Proceedings of the Sixth International Workshop on the 

Genetics of Host-Parasite Interactions in Forestry

Tree Resistance to Insects and Diseases: 

Putting Promise into Practice

Mt. Sterling, Ohio

August 5–10, 2018

United States Department of Agriculture

Forest Service

Research & Development

Southern Research Station

General Technical Report SRS–252
Cover Photos 

Front:

Top left and top right—Several provenances of North American and European ash trees are evaluated in field trials for 

resistance to the invasive emerald ash borer (Agrilus planipennis) at the U.S. Forest Service Northern Research Station, 

Delaware, OH. (photo courtesy of Ignazio Graziosi)

Bottom left—Emerald Ash Borer on an ash tree that has visible bark cracks from larval feeding.

Bottom center—The extensive emerald ash borer (Agrilus planipennis) feeding galleries visible on this European narrow leaf 

ash (Fraxinus angustifolia), part of a field trial along with other North American ash trees at the U.S. Forest Service Northern 

Research Station, Delaware, OH indicates that it is susceptible. (photo courtesy of Ignazio Graziosi)

Bottom right—Several tree species are grown in common gardens to be evaluated for resistance to various insect pests and 

pathogens at the U.S. Forest Service Northern Research Station, Delaware, OH. (photo courtesy of Ignazio Graziosi)

Back:

Top left—Dead Port-Orford-cedar (Chamaecyparis lawsoniana) in Redwood National Park, California, killed by the non-native 

pathogen Phytophthora lateralis. (photo by Richard Sniezko, U.S. Forest Service)

Top right—Dead Port-Orford-cedar along Highway 101 in southern Oregon. (photo by Richard Sniezko, U.S. Forest Service)

Second row left—Resistant parent tree surrounded by Port-Orford-cedar killed by P. lateralis in northern California. (photo 

courtesy of Chuck Frank)

Second row right—Breeding Port-Orford-cedar for resistance in containerized orchard at Dorena Genetic Resource Center. 

(photo by Richard Sniezko, U.S. Forest Service)

Third row center—Port-Orford-cedar (dead and alive) in field trial. (photo by Richard Sniezko, U.S. Forest Service)

Third row right—Resistance trial planted underneath dead Port-Orford-cedar at South Slough NERR in Oregon. (photo by 

Richard Sniezko, U.S. Forest Service)

Bottom left—Port-Orford-cedar field trial near Oregon coast. (photo by Richard Sniezko, U.S. Forest Service)

Disclaimer

Any opinions, findings, conclusions, or recommendations 

expressed in the material are those of the authors and do not 

necessarily reflect the policies and views of the U.S. Department 

of Agriculture Forest Service. 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.

December 2020

Southern Research Station

200 W.T. Weaver Blvd.

Asheville, NC 28804

www.srs.fs.usda.gov
Proceedings of the Sixth International Workshop on the 

Genetics of Host-Parasite Interactions in Forestry

Tree Resistance to Insects and Diseases: 

Putting Promise into Practice

Mt. Sterling, Ohio

August 5–10, 2018

Editors: C. Dana Nelson, Jennifer L. Koch, and Richard A. Sniezko

ABSTRACT

The Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree 

Resistance to Insects and Diseases: Putting Promise into Practice was held the first week of August 

2018 at Deer Creek State Park in Mt. Sterling, Ohio. The workshop provided a continuing forum for 

researchers, tree breeders, and forest managers to focus on perhaps the most salient action society can take 

to restore tree species imperiled by invasive pests and pathogens—developing and deploying resistant tree 

populations. The 95 presentations (oral and poster) covered a wide range of tree species and associated 

pathogens and pests from throughout the world. Ash species (genus Fraxinus) throughout Europe and 

North America face relatively new perils (ash dieback disease and emerald ash borer) and a special 

session was devoted to this genus. Screening for resistance is a fundamental component of resistance 

development and the topic of another full session. Additional presentations detailed efforts to develop 

genomics and biotechnology tools and resources that have the potential to increase the efficiencies of 

applied resistance programs. Other presentations provided inputs on collaborative breeding approaches, 

citizen science in forest health, the potential role of endophytes in managed forests, threats to agroforestry 

species, prioritization of species in need of resistance programs, considerations necessary for the use of 

new biotechnologies, and concepts for integrating genetic, phenotypic and environmental data across 

host-parasite systems. Several presentations provided updates on “the ultimate goal”—progress of applied 

resistance programs and actual restoration efforts—leading into discussions on key topics such as the 

durability, stability, and usability of resistance in forest tree species. Taken together the presentations and 

discussions provided ample evidence that developing resistant populations is a viable (and in some cases 

necessary) approach for society to use to ensure healthy forests for future generations. These proceedings 

document the presentations given as lightly reviewed full papers, extended abstracts, and standard 

abstracts. In concluding the workshop, the attendees approved Spain by acclamation as the host of the 

Seventh Tree Resistance Workshop to be held in 20201.

Keywords: Durable resistance, ecosystem restoration, forest health, genetics of resistance, insect and 

disease mitigation, invasive pests and pathogens, resistance breeding.

1This workshop has been postponed until 2021 due to the Covid-19 pandemic.
ii Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

ACKNOWLEDGMENTS

Many people and organizations helped to make 

this a successful workshop, including the workshop 

organizing and technical committees (listed below). 

We especially thank the local organizing committee 

and volunteers and the field tour presenters and guides 

(see listed below). We also thank Ellen Crocker, Reneé 

Williams, Leslie Queary, Briana Fortunato, Hannah 

Hollowell, and Sarah Hayes (University of Kentucky, 

Department of Forestry and Natural Resources) 

for administrative and logistic support including 

workshop program and abstract book preparation. 

We thank Monica Schwalbach and her colleagues 

(U.S. Forest Service, Southern Research Station) 

for coordinating the USFS’s meetings management 

program for the workshop. A special thank you to our 

Keynote speaker Dr. Stephen Woodward (University 

of Aberdeen), Banquet speaker Dr. Kim Steiner 

(Pennsylvania State University), and the Western 

Forestry and Conservation Association for on-line 

registration. Our sponsors were key to making 

the workshop happen and included: USDA Forest 

Service; INRA, France; Forest Health Research 

and Education Center (University of Kentucky); 

Departments of Plant Pathology and Entomology 

(The Ohio State University); New Phytologist 

Trust (and their Plants, People, Planet journal for 

publishing the special issue); the International 

Union of Forestry Research Organizations (IUFRO) 

Groups: 7.03.11—Resistance to Insects and Pathogens, 

2.02.15—Breeding and Genetic Resources of Fiveneedle Pines, Task Force on Forests and Biological 

Invasions; and the American Phytopathological 

Society (APS), Forest Pathology Committee. We also 

gratefully acknowledge the support provided by the 

Foundational Program on Pests and Beneficial Species 

in Agricultural Production Systems, grant no. 2018-

67013-28487/project accession no. 1016633, USDA 

National Institute of Food and Agriculture (NIFA).

Finally, we thank all the participants of the workshop 

for presenting their work and serving as moderators, 

discussion leaders, poster session organizers, and 

closing summary commenters; and we are grateful 

for the many interactions that made this workshop so 

successful and helping to keep resistance work in a 

prominent place in maintaining forest health into the 

future.

Workshop Organizing Committee:

• Jennifer Koch, Chair, USDA Forest Service, Northern Research Station, USA

• Richard Sniezko, co-Chair, USDA Forest Service, Pacific Northwest Region, USA

• C. Dana Nelson, co-Chair, USDA Forest Service, Southern Research Station, USA

• Albert Abbott, University of Kentucky, Forest Health Research and Education 

Center, USA

• Mark Coggeshall, USDA Forest Service, Northern Research Station, USA

• Ellen Crocker, University of Kentucky, Forest Health Research and Education 

Center, USA

• Richard Buggs, Kew Royal Botanic Garden, Queen Mary University of London, UK

• Arnaud Dowkiw, INRA, AGPF Research Unit, Orléans, France

• Jeanne Romero-Severson, University of Notre Dame, Department of Biological 

Sciences, USA
iii

Workshop Technical Committee:

• Enrico Bonello, Ohio State University, USA

• Stephen Woodward, University of Aberdeen, UK

• Acelino Couto Alfenas, Federal University of Viçosa, Brazil

• Caterina Villari, University of Georgia, USA

• Veronique Jorge, INRA AGPF, Orléans, France

• Rita Costa, INIAV, Oeiras, Portugal

• Jared LeBoldus, Oregon State University, USA

• Bruce Moltzan, USDA Forest Service, USA

• Anna Conrad, Ohio State University, USA

Local Organizing Committee: Kathleen Knight, Mary Mason, Leila Pinchot, 

Dave Carey, Aletta Doran

Local volunteers: Kathi Combs, Andrea Watros, Terri Herd

Field tour presenters: Mary Mason, Dave Carey, Kathleen Knight, Leila Pinchot, 

Charlie Flower, Jim Slavicek

Field tour guides: Aletta Doran, Kelly Rusin, Kirsten Lehtoma, Tim Fox, 

Stephen Kelleher, Ruby Stathers, Alan Coburn

Workshop website: https://treeresistance2018.ca.uky.edu/.

Acknowledgments
iv Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

CONTENTS

ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i

ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii

PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  xiii

A SHORT HISTORY OF TREE RESISTANCE SELECTION AND BREEDING 

THROUGH THE JOURNAL FOREST PATHOLOGY

Steve Woodward, Stuart Fraser, Tuğba Doğmuş, and Alberto Santini . . . . . . . . . . . . . . . . . . . . . 1

SESSION 1: BREEDING FOR RESISTANCE TO INSECTS . . . . . . . . . . . . . . . . . . 3

SCREENING FOR RESISTANCE AGAINST INSECT HERBIVORES USING CHEMICAL 

AND ANATOMICAL DEFENSES IN LODGEPOLE PINE AND WHITE SPRUCE 

Jennifer G. Klutsch, Chen. X. Kee, and Nadir Erbilgin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

ADULT HOST SELECTION TRIALS FOR SPRUCE BUDWORM RESISTANCE 

IN DOUGLAS-FIR: A CAUTIONARY TALE

Ward Strong . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 

SCREENING FOR GENETIC RESISTANCE TO THE HEMLOCK WOOLLY ADELGID

Ben C. Smith, Scott A. Merkle, and Fred P. Hain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 

A COLLABORATIVE MULTI-AGENCY PROGRAM TO DEVELOP RESISTANCE TO 

BEECH BARK DISEASE IN AMERICAN BEECH (FAGUS GRANDIFOLIA)

Title presented at workshop: BEECH BARK DISEASE RESISTANCE BREEDING 

PROGRAM IN AMERICAN BEECH

David W. Carey, Marjorie Allmaras, Paul Bloese, David Burke, Paul Berrang, 

Lindsay Dalton, Roger Gettig, Tom Hall, Andrea Hille, Jeff D. Kochenderfer, Scott Lint, 

Mary Mason, James McKenna, Scott Rogers, Jill Rose, Carol Young, Keith Konen, 

and Jennifer L. Koch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

SESSION 2: GENUS FRAXINUS—THREATENED BY 

EMERALD ASH BORER AND ASH DIEBACK DISEASE . . . . . . . . . . . . . . . . . . . 11

PHOENIX FROM THE ASHES: EXPLOITING HOST RESISTANCE TO CONSERVE 

AND RESTORE POPULATIONS OF EUROPEAN ASH

Michelle Cleary, Lars-Göran Stener, Pierluigi Bonello, and Caterina Villari . . . . . . . . . . . . . . . . . . 12 

RESTORING GREEN ASH (FRAXINUS PENNSYLVANICA): BREEDING FOR 

RESISTANCE TO THE EMERALD ASH BORER (AGRILUS PLANIPENNIS)

Jennifer L. Koch, David W. Carey, Mary E. Mason, Therese M. Poland, Kathleen S. Knight, 

Jeanne Romero-Severson, Charles Tubesing, and Roger Gettig . . . . . . . . . . . . . . . . . . . . . . . . . . 13

EVALUATION OF RESISTANCE OF ASIAN AND EUROPEAN ASH SPECIES AND 

CULTIVARS TO EMERALD ASH BORER (AGRILUS PLANIPENNIS)

Frederic Miller, Erin McMahan, and Kim Shearer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

THE PAST IS PROLOGUE: UNDERSTANDING THE CURRENT STATE OF 

TREE IMPROVEMENT IN THE UNITED STATES

Kim Steiner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

VARIATION AND GENOMIC BASIS OF FRAXINUS EXCELSIOR (COMMON ASH) 

SUSCEPTIBILITY TO HYMENOSCYPHUS FRAXINEUS (ASH DIEBACK) 

THROUGHOUT BRITAIN

Jonathan J. Stocks, Steve J. Lee, and Richard Buggs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Contents v

SESSION 3: BREEDING FOR RESISTANCE TO PATHOGENS . . . . . . . . . . . . . 17

PROGRESS OF AN APPLIED DISEASE SCREENING AND SELECTION PROGRAM FOR 

RESISTANCE TO VASCULAR WILT IN HAWAIIAN ACACIA KOA

Nick Dudley, Tyler Jones, Aileen Yeh, Richard Sniezko, and Phil Cannon . . . . . . . . . . . . . . . . . . . . 18

THE DEVELOPMENT OF A RESISTANCE SCREENING PROGRAM OF OHIA TO 

CERATOCYSTIS PATHOGENS CAUSING WIDESPREAD MORTALITY ON HAWAII ISLAND

Marc Hughes, Blaine Luiz, Lisa Keith, and Christian Giardina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 

SCREENING RANGE-WIDE BLACK WALNUT SEED FAMILIES FOR THOUSAND CANKERS 

DISEASE (TCD) RESISTANCE AND MEASURING THE REGIONAL ADAPTABILITY OF 

RESISTANT SEED SOURCES

James R. McKenna, Racheal A. Sitz, Ned Tisserat, Don Carlson, and Paul Bloese . . . . . . . . . . . . 20 

THE SEARCH FOR RESISTANCE IN LODGEPOLE PINE TO COMANDRA BLISTER RUST 

IN THE CENTRAL INTERIOR OF BRITISH COLUMBIA

Richard W. Reich . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

GENETIC RESISTANCE TO PHYTOPHTHORA LATERALIS IN PORT-ORFORD-CEDAR 

(CHAMAECYAPRIS LAWSONIANA)

Richard A. Sniezko, Douglas P. Savin, and Paul Reeser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

GENETIC DIVERSITY IN THE LAUREL WILT PATHOGEN, RAFFAELEA LAURICOLA, 

AND THE CONSEQUENCES FOR RESISTANCE BREEDING

Tyler J. Dreaden, Marc A. Hughes, Randy C. Ploetz, Adam Black, and Jason A. Smith . . . . . . . . . 24

FUSIFORM RUST RESISTANCE IN AN ELITE POPULATION OF LOBLOLLY PINE POPULATION

April Meeks, Steve McKeand, Ross Whetten, and Fikret Isik . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

GENETIC PARAMETER ESTIMATES FOR FUSIFORM RUST DISEASE FROM A 

MULTI-ENVIRONMENT TRIAL OF LOBLOLLY PINE (PINUS TAEDA L.)

Eddie Lauer, Steve McKeand, and Fikret Isik . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

USDA ADVANCES IN PLANT BREEDING AND INNOVATION: RESISTANCE BREEDING, 

GERMPLASM COLLECTIONS, AND GENETIC DIVERSITY

Sarah Federman and Paul Zankowski . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

SESSION 4: GENOMIC AND METABOLOMIC APPROACHES TO 

IDENTIFY RESISTANCE GENES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

GENOMICS OF FRAXINUS (OLEACEAE): A GENUS UNDER SEVERE THREAT

Laura J. Kelly, Steve Lee, Jennifer Koch, Stephen J. Rossiter, and Richard J.A. Buggs . . . . . . . . . . 30

EXPRESSION PROFILING OF CANDIDATE GENES OF RESISTANCE TO PHYTOPHTHORA 

CINNAMOMI DETERMINED IN DIFERENTE GENOTYPES OF CASTANEA SPP

Carmen Santos, Sofia Duarte, Sara Tedesco, Pedro Fevereiro, and Rita L. Costa . . . . . . . . . . . . . 31

EXPLOITING NATURAL VARIATION IN A TREE SPECIES TO COMBAT AN INVASIVE 

FOREST PATHOGEN

Jared M. LeBoldus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

GENOME-WIDE EXON-CAPTURE APPROACH IDENTIFIES GENETIC VARIANTS 

ASSOCIATED WITH SUSCEPTIBILITY OF NORWAY SPRUCE TREES TO 

HETEROBASIDION PARVIPORUM INFECTION

Mukrimin Mukrimin, Andriy Kovalchuk, Leandro G. Neves, Emad Jaber, Matti Haapanen, 

Matias Kirst, and Fred O. Asiegbu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

 
vi Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

METABOLOMIC COMPARISON OF EUROPEAN ASH GENOTYPES WITH DIFFERENT 

TOLERANCE LEVELS AGAINST ASH DIEBACK

Miguel Nemesio-Gorriz, Christian Paetz, Riya C. Menezes, Almuth Hammerbacher, 

Jonathan Gershenzon, and Gerry Douglas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

SESSION 5: USE OF GENOMICS TO DISSECT MECHANISMS 

OF HOST-PARASITE INTERACTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

CANDIDATE GENES FROM GWAS AND RNASEQ FOR BEECH BARK DISEASE 

RESISTANCE IN AMERICAN BEECH

John E. Carlson, Irina Ćalić, Jennifer Koch, David Carey, Charles Addo-Quaye, 

Donghwan Shim, and David B. Neale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 

FACTORS UNDERPINNING RESISTANCE AGAINST THE GALLING PEST, 

LEPTOCYBE INVASA IN EUCALYPTUS GRANDIS

Sanushka Naidoo, Caryn Oates, Lorraine Mhoswa, Marja ‘O Neil, Juan Acosta, 

Nanette Christie, Mmoledi Mphahlele, Kitt Payn, Alexander Myburg, 

Bernard Slippers, and Carsten Külheim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

IDENTIFICATION OF GENES INVOLVED IN THE RESISTANCE RESPONSE OF 

PINUS PINASTER TO THE PINEWOOD NEMATODE INFECTION

Inês Modesto, Lieven Sterck, Isabel Carrasquinho, Yves Van de Peer, and Célia Miguel . . . . . . . 53

IDENTIFYING AND VALIDATING NECROTROPHIC EFFECTORS IN THE POPULUS 

TRICHOCARPA-SPHAERULINA MUSIVA PATHOSYSTEM

Kelsey L. Søndreli, Robert Brueggeman, Jonathan Richards, Timothy Friesen, 

and Jared M. LeBoldus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

HOST-PATHOGEN INTERACTION IN LEAVES OF LAUREL WILT HOSTS 

Junli Zhang, Stephanie M. Adams, and Jason A. Smith . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

SESSION 6: GENETIC MAP-BASED APPROACHES TO 

IDENTIFY RESISTANCE GENES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

RESISTANCE TO HETEROBASIDION ANNOSUM S.L. IN PICEA ABIES IS ASSOCIATED WITH 

A MORE ACTIVE ALLELE OF PaLAR3, A LECUANTHOCYANIDIN REDUCTASE GENE

Title presented at workshop: DO ALLELE-SPECIFIC EXPRESSION PATTERNS CONTROL 

RESISTANCE TO HETEROBASIDION ANNOSUM IN PICEA ABIES?

Miguel Nemesio-Gorriz, Almuth Hammerbacher, Kerstin Dalman, Julia Wind, Åke Olson, 

Martin Lascoux, Bo Karlsson, Jan Stenlid, Ines Ezcurra, Jonathan Gershenzon, 

and Malin Elfstrand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

DEVELOPMENT AND APPLICATION OF HIGH DENSITY GENETIC MAP OF LIMBER PINE 

(PINUS FLEXILIS JAMES) FOR GENOMICS-BASED BREEDING

Jun-Jun Liu, Anna W. Schoettle, and Richard A. Sniezko . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

GENETIC MAPPING AND FUNCTIONAL GENOMICS ANALYSES OF THE RESISTANCE/

SUSCEPTIBLE RESPONSE IN CHESTNUT SEEDLINGS TO PHYTOPHTHORA 

CINNAMOMI INFECTION

Tetyana Zhebentyayeva, Andrew Gitto, Steven Jeffers, Paul Sisco, Taylor Perkins, 

Hill Craddock, Christopher Saski, Jared Westbrook, Frederick Hebard, Laura Georgi, 

Joseph James, Margaret Staton, C. Dana Nelson, and Albert Abbott . . . . . . . . . . . . . . . . . . . . . . 61

MULTIPLE APPROACHES TO DISSECT FUSIFORM RUST RESISTANCE IN PINUS TAEDA L.

Daniel Ence, Katherine E. Smith, Robin Paul, Matias Kirst, Jill L. Wegrzyn, 

C. Dana Nelson, and John M. Davis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

 

 
vii

SESSION 7: INTEGRATING GENOMICS/BIOTECHNOLOGY WITH 

TRADITIONAL TREE BREEDING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 

THE POTENTIAL FOR BIOTECHNOLOGY TO ADDRESS FOREST HEALTH

Title presented at workshop: OVERVIEW ON THE POTENTIAL FOR BIOTECHNOLOGY 

TO ADDRESS FOREST HEALTH-BASED ON PENDING NATIONAL ACADEMY OF SCIENCE, 

ENGINEERING AND MEDICINE REPORT

Kara N. Laney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 

RESTORATION OF AMERICAN CHESTNUT: A MARRIAGE OF BREEDING 

AND BIOTECHNOLOGY

Jared W. Westbrook, Jason A. Holliday, and William A. Powell . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

NEW FRONTIERS IN FORESTRY: COMBINING PHENOMICS, COMMON GARDENS, 

AND LANDSCAPE GENOMICS TO ADDRESS DISEASE RESISTANCE AND 

CLIMATE CHANGE

Jeremy S. Johnson, Sam Cushman, Andrew Eckert, Lluvia Flores-Rentería, 

Richard Sniezko, Christopher Still, Christian Wehenkel, Amy Whipple, Michael Wing, 

Justin Bagley, Emily Boes, Ethan Bucholz, Jonathan Burnett, Antonio Castilla, 

Jessica DaBell, Cory Garms, Marja Haagsma, Evan Heck, Erin Landguth, 

Alejandro Leal Sáenz, Jared Swenson, Brianna McTeague, Mitra Menon, Ehren Moler, 

Gerald Page, Andrew Shirk, and Kristen Waring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

WISE USE OF GENOMICS IN IMPROVING FOREST HEALTH

Jeanne Romero-Severson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

SESSION 8: SCREENING FOR RESISTANCE TO PATHOGENS: 

METHODS DEVELOPMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

VIRULENCE OF SPHAERULINA MUSIVA ISOLATES DETERMINED BY GENOME-WIDE 

ASSOCIATION MAPPING, HOST INFECTION, AND COLONIZATION

Susanna Keriö, Kelsey L. Søndreli, and Jared M. LeBoldus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

EARLY SCREENING POTENTIALLY BLIGHT-RESISTANT AMERICAN CHESTNUT 

USING SMALL STEM ASSAYS

Thomas Saielli and Jared Westbrook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

DEFENSE RESPONSES OF AUSTRIAN PINE TO TWO OPPORTUNISTIC PATHOGENS 

OF CONTRASTING AGGRESSIVENESS UNDER COMBINED DROUGHT AND 

TEMPERATURE STRESS

Pierluigi Bonello, Anna Conrad, Jason Slot, Erik Andrei Visser, 

and Sanushka Naidoo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

SESSION 9: MECHANISMS OF RESISTANCE TO INSECTS . . . . . . . . . . . . . . . . 73

ARE NOVEL PLANT CHEMICALS FRIENDS OR FOES OF NATIVE INVASIVE INSECT 

HERBIVORES? EXPLAINING SUCCESSFUL HOST RANGE EXPANSION OF MOUNTAIN 

PINE BEETLE IN CANADA’S BOREAL FORESTS

Nadir Erbilgin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

VOLATILES RELEASED BY PINUS PINASTER HALF-SIB GENETICALLY CONTRASTING 

FAMILIES FOR PINE WILT DISEASE SUSCEPTIBILITY BEFORE AND DURING 

MONOCHAMUS GALLOPROVINCIALIS INSECT-VECTOR FEEDING

Elsa Gonçalves, A. Cristina Figueiredo, Isabel Carrasquinho, José G. Barroso, 

Maria l. Inácio, Joana Henriques, Edmundo Sousa, and Luís Bonifácio . . . . . . . . . . . . . . . . . . . . . 75

 

 

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viii Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

HEMLOCK AND HEMLOCK WOOLLY ADELGID: PAST RESEARCH, CURRENT FINDINGS, 

AND FUTURE DIRECTIONS IN A TROUBLED NORTH AMERICAN TREE-PEST SYSTEM

Chad M. Rigsby, Richard A. Casagrande, Colin M. Orians, and Evan L. Preisser . . . . . . . . . . . . . . 76

A HINT LEFT BY MOUNTAIN PINE BEETLE ON ANATOMICAL DEFENSES OF 

LODGEPOLE PINE TREES: LARGER RESIN DUCTS ENHANCE TREE RESISTANCE

Shiyang Zhao and Nadir Erbilgin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

VARIATION ACROSS POPULATIONS IN RESISTANCE TO A KEY HERBIVORE AND ITS 

RELATIONSHIP WITH CONSTITUTIVE AND INDUCED SECONDARY METABOLITES IN 

A MEDITERRANEAN PINE TREE

Xosé López-Goldar, Caterina Villari, Pierluigi Bonello, Anna-Karin Borg-Karlson, 

Delphine Grivet, Rafael Zas, and Luis Sampedro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  8

SESSION 10: PRIORITIZING WORK AND PATHS TO SUCCESS . . . . . . . . . . . 85

BETTER FOREST HEALTH THROUGH TREE RESISTANCE—COLLABORATIVE APPROACHES

C. Dana Nelson, Jennifer Koch, Kevin Potter, Susan McCord, Adam Costanza, 

Mark Coggeshall, Margaret Staton, and Albert Abbott . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

TREEGENES: INTEGRATING GENOTYPIC, ENVIRONMENTAL, AND PHENOTYPIC DATA 

FOR FOREST HEALTH

Emily Grau, Nic Herndon, Sean Buehler, Taylor Falk, Peter Richter, Sumaira Zaman, 

Stephen Ficklin, Margaret Staton, Doreen Main, and Jill Wegrzyn . . . . . . . . . . . . . . . . . . . . . . . . 87 

A NATIONAL PRIORITIZATION OF UNITED STATES TREE SPECIES THREATENED BY 

INSECT AND DISEASE INFESTATION

Kevin M. Potter, Maria E. Escanferla, Barbara S. Crane, Robert M. Jetton, 

and Gary Man . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 

SEARCHING, DOWNLOADING AND ANALYZING TREE GENETIC AND GENOMIC DATA 

WITH THE HARDWOOD GENOMICS WEBSITE

Margaret E. Staton, Abdullah Almsaeed, Bradford Condon, Ming Chen, Jill L. Wegrzyn, 

Doreen Main, Stephen P. Ficklin, Albert G. Abbott, C. Dana Nelson, and John E. Carlson . . . . . . 89

DURABILITY OF RESISTANCE AND “USEFUL RESISTANCE” IN FOREST TREES TO 

NON-NATIVE PATHOGENS

Richard A. Sniezko, Douglas P. Savin, Angelia Kegley, and Robert Danchok . . . . . . . . . . . . . . . . . 90

SESSION 11: NOVEL TOOLS FOR IDENTIFYING RESISTANCE . . . . . . . . . . 91

TREESNAP: A CITIZEN SCIENCE PROJECT AND MOBILE APP TO TAG TREES FOR SCIENCE

Bradford Condon, Abdullah Almsaeed, Ellen Crocker, Albert Abbott, 

C. Dana Nelson, and Margaret Staton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

DEVELOPMENT OF A TOOL FOR RAPID IDENTIFICATION OF RESISTANT TREES IN 

SPECIES AFFECTED BY ALIEN INVASIVE PATHOGENS

Anna O. Conrad, Caterina Villari, Richard A. Sniezko, Luis Rodriguez-Saona, 

and Pierluigi Bonello . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

DETECTION OF NEAR INFRARED SPECTRAL DIFFERENCES IN PINUS RADIATA 

Judith S. Nantongo, Thomas Rodemann, Hugh Fitzgerald, Brad M. Potts, 

and Julianne O'Reilly-Wapstra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
ix

SESSION 12: RESISTANCE AND INTEGRATED 

PEST MANAGEMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

ENDOPHYTE ENHANCEMENT OF SPRUCE AND EASTERN WHITE PINE SEEDLINGS 

TO IMPROVE TOLERANCE TO INSECTS AND DISEASE

Gregory W. Adams, Andrew McCartney, J. David Miller, and Dan Quiring . . . . . . . . . . . . . . . . . . 96

MONITORING OF ASH MORTALITY PATTERNS INFORMS EMERALD ASH BORER (EAB) 

RESISTANCE BREEDING EFFORTS: INTEGRATED PEST MANAGEMENT FOR EAB

Kathleen Knight, Rachel H. Kappler, Karen V. Root, Jennifer L. Koch, 

and Charles E. Flower . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

EMERGING THREATS OF PRIORITY AGROFORESTRY TREES IN AFRICA: CHALLENGES 

AND OPPORTUNITIES 

Jane Njuguna, Eston Mutitu, Ely Mwanza, Alice Muchugi, and Ignazio Graziosi . . . . . . . . . . . . . 98

SESSION 13: INTERACTIONS BETWEEN RESISTANCE

AND ENVIRONMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

THE EFFECT OF SITE QUALITY ON PERFORMANCE OF AMERICAN CHESTNUT 

(CASTANEA DENTATA) SEEDLINGS BRED FOR BLIGHT (CRYPHONECTRIA 

PARASITICA) RESISTANCE

Title presented at workshop: INFLUENCE OF SITE QUALITY ON BLIGHT RESISTANCE 

AND GROWTH OF PLANTED HYBRID CHESTNUT SEEDLINGS

Cornelia C. Pinchot, Alejandro A. Royo, Scott E. Schlarbaum, Matthew P. Peters, 

Ami M. Sharp, and Sandra L. Anagnostakis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

DRIVERS OF DISEASE EMERGENCE IN BOREAL CONIFER FORESTS, 

IMPORTANCE OF PHENOTYPIC BALANCE

Jan Stenlid, Laura Brodde, J. Julio Camarero, Ángela Sánchez-Miranda, 

and Jonàs Oliva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 

INTRASPECIFIC GENETIC VARIATION IN DEFENSIVE INVESTMENT OF A MODEL 

MEDITERRANEAN PINE: A DENDROECOLOGICAL ANALYSIS OF RESIN DUCTS

Title presented at workshop: INTRASPECIFIC GENETIC VARIATION IN DEFENSIVE 

INVESTMENT VARIES ALONG THE ONTOGENY OF A MODEL MEDITERRANEAN PINE: 

A DENDROECOLOGICAL ANALYSIS OF RESIN DUCTS 

Carla Vázquez-González, Rafael Zas, Vicente Rozas, Ricardo Alía, and Luis Sampedro . . . . . . . . 109

POSTER SESSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

AN ANSWER AWAITING A QUESTION: THE UNITED KINGDOM NATIONAL 

TREE SEED PROJECT COLLECTIONS

Alice R. Hudson, Richard J.A. Buggs, and Clare Trivedi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

GENOTYPIC DIVERSITY AND REPRODUCTIVE BIOLOGY OF THEKOPSORA AREOLATA, 

THE CAUSAL AGENT OF CHERRY SPRUCE RUST IN NORWAY SPRUCE SEED ORCHARDS

Hernán Capador, Berit Samils, and Åke Olson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

AN IMPROVED CHINESE CHESTNUT GENOME

John E. Carlson, Margaret E. Staton, Charles Addo-Quaye, Nathaniel Cannon, 

Tetyana Zhebentyayeva, Nurul Islam-Faridi, Jiali Yu, Matthew Huff, Shenghua Fan, 

Anna O. Conrad, Stephan C. Schuster, Albert G. Abbott, Jared Westbrook, 

Jason Holliday, C. Dana Nelson, Laura Georgi, and Frederick V. Hebard . . . . . . . . . . . . . . . . . . . . 118

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x Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

ADVANCED PHENOTYPING USING FT-IR DISTINGUISHES DISEASE RESISTANCE IN 

FRAXINUS EXCELSIOR AGAINST HYMENOSCYPHUS FRAXINEUS

Caterina Villari, Arnaud Dowkiw, Rasmus Enderle, Marjan Ghasemkhani, 

Thomas Kirisits, Erik Kjaer, Diana Marčiulynienė, Lea McKinney, Berthold Metzler, 

Facundo Muñoz, Lene Rostgaard Nielsen, Alfas Pliūra, Lars-Göran Stener, 

Vytautas Suchockas, Luis Rodriguez-Saona, Pierluigi Bonello, and Michelle Cleary . . . . . . . . . . . 125

DELPHI EXPERT OPINION SURVEY TO ASSESS THREATS TO OAKS IN THE 

EASTERN UNITED STATES

Anna O. Conrad, Ellen V. Crocker, Xiaoshu Li, Billy Thomas, Thomas Ochuodho, 

and C. Dana Nelson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

TREESNAP: A CITIZEN SCIENCE APP CONNECTING TREE ENTHUSIASTS 

AND FOREST SCIENTISTS

Title presented at workshop: TREESNAP: A MOBILE APP TO HELP YOU ENGAGE THE 

PUBLIC IN RESEARCH

Bradford Condon, Ellen Crocker, Abdullah Almsaeed, C. Dana Nelson, 

Albert Abbott, and Margaret Staton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

PHYTOPHTHORA RAMORUM IN LARCH: FROM EPIDEMIOLOGY TO HOST RESISTANCE

Title presented at workshop: UNDERSTANDING SUDDEN LARCH DEATH—FROM EPIDEMIOLOGY 

TO HOST RESISTANCE

Heather Dun, John Mackay, and Sarah Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

GEOGRAPHIC AND LOCAL GENETIC VARIATION IN PACIFIC MADRONE LEAF BLIGHT

Laura E. DeWald, Marianne Elliott, Richard Sniezko, and Gary A Chastagner . . . . . . . . . . . . . . . . 132

BACK FROM THE BRINK: FOREST SERVICE EFFORTS TO CREATE DUTCH ELM DISEASE 

TOLERANT TREES FOR USE IN URBAN AND RURAL RESTORATION

Charles E. Flower, Cornelia C. Pinchot, Kathleen S. Knight, Keith Woeste, 

and James M. Slavicek . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

FIELD PERFORMANCE OF PREVIOUSLY SELECTED ROOT ROT TOLERANT 

TROPICAL ACACIAS

Abdul Gafur, Ching Y. Wong, and Mukesh Sharma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

BUILDING A CERATOCYSTIS RESISTANCE PROGRAM FOR METROSIDEROS IN HAWAII

Christian Giardina, Lisa Keith, Phil Cannon, Richard Sniezko, Douglas Jacobs, 

and J.B. Friday . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

THE IMMINENT INVASION OF THE EMERALD ASH BORER IN SOUTHERN EUROPE 

AND THE THREAT TO NATIVE OLEACEAE

Ignazio Graziosi and Lynne Rieske-Kinney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

A COLLABORATIVE RESEARCH APPROACH FOR DIAGNOSING AND EVALUATING 

THOUSAND CANKERS DISEASE IN WALNUT: CURRENT PROGRESS AND 

FUTURE DIRECTIONS

Emel Oren, William Klingeman, Romina Gazis, John Moulton, Paris Lambdin, 

Massimo Faccoli, Mark Coggeshall, Jiri Hulcr, Steven J. Seybold, 

and Denita Hadziabdic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

COLLABORATIVE RESEARCH FOR SUSTAINABLE MANAGEMENT OF 

SOUTHWESTERN WHITE PINE

Kristen Waring, Sam Cushman, Andrew Eckert, Lluvia Flores, Richard Sniezko, 

Christopher Still, Christian Wehenkel, Amy Whipple, Michael Wing, Justin Bagley, 

Emily Boes, Ethan Bucholz, Jonathan Burnett, Antonio Castilla, Jessica DaBell, 

Chris Friedline, Cory Garms, Evan Heck, Jeremy Johnson, Erin Landguth, 

Alejandro Leal Sáenz, Briana McTeague, Mitra Menon, Ehren Moler, 

Gerald Page, and Andrew Shirk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Contents xi

BUTTERNUT CONSERVATION AND BREEDING TO MITIGATE BUTTERNUT 

CANKER DISEASE

James R. McKenna, Paul Berrang, Aziz Ebrahimi, Scott Rogers, Stuart Seaborne, 

and James Jacobs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

DIE BACK SYNDROME CAUSED BY LASIODIPLODIA THEOBROMAE AND FUSARIUM 

PROLIFERATUM IN ELITE GENOTYPES OF TECTONA GRANDIS LF., COSTA RICA

Dawa Méndez-Álvarez, Arantxa Rodríguez, Yorleny Badilla-Valverde, 

and Olman Murillo-Gamboa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

SOMATIC EMBRYOGENESIS AND CRYOSTORAGE FOR RESTORATION OF ASH 

FORESTS DEVASTATED BY EMERALD ASH BORER

Scott A. Merkle, Paul M. Montello, A. Ryan Tull, Daniel A. Herms, 

and Kamal J.K. Gandhi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

QTL MAPPING OF SPHAERULINA MUSIVA CANKER RESISTANCE/SUSCEPTIBILITY 

IN A POPLAR T x D HYBRID POPULATION

Kyle Mondron, Susanna Keriö, Sandra Simon, Stephen DiFazio, 

and Jared M. LeBoldus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

THE RESISTANCE OF MULBERRY TREE SPECIES TO TWO FOLIAR DISEASES IN 

THE RAINFOREST ECOLOGICAL ZONE OF NIGERIA 

Oduntan, O. Akinlabi, Onilude Q. Ayansola, and Charles O. Ogunremi . . . . . . . . . . . . . . . . . . . . . 147

NURSERY PEST RESISTANCE OF MANSONIA ALTISSIMA SEEDLINGS TO GODASA 

SIDAE ATTACK IN THE RAINFOREST ECOLOGICAL ZONE OF NIGERIA

Oduntan, O. Akinlabi, Omotade R. Ogunremi, Onilude Q. Ayansola, 

and Awotedu B. Folake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

QTL MAPPING POPULATION DEVELOPMENT IN CORNUS FLORIDA USING 

OPEN POLLINATED SEEDLINGS AND SSR MARKERS

Erin Pfarr, Josh Honig, Jennifer Vaiciunas, Christine Kubik, John Capik, 

and Thomas J. Molnar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 

GENETIC BARCODES FOR IDENTIFICATION OF PURE AND HYBRID FRAXINUS SPECIES

William J. Plumb, Laura J. Kelly, Gerry Douglas, and Richard Buggs . . . . . . . . . . . . . . . . . . . . . . . 154

CONFOCAL MICROSCOPY CONFIRMS THE FEASIBILITY OF GENE SILENCING FOR 

EMERALD ASH BORER SUPPRESSION

Thais B. Rodrigues, Flavia Pampolini, Tomokazu Kawashima, and Lynne Rieske-Kinney . . . . . . . 155

VARIOUS PROPAGATION WAYS IN ALNUS GLUTINOSA WITH DIFFERENT SENSITIVITY 

TO PHYTOPHTHORA × ALNI

Jana Šedivá, Petra Štochlová, Kateřina Novotná, and Karel Černý . . . . . . . . . . . . . . . . . . . . . . . . 156

USING A POPLAR HYBRID TO EXPLORE HOST PLANT GENETIC CONTROL OF 

ASSOCIATING INSECT AND FUNGAL SPECIES

Sandra J. Simon, Stephen P. DiFazio, and David Macaya-Sanz . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

GENETIC RESISTANCE TO BLISTER RUST IN DECLINING LIMBER PINE 

(PINUS FLEXILIS) IN ALBERTA—THE PATH TO RESTORATION?

Richard A. Sniezko, Jodie Krakowski, Robert Sissons, Robert Danchok, 

Angelia Kegley, and Douglas P. Savin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

GENETIC RESISTANCE AND RESTORING A THREATENED SPECIES: WHITEBARK PINE 

AT CRATER LAKE NATIONAL PARK

Richard A. Sniezko and Jennifer S. Hooke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
xii Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

INVESTIGATING ANATAGONISTIC ACTIVITY OF FUNGAL ENDOPHYTES TOWARD 

PATHOGENS OF WESTERN WHITE PINE (PINUS MONTICOLA)

Emily Martin, Beau Larkin, Richard Sniezko, and Lorinda Bullington . . . . . . . . . . . . . . . . . . . . .  16

SUDDEN OAK DEATH IN SOUTHERN OREGON: COMPARING THE EU1 AND NA1 

LINEAGES OF PHYTOPHTHORA RAMORUM

Kelsey L. Søndreli, Alan Kanaskie, Richard Sniezko, Everett Hansen, 

and Jared M. LeBoldus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

SCREENING FOR WHITE PINE BLISTER RUST IN WHITEBARK PINE: 

BRITISH COLUMBIA, CANADA

Ward Strong . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 

DIFFERENTIAL RESISTANCE TO PHYTOPHTHORA CINNAMOMI IN TROJAN FIR

Alexander J. Trouern-Trend, William H. Kohlway, Lilian Matallana, Ross W. Whetten, 

John Frampton, and Jill L. Wegrzyn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16

PHENOLIC COMPONENT OF LOBLOLLY PINE DEFENSE RESPONSE TO BLUE-STAIN 

FUNGI ASSOCIATED WITH ROOT-FEEDING BEETLES

Zackary N. Parker, Brittany F. Barnes, and Caterina Villari . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16

BREEDING FOR RESISTANCE TO PATHOGENIC PINE WOOD NEMATODES

Kwan-Soo Woo, Donghwan Shim, Sung-Joon Na, Jinjoong Kim, and Il Hwan Lee . . . . . . . . . . .  16

VARIATIONS IN SUSCEPTIBILITY TO DOTHISTROMA NEEDLE BLIGHT AND 

PINE PITCH CANKER SCOTTISH POPULATIONS OF SCOTS PINE

Steve Woodward . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16

BREEDING AND DEPLOYMENT OF INSECT AND DISEASE RESISTANCE TREES IN 

BRITISH COLUMBIA: A 2018 UPDATE

Alvin Yanchuk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16

RESTORATION AFTER THE LAUREL WILT DISEASE EPIDEMIC

Katherine Smith, Junli Zhang, Marc Hughes, Craig Echt, Sedley Josserand, 

C. Dana Nelson, and Jason Smith....

THE EFFECTS OF LEAF WAX COMPOSITION AND RATIO ON 

RUST RESISTANCE OF POPLARS

Zhongdong Yu, Wei Zheng, Kuocheng Shen, Dan Yu, and Zhimin Cao . . . . . . . . . . . . . . . . . . . . . 170
xiii

PREFACE

In 2018, an engaging and energizing workshop on Resistance to Pests and Pathogens in Forest 

Trees (also known as the Sixth International Workshop on the Genetics of Host-Parasite 

Interactions in Forestry) was held at the Deer Creek State Park Lodge in Mt. Sterling, Ohio. This 

was the third workshop in this series since 2011. The 2011 workshop re-initiated the series that 

had started in the 1960s and last convened in 1980 (information and the rationale for the pre-2011 

workshops are included in the Preface to the proceedings of the 2011 workshop. We compiled 

and edited these proceedings to document the research presented and discussed at the “Sixth 

Workshop” and to encourage attendees and our readers to continue their efforts to develop forest 

trees that are resistant to pests and pathogens. Information about the Fourth, Fifth and Sixth 

Workshops can be found at the following web sites:

Fourth (2011)—https://ucanr.edu/sites/tree_resistance_2011conference/, 

Fifth (2015)—https://colloque.inra.fr/tree-parasite-interactions2015/, and

Sixth (2018)—https://treeresistance2018.ca.uky.edu/.

The Fourth Workshop (2011) workshop inspired several of its participants to call further attention 

to an underlying problem in forest health with a paper published in Frontiers in Ecology and the 

Environment. In the same manner, discussions at the Fifth Workshop (2015) helped crystallize 

some thoughts that led directly to a publication in Frontiers in Ecology and Evolution focused 

on the potential of tree resistance to mitigate invasive pathogens and pests. Following this, an 

IUFRO Task Force on Forests and Biological Invasions recognized the importance of including the 

resistance topic, and produced a special issue in Biological Invasions which included a paper that 

provided recent success stories in breeding for resistance. 

For the Sixth Workshop (2018), we partnered with New Phytologist Trust to produce a special 

issue on Tree Resistance in its journal Plants, People and Planet (PPP). This special issue

(PPP, volume 2, issue 1) includes full papers that are directly related to four of the abstracts 

presented at the Sixth Workshop. The four abstracts as submitted to the workshop are published 

in these proceedings and we annotated them with their reference in the special issue of PPP. A 

fifth presented abstract was published as a paper in New Phytologist and its reference is provided. 

In addition, the special issue includes three other papers that are related to various presentations 

given at the workshop.1 In total, the proceedings contain abstracts, extended abstracts or full 

papers on all 59 talks and 37 posters offered at the meeting. Of these, 14 are extended abstracts or 

full papers.

It is notable that Dr. Norman Borlaug (Nobel Peace Prize winner in 1970 for his world-wide 

contributions in agriculture) participated in the first two workshops (in 1964 and 1969). The 

proceedings and book resulting from the earlier workshops inspired both the re-initiation of this 

workshop series, and the format for the meeting—with an emphasis on discussion, debate, and 

the absence of concurrent sessions. This workshop series was envisioned as a melding of research 

Preface

1Showalter, D.N., et al. Resistance of European ash (Fraxinus excelsior) saplings to larval feeding by the emerald ash 

borer (Agrilus planipennis); Plumb, W.J., et al. The viability of a breeding programme for ash in the British Isles in the 

face of ash dieback; Buggs, R.A. Changing perceptions of tree resistance research.
xiv Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

with application, that is, the delivery of effective pest and pathogen resistance for restoration and 

reforestation. To do this, it was realized that we needed to retain the focus of the end point—the 

development and use of resistance—and bridge the divide that exists between researchers, tree 

breeders and forest managers. To facilitate this goal, we held a discussion session with a panel 

of forest health managers that specifically addressed this topic. Furthermore, we felt it was 

important to emphasize success stories to help dislodge both the myths around resistance and the 

fallacies that other management activities by themselves could save our forests. Success stories 

on the development of resistance are starting to emerge and this should facilitate support for 

ongoing and future efforts. Developing resistance is a concrete, attainable goal that can lead to 

substantial societal benefits. 

The need for action on efficiently developing genetic resistance to the excessively damaging pests 

and pathogens in forest trees is more apparent now than ever, and several examples presented at 

the Sixth Workshop and published in these proceedings show that it can be done. As new tools 

in genomics and biotechnology are developed in an integrated fashion with ongoing resistance 

breeding programs, there is evidence that they can increase efficiency and effectiveness of 

the production of resistant trees. The key is keeping the focus on the breeding objectives and 

integrating tools that can expedite progress. Success in developing resistance will require 

communication and partnerships between researchers, tree breeders, resource managers and the 

public, as well as a sharp and steady focus on the “ultimate goal”—restoration or reforestation of 

species of concern for forest health and human benefit. To fully succeed we also need to be aware 

of other key ecological factors that may play a role in the health of forest trees. 

These workshops have been well received and interest in the topic continues to grow. As an 

example, groups were volunteering to host the next two workshops before the 2018 workshop 

began. The 2018 attendees approved by consensus a proposal to host the Seventh Workshop in 

Spain in September 20202. Furthermore, the time between workshops has been reduced as both 

the urgency of action and the enthusiasm of the many visionaries in this field have coalesced. We 

are grateful that IUFRO and other groups that have supported these meetings and optimistic that 

the next generation of scientists and resource professionals will continue this important work for 

the benefit of our planet’s forests. In addition, we thank the participants and sponsors of the Sixth 

Workshop for their generous support and for adding to the vision that with Tree Resistance we 

are truly on the cusp of a paradigm change for restoring forest health. Finally, we thank those that 

came before us and organized and hosted the prior five workshops. To succeed, we need to be a 

community, all committed to a common cause – forest health, globally. 

Richard Sniezko, USDA Forest Service, Cottage Grove, Oregon

Jennifer Koch, USDA Forest Service, Delaware, Ohio

C. Dana Nelson, USDA Forest Service, Lexington, Kentucky

March 10, 2020

2 This workshop has been postponed until 2021 due to the Covid-19 pandemic.
xv

Participants in The Sixth International Workshop on 

the Genetics of Host-Parasite Interactions in Forestry. 

Mt. Sterling, Ohio, August 5–10, 2018.

Tree Resistance Workshop Organizing 

Committee. From left to right: 

Ellen Crocker, Mark Coggeshall, 

Dana Nelson, Jeanne Romero-Severson, 

Jennifer Koch, Richard Sniezko

Preface

1

A SHORT HISTORY OF TREE RESISTANCE SELECTION AND BREEDING 

THROUGH THE JOURNAL FOREST PATHOLOGY

Steve Woodward1, Stuart Fraser1,4, Tuğba Doğmuş2, and Alberto Santini3

Forest Pathology was first published in 1971, 

then under the title European Journal of Forest 

Pathology. The first editor-in-chief, Peter Schütt, 

assembled a board of like-minded individuals 

based in several European countries to present 

the progress of research on tree and forest health, 

initially focused on Europe itself. Within a 

short time, however, the journal was recognized 

internationally and attracted manuscripts from 

all forested continents. In 2000, under the second 

editor-in-chief, Ottmar Holdenrieder, the inclusion 

of increasing quantities of global science in the 

journal led to a change in the name to Forest 

Pathology, which is maintained to this day.

Since the outset, the journal has published papers 

on the potential use of resistance to pathogens 

in the management and control of tree diseases. 

Using “resistance” as the search term on the 

journal home page produces 1,131 “hits” for 

individual papers, although the precise nature of 

the “resistance” referred to is obscure in some 

cases. Host-pathogen systems reported on vary 

widely, from the expected (e.g., white pine blister 

rust, Dutch elm disease, chestnut canker) to less 

commonly known interactions (e.g., Microcyclus

on Hevea; Ralstonia on Casuarina). More recently, 

the focus has, to some extent, shifted to potential 

resistance against recently detected invasive 

pathogens, such as ash dieback and species of 

Phytophthora, but other longer-term problems still 

receive plenty of attention.

Subjects for examination have also varied 

from the traditional aspects of selection and 

breeding through the physiological mechanisms 

contributing to resistance, to, more recently, the 

molecular biology of tree-pathogen interactions.

In this review, we will present an analysis of 

the work on tree resistance published in Forest 

Pathology since the journal’s first issue, including 

data on the numbers of papers published on 

different host-pathogen interaction. We will 

evaluate, as far as is possible, the overall 

contribution of papers in this journal to our 

current understanding of the potential for host 

resistance in contributing to the production of 

trees resistant to pathogens.

1University of Aberdeen, Department of Plant and Soil Science, School of Biological Sciences, Cruickshank Building, Aberdeen AB24 3UU, 

United Kingdom, (s.woodward@abdn.ac.uk). 2 Faculty of Forestry, Süleyman Demirel University, Isparta, Turkey.

