray, R. Reich, D. Rusch, A. Woods, S. Zeglen Survival of MGR Resistance in Western White Pine in British Columbia 15+5 Instructions and Head over to NCGRP 30 NCGRP tours and Lunch Christina Walters; Pat Conine 115 Return to Hilton 20 1345‐1405 50 M. Pernaci, S. De Mita, B. Fabre, A. Andrieux, F. Halkett, and P. Frey On the Road to Mapping QTLs of Morphological and Aggressiveness Traits in the Poplar Rust Fungus 15+5 1405‐1425 51 B. Dhillon and R. Hamelin Comparative Genomics of the Cronartium genus 15+5 1425‐1445 52 S. Tavares, A.S. Pires, H.G. Azinheira, A.P. Ramos, C. Bispo, C. Andrade, A. Loureiro, D. Schmidt, T. Link, R.T. Voegele, R. Gardner, R. Abranches, M.C. Silva, J. Loureiro, P. Talhinhas Cytological Analyses Reveal Variations in Nuclear Content along the Urediniosporic Infection Cycle of Hemileia vastatrix and Other Rust Fungi 15+5 1445‐1505 53 M. Scholler, L. Beenken, M. Lutz, W. Maier, A. Tahir and M. Thines Barcoding the Rust Fungi of Germany 15+5 1505‐1535 Break Poster viewing 30 Announcements TOPIC: Genetics, Genomics & Barcoding Moderator 11: Sebastien Duplessis 1100‐1345 FIELDTRIP Moderator 10: Salvatore Moricca CONCURRENT SESSIONS (a) 5‐needle pines (b) Rusts TOPIC: Epidemiology & Population Genetics Moderator 9: Pascal Frey TOPIC: Etiology & Management TOPIC: Resistance
USDA Forest Service RMRS-P-76. 2018. 245 1535‐1555 54 P.Q. Thu, S. Kaneko and M. Imazu Stem Rust Diseases of Pinus kesiya in Vietnam 15+5 1555‐1615 55 S. Moricca, B. Ginetti, A. Lupo, A. Ragazzi and G. Assante Research on the Rust Hyperparasite Cladosporium tenuissimum : Status and Prospects 15+5 1620‐1635 56 W.B. Monahan, T. Cook, F. Melton, J. Connor, B. Bobowski, and F. Krist Forecasting Climate and Disease Impacts on Limber Pine in Rocky Mountain National Park 15 1635‐1650 57 J.E. Major, A. Mosseler, J. Malcolm and M. Cambell Comparative Photosynthetic Aadaptive Traits in Four Pinus species (P. strobus, P. resinosa, P. banksiana, P. rigida ) to CO2 x Moisture Stress Factorial 15 1650‐1705 58 E. Shanahan, K.M. Irvine, C. Hollimon, K. Legg, R. Daley, Greater Yellowstone Whitebark Pine Monitoring Working Group Monitoring Trends in White Pine Blister Rust Infection in the Greater Yellowstone Ecosystem 15 1705‐1720 59 D.M. Davíd‐Chavez, R.T. Belote, Matt Dietz, and G.H. Aplet Climate Change Alters Distribution of Whitebark Pine (Pinus albicaulis) with Respect to Land Management Status: are there Implications for Adaptation? 15 1720‐1735 Q&A 15 1830‐1900 Business meetings Dinner on your own Take posters down Thursday June 19 Field Trip 0730‐2130 FIELDTRIP to Rocky Mountain National Park; dinner at the Stanley Hotel, Estes Park (included) Friday June 20 Optional Field Trip 0700‐2000 Optional FIELDTRIP to Mount Goliath and Mt Evans; light dinner at Barrels & Bottles Brewery in Golden (included) Topic: Host ‐ Disease Climate Interactions on Landscapes Moderator 12: Diana Tomback Discussion Moderator 8: Richard Hamelin

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