3 Institute for Sustainable Plant Protection – C.N.R., Via Madonna del Piano, 10 - 50019 Sesto fiorentino, ITALY; 

4 Present address: Scion, Rotorua, New Zealand.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.

3

Session

BREEDING FOR RESISTANCE TO INSECTS

1

Session 1: Breeding for Resistance to Insects
4 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

SCREENING FOR RESISTANCE AGAINST INSECT HERBIVORES USING CHEMICAL AND 

ANATOMICAL DEFENSES IN LODGEPOLE PINE AND WHITE SPRUCE

Jennifer G. Klutsch1, Chen. X. Kee2, and Nadir Erbilgin2

Insect outbreaks and drought have increasingly caused widespread tree mortality worldwide. Host 

defenses are important factors that determine tree resistance to these insect disturbances. However, 

trade-offs between growth and defense may occur and vary with life history strategy of the host tree. 

We measured defense and growth patterns from mature lodgepole pine (Pinus contorta var. latifolia) 

and white spruce (Picea glauca) in two progeny trials in Alberta, Canada. These trees have different 

life history strategies, with lodgepole pine being shade intolerant and white spruce being shade tolerant. 

Furthermore, the major insect pests for these trees differ; the bark beetle Dendroctonus ponderosa

is a tree-killing insect on lodgepole pine and defoliation from Choristoneura fumiferana is the main 

disturbance for boreal white spruce. We measured anatomical defenses found in the xylem and 

monoterpene concentrations within phloem of the stem, which have been associated with resistance 

to bark beetle attack. Lodgepole pine and white spruce were found to differ in their growth-defense 

relationships. In addition, we did not find evidence of a trade-off between anatomical defenses and 

radial growth. Furthermore, we found an effect of the presence of the disease caused by Endocronartium 

harknessii on lodgepole pine on both anatomical and chemical defense traits. Our results preliminarily 

suggest that the major insect herbivore associated with tree species may influence their relative investment 

in defense and that understanding the variation in constitutive defense capabilities and the existence of 

genetic trade-offs between growth and defenses can be important factors for pest resistance screening.

1University of Alberta, Department of Renewable Resources, Edmonton, AB, Canada T6E 2E3, (klutsch@ualberta.ca). 2University of Alberta, Department of Renewable Resources, Edmonton, AB, Canada T6E 2E3.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
5

ADULT HOST SELECTION TRIALS FOR SPRUCE BUDWORM RESISTANCE 

IN DOUGLAS-FIR: A CAUTIONARY TALE

Ward Strong1

Session 1: Breeding for Resistance to Insects

Douglas-fir, Pseudotsuga menziesii (Mirb.) 

Franco, is a major commercial forestry species 

in British Columbia, Canada. Its range includes 

wet coastal areas and dryer Interior regions 

(Hermann and Lavender 1990) (fig. 1). In the 

interior range, it is subject to attack by the western 

spruce budworm, Choristoneura occidentalis

Freeman (Lepidoptera: Tortricidae). The larvae 

of this moth can cause up to 100 percent foliage 

loss, and tree death can occur after successive 

years of defoliation (Maclauchlan et al. 2006). 

During the latest outbreak, which peaked in 

2007, over 800 000 ha were infested (British 

Columbia Forest Service 2007). Current means of 

controlling C. occidentalis include aerial sprays 

of Bacillus thuringiensis, a bacterium toxic to 

Lepidoptera larvae. In 2012, 116 012 ha were 

sprayed to attempt control of this insect (British 

Columbia Forest Service 2012), at an estimated 

cost of CDN $2.51M (Personal communication. 

2018. Lorraine Maclauchlan, BC Ministry of 

Forests, Lands, Natural Resource Operations, 

Figure 1—Range of Douglas-fir (Pseudotsuga menziesii) in British Columbia. (courtesy photo by Province 

of British Columbia)

1BC Ministry of Forests, Lands, Natural Resource Operations, and Rural Development, Vernon, British Columbia, CANADA V1B 2C7, 

(ward.strong@gov.bc.ca).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
6 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

and Rural Development, Kamloops, BC Canada). 

A more cost-effective means of control might 

be to develop genetically-based resistance to C. 

occidentalis. 

The British Columbia Forest Service has been 

conducting genetics studies and breeding 

programs for commercially important trees since 

the 1960s (e.g., Roche 1969). Selective traits of 

importance have historically been volume, tree 

form, and wood quality, and more recently pest 

resistance has become a focus. One region of BC, 

where we have an advanced breeding program, 

is from the Thompson-Okanagan region, which 

suffered widespread C. occidentalis infestation 

during the 2007 outbreak. This breeding program 

is composed of 78 parent genotypes, and clones 

of these parents are held in a breeding arboretum 

in Vernon, BC, with 3 ramets per clone. We 

decided to examine these trees for C. occidentalis

resistance, with the objective of incorporating 

resistance into our breeding program, for eventual 

deployment across the landscape if gains can be 

made for this trait.

We started in 2012 by purchasing C. occidentalis 

pupae from an insectary (NRCan Insect 

Production services, Sault Ste-Marie, Ontario), 

derived from British Columbia stock. These 

were reared at 22 °C and 40 percent RH until 

eclosion. Moths were allowed to mate and females 

oviposited on wax paper. Paper tabs supporting 

10 eggs were cut with scissors and affixed with 

a paper clip to new shoots of Douglas-fir, shortly 

after budburst. One shoot on each ramet of 

each parent genotype was thus infested. Over 

each shoot a 53 x 63 cm Organza mesh bag was 

installed (Creative Bag, Toronto, Canada) to keep 

C. occidentalis in and predators or parasites out. 

In late June the bags were removed, remaining 

moths, pupae or larvae within counted, and foliage 

rated for proportion consumed. 

There was a wide range in variation in feeding 

performance (fig. 2), ranging from 0 percent of 

foliage consumed to over 90 percent. No larvae 

remained, but the number of pupae and moths that 

came out also varied, with generally the lowest 

numbers coming from the least palatable parents, 

and the highest numbers from the most palatable 

parents. This is a result typical of other studies 

(Cates 1983, McDonald 1981), though no other 

studies specifically measured the influence of 

genotype. However, the usefulness of these results 

might be limited, because while the larvae cause 

the damage, it is their parents, which are strong 

Figure 2—Proportion of new foliage consumed by captive spruce budworm larvae on 78 different Douglas-fir genotypes.
Session 1: Breeding for Resistance to Insects 7

flyers that choose the host tree that the larvae 

must then feed upon. We therefore decided to 

investigate adult host selection of C. occidentalis

on Douglas-fir.

Surprisingly, only two papers were found on 

adult budworm host selection: Städler (1974) 

described host plant characteristics, presumably 

under genetic control, that influence oviposition 

choice in eastern spruce budworm, C. fumiferana; 

and Leyva et al. (2000) investigated adult host 

preference of C. occidentalis among 3 host 

species, without reference to genotype within 

species. Another similar system in which adult 

host preference has been investigated is in fruit 

trees with other members of the same moth family, 

Tortricidae. Oriental fruit moth Grapholyta 

molesta females show oviposition preference 

between fruit tree species (Myers et al. 2006a), 

while obliquebanded leafroller Choristoneura 

rosaceana adult females prefer some host species 

over others (Carrier et al. 1995), both again 

without reference to genotype within species. 

Only one paper was found investigating withinspecies host preference: Myers et al. (2006b) found 

that C. rosaceana oviposited preferentially on 

certain apple cultivars. Discussion with Spruce 

Budworm researchers in Canada revealed no 

knowledge of adult host selection, within host 

species, in any Choristoneura in Canada. This is 

apparently a wide-open field.

In 2013, we collected shoots from each of 

the 78 parent genotypes, in early July when 

moths emerge, fly, and oviposit in the wild. 

We installed these shoots in water troughs in 

a small greenhouse, arranged in a randomized 

complete block design with 5 replicates. Insect 

mesh was installed inside the glass to contain 

adult moths. Into this enclosure we introduced 

500 C. occidentalis pupae, again purchased from 

the Canadian insectary, spread among 10 petri 

dishes. By the next morning, all pupae had been 

consumed by mice. Because the insectary had no 

more pupae available, we were shut down for the 

year. 

In 2014, we tried again with a similar 

experimental design and physical facilities, but 

with a mouse-proof release device. We also 

purchased an extra 500 pupae as a backup. The 

moths emerged within 5 days and immediately 

flew to the ground and laid their eggs on the 

concrete floor. The backup pupae produced moths 

that behaved in exactly the same manner. Our 

moths were highly confused. 

In discussion with several budworm experts in 

Canada, we identified three potential issues. 

• The cuttings were removed from the parent 

trees, they were not a part of the natural 

tree. In other systems, as soon as a shoot is 

removed from the parent plant, its volatiles 

change dramatically, potentially losing 

attractiveness, or even repelling adult 

C. occidentalis.

• The greenhouse environment might 

confuse the moths, by altering the light, 

temperature, humidity, or wind flow 

characteristics that moths outdoors use to 

detect potential hosts. 

• The C. occidentalis colony might have a 

highly restricted genetic makeup, having 

been selected through several generations 

for freedom from disease and good 

performance in a laboratory colony on 

artificial diet, ovipositing on wax paper. 

Their normal host-finding systems may 

have been affected, and even their ability to 

fly may have been impacted.

We decided to deal with these issues by grafting 

scion from each of the 78 parents onto rootstock 

growing in an outdoors compound. Four hundred 

and fifty Douglas-fir seedlings of random genetic 

origin were planted in 2015. Growth was not as 

expected by 2016, so we waited until 2017 to 

collect scion and make the grafts. Grafts were 

arranged in a 13 x 6 alpha-design (Fu et al. 1998) 

with five linearized replicates. In 2018, grafts 

were growing well, so one-half the rootstock 

foliage was removed. The objective is to remove 

all the rootstock by 2019 so that the only foliage 

remaining is from the parent genotype.

In 2019, a screenhouse will be constructed over 

the outdoor trees. This light structure, covered 

with a large-mesh insect screen, will provide very 

little shade or other light disturbance, and allow 

maximum airflow through the grafts, but contain 

the moths once they have emerged.
8 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

We plan to release wild-collected pupae. Wildcollected pupae might be more adapted to hostfinding in their native habitat and will have a 

broader genetic background. Though the greater 

genetic variation of the budworm pupae might add 

experimental error, any inferences will be more 

broadly applicable.

Though we have no data yet in the 4th year of 

our C. occidentalis adult host selection study, we 

have answered one question: why this has not 

been done before. It’s hard! This sort of study is 

not amenable to a graduate student program, so it 

remains to government researchers like our group 

to conduct it. We hope to have some results by the 

end of 2019. 

Addendum, April 2020. Results from the year 

2019. We constructed a screenhouse as planned 

over the grafted outdoor trees. We collected 

about 1,800 pupae from the wild in late June. 

These were placed in brass soil screens (20.3 cm 

diameter, 5 cm rim, 8–12 mesh) suspended by 

wire from the screenhouse structure. Soil screens 

were used so that rain would not accumulate and 

drown the pupae; they were suspended by wires, 

and not allowed to touch any trees, in order to 

prevent ants from discovering and preying upon 

the pupae. Pupal emergence was only 13.8 percent, 

and the moths that emerged died within 1 or 2 

days of emergence, without laying any eggs. We 

determined later that the brass soil screens heated 

in the sun to temperatures in excess of 45 0C, 

which likely killed most of the pupae. Those 

that did emerge encountered very dry conditions 

with no dew formation or wildflowers to provide 

moisture, so moths likely died of dehydration. We 

will try one last kick at the can in 2020, modifying 

the release devices to provide shade (so they do 

not heat up in the sun) and misting the plants daily 

to provide moisture. Maybe after 7 years of trying 

we will finally get some results.

LITERATURE CITED

British Columbia Forest Service. 2007. 2007 Overview of 

Forest Health in the Southern Interior Forest Region. 

Government of British Columbia, Ministry of Forests and 

Range. 

British Columbia Forest Service. 2012. 2012 Overview of 

Forest Health in the Southern Interior Forest Region. 

Government of British Columbia, Ministry of Forests, 

Lands, and Natural Resource Operations. 

Carrier, Y.; Paré, S.; Roitberg, B.D. 1995. Oviposition 

preference of a polyphagous moth, the Obliquebanded 

Leafroller, Choristoneura rosaceana (Harris) (Lepidoptera: 

Tortricidae). The Canadian Entomologist. 127: 577–586.

Cates, R.G.; Redak, R.; Henderson, C.B. 1983. Natural 

product defensive chemistry of Douglas-fir, Western Spruce 

Budworm success, and forest management practices. 

Journal of Applied Entomology. 96: 173–182.

Fu, Yong-Bi.; Peter, G.; Clarke, Y. [et al.]. 1998. Incomplete 

block designs for genetic testing: statistical efficiencies 

of estimating family means. Canadian Journal of Forest 

Research. 28: 977–986.

Hermann, R.K.; Lavender, D.P. 1990. Pseudotsuga menziesii 

(Mirb.) Franco Douglas-fir. In: Silvics of North America 

vol 1: Conifers. USDA Handbook 654. U.S. Department of 

Agriculture Forest Service, Washington, DC: 527–540.

Leyva, K.J.; Clancy, K.M.; Price, P.W. 2000. Oviposition 

preference and larval performance of the Western Spruce 

Budworm (Lepidoptera: Tortricidae). Environmental 

Entomology. 29: 281–289.

Maclauchlan, L.E.; Brooks, J.E.; Hodge, J.C. 2006. Analysis 

of historic western spruce budworm defoliation in south 

central British Columbia. Forest Ecology and Management. 

226: 351–356.

McDonald, G.I. 1981. Differential defoliation of neighboring 

Douglas-fir trees by Western Spruce Budworm. Research 

Note INT-306. Ogden, UT: U.S. Department of Agriculture 

Forest Service. Intermountain Forest and Range 

Experiment Station. 306 p.

Myers, C.T.; Hull, L.A.; Krawczyk, G. 2006a. Effects of 

orchard host plants on the oviposition preference of the 

oriental fruit moth (Lepidoptera: Tortricidae). Journal of 

Economic Entomology. 99: 1176–1183.

Myers, C.T.; Hull, L.A.; Krawczyk, G. 2006b. Seasonal and 

cultivar-associated variation in oviposition preference of 

oriental fruit moth (Lepidoptera: Tortricidae) adults and 

feeding behavior of neonate larvae in apples. Journal of 

Economic Entomology. 99: 349–358.

Roche, L. 1969. A genecological study of the genus Picea in 

British Columbia. New Phytologist. 68: 505–554.

Städler, E. 1974. Host plant stimuli affecting oviposition 

behaviour of the Eastern Spruce Budworm. Entomologia 

Experimentalis et Applicata. 17: 176–188.
Session 1: Breeding for Resistance to Insects 9

SCREENING FOR GENETIC RESISTANCE TO THE HEMLOCK WOOLLY ADELGID

Ben C. Smith1, Scott A. Merkle2, and Fred P. Hain3

The exotic hemlock woolly adelgid (HWA) (Adelges tsugae) has caused widespread decline and mortality 

in natural-occurring and ornamental eastern (Tsuga canadensis) and Carolina (T. caroliniana) hemlocks 

native to the Eastern United States. The Forest Restoration Alliance (FRA) seeks to develop and produce 

hemlocks resistant to or tolerant of HWA that are suitable for both species restoration and ornamental 

uses. FRA strategies include the identification of resistant and tolerant genotypes of native hemlocks 

for inclusion in a resistance breeding program, and creation of interspecific hybrids between native 

and HWA-resistant or tolerant exotic hemlocks. In June 2017, 2-year-old cuttings propagated from 24 

candidate trees were intentionally infested with HWA using a rain-down technique of crawlers hatching 

from suspended HWA-infested cut branches. Rooting success among clones was highly variable, so the 

number of ramets per clone ranged from 1 to 16 in the infested treatment (n = 271 plants) and 1 to 20 

in the non-infested control treatment (n = 118 plants). We evaluated the trees in July 2018 for adelgid 

presence, total height, growth of the dominant terminal branch, branch tip dieback, and overall plant vigor. 

Height and growth differed significantly among clones, but no measured trait was significantly affected 

by infestation. HWA survival and reproduction was poor over time, so additional artificial infestation 

and evaluation are needed to accurately assess levels of resistance. Through the in vitro propagation 

technique of somatic embryogenesis (SE), we have successfully produced somatic seedlings from eastern, 

Carolina, and hybrids between Carolina and Chinese (T. chinensis) hemlock and Carolina and southern 

Japanese (T. sieboldii) hemlock, verified by chloroplast DNA markers. We are utilizing SE to accelerate 

clonal replication for resistance/tolerance screening and deployment of tested genotypes. We have also 

attempted, without success yet, to create novel interspecific hybrids incorporating eastern hemlock using 

embryo rescue.

1 Forest Restoration Alliance, Department of Entomology and Plant Pathology, North Carolina State University, Waynesville, NC 28786, 

(bcsmith6@ncsu.edu). 2Warnell School of Forestry and Natural Resources, University of Georgia, Athens, GA 30602.

3 Forest Restoration Alliance, Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC 27695.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
10 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

A COLLABORATIVE MULTI-AGENCY PROGRAM TO DEVELOP RESISTANCE TO 

BEECH BARK DISEASE IN AMERICAN BEECH (FAGUS GRANDIFOLIA)

Title presented at workshop:

BEECH BARK DISEASE RESISTANCE BREEDING PROGRAM IN AMERICAN BEECH

David W. Carey1, Marjorie Allmaras2, Paul Bloese3, David Burke4, Paul Berrang5, Lindsay Dalton6, 

Roger Gettig4, Tom Hall7, Andrea Hille8, Jeff D. Kochenderfer9, Scott Lint10, Mary Mason11, 

James McKenna12, Scott Rogers13, Jill Rose14, Carol Young15, 

Keith Konen16, and Jennifer L. Koch1

American beech is typically not a desired timber species, but it is among the most ecologically important 

trees of the Eastern United States. Beech nuts, a common component of mature forests, provide an 

important source of food for many birds and mammals. Beech bark disease (BBD) has been killing 

American beech trees since the late 1890’s, but it is only over the last 15 years that significant progress 

has been made in developing techniques to identify, screen, propagate and breed resistant beech trees. 

Beech bark disease is caused when feeding of the invasive beech scale insect (Cryptococcus fagisuga) 

creates cracks in the bark, providing entry points for spores of Neonectria species of fungi. Although it is 

the growth of the fungus that weakens and may kill the tree, beech trees that remain disease-free despite 

long-term exposure to BBD have been shown to have heritable resistance to the scale insect. Genetic 

studies have shown that when two resistant parents are bred, about 50 percent of the progeny will be 

resistant. Working collaboratively with State and national forest managers, we have taken a participatory 

breeding approach to develop regional seed orchards of genetically diverse, grafted, BBD-resistant 

genotypes. Workshops are conducted to provide partner guidelines for the identification of resistant beech 

trees, training on the use of scale eggs to artificially inoculate candidate trees to confirm resistance, 

and instructions on the collection of beechnuts (to be used as root stock) and scion. After identifying 

and confirming scale-resistant trees, partners ship scion to U.S. Forest Service researchers in Ohio, or 

Region 9 personnel in Wisconsin, for grafting. A total of nine grafted regional beech seed orchards have 

been established or are underway in seven different States. Prior to seed orchards becoming productive, 

grafted containerized seed orchards are being used to accelerate seed production for genetic studies and 

restoration plantings.

1Northern Research Station, USDA Forest Service, Delaware OH 43015.

2 Hiawatha National Forest, USDA Forest Service, St. Ignace, MI 49781.

3Department of Forestry, Michigan State University, East Lansing, MI 48824.

4The Holden Arboretum, Kirtland, OH 44094.

5Region 9, USDA Forest Service, Milwaukee, WI 53202.

6 Green Mountain and Finger Lakes National Forests, USDA Forest Service, Rochester, VT 05767.

7Bureau of Forestry, Commonwealth of Pennsylvania Department of Conservation and Natural Resources, Harrisburg, PA 17105.

8 Allegheny National Forest, USDA Forest Service, Warren, PA 16365.

9 Monangahela National Forest, USDA Forest Service, Parsons, WV 26287.

10 Michigan Department of Natural Resources, Cadillac, MI 49601. 11 Department of Ecology, Evolution and Organismal Biology, The Ohio State University, Delaware, OH 43015. 12 Northern Research Station, USDA Forest Service, West Lafayette, IN 47907. 13 Oconto River Seed Orchard, USDA Forest Service, White Lake, WI 54491. 14West Virginia Department of Agriculture, Charleston, WV 25312.

15Huron-Manistee National Forest, USDA Forest Service, Cadillac, MI 49601. 16 Huron-Manistee National Forest, USDA Forest Service, Manistee, MI 49660, (jkoch@fs.fed.us).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 2: Genus Fraxinus—Threatened by Emerald Ash Borer and Ash Dieback Disease 11

Session

EMERALD ASH BORER AND ASH DIEBACK DISEASE

GENUS FRAXINUS—THREATENED BY 

2
12 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

PHOENIX FROM THE ASHES: EXPLOITING HOST RESISTANCE TO 

CONSERVE AND RESTORE POPULATIONS OF EUROPEAN ASH

Michelle Cleary1, Lars-Göran Stener2, Pierluigi Bonello3, and Caterina Villari4

The invasive ascomycete fungus (Hymenoscyphus fraxineus) has caused a serious, steady population 

decline of common ash (Fraxinus excelsior) across Europe. In Sweden, ash is an important species among 

the noble broad-leaved trees available to forest management. Since 2010, ash is a Red-listed species and 

its status has worsened during recent years to become critically endangered. This is concerning not only 

for this economically and culturally important species but also for its associated biodiversity given its 

role as a keystone species for natural communities throughout Europe. Several studies have shown large 

genotypic variation in the level of disease susceptibility among different individuals, and that a small 

proportion (1–5 percent) of the natural population of F. excelsior exhibits disease tolerance. Here we 

report on several activities aimed at selecting and testing F. excelsior genotypes including: (1) broad-scale 

mapping, identifying and selecting more than 500 disease-tolerant F. excelsior with respect to ash dieback 

damage in wild populations for further monitoring and testing, (2) screening resistance in F. excelsior

genotypes in clonal trials, and (3) early results of progeny testing from known susceptible and tolerant 

families. In addition, phenomics technologies show great promise to advance breeding efforts. Fouriertransform Infrared (FT-IR) spectroscopy coupled with chemometric model was able to successfully 

discriminate between resistant and susceptible ash genotypes. Collectively, these activities and the 

implementation of rapid phenotyping technologies in practice will help support the development of a more 

resistant population of F. excelsior restoration activities aimed at repopulating Swedish forests, cities, and 

landscapes.

1Southern Swedish Forest Research Centre, Swedish University of Agricultural Sciences, Alnarp, Sweden, (Michelle.Cleary@slu.se). 2 Skogforsk—The Forest Research Institute, Ekebo, Sweden.

3Department of Plant Pathology, Ohio State University, Columbus, OH 43210.

4Warnell School of Forestry & Natural Resources, University of Georgia, Athens, GA 30602.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 2: Genus Fraxinus—Threatened by Emerald Ash Borer and Ash Dieback Disease 13

RESTORING GREEN ASH (FRAXINUS PENNSYLVANICA): BREEDING FOR 

RESISTANCE TO THE EMERALD ASH BORER (AGRILUS PLANIPENNIS)

Jennifer L. Koch1, David W. Carey1, Mary E. Mason2, Therese M. Poland3, Kathleen S. Knight1, 

Jeanne Romero-Severson4, Charles Tubesing5, and Roger Gettig5

The invasion of emerald ash borer (EAB) (Agrilus planipennis) threatens the survival of green ash 

(Fraxinus pennsylvanica) in the United States, where it is extensively used for soil conservation, rural 

water management, and in urban green spaces. It is also one of the most common hardwood species in 

riparian forests of eastern North America. Long term monitoring of permanent plots in EAB infested 

natural forests identified surviving green ash trees, or “lingering” ash trees, that had maintained 

healthy canopies for at least 2 years after all other ash trees (>10 cm DBH) had died. EAB egg bioassay 

experiments confirmed that these trees possess an increased level of resistance due to multiple types 

of host defense responses, including mortality of early instar larvae, development of larvae having 

significantly lower weights, and reduced adult feeding preference of foliage. Fifty-five lingering green ash 

trees have been accessioned and a replicated clone test has been established to assess field performance of 

42 of these. We are employing a polycross breeding strategy and to date, 16 full-sibling families resulting 

from crosses between 8 mother trees and 15 different father trees have been produced. Additional families 

will be added as select parent trees begin to flower. Bioassay evaluation of seedling progeny from 

7 different lingering x lingering families, 2 susceptible x susceptible families, 1 susceptible x lingering 

family and 1 susceptible open-pollinated family demonstrated variation both within and between families. 

Between 15 to 40 percent of lingering x lingering progeny had a more effective defensive response to 

EAB than either parent, a result expected if the two parents employ different mechanisms based on 

allelic variants at more than one gene. Polycross progeny are being further evaluated in field trials that 

also include progeny from susceptible x susceptible and susceptible x lingering families as reference 

populations to allow for analysis of genetic gain.

1Northern Research Station, USDA Forest Service, Delaware, OH 43015, (jennifer.koch@usda.gov). 2 Department of Evolution, Ecology and Organismal Biology, The Ohio State University, Columbus, OH 43210.

3Northern Research Station, USDA Forest Service, East Lansing, MI 48910.

4 Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556.

5The Holden Arboretum, Kirtland, OH 44094.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
14 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

EVALUATION OF RESISTANCE OF ASIAN AND EUROPEAN ASH SPECIES AND 

CULTIVARS TO EMERALD ASH BORER (AGRILUS PLANIPENNIS)

Frederic Miller1, Erin McMahan1, and Kim Shearer1

Since its discovery in Michigan in 2002, the emerald ash borer (EAB) (Agrilus planipennis Fairmaire) 

has been a devastating exotic invasive pest of North American ash trees (Fraxinus spp.), killing millions 

of urban and rural ash trees. Many communities have lost or risk losing many valuable ash trees, and as 

part of reforestation efforts, it is imperative that new Fraxinus taxa less susceptible to EAB be developed. 

To identify possible Fraxinus taxa less susceptible to EAB, we performed a series of no-choice (NC) 

feeding bioassays with 24 different species and cultivars of Asian and European ash between 2009 and 

2017. Ash studies included laboratory NC adult leaf feeding studies, and laboratory and field larval 

colonization studies. The Asian ash taxa F. chinensis, F. chinensis var. rhynchophylla, F. mandschurica, 

and F. mandschurica var. japonica were the least preferred in the NC leaf feeding studies, with similar 

results found in the larval colonization studies. Of the European ash taxa tested, F. angustifolia, 

F. excelsior, F. pallisae, and F. longicuspus were the least preferred by adult EABs. Field evaluation of 

these same Asian and European taxa, exposed to a natural EAB population in an arboretum tree breeding 

nursery, was consistent with the aforementioned studies. In addition, since elm (Ulmus) is mentioned in 

the literature as a potential EAB host, we conducted adult NC leaf-feeding and larval colonization studies 

to determine the relative susceptibility and preference for elm taxa by EAB. Field and laboratory studies 

revealed that none of the 11 elm taxa tested were susceptible to feeding by adult EABs and were not 

colonized by larvae. To date, overall, six Asian and three European species appear to be less suitable and/

or preferred for adult feeding and larval colonization by EAB and may have potential for use in future ash 

breeding programs.

1Morton Arboretum, Lisle, IL 60532, (fmiller@jjc.edu).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 2: Genus Fraxinus—Threatened by Emerald Ash Borer and Ash Dieback Disease 15

THE PAST IS PROLOGUE: UNDERSTANDING THE CURRENT STATE OF 

TREE IMPROVEMENT IN THE UNITED STATES

Kim Steiner1

Forest genetics research and related tree improvement R & D in the United States has followed a long arc of 

interest and investment since pioneering efforts by the USDA Forest Service in the first decades of the last 

century. The scope and intensity of this work exploded between 1950 and 1985 as public agencies, private 

industry, and universities shifted resources to capitalize on optimism about the application of genetics to 

forestry and forest productivity. In the latter part of this period, there was a genetics or breeding specialist 

in every Forest Service experiment station, every National Forest Region, many individual National 

Forests, about half of all State forestry agencies, most large timber companies, and almost every forestry 

school. As the century ended, however, there followed a period of rapid, almost precipitous retrenchment 

in tree improvement work due to a “perfect storm” of circumstances that are described in the presentation. 

Forest tree improvement has contracted to natural core of work focused on productivity gains in species 

that are planted on a large scale. As a research discipline, forest genetics survives in a healthy, if somewhat 

diminished state, but the focus has shifted to lab-based genomic studies, and the scientists are often 

disengaged from real-world problems. Unfortunately, institutional disengagement from tree improvement 

has left a lingering reluctance to invest in long-term projects that may be essential for the protection 

and rescue of species threatened by invasive diseases or insects. Permanent solutions to some of these 

problems will require long-term breeding programs coupled with silvicultural opportunities to implement 

gains. 

1 Pennsylvania State University, University Park, PA 16802, (kcs@psu.edu).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
16 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

VARIATION AND GENOMIC BASIS OF FRAXINUS EXCELSIOR (COMMON ASH) 

SUSCEPTIBILITY TO HYMENOSCYPHUS FRAXINEUS

(ASH DIEBACK) THROUGHOUT BRITAIN

Jonathan J. Stocks1, Steve J. Lee2, and Richard Buggs3

Ash dieback (ADB), caused by Hymenoscyphus fraxineus, has severely damaged a large proportion of ash 

trees (Fraxinus excelsior) in continental Europe. In Britain, the disease was found only 6 years ago in the 

southeast, and is still spreading. A large-scale screening trial to evaluate ADB damage to provenances of 

F. excelsior sourced from throughout the British Isles was planted in 2013 in the southeast of England. 

Trees were scored in 2015 and 2016 for their level of ADB damage observed in field. Significant 

differences were found in average ADB damage among planting sites and seed source provenances 

in 2016. All provenances contained some healthy trees, so a breeding program to produce genetically 

variable native ash tree populations with lower ADB susceptibility may be feasible. In 2015, we undertook 

a pilot project, using Restriction Site Associated DNA sequencing (RADseq) on 95 samples, to search 

for alleles associated with low susceptibility to ash dieback. We are now sequencing whole genomes from 

1,400 individuals sampled across all provenances: 700 high-susceptibility and 700 low-susceptibility 

trees. Of these 1,250 are placed in 31 pools with 80X coverage per pool, and 150 are being sequenced as 

individuals with 20X coverage each. This provides data for a genome wide association study, searching for 

loci associated with low susceptibility, and genomic prediction of trees with low susceptibility.

1School of Biological and Chemical Sciences, Queen Mary University of London, London, E1 4NS, United Kingdom and Royal Botanic 

Gardens, Kew, Richmond, Surrey, TW9 3AE, United Kingdom, (j.j.stocks@qmul.ac.uk). 2 Forest Research, Northern Research Station, Roslin Midlothian, EH25 9SY, United Kingdom.

3 School of Biological and Chemical Sciences, Queen Mary University of London, London, E1 4NS, United Kingdom and Royal Botanic 

Gardens, Kew, Richmond, Surrey, TW9 3AE, United Kingdom, (r.buggs@kew.org).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 3: Breeding for Resistance to Pathogens 17

Session

BREEDING FOR RESISTANCE TO PATHOGENS

3
18 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

PROGRESS OF AN APPLIED DISEASE SCREENING AND SELECTION PROGRAM 

FOR RESISTANCE TO VASCULAR WILT IN HAWAIIAN ACACIA KOA

Nick Dudley1, Tyler Jones1, Aileen Yeh1, Richard Sniezko2, and Phil Cannon3

Koa (Acacia koa A.Gray) is a valuable tree species economically, ecologically, and culturally in Hawaii. 

A vascular wilt disease of koa caused by the fungal pathogen Fusarium oxysporum f. sp. koae (FOXY) 

causes high rates of mortality in field plantings and threatens native koa forests in Hawaii. The Hawaii 

Agriculture Research Center (HARC), with both public and private partners, operates a tree improvement 

program to develop koa wilt resistant populations in Hawaii. This applied program was started in 2003 

and has made progress including the establishment of first generation seed orchards, delineation of 

11 provisional seed zones, and release of the first seed with confirmed levels of genetic resistance for 

reforestation and restoration. Data from seedling inoculation trials and the first field trials suggest 

survival on infected sites may be expected to exceed 60 percent in planting using seed from the best 

parents compared to 30 percent or less survival in unimproved seedlings. One clonal and six seedling field 

trials were established between 2012–2016 using selections based on the inoculation trials. The screening 

method serves as a powerful tool to rapidly evaluate koa families prior to outplanting. Additional 

seedlings screening is on-going to identify parent trees for seed zones not yet established. This rapidly 

developing resistance program will need to continue the monitoring of field trials to further evaluate the 

durability of resistance.

1 Hawaii Agriculture Research Center, Kunia, HI 96759, (ndudley@harc-hspa.com). 2 Dorena Genetic Resource Center, USDA Forest Service, Cottage Grove, OR 97424.

3 Forest Health Protection, USDA Forest Service, Vallejo, CA 94592.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 3: Breeding for Resistance to Pathogens 19

THE DEVELOPMENT OF A RESISTANCE SCREENING PROGRAM OF OHIA 

TO CERATOCYSTIS PATHOGENS CAUSING WIDESPREAD 

MORTALITY ON HAWAII ISLAND

Marc Hughes1, Blaine Luiz2, Lisa Keith3, and Christian Giardina4

Ohia (Metrosideros polymorpha) is Hawaii’s most abundant and ecologically important native tree. 

Present on all major Hawaiian Islands, ohia trees account for roughly 50 percent of the stems and 

50 percent of total basal area of all forests (native or non-native) across the Hawaii Island. In 2014, high 

and expanding levels of ohia mortality were observed in the Puna district on the east side of Hawaii 

Island, spurring investigations to its cause. Pathological investigations revealed a complex of two 

xylem-affecting diseases caused by two novel fungal species, Ceratocystis lukuohia and C. huliohia. 

The mortality caused by these diseases, called Rapid Ohia Death (ROD), has quickly spread to most 

districts of the island affecting over 54 600 ha of forests. ROD is a primary concern to the health of ohia 

populations, forests, and the vast assemblages of organisms that rely on this keystone tree. To combat the 

expanding mortality associated with ROD, a resistance screening program is in development to screen the 

various varieties and ecotypes of ohia from Hawaii. Initial screening results of M. polymorpha varieties 

(glaberrima, incana, newellii, and polymorpha) found on the Hawaii Island show promise of differential 

susceptibility and the potential for host resistance. Selection will involve collection of seed and vegetative 

material for propagation and later screened for resistance by inoculation trials. In addition to targeting 

surviving M. polymorpha individuals both within and adjacent to sites of intense ROD mortality, we will 

also screen a diverse collection of M. polymorpha genotypes from a State-wide seed drive as well as four 

other Metrosideros species from Hawaii. 

1College of Tropical Agriculture and Human Resources, University of Hawaii Mānoa, Hilo, HI 96720, (mhughes7@hawaii.edu). 2College of Tropical Agriculture and Human Resources, University of Hawaii Mānoa, Hilo, HI 96720.

3 Pacific Basin Agricultural Research Center, USDA Agricultural Research Service, Hilo, HI 96720.

4 Institute of Pacific Island Forestry, USDA Forest Service, Hilo, HI 96720.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
20 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

SCREENING RANGE-WIDE BLACK WALNUT SEED FAMILIES FOR THOUSAND CANKERS 

DISEASE (TCD) RESISTANCE AND MEASURING THE REGIONAL 

ADAPTABILITY OF RESISTANT SEED SOURCES

James R. McKenna1, Racheal A. Sitz2, Ned Tisserat3, Don Carlson3, and Paul Bloese4

Black walnut (Juglans nigra), an economically 

important native species in the Eastern United 

States is threatened by a new disease thousand 

cankers disease (TCD) caused by a fungus 

(Geosmithia morbida) vectored by a bark beetle 

(Pityophthorus juglandis). Both organisms 

originate in the Southwestern United States but 

have spread to become endemic throughout the 

Western United States and the disease was first 

found in the eastern native range in 2010. We 

challenged a range-wide collection of native black 

walnut (Juglans nigra) families, collected in 

2012, to artificial stem inoculation of Geosmithi 

morbida (Gm) to determine the variation among 

regions and families of black walnut, to test the 

regional adaptability of any sources of resistance 

and to capture resistant material for future 

breeding and new seed orchards. In addition, we 

included a number of our select timber families 

from Indiana as an improved check-lot.

Up to 9 seedlings per family were shipped to 

Colorado State University (CSU) and planted 

in three complete blocks in the field. Families 

with more than 9 seedlings were planted into 

the common garden (CG) plots in Arkansas, 

Indiana, and Michigan. Each plot was planted 

on a 2.2 meter grid with 24 mixed families per 

provenance per block and 12 seedlings of three 

oak species (bur, northern red, and cherry bark) 

per block (36 trees total). Each plot was fenced to 

exclude deer and weeds were controlled for the 

first 2 years with herbicides and cultivation. 

Trees were inoculated with two Gm strains first 

in 2014 the summer after planting, and then again 

in the summer of 2016 at CSU. Plugs of two Gm

isolates and a sterile control were placed into live 

stems with a 6-mm cork-borer and the branches 

cut 90 days later and stored in a cooler until we 

could scrape off the outer bark and measure the 

length and width of cankers. The first year screen 

at CSU showed Gm isolate significantly affected 

canker size, but provenance did not. In the repeat 

screen of 2016, stem diameter significantly 

affected canker size as did family and provenance. 

Breeding values were used to estimate the percent 

difference among provenances in canker size 

relative to all trees in the study and we found 

western provenances had from -5.3 to -5.7 percent 

smaller Gm cankers while the eastern provenances 

had +5.5 percent to +5.8 percent larger Gm

cankers. The center provenance had cankers 

midway between both western and eastern 

provenances (+0.3 percent). The improved walnut 

families, selected for increased growth and timber 

quality, had the most variation and as a group 

produced the largest cankers +6.8 percent.

First year survival was poorest for both the SW 

provenance and the Arkansas CG plot. HTI 

families survived the best in all 3 CG plots and 

there was no significant plot × provenance effect. 

Three year height growth of the provenances in 

the CG plots shows that the selected HTI walnut 

outgrew all wild provenances in Indiana, growing 

nearly 70 percent taller than wild CTR families. 

1Northern Research Station, USDA Forest Service, West Lafayette, IN 53726, (james.mckenna@usda.gov). 2 Department of Plant Pathology, Colorado State University, Center, CO 81125.

3Department of Forestry and Natural Resources, Purdue University, West Lafayette, IN 47907-2061.

4 Department of Forestry, Michigan State University, East Lansing, MI 48824-6402.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
21

We had difficulties keeping deer out of the 

MI CG plot but were successful at eliminating 

deer browse in AR and IN. Thus, meaningful 

growth differences among the CG plots is not 

possible yet. 

Previous studies have demonstrated variation 

among black walnut genotypes by this type 

of screening which suggests that black walnut 

has co-evolved with Gm and hence variation in 

resistance exists. Our results suggest that western 

black walnut possesses more resistance than 

eastern black walnut. Ranking families by State 

show NC, PA, and TN families produced the 

largest Gm cankers and those States were the first 

three where TCD was found in the East after 2010. 

Whether or not this is a coincidence will require 

further study. Finally, black walnut selected for 

faster growth may unwittingly reduce resistance 

to TCD and should be addressed by improvement 

programs.

Session 3: Breeding for Resistance to Pathogens
22 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

THE SEARCH FOR RESISTANCE IN LODGEPOLE PINE TO COMANDRA BLISTER RUST 

IN THE CENTRAL INTERIOR OF BRITISH COLUMBIA

Richard W. Reich1

Comandra blister rust (Cronartium comandrae Peck) although indigenous to North America may result in 

severe epidemic levels of infection on lodgepole pine (Pinus contorta var. latifolia Engelm. ex. S. Watson). 

Proximity to the alternate host plant, bastard toadflax [Geocaulon lividum (Richardson) Fern] may act as 

a significant confounding factor in the evaluation of resistance in field trials. Incidence and severity of 

comandra was assessed at age 15 in a 130 seedlot (family) progeny trial (referred to as the Chief Lake site) 

designed to identify families with superior growth and wood quality traits within the tree improvement 

program in British Columbia, Canada. The distribution of the alternate host was mapped and individual 

plant stems counted to evaluate the degree of local infection gradients. The incidence and severity of 

other pine stem rust pathogens and Elytroderma foliage and shoot disease [Elytroderma deformans

(Weir) Darker] was assessed to evaluate the degree of cross resistance to other pathogens. The cumulative 

incidence of comandra was 52 percent, with cumulative family incidence ranging between 2 and 

95 percent. The cumulative severity of infection was 2.37 infections per tree, and the mean family severity 

ranged between 0.02 and 11.95 infections per tree (a difference of over 500 times between least susceptible 

and most susceptible family). The results of the Chief Lake site was contrasted with a previously reported 

upon assessment of a different 130 seedlot (family) comandra screening trial, which was replicated on 

three sites. Although the previous study showed that the majority of families in that trial appeared to be 

highly susceptible, the set of families tested at the Chief Lake site appeared to contain a considerably 

higher proportion of families with low susceptibility. As a result, it may now be possible to identify a 

sufficient number of families from this subsequent trial to establish a comandra resistant orchard.

1College of New Caledonia, Prince George, British Columbia, Canada. V2N 1P8, (reichr1@cnc.bc.ca).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 3: Breeding for Resistance to Pathogens 23

GENETIC RESISTANCE TO PHYTOPHTHORA LATERALIS IN 

PORT-ORFORD-CEDAR (CHAMAECYAPRIS LAWSONIANA)

Richard A. Sniezko1, Douglas P. Savin1, and Paul Reeser2

Native forest ecosystems as well as reforestation and restoration efforts in forest trees can be severely 

impacted by pathogens and insects, especially non-native ones. In many of these cases, the future viability 

and utility of a species may depend heavily upon efforts to harness any naturally occurring genetic 

resistance. Port-Orford-cedar (POC), Chamaecyparis lawsoniana, a long-lived conifer native to northwest 

California and southwest Oregon, and also used horticulturally internationally, is highly susceptible to a 

non-native root disease, caused by the oomycete pathogen Phytophthora lateralis. An applied program 

to develop populations of POC with genetic resistance has identified both qualitative and quantitative 

resistance based on seedling inoculation trials. Families from some parent trees show high survival, 

consistent with the expectation of resistance from a single major dominant gene. Seedlings from families 

with quantitative resistance show a differential rate of mortality within a family over 3 years. The level of 

quantitative resistance varies by family with some families reaching 100 percent mortality by the end of 

3 years, while other families still have low to moderate survival. Parents (or their seedling progeny) with 

each type of resistance are being incorporated into containerized seed orchards for each breeding zone, 

and breeding to increase the level of quantitative resistance is underway. Early data from field trials is 

encouraging, and restoration and reforestation with resistant seedlings is underway. The applied resistance 

program in POC can offer some guidance to other programs contemplating the development of resistant 

populations to help maintain viable populations of forest tree species affected by non-native pathogens or 

insects.

The full paper was published in Plants, People, Planet

https://nph.onlinelibrary.wiley.com/toc/25722611/2020/2/1

The reference for the published paper is:

Sniezko, R.A.; Johnson, J.S.; Reeser, P.; Kegley, A.; Hansen, E.M.; Sutton, W.; Savin, D.P. 2020. Genetic resistance to 

Phytophthora lateralis in Port‐Orford‐cedar (Chamaecyparis lawsoniana) – Basic building blocks for a resistance 

program. Plants, People, Planet. 2(1): 69–83. https://doi.org/10.1002/ppp3.10081.

1 Dorena Genetic Resource Center, USDA Forest Service, Cottage Grove, OR 97424, (richard.sniezko@usda.gov). 2 Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
24 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

GENETIC DIVERSITY IN THE LAUREL WILT PATHOGEN, RAFFAELEA LAURICOLA, 

AND THE CONSEQUENCES FOR RESISTANCE BREEDING

Tyler J. Dreaden1, Marc A. Hughes2, Randy C. Ploetz3, Adam Black4, and Jason A. Smith5

Laurel wilt is caused by Raffaelea lauricola, a fungal nutritional symbiont of its vector, Xyleborus 

glabratus, the redbay ambrosia beetle. Both the fungus and beetle are native to Asia but were found in 

Georgia in the early 2000s. The disease has spread to much of the Southeastern United States killing 

> 300 million host trees in the Lauraceae family. The objectives of this research were to elucidate 

the genetic structure of populations of R. lauricola, examine its reproductive strategy, and determine 

the number of USA introductions. A panel of 12 simple sequence repeat (SSR) markers identified 

15 multilocus genotypes (MLGs) in a collection of 59 isolates from the USA (34 isolates), Myanmar (18), 

Taiwan (6) and Japan (1). Limited diversity in the USA isolates and the presence of one MAT idiotype 

(mating type locus) indicated that R. lauricola was probably introduced into the country once. MLG 

diversity was far greater in Asia than the USA. Only three closely related MLGs were detected in the 

USA, the most prevalent of which (30 of 34 isolates) was also found in Taiwan. Although more work is 

needed, the present results suggest that a Taiwanese origin is possible for the population of R. lauricola

in the USA. Isolates of R. lauricola from Myanmar were distinct from those from Japan, Taiwan and the 

USA. Although both MAT idiotypes were present in Myanmar and Taiwan, only the population from 

Taiwan had the genetic structure of a sexually reproducing population. The results highlight the need 

to prevent the introduction of additional genotypes and the second mating type into the USA because 

this could allow the pathogen to rapidly overcome the resistance that has been developed. The pathogen 

population needs to be monitored so that new genotypes can be identified and incorporated into resistance 

screening trials if they are found.

1 Forest Health Research and Education Center, Southern Research Station, USDA Forest Service, Lexington, KY 40546, 

(tdreaden@usda.gov). 2College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, HI 96720.

3Tropical Research & Education Center, University of Florida, Homestead, FL 33031.

4 Peckerwood Garden Conservation Foundation, Hempstead, TX 77445.

5 School of Forest Resources and Conservation, University of Florida, Gainesville, FL 32611.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 3: Breeding for Resistance to Pathogens 25

FUSIFORM RUST RESISTANCE IN AN ELITE POPULATION 

OF LOBLOLLY PINE POPULATION

April Meeks1, Steve McKeand1, Ross Whetten1, and Fikret Isik1

Fusiform rust (caused by the fungus Cronartium 

quercuum f. sp. fusiforme) is the most critical 

disease affecting the health and productivity 

of loblolly pine (Pinus taeda L.), the most 

commercially important pine in the Southeastern 

United States. An Atlantic Coastal Elite (ACE) 

breeding population was developed in the N.C. 

State University Cooperative Tree Improvement 

Program to assess short-term genetic gain for 

the Coastal regions of the Southeast. Twentyfour elite Atlantic Coastal parents were mated 

to produce 76 crosses and were screened at the 

U.S. Forest Service Resistance Screening Center 

(RSC) in Asheville, NC. About 9,775 progeny of 

the 76 crosses were challenged with a broad-based 

inoculum from across the expected deployment 

region. A spore density of 50,000 spores per 

milliliter was used, and seedlings were assessed 

for gall presence or absence after 6 months. The 

overall rust incidence was 0.48, and full-sibling 

family means ranged from 0.11 to 0.83. The 

narrow-sense heritability of half-sibling family 

means was 0.95 (± 0.003), suggesting strong 

genetic differences among families. Broad-sense 

heritability of full-sibling family means was 

0.93 (± 0.03).

Based on the 6-month results, all seedlings 

with rust galls as well as some entire fullsibling families were discarded based on the 

expectation they would also be susceptible in 

the field. The remaining 2,362 seedling progeny 

of the 51 crosses (~45 per family) were clonally 

propagated via rooted cuttings, and ramets of 

the clones were planted across eight test sites. 

A half-sibling family that was not tested for rust 

resistance at the RSC was included in the test 

design as a checklot, and fusiform rust disease 

incidence was recorded for these seedlings in 

the field tests. Incidence of fusiform rust galls, 

survival, height, diameter at breast height, 

straightness, forking and ramicorn branching 

were all assessed at ages four and six. At age 

6 years, the overall rust incidence was 0.06. The 

checklot family had 0.19 rust incidence both at 

ages four and six. Two sites in Colleton county 

South Carolina had 56 percent and 71 percent rust 

incidence in the non-screened checklot compared 

to 0.11 and 0.15 in the screened rooted cuttings. 

These results indicate screening at the RSC with 

subsequent field-testing of survivors was effective.

1North Carolina State University, Raleigh, NC 27695, (allail@ncsu.edu).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
26 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

GENETIC PARAMETER ESTIMATES FOR FUSIFORM RUST DISEASE FROM A 

MULTI-ENVIRONMENT TRIAL OF LOBLOLLY PINE (PINUS TAEDA L.)

Eddie Lauer1, Steve McKeand1, and Fikret Isik1

Fusiform rust, caused by the fungus Cronartium quercuum f. sp. fusiforme, is an important disease of 

loblolly pine (Pinus taeda) plantations in the Southern United States, causing multimillion dollar losses to 

landowners. In controlled seedling inoculation trials, fusiform rust resistance segregates within families 

like a traditional Mendelian gene. However, at the population level in the field, rust resistance appears 

more quantitative in nature. This is due to the presence of multiple R genes segregating among different 

families, conferring resistance to different races of the pathogen. In this study, a dataset of 11,987 trees 

from 68 half-sib families of loblolly pine tested across 10 environments was analyzed to understand the 

inheritance and patterns of GxE interactions for fusiform rust resistance. Heritability of family means 

was 0.91, indicating that a substantial amount of phenotypic variation in the field can be explained by 

genetic differences between families. The type B genetic correlation was 0.77, indicating a small amount 

of GxE. When the rust resistance breeding values for each family within each environment were ranked, 

the most resistant and most susceptible genotypes tended to have the same rank across environments and 

families with moderate levels of resistance showed frequent changes in rank. The results of this study 

indicate that substantial gain can be made through traditional family selection by exploiting the additive 

genetic variance component.

1North Carolina State University, Raleigh, NC 27695, (elauer@ncsu.edu).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 3: Breeding for Resistance to Pathogens 27

USDA ADVANCES IN PLANT BREEDING AND INNOVATION: RESISTANCE BREEDING, 

GERMPLASM COLLECTIONS, AND GENETIC DIVERSITY

Sarah Federman1 and Paul Zankowski1

The Office of the Chief Scientist (OCS) assists in providing strategic coordination of the science that 

informs Departmental policies by facilitating discourse and the identification of areas of synergy. Major 

focal areas in OCS include plant health, production and plant products; and agricultural systems and 

technology. This talk presents a series of vignettes on plant breeding and innovation throughout the 

Department, with a focus on breeding for diversity and resilience; resistance breeding programs; the 

national plant germplasm database; and finally, examples of recent plant breeding coordination work at 

OCS to introduce the office as a resource and partner for facilitating the increased visibility of ongoing 

research and initiatives.

The full paper was published in Plants, People, Planet

https://nph.onlinelibrary.wiley.com/doi/10.1002/ppp3.10075

The reference for the published paper is:

Federman, S.; Zankowski, P. 2020. Strategic science planning for responsible stewardship and plant protection at the 

U.S. Department of Agriculture. Plants, People, Planet. 2: 53–56. https://doi.org/10.1002/ppp3.10075.

1USDA Office of the Chief Scientist, Washington, DC 20250, (sarah.federman@osec.usda.gov).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.

29

Session

GENOMIC AND METABOLOMIC APPROACHES

TO IDENTIFY RESISTANCE GENES 4

Session 4: Genomic & Metabolomic Approaches to Identify Resistance Genes
30 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

GENOMICS OF FRAXINUS (OLEACEAE): A GENUS UNDER SEVERE THREAT

Laura J. Kelly1, Steve Lee2, Jennifer Koch3, Stephen J. Rossiter1, and Richard J.A. Buggs1,4

Fraxinus (Oleaceae; olive family) is a genus of c. 48 tree and shrub species, including taxa of ecological 

and economic importance. Fraxinus species face severe threats from an invasive beetle species, emerald 

ash borer (EAB) in North America and a disease caused by an invasive fungal pathogen, ash dieback 

(ADB) in Europe. Susceptibility to these threats varies among ash species, and seems to be low in at 

least some Asiatic species. We have therefore sequenced and assembled whole genomes of 28 species 

or sub-species of Fraxinus (http://www.ashgenome.org/worldwide). We are taking a new approach for 

identifying genes conferring resistance to threats, based on detecting evidence of convergent molecular 

evolution among host species’ genomes. We are currently analyzing patterns of gene evolution between 

species highly susceptible to EAB (F. americana, F. latifolia, F. pennsylvanica, F. ornus and F. velutina) 

and species with low susceptibility (F. baroniana, F. chinensis, F. floribunda, F. mandshurica, and 

F. platyploda). Genes showing patterns of convergent sequence similarity within low susceptibility 

species are identified as candidate genes roles in susceptibility. We will soon extend these studies to 

further species and to the analysis of ADB susceptibility.

1School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, United Kingdom.

2 Forest Research, Northern Research Station, Roslin, Midlothian, EH25 9SY, United Kingdom.

3Northern Research Station, USDA Forest Service, Delaware, OH 43015.

4 Jodrell Laboratory, RBG Kew, Richmond, TW9 3DS, United Kingdom, (r.buggs@kew.org).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 4: Genomic & Metabolomic Approaches to Identify Resistance Genes 31

EXPRESSION PROFILING OF CANDIDATE GENES OF RESISTANCE TO 

PHYTOPHTHORA CINNAMOMI DETERMINED IN DIFERENTE 

GENOTYPES OF CASTANEA SPP

Carmen Santos1,2, Sofia Duarte1, Sara Tedesco1, Pedro Fevereiro2,3, and Rita L. Costa,1,4

Phytophthora cinnamomi is a hemibiotrophic that causes root rot, also known as ink disease. Little 

information has been acquired in chestnut on the molecular defense strategies against this pathogen. The 

expression of eight candidate genes potentially involved in the defense to P. cinnamomi was quantified by 

digital PCR in Castanea genotypes showing different susceptibility to the pathogen. Seven of the eight 

candidate genes displayed differentially expressed levels depending on genotype and time-point after 

inoculation. Cast_Gnk2-like revealed to be the most expressed gene across all experiments and the one 

that best discriminates between susceptible and resistant genotypes. Our data suggest that the pre-formed 

defenses are crucial for the resistance of Castanea crenata to P. cinnamomi. A lower and delayed 

expression of the eight studied genes was found in the susceptible Castanea sativa, which may be related 

with the establishment and spread of the disease in this species. A working model integrating the obtained 

results is presented.

INTRODUCTION

The aim of this study was to evaluate the early 

expression of candidate resistance genes to 

P. cinnamomi infection (0, 24, and 48 h) in 

C. sativa and a C. crenata, as well as in four 

hybrids (three C. sativa × C. crenata genotypes 

and a C. sativa × C. mollissima) with different 

susceptiblities to P. cinnamomi, produced from 

the breeding program on course, for resistance 

to P. cinnamomi, the causal agent of root rot, 

to understanding of the molecular mechanisms 

underlying the resistance that Asian species 

(C. crenata and C. mollissima) show to this 

pathogen. Among the different methods available 

to quantify gene expression in plants, digital PCR 

(dPCR) is emerging as an absolute quantification 

method with high precision, sensitivity and 

specificity (Majumdar et al. 2015). This new 

technology has been mainly used for biomedicine 

research, however, some studies in plant science 

using dPCR have also been recently released 

(Bahder et al. 2016; Ge et al. 2016; Stevanato and 

Biscarini 2016).

MATERIAL AND METHODS

Six chestnut genotypes showing different levels 

of resistance after inoculation with pathogen were 

used in this work. C. crenata (resistant), C. sativa

(susceptible) and four hybrid genotypes, selected 

from the on-going chestnut breeding program 

(Costa et al. 2011): three C. sativa × C. crenata

hybrids (SC55 – resistant, SC914 – intermediate, 

and SC903 – susceptible) and one C. sativa × 

C. mollissima hybrid (SM904), selected as a 

resistance control. All plant material used in 

this study was multiplied by micropropagation. 

P. cinnamomi root inoculation was performed 80 

days after plant acclimatization, under controlled 

conditions according to Santos et al. (2015). 

Genes were selected from the 283 C. crenata

differentially expressed genes (DEGs), previously 

identified by Serrazina et al. (2015). In this study, 

gene selection parameters were: (1) DEGs with the 

log2 of the ratio between C. crenata inoculated 

(Cci) and non-inoculated (Ccn) reads higher than 

1.5(Log2Cci/Ccn >1.5); (2) The correspondent 

1Molecular Biology Lab, Instituto Nacional de Investigação Agrária e Veterinária, I.P., Oeiras, Portugal.

2 Plant Cell Biotechnology Lab, Instituto de Tecnologia Química e Biológica António Xavier (Green-it Unit), Universidade Nova de Lisboa, 

Oeiras, Portugal.

3Departamento Biologia Vegetal, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, Portugal.

4Centro de Estudos Instituto Superior de Agronomia, Universidade de Lisboa—Tapada da Ajuda, Lisboa, Portugal, (rita.lcosta@iniav.pt).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
32 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

DEGs in C. sativa transcriptomes with Log2Csi/

Csn <1.5 or absent; (3) DEGs not involved in 

general biological processes, such as oxidative, 

metabolic and transporter activities; (4) DEGs 

involved in defense response and categorized 

in pathogen recognition which usually triggers 

resistance signalling pathways, anti-pathogen 

proteins, cell wall modification proteins, and 

transcription factors involved in the regulation of 

other defense related processes. QS3D digital PCR 

System (Life Technologies) was used to quantify 

gene expression of eight P. cinnamomi resistance 

candidate genes in the roots of the six chestnut 

genotypes under study.

RESULTS AND DISCUSSION

Physical and Chemical Barriers to 

P. cinnamomi Infection

Using the gene selection parameters defined, 

eight candidate genes were identified. These 

genes codify proteins potentially involved in 

the three layers of defense to P. cinnamomi

infection, previously described (Freeman and 

Beattie 2008) two pathogen recognition proteins 

(Cast_LRR-RLK and Cast_C2CD) which trigger 

resistance signaling pathways; three transcription 

factors (Cast_WRKY 31, Cast_ABR1, and Cast_

Myb4) involved in the regulation of other defense 

processes; a ubiquitination regulator (Cast_RNF5); 

a cell wall modification enzyme (Cast_PE-2) and 

an antifungal protein (Cast_Gnk2-like). All genes 

selected were up-regulated after inoculation in C. 

crenata root transcriptomes (Serrazina et al. 2015). 

The P. cinnamomi resistance candidate genes, 

their respective contig name (Serrazina et al., 

2015), primers and TaqManR - Probes sequences 

are listed in Santos et al. (2017).

Before inoculation there is a clear differentiation 

in gene expression between C. sativa and 

C. crenata. Except for Cast_ABR1 and Cast_

RINF5, the pre-inoculated expression of all 

other genes is significantly higher in C. crenata. 

This pattern, with some variation, holds for the 

two most resistant hybrids tested (SM904 and 

SC55). The secretion of toxic compounds is an 

effective defense mechanism against pathogens 

in plants (Montesinos 2007, Wittstock and 

Gershenzon 2002). Ginkbilobin-2 (Gnk2) is a 

protein secreted by Ginkgo biloba seeds that 

exhibits an antifungal activity (Sawano et al. 2007, 

Wang and Ng 2000). Gnk2 has a plant-specific 

cysteine-rich motif DUF26 (domain of unknown 

function 26, also known as stress-antifungal 

domain: PF01657) which belongs to cysteinerich receptor-like kinases (CRKs) (Miyakawa et 

al. 2014) not showing any similarity with other 

known antimicrobial proteins (Miyakawa et al. 

2014, Sawano et al. 2007). It was recently shown 

that Gnk2 can also activate actin-dependent cell 

death (Gao et al. 2016). Therefore, Cast_Gnk2-

like may prevent pathogen growth either by its 

chemical properties or by inducing HR-related 

cell death. The highest Cast_Gnk2-like expression 

registered in non-inoculation conditions suggests 

that C. crenata root surroundings may be a 

hostile environment for fungal and fungal-like 

pathogens, such as P. cinnamomi. On the other 

hand, C. sativa showed a very low Cast_Gnk2-

like expression level, even after pathogen 

inoculation. Considering the whole experiment, 

Cast_Gnk2-like was the most expressed gene 

and that best discriminates between susceptible 

and resistant genotypes (Santos et al. 2017). The 

isolation and purification of Cast_Gnk2- like 

protein may have biotechnological applications, 

such as the development of an antimicrobial 

phytopharmaceutical against P. cinnamomi. 

A crucial constitutive defense is the formation of 

wall appositions that comprise a physical barrier to 

pathogen growth (Hardham and Blackman 2010). 

The reinforcement of plant cell walls by calciumpectate gel apposition with the involvement 

of pectinesterases have been shown to confer 

resistance to Phytophthora species (Kieffer 2000, 

Wiethölter et al. 2003). In this study, expression 

levels of Cast_PE-2 show that this enzyme 

may have a role on P. cinnamomi resistance in 

chestnut. Compared with C. sativa, C. crenata

exhibited higher Cast_PE-2 expression levels 

in all time points, mainly in the non-inoculated 

samples (about 10x more), suggesting that their 
33

cell walls may be more resistant to pathogen 

penetration. After the first pathogen contact, 

Cast_PE-2 expression increases, suggesting a 

possible continuing apposition of pectates in cell 

walls, probably to inhibit further colonization. 

This seems to be more important in a late stage 

of infection (48 hpi) except for the C. sativa × 

C. mollissima hybrid. Possibly, other resistance 

mechanisms may be activated earlier in this hybrid 

and control the infection.

Pathogen Recognition and Successive Host 

Response Regulation

Generally, during pathogen infection, PAMPs 

are recognized by pattern-recognition receptors 

(PRRs) at the plant’s cell surface. The beststudied class of plant PRRs are receptor-like 

kinases (RLKs), which have an ectodomain of 

leucine-rich repeats (LRRs) involved in PAMP 

perception (Boller and Felix 2009; Jones and 

Dangl 2006; tenHove et al. 2011). Resistance 

related LRR proteins have been shown to be 

differentially expressed in global transcript 

profiling studies in Phytophthora spp. infection 

response (Ballvora et al. 2002, Boava et al. 2011, 

Coelho et al. 2011, Gao et al. 2005, Mahomed 

and Berg 2011, van der Vossen et al. 2003). 

Contrasting to C. sativa, C. crenata has a 

much higher (about 10x more) Cast_LRR-RLK 

expression before inoculation (Santos et al. 2017), 

which may mediate a fast and effective response 

against P. cinnamomi, suggesting that this earlier 

recognition is part of the resistance phenotype. 

Furthermore, Cast_LRRRLK expression 

increased after P. cinnamomi inoculation for all 

Castanea genotypes. Considering the previous 

studies on LRR biological functions in Fagaceae, 

Cast_LRR-RLK may recognize and interact with 

PAMPs molecules, secreted by P. cinnamomi, 

activating downstream signaling responses 

(Coelho et al. 2011). 

Cast_WRKY 31 may have a role in the 

response of chestnut to P. cinnamomi infection, 

since its expression increased in inoculated 

samples when compared with non-inoculated 

ones, probably regulating SA-responsive 

genes expression. This increase seems more 

consistent in the more resistant hybrids. 

The overexpression of WRKY 31 in rice 

seedlings after treatment with a hemibiotrophic 

fungus (Magnaporthe grisea) was associated 

with blockade of pathogen invasion (Zhang et 

al. 2008). The balance between SA and other 

phytohormones is increasingly recognized 

as central to the outcome of plant– pathogen 

interactions (de Torres-Zabala et al. 2009). Abcisic 

acid (ABA) disrupts SA-mediated response and 

suppresses the expression of many defense-related 

genes. The ethylene responsive transcription factor 

ABR1 is a negative regulator of ABA signaling 

pathway in Arabidopsis thaliana (Pandey et al. 

2005) and its expression allows SA and lignin 

accumulation (Boatwright and PajerowskaMukhtar 2013; de Torres-Zabala et al. 2009; Mohr 

and Cahill 2007). Cast_ABR1 expression was 

triggered after P. cinnamomi inoculation, earlier in 

the more resistant genotypes, suggesting that ABA 

may be repressed after pathogen perception. In the 

resistant C. crenata genotype the relatively low 

increase of Cast_ABR1 expression may be due to 

the efficiency of other resistant mechanisms that 

avoid pathogen colonization, or by independence 

of ABA suppression for SA signalling activation. 

Genes of the MYB transcription factor family 

are involved in the control of specific processes 

including responses to biotic stresses (Dubos 

et al. 2010). The expression balance of Cast_

Myb4 in Castanea genotypes may regulate SA 

accumulation vs. synthesis of phenylpropanoids. 

The ratio of Cast_Myb4 expression between 

24/48 hpi decreased progressively from the 

resistant C. crenata, to C. sativa × C. crenata

hybrids (the most resistant to the most susceptible) 

to the susceptible C. sativa. This indicates that 

SA signaling may be faster (24 hpi) in resistant 

genotypes than in susceptible ones. As mentioned 

before, elevated concentrations of endogenous 

SA will induce expression of Cast_Gnk2-like 

and Cast_WRKY31. For resistant genotypes, 

(C. crenata and SC55) after a probable early 

induction of SA pathways, expression of Cast_

Myb4 decreases at 48 hpi, which may allow the 

synthesis of lignin and other defense molecules.

Session 4: Genomic & Metabolomic Approaches to Identify Resistance Genes
34 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

Hypothetical P. cinnamomi Response Mechanism 

in Castanea

The expression profiles obtained suggest that 

susceptible and resistant plants may share the 

same response mechanisms. Despite, resistant 

plants show a much higher constitutive expression 

of the tested candidate genes before inoculation. 

A working model describing part of the molecular 

interaction of Castanea spp. to P. cinnamomi

infection was presented in Santos et al. (2017): 

Physiochemical barriers, antifungal proteins 

secretion (Cast_Gnk2-like) and stronger cell 

walls (by action of Cast_PE-2, Cast_ABR1) 

respectively, may inhibit P. cinnamomi growth 

and infection. If P. cinnamomi overcome those 

barriers, specific pathogen recognition may occur, 

by Cast_LRR-RLK. Hence, host transcription 

is reprogrammed via MAPK cascades and 

SA signaling. Cast_WRKY 31 should activate 

transcription of LRR-RLK. Cast_ABR1 regulate 

SA accumulation via ABA suppression. HR could 

be activated by many mechanisms: SA or calcium 

signaling, via Cast_Gnk2-like (actin-dependent) 

or by vital protein degradation (by Cast_RNF5). 

Cell walls not infected may be reinforced and 

antifungal proteins may be secreted in more 

abundance, inhibiting further colonization. 

Resistant genotypes present a higher expression 

of genes in non-inoculation conditions that may 

be part of a constitutive defense mechanism 

that prepare and protect the plant in advance to 

P. cinnamomi infection by secreting antifungal 

proteins and having stronger cell walls even before 

the contact with the pathogen. If P. cinnamomi

overcomes those chemical and physical barriers, 

specific pathogen recognition proteins are 

earlier and more expressed in the resistant 

genotypes when compared to the susceptible 

ones. Thereafter, the transcription of the host 

will probably be reprogrammed via signal 

transduction and SA signaling. HR-related cell 

death is probably activated and cell walls may 

be reinforced in non-infected tissues, preventing 

further colonization. 

In conclusion, the first layer of defense seems 

to be active and decisive in the resistance of 

C. crenata to P. cinnamomi. A lower and delayed 

expression of the eight studied genes was found 

in C. sativa, which may be related with the 

sensitivity of this species towards the disease. One 

probable explanation for this difference can be the 

allelic variation of the genes or gene-promoters 

that in C. sativa may condition the levels of gene 

expression before inoculation. C. mollissima, also 

a resistant species, may share with C. crenata

some of the allelic variants that allow an efficient 

level of resistance against P. cinnamomi. This will 

be the object of further research. Natural selection 

could have had an active role in keeping those 

allelic variants, since Asian species have evolved 

in contact with P. cinnamomi. This study is part 

of an ongoing Portuguese breeding program 

to introduce resistance to P. cinnamomi in 

C. sativa. This knowledge may contribute for the 

development of strategies to control ink disease 

in chestnut and other woody plants, which may 

include early selection of resistant genotypes.

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Session 4: Genomic & Metabolomic Approaches to Identify Resistance Genes 37

EXPLOITING NATURAL VARIATION IN A TREE SPECIES TO 

COMBAT AN INVASIVE FOREST PATHOGEN

Jared M. LeBoldus1

Invasive microbes, causing diseases such as sudden oak death negatively affect ecosystems and economies 

around the world. The deployment of resistant genotypes for combating introduced diseases typically 

relies on breeding programs that can take decades to complete. To demonstrate how this process can 

be accelerated, we employed a genome-wide association mapping of ca. 1,000 re-sequenced Populus 

trichocarpa trees individually challenged with Sphaerulina musiva, an invasive fungal pathogen. 

Among significant associations, three resistance loci were identified and predicted to encode a putative 

membrane-bound L-type receptor-like kinase and two receptor-like proteins. A susceptibility-associated 

locus was predicted to encode a putative G-type D-mannose-binding receptor-like kinase. Multiple 

lines of evidence including allele analysis, transcriptomics, binding assays, and overexpression support 

the function of these genes in mediating this interaction. Herein we demonstrated how the power of 

population-wide re-sequencing of undomesticated, non-model plant species could accelerate the mitigation 

of emerging diseases in native ecosystems.

1 Department of Botany and Plant Pathology & Forest Engineering, Resources and Management, Oregon State University, Corvallis, 

OR 97331, (jared.leboldus@science.oregonstate.edu).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
38 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

GENOME-WIDE EXON-CAPTURE APPROACH IDENTIFIES GENETIC VARIANTS 

ASSOCIATED WITH SUSCEPTIBILITY OF NORWAY SPRUCE TREES 

TO HETEROBASIDION PARVIPORUM INFECTION 

Mukrimin Mukrimin1,2, Andriy Kovalchuk1, Leandro G. Neves3, Emad Jaber1, 

Matti Haapanen4, Matias Kirst3, and Fred O. Asiegbu1

In boreal forests the root and butt rot caused by members of the Heterobasidion annosum species 

complex is the most economically important disease of conifer trees. In the infected trees, the wood 

decay dramatically decreases their value and causes considerable losses to forest owners. Trees vary in 

their susceptibility to Heterobasidion infection, but the genetic determinants underlying the variation 

in the susceptibility are not well understood. We performed the identification of Norway spruce genes 

associated with the resistance to Heterobasidion parviporum infection using genome-wide exon-capture 

approach. Sixty-four clonal Norway spruce lines were phenotyped, and their responses to H. parviporum

inoculation were determined by lesion length measurements. Afterwards, the spruce lines were genotyped 

by targeted resequencing and identification of genetic variants (SNPs). Genome-wide association analysis 

identified 36 SNPs located within 34 genes as significantly associated with the larger necrotic lesions in 

response to H. parviporum inoculation. The genetic variants identified in our analysis are potential marker 

candidates for future breeding programs.

1 Department of Forest Sciences, University of Helsinki, Latokartanonkaari 7, P.O. Box 27, 00014, Helsinki, Finland, (mukrimin.mukrimin@

helsinki.fi). 2 Department of Forestry, Faculty of Forestry, Hasanuddin University, Jln. Perintis Kemerdekaan Km. 10, 90245, Makassar, Indonesia.

3 School of Forest Resources and Conservation, University of Florida, Gainesville, FL 32611.

4 Natural Resources Institute Finland (Luke), Latokartanonkaari 9, 00790, Helsinki, Finland.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 4: Genomic & Metabolomic Approaches to Identify Resistance Genes 39

METABOLOMIC COMPARISON OF EUROPEAN ASH GENOTYPES WITH 

DIFFERENT TOLERANCE LEVELS AGAINST ASH DIEBACK

Miguel Nemesio-Gorriz1, Christian Paetz2, Riya C. Menezes2, Almuth Hammerbacher3, 

Jonathan Gershenzon2, and Gerry Douglas1

Ash dieback, a tree disease caused by the ascomycete Hymenoscyphus fraxineus, is currently decimating 

ash populations in Europe and poses a threat to other ash species in North America. Despite its severity, 

between 1 percent and 3 percent of the European ash trees show moderate to high tolerance to the disease. 

This study compares the biochemical profile of 20 highly tolerant and 20 highly susceptible Fraxinus 

excelsior genotypes from 16 populations in 7 European countries using metabolomics. In terms of 

intraspecific diversity, phenotyping, and number of individuals, this is the most complete metabolomics 

study in ash that has been ever attempted. Results show a very different biochemical profile for both 

groups and focus on specific compounds that may play a major role in determining the tolerance or 

susceptibility of ash genotypes.

1 Forest Development Department, Teagasc, Ireland, (miguel.nemesiogorriz@teagasc.ie). 2 Max Planck Institute for Chemical Ecology, Jena, Germany.

3Department of Zoology and Entomology, FABI, Pretoria, South Africa.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.

Session 5: Use of Transcriptomics/GWAS to Dissect Mechanisms of Host/Parasite Interaction 41

Session

USE OF GENOMICS TO DISSECT MECHANISMS 

OF HOST-PARASITE INTERACTION 5
42 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

CANDIDATE GENES FROM GWAS AND RNASEQ FOR BEECH BARK 

DISEASE RESISTANCE IN AMERICAN BEECH

John E. Carlson1, Irina Ćalić2,3, Jennifer Koch4, David Carey4, Charles Addo-Quaye5, 

Donghwan Shim1, and David B. Neale3

SUMMARY

American beech (Fagus grandifolia Ehrh.) is an aesthetically, ecologically, and economically important 

native component of the North American eastern hardwood forest. American beech is susceptible to 

beech bark disease (BBD), however, and has suffered high rates of mortality as the disease complex 

spreads. The invasive sap-feeding woolly beech scale insect (Cryptococcus fagisuga) is the pre-disposing 

factor for infection by introducing either Neonectria faginata or N. ditissima fungus species that result 

in extensive cankering of American Beech trees. A small percentage of trees survive BBD attack, and 

many of these show signs of natural resistance to the insect vector in the egg-inoculation tests used to 

assay for resistance. We have developed and applied genomics resources to learn more about the molecular 

genetic basis of gene expression and gene sequence variation associated with cases of natural resistance 

to the insect. Initially, transcriptome resources were developed, and differential gene expression analyses 

conducted from which candidate genes were selected. From the transcriptome, an SNP chip assay was 

developed to genotype an association population of 506 individuals from across the American beech 

range, 249 of which were resistant and 257 susceptible to BBD. We also constructed a genetic linkage map 

based on SNPs with a full-sib family of 115 individuals to locate BBD-resistance QTL. The GWAS project 

revealed four highly significant SNPs on Linkage Group 5 for a single gene encoding a metallothioneinlike protein. Metallothioneins are cysteine-rich metal chelator proteins that can moderate oxidative stress 

by coordinating metal atoms, which may provide a resistance mechanism against the woolly beech scale 

insect. 

OBJECTIVES

The primary goal of this research was to gain a 

better understanding of the molecular mechanisms 

underlying resistance and susceptibility to beech 

bark disease (BBD) in American beech. Our 

objectives towards this goal were to:

1. Develop genomics tools for molecular genetic 

studies in American beech.

2. Conduct differential gene expression analyses 

to identify candidate genes for future 

research on BBD resistance in beech.

3. Conduct a genome-wide association study 

(GWAS) to identify which genes or alleles 

have the greatest contribution to BBD 

resistance in natural stands of American 

beech. 

METHODS AND RESULTS

Development of Genomics Tools for American 

Beech

Our development of genomic resources for 

American beech began with support from 

the National Science Foundation’s Plant 

Genome Resources Program for the “Fagaceae 

Genomic Tool Development Project”, which 

ran from 2006 to 2009. This project was led 

by Ronald Sederoff (North Carolina State 

University), with participants from Pennsylvania 

State University (John Carlson, Haiying 

Liang, Abdelali Barakat, Stephan Schuster), 

SUNY ESF (William Powell, Kathleen Baier, 

Charles Maynard), Clemson University 

(Albert Abbott, Margaret Staton, Jeff Tomkins, 

1Schatz Center for Tree Molecular Genetics, Pennsylvania State University, University Park, PA, 16802, (jec16@psu.edu). 2 Department of Plant Biology, University of Georgia, Athens, GA 30602.

3Department of Plant Sciences, University of California, Davis, CA 95616.

4 Northern Research Station, USDA Forest Service, Delaware, OH 43015.

5Division of Natural Sciences and Mathematics, Lewis-Clark State College, Lewiston, ID 83501.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 5: Use of Transcriptomics/GWAS to Dissect Mechanisms of Host/Parasite Interaction 43

Steven Ficklin, Barbara Blackman, Eric Fang), 

North Carolina State University (Nick Wheeler, 

Chris Smith, Dahlia Nielsen, Ron Sederoff), The 

Connecticut Agricultural Experiment Station 

(Sandra Anagnostakis, Lila Pinchot), USDA 

Forest Service (Tom Kubisiak, Dana Nelson), The 

American Chestnut Foundation (Fred Hebard, 

Paul Sisco), and Science Advisory Board members 

(Doug Cook of UC-Davis, Jennifer Koch of USDA 

Forest Service, and Jeanne Romero-Severson of 

Notre Dame University). 

The “Fagaceae Project” focused on Chinese 

chestnut (Castanea mollissima), American 

chestnut (C. dentata), Northern red oak (Quercus 

rubra), White oak (Q. alba), and American beech. 

For these five species we developed expressed 

sequence tag (EST) databases by next generation 

sequencing (NGS) with 454 technology (Roche) 

for use in gene discovery, DNA markers for 

genetic mapping, and in some species BAC 

libraries for gene cloning and physical mapping. 

In addition, work on high density genetic 

mapping and physical mapping was initiated. 

In total, 16 cDNA libraries were constructed 

and sequenced, including two for American 

beech. The beech libraries were prepared from 

poly-A RNA isolations from tissue collections 

designed to represent gene expression plantwide, including roots, bark, buds, twigs, leaves, 

petioles, and flowers. The tissues were collected 

from USDA Forest Service genotype 1504—a 

healthy American beech tree, and genotype 

1506—a BBD-infected American beech tree. 

For American beech, a total of 14.6 million 

bases of RNA sequence data (ESTs) were 

generated and assembled into 8,319 transcripts. 

From the assembled transcripts, 2,383 proteins 

could be predicted based on protein databases 

(such as InterProScan) of which 2,231 were 

assigned putative annotations and functions 

based on similarities to known genes in the 

NCBI databases. The EST sequences for each of 

the American beech trees 1504 and 1506 were 

deposited at the NCBI Short Read Database 

under accession numbers SRX001797 and 

SRX001798, respectively. The American beech 

EST sequences, transcripts, annotations, and 

DNA markers from the Fagaceae Project are also 

available for download and query at the Hardwood 

Genomics Database entry for Fagus grandifolia

(https://www.hardwoodgenomics.org/organism/

Fagus/grandifolia) that also includes a Gene 

Ontology Browser of 629 biological, 242 cellular, 

and 830 molecular functions predicted from the 

transcript annotations, as well as a KEGG Brower 

with the placement of the beech transcripts into 

enzymatic pathways.

After completion of the Fagaceae Project, genomic 

resources targeted to BBD-resistance in American 

beech were greatly expanded through a grant to 

Jennifer Koch from the USDA Forest Service 

Forest Health Protection’s Special Technology 

Development Program. The project “Development 

of DNA-based markers to identify beech bark 

disease (BBD) - resistant trees in natural stands” 

ran from 2009 to 2012, with Jennifer Koch as 

Principal Investigator and participants from 

Pennsylvania State University (John Carlson, 

Donghwan Shim, Charles Addo-Quaye, Tyler 

Wagner, Lynn Tomsho), UC-Davis (David 

Neale, Irina Ćalić, Randi Famula, Mirko Ledda, 

Christopher Campbell), and USDA Forest Service 

Northern Research Station lab in Delaware, OH 

(David Carey). 

The “BBD-resistance project” focused on 

gaining a better understanding of the molecular 

mechanisms underlying BBD-resistance 

in American beech. We studied a set of 

BBD-resistant and BBD-susceptible trees 

identified in natural stands by Forest Service 

researchers, some of which were accessioned as 

part of the beech bark disease genetic resistance 

improvement program (Koch et al. 2010; Houston 

and Houston 2000). RNA sequence data was 

produced by 454 NGS technology from each of 

10 cDNA libraries prepared from poly-A RNA 

isolated from bark tissues of 5 BBD-resistant trees 

(USDA Forest Service accessions 1228, 1208, 

2692, 1504, 2776) and from 5 BBD-susceptible 

trees (accessions 726, 3128, 1973, 2143, “Holden”) 

following treatment of clonal replicates with 

larvae of the woolly beech scale under greenhouse 

conditions. The relative resistance of nine of 

the 10 clonal accessions (genotypes) (fig. 1) was 

assayed by determining the number of adult scale 

insects produced from genotypes following egg 

inoculations (the Holden genotype did not survive 

the inoculation test).
44 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

Figure 1—Average number of scale insects produced from clones 

of American beech genotypes following egg inoculation under 

greenhouse conditions. A range of resistance and susceptibility to 

BBD are demonstrated.

Figure 2—Comparison of scale insect development on BBD-resistant 

vs. BBD-susceptible genotypes 1228R and 2143S.

In total we obtained 257.5 Mb of RNA sequence 

data from the BBD-resistant trees and 508.7 Mb 

from the susceptible trees. The RNA sequences 

from the BBD-resistant trees were pooled as were 

the sequences from the BBD-susceptible trees, 

and then assembled into two sets of transcripts 

from which over 26,000 Single Nucleotide 

Polymorphism (SNP) DNA markers were 

identified for use in genetic linkage mapping and 

genome-wide association (GWAS) studies (Ćalić 

et al. 2017). The sequences from this study are 

available in the Short Read Archive at the National 

Center for Biotechnology Information, genotype 

numbers SRX1781388 to SRX1781397 (NCBI 

BioProject genotype PRJNA321730; http://www.

ncbi.nlm.nih.gov).

From the 10 beech tree genotypes, we chose 

genotypes 1228 (‘1228R’) and 2143 (‘2143S’) 

to represent the putatively most BBD-resistant 

and BBD-susceptible (fig. 1) for an initial study 

of the response of gene expression in beech to 

attack by the scale insect. The results of a time 

course comparing insect viability at 2, 8, and 

40 weeks, confirmed the pronounced differences 

in susceptibility between genotypes 1228R and 

2143S (fig. 2). 

The workflow devised for analysis and 

comparison of gene expression in genotypes 

1228R and 2143S is presented in figure 3. Steps 

completed during this project are indicated by 

solid lines, while dotted lines suggest steps that 

should be taken in future projects, based on results 

gained in the BBD-resistance project. 

The RNA sequence yields and statistics from 

the cDNA libraries from genotypes 1228R and 

2143S are shown in table 1. A total of 1,021,530 

sequence reads corresponding to 403,362,357 

bases were obtained from the two outer bark tissue 

RNA samples. The RNA sequences from the two 

libraries were assembled separately and after 

pooling (using Trinity software). A total of 22,463 

transcripts were assembled for genotype 1228R 

and 21,957 for genotype 2143S, and 31,525 from 

the pooled RNA sequence data. 

Of the 31,525 transcripts in the reference set of 

transcripts from pooling the two sets of RNA 

sequences prior to assembly (table 1 “Both”), 

26,784 transcripts were for unique genes (table 1 

“Total trinity components”), while 4,741 were 

predicted to be isoforms from RNA-splicing. Of 

the 26,527 unique transcripts, 22,015 genes were 

expressed in both genotypes 1228R and 2143S, 

while 2,352 transcripts could only be detected 

in genotype 1228R and 2,160 transcripts were 

unique to genotype 2143S (fig. 4A). The combined 

assembly served as a reference against which 

the number of individual sequence reads for 

each transcript from each library was aligned 
Session 5: Use of Transcriptomics/GWAS to Dissect Mechanisms of Host/Parasite Interaction 45

Figure 3—RNA sequence analysis workflow. Dashed arrows are steps which are recommended for future research 

projects.

Table 1—Statistics of 454 pyrosequencing reads and assembled contigs

derived for BBD‐resistant and BBD‐susceptible F. grandifolia trees

Resistant tree

(1228R)

Susceptible tree

(2143S)

Raw reads

Num. sequences 530,622 493,807

Total nucleotides 211,316,501 191,666,186

Min. length 27 27

Max. length 1,568 1,558

Ave. read length 398.24 388.14

Q20 85.07 84.99

Q30 57.82 57.02

Ave. quality score 30.47 30.33

      Cleaned reads

Num. sequences 408,108 380,425

Total nucleotides 83,041,684 77,164,051

Min. length 50 50

Max. length 620 603

Ave. read length 203.48 202.84

Q20 98.1 98.15

Q30 87.62 87.11

Ave. quality score 36.29 36.14

Resistant tree

(1228R)

Susceptible tree

(2143S) Both

          Assembled contigs

Total trinity transcripts 22,463 21,957 31,525

Total trinity components 20,574 20,238 26,784

Percent GC 43.23 43.35 42.8

Contig N50 754 735 921

Average contig 606.89 599.17 688.78

Total assembled bases 13,632,611 13,155,905 21,713,754

Table 1—Statistics of 454 pyrosequencing reads and assembled contigs derived 

for BBD‐resistant and BBD‐susceptible F. grandifolia trees
46 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

Figure 4—Distribution of differentially expressed genes (DEGs) in the BBD-resistant (1228R) and BBD-susceptible (2143S) 

American beech trees. (A) Venn diagram showing proportions of DEGs expressed in common and expressed uniquely in 1228R 

and 2143S trees; (B) Log-fold changes observed in up-regulated (induced) and down-regulated (suppressed) genes overall; 

(C) Venn diagrams showing numbers and proportions of Gene Ontology determined functional categories of DEGs in biological 

processes, cell components, and molecular functions. 

and counted (using Qiagen/CLCBio Genomics 

Workbench software). Expression analysis of 

the RNA-Seq data revealed a total of 860 genes 

that were upregulated (induced) and 934 that 

were downregulated (suppressed) across the 

two genotypes, 1228R and 2143S (fig. 4B). Gene 

ontology (GO) analysis of putative functions 

showed a wide range of functional classifications 

represented in the transcriptomes of each 

genotype (fig. 4C). Twenty-two candidate genes, 

for research on BBD resistance in American 

beech, were selected based on the magnitudes 

of upregulation of expression in 1228R and 

alignment scores to known proteins (table 2).

To determine which of the 4,458 uniquely 

expressed transcripts were significantly different 

in expression between genotypes 1228R and 

2143S, we conducted a differential gene expression 

analysis using the combined transcriptome 

referenced as above (using Qiagen/CLCBio 

Genomics Workbench software). A total of 608 

genes were identified as significantly differentially 

expressed between genotypes 1228R and 2143S 

(at P-value < 0.01, unique reads > 5), of which 

303 were up-regulated at least 2-fold and 305 

were down-regulated by 2-fold or more. Among 

these differentially expressed genes (DEGs), 

313 transcripts could be putatively identified by 
Session 5: Use of Transcriptomics/GWAS to Dissect Mechanisms of Host/Parasite Interaction 47

Table 2—Candidate genes for BBD resistance in American beech based on relative strengths (fold

change) of BBD‐induced gene expression differences, rather than GO functional classifications.

Only genes with the highest BLAST sequence alignment scores (E‐values) to known genes in the

model plants Arabidopsis and poplar in the NCBI Database are shown

homology to genes in the NCBI non-redundant 

protein database, while 295 transcripts were 

“unknown”, i.e. with no sequence matches in the 

database.

The number of annotated (putative protein 

function) DEGs in the major GO functional 

classifications observed between the 

BBD-resistant vs. BBD-susceptible genotypes 

following greenhouse inoculations is shown 

by histogram comparisons in figure 5. In the 

BBD-resistant genotype 1228R, most DEGs 

were observed in functional categories related 

to stress-response - “response to abiotic stress” 

(Heidel‐Fischer et al. 2018, Marwal et al. 2019, 

Tajima et al. 2020, Waterman et al. 2019), 

“polysaccharide and glucan related responses” 

(Piršelová and Matušíková 2013; Pogorelko et al 

2013), “programmed cell death” (Locato and De 

Gara 2018), and “potassium ion transport” (Wang 

et al. 2013). In sharp contrast, the DEGs observed 

in the BBD-susceptible genotype 2143S were 

classified into GO functional categories that were 

in general more related to normal metabolic and 

developmental processes. These stark differences 

in gene expression in response to the scale insect 

indicate that genotype 1228R has the ability to 

recognize and mount a strong molecular defense 

against BBD, while genotype 2143S is susceptible 

due to lack of response at the gene expression level 

to BBD. We selected 17 candidate genes (table 3) 

for future studies, based on the four GO functional 

classifications and the putative gene assignments 

of DEGs observed in BBD-response by the 

resistant genotype. 

Table 2—Candidate genes for BBD resistance in American beech based on relative strengths (fold 

change) of BBD‐induced gene expression differences, rather than GO functional classifications. 

Only genes with the highest BLAST sequence alignment scores (E‐values) to known genes in the 

model plants Arabidopsis and poplar in the NCBI Database are shown
48 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

Figure 5—Gene Ontology functional categories of differentially expressed genes.

Table 3—Gene Ontology predicted descriptions for differentially expressed candidate 

genes in the stress response and apoptosis functional categories

Term Description Count P-value

GO:0009628 response to abiotic stimulus 34 2.21E-04

GO:0048585 negative regulation of response to stimulus 6 2.79E-03

GO:0055114 oxidation reduction 29 6.53E-03

GO:0009266 response to temperature stimulus 11 3.29E-02

GO:0009416 response to light stimulus 13 3.80E-02

GO:0009414 response to water deprivation 7 4.29E-02

GO:0009314 response to radiation 13 4.70E-02

GO:0005976 polysaccharide metabolic process 10 4.55E-03

GO:0000272 polysaccharide catabolic process 5 2.21E-02

GO:0044042 glucan metabolic process 7 2.23E-02

GO:0009250 glucan biosynthetic process 5 2.31E-02

GO:0006073 cellular glucan metabolic process 6 3.38E-02

GO:0012501 programmed cell death 10 5.12E-03

GO:0016265 cell death 10 1.17E-02

GO:0008219 cell death 10 1.17E-02

GO:0006915 Apoptosis 8 1.26E-02

GO:0006813 potassium ion transport 4 4.29E-02
Session 5: Use of Transcriptomics/GWAS to Dissect Mechanisms of Host/Parasite Interaction 49

Genome-Wise Association Study

The GWAS revealed four highly significant SNPs 

on Linkage Group 5 for a single gene encoding 

a metallothionein-like protein (Ćalić et al. 2017). 

Metallothioneins are cysteine-rich metal-chelating 

proteins. Metallothioneins and metallothioneinlike proteins respond to abiotic and biotic stresses 

in plants, including heavy metals, insect herbivory 

and fungal infections (reviewed by Leszczyszyn 

et al. 2013). A major role of metallothioneins and 

metallothionein-like proteins is their anti-oxidant 

effects that moderate damage from oxidative 

stress through scavenging of cofactors needed by 

reactive oxygen generating enzymes. One of the 

largest groups of pathogenesis-related proteins 

are also upregulated after heavy metal treatment, 

suggesting cross-talk between the heavy metal 

(abiotic) and biotic stress responses in plants 

(Joshi et al. 2019). Metallothionein-like class II 

proteins have previously been identified in Fagus 

sylvatica (NCBI accession number CAA10232) 

and Picea abies (Yakovlev et al. 2006). Type 

4 metallothionein-like protein genes were 

reported to be expressed in inner bark tissues of 

Cryptomeria japonica (Leszczyszyn et al. 2013). 

CONCLUSIONS, CAVEATS AND 

FUTURE RESEARCH 

It is important to point out that certain caveats 

existed with the GWAS approach taken in 

this study. First, at the time the GWAS study 

(Ćalić et al. 2017) was undertaken, and still at 

the time of this presentation and manuscript, 

a reference genome for American beech was 

not available. Thus, a SNP chip was used as 

the GWAS reference, based on the combined 

bark transcriptomes from BBD-resistant and 

BBD-susceptible trees, to target genes in the tissue 

and biotic stress of interest. However, unlike a full 

genome sequence, transcriptomes never include all 

possible genes of interest nor all the genome-wide 

variation that exists for a trait, especially outside 

of coding regions. For example, transcriptomes 

are limited to the specific time points selected to 

sample after treatment, which for practical reasons 

are always limited and cannot encompass all the 

cascades of gene expression that are qualitatively 

and quantitatively important in accomplishing 

resistance. In addition, when dealing with a 

complex disease such as BBD that involves an 

insect vector and multiple fungal pathogens, it 

cannot necessarily be presupposed that using a 

transcriptome reference based on only the insect 

establishment, feeding, and reproduction will 

provide any markers for resistance mechanisms 

to the fungal infection process. Furthermore, 

other loci or alleles that fall below a selected 

significance threshold may also collectively 

(quantitatively) be important in BBD-resistance, 

or important in specific populations but not across 

the range of populations sampled.

Validations of our GWAS results still need to be 

completed. Ideally, validation populations should 

be collected from the same or similar populations 

so that both resistant and susceptible individuals 

are included, but without advance knowledge of 

resistance prior to conducting the GWAS and not 

including samples used in the original study. This 

is a difficult criterion to meet when dealing with 

natural populations that are already experiencing 

mortality from BBD, and/or that are under other 

biotic or abiotic stresses affecting population 

structure.

Another powerful approach to validating GWAS 

results is to test whether the loci with significantly 

associated alleles map in or near QTL of the trait 

on genetic linkage maps. The Forest Service’s 

beech bark disease genetic resistance improvement 

program is evaluating 118 individuals from the 

1505 x 1504 full-sib family used to construct the 

SNP-based genetic linkage map in the GWAS 

study (Ćalić et al. 2017). At the time of this 

presentation, only part (46 trees) of the mapping 

population had so far been evaluated, for which 

two QTL for resistance were found at regions 

other than the metallothionein locus detected by 

GWAS. This negative result, if it holds up in the 

whole mapping population, does not necessarily 

invalidate the GWAS result which derived from 

the strongest genetic signal that could be detected 

in 327 genetically distinct individuals sampled 

from several populations, rather than inheritance 

from only two genotypes (parents of the mapping 

family which could be fixed at the GWAS 

locus). It could however suggest that multiple 

mechanisms may result in BBD-resistance, for 
50 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

example constitutive resistance traits versus 

induced-resistance traits, and differences in the 

natural histories of population exposures to past 

environmental stresses.

The metallothionein-like protein gene detected 

in the GWAS study was not one of the candidate 

genes selected from our transcriptome studies 

following BBD treatments. Changes in expression 

of the gene are not necessarily expected from 

variants in the coding sequence. However, the 

transcriptome overall did contain 12 transcript 

contigs which aligned well by BLAST with 

two metallothionein protein genes in the plant 

model Arabidopsis thaliana—a metallothionein 

2A gene (NCBI accession AT3G09390.1) and 

metallothionein 3 (NCBI accession AT3G15353.1). 

It would be interesting to conduct a qRT-PCR 

study of expression of metallothionein or 

metallothionein-like genes, and other candidate 

genes, during a detailed time course of response 

to inoculations of well-established resistant 

and susceptible genotypes. In the study on 

differential gene expression reported here, 

we directly compared only the two extreme 

genotypes. A detailed gene network analysis 

study including the 8 genotypes with intermediate 

resistance/susceptibility phenotypes might reveal 

additional candidate genes, perhaps of smaller 

individual effects, which might collectively 

still be quantitatively important. However, 

the ultimate validation of the metallothioneinlike protein detected by GWAS will require 

functional genomics studies to confirm its role 

in BBD-resistance. That awaits development of 

a gene transfer system for American beech with 

which knock-out, suppression, or gene editing 

studies can be conducted. 

Beyond basic knowledge of how beech trees 

respond to BBD, such transcriptome and GWAS 

studies aim to contribute to the restoration of the 

species by providing molecular tools for selection 

and breeding. Markers that exhibit a significant 

association with the resistance phenotype can 

provide the basis of efficient indirect selection 

techniques such as marker assisted selection 

(MAS) and genomic selection (GS). For simply 

inherited, single-gene resistance traits, having 

several DNA markers within the gene increases 

the power of MAS, as individuals within a 

population may vary as to how many or which of 

the markers they carry for the gene. The presence 

of several markers and thus possibly several alleles 

for resistance may also slow the breakdown of 

resistance that can be a concern with single-gene 

traits in long-lived organisms like American 

beech. It should be acknowledged, however, that 

individual markers or alleles for a trait obtained 

from QTL mapping or transcriptome studies with 

one family or a small number of genotypes may 

contribute to only a small amount of phenotypic 

variation within and between large natural 

populations (Korte and Fallow 2013). Thus, 

MAS conducted with a few markers may not 

accomplish substantial advancements of selection 

and breeding goals, if not incorporated into 

genome-wide approaches. Genome-wide selection 

models that aggregate the small effects of many 

markers or alleles for a wider range of traits have 

proven to be powerful in representing a substantial 

fraction of genetic and phenotypic variation in tree 

breeding programs (Resende et al. 2017a, 2017b; 

Müller et al. 2017; Tan et al. 2017). The costs and 

benefits should be carefully considered prior to 

undertaking such approaches, as costs (real and 

opportunity) may be prohibitive, particularly in 

trees of ecological value that lack commercial 

value.

FUNDING

This research was supported by grant #DBI0605135 to R. Sederoff from the National Science 

Foundation’s Plant Genome Resources Program 

(for the “Fagaceae Genomic Tool Development 

Project”), grant number 09-CA-11242304-100 

to Jennifer Koch from the USDA Forest Service 

Forest Health Protection Special Technology 

Development Program, and support to John E. 

Carlson through the USDA National Institute of 

Food and Agriculture Federal Appropriations 

under Project PEN04532, Accession number 

1000326. 
51

ACKNOWLEDGMENTS

We would like to acknowledge our many 

collaborators who participated in the development 

of resources and in conducting various aspects 

of the research described here. These include, 

but are not limited to, Abdelali Barakat, Albert 

Abbott, Barbara Blackman, Charles Maynard, 

Chris Smith, Christopher Campbell, Claude 

dePamphilis, Dahlia Nielsen, Dan Houston, 

Dana Nelson, David Carey, David Houston, Eric 

Fang, Haiying Liang, Jeff Tomkins, Ji Qi, John 

Bowers, Josh Herr, Kathleen Baier, Kerr Wall, 

Lynn Tomsho, Margaret Staton, Mirko Ledda, 

Nick Wheeler, Nicole Zembower, Randi Famula, 

Ron Sederoff, Stephan Schuster, Steven Ficklin, 

Tom Kubisiak, Tyler Wagner, Tyler Wagner, and 

William Powell. 

REFERENCES

Ćalić, I.; Koch, J.; Carey, D. [et al.]. 2017. Genome-wide 

association study identifies a major gene for beech bark 

disease resistance in American beech (Fagus grandifolia

Ehrh.). BMC Genomics. 18(1): 547.

Heidel‐Fischer, H.M.; Musser, R.O.; Vogel, H. 2018. Plant 

transcriptomic responses to herbivory. Annual Plant 

Reviews: 155–196.

Houston, D.B.; Houston, D.R. 2000. Allozyme genetic 

diversity among Fagus grandifolia trees resistant or 

susceptible to beech bark disease in natural populations. 

Canadian Journal of Forest Research. 30(5): 778–789. 

doi:1O.1139/cjfr-30-5-778.

Joshi, R.; Dkhar, J.; Singla-Pareek, S.L.; Pareek, A. 2019. 

Molecular mechanism and signaling response of heavy 

metal stress tolerance in plants. In: Srivastava S.; 

Srivastava A.; Suprasanna P., eds. Plant-Metal Interactions. 

Springer, Cham: 29–47.

Koch, J.L.; Carey, D.W.; Mason, M.E.; Nelson, C.D. 2010. 

Assessment of beech scale resistance in full- and halfsibling American beech families. Canadian Journal of 

Forest Research. 40: 265–272.

Korte, A.; Farlow, A. 2013. The advantages and limitations of 

trait analysis with GWAS - a review. Plant Methods. 9: 29.

Leszczyszyn, O.I.; Imam, H.T.; Blindauer, C.A. 2013. 

Diversity and distribution of plant metallothioneins: a 

review of structure, properties and functions. Metallomics. 

5(9): 1146–1169.

Locato, V.; De Gara, L. 2018. Programmed cell death in 

plants: an overview. New York: In: Plant Programmed Cell 

Death. Humana Press: 1–8.

Marwal, A.; Kumar, R.; Verma, R.K. [et al.]. 2019. Genomics 

and molecular mechanisms of plant’s response to abiotic 

and biotic stresses. In: Plant Biotechnology: Progress in 

Genomic Era. Springer, Singapore: 131–146.

Müller, B.S.; Neves, L.G.; de Almeida Filho, J.E. [et al.]. 

2017. Genomic prediction in contrast to a genomewide association study in explaining heritable variation 

of complex growth traits in breeding populations of 

Eucalyptus. BMC Genomics. 18(1): 524.

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polysaccharide with multiple biological functions. Acta 

Physiologiae Plantarum. 35(3): 635–644.

Pogorelko, G.; Lionetti, V.; Bellincampi, D.; Zabotina, O. 

2013. Cell wall integrity: targeted post-synthetic 

modifications to reveal its role in plant growth and defense 

against pathogens. Plant Signaling and Behavior. 8(9): 

e25435.

Resende, R.T.; Resende, M.D.V.; Silva, F.F. [et al.]. 2017a. 

Assessing the expected response to genomic selection of 

individuals and families in Eucalyptus breeding with an 

additive-dominant model. Heredity. 119(4): 245–255.

Resende, R.T.; Resende, M.D.V.; Silva, F.F. [et al.]. 

2017b. Regional heritability mapping and genome‐wide 

association identify loci for complex growth, wood and 

disease resistance traits in Eucalyptus. New Phytologist. 

213(3): 1287–1300.

Tajima, Y.; Loo, E.P.I.; Saijo, Y. 2020. Plant physiological and 

molecular mechanisms in cross-regulation of biotic-abiotic 

stress responses. In: Priming-Mediated Stress and CrossStress Tolerance in Crop Plants. Academic Press: 21–34. 

Tan, B.; Grattapaglia, D.; Martins, G.S. [et al.]. 2017. 

Evaluating the accuracy of genomic prediction of growth 

and wood traits in two Eucalyptus species and their F 1 

hybrids. BMC Plant Biology. 17(1): 110.

Wang, M.; Zheng, Q.; Shen, Q; Guo, S. 2013. The critical role 

of potassium in plant stress response. International Journal 

of Molecular Sciences. 14(4): 7370–7390.

Waterman, J.M.; Cazzonelli, C.I.; Hartley, S.E.; Johnson, S.N. 

2019. Simulated herbivory: the key to disentangling plant 

defense responses. Trends in Ecology Evolution. 34(5): 

447–458.

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Analysis of gene expression during bud burst initiation in 

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Session 5: Use of Transcriptomics/GWAS to Dissect Mechanisms of Host/Parasite Interaction
52 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

FACTORS UNDERPINNING RESISTANCE AGAINST THE GALLING PEST, 

LEPTOCYBE INVASA IN EUCALYPTUS GRANDIS

Sanushka Naidoo1, Caryn Oates1, Lorraine Mhoswa1, Marja ‘O Neil1, Juan Acosta2, 

Nanette Christie1, Mmoledi Mphahlele3, Kitt Payn3, Alexander Myburg1, 

Bernard Slippers1, and Carsten Külheim4

The blue gum chalcid, Leptocybe invasa is considered a serious threat to the production of Eucalyptus 

species and clones in plantations. This pest induces galls on the midrib, petiole, and shoot tip of young 

Eucalyptus leaves leading to deformation, stunting and in severe cases, tree death. We adopted a multipronged approach to uncover factors contributing to resistance in E. grandis. We performed a genome 

wide association study for resistance against the insect pest, in a breeding population of E. grandis, 

and identified seven genomic regions. Transcriptomics in resistant and susceptible interactions with 

the pest showed the expression of several candidate genes within 50Kb windows of the significantly 

associated SNP markers, including putative R-genes. We also investigated whether plant specialized 

metabolites such as the terpenes would be associated with resistance against the insect pest. Based on near 

infra-red spectral models we could predict specific terpene content with a prediction ability of 0.67. The 

terpenes α-pinene, γ-terpinene and iso-pinocarveol were important for predicting L. invasa infestation. 

Susceptibility was associated with increased γ-terpinene and α-pinene. Resistance was associated with 

iso-pinocarveol. The attractant or repellent properties of these terpenes remain to be tested in E. grandis. 

In the next phase of our study, the markers will be translated to other E. grandis populations for breeding 

purposes and we will focus on characterizing the functional role of the candidate genes. Together, these 

findings contribute to improving resistance against L. invasa.

1 Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Private bag x20, Pretoria, 0028, 

South Africa, (sanushka.naidoo@up.ac.za). 2Camcore, Department of Forestry and Environmental Resources, University of North Carolina, Raleigh NC 27695-8008.

3Mondi Forests, Trahar Technology Centre, P.O. Box 12, Hilton, 3245, South Africa.

4Research School of Biology, Australian National University, 116 Daley Road, Canberra, 0200, ACT, Australia.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 5: Use of Transcriptomics/GWAS to Dissect Mechanisms of Host/Parasite Interaction 53

IDENTIFICATION OF GENES INVOLVED IN THE RESISTANCE RESPONSE OF 

PINUS PINASTER TO THE PINEWOOD NEMATODE INFECTION

Inês Modesto1,2,3, Lieven Sterck3,4, Isabel Carrasquinho5,6, 

Yves Van de Peer3,4,7, and Célia Miguel1,2,8

Pine wilt disease (PWD), caused by the pathogenic nematode Bursaphelenchus xypophilus, is a serious 

threat to conifer forests in Asia and Europe. In the Iberian Peninsula, Pinus pinaster is the main 

species affected by this disease, and trees start to wilt and die soon after the infection. Despite the high 

susceptibility of most P. pinaster trees, a previous study has shown that some individuals display a 

resistance phenotype after inoculation and that this trait is heritable. Understanding the genetic basis 

of this resistance to PWD can be of extreme relevance for future programs aiming at reducing PWD in 

P. pinaster forests. In order to test if contrasting phenotypes are consistent with differential responses of 

gene expression after infection with B. xypophilus, RNA-seq was used to compare transcriptional changes 

between resistant and susceptible seedlings. Our analysis showed a more intense defense response in 

plants resistant to B. xypophilus infection, with a higher number of differential expressed genes (DEGs) 

in resistant plants (1281) than in susceptible ones (773). Although part of the defense response is shared 

between resistant and susceptible plants, gene set enrichment analysis highlighted biological processes and 

molecular functions that may interfere with nematode feeding, growth, and reproduction. This is the first 

work that enlightens the mechanisms involved in P. pinaster resistance to PWD.

1 ITQB NOVA, Universidade Nova de Lisboa, Oeiras, Portugal, (ismodesto@itqb.unl.pt).

2 iBET, Oeiras, Lisboa, Portugal.

3Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.

4VIB-UGent Center for Plant Systems Biology, Ghent, Belgium.

5 INIAV, Oeiras, Portugal.

6LEAF—Linking Landscape, Environment, Agriculture and Food, Instituto Superior de Agronomia, Universidade de Lisboa.

7Department of Biochemistry, Genetics and Microbiology, University of Pretoria, Pretoria, South Africa.

8Biosystems & Integrative Sciences Institute, Faculdade de Ciências, Universidade de Lisboa (FCUL), Lisboa, Portugal.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
54 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

IDENTIFYING AND VALIDATING NECROTROPHIC EFFECTORS IN THE 

POPULUS TRICHOCARPA-SPHAERULINA MUSIVA PATHOSYSTEM

Kelsey L. Søndreli1, Robert Brueggeman2, Jonathan Richards2, 

Timothy Friesen3, and Jared M. LeBoldus1,4

Sphaerulina musiva causes stem cankers on Populus species in North America. The literature indicates 

that the use of resistance genotypes is the best approach to manage S. musiva, so it is imperative that 

we know more about the mechanisms that underlie resistance/susceptibility in the S. musiva–Populus

pathosystem. RNA-seq was used to identify potential S. musiva necrotrophic effectors involved in stem 

canker formation at two and three weeks post-inoculation. Approximately 20 percent of the total reads 

were aligned to the S. musiva reference genome with the remaining reads aligning to the reference genome 

of Populus trichocarpa. There were 70 genes identified at two weeks post-inoculation and 110 genes 

identified at three weeks post-inoculation differentially expressed between the inoculated trees and the 

control. The candidate genes were selected for transformation into Pichia pastoris, subsequent protein 

expression, and infiltration of the proteins into P. trichocarpa leaves. This study provides the first 

evidence that low coverage RNA-seq can be used to identify putative necrotrophic effectors facilitating 

the study of host-pathogen interactions in the P. trichocarpa-S. musiva pathosystem.

1 Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, (Kelsey.Sondreli@oregonstate.edu). 2 Department of Plant Pathology, North Dakota State University, Fargo, ND 58102.

3Red River Valley Agricultural Research Center, Cereal Crops Research Unit, U.S. Department of Agriculture–Agricultural Research 

Service, Fargo, ND 58102.

4 Forest Engineering Resources and Management Department, Oregon State University, Corvallis, OR 97331.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 5: Use of Transcriptomics/GWAS to Dissect Mechanisms of Host/Parasite Interaction 55

HOST-PATHOGEN INTERACTION IN LEAVES OF LAUREL WILT HOSTS

Junli Zhang1, Stephanie M. Adams2, and Jason A. Smith3

Laurel wilt is devastating to trees in the Lauraceae family. Since 2003, laurel wilt has caused death to over 

30 percent of all the redbay (Persea borbonia) trees in the Southeastern United States. And in 2012, laurel 

wilt on Avocado (P. americana Miller) was discovered. The disease is caused by the fungus Raffaelea 

lauricola and is transmitted by its ambrosia beetle symbiont (Xyleborus glabratus) or root grafting. It has 

been reported that the accumulation of gel and tyloses induced by R. lauricola in the xylem was related to 

disease symptom development. However, movement of R. lauricola inside host trees has not been clearly 

elucidated. Whether the fungal pathogen moves to host leaves and reversibly move from leaves to other 

parts of a host tree is still unknown. We demonstrated that the fungus moves to tree leaves from stems, 

which is evidenced by the fact that the fungus was isolated from the leaves of stem-inoculated trees. At the 

same time, we also discovered that R. lauricola is able to be translocated from leaf to leaf and further to 

the entire tree. We isolated the fungus from non-inoculated leaves and induced systemic wilt in trees after 

inoculation of R. lauricola into tree leaves. This is the first report on bilateral movement of R. lauricola

inside host trees. This study further sheds light on mechanisms of laurel wilt disease development.

1School of Forest Resources and Conservation, University of Florida, Gainesville, FL 33620, (zjl198@ufl.edu). 2Research Fellow at The Morton Arboretum, Lisle, IL 60532.

3 School of Forest Resources and Conservation, University of Florida, Gainesville, FL 33620, (jasons@ufl.edu).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.

Session 6: Genetic Map-Based Approaches to Identify Resistance Genes 57

Session

GENETIC MAP-BASED APPROACHES TO 

IDENTIFY RESISTANCE GENES 6
58 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

RESISTANCE TO HETEROBASIDION ANNOSUM S.L. IN PICEA ABIES IS 

ASSOCIATED WITH A MORE ACTIVE ALLELE OF PaLAR3,

A LECUANTHOCYANIDIN REDUCTASE GENE

Title presented at workshop:

DO ALLELE-SPECIFIC EXPRESSION PATTERNS CONTROL RESISTANCE TO 

HETEROBASIDION ANNOSUM IN PICEA ABIES?

Miguel Nemesio-Gorriz1,7, Almuth Hammerbacher 2, Kerstin Dalman1, Julia Wind3,

Åke Olson1, Martin Lascoux4, Bo Karlsson5, Jan Stenlid1, Ines Ezcurra3,

Jonathan Gershenzon6, and Malin Elfstrand1

Norway spruce [Picea abies (L.) Karst.] wood is 

economically important to the European forest 

industry and infections by the Heterobasidion 

annosum s.l. species complex, and particularly 

Heterobasidion parviporum the major forest 

pathogen on Norway spruce cause substantial 

losses. The economic losses to this pathogen 

amounts to approximately 200 0000 USD every 

day, only in Sweden. One major route for infection 

is carryover between rotations on already 

infected forest sites. Thus, replantation material 

with improved resistance could be a valuable 

resource in the management of H. annosum s.l. 

There is sufficient genetic variation in resistance 

against H. annosum s.l in Norway spruce for 

substantial progress to be made through genetic 

selection. The variation in resistance in Norway 

spruce is quantitative in its nature, and the 

genetic component explains 10–46 percent of the 

variation (Arnerup et al. 2010; Chen et al. 2018). 

No adverse correlations have been found between 

growth and resistance which makes resistance an 

attractive trait to implement in breeding programs 

(Chen et al. 2018). Resistance of P. abies to 

H. annosum s.l., is linked to a number quantitative 

trait loci (QTL) in the host (Lind et al. 2014). 

We have examined one of the QTLs for variation 

in fungal growth in sapwood (FGS) reported 

by Lind et al. (2014) in 14 unrelated Norway 

spruce families from Northern Europe. The 

QTL includes PaLAR3, a leucoanthocyanidin 

reductase gene, (Nemesio-Gorriz et al. 2016). 

We found two conserved PaLAR3 allelic 

lineages in Norway spruce and higher resistance 

to H. parviporum was associated with the 

minor allele PaLAR3B in all studied families. 

Trees carrying at least one copy of PaLAR3B

showed a significant reduction in FGS after 

inoculation with H. parviporum compared to 

their half-siblings homozygous for the major 

allele. Progenies homozygous for PaLAR3B

have significantly higher levels of (+)-catechin, 

the catalytic product of PaLAR3, in their bark 

than their siblings homozygous for PaLAR3A. 

Differences in transcriptional regulation between 

alleles is a likely explanation for the resistance 

associated with PaLAR3B. The two isoforms 

of the PaLAR3 protein showed similar in vitro 

catalytic properties, but allele-specific transcript 

levels were significantly higher for PaLAR3B

in the heterozygous progenies (Nemesio-Gorriz 

et al. 2016). Expression profiling suggests that 

PaLAR3 is part of a wider transcriptional network 

responding to H. annosum s.l. infection in Norway 

spruce involving a number of transcription factors 

possibly interacting differently with promoters of 

the two PaLAR3 allelic lineages in Norway spruce 

(Dalman et al. 2017, Nemesio-Gorriz et al. 2017).

1 Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, 75007 Uppsala, Sweden, 

(miguel.nemesiogorriz@teagasc.ie). 2 Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute, University of Pretoria, 0002 Pretoria, 

South Africa.

3KTH Biotechnology, Royal Institute of Technology, AlbaNova University Centre, Stockholm, Sweden.

4 Department of Ecology and Genetics, Evolutionary Biology Centre, Uppsala University, 75236 Uppsala, Sweden.

5 Skogforsk, Ekebo, 268 90 Svalöv, Sweden.

6 Department of Biochemistry, Max Planck Institute for Chemical Ecology, D-07745 Jena, Germany.

7Teagasc Food Research Centre, Dublin 15, D15 KN3K, Ireland.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 6: Genetic Map-Based Approaches to Identify Resistance Genes 59

LITERATURE CITED

Arnerup, J.; Swedjemark, G.; Elfstrand, M. [et al.]. 2010. 

Variation in growth of Heterobasidion parviporum in a fullsib family of Picea abies. Scandinavian Journal of Forest 

Research. 25: 106–110.

Chen, Z-Q.; Lundén, K.; Karlsson, B. [et al.]. 2018. Early 

selection for resistance to Heterobasidion parviporum in 

Norway spruce is not likely to adversely affect growth 

and wood quality traits in late-age performance. European 

Journal of Forest Research. 137: 517–525.

Dalman, K.; Wind, J.J.; Nemesio-Gorriz, M. [et al.]. 2017. 

Overexpression of PaNAC03, a stress induced NAC 

gene family transcription factor in Norway spruce leads 

to reduced flavonol biosynthesis and aberrant embryo 

development. BMC Plant Biology. 17: 6.

Lind, M.; Källman, T.; Chen, J. [et al.]. 2014. A Picea abies

linkage map based on SNP markers identifies QTLs for 

four aspects of resistance to heterobasidion parviporum

Infection. PLoS ONE. 9: e101049.

Nemesio-Gorriz, M.; Hammerbacher, A.; Ihrmark, K. 

[et al.]. 2016. Different alleles of a gene encoding 

Leucoanthocyanidin Reductase (PaLAR3) influence 

resistance against the fungus Heterobasidion parviporum in 

Picea abies. Plant Physiology. 171: 2671–2681.

Nemesio-Gorriz, M.; Blair, P.B.; Dalman, K. [et al.]. 2017. 

Identification of Norway Spruce MYB-bHLH-WDR 

transcription factor complex members linked to regulation 

of the flavonoid pathway. Frontiers in Plant Science. 8: 

305. 
60 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

DEVELOPMENT AND APPLICATION OF HIGH DENSITY GENETIC MAP OF LIMBER PINE 

(PINUS FLEXILIS JAMES) FOR GENOMICS-BASED BREEDING

Jun-Jun Liu1, Anna W. Schoettle2, and Richard A. Sniezko3

Development and adaptation of genomics-based tools, such as high-throughput genotyping, high-density 

genetic maps, marker-assisted selection, genome-wide association of genotype versus phenotype, 

breeding by design, breeding without breeding, and genomic selection is moving conventional breeding 

into a new era. Next-generation sequencing (NGS)-based RNA-seq, exome-seq, and related technologies 

provide very effective approaches for detection of genome-wide variations and identification of DNA 

markers for marker-assisted selection in breeding of forest resistance against pathogens and pests. 

Genome-wide mapping of functional genes enhances our understanding of genetic resistance and local 

adaptation to changing climates in living organisms. Limber pine is one of the keystone conifer species 

of high elevation forest ecosystems in North America, and its populations in many regions are seriously 

impacted by the invasive, exotic fungal pathogen Cronartium ribicola, which causes white pine blister 

rust (WPBR) on five-needle pines of the subgenus Strobus. In the present study, we constructed limber 

pine high-density genetic maps by exome-seq, and developed TaqMan arrays for marker-assisted selection 

for breeding programs and field applications. Base on a limber pine reference transcriptome de novo

assembled previously (Liu et al. 2016), hybridization probes were designed to enrich the exome from the 

complex genome. Genetic variations and genotypes were explored by exome-seq in two seed families 

with phenotypic segregation of major gene (Cr4) resistance. Single nucleotide polymorphism (SNP) 

markers were used to construct genetic maps. A total of 9.5K expressed genes, including > 600 NBS-LRR 

genes and > 200 RLK genes, were mapped on 12 linkage groups for a consensus genetic map. Based on 

SNP markers at the Cr4 locus, TaqMan SNP arrays were developed for marker-assisted selection. Using 

these genomic tools, we revealed genetic relationships of major gene resistance between limber pine seed 

families originated in Canada and the United States, as well as between limber pine (Cr4) and other fiveneedle pines (including southwestern white pine Cr3 and western white pine Cr2). Genomic resource and 

practical tools developed here will benefit breeding and genetic conservation programs and facilitate the 

genome-wide association study and assembly of the full-length genomes in limber pine and related Pinus

species. 

LITERATURE CITED

Liu J-J; Schoettle, A.W.; Sniezko, R.A. [et al]. 2016. Genetic mapping of Pinus flexilis major gene (Cr4) for resistance 

to white pine blister rust using transcriptome-based SNP genotyping. BMC Genomics. 17: 753.

1Canadian Forest Service, Natural Resources Canada, 506 West Burnside Road, Victoria, British Columbia, V8Z 1M5, Canada, (jun-jun.liu@

canada.ca). 2Rocky Mountain Research Station, USDA Forest Service, Fort Collins, CO 80526.

3Dorena Genetic Resource Center, USDA Forest Service, Cottage Grove, OR 97424.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 6: Genetic Map-Based Approaches to Identify Resistance Genes 61

GENETIC MAPPING AND FUNCTIONAL GENOMICS ANALYSES OF THE 

RESISTANCE/SUSCEPTIBLE RESPONSE IN CHESTNUT SEEDLINGS 

TO PHYTOPHTHORA CINNAMOMI INFECTION

Tetyana Zhebentyayeva1,2, Andrew Gitto3, Steven Jeffers3, Paul Sisco4, Taylor Perkins5, Hill Craddock5, 

Christopher Saski1,2, Jared Westbrook6, Frederick Hebard6, Laura Georgi 6, Joseph James7, 

Margaret Staton8, C. Dana Nelson9,10, and Albert Abbott10

Due to availability of resistant Asian and susceptible American and European chestnut species, Castanea

is ideally suited for studying plant- Phytophthora cinnamomi (Pc) interactions. We employed a genetic 

approach to map resistance to Pc in interspecific crosses using resistant Chinese chestnut, Castanea 

mollissima (“Mahogany” and “Nanking” resistance donors) and susceptible American chestnut C. dentata

(multiple parents). Five BC1, BC3 and F2 populations (1,435 individuals in total) were phenotyped 

multiple years for root rot severity and genotyped by reduced genome-representative sequencing. Using 

extensive sets of markers anchored to the C. mollissima genome v1.0 assembly, we constructed genetic 

maps and identified genomic regions underlying resistance to Pc in multiple crosses. The most consistent 

QTL signals in crosses with both Mahogany and Nanking backgrounds were detected in the lower part of 

LG E. Based on performance in years 2015 and 2016, we selected Nanking-derived F2 reciprocal crosses 

NK5 and NK6 segregating for healthy:unhealthy:dead trees in a ratio 1:1:1 (P < 0.05) for further studies 

of plant-Pc interaction using transcriptomics and metabolomics approaches. In a pilot experiment we 

challenged 1-year old NK5 progeny with Pc and harvested root tissue at 7 time-points (0, 1, 2, 3, 5, 7 and 

14 days) post inoculation. Non-inoculated plants were used as controls along with inoculated Chinese and 

American chestnut seedlings. Inoculated plants were monitored for progression of root rot symptoms and 

the three most resistant and three most susceptible plants were chosen for RNAseq analysis and metabolite 

profiling. Results of these experiments and the initial mapping experiments will be presented.

1 Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, (tnz4@psu.edu).

2 Laboratory of Genomics & Computational Biology, Clemson University, Clemson, SC 29634.

3 Plant and Environmental Sciences Department, Clemson University, SC 29634.

4The American Chestnut Foundation, Carolinas Chapter, Asheville, NC 28804.

5Department of Biology, Geology, and Environmental Science, University of Tennessee Chattanooga, TN 37403.

6The American Chestnut Foundation, Asheville, NC 28804.

7The Chestnut Return Farm, Seneca, SC 29678.

8 Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN 37996.

9 Southern Institute of Forest Genetics, Southern Research Station, USDA Forest Service, Saucier, MS 39574.

10 Forest Health Research and Education Center, Southern Research Station, USDA Forest Service, Lexington, KY 40506. 

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
62 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

MULTIPLE APPROACHES TO DISSECT FUSIFORM RUST 

RESISTANCE IN PINUS TAEDA L.

Daniel Ence1, Katherine E. Smith1,2, Robin Paul4, Matias Kirst1, 

Jill L. Wegrzyn3, C. Dana Nelson2, and John M. Davis1

Mapping the specific loci that regulate phenotypic traits in conifers is a major undertaking because of 

their very large genomes. However, the release of the annotated loblolly pine (Pinus taeda L.) genome may 

allow fine-mapping of Mendelian traits that are economically critical, such as for disease-resistance. Here 

we present the results of work mapping Fusiform rust resistance locus 1 (Fr1) in elite rust resistant loblolly 

pine trees. Fusiform rust is a disease incited by the fungus Cronartium quercuum f.sp. fusiforme (Cqf) 

on southern pines (where it causes galls on stems and branches) and on oaks (where it causes minimal 

leaf damage). Fusiform rust is a major disease threat to the timber industry in the United States. Rust 

galls cause yield losses that exceed US$100M/year. During the genome annotation process, an expressed 

sequence tag (EST) was identified that contains a single nucleotide polymorphism (SNP) mapping to 

the locus (Fr1) that interacts with the fungal avirulence gene, Avr1. This EST aligns to a transcript from 

RNA-sequencing data and a TIR-NB-LRR protein, thus identifying it as a candidate Fr1 gene. In order to 

further characterize the Fr1 locus, we assembled the transcriptomes of 92 elite rust-resistant loblolly pine 

genotypes from five pine-growing regions, identifying candidate resistance genes in the process. Next we 

aligned these transcripts to the loblolly pine genome and calculated population genetic parameters. These 

results enable analysis of the diversity and conservation of resistance genes that interact with Cqf and 

present a foundation for further characterization of Fr1 and other resistance loci.

1School of Forest Resources and Conservation, University of Florida, Gainesville, FL 33024.

2 Southern Institute of Forest Genetics, Southern Research Station, USDA Forest Service, Saucier, MS 39574.

3Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT 06269.

4 Department of Medicine, School of Medicine, Indiana University, Indianapolis, IN 46202, (jmdavis@ufl.edu).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 7: Integrating Genomics/Biotechnology with Traditional Tree Breeding 63

Session

INTEGRATING GENOMICS/BIOTECHNOLOGY 

WITH TRADITIONAL TREE BREEDING 7
64 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

THE POTENTIAL FOR BIOTECHNOLOGY TO ADDRESS FOREST HEALTH

Title presented at workshop

OVERVIEW ON THE POTENTIAL FOR BIOTECHNOLOGY TO ADDRESS FOREST HEALTH— 

BASED ON PENDING NATIONAL ACADEMY OF SCIENCE, ENGINEERING AND MEDICINE REPORT

:

Kara N. Laney1

The National Academies of Sciences, Engineering, and Medicine is conducting a consensus study on the 

potential for biotechnology to address forest health. A committee of 13 experts from diverse disciplines 

have collaborated to examine the potential use of biotechnology to mitigate threats to forest tree health; 

identify the ecological, ethical, and social implications of deploying biotechnology in forests; and develop 

a research agenda to address knowledge gaps about its application. In particular, the committee has 

considered the use of biotechnology to prevent the extirpation of a tree species by an insect or disease that 

could have negative consequences for forest health. The study includes the committee’s definition of forest 

health, a review of the state of the science for tree biotechnology and other tools for improving forest 

health, and an overview of the unique challenges and opportunities of using biotechnology to address 

forest health. To accomplish the goals of the study, the committee has held information-gathering meetings 

on a wide range of topics, including the ethics of using biotechnology in conservation, Native American 

perspectives on using biotechnology in forests, and how forest trees modified with biotechnology 

are handled in the U.S. regulatory system. The study will culminate in a report, Forest Health and 

Biotechnology: Possibilities and Considerations, published in early 2019. The final report can be found at 

https://doi.org/10.17226/25221. Funding for the study was provided by the U.S. Department of Agriculture, 

the U.S. Environmental Protection Agency, and the U.S. Endowment for Forestry and Communities. More 

information can be found at nas.edu/forest biotech.

1The National Academies of Sciences, Engineering, and Medicine, Washington, DC 20001, (klaney@nas.edu).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 7: Integrating Genomics/Biotechnology with Traditional Tree Breeding 65

RESTORATION OF AMERICAN CHESTNUT: A MARRIAGE 

OF BREEDING AND BIOTECHNOLOGY

Jared W. Westbrook1, Jason A. Holliday2, and William A. Powell3

Efforts to restore the American chestnut are approaching major milestones. Selection of the most blightresistant backcross trees in The American Chestnut Foundation’s (TACF’s) seed orchards at Meadowview 

Research Farms is expected to be complete between 2020 and 2025. Progeny testing with small stem 

assays and genomic selection are being used to increase the speed and accuracy of selection within seed 

orchards. The offspring from selected parents are predicted to have blight resistance that is, on average, 

intermediate between American chestnut and Chinese chestnut. Meanwhile, collaborators at the State 

University of New York have demonstrated that transgenic American chestnut containing the oxalate 

oxidase (OxO) gene has blight resistance that equals that of Chinese chestnut. Preparation for federal 

regulatory review for the release of transgenic American chestnut is ongoing. Beyond these milestones, 

blight resistance in backcross populations will be improved through two additional generations 

intercrossing and selection. Pending regulatory approval, the transgenic founder tree will be outcrossed 

to pure American chestnut and backcross trees over three generations with the objectives of (1) stacking 

blight resistance alleles from backcross trees with OxO, (2) combining OxO with backcross resistance to 

Phytophthora root rot (PRR), (3) creating a restoration population with an effective population size > 500. 

American chestnuts will be conserved in orchards for eventual outcrossing with transgenic trees by 

collecting seeds and transplanting from regions that are most genetically diverse and underrepresented 

by TACF’s breeding program. A diversified American chestnut population in which transgenic blight 

resistance is combined with backcross blight and PRR resistance is expected to be available for restoration 

by 2050. While backcross seed from selected seed orchards and first generation transgenic progeny are 

expected to be available for restoration trials in the next 5 years, long-term commitment to continued 

breeding and selection is required for full-scale restoration.

The full paper was published in Plants, People, Planet

https://nph.onlinelibrary.wiley.com/doi/full/10.1002/ppp3.10061

The reference for the published paper is:

Westbrook, J.W,; Holliday, J.A.; Newhouse, A.E,; Powell, W.A. 2020. A plan to diversify a transgenic blight‐tolerant 

American chestnut population using citizen science. Plants, People, Planet. 2: 84–95. https://doi.org/10.1002/

ppp3.10061.

1The American Chestnut Foundation, Asheville, NC 28804, (jared.westbrook@acf.org). 2 Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.

3 State University of New York-Environmental Science and Forestry, Syracuse, NY 13210.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
66 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

NEW FRONTIERS IN FORESTRY: COMBINING PHENOMICS, COMMON GARDENS, 

AND LANDSCAPE GENOMICS TO ADDRESS DISEASE RESISTANCE 

AND CLIMATE CHANGE

Jeremy S. Johnson1,2, Sam Cushman3, Andrew Eckert4, Lluvia Flores-Rentería5, Richard Sniezko2,

 Christopher Still6, Christian Wehenkel7, Amy Whipple8, and Michael Wing6, Justin Bagley4, 

Emily Boes2, Ethan Bucholz1, Jonathan Burnett6, Antonio Castilla2, Jessica DaBell1, 

Cory Garms6, Marja Haagsma9, Evan Heck2, Erin Landguth10, Alejandro Leal Sáenz7, 

Jared Swenson1, Brianna McTeague2, Mitra Menon4, Ehren Moler8, 

Gerald Page6, Andrew Shirk11, and Kristen Waring1

A collaborative team of researchers from the United States and Mexico have begun a project funded by 

The National Science Foundation’s Macrosystems Biology program. The project is to study ecological 

and evolutionary processes affecting the distribution of southwestern white pine, an important tree 

species of mixed conifer forests in the Southwest United States and Mexico. Southwestern white pine 

(Pinus strobiformis) sustainability is threatened by changing climate, and an invasive non-native tree 

disease, white pine blister rust. White pine blister rust causes extensive tree decline and mortality where 

it occurs in North America, including where it overlaps with southwestern white pine, an ever-expanding 

area. Climate may advance too rapidly for southwestern white pine to move or adapt. The dual threats of 

climate change and an invasive species make forecasting future tree distributions across continental scales 

an urgent challenge. The goal of this project is to determine how gene movement among populations, 

adaptation to disease and drought, epigenetic changes, and a changing environment interact to govern 

the success of southwestern white pine. This project is developing tools to help forecast and manage the 

future of the species, including genomics, common gardens, disease resistance testing, phenomics, and 

computer modeling in landscape ecology and genomics. We show how the project is combining genomics 

and traditional common garden approaches and its potential management applications for southwestern 

white pine.

1School of Forestry, Northern Arizona University, Flagstaff, AZ 86011, (Jeremy.johnson@nau.edu). 2 Dorena Genetic Resource Center, USDA Forest Service, Cottage Grove, OR 97424.

3Rocky Mountain Research Station, USDA Forest Service, Flagstaff, AZ 86001.

4 Department of Biology, Virginia Commonwealth University, Richmond, VA 23284.

5 Department of Biology, San Diego State University, San Diego, CA 92182.

6 Department of Forest Ecosystems and Society, Oregon State University, Corvallis, OR 97331.

7Universidad Juárez del Estado de Durango, Instituto de Silvicultura e Industria de la Madera, Durano, MX.

8 Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011.

9Water Resources Engineering, Oregon State University, Corvallis, OR 97331.

10 Division of Biological Sciences, University of Montana, Missoula, MT 59812. 11Climate Impacts Group, University of Washington, Seattle, WA 98195.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 7: Integrating Genomics/Biotechnology with Traditional Tree Breeding 67

WISE USE OF GENOMICS IN IMPROVING FOREST HEALTH

Jeanne Romero-Severson1

Over the last several hundred years, colonialism and international trade have had the unintended 

consequence of introducing destructive insects, microbial pathogens and invasive weeds into native 

forests throughout the world. Programs intended to save native forest species from destructive pests 

are often led by single discipline investigators who focus first on the insect or the pathogen, then on 

methods of external control (i.e., containment, pesticides and biocontrol agents). Investigation of genetic 

mechanisms using structured populations may be dismissed as “too expensive” while genomics costing 

at least as much if not more may be embraced without serious evaluation of the likelihood of success. The 

interdisciplinary approach to pests and pathogens used by the most successful private sector companies 

frequently succeeds and not for the reason you might think. These successful approaches include costbenefit analysis of all strategies, including traditional breeding and genomics.

1Notre Dame University, South Bend, IN 46556, (Jeanne.Romero-Severson.1@nd.edu).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.

Session 8: Screening for Resistance to Pathogens: Methods Development 69

Session

PATHOGENS: METHODS DEVELOPMENT

SCREENING FOR RESISTANCE TO 

8
70 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

VIRULENCE OF SPHAERULINA MUSIVA ISOLATES DETERMINED BY GENOME-WIDE 

ASSOCIATION MAPPING, HOST INFECTION, AND COLONIZATION 

Susanna Keriö1, Kelsey L. Søndreli1, and Jared M. LeBoldus1,2

The ascomycete Sphaerulina musiva causes Septoria leaf spot and stem canker of Populus, and is 

considered to be the most serious pathogen affecting poplar cultivation in North America. Knowledge 

of S. musiva virulence factors can promote the development of efficient control strategies against the 

disease, and improve the accuracy of risk analysis. In this project, we characterized the virulence of 

120 S. musiva isolates from geographically distinct populations in a non-wounding spray inoculation 

experiment. The isolates were sequenced (Illumina 150bp Paired End HiSeq 3000) and used for genomewide association mapping. In a second set of experiments, we selected a subset of six S. musiva isolates 

with either high or low virulence, which were used to inoculate two genotypes (GW9823 and GW11026) 

of black cottonwood (Populus trichocarpa). The degree of host colonization was estimated by qPCR, and 

qualitative differences in host colonization were analyzed by microscopic inspection. We will report the 

genes associated with the observed virulence differences, and discuss the significance of the results from 

the second set of experiments in the context of disease resistance breeding.

1 Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, (kerios@oregonstate.edu). 2 Department of Forest Engineering, Resources and Management, Oregon State University, Corvallis, OR 97331.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 8: Screening for Resistance to Pathogens: Methods Development 71

EARLY SCREENING POTENTIALLY BLIGHT-RESISTANT 

AMERICAN CHESTNUT USING SMALL STEM ASSAYS

Thomas Saielli1 and Jared Westbrook2

Restoration of the American chestnut (Castanea dentata) depends on producing a founder population of 

trees that are sufficiently disease-resistant to provide self-sustaining populations that have the timbertype growth and form of American chestnut. For 30 years, The American Chestnut Foundation (TACF) 

has pursued backcross breeding to introgress genetic resistance to chestnut blight (caused by the fungal 

pathogen Cryphonectria parasitica) from Chinese chestnut (C. mollissima) into a genetically diverse 

population of American chestnut. The final step in TACF’s backcross breeding program, prior to species 

restoration, is to establish multiple, large-scale seed orchards throughout the Eastern United States. 

Although many thousands of seedlings are planted in each seed orchard, once seedlings are artificially 

inoculated and assessed, only the few hundred trees with demonstrated high levels of resistance are kept. 

Initial screening may be performed by visual inspection of cankers, but the final candidates must be 

progeny tested in order to select the best trees in the orchards. The small stem assay (SSA) is one method 

being tested to progeny test large numbers of chestnut seedlings for resistance, which, when compared to 

orchard progeny tests, may allow for a greater number of seedlings to be tested in less time. In the small 

stem assay, the stems of chestnut seedlings > 3 mm in diameter are inoculated with C. parasitica and 

canker length is measured every 6 to 8 weeks for several months. In 2017, TACF tested approximately 

5,180 seedlings among 95 hybrid families. Results indicate that canker length was heritable among test 

families (h2 = 0.30 ± 0.09) and variation in canker length was significantly correlated between SSA tests 

and orchard progeny tests (rgenetic = 0.84 ± 0.74). TACF will continue to experiment with SSA techniques, 

as many tactical questions remain and results were not consistent throughout all tests. 

1The American Chestnut Foundation, Charlottesville, VA 22901, (tom.saielli@acf.org). 2The American Chestnut Foundation, Asheville, NC 28804.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
72 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

DEFENSE RESPONSES OF AUSTRIAN PINE TO TWO OPPORTUNISTIC PATHOGENS 

OF CONTRASTING AGGRESSIVENESS UNDER COMBINED 

DROUGHT AND TEMPERATURE STRESS

Pierluigi Bonello1, Anna Conrad1, Jason Slot1, 

Erik Andrei Visser 2, and Sanushka Naidoo2

Understanding the effects of climate change on host pathogen relationships is key to managing diseases 

in new weather extremes expected in the future, especially among long-lived perennials like trees. For 

example, trees under drought stress become more susceptible to pathogens as resources are allocated 

towards basic survival rather than chemical defenses, thus creating more favorable conditions for disease 

development. Our study investigates the defense mechanisms underlying the responses of Austrian 

pine (Pinus nigra) to the tip blight and canker pathogens Diplodia sapinea and D. scrobiculata under 

combined, elevated temperature, and drought; two conditions that are projected to become the norm in 

many areas of the world. Among the defense mechanisms likely to be affected by temperature and water 

restrictions are those involving specialized metabolites, specifically phenolics, which are modulated, in 

part, by the particular amino acid pathways associated with the response to drought. We are analyzing 

gene expression and amino acid metabolism, as well as accumulation/depletion of soluble phenolics and 

lignin. Increased understanding of the interactions between hosts and pathogens undergoing climate stress 

will contribute to the development of integrated management strategies, such as implementing updated 

monitoring/detection programs, creating predictive models, and furthering our understanding of host 

resistance mechanisms.

1 Department of Plant Pathology, The Ohio State University, Columbus, OH 43210, (bonello.2@osu.edu). 2 FABI, Department of Biochemistry, Genetics & Microbiology, University of Pretoria, Hatfield, South Africa 0028.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 9: Mechanisms of Resistance to Insects 73

Session

MECHANISMS OF RESISTANCE TO INSECTS

9
74 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

ARE NOVEL PLANT CHEMICALS FRIENDS OR FOES OF NATIVE INVASIVE INSECT 

HERBIVORES? EXPLAINING SUCCESSFUL HOST RANGE EXPANSION OF 

MOUNTAIN PINE BEETLE IN CANADA’S BOREAL FORESTS

Nadir Erbilgin1

Mountain pine beetle has recently breached the geo-climatic barrier of the northern Rocky Mountains and 

invaded in the jack pine (Pinus banksiana) forests in Canada. The jack pine forest ecosystem is considered 

a novel habitat. However, the mechanism underlying this successful host expansion is unknown, but 

likely involves phytochemicals that play significant roles in the acceptance and colonization of hosts by 

insect herbivores and their microbial symbionts. We have focused on whether compatibility of jack pine 

chemicals with beetles and their symbiotic fungi has facilitated the colonization of this novel host and 

identified five mechanisms that have likely facilitated the beetle host range expansion. First, jack pine 

trees appeared to have less pronounced chemical defenses than a historical host of MPB (lodgepole pine, 

P. contorta). Second, prior to the arrival to the jack pine forests, invasion of a zone of hybrids of jack 

and lodgepole pine trees in Alberta by beetles likely improved their success in the jack pine as hybrids 

show chemical characteristics of both novel and historical hosts. Third, fungal and bacterial symbionts of 

beetles likely contributed beetle performance in the jack pine by detoxifying toxic secondary compounds 

as jack pine was compatible for the growth of both the fungi and bacteria. Fourth, similarity of secondary 

compounds and fatty acids between the novel and historical hosts may have facilitated the host expansion 

of beetles because of compatibility of these chemical compounds for pheromone production, aggregation 

on the host trees, larval development, and the growth of its fungal symbionts. Finally, jack pine contained 

low concentrations of defense and inhibitory compounds, and high concentrations of precursor and 

synergistic compounds that make historical host trees susceptible to beetles. I conclude that compatibility 

of primary and secondary chemical composition of jack pine to MPB and its symbiotic fungi has likely 

facilitated the host range expansion.

The full paper was published in New Phytologist.

The reference for the published paper is:

Erbilgin, N. 2018. Phytochemicals as mediators for host range expansion of a native invasive forest insect herbivore. 

New Phytologist. 1(3): 1268–1278 doi: https://10.1111/nph.15467.

1 Department of Renewable Resources, University of Alberta, Edmonton, Alberta Canada, (erbilgin@ualberta.ca).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 9: Mechanisms of Resistance to Insects 75

VOLATILES RELEASED BY PINUS PINASTER HALF-SIB GENETICALLY CONTRASTING 

FAMILIES FOR PINE WILT DISEASE SUSCEPTIBILITY BEFORE AND DURING 

MONOCHAMUS GALLOPROVINCIALIS INSECT-VECTOR FEEDING

Elsa Gonçalves1, A. Cristina Figueiredo1, Isabel Carrasquinho2,3, José G. Barroso1, 

Maria l. Inácio2, Joana Henriques2, Edmundo Sousa2, and Luís Bonifácio2

The pine wilt disease (pwd), caused by the pinewood nematode (PWN) bursaphelenchus xylophilus, is a 

major threat to conifer trees worldwide. The PWN is transmitted from infected to healthy trees by insect 

vectors in the cerambycid beetle genus monochamus. The PWD cycle involves the movement of the 

nematode between the insect vector and the host tree. PWN leaves the insect and enters the host through 

feeding wounds made by the insect. This study analyzed the volatiles released by genetically contrasting 

maritime pine half-sib families with susceptibility to PWD, before and during insect vector feeding 

activity, which can act as signals for the nematode to move into healthy trees. Thirty-eight pine trees 

from four half-sib families were selected among 96 different families. The canopy of each of the 7 year 

old pines was covered by a metallic net and foliage was enclosed in a plastic bag to collect the headspace 

volatiles, before and during 24 h exposure to feeding by a pair of newly emerged m. galloprovincialis

males and females. Volatiles were collected by solid phase microextraction and analyzed by gas 

chromatography-mass spectrometry for component identification, and by gas chromatography for 

component quantification. For comparing the different families, the non-parametric kruskal-wallis test 

and dunn’s test with bonferroni-type adjustment were used. Seventeen volatile compounds were detected 

both before and during feeding. Before insect feeding, β-pinene, β-myrcene, β-caryophyllene, phenethyl 

2-methylbutyrate, and phenethyl 3-methylbutyrate showed significant differences among the contrasting 

families. During feeding, only β-myrcene and germacrene d showed different emission patterns among 

them. In this case, the most pwn susceptible family released significantly higher amounts of β-pinene and 

β-caryophyllene, and lower amounts of β-myrcene than the resistant cultivars.

1Centro de Estudos do Ambiente e do Mar (CESAM Lisboa), Faculdade de Ciências da Universidade de Lisboa (FCUL), Centro de 

Biotecnologia Vegetal (CBV), Departamento de Biologia Vegetal (DBV), C2, Campo Grande, 1749-016, Lisboa, Portugal.

2 Instituto Nacional de Investigação Agrária e Veterinária (INIAV), Quinta do Marquês, 2780-159 Oeiras, Portugal, (isabel.carrasquinho@

iniav.pt). 3Linking Landscape, Environment, Agriculture and Food (LEAF). Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da 

Ajuda, 1349-017 Lisboa, Portugal.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
76 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

HEMLOCK AND HEMLOCK WOOLLY ADELGID: PAST RESEARCH, CURRENT FINDINGS, 

AND FUTURE DIRECTIONS IN A TROUBLED NORTH AMERICAN 

TREE-PEST SYSTEM

Chad M. Rigsby1, Richard A. Casagrande2, Colin M. Orians3, and Evan L. Preisser1

Hemlock woolly adelgid (HWA) (Adelges tsugae) was first introduced to the East Coast of North America 

in the 1950’s and has since spread throughout roughly half of the native range of its primary host, 

eastern hemlock (hemlock; Tsuga canadensis), which is under threat of extirpation due to this insect. 

Here, we summarize past and current work that our research group has undertaken and address future 

research directions and management concerns in this system. Over the past several years, one aspect 

of our research has been investigating HWA-hemlock interactions and HWA interactions with other 

hemlock herbivores. Adelgid infestation results in a “hypersensitive-like” response in hemlock foliage, 

with the accumulation of reactive oxygen species, certain amino acids, hormones, defensive metabolites 

and enzymes, and increased volatile emissions, a response that more resembles pathogen infection than 

herbivore attack. The infestation of HWA additionally results in deleterious phenological, growth, and 

belowground effects on hemlock. Adelgid interacts with other native and exotic hemlock herbivores, 

presumably due to the manipulation of host physiology, and appears to make hemlock more attractive 

to certain herbivores. Our research group has also led efforts to develop HWA-resistance in hemlock. 

Unfortunately, decades-long efforts to develop effective biological controls have not yet proven effective 

on highly susceptible eastern hemlocks. However, hundreds of hemlock trees have been found persisting 

in HWA-decimated forests throughout the East Coast, and some have remained healthy for decades. We 

are leading efforts to screen persisting trees for elevated levels of resistance and to propagate trees to 

supply stakeholders, tree breeders, and scientists with resistant germplasm. We also utilize these trees to 

develop phenotyping assays for the rapid identification of resistance in the field. We strongly advocate for 

the development of resistance and the breeding of resistant hemlocks as part of a long-term management 

strategy for hemlock.

1 Department of Biological Sciences, The University of Rhode Island, Kingston, RI 02881, (rigsby@uri.edu). 2 Department of Plant Sciences and Entomology, The University of Rhode Island, Kingston, RI 02881.

3Department of Biology, Tufts University, Medford, MA 02155.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 9: Mechanisms of Resistance to Insects 77

A HINT LEFT BY MOUNTAIN PINE BEETLE ON ANATOMICAL DEFENSES OF LODGEPOLE PINE 

TREES: LARGER RESIN DUCTS ENHANCE TREE RESISTANCE

Shiyang Zhao1 and Nadir Erbilgin2

Mountain pine beetle (MPB) [Dendroctonus 

ponderosae Hopkins (Coleoptera: Curculionidae)] 

populations have increased in Alberta since 

they have crossed the Rocky Mountains and 

colonized pine forests in western Alberta in 

2006. Millions of mature lodgepole pine (Pinus 

contorta var. latifolia) trees have been killed, 

leaving only a low number of residual overstory 

mature lodgepole pine trees and non-host tree 

species, such as spruce and Populus, remaining in 

beetle-killed stands (Taylor et al. 2006, Dhar et al. 

2016). The reason behind the survival of residual 

trees remains unknown. The change in nutrient 

cycling, underground soil communities, forest 

structure, and other factors in post-MPB stands 

can certainly affect the health condition of residual 

pine trees and potentially change their suitability 

to other insects and tree pathogens (McIntosh and 

Macdonald 2013b, Cigan et al. 2015, Karst et al. 

2015, Pec et al. 2015). Considering the low pine 

recruitment and regeneration in MPB-affected 

stands (Astrup et al. 2008, McIntosh and 

Macdonald 2013a), the residual overstory pine 

trees might be the only seed source for the future 

pine regeneration in Alberta. Thus, studies on the 

future health conditions of residual pine trees in 

these disturbed landscapes are urgently needed. 

While concentration of chemical compounds 

in tree phloem can vary between resistant and 

susceptible lodgepole pine trees (Erbilgin et 

al. 2017), anatomical defense represents tree 

defense capacities over a longer time. Here we 

analyzed patterns in anatomical defenses and tree 

increment growth to understand (1) how residual 

trees survived the MPB outbreak, (2) whether the 

outbreak altered growth/defense relationships, 

and (3) identify relationships with current health 

conditions of residual trees. 

We selected 31 sites in post-MPB stands in 

western Alberta, Canada. At most of the selected 

sites we established 2 plots (n = 61 plots in total). 

In plots, we sampled the wood from 140 beetlekilled trees using wedges and 210 residual trees 

using increment cores at breast height (1.4 m). 

Samples were collected in 2016. All trees had a 

diameter at breast height (DBH) over 15 cm and 

had a crown class of intermediate, codominant, 

or dominant. We confirmed MPB-killed tree by 

the presence of MPB attack signs such as beetle 

entrance holes (pitch tubes) and extensive beetle 

galleries under bark. The sampled residual trees 

were classified into three groups based on tree 

health conditions and included 76 healthy, 62 

declining due to biotic agents other than MPB, and 

72 trees that survived MPB with signs of attack 

but appeared vigorous. 

We measured ring width (mm yr-1) from bark 

to pith on all samples by using WinDendroTM 

(Regent Instruments 2008). A master chronology 

was developed based on the ring width series of 

cores from healthy residual trees. The strength 

of cross-dating was confirmed by COFECHA 

(Grissino-Mayer 2001). This master chronology 

was used to justify any missing or false rings on 

cores before any calendar year was assigned to 

each ring. The year of death for beetle-killed trees 

was adjusted by visual cross-dating due to the 

low number of years sampled on wedges. Since 

most sites experienced beetle mortality at multiple 

years, the start of an outbreak was considered as 

the year of the earliest death caused by MPB that 

occurred in a site. 

A sampling area of 0.9 mm for cores and wedges 

was used to count and measure resin ducts. The 

resin duct characteristics that were measured 

1 Department of Renewable Resources, University of Alberta, Edmonton, AB, Canada T6G 2E3, (shiyang1@ualberta.ca). 2 Department of Renewable Resources, University of Alberta, Edmonton, AB, Canada T6E 2E3, (erbilgin@ualberta.ca).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
78 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

from the sampling area included annual resin 

duct production (no. year-1) and individual resin 

duct area (mm2 year-1), which were determined 

by ImageJ (version 1.50i, National institutes of 

Health, USA). Two standardized variables of resin 

duct characteristics were also calculated on each 

core or wedge: resin duct density (no. mm-2 year-1) 

and relative resin duct area (percent year-1). 

There were no differences on mean growth rate 

between beetle-killed and residual trees within 

the first 10-year preceding outbreak. However, 

beetle-killed trees produced 127–131 percent more 

xylem axial resin ducts than residual trees, while 

the size of individual duct in residual trees was 

18–27 percent larger than those in MPB-killed 

trees. A logistic model was built to predict the 

survival probability of lodgepole pine trees 

during the MPB outbreak based on the resin duct 

production and resin duct size within 10 years 

before the MPB outbreak. The survival probability 

increased with larger resin ducts, but less numbers 

of resin ducts. Meanwhile, trees that survived 

attack responded to MPB outbreaks in the stand 

with a lower mean growth rate in the first 3- and 

5-year following outbreak, and higher mean resin 

duct production for up to 10 years after outbreak, 

while healthy trees only showed increased resin 

duct production in the total growth period after 

the MPB outbreak. Furthermore, we found that 

healthy trees had larger individual resin ducts 

than declining trees in the recent 20 years (1996 

to 2015), while survived trees also had larger resin 

ducts than declining trees most of the time from 

1996 to 2015. 

Our results indicated that anatomical defenses 

were critical components of lodgepole pine 

survival during beetle attacks as well as they 

might continue to play major roles in tree defense 

to bark beetles in post-outbreak stands. Thus, pine 

anatomical defenses appear to be important traits 

for understanding tree resistance to bark beetles, 

supporting earlier studies (Kane and Kolb 2010, 

Hood and Sala 2015). Although it is not clear 

whether resin duct size is heritable, but if so, we 

expect that the next generation of lodgepole pine 

forests in western Alberta would be resistant 

to future bark beetle attacks. Using the logistic 

model generated in the current study, the survival 

probability of pine trees during MPB attacks 

could be calculated by determining resin duct 

production and resin duct size. Thus, keeping, and 

not harvesting, these residual trees should be the 

highest priority for land managers to assure the 

future sustainability of lodgepole pine forests in 

western North America.

Funding for this research was provided by fRI 

Research, Alberta Innovates-BioSolutions, 

NSERC–Discovery Award, Forest Resource 

Improvement Association of Alberta and Alberta 

Agriculture and Forestry. Caroline Whitehouse, 

Devin Letourneau, Ryan Hermanutz, Allison 

Brown, and Geoff Atkinson from Alberta 

Agriculture and Forestry provided us with help 

onsite selection and permitting. We also want to 

thank Tolko Industries Ltd. (High Prairie), West 

Fraser Timber Co. Ltd, Blue Ridge Lumber Inc. 

Daishowa-Marubeni International Ltd. (West) and 

Canfor Corporation who gave us permission to set 

up sites in their forest management units. 

LITERATURE CITED

Astrup, R.K.; Coates, K.D.; Hall, E. 2008. Recruitment 

limitation in forests: Lessons from an unprecedented 

mountain pine beetle epidemic. Forest Ecology and 

Management. 256: 1743–1750.

Cigan, P.W.; Karst, J.; Cahill, J.F. [et al.]. 2015. Influence of 

bark beetle outbreaks on nutrient cycling in native pine 

stands in western Canada. Plant Soil. 390: 29–47.

Dhar, A.; Parrott, L.; Hawkins, C.D.B. 2016. Aftermath of 

mountain pine beetle outbreak in British Columbia: stand 

dynamics, management response and ecosystem resilience. 

Forests. 7: 1–19.

Erbilgin, N.; Cale, J.A.; Hussain, A. [et al.]. 2017. Weathering 

the storm: How lodgepole pine trees survive mountain pine 

beetle outbreaks. Oecologia. 184: 469–78.
Session 9: Mechanisms of Resistance to Insects 79

Grissino-Mayer, H.D. 2001. Evaluating crossdating 

accuracy: a manual and tutorial for the computer program 

COFECHA. Tree-Ring Research. 57: 205–221.

Hood, S.; Sala, A. 2015. Ponderosa pine resin defenses and 

growth: Metrics matter. Tree Physiology. 35: 1223–1235.

Kane, J.M.; Kolb, T.E. 2010. Importance of resin ducts in 

reducing ponderosa pine mortality from bark beetle attack. 

Oecologia. 164: 601–609.

Karst, J.; Erbilgin, N.; Pec, G.J. [et al.]. 2015. 

Ectomycorrhizal fungi mediate indirect effects of a bark 

beetle outbreak on secondary chemistry and establishment 

of pine seedlings. New Phytologist. 208: 904–914.

McIntosh, A.C.S.; Macdonald, S.E. 2013a. Potential for 

lodgepole pine regeneration after mountain pine beetle 

attack in newly invaded Alberta stands. Forest Ecology and 

Management. 295: 11–19.

McIntosh, A.C.S.; Macdonald, S.E. 2013b. Short-term 

resistance of ecosystem properties and processes to 

simulated mountain pine beetle attack in a novel region. 

Ecosphere. 4: 1–28.

Pec, G.J.; Karst, J.; Sywenky, N. [et al.]. 2015. Rapid 

increases in forest understory diversity and productivity 

following a mountain pine beetle (Dendroctonus 

ponderosae) outbreak in pine forests. PLoS One. 10: 1–16.

Regent Instruments. 2008. WinDendro. Quebec, Canada: 

Regent Instruments.

Taylor, S.W.; Carroll, A.L.; Alfaro, R.I. [et al.]. 2006. Forest, 

climate and mountain pine beetle outbreak dynamics 

in western Canada. In Safranyik, L.; Wilson, B., eds. 

Mountain pine beetle a synthesis: biological management 

impacts lodgepole pine. Victoria, British Columbia, 

Canada: Pacific Forestry Centre: 67–94.
80 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

VARIATION ACROSS POPULATIONS IN RESISTANCE TO A KEY HERBIVORE AND ITS 

RELATIONSHIP WITH CONSTITUTIVE AND INDUCED SECONDARY METABOLITES 

IN A MEDITERRANEAN PINE TREE

Xosé López-Goldar1,2,4,5, Caterina Villari2,3, Pierluigi Bonello2, Anna-Karin Borg-Karlson4, 

Delphine Grivet5,6, Rafael Zas1, and Luis Sampedro2

Resistance to herbivores and pathogens has been 

recognized as an adaptive key plant trait (Futuyma 

and Agrawal 2009), particularly at early stages 

of plant development (Goodger et al. 2013). In 

long-lived organisms like trees, resistance against 

enemies usually relies on high concentrations of 

a diverse array of plant secondary metabolites 

(PSM) (Wiggins et al. 2016). These compounds 

reduce enemy performance and/or plant 

damage in a dose-dependent manner (Zhao and 

others 2011). PSM production is highly plastic, 

involving substantial changes in the profile 

and concentrations of PSM upon enemy attack 

(Heil 2009, Karban et al. 1999). This biochemical 

plasticity, referred to as inducibility, is a source 

of functional phenotypic variation in resistance 

and is commonly considered a trait under 

selection (Agrawal et al. 2015). However, very few 

studies have addressed the relationship between 

inducibility of defences with effective resistance 

against herbivores (Bingham and Agrawal 2010, 

Rasmann et al. 2015), and much less in longlived plants (Moreira et al. 2014, Ward et al. 

2012). One of the main reasons for this scarcity 

in the literature is that exploring relationships 

between inducibility of PSM and the effective 

resistance against herbivory is challenging. First, 

it requires knowing the concentration of PSM 

before and after exposure to herbivory when 

measuring plant damage or insect performance. 

Moreover, the mere fact of sampling plant tissues 

also induces plant defences, biasing inducibility 

estimation (Moreira et al. 2012). For small plants 

that require destructive sampling to evaluate 

defensive traits, this issue can only be solved using 

clonal replicates of the same genotype. Second, 

most studies on this topic have reported positive 

associations between few defensive traits and 

resistance (Pratt and Mooney 2013, Stevens et al. 

2007), while plant resistance are expected to be 

multivariate (Agrawal 2005, Lason et al. 2011, 

O’Reilly-Wapstra et al. 2014).

In this study, we aimed at investigating whether 

intraspecific variation in effective resistance of 

maritime pine (Pinus pinaster) to the pine weevil 

Hylobius abietis (a harmful insect herbivore) may 

be predicted by variation in inducibility of PSM. 

To do so, we used clonally replicated genotypes 

with known family structure from 10 populations 

representative of the main distribution range of 

maritime pine, and that belonged to the clonal 

collection ʻCLONAPIN Bank 1ʼ [10 populations × 

5 families per population × 5 genotypes per 

family = 250 genotypes in total] (López-Goldar et 

al. 2018). We performed a greenhouse experiment 

where 2-years old pines were distributed in a 

split-plot design replicated in 5 blocks, with 

population as the whole plot factor and the 

factorial combination of family (3–5 families 

per population) and the induction treatment 

[2 levels: control and induced] as the split factor. 

For the induced treatment, we exogenously 

applied 25 mM methyl jasmonate (MJ) to half 

of the plants, where the other half were left 

untreated. MJ is a plant hormone involved in 

herbivore damage signalling that has been widely 

accepted as a chemical elicitor (Sampedro et al. 

2011, Zas et al. 2014). One month after induction, 

two clonal replicates not subjected to herbivory 

(one control and one MJ-induced) were used to 

measure the concentration of constitutive terpenes 

and phenolics in the stem phloem, as well as 

their inducibility, by gas chromatography-mass 

1Misión Biológica de Galicia, Consejo Superior de Investigaciones Científicas. Apdo. 28, 36080, Pontevedra, Spain, (xlgoldar@mbg.csic.es).

2 Department of Plant Pathology, The Ohio State University, Columbus, OH 43210.

3Daniel B. Warnell School of Forestry and Natural Resources, University of Georgia, Athens, GA 30602.

4Ecological Chemistry Group, Department of Chemistry, Royal Institute of Technology, 100 44 Stockholm, Sweden.

5Department of Forest Ecology & Genetics, Forest Research Centre, INIA-CIFOR, Carretera de la Coruña km 7.5, 28040 Madrid, Spain.

6 Sustainable Forest Management Research Institute, INIA-University of Valladolid, Avda. Madrid 44, 34004 Palencia, Spain.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 9: Mechanisms of Resistance to Insects 81

spectrometry (GC-MS) and GC-flame 

ionization detection (GC-FID), and by ultrahigh performance liquid chromatography-mass 

spectrometry (UHPLC-MS) and UHPLC-diode 

array detection (UHPLC-DAD). We identified and 

quantified (in mg·g-1 dry weight of stem tissue) 

98 chemical compounds that were classified 

into eight chemical groups [monoterpenes, 

sesquiterpenes, diterpenes, flavonoids, 

hydroxycinnamic acids (HCAs), lignans, eugenols, 

and fatty acids]. Another control and MJ-induced 

clonal plants were exposed to a couple of insects 

for 48 hours to measure plant damage (in mm2) as 

a proxy of resistance (i.e., the greater the damage, 

the lower the resistance). Prior to herbivory 

treatment, insects were starved for 48 hours and 

then weighed, and plant diameter was measured.

Variation in H. abietis damage was analyzed by 

fitting a mixed model using the PROC MIXED 

procedure in SAS v9.4 (Littell et al. 2006). For the 

statistical analyses, the induction treatment (MJ), 

pine population (P), pine family within population 

[F(P)] and MJ×P and MJ×F(P) interactions were 

considered as fixed factors. Block (B) and the B×P 

interaction were considered as random factors to 

analyze the main effect of P with the appropriate 

error term (Littell et al. 2006). The genotype was 

also included as a random factor to account for 

the dependence between the two clonal replicates 

subjected to herbivory within each block. Weevil 

weight and stem diameter of the plant were 

included as covariates. Because structured genetic 

variation among populations was previously found 

for this species (Jaramillo-Correa et al. 2015, 

Serra-Varela et al. 2015), the same model was fit 

to explore the genetic variation among genetic 

clusters of populations (Atlantic France, Atlantic 

Spain, Central Spain, South Spain, and Morocco) 

and among families within clusters. In order to 

estimate the variance contribution of each factor 

to the genetic variation among populations and 

families within populations in resistance to the 

pine weevil, the previous mixed model was run 

again assuming all factors as random factors.

To explore the relationships between plant 

resistance and PSM in the stem for the constitutive 

and inducibility states, we performed pairwise 

correlations and stepwise multiple regression 

analyses between the weevil damage and PSM 

concentration at the genotypic level. Prior the 

analyses, we accounted for the non-independence 

among genotypes within families and populations 

by incorporating the population structure (Q) and 

kinship (K) matrices in mixed models (LópezGoldar et al. 2018). We fitted a mixed model for 

constitutive (C) and for the inducibility (MJ – C) 

of each total and individual PSM and weevil 

damage. For each defensive state, population 

(P) and family within population [F(P)] were 

considered as fixed factors. Block (B) and the B×P 

interaction were considered random factors as 

above. The predicted values of PSM variables and 

weevil damage from each plant were extracted 

from each mixed model and used for the analyses. 

Plant resistance to weevil herbivory in the stem 

significantly varied among plant populations 

(fig. 1) and genetic groups of populations 

(F4, 140 = 4.73, P = 0.001), but not between families 

within populations (fig. 1) or within genetic 

groups of populations (F43, 140 = 1.36, P = 0.096). 

Spanish Atlantic populations showed the greatest 

constitutive resistance (61.5 ± 7.3 mm2

 of damage; 

figure 2), whereas the French Atlantic populations 

were the most susceptible (94.9 ± 12.3 mm2

 of 

damage; fig. 2). The MJ-induction treatment 

significantly increased plant resistance to 

weevil herbivory in the stem (figs. 1 and 2). The 

inducibility of resistance was similar for all plant 

populations and families [no significant MJ×P and 

MJ×F(P) interactions, fig. 1], but differed among 

genetic groups (MJ×GP interaction, F4, 140 = 3.36, 

P = 0.012). In fact, greater inducibility of 

resistance after induction was found in both 

Atlantic genetic groups (71 and 67 percent in 

Atlantic France and Atlantic Spain, respectively) 

than in the other genetic groups (Central Spain, 

37 percent; South Spain, 34 percent; and Morocco, 

45 percent). Among the main effects of our 

study, that accumulated an explained variance 

of 36 percent of the total variance in resistance, 

MJ-induction effect accounted for > 80 percent 

of that explained variance in resistance, with 

the contribution of all remaining factors being 

comparatively much lower (fig. 1).

When exploring pairwise relationships at the 

genotypic level, we found that neither total 

nor individual PSM at the constitutive level 
82 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

MJ

30.95%

P (2.16%)

F(P) (2.15%)

MJ P

MJ F(P)

Weevil

Plant 

Residual

61.53%

P (2.16%)

F(P) (2.15%)

MJ P (0.72%)

MJ F(P) (~0.00%)

Weevil weight (2.31%)

Plant diameter (0.18%)

Fixed effect DF (n, d) F P-value

MJ-Induction (MJ) 1, 140 104 <0.0001

Population (P) 9, 36 2.32 0.0353

Family [F(P)] 38, 140 1.33 0.1205

MJ×P 9, 140 1.62 0.1159

MJ×F(P) 38, 140 0.92 0.6032

Weevil weight 1, 140 18.5 <0.0001

Plant diameter 1, 140 4.66 0.0326

Figure 1—Summary of the mixed model testing the effects of the induction treatment with methyl-jasmonate (MJ), population (P), 

family within population [F (P)] and the MJ×P and MJ×F(P) interactions on the early resistance to herbivory by the pine weevil on 

plants from 10 maritime pine populations. Variance components for each effect and the residual are shown in the companion pie 

chart. Weevil weight and plant diameter were included as covariates. Significant p-values (p < 0.05) are highlighted in bold.

Figure 2—Intraspecific genetic variation in constitutive resistance (white bars) and MJ-induced 

resistance (grey bars) to the pine weevil for 10 maritime pine populations along its main 

distribution range, grouped by the main genetic groups in the species. The inset panel shows 

the main effect of the MJ-induction on the resistance to weevil herbivory. Bars represent the 

least square mean ± SE (N = 8 to 25 plants for each population; N = 197 to 205 plants for each 

induction treatment in the small panel).

were significantly associated with weevil 

damage. Contrarily, we found significant 

negative relationships between inducibility of 

total flavonoids and total lignans with weevil 

damage (r = -0.33, P = 0.001 and r = -0.36, 

P < 0.001, respectively). When exploring 

individual PSMs, we found significant negative 

relationships between the inducibility of lignan 

hexoside derivative 1 and 2 with weevil damage 

(r = -0.30, P = 0.003; r = -0.33, P = 0.001, 

respectively). In the multiple regression model, 

concentration of constitutive PSM significantly 

predicted 70 percent of the total variation in 

weevil damage, but only 6 PSM were negatively 

related to weevil damage (table 1). Inducibility 

of PSM significantly predicted 68 percent of the 
Session 9: Mechanisms of Resistance to Insects 83

total variation of weevil damage, and 8 PSM 

were negatively related to weevil damage after 

MJ-induction (table 1).

This study represents a good example of the 

relevance of using clonal replicates of genotypes, 

belonging to families within populations along 

the distribution range of a single conifer species, 

to evaluate the role of inducibility of PSM in 

resistance. Moreover, our findings are based on 

a rigorous framework by accounting for spurious 

associations due to genetic relatedness, often 

overlooked in intraspecific studies. Here we 

provide evidences that multivariate analyses of 

PSM, rather than bivariate correlations, provide 

more realistic information about the potentially 

causal relationships between PSM and resistance 

to herbivory in pine trees. 

Table 1—Summary of the multiple regression analysis explaining the resistance to 

the pine weevil using the concentration of individual PSM as predictor variables 

in constitutive (upper part of the table) and in inducibility (lower part of the 

table) defensive states in 102 genotypes from 10 maritime pine populations. 

The regression coefficients (β) and partial R2 of PSM included in the model after 

stepwise selection method are shown

Defensive mode Plant secondary chemicals β Partial R2

Constitutive PSM β-Phellandrene + Limonene -0.98 0.150

(N = 87) Lignan hexoside derivative 1 -1.58 0.077

Intercept = 7.908 Ferulic acid -0.33 0.028

Model adjusted R2 = 0.70 α-Copaene -1.89 0.025

F18,62 = 18.97 Unk P11 -0.35 0.024

P < 0.001 α-Cubebene + α-Longipinene -0.76 0.016

Coumaric acid hexoside 0.60 0.079

Methyl thymyl ether 0.57 0.054

α-Phellandrene 0.52 0.040

Methyl eugenol 0.87 0.035

Elemol 0.75 0.031

Eugenol 0.36 0.029

Bicyclosesquiphellandrene 1.31 0.028

Myrcene 0.56 0.022

Lignan xyloside derivative 2 0.40 0.021

Citronellyl propionate 0.46 0.018

Sabinene 0.53 0.015

Unk P6 0.39 0.011

Inducibility of PSM Unk P5 -22.46 0.234

(N = 93) Lignan hexoside derivative 2 -19.17 0.060

Intercept = -36.03 Isopimaric acid -10.05 0.060

Model adjusted R2 = 0.68 Elemol -12.11 0.058

F12,78 = 17.32 cis-β-Ocimene -9.04 0.048

P < 0.001 Methyl thymyl ether -10.36 0.040

Pimaric acid -11.76 0.029

trans-Pinocamphone -10.15 0.022

Oleic acid C18:1 10.03 0.048

Abietic acid 31.24 0.039

Ferulic acid hexoside 8.03 0.024

trans-β-Ocimene 4.78 0.019

PSM associated with resistance to weevil damage (negative β) are typed in bold. Linear 

regression model at each defensive mode is significant at p < 0.05. Genotypic sample size of 

each defensive state is indicated in parentheses. Given that some clonal replicates (control or 

MJ-induced) were not available for several genotypes, sample size for the inducibility dataset 

was slightly lower than the original number of genotypes used. Unk P# = unidentified phenolic 

compound.
84 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

LITERATURE CITED

Agrawal, A.A. 2005. Natural selection on common milkweed 

by a community of specialized insect herbivores. 

Evolutionary Ecological Research. 7: 17.

Agrawal, A.A.; Hastings, A.P.; Bradburd, G.S. [et al.]. 2015. 

Evolution of plant growth and defense in a continental 

introduction. The American Naturalist. 186(1): E1–E15.

Bingham, R.A.; Agrawal, A.A. 2010. Specificity and tradeoffs in the induced plant defence of common milkweed 

Asclepias syriaca to two lepidopteran herbivores. Journal 

of Ecology. 98(5): 1014–1022.

Futuyma, D.J.; Agrawal, A.A. 2009. Macroevolution and the 

biological diversity of plants and herbivores. Proceedings 

of the National Academy of Sciences U S A. 106(43): 

18054–18061.

Goodger, J.Q.; Heskes, A.M.; Woodrow, I.E. 2013. 

Contrasting ontogenetic trajectories for phenolic and 

terpenoid defences in Eucalyptus froggattii. Annals of 

Botany. 112(4): 651–659.

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85

Session

PRIORITIZING WORK AND PATHS TO SUCCESS

10

Session 10: Prioritizing & Paths to Success
86 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

BETTER FOREST HEALTH THROUGH TREE RESISTANCE—

COLLABORATIVE APPROACHES

C. Dana Nelson1, Jennifer Koch2, Kevin Potter3, Susan McCord4, Adam Costanza4, 

Mark Coggeshall 5, Margaret Staton6, and Albert Abbott7

Exotic pests and pathogens are causing wide-spread decline and possible extirpation of several of our 

foundational forest tree species. The decline of such species has large-scale effects on their associated 

ecosystems and services they provide. A steady barrage of new threats to important forest species is 

anticipated to continue as a by-product of increasing global trade. The Forest Health Initiative (FHI) was 

conceived in recognition of these issues and the challenges they present to biodiversity and ecosystem 

sustainability. The FHI concept is a new paradigm that engages social, regulatory, and biological science 

experts and various stakeholders in developing and evaluating options for solving our most critical forest 

health problems. Blight resistance in American chestnut was selected as a test case for FHI, and although 

not completely solved, significant progress is being made and the promise of effective resistance and 

chestnut restoration has been advanced. Clearly additional work remains for American chestnut as well 

as the many other severely threatened forest tree species. To address these additional species, we are 

building on the FHI experience and developing two complementary initiatives designed to improve the 

ability of the research community to identify, produce, and deploy effective tree resistance. A projectoriented initiative, Forest.Health, will prioritize the most seriously threatened species, bring researchers 

and stakeholders together to reach consensus on a science-based solution, and seek collaborators and 

funding to carry out the work. At the same time, a network-based participatory tree breeding consortium 

is proposed to ensure long-term development of publically available, genetically improved forest trees.

1 Forest Health Research and Education Center, Southern Research Station, USDA Forest Service, Lexington, KY 40546, 

(charles.d.nelson@usda.gov). 2 Northern Research Station, USDA Forest Service, Delaware, OH 43015.

3North Carolina State University, Raleigh, NC 27607.

4 Institute of Forest Biosciences, Cary, NC 27513.

5Hardwood Tree Improvement and Regeneration Center, Northern Research Station, USDA Forest Service, West Lafayette, IN 53726.

6 Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN 27996.

7Forest Health Research and Education Center, University of Kentucky, Lexington, KY 40506.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 10: Prioritizing & Paths to Success 87

TREEGENES: INTEGRATING GENOTYPIC, ENVIRONMENTAL, 

AND PHENOTYPIC DATA FOR FOREST HEALTH

Emily Grau1, Nic Herndon1, Sean Buehler1, Taylor Falk1, Peter Richter1, Sumaira Zaman1, 

Stephen Ficklin2, Margaret Staton3, Doreen Main2, and Jill Wegrzyn1

TreeGenes (https://treegenesdb.org/) is a 

web-based information resource designed to serve 

the diverse needs of the forest tree genomics 

research community by uniting information 

resources with visualization and analysis tools. 

TreeGenes has recently undergone a complete 

redesign using Tripal, a tool to create and manage 

genomic database websites. An open source 

project, Tripal allows developers the flexibility to 

create and share their own tools (modules) as well 

as open communication among Tripal supported 

repositories.

TreeGenes hosts a range of modules that have 

expanded functionality to deliver genomic and 

phenotypic data on >1,700 forest tree species. 

The new Tripal Galaxy module allows users to 

execute analytical workflows with next generation 

sequencing datasets on high performance 

computing (HPC) resources with the click of a 

button. The Tripal Elasticsearch module allows 

flexible searching and data retrieval between 

sites such that TreeGenes can share data with 

Hardwood Genomics Project and the Genome 

Database for Roseaceae. 

TreeGenes is also developing new Tripal modules 

including the Tripal Plant PopGen Submit (TPPS) 

module, the TreeGenes TSeq module, TreeGenes 

OrthoQuery, and Cartogratree. TPPS is a pipeline 

for accepting direct submissions from researchers. 

This module collects relevant metadata/data 

for studies focused on the interactions between 

genotype, phenotype, and the environment 

while reducing the burden on the researcher for 

submission. The new Tripal Sequence Similarity 

(TSeq) Module module offers speed improvements 

over BLAST via DIAMOND and allows 

sequence similarity searches across numerous 

pre-indexed genomes and transcriptomes. 

TreeGenes OrthoQuery permits gene family and 

phylogenetic analysis for single sequences and/

or entire proteomes with a robust and interactive 

visualization.

CartograTree (https://cartogratree.org) is a 

map-based open-source analytic module integrates 

across data that is curated through TPPS to 

facilitate association mapping and landscape 

genetics analysis. It uses genotypic and phenotypic 

data provided by two clade organism databases: 

TreeGenes via TPPS and Hardwood Genomics. 

Environmental data currently includes climate 

variables, land cover, canopy density, atmospheric 

metrics, and soil types. CartograTree leverages 

Galaxy workflows to support analysis in the web 

framework with High Performance Computing 

(HPC) resources.

1University of Connecticut, Storrs, CT 06269.

2Washington State University, Pullman, WA 99164.

3University of Tennessee, Knoxville, TN 37996, (jill.wegrzyn@uconn.edu).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
88 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

A NATIONAL PRIORITIZATION OF UNITED STATES TREE SPECIES 

THREATENED BY INSECT AND DISEASE INFESTATION

Kevin M. Potter1, Maria E. Escanferla1, Barbara S. Crane2, 

Robert M. Jetton1, and Gary Man3

Insect and disease infestations pose major threats to several North American forest tree species. 

Scientists and managers from throughout the U.S. Forest Service developed a conservation prioritysetting framework for forest tree species at risk from insects and disease and other threats. The Project 

CAPTURE (Conservation Assessment and Prioritization of Forest Trees Under Risk of Extirpation) 

framework is data-driven and guided by expert opinion, allowing the quantitative grouping of species 

into vulnerability classes that may require different management and conservation strategies. We applied 

this framework to categorize and prioritize 419 native North American tree species for conservation, 

monitoring, and management using trait data and insect and disease threat data for each host tree 

species. The categorization is based on vulnerability factors relating to each tree species’ (1) insect and 

disease threat severity, (2) sensitivity to insect and disease infestation, and (3) capacity to adapt to insect 

and disease infestation. We used K-means clustering to group species into 11 classes based on these 

vulnerability dimensions. The three most vulnerable classes encompassed 15 species which require the 

most immediate conservation intervention. Two additional classes face less severe insect and disease 

threats and may be good candidates for resistance breeding efforts. Other groups had traits associated 

with high sensitivity and/or low adaptive capacity to potential future insect and disease threats, suggesting 

that these species need close monitoring. This assessment tool should be valuable for decisionmakers 

determining which species and populations to target for monitoring efforts and for pro-active gene 

conservation and management activities.

1 Department of Forestry and Environmental Resources, North Carolina State University, Research Triangle Park, NC 27709, (kpotter@ncsu.edu). 2 Southern Region, National Forest System, USDA Forest Service, Atlanta, GA, 97424.

3 Forest Health Protection, USDA Forest Service, Washington, DC 20024.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International Workshop on 

the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. e-Gen. Tech. Rep. 

SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 10: Prioritizing & Paths to Success 89

SEARCHING, DOWNLOADING AND ANALYZING TREE GENETIC AND 

GENOMIC DATA WITH THE HARDWOOD GENOMICS WEBSITE

Margaret E. Staton1, Abdullah Almsaeed1, Bradford Condon1, Ming Chen1, 

Jill L. Wegrzyn2, Doreen Main3, Stephen P. Ficklin3, Albert G. Abbott 4, 

C. Dana Nelson4, and John E. Carlson5

The Hardwood Genomics Website (HWG) provides access to genetic and genomic data from woody 

plant species, including many ecologically important and threatened forest trees such as American 

chestnut, green ash, black walnut, and American beech. The site has tree genomes and transcriptomes 

that are not housed elsewhere or lack publicly available annotation. It provides searchable annotation for 

all genes and transcripts, including BLAST results to curated plant protein databases, protein domains, 

and gene ontology terms. To further characterize gene sequences, HWG houses expression values from 

high throughput RNASeq studies. We identify simple sequence repeats (SSRs) and flanking primers 

from genome and gene sequences and make these available on HWG for use as genetic markers. Where 

available, HWG also provides access to published microsatellite markers. To facilitate access to these 

datasets, we have a number of tools for researchers, including a powerful keyword search, JBrowse, and 

BLAST. Most recently we have added access to bioinformatics analysis workflows powered by Galaxy 

software. Users can select from a number of common analysis types, including mapping DNA or RNA 

reads, performing differential gene expression analysis, calling genetic variants from reads, etc. A user 

can upload their own data for analysis, select HWG site data, or use both as input to a workflow. To meet 

the growing need for forest tree breeding programs and genetic research, we are beginning a new effort to 

add additional genetic markers, genetic maps including association and QTL studies, and high throughput 

genotyping and phenotyping data. The site will also be adding descriptions of physical collections such as 

germplasm or mapping populations with contact information for the maintainer. The HWG is a growing 

resource for tree scientists, and we welcome feedback, data submissions, or new partnerships to continue 

site development. HWG is supported by the National Science Foundation (NSF) awards numbers 1443040 

and 1444573.

1University of Tennessee, Knoxville, TN 37996, (mstaton1@utk.edu). 2University of Connecticut, Storrs, CT 06260.

3Washington State University, Pullman, WA 99164.

4 Forest Health Research and Education Center, Southern Research Station, USDA Forest Service, Lexington, KY 40536.

5 Pennsylvania State University, State College, PA 16802, (jec16@psu.edu).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
90 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

DURABILITY OF RESISTANCE AND “USEFUL RESISTANCE” 

IN FOREST TREES TO NON-NATIVE PATHOGENS

Richard A. Sniezko1, Douglas P. Savin1, Angelia Kegley1, and Robert Danchok1

Many tree species are extremely susceptible to one or more non-native pathogens or insects. For some, the 

level of susceptibility can be so severe that they are essentially extirpated from many forest ecosystems, 

and their use for restoration, reforestation, or urban forestry is lost. Applied programs to find, enhance, 

and utilize genetic resistance in some tree species are well underway, and more are being considered. 

Some people equate genetic resistance with 100 percent immunity in planting stock, but in most forest 

situations this will not be the case. Resistance can also refer to having fewer, less severe, or incompatible 

infections, or being able to survive and reproduce, and it may be present in only a portion of the planting 

stock. Trees are long-lived organisms, and resistance has to be effective for decades (in managed 

plantations) to centuries (in native forests) to be utilitarian. Resistance must not only be durable and stable 

but also at a useful level, which may vary by species and application. A seedlot with ‘useful resistance’ 

(UR) is expected to meet management objectives for long-term deployment, often over many sites and 

for decades after planting. Field plantings provide the best knowledge of durability of resistance and its 

stability across a range of environments. We examine the durability, stability, and resistance levels of two 

white pine species (western white pine, Pinus monticola and sugar pine, P. lambertiana) from some of the 

oldest existing white pine blister rust (caused by the fungal pathogen Cronartium ribicola) resistance field 

trials. Genetic resistance will be key to retaining many prominent species in forest ecosystems as well as 

for use in reforestation and urban forestry. The examples provided here can provide guidance to managers 

on early expectations of resistance in application to a range of other non-native pathogens or insects in 

forest trees.

The full paper was published in Plants, People, Planet

https://nph.onlinelibrary.wiley.com/toc/25722611/2020/2/1

The reference for the published paper is:

Sniezko, R.A.; Johnson, J.S.; Savin, D.P. 2020. Assessing the durability, stability, and usability of genetic resistance to 

a non‐native fungal pathogen in two pine species. Plants, People, Planet. 2(1): 57-68. DOI: https://doi.org/10.1002/

ppp3.49.

1 Dorena Genetic Resource Center, USDA Forest Service, Cottage Grove, OR 97424, (richard.sniezko@usda.gov).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 11: Novel Tools for Identifying Resistance 91

Session

NOVEL TOOLS FOR IDENTIFYING RESISTANCE

11
92 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

TREESNAP: A CITIZEN SCIENCE PROJECT AND MOBILE APP 

TO TAG TREES FOR SCIENCE

Bradford Condon1, Abdullah Almsaeed1, Ellen Crocker 2, Albert Abbott 2, 

C. Dana Nelson3, and Margaret Staton1

To help scientists find forest trees of particular species and characteristics, we have built a new mobile app 

and citizen science outreach project, TreeSnap. TreeSnap uses the ubiquity of smartphones in our society 

to engage the public in scouting for trees affected by invasive insects and diseases, including North 

American ashes, American chestnut, eastern and Carolina hemlocks, and American elm. This strategy 

provides tree locations, images, and characteristics to tree research programs while also engaging citizens 

and promoting public awareness of forest health threats and the benefits of forest health. The TreeSnap 

mobile app is available for free on both iOS and Android. The list of highlighted tree species may be 

updated to reflect current needs across many forest health research programs. To increase the utility of 

the collected data for scientists, we ask a set of customized questions for each species, such as a list of 

signs of Emerald Ash Borer for ash trees and presence of cones for hemlocks. This information is paired 

with photos taken by the user and GPS coordinates automatically detected by the mobile device. TreeSnap 

has an accompanying website where users can view their own and others’ tagged trees. The website also 

serves as a data curation and outreach workspace for scientists. They can login to sort and filter trees, 

download user observations, and contact individual users for more information. Also, scientists can set 

up an email alert to be notified when new observations fitting their criteria are submitted (i.e., American 

Chestnuts in Tennessee with a diameter of at least 10 inches). We envision TreeSnap as a crucial long term 

research tool for threatened forest trees, which often lack a large pool of lingering genotypes to study. 

We are actively seeking new partner scientists to expand the trees available in TreeSnap and partner 

forest outreach groups that can promote the App to interested citizens, or incorporate it into their existing 

activities.

The full paper was published in Plants, People, Planet

https://nph.onlinelibrary.wiley.com/toc/25722611/2020/2/1.

The reference for the published paper is:

Crocker, E.; Condon, B.; Almsaeed, A.; Jarret, B.; Nelson, C.D.; Abbott, A.G.; Main, D.; Staton, M. 2019. TreeSnap: 

A citizen science app connecting tree enthusiasts and forest scientists. People, Plants, Planet. 2(1): 47–52. DOI: 

https://10.1002/ppp3.41.

1University of Tennessee, Knoxville, TN 37996.

2University of Kentucky, Lexington, KY 40536, (e.crocker@uky.edu). 3 Forest Health Research and Education Center, Southern Research Station, USDA Forest Service, Lexington, KY 40536.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 11: Novel Tools for Identifying Resistance 93

DEVELOPMENT OF A TOOL FOR RAPID IDENTIFICATION OF RESISTANT TREES 

IN SPECIES AFFECTED BY ALIEN INVASIVE PATHOGENS

Anna O. Conrad1, Caterina Villari2, Richard A. Sniezko3, 

Luis Rodriguez-Saona4, and Pierluigi Bonello1

Alien invasive pathogens continue to threaten the health of forests across the United States and globally. 

Identifying and breeding resistant trees is a viable strategy for disease management and may be used 

as part of efforts aimed at restoring disturbed habitats. However, identifying disease resistant trees 

requires artificial inoculation or natural infection with a pathogen, and time needed for symptoms to 

develop. This often comes after significant resources have already been invested in collecting seeds and 

growing seedlings. Therefore, the objective of our work is to develop a more rapid phenotyping method 

for disease resistance using Fourier transform infrared (FT-IR) and Raman spectroscopy combined with 

chemometrics. Currently, we are focusing our efforts on two forest pathosystems: root rot of Port-Orfordcedar (Chamaecyparis lawsoniana) caused by Phytophthora lateralis and white pine blister rust on 

whitebark pine (Pinus albicaulis) caused by Cronartium ribicola. In Port-Orford-cedar, we are analyzing 

material with origins across the geographical range of the species, while with whitebark pine our efforts 

are focused on populations of the Pacific Northwest. Our goals include evaluating whether the tool can be 

used to predict resistance within and between families, and the impact of geographical origin on model 

predictions. We ultimately aim to develop protocols for rapid, in-field analysis of intact plant materials that 

can be used to facilitate and expedite current breeding and restoration efforts.

1 Department of Plant Pathology, Ohio State University, Columbus, OH 43210, (conrad.245@osu.edu). 2Warnell School of Forestry and Natural Resources, University of Georgia, Athens, GA 30602.

3Dorena Genetic Resource Center, USDA Forest Service, Cottage Grove, OR 97424.

4 Department of Food Science and Technology, Ohio State University, Columbus, OH 43210.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
94 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

DETECTION OF NEAR INFRARED SPECTRAL DIFFERENCES IN PINUS RADIATA

Judith S. Nantongo1, Thomas Rodemann2, Hugh Fitzgerald1,

Brad M. Potts1, and Julianne O'Reilly-Wapstra1

Selective bark stripping of P .radiata by wallabies and pademelons may be explained by differences 

in constitutive or induced chemistry (Miller et al. 2014). The potential of near infra-red spectroscopy 

(NIRS) to detect differences between the less and more susceptible genotypes was evaluated. Nine 

families classified as less susceptible (R) and more susceptible (S) were selected. Three families of each 

were treated with stress inducing Methyl jasmonate (MJ), mechanical stripping (strip) or no treatment 

(control) in a randomised block design. Physio-chemical changes were monitored by NIRS for 4 weeks. 

Partial least squares (PLS) regression was used to group the principle components (PC) of the spectra. The 

results showed clearly that NIRS distinguished less and more susceptible genotypes. NIRS also separated 

individuals subjected to different treatments. In conclusion, there is evidence of differences in bark 

physio-chemical attributes of the less and more resistant genotypes. NIRS provides a powerful tool for 

detecting physio-chemical differences in P. radiata.

REFERENCES

Miller, A.; O’Reilly-Wapstra, J.; Potts, B. 2014. Genetic variation in bark stripping among Pinus radiata. Internal 

Report. Submitted to the National Centre for Future Forest Industries (NCFFI) and Timberlands Pacific Pty Ltd.

1School of Natural Sciences, University of Tasmania (UTAS), Australia, (Judith.Nantongo@utas.edu.au). 2Central Science Laboratories, University of Tasmania (UTAS), Australia.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 12: Resistance & Integrated Pest Management 95

Session

RESISTANCE AND INTEGRATED

PEST MANAGEMENT12
96 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

ENDOPHYTE ENHANCEMENT OF SPRUCE AND EASTERN WHITE PINE 

SEEDLINGS TO IMPROVE TOLERANCE TO INSECTS AND DISEASE

Gregory W. Adams1, Andrew McCartney2, J. David Miller3, and Dan Quiring4

Research over the past two decades has focused on endophytic fungi isolated from the needles of conifer 

trees, forming mutualistic associations with their hosts. We have identified native strains of endophytic 

fungi that produce secondary metabolites which act antagonistically towards certain forest pests, 

namely eastern spruce budworm (Choristoneura fumiferana) and white pine blister rust (Chronatium 

ribicola). Spruce budworm, which is the most significant insect pest in Canada and parts of the United 

States, defoliates balsam fir and white spruce and to a lesser extent red spruce and black spruce. In 2017, 

7 million ha of forests in the Province of Québec experienced defoliation from the current outbreak. White 

spruce is the most widely planted tree species in Canada and was the subject of early studies on potentially 

useful foliar endophytes. Research was initiated in 1998 to determine if white spruce seedlings could be 

inoculated with selected endophyte strains. Field tests were established to study the persistence of the 

endophytes in trees, to understand the transmission of the endophyte, and to provide trees for challenging 

experiments with the spruce budworm. Challenging studies with spruce budworm have demonstrated a 

reduction of spruce budworm survival through to adulthood and a reduction of defoliation in the range 

of 10–30 percent. Based on positive results of this work, research was initiated in 2008 to test the same 

approach on white pine blister rust, a devastating non-native disease affecting all five-needle pines in 

North America. Through screening many strains, endophytes have been selected which produce potent 

anti-fungal secondary metabolites and inoculation procedures have been developed. Lab assays using 

the disease pathogen and secondary metabolites at relevant concentrations have demonstrated inhibition 

of the disease and field testing is underway. Twenty years of research has demonstrated that selected 

endophytes can play a role in improving tolerance of planted trees to pests as one component of integrated 

pest management. J.D. Irving, Limited has implemented endophyte enhancement in nursery seedling 

production at a large scale since 2008.

1Research and Development, J.D. Irving, Limited, 181 Aiton Road, Sussex East, New Brunswick, Canada E4G 2V5, (adams.greg@

jdirving.com). 2 Maritime Innovation Limited, 181 Aiton Road, Sussex East, New Brunswick, Canada E4G 2V5.

3Carleton University, 1125 Colonel By Drive, Ottawa, Ontario K1S 5B6.

4 Faculty of Forestry and Environmental Management, University of New Brunswick, Fredericton, New Brunswick, Canada E3B 6C2.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 12: Resistance & Integrated Pest Management 97

MONITORING OF ASH MORTALITY PATTERNS INFORMS EMERALD ASH BORER (EAB) 

RESISTANCE BREEDING EFFORTS: INTEGRATED PEST MANAGEMENT FOR EAB

Kathleen Knight1, Rachel H. Kappler 2, Karen V. Root2, Jennifer L. Koch1, and Charles E. Flower1

Integrated pest management (IPM) is a framework that combines multiple different strategies to reduce 

the impacts of pests and pathogens. Strategies include preventative measures; cultural, biological, and 

chemical control; monitoring of host and pest populations; and mitigation of impacts. Pest- or pathogenresistant trees are cultural control techniques that, even standing alone, can reduce impacts by increasing 

survival and health of the host tree species. By combining tree resistance breeding with other strategies 

within an integrated pest management framework, even greater reductions in pest and pathogen impacts 

may be achieved and may last over a much longer term. We present a case study of the insect pest emerald 

ash borer (Agrilus planipennis) (EAB) and its impacts on ash trees (Fraxinus spp.) in the United States. 

Ten years of monitoring data on ash and EAB populations has clearly shown the typical trajectory of ash 

mortality and subsequent crash in EAB populations, and has also shown the factors that affect the rate of 

mortality of ash trees. Intensive monitoring of “lingering” ash populations after the EAB mortality wave 

has revealed how these factors that affect mortality may change in this post-infestation landscape. While 

areas with higher ash density exhibit slower mortality during the initial mortality wave, ash trees with 

neighboring ash trees exhibit greater decline and mortality in a post-infestation time period. This finding 

suggests that cultural control methods, while not useful prior to or during the initial wave of EAB, may 

be helpful in maintaining lingering ash populations after EAB has killed most of the trees. The remaining 

healthy trees, many exhibiting resistance to EAB, may benefit from reductions in susceptible declining 

neighbors. Consideration of natural patterns of mortality may inform resistance breeding programs: not 

only which trees to choose for breeding programs and the appropriate timing to choose them, but how to 

deploy them using appropriate cultural control measures that may influence their success.

1Northern Research Station, USDA Forest Service, Delaware, OH, 43015, (ksknight@fs.fed.us). 2 Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
98 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

EMERGING THREATS OF PRIORITY AGROFORESTRY TREES IN AFRICA: 

CHALLENGES AND OPPORTUNITIES

Jane Njuguna1, Eston Mutitu1, Ely Mwanza1, Alice Muchugi2, and Ignazio Graziosi2

Agroforestry has a strategic role for Africa’s smallholders, as it builds resilient agro-ecosystems and 

provides enduring benefits to farmers. The World Agroforestry Centre is conserving over 18,000 

germplasm accessions of more than 200 tree species in globally established field genebanks and seed 

banks. Emerging biotic threats are targeting the cultivation of crucial tree species in Sub-Saharan 

Africa, thus frustrating local benefits of agroforestry and forestry. A stem canker disease caused by 

Botryosphaeriaceae pathogens is triggering extensive dieback to the Australian silk oak Grevillea robusta, 

widely used in Africa as highly adaptable, multi-purpose, and fast growing tree. Botryosphaeriaceae 

disease complexes are also involved in the newly detected dieback of native baobab Adansonia digitata, 

and, along with Teratosphaeriaceae species, in cankers of Eucalyptus spp. In addition, Eucalyptus

planted forests in Southern and Eastern African countries are under attack from non-native bronze 

bug Thaumastocoris peregrinus (Hemiptera: Thaumastocoridae), blue gum chalcid Leptocybe invasa

(Hymenoptera: Eulophidae), and red gum lerp psyllid Glycaspis brimblecombei (Hemiptera: Psyllidae). 

We developed laboratory protocols to detect fungal pathogens affecting G. robusta and A. digitata seeds 

and seedlings with the aim of distributing clean agroforestry material and reducing further spread of these 

organisms. We also implemented a countrywide survey to assess distribution, incidence, and population 

levels of Eucalyptus insect pests in Kenya. As the next step, we plan to determine variation in disease 

resistance within germplasm collections for G. robusta (40 accessions from Australia and Kenya) and 

A. digitata (297 accessions from Tanzania Burkina Faso, Kenya, Mali), and investigate the occurrence of 

natural resistance to insect pests in Kenya’s Eucalyptus planted forests. Developing management strategies 

that include selecting for resistance to pests and diseases may be the key to mitigate biotic threats to 

agroforestry in Sub-Saharan Africa.

1 Kenya Forestry Research Institute, P.O. Box 20412 - 00200 Nairobi, Kenya, (jwknjuguna@yahoo.com). 2World Agroforestry Centre, P.O. Box 30677, 00100, Nairobi, Kenya, (ignazio.graziosi@fulbrightmail.org).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 13: Interactions Between Resistance and Environment 99

Session

RESISTANCE AND ENVIRONMENT

INTERACTIONS BETWEEN 

13
100 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

THE EFFECT OF SITE QUALITY ON PERFORMANCE OF AMERICAN CHESTNUT 

(CASTANEA DENTATA) SEEDLINGS BRED FOR BLIGHT 

(CRYPHONECTRIA PARASITICA) RESISTANCE 

Title presented at workshop:

INFLUENCE OF SITE QUALITY ON BLIGHT RESISTANCE AND GROWTH 

OF PLANTED HYBRID CHESTNUT SEEDLINGS

Cornelia C. Pinchot1, Alejandro A. Royo2, Scott E. Schlarbaum3, Matthew P. Peters1, 

Ami M. Sharp3, and Sandra L. Anagnostakis4

ABSTRACT

Efforts to produce American chestnuts (Castanea dentata) resistant to chestnut blight fungus 

(Cryphonectria parasitica) have spurred an interest in reintroducing the species to managed forests. 

Understanding how site characteristics impact chestnut performance will inform appropriate site selection. 

This study was designed to evaluate the impact of site quality on survival, growth, competitive ability, 

and blight resistance durability of American, Chinese, and three families of backcross hybrid chestnuts. 

Chestnuts were planted in xeric, intermediate, and sub-mesic sites in Pennsylvania. Three years after 

planting, survival was 86 percent across all treatments. American chestnuts were taller on intermediate 

than xeric sites, and two backcross hybrid families were taller on intermediate as compared to mesic sites. 

Intermediate sites may offer enough soil moisture to optimize growth, without the intense competition 

characteristic of more mesic sites. Incidence of blight infection was too low to assess differences among 

treatments, though is expected to increase over time. 

INTRODUCTION

Introductions of non-native invasive pests and 

pathogens have caused the loss or decline of 

an increasing number of tree species globally 

(Campbell and Schlarbaum 2014, Loo 2008, 

Santini et al. 2013). These losses can cause 

significant alterations to ecosystem processes and 

functions and threaten forest resilience to future 

pressures (Ellison et al. 2005, Flower et al. 2013). 

In response, there is a growing interest to identify 

or breed populations of tree species resistant to 

their respective pests and pathogens (Sniezko 

2006), with the goal of reintroducing these species 

to managed landscapes. Reintroducing extirpated 

tree species may restore altered ecosystem 

dynamics, functions, and services, thereby 

enhancing resilience, and contributing to larger 

ecosystem restoration goals (Knight et al. 2011, 

2017). Understanding the durability of resistance 

(Sniezko, 2006) and performance of improved 

genotypes in field settings (Clark et al. 2014, 

Seddon 2010) is essential for successful species 

reintroduction, however long term studies on these 

considerations are limited (Thompson et al. 2006).

Extensive efforts have gone into developing 

American chestnuts (Castanea dentata) that are 

resistant to chestnut blight disease (caused by 

the fungus Cryphonectria parasitica), and more 

recently ink disease (caused by the oomycete, 

(Phytophthora cinnamomi),(Anagnostakis 2012, 

Steiner et al. 2017). The principal strategy has 

involved hybridizing American chestnuts with 

blight resistant chestnut species (primarily 

Chinese chestnut, C. mollissima), followed by 

repeated backcrossing and intercrossing to recover 

American chestnut traits (Anagnostakis 2012, 

Hebard 2005). More recently, The American 

Chestnut Foundation, one of the primary 

organizations working to develop blight-resistant 

American chestnuts, has incorporated the use 

of genomic selection to accelerate the breeding 

program (Steiner et al. 2017). 

1Northern Research Station, USDA Forest Service, Delaware, OH 43015, (corneliapinchot@fs.fed.us). 2 Northern Research Station, USDA Forest Service, Irvine, PA 16329.

3Department of Forestry, Wildlife, and Fisheries, The University of Tennessee, Knoxville, TN 37996.

4 Department of Plant Pathology, The Connecticut Agricultural Experiment Station, New Haven, CT 06511.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 13: Interactions Between Resistance and Environment 101

Once blight-resistant American chestnuts are 

available, planting them into managed forests 

will be the next step toward restoring the species. 

Understanding the interacting effects of genotype 

and environment on long-term performance 

and durability of resistance of planted chestnuts 

will inform operational reintroduction of the 

species, including site selection. Selecting sites for 

American chestnut reintroduction that optimize 

growth, competitive ability, and blight control 

will help maximize limited resources available 

for restoration efforts. Site characteristics may 

affect these metrics of establishment success 

differently, however (Griffin et al. 2006). For 

example, Rhoades et al. (2009) found that planted 

American chestnut had better growth rates and 

lower incidence of blight on mesic than xeric sites 

2 years after planting. McNab (2003), however, 

found that planted chestnuts were outcompeted 

by faster growing species on mesic sites, a trend 

that Griffin et al. (1991) also noted for naturally 

occurring chestnut sprouts growing on mesic sites. 

Bauman et al. (2014) found that site preparation 

treatments that led to greater growth of planted 

chestnut also yielded higher blight infection rates, 

by decreasing the time required for bark splits and 

subsequent infection. Gao and Shain (1995) found 

that chestnut blight canker expansion on American 

chestnut stem segments was negatively related 

to moisture availability, which suggests blight 

resistance may be influenced by soil moisture 

availability in situ. Here we present early results 

from a study to evaluate the long-term impact of 

site quality on field performance and resistance 

durability of hybrid backcross American chestnuts 

planted in recent shelterwood harvests.

METHODS 

Study Area

We planted chestnut seedlings at 15 northern 

hardwood forest sites distributed throughout 

a 6500-km2

 area of northern Pennsylvania, 

USA, spanning from Warren County to the 

west and Potter County to the east. Major tree 

species found at the sites include red maple 

(Acer rubrum), sugar maple (Acer saccharum), 

black cherry (Prunus serotina), and American 

beech (Fagus grandifolia), with northern red 

oak (Quercus rubra), eastern hemlock (Tsuga 

canadensis), birch (Betula spp.), and white ash 

(Fraxinus americana) found in lesser abundance. 

The 15 sites used in this study were selected from 

a larger set of 25 sites (Royo et al. 2016, 2017) to 

capture variation in soil moisture availability. To 

do this we calculated the integrated moisture index 

(IMI), (Iverson et al. 1997) for all 25 available 

sites. The integrated moisture index combines 

GIS-derived topographic and soil features of the 

landscape into a single index that models potential 

soil moisture capacity (Iverson et al. 1997). We 

then ran a cluster analysis to group sites based on 

their IMI values (PROC CLUSTER, SAS Institute 

Inc. 2011). The analysis distributed the 25 sites 

among four clusters, one containing only one site 

with the highest IMI value (most mesic), which 

we merged with the cluster with the next highest 

IMI values to produce three clusters representing 

xeric, intermediate and sub-mesic site types. We 

chose five sites within each cluster to maximize 

variability across treatments. The integrated 

moisture index for the selected xeric sites averaged 

31.7 (range 29.4–34.7), 40.3 for the intermediate 

sites (range 39.5–40.8), and 46.9 for the sub-mesic 

sites (range 43.6–55.6).

In 14 of the 15 sites, managers conducted the 

initial cut of a shelterwood sequence to reduce 

stand relative density (the residual relative 

density was 31–61.5 percent and the residual 

basal area was 13.9–26.1 m2

/ha) and applied 

broadcast herbicides (tank mix of glyphosate and 

sulphometuron methyl), (Marquis et al. 1992) to 

control interfering plant species in the mid- and 

understory layers 3–6 years prior to planting. The 

site that did not receive a harvest treatment was 

dropped from the analysis, leaving four sites in the 

intermediate treatment and five each in the xeric 

and sub-mesic treatments.

Experimental Materials

Four-hundred and eighty seven chestnut seedlings; 

83 American, 84 Chinese, and 320 backcross 

hybrid chestnuts (Anagnostakis 2012, Hebard 

2005) from three families were used in this study 

(table 1). The backcross hybrid chestnuts were 

from the BC3F2 generation—second generation 

of the third backcross —or were crosses between 
102 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

BC3F1 and BC2F1 parents. All chestnuts were from 

open-pollinated seed collections made in the 

fall of 2013 and grown for 1 year at commercial 

or State tree nurseries. The American chestnuts 

were collected from one mother tree in Maryland 

and grown at the Kentucky State tree nursery 

(Grassy Creek, KY). The Chinese chestnuts 

were collected from multiple mothers from one 

orchard and grown at the Forrest Keeling Nursery 

(Elsberry, MO). The backcross hybrid American 

chestnuts (three families, table 1) were collected 

at the Windsor Locks orchard of the Connecticut 

Agricultural Experiment Station (Anagnostakis 

2012) and grown at the Vallonia Nursery in 

Vallonia, IN (fig. 1A). All seedlings were lifted as 

1-0 bare-root seedlings in the early spring of 2015 

and stored in a cold room (~1° C). The seedlings 

were processed for planting in March, with roots 

trimmed to 15 cm from the main tap root to 

facilitate planting (fig. 1B). Seedlings were planted 

April 10–14, 2015 with a Jim Gem KBC© bar, 

modified by adding 5 cm to each side of the blade, 

creating a blade 20 cm at the top, tapering to the 

tip. 

Experimental Design and Data Collection

Site type (xeric, intermediate, sub-mesic) was 

analyzed as a completely randomized design. 

Chestnut species and hybrids were analyzed 

as one treatment factor, called “family”, with 

five total treatment levels (table 1). Within each 

site type replicate, the chestnut seedlings were 

arranged using an incomplete block design, each 

Table 1—Chestnut species/generations and 

families used in this study 

Chestnut species/hybrid 

generation Family N

American chestnut American 83

Chinese chestnut Chinese 84

BC3F2 backcross hybrid W1-100 83

BC2F1xBC3F1 backcross hybrida W3-20 84

BC2F1xBC3F1 backcross hybrida W4-75 153

Total — 487

aAll chestnuts in the Windsor orchard except for one family 

are male-sterile, therefore, the father family is known, even 

though the crosses were open-pollinated.

Figure 1—(A) backcross hybrid chestnuts growing at Vallonia Nursery (Vallonia, IN), and (B) Trimming the roots of and 

tagging the lifted seedlings at the Tennessee Tree Improvement Center (Knoxville, TN). (Forest Service photo by Cornelia 

Pinchot) 
Session 13: Interactions Between Resistance and Environment 103

block containing four seedlings from different 

family treatments. At each site, between 34 and 

36 chestnut seedlings were planted within a 

0.42 ha deer exclosure. Seedlings were planted 

in grids, 3.7 m spacing within and 6 m spacing 

between rows. 

Seedling height and ground-level diameter (GLD) 

were measured at the time of planting and towards 

the end of the first three growing seasons (August 

or September, once the buds were set) since 

planting. Mortality and incidence of chestnut 

blight was also assessed at this time. 

A camera equipped with a hemispherical lens 

was used to evaluate canopy openness above each 

planted chestnut seedling during the 2017 (year 3) 

field season. To evaluate competing vegetation, a 

2.6 m diameter competition plot was centered on 

each chestnut seedling and competition data on 

height and species of the tallest understory woody 

plants (DBH <10 cm) collected towards the 2017 

growing season.

All analyses for this study were processed using 

SAS 9.3 software (SAS Institute 2011). Seedling 

height and GLD were analyzed using a mixedmodel analysis of covariance (ANACOVA) to 

determine significant differences among the 

fixed effects of site type, chestnut family, and 

their interactions for year 3 after planting. Initial 

height, GLD, and percent canopy openness were 

tested as covariates in the ANACOVA models for 

height and GLD, respectively. Generalized linear 

mixed model with binomial distribution was used 

to analyze third year survival (1 = alive, 0 = dead) 

and dominance of the seedlings. Seedlings that 

attained at least 80 percent of the height of the 

tallest competitor within the competition plot 

were defined as dominant (Spetich et al. 2002). 

Data were checked for homogeneity of variance 

and normality. Unequal variance was added to the 

model if the Akaike Information Criterion (AICc) 

was significantly improved. Least-significantdifference tests were performed to identify 

differences among means (α = 0.05). Incidence 

of blight was too low to analyze for statistically 

significant differences among treatments. 

RESULTS

Survival

Three growing seasons after planting, 

86 percent (±2) of the chestnut seedlings were 

alive. There was no difference in survival among 

site types (P = 0.99), while family did differ 

(P = 0.01). Chestnuts in the W4-75 backcross 

family had greater survival (99 percent ± 2) than 

all other chestnuts except those in the W1-100 

backcross family (97 percent ± 2). All other 

chestnuts were statistically similar in survival (and 

did not differ from the W1-100 backcross family); 

87 percent ± 4 for the W3-20 backcross family; 

84 percent ± 5 for Chinese, and 83 percent ± 5 

for American chestnuts. The interaction between 

site and family treatments was not significant 

(P = 0.24). 

Height and Ground Level Diameter

Height after three growing seasons did not 

differ among site or family treatments (P = 0.20, 

P = 0.19, respectively) (fig. 2), and averaged 

162 cm across all treatments. However, the 

interaction between the two treatment factors was 

significant (P = 0.03) (fig. 3). American chestnuts 

were taller on intermediate than xeric sites; while 

W1-100 and W4-75 backcross hybrid families 

were taller on intermediate than sub-mesic sites. 

There were no differences in height for W3-20 

backcross hybrid or Chinese chestnuts across the 

site types. Height at planting and percent canopy 

openness were both significant covariates in this 

model (P < 0.0001 and P = 0.001, respectively). 

Ground level diameter after three growing seasons 

was similar across the site types (P = 0.41), but 

differed among families (P < 0.0001). Diameter 

was greatest for backcross hybrid families W1-100 

and W4-75 (17.5 mm ± 0.8 and 17.4 mm 0.7, 

respectively). Diameter was similar among the 

remaining families; 14.7 mm ± 0.8 for W3-20, 

14.6 mm ± 0.8 for American, and 13.7 mm ± 0.8 

for Chinese chestnuts. The interaction between 

site and family treatments was not significant 

(P = 0.23). Ground level diameter at planting and 

percent canopy openness were both significant 

covariates in the model (P < 0.0001 for each). 
104 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

Figure 2—Chestnut seedlings in a xeric (A), intermediate (B), and sub-mesic (C) sites 3 years after planting. (Forest Service photo by 

Cornelia Pinchot)

Figure 3—Mean height (± standard error) of chestnut families among site types. Different 

letters indicate height differences among treatments (α = 0.05).

Dominance

Dominance was statistically similar across the 

site types (P = 0.33) see figure 4 for examples 

of dominant and suppressed seedlings), but 

differed both among families (P = 0.004) and the 

interaction between site and family treatments 

(P = 0.04). Dominance was greatest for backcross 

family W4-75 (42 percent) and family W1-100 

(36 percent) and lowest for Chinese chestnuts 

(17 percent) (table 2). Family W4-75 was the only 

family to differ in dominance among site types; 

Table 2—Dominance probability 

among chestnut families

Chestnut 

family

Dominance after 

3 years (%)

American 22 ± 7 BC

Chinese 17 ± 6 C

W1-100 36 ± 9 AB

W3-20 27 ± 7 BC

W4-75 42 ± 9 A

Note: Means followed by the same 

letter are not statistically different.
Session 13: Interactions Between Resistance and Environment 105

Figure 4—(A) dominant and (B) suppressed chestnut seedlings, 3 years after planting. Arrows point to the top of each seedling. 

(Forest Service photo by Cornelia Pinchot)

54 percent of W4-75 seedlings growing in xeric 

sites were dominant, compared with 60 percent 

(± 15) in intermediate sites, and 18 percent (± 9) in 

sub-mesic sites. 

Blight Incidence

Chestnut blight was identified on 23 individual 

chestnut seedlings throughout the first three 

growing seasons; nine in xeric, and seven each 

in intermediate and sub-mesic sites. Blight was 

identified in three of five xeric, three of four 

intermediate, and four of five sub-mesic sites. 

Nine of the seedlings with blight symptoms were 

American chestnut, one Chinese, five W1-100, 

five W4-75, and three were W3-20 backcross 

hybrid chestnuts. 

DISCUSSION

Early survival of planted chestnut seedlings 

was high across site and family treatments. Two 

of the backcross families (W1-100 and W4-75) 

demonstrated superior results in at least one 

performance metric (diameter, survival, and/or 

dominance). Differences in performance among 

chestnut families has been found in other studies 

evaluating outplanting performance in forested 

settings (Clark et al. 2016, Pinchot et al. 2017, 

Thomas-Van Gundy et al. 2017). Of greater 

interest is the significant interaction between site 

and family treatments for height and dominance. 

American chestnuts demonstrated inferior height 

in xeric, compared to intermediate sites, while 

two of the backcross hybrid families demonstrated 

inferior growth in sub-mesic sites. Intermediate 

sites may offer enough soil moisture to optimize 

growth, without the intense competition 

characteristic of more mesic sites (Loftis 1983). 

Continued monitoring of the seedlings over time is 

necessary to determine if differences across sites 

will become more pronounced across all families, 

or if there is indeed a genotype by environment 

interaction for these variables. While incidence 

of blight was too low for statistical analysis, more 

American chestnuts were infected than Chinese or 

backcross hybrid chestnuts. Incidence and severity 
106 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

of blight infection will likely increase over time, 

as Hebard (1982) and Griffin (1989) have found 

for natural American chestnut sprout populations 

following canopy disturbance. Understanding if 

site and family treatments effect blight severity, 

particularly in the context of growth and 

competitive ability among treatments, will help 

inform a holistic American chestnut reintroduction 

strategy. 

ACKNOWLEDGMENTS

We thank Michael French and The American 

Chestnut Foundation for providing the American 

chestnut seedlings and Bob Hawkins and Rob 

Winks, of the Indiana Division of Forestry, for 

growing the backcross hybrid chestnuts used 

in this study. We acknowledge the Allegheny 

National Forest, the Pennsylvania Bureau of 

Forestry, Bradford Water Authority, Forest 

Investment Associates, Generations Forestry, 

Hancock Forest Management, Landvest, and 

Kane Hardwoods for the use of their field sites. 

We thank Charles Vandever for assistance with 

data collection. Todd Hutchinson, Stacy Clark, 

and Tom Hall provided useful feedback on earlier 

versions of this paper. 

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144–150. 

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invasive fungi as forest pathogens. Biological Invasions. 

11(1): 81–96. https://doi.org/10.1007/s10530-008-9321-3.

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silvicultural treatments in hardwood stands of the 

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American chestnut seedlings under a forest canopy. Journal 

of the American Chestnut Foundation. 16(2): 32–41.

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on American chestnut (Castanea dentata) seedling 

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canary in the coal mine: Sprouts as a rapid indicator of 

browse impact in managed forests. Ecological Indicators. 

69: 269–275. https://doi.org/10.1016/j.ecolind.2016.04.030.

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colonization: Moving along the conservation translocation 

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org/10.1111/j.1526-100X.2010.00724.x.

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S270–S279. https://doi.org/10.1080/07060660609507384.

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org/10.1111/j.1526-100X.2006.00131.x.
108 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

DRIVERS OF DISEASE EMERGENCE IN BOREAL CONIFER FORESTS, 

IMPORTANCE OF PHENOTYPIC BALANCE

Jan Stenlid1, Laura Brodde1, J. Julio Camarero2, 

Ángela Sánchez-Miranda2, and Jonàs Oliva1,3

We consider host–tree imbalances from a phenotypic perspective, the lack of co-evolutionary and 

evolutionary history with the pathogen and the environment, respectively. Phenotypic plastic responses 

to environmental shifts may become maladaptive when hosts are faced with novel pathogens. Complex 

interactions involving the interaction with the environment (host or pathogen phenotype) are in theory 

possible, but are still understudied. Following an increase of records in Northern Europe, the first 

large scale outbreak of Diplodia pinea was discovered in Southern Sweden in 2016. By microsatellite 

markers, we saw that Diplodia strains from the outbreak area did not differ from strains belonging to the 

pre-existing innocuous Diplodia population, rejecting the hypothesis of disease driven by a new strain of 

the pathogen. Disease increased steadily over time, but new infections were more frequent in anomalously 

dry years. Tree-ring and isotope (δ C) analyses showed that highly infected trees produced more latewood 

and had lower water-use efficiency than their non-infected counterparts prior to the outbreak, pointing 

to a phenotypic predisposition and increased susceptibility. However, following disease outbreak, the 

highly infected trees produced practically no latewood while more healthy trees maintained latewood 

formation. We speculate that infected trees, by forming more wood in the late growing-season show a less 

conservative water use and may have experienced stronger summer drought stress, making them more 

susceptible to the pathogen. Host phenotype plays a relevant role driving disease emergence in boreal 

forests. Since current climatic conditions in Sweden are suitable for Diplodia survival, management efforts 

should aim to minimize the spread of the pathogen. Trees with a conservative use of water may be more 

suitable for areas in which Diplodia is present and where climate models forecast warmer and possibly 

drier conditions.

1 Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Box 7026, 75007, Uppsala, Sweden, 

(jan.stenlid@slu.se). 2 Pyrenean Institute of Ecology (IPE-CSIC), Avda Montañana 1005, 50059 Zaragoza, Spain.

3Department Crop and Forest Sciences, University of Lleida, Alcalde Rovira Roure 191, 25198 Lleida, Spain.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Session 13: Interactions Between Resistance and Environment 109

INTRASPECIFIC GENETIC VARIATION IN DEFENSIVE INVESTMENT OF A MODEL 

MEDITERRANEAN PINE: A DENDROECOLOGICAL ANALYSIS OF RESIN DUCTS 

Title presented at workshop:

INTRASPECIFIC GENETIC VARIATION IN DEFENSIVE INVESTMENT VARIES 

ALONG THE ONTOGENY OF A MODEL MEDITERRANEAN PINE: 

A DENDROECOLOGICAL ANALYSIS OF RESIN DUCTS 

Carla Vázquez-González1, Rafael Zas1, Vicente Rozas2, 

Ricardo Alía3, and Luis Sampedro1

Conifers, the largest and longest-lived organisms 

on the planet, have evolved a sophisticated 

defensive system which has contributed to their 

colonization of diverse habitats (Franceschi et 

al. 2005). The defensive system of conifer trees 

heavily relies on the production of oleoresin, 

a terpenoid-based viscous fluid which is toxic 

for insect herbivores and pathogens (Phillips 

and Croteau 1999). Oleoresin is produced 

and accumulated in structures of different 

specialization degree, from single cells to a 

complex network of resin ducts in some genera 

such as Picea or Pinus (Bannan 1936, Wu and 

Hu 1997). Resin ducts protect the tree against 

pests by flushing out resin upon wounding, 

but also by acting as a mechanical barrier that 

prevents invasion by tree enemies (Franceschi et 

al. 2005). Importantly, resin duct characteristics 

(e.g., abundance and size) have been reported to 

be strongly associated with tree resistance against 

insect pests and pathogens in several conifer 

species. For instance, previous studies in different 

Pinus species have shown trees that survived after 

bark beetle attacks had more and/or larger xylem 

resin ducts than trees that died (Ferrenberg et al. 

2014, Hood et al. 2015, Kane and Kolb 2010). 

As other plant defensive traits, genetic and 

environmental factors mediate resin duct 

production. On the one hand, genetic variation in 

resin duct production has been reported in several 

conifer species (Esteban et al. 2012, Martin et al. 

2010, Zas et al. 2015). Such differences among 

populations are expected to be, at least partially, 

the result of local adaptation. Supporting this idea, 

Esteban et al. (2012) found that geographic and 

climatic variables at population origin explained 

intraspecific genetic variation in resin duct 

characteristics in P. nigra. On the other hand, 

environmental conditions that limit plant growth 

may enhance resource allocation to defences in 

trees due to the existence of trade-offs between 

both plant functions (Endara and Coley 2011, 

Herms and Mattson 1992). In this sense, previous 

studies have shown that trees growing under low 

resource (e.g., nutrients, light, water) conditions 

tend to growth less and increase their resin duct 

production (Moreira et al. 2015, Moreira et al. 

2008). In addition to the individual effects of plant 

genotype (G) and environment (E), there may also 

be interactive effects of these two major sources 

of variation (G×E interactions). An increasing 

number of studies have addressed G×E effects 

on resin duct production with contrasting results 

(Hannrup et al. 2004, Moreira et al. 2015, Rosner 

and Hannrup 2004, Westbrook et al. 2013). Such 

interactions may have important implications 

for tree breeding, such as unstable selection for 

resistance when considering global warming 

scenarios. More research is thus needed to derive 

general patterns. 

Plant defence allocation can be also determined 

by plant ontogeny (Barton and Koricheva 2010, 

Boege and Marquis 2005). In particular, previous 

studies have reported both positive and negative 

trends in defensive investment with tree age 

(Boege and Marquis 2005). However, none of 

1Misión Biológica de Galicia-Consejo Superior de Investigaciones Científicas, Pontevedra, Spain 36143, (cvazquez@mbg.csic.es). 2 Departamento de Ciencias Agroforestales, Universidad de Valladolid, Campus Duques de Soria, Soria, Spain 42004.

3Centro de Investigación Forestal CIFOR – INIA, Madrid, Spain 28040.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
110 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

these previous studies have investigated the 

effect of plant ontogeny on resin duct production. 

Axial resin ducts are recorded in growth rings, 

representing thus an annually resolved archive of 

plant defences. This special feature allows their 

quantification by standard dendrochronological 

procedures (Fritts 2012) to retrospectively assess 

the existence of age-related effects on defensive 

investment. 

In this study, we aimed at disentangling the 

independent and interactive effects of plant 

genotype and environment on annual resin duct 

production across the last 30 years of growth 

of a model Mediterranean pine (P. pinaster). 

Additionally, we explored the adaptive origin of 

this anatomical defensive trait by testing whether 

climatic gradients at the population origin explain 

intraspecific genetic variation in resin duct 

production. 

We sampled P. pinaster trees from two replicated 

genetic trials established in 1967 located in 

two different sites (Cabañeros and Riofrío). 

Cabañeros was located on a plain top-hill 

while Riofrío was on a mid-hill with irregular 

topography and stonier soil. These contrasting 

abiotic conditions caused that tree growth was 

significantly higher in Cabañeros than in Riofrío. 

In each genetic trial, we sampled stem cores from 

trees belonging to nine different populations 

(N = 10 trees per population) covering most of 

the natural distribution of this species. Stem cores 

were extracted at breast height with a Pressler 

borer, obtaining four cores per tree, each from a 

cardinal point. Wood cores were progressively 

sanded and visually crossdated following standard 

dendrochronological procedures (Fritts 2012). 

We measured tree-ring width and statistically 

validated the crossdating applying a crosscorrelation analysis in COFECHA software 

(Grissino-Mayer 2001). We counted axial resin 

ducts (RD) in each annual growth ring in the 

latewood (LW), the earlywood (EW) and the 

total ring (TR) for the last 30 years of growth. 

Annual RD density was calculated as the number 

of RD per squared millimetre of wood and year 

(RD mm-2 yr-1). We ran linear mixed models to 

test for the effects of site, population and their 

interaction (all fixed factors) on annual RD 

density. To test for ontogenetic effects on resin 

duct production during three 10-year periods 

along the tree lifespan (10–20, 20–30, 30–40 years 

old), we also included the effect of year as a fixed 

categorical factor in the model. Block within 

site and population by block within site were 

included as random factors. Finally, to assess the 

existence of climatic clines explaining among 

population variation, we performed correlations 

between climatic variables at the population origin 

and annual RD density. The climatic data was 

obtained according to a climatic model detailed 

in Gonzalo (2008). All statistical procedures were 

performed in R software version 3.4.3 (RStudio 

Team 2016).

Our results showed no significant differences 

in mean annual RD density in the TR among 

pine populations and sites (table 1). However, 

when we separately analysed EW and LW, we 

found significant variation in annual RD density 

in the EW and LW among pine populations 

(table 1). A number of studies have also reported 

intraspecific genetic variation in RD production 

among populations in different Pinus species 

(Esteban et al. 2012, Martin et al. 2010, Zas et al. 

2015). However, none of the previous studies have 

measured RD production separately in both ring 

compartments. Similarly, we found significant 

differences in RD density in the EW across sites 

(table 1), being such RD density markedly higher 

in the low quality site (Riofrío). These findings 

agree with a long-standing ecological paradigm 

which predicts that trees growing under poorresource environments tend to invest less in 

growth and more in defence (Endara and Coley 

2011, Herms and Mattson 1992). 

Our results also showed that the G×E interaction 

did not significantly affect RD density (fig. 1, 

table 1), indicating that the relative performance of 

the populations regarding resin duct differentiation 

is stable across environments. Our results 

agree with those reported previously in Picea 

abies under drought conditions (Hannrup et al. 

2004, Rosner and Hannrup 2004). Contrary to 

our findings, Moreira et al. (2015) found that 

P. pinaster genotypes markedly differed in the 

phenotypic plasticity of resin duct production 

in response to soil nutrient availability (i.e., a 
111

Table 1—Effect of Population (Pop), site (S), year and their 

interactions on Pinus pinaster resin duct (RD) density 

(RD mm-2 yr-1) in total ring, the earlywood and the latewood, 

F-ratio and associated p-values are shown

Total ring Earlywood Latewood

F p-value F p-value F p-value

Pop 2.1 0.06 2.9 0.011 4.40 < 0.01

S 4.2 0.09 18.8 < 0.01 5.14 0.06

Year 39.1 < 0.01 74.2 < 0.01 68.49 < 0.01

Pop × S 0.7 0.72 0.7 0.71 0.55 0.81

Pop × Year 2.6 < 0.01 1.4 < 0.01 1.50 0.01

S × Year 10.7 < 0.01 12.6 < 0.01 10.93 < 0.01

Linear mixed model results. Significant p-values are indicated in bold.

Figure 1—Relationship between annual RD density in earlywood and latewood in both common gardens 

(Cabañeros and Riofrío). Each dot represents a Pinus pinaster population (N = 9). R-squared coefficients 

(R2

) are shown. Asterisks indicate significant (+P < 0.1, *P < 0.05, **P < 0.01) P-values.

significant G×E interaction). However, these 

contrasting findings could be explained by 

ontogenic effects. In particular, these autors 

measured resin duct production in juvenile trees 

(Moreira et al. 2015), whereas we focused our 

study in adult (reproductive) trees. Supporting this 

idea, we found that plant ontogeny significantly 

affected resin duct production (significant effect 

of year; table 1), where RD density in the EW was 

significantly greater at younger stages and RD 

density in LW was significantly greater at older 

stages (fig. 2). Previous studies have commonly 

reported increased resin production with tree age 

in other tree species (Bryant and Julkunen-Tiitto 

1995), but to our best knowledge this is the first 

study reporting plant ontogenetic effects on resin 

duct production. 

Finally, our results showed that local climate at 

the population origin significantly explained resin 

duct differences among populations. Specifically, 

RD density in EW was negatively correlated with 

summer temperature, whereas RD density in LW 

was positively correlated with spring temperature 

(r = -0.89 and r = 0.72 respectively; p < 0.05). 

Accordingly, our results suggest that pine trees 

from populations at warmer regions produce 

more RD in the late growing season, while resin 

duct development takes place earlier in trees 

from populations at colder regions. Supporting 

our findings, Esteban et al. (2012) found that 

temperature at the population origin negatively 

affected resin duct investment among P. nigra

populations. 

Despite the implications of G×E interactions for 

tree breeding, to date most breeding programs 

have been developed based on genetic or 

environmental factors, disregarding the effect of 

their interaction. Our results indicate that, despite 

we observed significant effects of plant ontogeny 

and environment on resin duct production, 

Session 13: Interactions Between Resistance and Environment
112 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

Figure 2—Mean annual RD density (RD mm-2 yr-1) in the earlywood and latewood of Pinus pinaster

trees in both common gardens (white dots for Cabañeros and black dots for Riofrío). Values represent 

mean annual RD density in three different 10-year periods along tree lifespan.

intraspecific genetic variation in this anatomical 

defence can be exploited in breeding programs 

for stable resistance, due to the absence of G×E 

effects. Moreover, the contribution of climatic 

clines explaining among population differentiation 

highlights the importance and putatively adaptive 

value of this trait. However, other processes 

that generate non-adaptive intraspecific genetic 

variation (e.g., genetic drift and demographic or 

migratory patterns) can also contribute to explain 

phenotypic differences among populations (Grivet 

et al. 2010). For this reason, in order to fully 

understand the adaptive value of this anatomical 

defensive trait, further work should also take into 

account the effect of neutral genetic variation.

LITERATURE CITED

Bannan, M. 1936. Vertical resin ducts in the secondary wood 

of the Abietineae. New Phytologist. 35: 11–46.

Barton, K.E.; Koricheva, J. 2010. The ontogeny of plant 

defense and herbivory: characterizing general patterns 

using meta-analysis. The American Naturalist. 175: 481–

493.

Boege, K.; Marquis, R.J. 2005. Facing herbivory as you grow 

up: the ontogeny of resistance in plants. Trends in Ecology 

& Evolution. 20: 441–448.

Bryant, J.P.; Julkunen-Tiitto, R. 1995. Ontogenic development 

of chemical defense by seedling resin birch: energy cost of 

defense production. Journal of Chemical Ecology. 21(7): 

883–896.

Endara, M.J.; Coley, P.D, 2011. The resource availability 

hypothesis revisited: a meta‐analysis. Functional Ecology. 

25: 389–398.

Esteban, L.G.; Martin, J.A.; de Palacios, P.; Fernandez, 

F.G. 2012. Influence of region of provenance and climate 

factors on wood anatomical traits of Pinus nigra Arn. subsp 

salzmannii. European Journal of Forest Research. 131: 

633–645.

Ferrenberg, S.; Kane, J.M.; Mitton, J.B. 2014. Resin duct 

characteristics associated with tree resistance to bark 

beetles across lodgepole and limber pines. Oecologia. 174: 

1283–1292.

Franceschi, V.R.; Krokene, P.; Christiansen, E.; Krekling; T. 

2005. Anatomical and chemical defenses of conifer bark 

against bark beetles and other pests. New Phytologist. 

167(2): 353–376.

Fritts, H. 2012. Tree rings and climate. Elsevier. 582 p.

Gonzalo, J. 2008. Phytoclimatic analysis of the Spanish 

Peninsula: update and geostatistical analysis. University of 

Valladolid; Palencia; Spain. Ph.D. dissertation.

Grissino-Mayer, H.D. 2001. Evaluating crossdating 

accuracy: a manual and tutorial for the computer program 

COFECHA. Tree-Ring Research. 

Grivet, D.; Sebastiani, F.; Alía, R. [et al.]. 2010. Molecular 

footprints of local adaptation in two Mediterranean 

conifers. Molecular Biology and Evolution. 28: 101–116.

Hannrup, B.; Cahalan, C.; Chantre, G. [et al.]. 2004. Genetic 

parameters of growth and wood quality traits in Picea 

abies. Scandinavian Journal of Forest Research. 19: 14–29.
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Martin, J.A.; Esteban, L.G.; de Palacios, P.; Fernandez, 

F.G. 2010. Variation in wood anatomical traits of Pinus

sylvestris L. between Spanish regions of provenance. TreesStructure and Function. 24: 1017–1028.

Moreira, X.; Zas, R.; Solla, A.; Sampedro, L. 2015. 

Differentiation of persistent anatomical defensive structures 

is costly and determined by nutrient availability and genetic 

growth-defence constraints. Tree Physiology. 35: 112–123.

Moreira, X. Q.; Sampedro, L.; Zas, R.; Solla, A. 2008. 

Alterations of the resin canal system of Pinus pinaster

seedlings after fertilization of a healthy and of a Hylobius 

abietis attacked stand. Trees-Structure and Function. 22: 

771–777.

Phillips, M.A.; Croteau, R.B. 1999. Resin-based defenses in 

conifers. Trends in Plant Science. 4: 184–190.

Rosner, S.; Hannrup, B. 2004. Resin canal traits relevant 

for constitutive resistance of Norway spruce against bark 

beetles: environmental and genetic variability. Forest 

Ecology and Management. 200: 7–87.

R Studio Team. 2016. R Studio: Integrated Development for 

R. Boston: R Studio, Inc. http://www.rstudio.com/. [Date 

accessed: 5/1/20].

Westbrook, J.W.; Resende, M.F.R.; Munoz, P. [et al.]. 2013. 

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discovering genes and predicting phenotype for improved 

resistance to bark beetles and bioenergy potential. New 

Phytologist. 199: 89–100.

Wu, H.; Hu, Z.H. 1997. Comparative anatomy of resin ducts 

of the Pinaceae. Trees. 11: 135–143.

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Trees-Structure and Function. 29: 663–673.

Poster Session 115

Poster Session
116 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

AN ANSWER AWAITING A QUESTION: THE UNITED KINGDOM 

NATIONAL TREE SEED PROJECT COLLECTIONS

Alice R. Hudson1, Richard J.A. Buggs 2,3, and Clare Trivedi4

Forest trees are facing an increasing range of threats from pests and pathogens globally. Because of this, 

research into both natural and induced resistance is more important than ever to provide a solid foundation 

for informed management decisions. Despite this, there are a wide range of challenges researchers face 

including easy access to suitable native plant material. Here we present a forest tree resource developed 

by the Royal Botanic Gardens (RBG) Kew Millennium Seed Bank Partnership. The UK National Tree 

Seed Project (UKNTSP) was established in 2013 with the aim of collecting and storing seeds from all UK 

trees across their native UK distribution to capture as much the genetic diversity as possible. Now in its 

fifth year, this collection totals over 9.6 million seeds, with multi-provenance collections for 68 native UK 

species. For 22 species, we have between 8 and 53 seed collections banked by individual maternal trees 

many of which are georeferenced. This broad suite of species covers many facing particular plant health 

threats in the UK and elsewhere such as Fraxinus excelsior, Juniperus communis, and Ulmus glabra. This 

unique resource offers the potential to study natural variability in pest and pathogen resistance, pest-host 

relationships, as well as the possibility to test control mechanisms. This resource is ready and available 

for research via the Millennium Seed Bank (MSB) Seed List along with accompanying field data and 

herbarium specimens.

1RBG Kew Millennium Seed Bank Partnership, Wakehurst Place, Ardingly, United Kingdom RH17 6TN, (alice.hudson@kew.org). 2RBG Kew, Richmond, United Kingdom TW9 3AB.

3 Queen Mary University, London, United Kingdom E1 4NS.

4RBG Kew Millennium Seed Bank Partnership, Wakehurst Place, Ardingly, United Kingdom RH17 6TN.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Poster Session 117

GENOTYPIC DIVERSITY AND REPRODUCTIVE BIOLOGY OF THEKOPSORA AREOLATA, 

THE CAUSAL AGENT OF CHERRY SPRUCE RUST IN NORWAY SPRUCE SEED ORCHARDS

Hernán Capador1, Berit Samils1, and Åke Olson1

Swedish forestry is sustained by planting about 200 million Norway spruce (Picea abies) seedlings 

every year. Most of these seedlings come from seed orchards, which have been established to transfer 

the genetic gain in growth and yield achieved through plant breeding since the 1940s. However, today 

there is a deficit of seeds coming from Swedish seed orchards mainly caused by irregular flowering, but 

also due to pest and pathogen infections such as Thekopsora areolata—the causal agent of cherry spruce 

rust, a fungus that significantly reduces seed production in Fennoscandia. Here, we aim to investigate 

the reproduction mode and population structure of the pathogen using a newly developed microsatellite 

marker and a hierarchical sampling strategy. Sampling was done at different locations: one in Norway, one 

in Finland and five in Sweden. For each location, one aecium per infected cone was analyzed. In addition, 

multiple aecia per scale and cone were sampled at two locations in Sweden. The results show an overall 

high genetic diversity in T. areolata at all hierarchical levels with no genetic structure, an indication of 

high gene flow and random mating. However, at the cone/scale level non-random mating was observed. 

These results suggest that T. areolata has long distance spore dispersal in Fennoscandia with common 

recombination events and vegetative spread in cones and scales.

1 Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden, 

(hernan.capador@slu.se).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
118 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

AN IMPROVED CHINESE CHESTNUT GENOME

John E. Carlson1, Margaret E. Staton2, Charles Addo-Quaye1,3, Nathaniel Cannon1, 

Tetyana Zhebentyayeva4, Nurul Islam-Faridi 5, Jiali Yu2, Matthew Huff 2, Shenghua Fan6, 

Anna O. Conrad6, Stephan C. Schuster1,7, Albert G. Abbott6, Jared Westbrook8, Jason Holliday9, 

C. Dana Nelson10, Laura Georgi11, and Frederick V. Hebard11

SUMMARY

The introduction of the chestnut blight fungus (Cryphonectria parasitica) to North America in 1904 

devastated American chestnut populations. Asian Chestnut species, which evolved resistance to the 

sympatric chestnut blight fungus, are being used as donor species for the transfer of resistance genes to 

C. dentata and C. sativa via hybridization. In the United States, Chinese chestnut (Castanea mollissima) 

genotypes are the source of blight-resistance genes for introgression into American chestnut by backcross breeding. To better understand the genetic basis of blight resistance and to provide tools for chestnut 

breeding, we sequenced the genome of Chinese chestnut, with a particular focus on the major blight 

resistance quantitative trait locus (QTL). A draft genome (v.1.1) covering app. 90 percent of the genome 

of The American Chestnut Foundation’s Chinese chestnut cultivar “Vanuxem” was released to the public 

in January 2014. Recombinant DNA clones covering the three major blight resistance QTL were also 

sequenced to great depth. Over 780 genes were identified in the 3 blight resistance QTLs, including 15 

known “defense response” genes. We are developing a new version of the Chinese chestnut genome with 

chromosome-scale assemblies of genome scaffolds anchored to the 12 chestnut linkage groups. This will 

serve as a reference for genome-wide selection in advanced generations of backcross breeding programs, 

and for basic research on genome structure and function in woody plants. 

GOALS

The overarching goals of the public Chinese 

chestnut genome project were to:

(1) Construct a complete genome sequence for 

Castanea mollissima

(2) Identify candidate genes for Chryphonectria 

parasitica (chestnut blight) resistance.

(3) Provide tools to accelerate breeding 

and restoration of C. dentata (American 

chestnut) 

PROGRESS

Version 1 of the Chinese Chestnut Genome 

The first version of the genome assembly was 

produced for the TACF cultivar Vanuxem (fig. 1). 

Derived from 60 Gb of 454 and Illumina NGS 

sequence data, the assembly comprised 724.4 Mb 

in 41,270 scaffolds which averaged app. 40,000 

bp in length (table 1). This represented 91 percent 

of the predicted size of the Chinese chestnut 

genome, based on estimates from previous 

1Schatz Center for Tree Molecular Genetics, Pennsylvania State University, University Park, PA 16802, (jec16@psu.edu).

2University of Tennessee, Knoxville, TN 37996.

3Division of Natural Sciences and Mathematics, Lewis-Clark State College, Lewiston, ID 83501.

4Clemson University, Clemson, SC 29634.

5USDA Forest Service, Saucier, MS 39574.

6 Forest Health Research and Education Center, University of Kentucky, Lexington, KY 40506.

7 Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technology University, Singapore 639798.

8The American Chestnut Foundation, Meadowview, VA 24361.

9Virginia Polytechnic and State University, Blacksburg, VA 24061.

10 Forest Health Research and Education Center, Southern Research Station, USDA Forest Service, Lexington, KY 40546. 11Retired from The American Chestnut Foundation, Meadowview, VA 24361.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Poster Session 119

Figure 1—Fred Hebard and Laura Georgi with a clone of the 

cultivar “Vanuxem” at Meadowview, VA.

Table 1—Sequencing and assembly statistics for the first genome assembly, v1.1
120 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

cytometry studies. A total of 36,146 likely genes 

were identified. The quality and contiguity of 

the version 1.1 genome for Chinese chestnut 

proved suitable for genome-wide studies of 

genetic diversity, gene expression, DNA marker 

development, and evolutionary studies. LaBonte 

et al. (2018) used the version 1.1 genome to 

detect signatures of selection for genes related 

to flower phenology and development, fruit 

maturation, and secondary metabolism, and genes 

potentially impacted by domestication in the 

Chinese Chestnut genome. We conducted initial 

genome-wide comparative genome structure and 

evolution analyses, which revealed the levels of 

similarities with genes from other plants, and 

the extent to which large blocks of syntenies are 

shared between chestnut and model genome such 

as peach, grape, and poplar (fig. 2). 

Version v1.1 of the Chinese chestnut genome 

was released to the public as a genome 

browser in January 2014 at the website https://

hardwoodgenomics.org/chinese-chestnut-genome, 

developed and curated by Margaret Staton, 

along with browsers for the three chestnut-blight 

resistance QTL. For the blight resistance QTL, 

sets of overlapping BAC recombinant DNA clones 

that physically spanned each QTL region were 

separately sequenced and assembled into a total 

of 395 scaffolds covering 13.8 Mb (table 2). Over 

1,900 genes were found in the QTLs, including 

194 known stress-response genes, from which 

15 candidate genes for blight resistance were 

selected for further study (Fang et al. 2013). 

Annotations of the QTL gene sequences were used 

to conduct GO functional category analyses. From 

Figure 2—Examples of synteny analyses to model plant genomes and plant gene sequence databases.
Poster Session 121

QTL LG # 

scaffolds 

# bases / 

QTL 

Total # 

Genes 

# Genes / 

Mbases 

# Stressresponse genes 

cbr1 B 214 6.77Mb 994 151 98 

cbr2 F 128 4.12Mb 548 137 58 

cbr3 G 53 2.99Mb 410 139 38 

Table 2—Chestnut blight-resistance QTL assembly and gene content information

the genes classified as disease- or stress-response, 

15 of the most likely candidate genes for chestnut 

blight resistance were identified (table 3) for future 

transgenic studies. The genome and the QTL 

browsers have had thousands of visits from across 

the globe, for gene searches and downloads of 

genome and QTL scaffolds, genes, transcripts, and 

predicted protein data. 

Improvements to the Chinese Chestnut Genome

For application of the chestnut reference genome 

in Genome-Wide-Selection to advance back-cross 

breeding and disease resistance introgression 

programs, such as the TACF is conducting, 

chromosome-scale sequences assemblies of 

scaffolds are required. For the past 4 years, 

Table 3—Fifteen candidate disease resistance genes in Chinese chestnut blight-resistance QTL selected for 

future functional studies
122 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

we worked on producing such an improved, 

chromosome-scale version of the Chinese chestnut 

genome. The approach taken to accomplish this 

involved merging assembled sequence contigs 

and scaffolds from the v1.1 genome using longer 

genome sequences, followed by the anchoring 

of the larger scaffolds to positions on genetic 

linkage maps based on alignment to DNA 

marker sequences defining loci on the genetic 

maps (fig. 3). Taking this approach, we used 

long PACBio sequences (over 10 Kb in length) 

to bridge contigs into scaffolds and close gaps 

within scaffolds, reducing the number of genome 

scaffolds to 12,684, covering 784 Mb of genome 

sequence (table 4). By aligning sequences from 

BAC clones distributed across the physical length 

of the genome (Fang et al 2013), we estimated 

that app. 98 percent of the estimated genome 

size was included in the new set of scaffolds. 

Margaret Staton’s group used our RNA sequence 

resources for chestnut to find and annotate 30,832 

high quality gene models in the new assembly. 

Pseudo-chromosome sequences were assembled 

by anchoring 4,099 of the scaffolds to DNA 

markers in the reference genetic linkage map 

for chestnut (Kubisiak et al. 2013) (fig. 3D), and 

the integrated genetic-physical map for Chinese 

chestnut (Fang et al., 2013) (fig. 3A). The pseudochromosome sequences accounted for 421.3Mb, 

representing about 60 percent of the full genome. 

A B C D 

Figure 3—Illustration of approach taken in building pseudo-chromosome sequences by alignment and 

anchoring to genetic, physical, and cytology maps. (A) Example of physical map integrated with linkage 

group, (B) a chromosome-scale assembly of scaffolds, (C) cytogenetic map of linkage group DNA markers, 

and (D) chestnut genetic linkage map alignment example.
123

METRIC Chromosome-scale assembly of the Chinese chestnut genome 

Contig Assembly • 421.3Mb (4,099 contigs) assembly of scaffolds anchored to C. 

mollissima chromosomes (genetic linkage groups) 

• Anchored sequence per chromosome ranged from 25.9Mb (LG_L) 

to 60.2 Mb (LG_A) 

• 303.9Mb (10,011 contigs) unanchored contig sequences 

• 57.8Mb estimated gaps (based on estimated genome size of 794Mb) 

Gene models: 

• 30,832 high quality gene models identified in V3.2 

• 20,770 high quality gene models in anchored contigs 

Validations: • BUSCO reported 1,355 of 1,440 expected single-copy genes are 

complete and present within the C. mollissima genome 

• Alignments of BAC end sequences from the C. mollissima physical 

map confirmed order of contigs in the pseudochromosomes 

Table 4—Statistics for assembly improvements and initial anchoring of scaffolds to genetic maps

Poster Session

Gene positions and overall genome organization 

of the assembly were determined and compared to 

the genomes of other related trees and model plant 

systems. A manuscript on this chromosome-scale 

assembly (table 4) was published as a preprint on 

April 22, 2019 (Staton et al. 2019). Submission of 

a peer-reviewed manuscript is planned, following 

efforts to anchor more scaffold to increase gene 

and genome coverage in the pseudo-chromosomes. 

FUTURE RESEARCH DIRECTIONS

The current assembly, although an incomplete 

draft, does by virtue of chromosome-scale 

sequences provide a significant advancement in 

our ability to investigate genome organization 

and the evolution and genetic structure of 

important traits such as disease resistance, 

as well as applications such as genome-wide 

selection. Future improvement of the assembly 

may be achieved through the use of more recent 

long-read technologies, such as Nanopore 

(Madoui et al. 2015), and/or scaffolding with 

chromatin-interaction data, such as Hi-C 

(Jiao and Schneeberger 2017). However even 

these approaches may result in less than fullgenome assembly given the challenges of high 

heterozygosity levels and an inability to generate 

dihaploid individuals in Chinese chestnut. The 

recently published Quercus robur genome 

(Plomion et al. 2018) utilized synteny with the 

Prunus persica genome to order incorporate 

contigs and scaffolds that had not been assembled 

de novo nor scaffolded with oak genetic map 

markers. This approach assumes that micro-level 

syntenies follow known macro-syntenies based 

on genetic maps, which may not always hold true. 

However, the hybrid synteny approach could also 

complement long-read technologies in future 

Chinese chestnut genome improvements. 

Data Availability

The contigs, scaffolds, and pseudochromosome 

sequences are available at the NCBI BioProject 

No. PRJNA46687, and are also available for 

download and query at the Hardwood Genomics 

Project website (https://www.hardwoodgenomics.

org/genomes). 
124 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

FUNDING

Funding was provided by the Forest Health 

Initiative through grant # 137RFP#2008-011 to 

JEC. Support was also provided by the USDA 

National Institute of Food and Agriculture grant 

2016-67013-24581 to The American Chestnut 

Foundation, grants-in-aid to JEC from The 

American Chestnut Foundation, and to JEC 

through the USDA National Institute of Food 

and Agriculture Federal Appropriations under 

Project PEN04532, Accession number 1000326. 

Bioinformatics was supported by NSF Award 

#1444573 to MS (Main, PI).

REFERENCES

Fang, G.C.; Blackmon, B.P.; Staton, M.E. [et al.]. 2013. 

A physical map of the Chinese chestnut (Castanea 

mollissima) genome and its integration with the genetic 

map. Tree Genetics & Genomes. 9(2): 525–537.

Jiao, W.B.; Schneeberger, K. 2017. The impact of third 

generation genomic technologies on plant genome 

assembly. Current Opinion in Plant Biology. 36: 64–70.

Kubisiak, T.L.; Nelson, C.D.; Staton, M.E. [et al.]. 2013. 

A transcriptome-based genetic map of Chinese chestnut 

(Castanea mollissima) and identification of regions of 

segmental homology with peach (Prunus persica). Tree 

Genetics and Genomes. 9(2): 557–571.

LaBonte, N.R.; Zhao, P.; Woeste, K. 2018. Signatures of 

selection in the genomes of Chinese chestnut (Castanea 

mollissima Blume): the roots of nut tree domestication. 

Frontiers in Plant Science. 9: 810.

Madoui, M.A.; Engelen, S.; Cruaud, C. [et al.]. 2015. Genome 

assembly using Nanopore-guided long and error-free DNA 

reads. BMC Genomics. 16(1): 327.

Plomion, C.; Aury, J.M.; Amselem, J. [et al.]. 2018. Oak 

genome reveals facets of long lifespan. Nature Plants. 4(7): 

440–452.

Staton, M.; Addo-Quaye, C.; Cannon, N. [et al.]. 2019. 

The Chinese chestnut genome: a reference for species 

restoration. BioRxiv. 615047.
Poster Session 125

ADVANCED PHENOTYPING USING FT-IR DISTINGUISHES DISEASE RESISTANCE IN 

FRAXINUS EXCELSIOR AGAINST HYMENOSCYPHUS FRAXINEUS

Caterina Villari1, Arnaud Dowkiw2, Rasmus Enderle3, Marjan Ghasemkhani4, Thomas Kirisits5, 

Erik Kjaer 6, Diana Marčiulynienė7, Lea McKinney6, Berthold Metzler3, Facundo Muñoz2, 

Lene Rostgaard Nielsen6, Alfas Pliūra7, Lars-Göran Stener 8, Vytautas Suchockas7, 

Luis Rodriguez-Saona9, Pierluigi Bonello10, and Michelle Cleary4

The invasive ascomycete fungus Hymenoscyphus fraxineus has caused large scale population decline of 

European ash (Fraxinus excelsior) throughout much of its distribution range in Europe. Large genotypic 

variation in susceptibility to the pathogen exists in the natural population, albeit resistance occurs at a 

low level. Damage traits associated with the pathogen are strongly genetically controlled, which suggests 

that considerable gain can be achieved through selection and breeding. Large potential exists for utilizing 

modern approaches or tools that can quantitatively identify phenotypes and help explain the genetic 

basis of important plant traits, such as disease resistance. One main obstacle to making such advances is 

the lack of viable tools for rapid resistance phenotyping. Vibrational spectroscopy is one approach that 

has been used successfully for rapid phenotyping for plant resistance. In this study, we used Fouriertransform Infrared (FT-IR) spectroscopy coupled with a chemometric model to discriminate between 

resistant and susceptible European ash genotypes. Non-infected leaves and stem samples from known 

susceptible and resistant F. excelsior genotypes were collected from six European countries. Purified 

phenolic extracts were analyzed on a FT-IR spectrometer and soft independent modeling of class analogy 

(SIMCA) was used to discriminate between resistant and susceptible trees. The model built with stem 

samples, and subsequently validated using random blind samples, gives powerful evidence that FT-IR can 

clearly discriminate between susceptible and resistant genotypes. These results suggest that spectroscopic 

phenotyping tools may allow for superior genotypes to be quickly identified and employed in restoration 

efforts.

1Warnell School of Forestry & Natural Resources, University of Georgia, Athens, GA 30602.

2 Institut National de la Recherche Agronomique (INRA) 2163 Avenue de la Pomme de Pin, 45075 Ardon, Olreans, France.

3 Forest Research Institute BadenWurttemberg, Department Forest Protection Wonnhaldestrasse 4, 79100 Freiburg, Germany.

4 Swedish University of Agricultural Sciences (SLU), Southern Swedish Forest Research Centre Sundsvägen 3, 23053 Alnarp, Sweden.

5 Institute of Forest Entomology, Forest Pathology and Forest Protection (IFFF), Department of Forest and Soil Sciences, University of Natural 

Resources and Life Sciences, (BOKU), Peter-Jordan-Straße 82, 1190 Vienna, Austria.

6 Department of Geosciences and Natural Resource Management, University of Copenhagen Rolighedsvej 23, 1958 Frb Copenhagen, 

Denmark.

7Lithuanian Research Centre for Agriculture and Forestry, Institute of Forestry Liepu 1, LT53101 Girionys, Kaunas district, Lithuania.

8 SKOGFORSK - The Forest Research Institute, Ekebo 2250, 26890 Svalöv, Sweden.

9 Department of Food Science and Technology, Ohio State University, Parker Food Science and Technology Columbus, OH 43210.

10 Department of Plant Pathology, Ohio State University, 201 Kottman Hall Columbus, OH 43210, (Michelle.Cleary@slu.se).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
126 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

DELPHI EXPERT OPINION SURVEY TO ASSESS THREATS TO 

OAKS IN THE EASTERN UNITED STATES

Anna O. Conrad1, Ellen V. Crocker1, Xiaoshu Li1, Billy Thomas1, 

Thomas Ochuodho1, and C. Dana Nelson2

Oaks are important fixtures of many Eastern United States forests, providing both ecological and 

economic benefits. While regeneration is a major issue impacting oaks currently, biotic (e.g., pests and 

pathogens) and abiotic (e.g., abnormal weather and climate change) stressors, may also threaten oaks in 

this region. The goal of our Delphi expert opinion survey is to identify the most significant threats (biotic 

and abiotic) to oaks in the Eastern United States (as defined by the eastern and southern regions of the 

U.S. Forest Service), and to gauge the potential impact of these threats on oaks. To accomplish this, we 

initiated a three-part Delphi expert opinion survey. The iterative Delphi approach is useful for evaluating 

consensus (or lack thereof) among experts on a specific topic. In the course of this survey series, we asked 

experts to identify current and future biotic and abiotic threats to oaks, and then based on expert opinions, 

gauged the current and potential impact of these threats by asking a series of questions concerning, for 

example, their spatial and temporal manifestation. Data collected as part of this Delphi survey series will 

be used to support subsequent analyses aimed at assessing the economic impact of these threats, and may 

be useful for prioritizing the management of these threats within the Eastern United States. 

1 Forest Health Research and Education Center, Department of Forestry and Natural Resources, University of Kentucky, Lexington, KY 

40506, (e.crocker@uky.edu). 2 Forest Health Research and Education Center, Southern Research Station, USDA Forest Service, Lexington, KY 40506.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Poster Session 127

TREESNAP: A CITIZEN SCIENCE APP CONNECTING 

TREE ENTHUSIASTS AND FOREST SCIENTISTS

Title presented at workshop:

TREESNAP: A MOBILE APP TO HELP YOU ENGAGE THE PUBLIC IN RESEARCH

Bradford Condon1, Ellen Crocker 2, Abdullah Almsaeed3, 

C. Dana Nelson3, Albert G. Abbott2, and Margaret Staton1

SUMMARY

(from Plants, People, Planet PPP article)

• TreeSnap is a citizen science project that enables members of the public to observe and report trees of 

interest to scientists with a focus on tree species impacted by invasive pests and pathogens.

• TreeSnap includes a free mobile app that guides users in creating an observation, including answering 

questions customized for each tree species, taking relevant photos, and automatically collecting the 

GPS coordinates.

• This utility of a mobile app and online database for tree observations has been successful beyond the 

initial project plan, facilitating scientific data collection by both citizen scientists and professional 

scientists.

• Technology designed to be intuitive and comprehensive for the needs of both professional and 

nonprofessional scientists, as demonstrated by TreeSnap, can greatly expand the reach of tree research 

programs and engage the public in forest health research.

Note: For complete abstract, please see paper 49 (page 92).

The full paper was published in Plants, People, Planet

https://nph.onlinelibrary.wiley.com/toc/25722611/2020/2/1.

The reference for the published paper is:

Crocker, E.; Condon, B.; Almsaeed, A.; Jarret, B.; Nelson, C.D.; Abbott, A.G.; Main, D.; Staton, M. 2019. TreeSnap: 

A citizen science app connecting tree enthusiasts and forest scientists. People, Plants, Planet. 2(1): 47–52. DOI: 

https://10.1002/ppp3.41.

1University of Tennessee, Knoxville, TN 37996.

2University of Kentucky, Lexington, KY 40536, (crocker@uky.edu). 3 Forest Health Research and Education Center, Southern Research Station, USDA Forest Service, Lexington, KY 40536.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
128 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

PHYTOPHTHORA RAMORUM IN LARCH: FROM EPIDEMIOLOGY TO HOST RESISTANCE

Title presented at workshop:

UNDERSTANDING SUDDEN LARCH DEATH—FROM EPIDEMIOLOGY TO HOST RESISTANCE

Heather Dun1,2, John Mackay1, and Sarah Green2

Phytophthora ramorum is a forest pathogen 

known for two major epidemics, sudden oak death 

in the USA, which began in the 1990s (Rizzo et 

al. 2002) and, more recently, sudden larch death 

in the United Kingdom in which P. ramorum was 

first found infecting Japanese larch in south-west 

England in 2009. This was the first record of 

P. ramorum infecting a conifer species in a natural 

situation (Webber et al. 2010). In spring 2013 there 

was an observed dramatic increase in the spread 

and severity of P. ramorum in larch stands across 

western Britain, including in the Galloway Forest. 

The Galloway Forest is the largest forest in the UK 

and provides a particularly interesting field site for 

studying the epidemiology of P. ramorum in larch 

as disease control legislation for the 2013 epidemic 

resulted in a management zone where infected 

stands could be retained for research rather 

than being immediately felled. The Galloway 

Forest outbreak was caused by the EU2 lineage 

of P. ramorum which is particularly virulent on 

Japanese larch (Larix kaempferi) and European 

larch (Larix decidua) (King et al. 2014), two 

important timber species in the UK accounting for 

10 percent of all UK conifer plantations.

A better understanding of the epidemiology 

of sudden larch death is needed to inform 

management decisions and maintain larches as 

commercial timber species. Survivor trees within 

high mortality stands in the Galloway Forest 

suggest the possibility of natural resistance within 

the Japanese larch population. Understanding 

how the pathogen spreads within and between 

individuals will help to predict if survivor trees 

have escaped infection by chance or may actually 

be resistant.

In order to understand disease spread, 21 

individual larch trees in each of 21 plots located 

in larch stands of varying ages were surveyed 

for disease symptoms each May and September 

from 2016 to 2018. In addition to recording 

symptoms on needles, shoots, branches, and main 

stem, samples of lesion material were taken from 

central trees in each plot for pathogen isolation by 

selective media and qPCR to confirm infection 

by P. ramorum. In September 2017 and 2018 

symptomatic foliage was collected and 20 needles 

were stained with lactophenol blue and the abaxial 

and adaxial surfaces examined for P. ramorum

sporulation. 

Climate records from the Met Office Hadley 

Centre Observations provide a homogeneityadjusted series of averaged precipitation across 

areas of the UK. Our study sites lie within the 

South Scotland area, which uses records from 

eight weather stations. Three of these stations are 

located within 50 km of our study sites. 

Between the 2013 epidemic and the start of our 

surveys in September 2016 disease levels had been 

stable and spread both within and between stands 

was limited. Observations in our plots between 

2016–2018 showed that disease spread varied 

between years. In the first survey in September 

2016 all disease symptoms were recorded at low 

levels, with on average < 5 percent of each tree 

being affected. The main stem mortality was 

on average higher at 8 percent as this included 

the trees that had died and were recorded as 

100 percent mortality (fig. 1). 

1 Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, United Kingdom, (heather.dun@

linacre.ox.ac.uk). 2Centre for Ecology, Society and Biosecurity, Forest Research, Northern Research Station, Roslin, EH25 9SY, United Kingdom.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Poster Session 129

0

5

10

15

20

Needle mortality Needle

discolouration

Main stem

mortality

Average symptoms over the 

tree (%)

Disease symptoms across 15 plots (n=315)

Sep-16

Shoot mortality Branch mortality

May-17 Sep-17 May-18

Figure 1—Results of field surveys of P. ramorum disease symptoms in Japanese larch in the Galloway Forest. 

(n = 315) between September 2016 and May 2018. Main stem mortality is a measure of mortality of the main 

trunk from the apex of the tree down.

Needle mortality and discolouration were similar 

between September 2016 and May 2017 but fine 

shoot and branch mortality increased slightly. 

Needle discolouration was notably higher in 

September 2017 compared with the previous 

September, this was followed by a doubling in 

the average shoot and branch mortality in May 

2018 and we infer that this discoloration may have 

been caused by needle infection by P. ramorum. 

The adaxial and abaxial sides of sample sets 

of 40 needles from 5 plots were examined for 

sporangia production but no sporangia were 

observed on the needles at that assessment time. 

Successful isolations from lesion material have 

been made from trees in each plot thus confirming 

that P. ramorum is present across all the plots.

The May 2018 survey found a large increase in 

P. ramorum infection within and between stands 

(which was also apparent in larch across the wider 

region) with many trees showing symptoms for 

the first time in this study and stands that were 

previously uninfected becoming infected. 

The most obvious symptom across a range of 

plots was a frequently occurring dieback of the 

fine shoots; these shoots had not flushed at all 

since spring and were prominent across the crown 

(fig. 2). These aerial infections appeared to have 

largely started in the fine, outer shoots of the 

upper crown and extended down branches towards 

the main stem. Suggesting significant aerial 

spread of infection, possibly through windborne 

inoculum. 

There were similarities in disease expansion in 

the outbreaks in 2013 and 2018 but subsequent 

lulls in infection in the intervening years. The 

increase in infection seen in May 2018 brought 

up the question of why disease spread was much 

slower between the May 2013 epidemic and May 

2018. Comparing our survey results over time 

with climate records also allows us to consider the 

possible links of climatic conditions to epidemic 

spread of P. ramorum. 

Figure 2—Fine shoot and branch dieback on 

Japanese larch in Galloway Forest in May 

2018. (Courtesy photo by Heather Dun)
130 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

Comparison to climate records suggests that the 

disease expansions observed in 2013 and 2018 

were preceded by record wet summers (fig. 3). 

Monthly rainfall between 2012 and 2018 is highly 

variable compared to the 18-year average rainfall 

between 2000 and 2018. 

Of particular interest is June rainfall in 2012 and 

2017, with double average precipitation preceding 

both the 2013 and 2018 epidemics whereas the 

intervening years, in which disease expansion 

was much slower, had lower than average June 

rainfall. Less obvious but also worth noting is the 

higher than average rainfall in July, August, and 

September in both 2012 and 2017.

We observed an increase in infection of larch 

characterized by shoot mortality in our field sites 

in the spring of 2018 and this was also observed 

across the wider region (Forestry Commission 

Scotland 2018). Our observations of infection 

spread over the scale of individual trees allowed us 

to develop a more detailed understanding of how 

disease progresses. Previous reports of infection 

in larch focused on main stem lesions that caused 

girdling leading to whole tree mortality together 

with sporangia production on needles (Webber 

et al. 2010). Shoot dieback after infection with 

P. ramorum has been recorded in multiple woody 

species, including both deciduous and evergreens 

in the USA (Hansen et al. 2005). Our observations 

indicate that fine shoot infection is a mechanism 

of rapid disease progression in P. ramorum in 

larch. 

We propose that high summer rainfall might 

influence the levels of infection observed at 

flushing in the following season. It is possible 

that high summer humidity would facilitate the 

prolific needle sporulation needed for subsequent 

epidemic spread. The positive relationship 

between infection and humidity has been well 

documented in P. infestans (Harrison and Lowe 

1989) (Hirst and Stedman 1960). In sudden oak 

death in the USA sporangia in soil remained 

infectious over summer for 3 months after a rain 

event (Fichtner et al. 2007). It is possible that 

summer rainfall induces sporangia production 

on needles which then infect shoots over winter, 

becoming obvious with lack of spring flush. 

Alternatively the sporangia, if able to overwinter, 

might infect fine shoots directly in spring. 

This study proposes that disease spread involves 

fine shoot infections and the influence of rainfall 

in the preceding season. Inoculation experiments 

would be a useful approach to further test this 

infection pathway of P. ramorum in larch and 

investigate the effects of climate on epidemiology. 

0

50

100

150

200

250

300

350

Rainfall (mm)

Monthly rainfall in South West Scotland 

2012 2013 2014 2015 2016 2017 2018 18 year avg.

Figure 3—Monthly precipitation in south-west Scotland between 2012–2018 and 18-year average precipitation for each month.
Poster Session 131

LITERATURE CITED

Fichtner, E.J.; Lynch, S.C.; Rizzo, D.M. 2007. Ecology 

and epidemiology detection, distribution, sporulation, 

and survival of Phytophthora ramorum in a California 

Redwood-Tanoak forest soil. Phytopathology. 10: 1366–

1375. https://doi.org/10.1094/PHYTO-97-10-1366.

Forestry Commission Scotland. 2018. 04 December 

2018 statutory plant health notices (SPHNs) served 

for Phytophthora ramorum on Larch sites in woodland 

settings. https://scotland.forestry.gov.uk/images/

corporate/pdf/Phytophthora-ramorum-in-Scotland-map-4-

December-2018.pdf. [Date accessed: December 19, 2018]. 

Hansen, E.M.; Parke, J.L.; Sutton, W. 2005. Susceptibility of 

Oregon forest trees and shrubs to Phytophthora ramorum : 

A comparison of artificial inoculation and natural infection. 

Plant Disease. 89(1): 63–70. https://doi.org/10.1094/PD89-0063.

Harrison, J.G.; Lowe, R. 1989. Effects of humidity and air 

speed on sporulation of Phytophthora infestans on potato 

leaves. Plant Pathology. 38: 585–591. https://onlinelibrary.

wiley.com/doi/pdf/10.1111/j.1365-3059.1989.tb01455.x. 

Hirst, J.M.; Stedman, 0.J. 1960. Annals of Applied Biology. 

48(3): 471–488.

King, K.; Harris, A.; Webber, J. 2014. Current distribution 

of the new EU2 lineage of Phytophthora ramorum (PDF 

Download Available). In: Proceedings of the Sudden 

Oak Death symposium. https://www.researchgate.net/

publication/265468027_Current_distribution_of_the_new_

EU2_lineage_of_Phytophthora_ramorum. [Date accessed: 

April 7, 2020].

Rizzo, D.M.; Garbelotto, M.; Davidson, J.M. [et al.]. 2002. 

Phytophthora ramorum as the cause of extensive mortality 

of Quercus spp. and Lithocarpus densiflorus in California. 

Plant Disease. 86(3): 205–214. https://doi.org/10.1094/

PDIS.2002.86.3.205.

Webber, J.F.; Mullett, M.; Brasier, C.M. 2010. Dieback and 

mortality of plantation Japanese larch (Larix kaempferi) 

associated with infection by Phytophthora ramorum. New 

Disease Reports. 22(19): 2044–0588. https://doi.org/10.519

7/j.2044-0588.2010.022.019.
132 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

GEOGRAPHIC AND LOCAL GENETIC VARIATION 

IN PACIFIC MADRONE LEAF BLIGHT

Laura E. DeWald1, Marianne Elliott2, Richard Sniezko3, and Gary A Chastagner 2

Pacific madrone (Arbutus menziesii) is an 

evergreen hardwood species in western North 

America that occurs from British Columbia to 

southern California. The species has high cultural 

and specialty wood values, and is also ecologically 

important for wildlife habitat, wildfire 

revegetation, and creating midstory canopy 

biodiversity in mixed conifer forests. Pacific 

madrone is experiencing unsustainable mortality 

(Lintz et al. 2016), but reasons for the decline are 

unknown. Foliar pathogens such as described by 

Zeller (1934) have been implicated because of their 

high visibility and association with defoliation. 

However, very little is known about the genetics 

of this species, including variation in resistance to 

pathogens and other adaptive traits.

Range-wide common garden tests were 

established to examine patterns of genetic 

variation in traits such as height growth and 

survival, to identify relative contributions of 

abiotic, biotic and genetic causes of health 

declines, and for identifying resistant seed 

sources. The Washington State University Pacific 

madrone seed collection contains seed from 

320 families in seven ecoregions. Using this 

seed collection, common gardens were planted 

at seven locations in 2011 in California (1 site), 

Oregon (2), Washington (2), and in 2013 in British 

Columbia (2). These common garden sites are 

located in four of the seven ecoregions where 

seed was collected (fig. 1) and consist of 105 

half-sib families representing 42 seed sources. 

Survival and performance data were collected 

annually and leaf blight, caused by a complex of 

fungal pathogens, was also rated annually for the 

first 3 to 5 years, depending on site. In addition 

to leaf blight, assessments have been made of 

growth, other diseases, cold damage, flowering, 

and phenology. Here we summarize variation in 

leaf blight severity in the OR and WA common 

gardens.

Across all years and common gardens, average 

blight severity was slight to moderate (< 25 to 

25–50 percent of current leaves affected) with 

an average incidence of 25–50 percent. Blight 

symptoms were more severe in years where a 

common garden test was particularly stressed 

by drought and/or cold winter temperatures and 

tended to be more severe at the WA sites (PH, 

PV) compared to the OR sites (SF, SO). Patterns 

of blight severity among sites might be related to 

site conditions such as moisture and temperature. 

Typically precipitation decreases and temperatures 

increase from north to south latitude, thus the WA 

sites would be cooler and wetter than the SF site, 

which would be cooler and wetter than the SO 

site. However, the size of the differences in blight 

symptoms may be magnified since the OR sites 

were generally assessed in late fall/early winter 

while the WA sites were assessed in late winter. 

Subsequent observations in another year on a 

subset of trees confirm an increasing degree of 

blight over this time period.

Fungi associated with severe leaf blight, such as 

Phacidiopycnis washingtonensis (Elliott et al. 

2014) and Phomopsis sp., were not found at the 

southernmost site (SO) in 2018 samples (fig. 2).

Blight severity within sites varied significantly 

(p < 0.01) among seed sources for all years 

at all sites except for SO where sources only 

differed in 2012 (data not shown). Blight severity 

among half-sib families within sources also 

varied significantly (p < 0.01) for all years and 

at all sites except at SF where families only 

1University of Kentucky, Lexington, KY 40546, (Laura.DeWald@uky.edu). 2Washington State University, Puyallup Research and Extension Center, Puyallup WA 98371.

3Dorena Genetic Resource Center, USDA Forest Service, Cottage Grove, OR 97424.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Poster Session 133

PV

PH

TI

SF

SO

LC

BL

Figure 1—Locations of madrone common garden sites, seed sources, and ecoregions. (Insert: full range of Pacific madrone). Sites used in this 

study: PV, PH (WSU Puyallup), SF (Starker Forest), and SO (Sprague Orchard).
134 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

Oregon Washington

SO SF PH PV

Large leaf spots

Cryptostictis arbuti x

Didymosporium arbuticola x x x x

Rhytisma arbuti x

Small leaf spots

Epicoccum nigrum x x x x

Mycosphaerella sp. x

Shoot and leaf blight

Diaporthe spp. (=Phomopsis spp.) x x

Phacidiopycnis washingtonensis x x x

Endophytes

Allantophomopsis cytisporea x

Aureobasidum pullulans x x x x

Colletotrichum clavatum x

Fusarium sp. x x

Stemphylium sp. x

Sydowia polyspora x x x

Figure 2—Fungi associated with leaf blight. Fungi were identified from madrone foliage samples at the common garden sites using 

morphological and molecular techniques, from south (SO) to north (PV) in 2018.

differed in 2015. Averaged across all sites, 

variation in blight severity among sources 

was not related to longitude of origin but was 

significantly (p < 0.01) related to latitude of 

origin, (R2

 = 0.09- 0.69), and elevation of origin 

(R2

 = 0.13-0.16). When analyzed separately by 

site, variation in blight severity among sources 

was not related to longitude at any site, and at 

SO variation was also not related to latitude 

or elevation. Relationships with latitude were 

strongest for PH (R2 = 0.48-0.65) but at PV 

latitude was only significantly related in 2014. 

The strongest relationships between seed source 

origin and site were at the most northern common 

gardens (PH & PV). Averaged across sites, trends 

in table 1 show that in general, southern sources 

had higher blight severity than northern sources. 

Sources with the lowest blight severity were from 

the north: OH, CL (Oak Harbor and Clinton, WA), 

OR (Cornelius, OR). Sources with the highest 

blight severity were from the south: e.g., HC, HP, 

LA (Humboldt County, Hopland, Los Altos, CA). 

However, some northern sources such as PA, DE 

(Port Angeles, WA , Detroit, OR) had relatively 
Poster Session 135

Table 1—Blight severity score averaged across common garden sites for each Pacific 

madrone seed source for each year

Source

State or

Province Latitude 2012 2013 2014 2015

Mean, all 

years

SA California 37.02010 14.1 37.0 34.5 26.5 28.1

SC California 37.21705 16.2 28.2 26.9 21.2 23.1

LA California 37.29174 18.2 31.3 31.1 42.3 30.7

CC California 38.00579 14.6 26.3 30.4 38.2 27.4

SR California 38.01276 11.8 29.8 32.9 26.0 25.1

CA California 38.60876 12.8 24.0 22.7 29.2 22.2

PL California 38.75026 13.2 22.2 16.5 38.0 22.5

HP California 39.00000 22.3 31.9 31.5 44.0 32.4

BL California 39.16684 17.5 33.1 26.1 40.2 29.2

RV California 39.25356 17.1 29.0 30.7 42.7 29.9

SN California 39.35246 14.1 27.7 21.0 37.9 25.2

GA California 40.08990 18.2 31.0 26.7 41.4 29.3

HC California 40.29190 17.2 37.7 31.9 44.4 32.8

KL California 41.69240 16.2 27.3 18.6 40.7 25.7

RA Oregon 42.19585 15.4 25.1 18.7 40.6 24.9

BK Oregon 42.21448 12.2 23.3 25.4 37.3 24.6

CP Oregon 42.26213 21.0 33.6 17.1 41.6 28.3

GP Oregon 42.47669 18.0 28.1 17.0 40.5 25.9

CY Oregon 42.91553 15.4 35.5 16.6 40.4 27.0

BA Oregon 43.01794 12.4 20.5 17.7 33.1 20.9

OK Oregon 43.67947 13.3 27.9 15.0 40.0 24.1

VE Oregon 44.03646 11.6 21.2 12.3 32.2 19.3

DE Oregon 44.69452 20.3 30.3 17.4 44.4 28.1

AK Oregon 44.82509 13.4 22.5 14.0 38.0 22.0

ZE Oregon 45.01889 10.2 19.0 11.8 31.2 18.0

WL Oregon 45.35004 7.2 23.8 10.4 32.6 18.5

WV Oregon 45.36323 15.5 24.8 12.4 33.0 21.4

OR Oregon 45.44143 10.8 18.6 10.5 28.0 17.0

CR Washington 46.06361 14.4 20.1 13.2 29.6 19.3

PF Washington 46.84067 15.7 24.4 13.2 37.7 22.7

OL Washington 47.05435 10.5 15.7 10.9 33.6 17.7

FW Washington 47.30014 16.1 23.9 13.6 33.2 21.7

KM Washington 47.30751 14.0 23.1 16.0 35.7 22.2

BR Washington 47.55823 14.2 23.3 13.6 33.8 21.2

CL Washington 47.97990 8.6 17.5 10.7 28.6 16.4

LC Washington 48.06585 14.9 22.4 15.8 40.5 23.4

PA Washington 48.10687 20.2 29.2 15.8 37.8 25.8

OH Washington 48.30342 8.4 16.7 10.8 28.5 16.1

FB Washington 48.48699 8.7 22.1 14.0 31.1 19.0

VI British Columbia 48.51078 16.5 26.4 16.7 29.0 22.1

CB Washington 48.64940 14.7 31.1 13.7 32.4 23.0

BC British Columbia 49.18983 10.7 23.6 13.4 30.0 19.4

Note: Blight severity was scored as a percentage of blighted leaf area on the most severely impacted 

current season leaves. Blight incidence was based on percent of the whole tree with leaves having the 

severity rating described above. Severity and incidence scores were multiplied together for an overall 

severity rating score ranging from 0 to 100, with 100 being highly susceptible.
136 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

higher blight severity while some southern 

sources such as PL, CA (Placerville & Calistoga, 

CA) had lower blight severity. These differences 

may be related to the climate at the site where 

the seed was collected being similar to that of a 

southern or northern site, respectively, and will be 

investigated further (Wilhelmi et al. 2017). The 

source by common garden site interaction (i.e., 

genotype X environment) was significant for all 

years ( p < 0.001). 

These results suggest that wetter, cooler conditions 

at a site seem to increase blight severity. Sources 

that moved the farthest north generally had more 

severe blight. These sources may have been less 

well adapted, thus more stressed and less able to 

resist leaf blight. In addition, the results suggest 

that resistance to leaf blight might exist. Madrone 

seed sources collected within the ecoregion of 

a common garden had both high and low blight 

severity. Blight severity also varied among halfsibling families within seed sources. Relative 

blight severity of some sources was not consistent 

across all common garden sites. This indicates that 

blight symptoms are likely caused by a complex of 

biotic and abiotic factors. Relatively few details of 

the blight dynamics were known and assessments 

of these trials increase it substantially. Detailed 

observations in 2018 on a small subset of seedlings 

indicate that blight severity continues to increase 

from November through May. In addition, 

some trees double or triple flush and the later 

flushes tended to have lower blight severity. This 

information will be useful in planning the timing 

of future assessments to examine genetic variation 

in blight. Further study should also include a more 

refined study of the individual causes of leaf blight 

in Pacific madrone.

ACKNOWLEDGMENTS

The authors wish to thank CalFire, BC Ministry 

of Forests, Bureau of Land Management and 

Starker Forests for providing the sites, and to the 

people at Dorena Genetic Research Center, WSU, 

and OSU who helped with annual assessments. 

In addition, we thank the volunteers who assisted 

with seed collection, including Marin County Mid 

Peninsula Open Space, Ellen Goheen, Brenda 

Callan, Alan Kanaskie, and many others. We also 

thank Connie Harrington (USFS) for the use of 

the cameras documenting blight, and Doug Savin 

(U.S. Forest Service) for statistical help. Funding 

for establishing common garden sites and growing 

seedlings is from U.S. Forest Service Forest 

Health Protection Grant #2010-DG-11062754-015. 

Funding supporting data analyses and ongoing 

disease assessment is from U.S. Forest Service 

Forest Health Monitoring grant #EM-2018-WC-2. 

LITERATURE CITED

Elliott, M.; Chastagner, G.A.; Coats, K.P. [et al.]. 2014. 

First report of a new leaf blight caused by Phacidiopycnis 

washingtonensis on Pacific madrone (Arbutus menziesii) in 

Western Washington and Oregon. Plant Disease. 98: 1741.

Lintz, H.E.; Gray, A.N.; Yost, A. [et al.]. 2016. Quantifying 

density-independent mortality of temperate tree species. 

Ecological Indicators. 66: 1–9. 

Wilhelmi, N.P.; Shaw, D.C.; Harrington, C.A. [et al.]. 2017. 

Climate of seed source affects susceptibility of coastal 

Douglas‐fir to foliage diseases. Ecosphere. 8(12): e02011. 

10.1002/ecs2.2011.

Zeller, S.M. 1934. Some new or noteworthy fungi on 

Ericaceous hosts in the Pacific Northwest. Mycologia. 

26(4): 291–304.
Poster Session 137

BACK FROM THE BRINK: FOREST SERVICE EFFORTS TO CREATE 

DUTCH ELM DISEASE TOLERANT TREES FOR USE IN 

URBAN AND RURAL RESTORATION

Charles E. Flower1, Cornelia C. Pinchot1, Kathleen S. Knight1, 

Keith Woeste2, and James M. Slavicek3

Introduced into North America in the 1920’s, the causal agent of Dutch elm disease (Ophiostoma ulmi) 

and the more aggressive variant of the pathogen (O. novo-ulmi, collectively referred to herein as DED) 

swept across North America resulting in a widespread decline of elms across urban and rural landscapes. 

Restoration of American elm (Ulmus americana L.) necessitates the development of new selections that 

are DED tolerant, in addition to enhancing the genetic variability of tolerant elms across the landscape. 

Ongoing research and breeding efforts have focused on (1) identifying survivor trees that have likely been 

exposed to DED yet have thrived, (2) crossing these large survivor elms with DED-tolerant American 

elms, (3) testing cloned survivor trees and progeny trees with DED inoculations, and (4) identifying 

genes which confer tolerance. This study examined the 2-year DED-induced canopy decline response 

of 27 American elm cultivars planted at the U.S. Department of Agriculture Forest Service Laboratory 

in Delaware, OH. Results suggest differences in DED-induced canopy decline among clones of large 

survivor trees collected across the Midwestern United States, indicating that unique tolerance mechanisms 

may be present in the natural elm population.

1USDA Forest Service, Delaware, OH 43015, (charlesflower@fs.fed.us). 2USDA Forest Service, West Lafayette, IN 47907.

3USDA Forest Service, Delaware, OH 43015.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
138 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

FIELD PERFORMANCE OF PREVIOUSLY SELECTED 

ROOT ROT TOLERANT TROPICAL ACACIAS

Abdul Gafur1,2, Ching Y. Wong1, and Mukesh Sharma1

Reforestation programs have been established in some countries to anticipate the increase of the global 

demand for wood. The effort was initiated to sustain the supply of forest products while preserving the 

natural forests. In line with the policy, areas of industrial plantation forests of fast-growing species in 

Indonesia, especially acacias and eucalypts, have been increased to meet the ever-increasing demands. 

Disease infection has occurred since the establishment of the plantation forests. Root rots caused by red 

root rot (Ganoderma philippii) and Phellinus noxius (brown root rot) are among the most economically 

important diseases on the two fiber trees. Research on field controls of the diseases has focused on 

certain components of integrated disease management such as inoculum reduction, silviculture practices, 

application of biological control agents, and tolerant genotypes. We have developed a method of screening 

for root rot tolerance in acacia seedlings. Using the method, identification of variations in root rot 

tolerance is possible. Field experiments and commercial plantations indicated that tolerant materials 

previously selected in the nursery screening had less root rot incidence, paving the way for plant tolerance 

to be incorporated as the core component of integrated management of the diseases in tropical acacia 

plantation forests.

1 AAA Fiber Research and Development, PT RAPP, Pangkalan Kerinci 28300, Indonesia.

2 Present address: Jalan Yasmin Raya No. 131, Taman Yasmin Sektor II, Bogor 16112, Indonesia, (gafur@uwalumni.com).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Poster Session 139

BUILDING A CERATOCYSTIS RESISTANCE PROGRAM 

FOR METROSIDEROS IN HAWAII

Christian Giardina1, Lisa Keith2, Phil Cannon3, 

Richard Sniezko4, Douglas Jacobs5, and J.B. Friday6

The loss of ‘Ōhi‘a to Ceratocystis wilt is an unprecedented threat to the ecological integrity of native 

Hawaiian forests and to native Hawaiian culture. The genetic basis for disease resistance by ‘Ōhi‘a against 

rapid ‘Ōhi‘a death (ROD) is poorly understood, yet represents a centrally important piece of any longterm strategy for perpetuating ‘Ōhi‘a as a dominant component of Hawaii’s native forests. Specifically, 

because ROD is infecting and killing trees across Hawai‘i Island, with infection in some areas causing 

high rates of mortality, quantifying the presence and strength of resistance for various genotypes of 

Hawai‘i Island ‘Ōhi‘a is critical to anticipating long-term consequences of ROD for forest biodiversity 

and conservation in Hawaii, as well as informing restoration strategies including decisions about whether 

‘Ōhi‘a should be considered for restoration, and if so what genotypes. Questions about resistance need 

to be addressed for ‘Ōhi‘a genotypes and species across the Hawaiian Islands, and so we have initiated 

an ‘Ōhi‘a ROD resistance program that is relying a variety of nursery and in-field methods, focusing on 

Hawai‘i Island genotypes and varieties initially, but if indicated, expanding to genotypes, varieties and 

other Metrosideros species on other Hawaiian Islands. Selections are being made based on morphological 

and geographic features. Molecular tools are being used to examine the genetic basis for and distribution 

of resistance. This work aligns with the State of Hawaii 2017–2019 ROD Strategic Response Plan 

(www.ctahr.hawaii.edu/dl/rod/strategicresponseplanfinal.pdf) which identifies ROD resistance research 

as a priority investment. This research will lead to improved understanding of ‘Ōhi‘a disease resistance, 

identify the most resistant genotypes for propagation, and create a source of trees that will optimize the 

restoration of ROD impacted Hawaiian forests.

1 Pacific Southwest Research Station, USDA Forest Service, Albany, CA 94710-0011, (cgiardina@fs.fed.us). 2 Pacific Basin Agricultural Research Center, USDA Agricultural Research Service, Hilo, HI 96720.

3Region 5, USDA Forest Service, Vallejo, CA 94592.

4Region 6, USDA Forest Service, Cottage Grove, OR 91424.

5Tropical Hardwood Tree Improvement and Generation Center, Purdue University, West Lafayette, IN 47906.

6University of Hawaii at Manoa, Hilo, HI 96720.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
140 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

THE IMMINENT INVASION OF THE EMERALD ASH BORER IN SOUTHERN EUROPE 

AND THE THREAT TO NATIVE OLEACEAE

Ignazio Graziosi1 and Lynne Rieske-Kinney2

European countries are increasingly under invasion by non-native insects that are highly invasive in 

North America. For instance, the Japanese beetle Popillia japonica, the brown marmorated stink bug 

Halyomorpha halys and the Asian chestnut gall wasp Dryocosmus kuriphilus, notorious non-native 

pests of crops and forests in North America, have been recently introduced in Europe, with devastating 

consequence. The globally invasive emerald ash borer (EAB) Agrilus planipennis is currently expanding 

its range in both North America and European Russia, and its introduction in Southern Europe appears 

imminent. Furthermore, the spread of forest pathogens such as the ash dieback caused by Hymenoscyphus 

fraxineus, are adding concerns to the conservation of native forest habitats and Fraxinus species in 

particular. In addition, the recently reported utilization of non-Fraxinus hosts in the family Oleaceae 

is opening new scenarios for EAB invasion dynamics and future management challenges in Southern 

Europe. We seek to: (a) resume most recent knowledge on the pest’s current distribution in Europe, (b) 

present natural distributions of native Oleaceae in the southern part of the continent, and (c) propose tools 

for predicting EAB invasion pathways and impact in Southern Europe.

1World Agroforestry Centre, PO Box 30677, Nairobi, 00100, Kenya, (ignazio.graziosi@fulbrightmail.org). 2 Department of Entomology, University of Kentucky, S-225 Agricultural Science Center North, Lexington, KY 40546-0091.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Poster Session 141

A COLLABORATIVE RESEARCH APPROACH FOR DIAGNOSING AND 

EVALUATING THOUSAND CANKERS DISEASE IN WALNUT: 

CURRENT PROGRESS AND FUTURE DIRECTIONS

Emel Oren1, William Klingeman2, Romina Gazis3, John Moulton1, Paris Lambdin1, 

Massimo Faccoli4, Mark Coggeshall5, Jiri Hulcr 6, Steven J. Seybold7, 

and Denita Hadziabdic1

Thousand Cankers Disease (TCD) results from interactions between the canker-producing fungal 

pathogen Geosmithia morbida, an insect vector Pityophthorus juglandis (walnut twig beetle), and 

the susceptible plant hosts, Juglans spp. (walnuts) and Pterocarya spp (wingnuts). In the past two 

decades, TCD has expanded from the western to the native range of black walnut (J. nigra) in the 

Eastern United States. In 2013, TCD was discovered in northwestern Italy affecting both black and 

native English walnuts (J. regia). Although TCD has caused significant mortality among native and 

non-native walnut tree populations, our understanding regarding the genetic diversity of the insect 

vector and the fungal pathogen remains limited. One factor that may be contributing to the spread 

of the disease is the difficulty of its diagnosis due mainly to non-unique external symptoms. Based 

on the speed at which TCD has expanded its range and the potential global spread and invasion of 

areas where susceptible host plants are commercially grown, there is a critical need to understand the 

genetic diversity presented by the causal agent and its primary vector. Moreover, to limit the spread 

of the disease we need to improve our detection methods by utilizing specific and sensitive molecular 

tools that enable quick identification. Rapid and accurate detection of TCD will facilitate quarantine 

implementation in infested areas. Collaborative research presented here showcases our current 

understanding of the population structure of both pathogen and the vector of this disease, as well as 

a recently developed molecular protocol to rapidly detect G. morbida and P. juglandis directly from 

woody tissue samples. Our results indicate high genetic diversity, presence of population structure, 

and evidence of gene flow among subpopulations of P. juglandis and G. morbida. In particular, our 

work reveals that human mediated movement of infested plant material from multiple sources and 

on multiple occasions, has significantly contributed to TCD range expansion. Our results support 

an earlier hypothesis that the disease has been established in western TCD-affected areas for a 

long period of time and can’t be considered a recent introduction. An important by-product of our 

population genetics research was the development of a set of specific microsatellite regions that have 

been extremely useful in the detection of G. morbida directly from infested wood tissue, even at low 

concentrations. Here, we address how we developed this novel molecular detection tool and explain 

how this approach can serve as a model for future research on disease outbreaks caused by similar 

disease complexes. 

1 Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN 37996; Current address Diyarbakõr Plant 

Protection Research Institute, Diyarbakõr, Turkey, (dhadziab@utk.edu).

2 Department of Plant Sciences, University of Tennessee, Knoxville, TN 37996.

3Department of Plant Pathology, University of Florida, Tropical Research and Education Center, Homestead, FL 33031.

4 Department of Agronomy, Food, Natural Resources, Animals and Environment (DAFNAE), University of Padua, Legnaro, PD, Italy.

5USDA Forest Service, West Lafayette, IN 47906.

6 School of Forest Resources and Conservation, University of Florida, Gainesville, FL 32611.

7USDA Forest Service, Davis, CA 95616.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
142 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

COLLABORATIVE RESEARCH FOR SUSTAINABLE MANAGEMENT 

OF SOUTHWESTERN WHITE PINE

Kristen Waring1, Sam Cushman2, Andrew Eckert3, Lluvia Flores4, Richard Sniezko5, Christopher Still6, 

Christian Wehenkel7, Amy Whipple1, Michael Wing6, Justin Bagley3, Emily Boes5, Ethan Bucholz1, 

Jonathan Burnett6, Antonio Castilla1,5, Jessica DaBell1, Chris Friedline3, Cory Garms6, Evan Heck5, 

Jeremy Johnson1,5, Erin Landguth8, Alejandro Leal Sáenz7, Briana McTeague5, 

Mitra Menon3, Ehren Moler1, Gerald Page6, and Andrew Shirk9

A collaborative team of researchers from the United States and Mexico have begun an exciting new 

research project funded by The National Science Foundation’s Macrosystems Biology program. The 

project is to study ecological and evolutionary processes affecting the distribution of southwestern white 

pine, an important tree species of mixed conifer forests in the Southwest and Mexico. Southwestern 

white pine sustainability is threatened by changing climate, and a non-native tree disease, white pine 

blister rust. White pine blister rust causes extensive tree decline and mortality where it occurs in North 

America, including where it overlaps with southwestern white pine, an ever-expanding area. The climate 

may change too rapidly for southwestern white pine to adapt. The dual threats of climate change and 

invasive species make forecasting future tree distributions across continental scales an urgent challenge. 

The goal is to determine how gene movement among populations, adaptation to disease and drought, 

heritable changes beyond DNA mutations, and a changing environment interact to govern the success 

of southwestern white pine. This project will develop tools to help forecast and manage the future of the 

species, including genomics, common gardens, tree disease resistance testing, engineering and technology 

innovation to measure drought tolerance, and computer modeling in landscape ecology and genomics. The 

research team is using the Southwest Experimental Garden Array, set of common gardens, that allows 

scientists to quantify the ecological and evolutionary responses of species to changing climate conditions.

1Northern Arizona University, Flagstaff, AZ 86011, (kristen.waring@nau.edu). 2Rocky Mountain Research Station, USDA Forest Service, Fort Collins, CO 80526.

3 Virginia Commonwealth University, Richmond, VA 23284.

4 San Diego State University, San Diego, CA 92182.

5 Dorena Genetic Resource Center, USDA Forest Service, Dorena, OR 34963, (richard.sniezko@usda.gov). 6 Oregon State University, Corvallis, OR 97331.

7Universidad Juarez del Estado de Durango, Constitución 404 sur. Zona centro. C.P. 34000. Durango, Dgo. México.

8 University of Montana, Missoula, MT 59812.

9 University of Washington, Seattle, WA 98195.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
143

BUTTERNUT CONSERVATION AND BREEDING TO 

MITIGATE BUTTERNUT CANKER DISEASE

James R. McKenna1, Paul Berrang2, Aziz Ebrahimi3, Scott Rogers4, 

Stuart Seaborne5, and James Jacobs6

Poster Session

Butternut (Juglans cinerea), a valuable fine 

hardwood in North America, is no longer 

regenerating in the wild due its demise from an 

exotic fungal stem canker pathogen butternut 

canker disease (BCD) caused by the Ascomycete 

(Ophiognomonia clavigignenti-juglandacearum

[O-cj]). The most effective way to mitigate 

BCD, will be to develop genetically resistant 

trees. Natural hybrids with the Japanese walnut 

(J. ailantifolia) have been found to have much 

wider resistance than pure J. cinerea; however, 

some J. cinerea families have shown a significant 

level of resistance but will require more breeding. 

Seed from our clone banks and orchards has 

become available in enough quantity to support 

conservation plantings. Our project aims to deploy 

and conserve regionally adapted and genetically 

diverse pure butternut seedlings. Conserving 

genetically pure butternut is important since 

natural selection over the last decades has left only 

hybrid trees alive in many places. 

Dramatic differences in disease between upland 

and bottomland sites has been observed for nearly 

a decade. Artificial inoculation on bottomland 

sites has led to complete disease across the entire 

planting within 8–10 years. In contrast, two 

inoculated upland sites have shown as little as 

3 to 10 percent natural disease spread. We will 

present new results from two new bottomland and 

one new upland site in Union County, Indiana 

established in 2013. Resistant stock combined 

with site selection and other cultural or biological 

controls can integrate several pest management 

strategies to minimize butternut canker disease. 

From a large seed crop from our regional seed 

orchards and clone banks in 2013, we planted 

a new screening block in 2015 under naturally 

diseased trees. This extreme screening produced 

significant BCD in the first year after planting 

rapidly eliminating highly susceptible trees. New 

data will be presented on this and on our 10- and 

9-year canker ratings from our 2nd screening 

blocks established in 2008–09. Our most 

productive seed orchard, located in the Pacific 

Northwest in Walla Walla, WA, comprised of 

26 grafted trees of 15 genotypes, has yielded over 

6,000 seeds the last few years. This past winter, 

we used DNA markers to determine each trees 

identity and hybridity and to thin it down to one 

individual J. cinerea tree per genotype.

Conserving pure and largely susceptible J. cinerea

began last year and is continuing through 2019. 

We are providing seedlings from our Walla 

Walla, WA and Hoosier National Forest seed 

orchards to state and private foresters and 

interested woodland owners to plant in southern 

Indiana and Ohio, the geographic region of these 

orchard clones. 2018 marks the 10th consecutive 

year that our collaborators in the Indiana DNR 

have sold random hybrid butternut seedlings in 

Indiana. We harvested our first seed crop from a 

second generation resistant orchard in the fall of 

2017 (300 nuts) which contain a mix of the most 

resistant hybrid and pure selections from our 

screening. Such seedlings, or supplemental pollen, 

can be a source of resistance to add into these 

new conservation plantings over the next decade. 

Our presentation will show seed production over 

the last 10 years, the new conservation sites being 

established, and discuss our various breeding and 

screening strategies.

1Northern Research Station, USDA Forest Service, West Lafayette, IN 47907, (james.mckenna@usda.gov). 2Region 9, USDA Forest Service, Milwaukee, WI 53202.

3 Purdue University, Forestry & Natural Resources, West Lafayette, IN 47907.

4 OCONTO River Seed Orchard, Region 9, USDA Forest Service, Whitelake, WI 54491.

5University of Wisconsin, Madison, WI 53706.

6 Northern Area, Region 9, USDA Forest Service, St. Paul, MN 55108.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
144 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

DIE BACK SYNDROME CAUSED BY LASIODIPLODIA THEOBROMAE AND 

FUSARIUM PROLIFERATUM IN ELITE GENOTYPES 

OF TECTONA GRANDIS LF., COSTA RICA

Dawa Méndez-Álvarez1, Arantxa Rodríguez1, Yorleny Badilla-Valverde1, 

and Olman Murillo-Gamboa1

Tectona grandis L. (teak) is native to the tropical and subtropical regions of India, Burma, Thailand, 

and Laos in Asia and it is one of the most important timber species in the tropics because of its 

economic use. Costa Rica has been a pioneer in teak intensive cultivation, and a regional leader in tree 

improvement and conservation programs with GENFORES (international tree improvement cooperative). 

Forest plantation productivity and economical value has risen significantly; however, teak’s dieback 

syndrome in commercial plantations become worst year by year. The main objective of this research is 

to contribute with the development of prevention options through putative tolerant genotypes utilization, 

as a transdisciplinary approach between pathology and tree breeding. This research used plant material 

from GENFORES. Syndromes causal agents were isolated, characterized and identified from five 

trees (four with symptoms and one healthy) of three different sites. Affected vascular tissue segments 

were cultivated in potato dextrose agar (PDA) to isolate pure cultures. According to the morphological 

characteristics, the fungus was identified as Lasiodiplodia theobromae and Fusarium proliferatum. To 

confirm the morphological identification, portions of the DNA were sequenced. Later, GENFORES elite 

genotypes teak were inoculated with Lasiodiplodia theobromae and Fusarium proliferatum (separated and 

together) to fulfill Koch’s postulates and to screen out potential tolerant clones. Inoculation was carried 

out on 6 month old teak’s clones, by placing 1.5 mm diameter mycelial plugs of the isolates grown on 

PDA in artificially made wounds (2 cm length) in the vascular cambium tissue. All elite clones displayed 

symptoms 120 days after inoculations. The fungus was re-isolated from all inoculated plants, fulfilling 

Koch’s postulates. The research continues in order to obtain resistant or tolerant clones.

1School of Forest Engineering, Instituto Tecnológico de Costa Rica, 159-7050 Cartago, Costa Rica, (dawamendez02@gmail.com).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Poster Session 145

SOMATIC EMBRYOGENESIS AND CRYOSTORAGE FOR RESTORATION 

OF ASH FORESTS DEVASTATED BY EMERALD ASH BORER

Scott A. Merkle1, Paul M. Montello1, A. Ryan Tull1, 

Daniel A. Herms2, and Kamal J.K. Gandhi1

Emerald ash borer (EAB), Agrilus planipennis has devastated populations of ash trees in at least 

20 U.S. States and Canada over the past decade. To date, control measures have had minimal impact 

on halting the infestation. However, there is some evidence of genetic resistance or tolerance to EAB 

in natural populations of white ash (Fraxinus americana) and green ash (Fraxinus pennsylvanica) 

trees, as demonstrated by the continued survival and growth of scattered individual trees for several 

years following infestations that killed over 99 percent of the ash trees in infested populations. 

These “lingering” or “surviving” ash individuals may form the basis for reforestation programs in 

EAB-impacted areas, if these genotypes or their progeny can be mass-propagated. We initiated cultures 

from seeds collected from several surviving white ash trees in southeastern Michigan. Cultures were 

initiated by dissecting immature seeds and culturing the developing zygotic embryos on a semisolid 

modified Woody Plant Medium (induction-maintenance medium; IMM) with 2 mg/l 2,4-D. Multiple 

highly productive embryogenic culture lines representing six different lingering ash parents were 

obtained. Embryogenic cultures were grown in suspension culture in liquid IMM, followed by size 

fractionation on stainless steel sieves and plating on nylon mesh overlaid on semisolid basal medium to 

produce populations of somatic embryos. Somatic embryo germination and conversion were improved 

by a combination of pre-germination cold treatment for eight weeks and addition of gibberellic acid to 

the germination medium. Ash somatic seedlings grew rapidly following transfer to potting mix and 

almost 100 trees representing 6 white ash clones have been acclimatized and grown in the greenhouse in 

preparation for clonal screening. Future research will focus on expanding the embryogenic work on other 

surviving ash populations, cryostoring copies of all ash embryogenic cultures and planting and testing of 

the clonal ash saplings for EAB resistance in the field.

1School of Forestry and Natural Resources, University of Georgia, Athens, GA 30602,, (smerkle@uga.edu). 2 Davey Tree Expert Company, Kent, OH 44240.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
146 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

QTL MAPPING OF SPHAERULINA MUSIVA CANKER RESISTANCE/SUSCEPTIBILITY 

IN A POPLAR T x D HYBRID POPULATION

Kyle Mondron1, Susanna Keriö2, Sandra Simon3, 

Stephen DiFazio3, and Jared M. LeBoldus1,2

Poplar trees impact our ecological and economic well-being. Poplar hybrids benefit from heterosis, but 

the P. trichocarpa x P. deltoides (T x D) hybrid is very susceptible to infection by the fungal pathogen 

Sphaerulina musiva, which can cause premature mortality due to breakage or stem-girdling cankers. 

Breeding for resistance is currently the best technique for disease management. A prior Genome-Wide 

Association Study (GWAS) of P. trichocarpa found three genes that are thought to confer resistance 

(RLP1, RLP2, L-type lecRLK) and one gene that is thought to confer susceptibility (G-type lecRLK). 

A conidia-based greenhouse inoculation experiment was performed on T x D poplar hybrids using three 

isolates of S. musiva. Stem cankers were counted and disease severity scores (1–5) were assigned. The 

resulting phenotypes were analyzed using the R/qtl package with two models and a QTL map was created 

along with corresponding gene intervals. The four genes from the GWAS experiment were not found in 

the QTL gene intervals, nor were homologs found using BLAST analysis methods. Several candidate 

genes are listed and may be instrumental for further resistance breeding improvements.

1 Department of Forest Engineering, Resources and Management, Oregon State University, Corvallis, OR 97331, (kyle.mondron@oregonstate.edu). 2 Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331.

3Department of Biology, West Virginia University, Morgantown, WV 26506.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International Workshop on 

the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. e-Gen. Tech. Rep. 

SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Poster Session 147

THE RESISTANCE OF MULBERRY TREE SPECIES TO TWO FOLIAR DISEASES 

IN THE RAINFOREST ECOLOGICAL ZONE OF NIGERIA

Oduntan, O. Akinlabi1, Onilude Q. Ayansola1, and Charles O. Ogunremi2

The capability of mulberry tree species to produce high quality leaf materials for silkworm growth and 

cocoon production has been dampened by incidence of insect and pest diseases. This study was carried 

out at the Sericulture plantation unit, Forestry Research Institute of Nigeria, located in the rainforest 

ecological zone to assess the resistance of six different mulberry tree accessions (S30, S14, S41, K2, 

N17, S54) to two foliar diseases (Leaf spot and powdery mildew). The study laid out in a Randomized 

Complete Block Design (RCBD) with five replications and Analysis of variance (ANOVA) used to test 

significance of the different means. The result revealed the percent incidence for leaf spot was highest in 

S14 (56.23 percent) and lowest in K2 (2.32 percent) where as in powdery mildew, it was highest in S40 

(67.34 percent) and lowest in K2 (3.56 percent). It was also observed that the disease severity was ranging 

from a few individual spots to numerous lesions nearly covering the entire leaves. The results of different 

foliar anatomical characteristics showed that it plays a significant role in inhibiting the spread of the 

pathogen causing the diseases. Varietal rating to insect pest susceptibility showed that K2 accession was 

least affected or invested. It was concluded that K2 accession has high potentials that could be exploited 

for mass production of mulberry germplasm for silkworm, livestock, and human consumption. However, 

recommendations were also suggested for the study.

1 Forestry Research Institute of Nigeria, P.M.B 5054, Jericho Hill, Ibadan, Oyo State, Nigeria, (harkinlarbee@gmail.com). 2 National Biotechnology Development Agency, P.M.B 5118, Wuse, Abuja, Nigeria.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
148 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

NURSERY PEST RESISTANCE OF MANSONIA ALTISSIMA SEEDLINGS TO GODASA SIDAE

ATTACK IN THE RAINFOREST ECOLOGICAL ZONE OF NIGERIA

Oduntan, O. Akinlabi1, Omotade R. Ogunremi2, 

Onilude Q. Ayansola1, and Awotedu B. Folake1

The establishment of plantations of Mansonia altissima has been constrained by the insect pest Godasa 

sidae that causes extensive damage to the seedlings in the nursery. This study was carried out at Forestry 

Research Institute of Nigeria nursery site, using 120 seedlings with the aim to prevent Godasa sidae attack 

on Mansonia altissima seedlings in order to achieve an ecologically based pest resistance management 

strategy. Seedlings were placed in a screen house (SHS); in the open treated with a methanoic extract from 

Gliricidia sepium once a week (MTH); also, in the open treated with a low concentration (0.05 percent) 

of water-based insecticide (Lambda-Cyhalothrin) once a week (CHM), all replicated 30 times. Growth 

data recorded were subjected to ANOVA and DMRT (post mortem). The results revealed that the SHS, 

MTH, and CHM were all effective in the control of the studied insect pests except the experimental 

control. However, the results revealed a significant difference at 5 percent level of probability among 

the treatments in terms of seedling height, collar diameter, and leaf production with highest mean value 

of 15.41 ± 2.36 cm observed for seedling height for SHS, followed by CHM (14.11 ± 2.18 cm) and MTH 

(13.87 ± 2.16 cm). The values of collar diameter were 0.49 ± 0.05 mm, 0.43 ± 0.03 mm and 0.40 ± 0.03 

mm for SHS, CHM, and MTH respectively. Also, the mean number of leaves in each treatment was 

23 ± 3.23, 20 ± 3.15 and 19 ± 3.10 for SHS, CHM and MTH, respectively. It was concluded that the screen 

house (SHS) performed best. However, because of cost implications, an alternative is suggested. Due to 

the hazardous effects of the chemical on the environment, the methanoic extract which can be a potent 

pesticide should be adopted for the control of insect pest of Mansonia altissima at nursery stage.

1 Forestry Research Institute of Nigeria, P.M.B 5054, Jericho Hill, Ibadan, Oyo State, Nigeria, (harkinlarbee@gmail.com). 2 Department Of Microbiologist, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Poster Session 149

QTL MAPPING POPULATION DEVELOPMENT IN CORNUS FLORIDA USING 

OPEN POLLINATED SEEDLINGS AND SSR MARKERS

Erin Pfarr1, Josh Honig1, Jennifer Vaiciunas1, Christine Kubik1, 

John Capik1, and Thomas J. Molnar1

Flowering dogwood (Cornus florida) is an 

important understory tree in deciduous mesic 

hardwood forests of the Eastern United States, 

from Massachusetts to Florida and as far west as 

Texas (Fulcher et al. 2012). In addition, flowering 

dogwood is a popular tree in managed landscapes 

thanks to its graceful form and spring blooms with 

showy bracts. It is honored as the State flower or 

tree of North Carolina, Virginia, and Missouri. 

However, since the early 1990s, powdery mildew 

(PM) (Erysiphe pulchra) has been one of the most 

problematic diseases for this species (Li et al. 

2009). Infected leaves can be curled and stunted 

with unsightly white fungal growth or increased 

red pigmentation (Li et al. 2009). In the seedling 

stage, a heavy infection can be fatal (Parikh et 

al. 2016). For mature trees, the disease negatively 

affects bloom and repeated severe infections can 

stunt a tree’s growth and reduce its appeal in the 

landscape (Parikh et al. 2016). 

One selection from the Rutgers dogwood breeding 

program, C. florida H4AR15P25, shows excellent 

resistance to powdery mildew. The resistance 

holds up in clones across multiple locations and 

appears to be heritable. The quantitative trait 

loci (QTL) underlying this resistance will be 

investigated and mapped in a pseudo F2 population 

using genotyping by sequencing (GBS)-derived 

single nucleotide polymorphism (SNP) markers. 

To expedite population development, openpollinated (OP) seeds were harvested from 

H4AR15P25 grown in a crossing block of limited 

PM-susceptible male parents. The purpose of 

this study is to determine the pollen parents of 

the seedlings using simple sequence repeat (SSR) 

markers and assemble a full-sibling population 

of 150 seedlings from the same PM-susceptible 

pollen parent for the future QTL PM resistance 

study.

Mature fruit were harvested from C. florida

H4AR15P25 at Rutgers University New 

Brunswick Agricultural Experiment Station in 

October 2017. Seeds were cleaned, stratified, and 

germinated in the greenhouse, resulting in ~650 

healthy seedlings. 

Newly expanding leaves were collected from 

H4AR15P25, 16 possible pollen parents, and a 

subset of 37 seedlings. DNA was extracted using 

the Qiagen DNeasy Plant Kit. The samples were 

genotyped with 11 SSR primer pairs developed by 

Wang et al. (2008) and Wadl et al. (2008) (table 1) 

(Wadl et al. 2008, Wang et al. 2008).

Briefly, the primer pairs were synthesized by 

Integrated DNA Technologies (Coralville, IA) with 

a 7 bp PIG-tailing sequence added to the reverse 

primers to aid in scoring true vs. plus A alleles, 

Table 1—SSR marker summary statistics for Cornus 

florida H4AR15P25, 16 possible pollen parents, and 

37 open pollinated progeny

Locus k N HObs HExp PIC F(Null)

CF020 8 54 0.630 0.620 0.582 -0.002

CF273 8 52 0.808 0.759 0.712 -0.039

CF125 4 54 0.889 0.639 0.566 -0.185

CF701 7 54 0.870 0.814 0.778 -0.043

CF048 5 54 0.667 0.589 0.550 -0.071

CF597 8 54 0.722 0.673 0.615 -0.041

CF1045 3 54 0.333 0.334 0.287 -0.010

CF055 4 54 0.630 0.485 0.413 -0.156

CF646 5 54 0.870 0.765 0.720 -0.067

CK040 3 54 0.222 0.283 0.246 0.114

CK015 4 54 0.556 0.553 0.448 -0.005

Average 5.4 53.8 0.654 0.592 0.538 -0.046

k = the number of alleles for each SSR marker.

N = the number of individuals genotyped at a locus.

HObs = the Observed Heterozygosity.

HExp = the Expected Heterozytosity.

PIC = Polymorphic Information Content. 

F(Null) = the null allele frequency estimate.

1Rutgers University, Department of Plant Biology, New Brunswick, NJ 08901, (erin.pfarr@rutgers.edu).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
150 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

and an 18 bp M13 sequence added to the forward 

primers for economic PCR florescent labeling 

(Brownstein et al. 1996, Schuelke 2000). 

The following were combined in a 96 well PCR 

plate with 13 μl reaction volumes: 0.5 pmol 

forward primer, 1 pmol reverse primer, 1 pmol 

florescent dye M13 primer (FAM, NED, PET, 

or VIC), 5 ng DNA, and 10xRamp-Taq PCR 

buffer (Denville Scientific, Metuchen, NJ), 2 mM 

MgCl2, and 0.25 mM of each dNTP (Denville 

Scientific). PCR was performed on GeneAmp 

9700 thermalcyclers (Applied Biosystems, Foster 

City, CA) with the following parameters: initial 

denaturation of 94 °C for 5 min followed by 

30 cycles of 94 °C for 30 s, 55 °C for 45 s, and 

72 °C for 45 s, followed by 20 cycles of 94 °C for 

30 s, 53 °C for 45 s, and 72 °C for 45 s, followed 

by a final extension of 72 °C for 10 min. The PCR 

products were run with LIZ 600 size standard 

on a capillary electrophoresis genetic analyzer 

(ABI 3500xl; Applied Biosystems). SSR alleles 

were binned and assigned with Genemapper 4.0 

(Applied Biosystems). Allele frequency analysis 

and paternity parentage analysis was performed 

with CERVUS 3.0 (Kalinowski and others 

2007), which uses a likelihood-based approach 

and parentage analysis simulation to determine 

confidence levels for parentage assignments.

The SSRs were highly polymorphic with 3 to 

8 alleles per marker and polymporphic information 

content (PIC) values between .25 and .78 (table 1.). 

The SSR marker data confirmed the pedigrees 

of C. florida H4AR17P05 and H4AR17P48, two 

of the possible pollen parents, as progeny of 

C. florida forma rubra × C. florida “Red Beauty”, 

which were also genotyped for this study. 

H4AR15P28, the closest tree to H4AR15P25, 

was identified as the pollen parent of 10 of the 

seedlings, the most of any other possible parent 

(fig. 1 and table 2.). H4AR15P28 would be the 

best susceptible pollen parent for the mapping 

population because it is the most PM susceptible 

tree in the breeding block. With slightly over 

a fourth of genotyped seedlings in this subset 

assigned to H4AR15P28, we are optimistic that 

upon screening all 650 OP seedlings, we will find 

150 full siblings from this pollen parent for the 

bi-parental QTL mapping population. 

Six plants, H4AR17P05, H4AR15P49, 

H4AR15P58, forma rubra, H4AR17P48, “Red 

Beauty”, and H4AR15P40, did not have any 

seedlings in this subset (fig. 1). This may be due 

to a variety of factors, such as increased distance 

from and asynchronous flowering with the 

mother plant, or limited flower set in the case of 

H4AR15P40 (Rhoades et al. 2011). 

Interestingly, 7 seedlings could not be assigned 

with 80 percent confidence to any of the possible 

pollen parents. F-003, F-014, and F-106 had one 

or more alleles that were not present in any of the 

possible parents. It is unclear where this pollen 

flow is coming from as this breeding block is 

separated from the closest C. florida trees by 

1,100 feet (including a 400 foot-wide river).

The set of 11 markers used here will be paired 

down to the 8 most informative for more efficient 

screening. They will be used to screen the rest of 

the OP seedlings from H4AR15P25 to assemble 

the 150-individual mapping population. This 

C. florida population will be genotyped using 

GBS and evaluated for PM response at 2 and 

3 years of age. The final goals are to better 

understand PM resistance inheritance and to 

subsequently use QTLs for PM resistance to aid in 

marker assisted selection in the Rutgers dogwood 

breeding program. This would allow for early 

culling of PM susceptible trees, maximizing space 

and resources for a plant with a relatively slow 

generation time.
Poster Session 151

Figure 1—Approximate spatial relationships of C. florida H4AR15P25 (blue circle) and the possible 

pollen parents (green circles). Numbers inside of the circles represent number of progeny assigned 

to trees with 95% trio confidence or greater by CERVUS.
152 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

Table 2—CERVUS program parentage assignments for 37 open pollinated Cornus florida seedlings

Offspring 

ID

Loci 

typed

Candidate 

mother ID

Pair loci 

mismatch

Candidate 

father ID

Trio loci 

mismatch

Trio LOD 

score

Trio 

Delta

Trio 

confidence

F-006 11 H4AR15P25 0 H4AR15P28 1 4.23 4.23 *

F-011 11 H4AR15P25 0 H4AR15P28 1 5.12 5.12 *

F-015 11 H4AR15P25 0 H4AR15P28 1 4.78 4.78 *

F-038 11 H4AR15P25 0 H4AR15P28 0 9.72 6.51 *

F-047 11 H4AR15P25 0 H4AR15P28 1 4.52 4.52 *

F-073 11 H4AR15P25 0 H4AR15P28 1 4.74 4.74 *

F-075 11 H4AR15P25 0 H4AR15P28 0 10.5 10.5 *

F-096 11 H4AR15P25 0 H4AR15P28 0 9.73 6.51 *

F-104 11 H4AR15P25 0 H4AR15P28 1 4.52 4.52 *

F-129 11 H4AR15P25 0 H4AR15P28 1 4.51 4.51 *

F-020 11 H4AR15P25 0 H4AR15P30 0 12.4 12.4 *

F-024 11 H4AR15P25 0 H4AR15P30 1 5.33 5.33 *

F-029 11 H4AR15P25 0 H4AR15P30 0 10 10 *

F-018 11 H4AR15P25 0 H4AR15P31 0 14.1 14.1 *

F-004 11 H4AR15P25 0 H4AR15P33 0 9.69 9.69 *

F-016 11 H4AR15P25 0 H4AR15P35 0 8.15 8.15 *

F-021 11 H4AR15P25 0 H4AR15P35 1 3.29 3.29 *

F-026 11 H4AR15P25 0 H4AR15P35 1 5.25 5.25 *

F-042 11 H4AR15P25 0 H4AR15P35 0 2.54 2.54 *

F-101 11 H4AR15P25 0 H4AR15P35 0 8.47 8.47 *

F-109 10 H4AR15P25 0 H4AR15P35 0 1.16 1.16 *

F-098 11 H4AR15P25 0 H4AR15P41 0 4.26 1.51 *

F-110 11 H4AR15P25 0 H4AR15P41 0 6.39 6.39 *

F-007 11 H4AR15P25 0 H4AR15P42 0 8.24 8.24 *

F-113 11 H4AR15P25 0 H4AR15P46 1 0.79 0.79 *

F-128 11 H4AR15P25 0 H4AR15P46 0 2.48 2.48 *

F-009 11 H4AR15P25 0 H4AR15P50 0 7.37 4.89 *

F-017 11 H4AR15P25 0 H4AR15P50 0 7.08 7.08 *

F-095 11 H4AR15P25 0 H4AR15P50 0 7.88 7.88 *

F-022 11 H4AR15P25 0 H4AR15P28 1 5.37 0.16 +

F-071 11 H4AR15P25 0 H4AR15P28 2 ####### 0

F-106 10 H4AR15P25 0 H4AR15P41 2 -0.167 0

F-003 11 H4AR15P25 0 H4AR15P46 5 ####### 0

F-013 11 H4AR15P25 0 H4AR15P46 1 -0.11 0

F-005 11 H4AR15P25 0 H4AR15P50 3 ####### 0

F-014 11 H4AR15P25 0 H4AR15P50 5 ####### 0

F-136 11 H4AR15P25 0 H4AR15P50 2 ####### 0

Summary statistics for CERVUS program parentage assignments. 

The most likely pollen parent ID is listed for each seedling. 

For trio confidence levels * = 95 percent; + = 80 percent.
153

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genotyping. Biotechniques. 20(6): 1004–1006, 1008–1010.

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deciduous trees in southeastern U.S. nursery production. 

Knoxville, TN: Southern Nursery IPM Working Group: 

137–158.

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how the computer program cervus accommodates 

genotyping error increases success in paternity assignment. 

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Li, Y.; Mmbaga, M.T.; Windham, A.S. [et al.]. 2009. Powdery 

mildew of dogwoods: Current status and future prospects. 

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Parikh, L.; Mmbaga, M.; Kodati, S. [et al.]. 2016. Broadsense heritability and genetic gain for powdery mildew 

resistance in multiple pseudo-F2 populations of flowering 

dogwoods (Cornus florida L.). Scientia Horticulturae. 213.

Rhoades, P.; Wadl, P.A.; Klingeman, W.E. [et al.]. 2011. Using 

simple sequence repeats (SSRs) to measure pollen flow in 

a flowering dogwood orchard. Journal of Environmental 

Horticulture. 29(4): 175–179.

Schuelke, M. 2000. An economic method for the fluorescent 

labeling of PCR fragments. Nature Biotechnology. 18: 233.

Wadl, P.A.; Wang, X.; Scheffler, B.E. [et al.]. 2008. 

Microsatellites from kousa dogwood (Cornus kousa). 

Molecular Ecology Resources. 8(4): 780–782.

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4(3): 461–468.

Poster Session
154 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

GENETIC BARCODES FOR IDENTIFICATION OF 

PURE AND HYBRID FRAXINUS SPECIES

William J. Plumb1,2, Laura J. Kelly1, Gerry Douglas3, and Richard Buggs1,2

Native populations of species in the genus Fraxinus (ash) are being devastated in North America by the 

emerald ash borer, and in Europe by ash dieback. There is evidence that some Asiatic species of Fraxinus

have low susceptibility to both of these threats. It is therefore possible that hybridisation between Asiatic 

species and congeners native to Europe and North America may be one way of retaining ash as a viable 

forestry species on those continents. We have developed a set of genetic barcodes for the confirmation 

and identification of ash hybrids. Using whole genome assemblies for 29 individuals from 28 Fraxinus

taxa (www.ashgenome.org), we identified and aligned putative orthologs from low copy number genes. 

A subset of 1396 variable gene alignments were analyzed using the statistical programme CONTEXT to 

identify candidates with a pattern of variability suitable for barcoding the individual species of Fraxinus. 

Primers were designed for the selected barcoding regions, and tested using several example species from 

the living collection at the Royal Botanic Gardens, Kew, UK and hybrids developed at Ashtown Research 

Station (Teagasc, Ireland). Sequence data were generated for all regions using Polymerase Chain Reaction 

(PCR) amplification and Sanger sequencing. The results of this study show that these barcodes are capable 

of identifying various different Fraxinus species, as well as being suitable for both the confirmation and 

identification of hybrids.

1School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom.

2Royal Botanic Gardens Kew, Richmond, Surrey TW9 3AB, United Kingdom, (w.plumb@qmul.ac.uk). 3Teagasc Food Research Centre, Dublin, Ireland.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Poster Session 155

CONFOCAL MICROSCOPY CONFIRMS THE FEASIBILITY OF GENE SILENCING 

FOR EMERALD ASH BORER SUPPRESSION

Thais B. Rodrigues1, Flavia Pampolini1, Tomokazu Kawashima2, and Lynne Rieske-Kinney1

Emerald ash borer (EAB) (Agrilus planipennis) is an aggressive, invasive forest and urban tree pest that 

feeds and develops beneath bark, killing trees rapidly. We are investigating the use of RNA interference 

(RNAi) as a tool for managing EAB. Previously, we showed that oral ingestion of double-strand RNA 

(dsRNA) can silence essential and specific genes, and subsequently found in droplet assays that silencing 

heat shock protein and shibire genes kills up to 90 percent of neonates and adults after 1–2 weeks of 

exposure. To evaluate our findings in vivo, we assessed dsRNA uptake through plant material and 

through the egg chorion. Twigs of greenhouse-grown tropical ash, Fraxinus uhdei, were treated with 

labeled dsRNA and infested with EAB eggs; twigs treated with unlabeled dsRNA were used as controls. 

Confocal microscope images were then taken and analyzed to detect fluorescence in both plant and insect 

material. Additionally, EAB eggs were soaked in labeled dsRNA, and eggs and hatched neonates analyzed 

by confocal microscopy. Gene silencing was performed to corroborate imaging results. After 8 days of 

exposure, we detected fluorescence in ash bark and xylem, and larvae fed on these plants showed strong 

fluorescence inside their alimentary canal. When eggs were soaked with labeled dsRNA, fluorescence 

was detected in eggs and the hatched larvae, confirmed by gene expression. Our twig assay demonstrated 

that dsRNA is taken up through plant material, suggesting that trunk injections or soil treatments with 

dsRNA may potentially be an efficient delivery method for this technology. Absorption of dsRNA by eggs 

demonstrated that spray-able dsRNA may be a potential delivery method. EAB is rapidly expanding its 

geographic range, and innovative means of management are essential to mitigate its impacts. We’ve shown 

that RNAi in EAB is feasible; here we demonstrate the potential of topical applications of this technology.

1Department of Entomology, University of Kentucky, Lexington, KY 40546.

2Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY 40546, (tomo.k@uky.edu).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
156 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

VARIOUS PROPAGATION WAYS IN ALNUS GLUTINOSA WITH 

DIFFERENT SENSITIVITY TO PHYTOPHTHORA × ALNI

Jana Šedivá1, Petra Štochlová1, Kateřina Novotná1, and Karel Černý1

The hardwood tree Alnus glutinosa L. (black alder) is a typical riparian plant and originally ranged 

throughout most of Europe and also into North Africa, Asia Minor, and Western Siberia. One of the most 

serious pathogens of Alnus is Phytophthora × alni causes a rapid necrosis of the inner bark of the collar, 

stems, and roots of infected trees. The identification of black alder genotypes resistant to the disease 

and successful propagation methods would be very beneficial in the rejuvenation of damaged riparian 

ecosystems and the prevention of possible losses in the future. In seeking to rapidly propagate plants 

possessing a rare but highly desirable character, it is more appropriate to use vegetative means. The 

feasibility of propagating mature A. glutinosa trees by vegetative means that could be used to propagate 

trees resistant to P. × alni was examined. Both softwood and hardwood cuttings were tested. The different 

treatments were employed, such as growth regulator concentration and method of application. The 

rooting success was highly dependent on the type of cuttings and treatments used. The softwood cuttings 

collected in the middle of July and then treated with 1 percent IBA rooted the best of all, with 42.5 percent 

of cuttings rooting successfully. Micropropagation is an effective and rapid propagation method for 

valuable genotypes of trees. An important role in the establishment of in vitro cultures of black alder plaid 

the physiological state of the donor trees, the time of explant collection and the composition of the nutrient 

medium, especially in terms of the content of the growth regulators.

1 Department of Plant Biotechnology, The Silva Tarouca Research Institute for Landscape and Ornamental Gardening, Průhonice 252 43, 

Czech Republic, (sediva@vukoz.cz).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Poster Session 157

USING A POPLAR HYBRID TO EXPLORE HOST PLANT GENETIC CONTROL 

OF ASSOCIATING INSECT AND FUNGAL SPECIES

Sandra J. Simon1, Stephen P. DiFazio1, and David Macaya-Sanz1

Community genetics is the study of the influence of host genotype on associating arthropods, fungi, 

pathogens, and predators, such that related individuals support similar, heritable communities. 

Understanding how shifts in the genes of a population contribute to ecosystem function effectively 

links ecology and evolutionary biology. Observing these interactions allows genes associated with pest 

resistance and susceptibility to be identified within a host plant. This in turn could aid in the cross design 

of commercially valuable biofuel crops such as those in the Salicaceae family, which include poplar and 

willow species. To test the extent to which genes of Populus have extended phenotypes on arthropod 

communities, surveys were conducted in a P. trichocarpa x P. deltoides pseudo-backcross plantation. 

Trees were observed for the arthropods Pemphigus populitransversus, Mordwilkoja vagabunda, 

Phyllocolpa bozemani, and Phyllocnistis populiella. Individuals were also scored for Melampsora 

leaf rust, Septoria musiva leaf spot, and Septoria musiva stem canker fungal infections. Broad-sense 

heritability (H2

c) revealed a significant genetic factor controlling both insect and fungal pests. Several 

quantitative trait loci (QTL) intervals were discovered to be associated with these biotic stressors that 

contain numerous candidate genes which mediate their interactions. There was also an elevated number 

of tandem duplications and chromosome rearrangements in the intervals for several biotic phenotypes. 

Future work will be aimed at understanding the contribution of structural variation in mediating biotic 

interactions.

1West Virginia University, Morgantown, WV 26505, (ssimon4@mix.wvu.edu).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
158 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

GENETIC RESISTANCE TO BLISTER RUST IN DECLINING LIMBER PINE 

(PINUS FLEXILIS) IN ALBERTA—THE PATH TO RESTORATION? 

Richard A. Sniezko1, Jodie Krakowski2, Robert Sissons3, Robert Danchok1, 

Angelia Kegley1, and Douglas P. Savin1

Limber pine (Pinus flexilis) is a long-lived conifer native to western North America. The northernmost 

populations of limber pine are in Canada, of which over 90 percent are in Alberta. These populations 

are heavily infected by white pine blister rust (WPBR), caused by the non-native pathogen Cronartium 

ribicola. Many natural populations are more than 70 percent infected, with some areas having 

> 95 percent infection. Limber pine is listed as endangered in Alberta and has been recommended as 

endangered by the Committee on the Status of Wildlife in Canada. Efforts to restore the species are 

underway, such as finding a diverse complement of genetically resistant parent trees and assembling 

seed collections for genetic conservation and restoration. Two small tests of Alberta parent tree progeny 

at the USDA Forest Service’s Dorena Genetic Resource Center (DGRC) detected complete resistance 

to WPBR major gene resistance (MGR). Only low levels of partial resistance have been detected to 

date. Currently, Alberta Agriculture and Forestry and Waterton Lakes National Park are working with 

DGRC to greatly expand the number of seedling families being screened for resistance, using progeny of 

putatively resistant field selections. Sixty half-sib families were inoculated with WPBR spores in fall 2017, 

50 additional families inoculated in 2018, and additional families are scheduled for inoculation in 2019 

and 2020. Resistant seedlings from the first MGR tests at DGRC were included in restoration plantings 

in 2018. These seedlings and their parent trees will serve as sentinels to monitor changes in the rust 

fungus or environmental conditions that may affect the durability or stability of resistance. Within 5 years 

we anticipate having an increased knowledge of the frequency, type, and level of genetic resistance in 

northern Canadian populations, as well as establishment of more restoration plantings. 

1 Dorena Genetic Resource Center, USDA Forest Service, Cottage Grove, OR 97424, (richard.sniezko@usda.gov). 2 Alberta Tree Improvement and Seed Centre, Alberta Agriculture and Forestry, Edmonton, Alberta T5K 2M4, Canada.

3Waterton Lakes National Park, Waterton Park, Alberta, T0K 2M0, Canada.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Poster Session 159

GENETIC RESISTANCE AND RESTORING A THREATENED SPECIES: 

WHITEBARK PINE AT CRATER LAKE NATIONAL PARK

Richard A. Sniezko1 and Jennifer S. Hooke2

Whitebark pine (WBP), Pinus albicaulis is a wide-ranging conifer species found at high elevations in 

western North American forests. It is a keystone species, but is facing serious challenges, including high 

mortality in many parts of its range due to the presence of white pine blister rust (WPBR), caused by 

the non-native pathogen Cronartium ribicola, as well as the impacts of mountain pine beetle infestation, 

climate change, and altered fire regimes. Due to impacts of these daunting challenges WBP has been 

listed as endangered in Canada, while in the United States it is being considered for listing under the 

Endangered Species Act. The consensus among scientists working with WBP is that for restoration efforts 

to be successful in most areas, they will have to include seedlings with genetic resistance to WPBR. 

Dorena Genetic Resource Center has been evaluating resistance of WBP from Crater Lake National Park 

(CRLA) since 2004. These trials show that at least some populations, including CRLA populations, have 

genetic resistance that will allow restoration to proceed successfully. From 2009 to 2016, six restoration 

plantings have been established using seedlings from resistant CRLA parent trees. Through fall 2013, 

survival in the four oldest restoration plantings ranged from 77.6–90.6 percent. Rust infection has been 

very low to this point. Each planting has documented family identification of each seedling, which allows 

for WBP restoration and conservation, along with facilitating further research on how survival, rust 

resistance, growth, and specific traits vary among and within family groups. These restoration plantings 

also serve as long-term source populations of resistance alleles to help the natural regeneration of resistant 

WBP throughout neighboring areas. In addition, the current living rust-resistant parent trees in CRLA are 

being protected from mountain pine beetle infestation using verbenone pouches, to further help spread 

genetic resistance. The CRLA plantings provide a successful example of the use of genetic resistance to 

begin the restoration of a non-commercial forest tree species. They also provide a conservation education 

tool to raise public awareness of the potential for restoration using genetic resistance.

1 Dorena Genetic Resource Center, USDA Forest Service, Cottage Grove, OR 97424, (richard.sniezko@usda.gov). 2Crater Lake National Park, Crater Lake, OR 97604.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
160 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

INVESTIGATING ANATAGONISTIC ACTIVITY OF FUNGAL ENDOPHYTES TOWARD 

PATHOGENS OF WESTERN WHITE PINE (PINUS MONTICOLA)

Emily Martin1, Beau Larkin1, Richard Sniezko2, and Lorinda Bullington1

North American white pine populations have experienced increased mortality due to the invasive fungal 

pathogen Cronartium ribicola. Research has shown that white pines may defend against C. ribicola

infection by hosting diverse fungal endophyte communities. Tree genetics appear to influence these 

endophyte communities and some fungal endophytes can be vertically transmitted, through seeds, from 

parent to offspring. Vertically transmitted fungi are often mutualists of plants and can facilitate host 

defenses against pathogens as seedlings mature. We plan to isolate and screen vertically transmitted fungi 

from resistant western white pines (Pinus monticola), for antagonistic activity that may inhibit infection, 

survival, and growth of C. ribicola. Western white pine seeds and needles will be collected from healthy 

trees throughout the Pacific Northwest and MPG North, Swan Valley, MT. We will culture tissue to isolate 

fungal endophytes and test for their ability to inhibit pathogen growth with microspectrophotometry, 

microscopy, and dual cultures. Fungi previously found to occur more often in resistant white pines will 

also be screened against pathogens. Microspectrophotometry will allow us to monitor pathogen growth 

and measure the inhibitory effects of toxic antimicrobial compounds produced by fungal endophytes. Dual 

cultures and microscopy will allow us to observe competition and mycoparasitism between pathogens 

and endophytes. With these methods we can screen many fungal endophytes at a time and quantify and 

compare their inhibitory effects. Fungal endophytes exhibiting the most effective antagonistic activity will 

be selected and used in western white pine seedling inoculation trials in 2019.

1MPG Ranch, Missoula, MT, 59801, (www.mpgranch.com, www.mpgnorth.com). 2 Dorena Genetic Resource Center, USDA Forest Service, Cottage Grove, OR 97424, (lbullington@mpgranch.com).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Poster Session 161

SUDDEN OAK DEATH IN SOUTHERN OREGON: COMPARING THE 

EU1 AND NA1 LINEAGES OF PHYTOPHTHORA RAMORUM

Kelsey L. Søndreli1, Alan Kanaskie2, Richard Sniezko3, 

Everett Hansen1, and Jared M. LeBoldus1

Phytophthora ramorum is the cause of Sudden Oak Death and is an invasive pathogen in California and 

southwestern Oregon forests. Until recently, forest infestations in Oregon and California have all been 

the NA1 clonal lineage. However in 2015, the EU1 lineage of P. ramorum was isolated from a tanoak 

tree in the forest in Curry County, Oregon. In this region, tanoak (Notholithocarpus densiflorus) is the 

most susceptible species to P. ramorum, developing lethal stem cankers and sporulating on infected 

leaves and branches, spreading the disease. A variety of experiments were conducted, including field and 

greenhouse inoculations, in order to determine the relative threat of the new EU1 lineage compared to 

the NA1 lineage in Oregon forests. Overall, EU1 was shown to be more aggressive on Oregon trees than 

NA1. A sporulation assay demonstrated a 10-fold increase in sporulation of the EU1 isolates compared to 

NA1 isolates on tanoak. Results from a preliminary field experiment suggest that greater infection rates 

of a larger number of species occur under EU1 infested trees. An analysis of resistance in tanoak families 

inoculated with EU1 and NA1 isolates of P. ramorum indicated significantly larger cankers developing 

on EU1 inoculated trees compared to NA1 in 3/14 families. No trees were completely resistant to the 

pathogen. The accumulated evidence indicates that the EU1 lineage of P. ramorum is more aggressive and 

potentially poses a greater risk to Oregon forests than the NA1 lineage.

1 Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, (Kelsey.Sondreli@oregonstate.edu). 2 Oregon Department of Forestry, Salem, OR 97310.

3Dorena Genetic Resource Center, U.S. Forest Service, Cottage Grove, OR 97424.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
162 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

SCREENING FOR WHITE PINE BLISTER RUST IN WHITEBARK PINE: 

BRITISH COLUMBIA, CANADA

Ward Strong1

Whitebark pine, Pinus albicaulis, is a high-altitude 5-needle pine native to Western North America. It is a 

keystone ecological species, essential to ecosystem functioning in many subalpine and treeline forests. In 

2012, this species was identified as Endangered by the Canadian Species at Risk Act, due in large part to 

mortality by White Pine Blister Rust (Cronartium ribicola). This Old World disease was introduced into 

North America at the beginning of the 20th century; there is very little genetic resistance to it in endemic 

5-needle pine populations. The British Columbia government has started a screening program to identify 

genetic resistance in Whitebark pine, with the objective of inter-situ gene conservation and in-situ species 

recovery. The process starts by identifying surviving trees in high-mortality areas. The progeny of these 

parent trees are artificially inoculated with blister rust basidiospores in a climate-controlled room, then 

scored over the next 2–4 years. We have now made our first rust-resistant parent selections, which have 

been grafted for deployment in clone banks or seed orchards. 2018 will be our 7th year of parent tree seed 

collections in this ongoing, long-term project.

1BC Ministry of Forests, Lands, Natural Resource Operations, and Rural Development, Kalamalka Forestry Center, 3401 Reservoir Rd., 

Vernon, British Columbia, CANADA V1B 2C7, (ward.strong@gov.bc.ca).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Poster Session 163

DIFFERENTIAL RESISTANCE TO PHYTOPHTHORA CINNAMOMI IN TROJAN FIR

Alexander J. Trouern-Trend1, William H. Kohlway2, Lilian Matallana2, 

Ross W. Whetten2, John Frampton2, and Jill L. Wegrzyn1

RNA was sequenced from roots tissue samples of Abies equi-trojani that demonstrated differential 

susceptibility to dieback (root rot) when experimentally inoculated with the oomycete pathogen 

Phytophthora cinnamomi. Eight pooled RNA libraries of 10 inoculated individuals based on the 

progression of tree morbidity over the course of the inoculation experiment represented three conditions, 

uninoculated control (2 libraries), resistant (4 libraries), and susceptible (2 libraries). These data were used 

for differential expression (DE) analysis, which uncovered 12,680 instances of differential expression 

among the three groups; 326 upregulated and 1,219 downregulated in resistant relative to control, 

5,955 upregulated and 343 downregulated in resistant relative to susceptible, and 4,722 upregulated and 

115 downregulated in control relative to susceptible. Our analysis of DE genes leveraged a new functional 

annotation pipeline, Eukaryotic Non-Model Transcriptome Annotation Pipeline (EnTAP), which 

automates similarity searches across multiple databases and includes protein domain, gene ontology, and 

gene family assignment, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation and contaminant 

filtering. Functional annotation clustering of DE genes yielded patterns of increased expression of stress, 

metabolic and apoptosis related genes in resistant trees.

1 Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT 06269.

2 Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC 27695, (jill.wegrzyn@uconn.edu).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
164 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

PHENOLIC COMPONENT OF LOBLOLLY PINE DEFENSE RESPONSE TO 

BLUE-STAIN FUNGI ASSOCIATED WITH ROOT-FEEDING BEETLES

Zackary N. Parker1, Brittany F. Barnes1, and Caterina Villari1

Southern pine species are integral components 

to southeastern forests, comprising a significant 

percentage of the forested lands. Among these 

species, loblolly pine (Pinus taeda L.) is the 

dominant timber species grown commercially, 

thus being extremely important for both the 

economy and the ecology of southeastern regions. 

Extensive studies on pathogens and insect pests 

affecting this species have been conducted, 

but for obvious reasons, most of them have 

focused on major tree-killing species, such as 

the southern pine beetle (SPB) [Dendroctonus 

frontalis Zimmerman (SPB)] and its associated 

fungal complex. However, many other beetles 

and associated fungi colonize the species, and 

are still worth being investigated, as they too 

are integral components of the loblolly pine 

planation system. The root-feeding beetle system 

and associated fungi, for instance, have largely 

been overlooked in the past, and only recently 

become of interest (Coyle et al. 2015, Eckhardt et 

al. 2007). These beetles are generally secondary 

invaders, colonizing tree hosts only after their 

defenses have already been weakened (Matusick 

et al. 2013). Additionally, their associated 

ophiostomatoid fungi, which are carried externally 

on the exoskeleton, are usually considered weak 

pathogens (Jacobs and Wingfield 2001). However, 

there is still relatively too little known about the 

system, and in particular, nothing is known about 

how loblolly pine responds to colonization by the 

fungal species. 

In this study, we focused on the phenolic 

metabolisms of loblolly pine, which, together 

with the terpenoid component, is one of the main 

defense mechanisms of the species. In fact, while 

terpenoids of loblolly pine have been widely 

explored, surprisingly, phenolics are currently 

totally unexplored. Objectives of the study were: 

(1) to characterize the constitutive phenolic profile 

of loblolly pine phloem, and (2) to investigate the 

qualitative and quantitative induced responses of 

the phenolic metabolism to inoculation with bluestain fungi commonly associated with root feeding 

beetles in the region. 

Using a nested randomized complete blocked 

design, 45 mature loblolly pines were selected 

from a planted stand in Whitehall Forest (Athens, 

GA). Trees were assigned to one of five different 

treatments: (1) inoculation with Grosmannia 

alacris, (2) G. huntii, (3) or Leptographium 

profanum, (4) sterile wounding and 

(5) non-wounded control. For inoculations, four 

plugs of phloem were removed from each tree 

with a cork borer, and then substituted with 

another bark plug that had been previously 

sterilized and then colonized by the fungi (fig. 1). 

All fungal isolates used in this study had been 

isolated from the surface of root-feeding beetles 

collected in the area. For sterile wounding, the 

plug was not replaced after removal, while control 

trees were left unwounded. Plugs collected at the 

time of treatment (September 2017) were used for 

the characterization of the constitutive phenolic 

profile. Four weeks after treatment, the outer 

bark surrounding inoculation sites was removed 

and vertical length of each induced lesion (fig. 2) 

was measured. Phloem tissues surrounding 

the inoculation sites and sterile wounding sites 

were collected and immediately stored in liquid 

nitrogen. Also at this time, phloem samples 

from non-wounded control trees were collected. 

All samples were then transported to the lab 

and stored at -80 ̊C until further processing. 

1 D.B. Warnell School of Forestry and Natural Resources, University of Georgia, Athens, GA 30602, (cvillari@uga.edu).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
165

Figure 1—Loblolly pine bark plugs previously sterilized and then 

colonized by one of the fungal species used in this experiment. 

Inoculations consisted in removing bark plugs from the 

experimental trees and substituting them with colonized ones. 

(courtesy photo by Caterina Villari)

Poster Session

Collected tissues were ground to powder in 

liquid nitrogen, and phenolics were extracted in 

methanol. Extracts are currently being analyzed 

using a combination of ultra-high pressure 

liquid chromatography—diode array detector 

(UHPLC-DAD) and high-pressure liquid 

chromatography—mass spectrometry (HPLC-MS) 

approaches.

Preliminary results show that all fungal 

inoculations produced significantly longer lesions 

than sterile wounding alone, and that inoculations 

with G. huntii produced the longest lesions. 

Additionally, preliminary observations of the 

phenolic profile indicate distinct quantitative 

changes in specific compounds after inoculation. 

Results from this study will provide valuable 

insights into the phenolic response of loblolly 

pine to fungal infections. Findings will also allow 

for further investigations regarding the effects of 

those phenolic compounds most responsive to the 

fungal induction on the survival and fitness of the 

same inoculated fungal species. 

LITERATURE CITED

Coyle, D.R.; Klepzig, K.D.; Koch, F.H. [et al.]. 2015. A 

review of southern pine decline in North America. Forest 

Ecology and Management. 349: 134–148. 

Eckhardt, L.G.; Weber, A.M.; Menard, R.D. [et al.]. 2007. 

Insect-fungal complex associated with loblolly pine decline 

in central Alabama. Forest Science. 53: 84–92. 

Jacobs, K.; Wingfield, M.J. 2001. Leptographium Species: 

tree pathogens, insect associates, and agents of blue-stain. 

American Phytopathological Society Press. 207 p.

Matusick, G.; Menard, R.D.; Zeng, Y.; Eckhardt, L.G. 2013. 

Root-inhabiting bark beetles (Coleoptera: Curculionidae) 

and their fungal associates breeding in dying loblolly pine 

in Alabama. Florida Entomologist. 96: 238–241. 

Figure 2—Fungal induced lesion in loblolly pine phloem. 

Lesions were measured in centimeters and used to estimate 

virulence of pathogens. (courtesy photo by Zack Parker)
166 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

BREEDING FOR RESISTANCE TO PATHOGENIC PINE WOOD NEMATODES

Kwan-Soo Woo1, Donghwan Shim1, Sung-Joon Na1, 

Jinjoong Kim1, and Il Hwan Lee1

We conducted an inoculation test using 231 open pollinated families (total 6,869 seedlings) of Pinus 

densiflora Sieb. et Zucc. and 301 open pollinated families (total 9,020 seedlings) of Pinus thunbergii Parl. 

to evaluate their susceptibility and mortality with pathogenic nematodes, Bursaphelenchus xylophilus. 

The 2-year-old seedlings were inoculated with the 3,000 nematodes/seedling in a nursery bed from 

July 5 to 20, 2017. The susceptibility of pine species to infection by B. xylophilus varied between pine 

species and among families. The average survival rate of P. densiflora at the time of the final sampling 

(about 60 days after inoculation) was 48.3 percent, and that of P. thunbergii was 12.5 percent. The average 

number of B. xylophilus recovered from 11 stems of P. densiflora and 12 stems of P. thunbergii was 

ranged from 40 to 398 and from 59 to 470 at 60 days after inoculation, respectively. Additional inoculation 

tests are suggested to identify tolerant families and individuals, particularly those that prevent nematode 

reproduction.

1 Department of Forest Genetic Resources, National Institute of Forest Science, Suwon 16631, Republic of Korea, (woo9431@korea.kr).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
167

VARIATIONS IN SUSCEPTIBILITY TO DOTHISTROMA NEEDLE BLIGHT AND 

PINE PITCH CANKER SCOTTISH POPULATIONS OF SCOTS PINE

Stuart Fraser1,5, Steve Woodward1, Anna Brown2, Eugenia Iturritxa3, 

Julio Casero Diez 4, and Jorge Martin-Garcia4

Poster Session

Pinus sylvestris is the national tree of Scotland, 

where it occurs naturally in a number of 

fragmented remnant populations. These 

populations show local adaptation in several traits, 

including susceptibility to pests and pathogens. 

Experiments were undertaken to investigate 

among-population variation in susceptibility to 

two important diseases. The first, dothistroma 

needle blight (DNB), caused by Dothistroma 

septosporum, is already an important disease in 

British pine plantations. The second, pine pitch 

canker (PPC), caused by Fusarium circinatum, is 

an important future threat to UK forestry, being 

established in pine plantations and forest nurseries 

on the Iberian Peninsula. 

Artificial inoculations with D. septosporum

suggested that among-population variation 

in susceptibility exists within native Scottish 

P. sylvestris. Natural infection experiments at two 

field sites in Scotland, however, indicated that 

apparent relative susceptibility of P. sylvestris

populations to DNB was not stable between 

sites or between years. When DNB severity was 

greatest in the field, the relative susceptibilities 

of populations correlated with those seen in 

artificial inoculation experiments. This variation 

was unrelated to the relative-continentality of 

populations.

Variations in susceptibility to PPC of 2–3 year 

old plants was examined in inoculations made 

in Phytotrons in Palencia and in glasshouses 

in Vitoria, Spain. Inoculation resulted in 

resin bleeding in most plants. Variations 

in susceptibility to F. circinatum between 

populations was unclear: most plants died within 

80 days of inoculation. Numbers of survivors 

were greatest in the North Central population. 

Small numbers from 4 populations remained 

alive at 132 days after inoculation. Glasshouse 

inoculations largely corroborated the results of 

the Phytotron experiments: a few plants survived 

inoculations, but most died within the first 90 days 

after inoculation. Field inoculations, carried out 

in an area of Cantabria with plantations of radiata 

pine badly affected by PPC, gave inconclusive 

results, with inoculated plants often re-growing 

from points below the infection. 

Very low numbers of F. circinatum conidiospores 

(50 in total) were required to kill most germinating 

P. sylvestris seed; greater losses occurred with 

increasing spore numbers. Highest rates of 

survival occurred in the Ballochbuie population.

Further work is required to determine the reasons 

for between-site variation seen in Scots pine 

responses to DNB and the roles of potential 

resistance mechanisms in this host-pathogen 

system. The findings presented are guiding 

improved DNB management in Britain. For the 

potential impacts of PPC, future research should 

focus on whether the lower susceptibility of some 

plants to the pathogen was due to genetic diversity 

in the Scottish provenance of P. sylvestris. 

Moreover, the susceptibility of Scots pine to 

multiple pathogens must be considered in future 

work.

1University of Aberdeen, Department of Plant and Soil Science, School of Biological Sciences, Cruickshank Building, Aberdeen AB24 3UU, 

United Kingdom.

2 Forestry Commission Scotland, Silvan House, 231 Corstorphine Road, Edinburgh, EH12 7AT, United Kingdom.

3Neiker Tecnalia, Campus Agroalimentario de Arkaute - E-01080 Vitoria-Gasteiz (Araba), Spain.

4 Universidad de Valladolid, Departamento Producción Vegetal y Recursos Forestales, Campus Yutera Edificio E, 34071 Palencia, Spain.

5 Present address: Scion, Whakarewarewa, Rotorua 3010, New Zealand.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International Workshop on 

the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. e-Gen. Tech. Rep. 

SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
168 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

BREEDING AND DEPLOYMENT OF INSECT AND DISEASE RESISTANCE 

TREES IN BRITISH COLUMBIA: A 2018 UPDATE

Alvin Yanchuk1

At the 2011 meeting in Eugene, OR, I provided a “thought piece” where I assumed we won’t have much 

success if we continue to breed trees for any individual pests or diseases, one at time. But most of us 

were somewhat overwhelmed of what that could mean, in terms of our research program, building new 

partnerships, obtaining funding for something that may not exist (i.e., the pest or disease) at this time, and 

the complexity in what to screen or test for. We are making small steps in the respect, and our program 

is expanding to include more pest and disease traits. For instance, (1) Swiss Needle Cast in Douglas-fir, 

(2) lodgepole pine rusts (western gall rust, commandra), and needle disease (Dothistroma), (3) a new cedar 

leaf rust, (4) western tent caterpillar in alder, and (5) western spruce budworm in interior Douglas-fir (see 

Strong presentation), to mention a few. We are still actively trying to improve resistance in our long-term 

programs (i.e., blister rust, spruce weevil, etc.), and hopefully over time, with more research, we might 

uncover more traits that could provide more durable trees (i.e., trees with “cross resistance”). Without a 

doubt, climate change, new exotic pests, the outbreak of our current pests, and major disturbances by fires, 

haven’t gone away and forest tree improvement programs will continue to show they have a large part to 

play in managing our forests.

1BC Ministry of Forests, Lands, Natural Resource Operations, and Rural Development, Saanichton Seed Orchard, 7380 Puckle Road, 

Saanichton, British Columbia, V8M 1W4 CANADA, (alvin.yanchuk@gov.bc.ca).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
Poster Session 169

RESTORATION AFTER THE LAUREL WILT DISEASE EPIDEMIC

Katherine Smith1, Junli Zhang2, Marc Hughes 3, Craig Echt1, 

Sedley Josserand1, C. Dana Nelson4, and Jason Smith2

The causative fungus of laurel wilt disease, Raffaelea lauricola, is a symbiont of the redbay ambrosia 

beetle, Xyleborus glabratus. The beetle was likely introduced to the United States through the port of 

Savannah in 2002. Since then the disease has spread across the southeast causing massive losses to 

redbay (Persea borbonia) and swamp bay (Persea palustris) populations. Restoration efforts will involve 

replanting of resistant genotypes and preservation of germplasm at botanical gardens and on public 

lands. Beginning in 2007, redbay survivor trees were collected from epidemic areas and propagated with 

rooted cuttings. This material was then screened for resistance using fungal inoculation. In this work, 

22 genotypes with 3 to 21 replicates were tested. Least squared means rankings for 12 of the genotypes 

were compared to previous inoculation experimental rankings. This showed that most genotypes are 

highly susceptible to fungal inoculation, while only one genotype showed high tolerance in multiple 

experiments. In addition, many other Lauraceae species, including silkbay, sassafras, and avocado have 

been affected by laurel wilt disease. In further work, the population genetic structure of three ecologically 

important Persea species redbay, swamp bay, and silk bay (Persea humilis), from five different Florida 

sandhill habitat locations were studied. Twenty trees were sampled per species at each location and data 

for 16 microsatellite loci were determined for each sample. Results show genetic differences between 

species and the strongest location differences among redbay. Taken together these data can be used to aid 

restoration efforts of both the Persea species in Florida and redbay across the southeast.

1Southern Institute of Forest Genetics, Southern Research Station, USDA Forest Service, Saucier, MS 39574, (smithk@ufl.edu). 2 School of Forest Resources and Conservation, University of Florida, Gainesville, FL 32611.

3University of Hawai'i at Mānoa, College of Tropical Agriculture and Human Resources, HI 96822.

4 Forest Health and Education Center, Southern Research Station, USDA Forest Service, Lexington, KY 40506.

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.
170 Proceedings of the Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry

THE EFFECTS OF LEAF WAX COMPOSITION AND RATIO 

ON RUST RESISTANCE OF POPLARS

Zhongdong Yu1, Wei Zheng1, Kuocheng Shen1, Dan Yu1, and Zhimin Cao1

Populus tomentosa, Populus deltoides Marsh and Populus purdomii Rehd are three typical poplar 

which show different resistant type to Melampsora larici-populina. Leaf surface waxes were extracted 

by chloroform, and detected by gas chromatography-mass spectrometry (GC-MC), scanning electronic 

microscope (SEM). Leaves of Populus tomentosa fixed the highest wax content of 5.6628μg · cm-2, 

followed by Populus deltoides 4.1371μg · cm-2, and then Populus purdomii Rehd with 1.6667μg .cm-2, 

which conformed to the thickness under the SEM and the phenotype of rust resistant poplars. Alkanes, 

alcohols, aldehydes, fatty acids, esters and a small amount of some phenols, ethers, ketones, amides 

substances are the main composition of leave surface wax. Among them, C12 and C15 phenols, C8 and 

C18 amides, are both directly correlated with poplar tree rust resistance. Additionally, amides show higher 

rights than phenol substances for evaluation of poplar rust resistance. Wax content ratio is an important 

index for evaluating poplar rust resistance, especially the C18 amides ratio can directly be used for 

Melampsora resistance in poplar breeding.

1 Forestry College, Northwest A&F University, Shaanxi Yangling, 712100, China, (yu-10083@163.com).

Citation for proceedings: Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and Diseases: Putting Promise into Practice. 

e-Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of Agriculture Forest Service, Southern Research Station. 170 p.

Nelson, C. Dana; Koch, Jennifer L.; Sniezko, Richard A., eds. 2020. Proceedings of the Sixth International 

Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance to Insects and 

Diseases: Putting Promise into Practice. Gen. Tech. Rep. SRS–252. Asheville, NC: U.S. Department of 

Agriculture Forest Service, Southern Research Station. 170 p.

The Sixth International Workshop on the Genetics of Host-Parasite Interactions in Forestry—Tree Resistance: 

Putting Promise into Practice was held the first week of August 2018 at Deer Creek State Park in Mt. Sterling, 

Ohio. The workshop provided a continuing forum for researchers, tree breeders, and forest managers to focus 

on perhaps the most salient action society can take to restore tree species imperiled by invasive pests and 

pathogens—developing and deploying resistant tree populations. The 95 presentations (oral and poster) covered 

a wide range of tree species and associated pathogens and pests from throughout the world. Ash species (genus 

Fraxinus) throughout Europe and North America face relatively new perils (ash dieback disease and emerald 

ash borer) and a special session was devoted to this genus. Screening for resistance is a fundamental component 

of resistance development and the topic of another full session. Additional presentations detailed efforts to 

develop genomics and biotechnology tools and resources that have the potential to increase the efficiencies 

of applied resistance programs. Other presentations provided inputs on collaborative breeding approaches, 

citizen science in forest health, the potential role of endophytes in managed forests, threats to agroforestry 

species, prioritization of species in need of resistance programs, considerations necessary for the use of new 

biotechnologies, and concepts for integrating genetic, phenotypic and environmental data across host-parasite 

systems. Several presentations provided updates on “the ultimate goal”—progress of applied resistance 

programs and actual restoration efforts—leading into discussions on key topics such as the durability, stability, 

and usability of resistance in forest tree species. Taken together the presentations and discussions provided 

ample evidence that developing resistant populations is a viable (and in some cases necessary) approach for 

society to use to ensure healthy forests for future generations. These proceedings document the presentations 

given as lightly reviewed full papers, extended abstracts, and standard abstracts. In concluding the workshop, 

the attendees approved Spain by acclamation as the host of the Seventh Tree Resistance Workshop to be held in 

2020.

Keywords: Durable resistance, ecosystem restoration, forest health, genetics of resistance, insect and disease 

mitigation, invasive pests and pathogens, resistance breeding. 
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