ASSESSING STATUS OF AND RESISTANCE TO PHYTOPHTHORA ROOT ROT ON TRUE FIR (ABIES SPP.) By KATHLEEN MARY MCKEEVER A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY WASHINGTON STATE UNIVERSITY Department of Plant Pathology DECEMBER 2016 © Copyright by KATHLEEN MARY MCKEEVER, 2016 All Rights Reserved © Copyright by KATHLEEN MARY MCKEEVER, 2016 All Rights Reserved To the Faculty of Washington State University: The members of the Committee appointed to examine the dissertation of KATHLEEN MARY MCKEEVER find it satisfactory and recommend that it be accepted. _____________________________________ Gary A Chastagner, Ph.D., Chair _____________________________________ Dennis A. Johnson, Ph.D. _____________________________________ Ned B. Klopfenstein, Ph.D. _____________________________________ Mark Mazzola, Ph.D. ii ACKNOWLEDGEMENT Thank you Dr. Gary Chastagner, my advisor through two degrees and eight years, for sharing with me your incredible wisdom and interminable curiosity, unequivocal patience and diplomacy, and commitment to scholastic and professional achievement. You’ve provided me with the skills to excel as a pathologist, extension educator, critical thinker, and a burgeoning human being. Thanks to my doctoral committee, Dr. Dennis Johnson (WSU), Dr. Mark Mazzola (USDA ARS), and Dr. Ned Klopfenstein (USDA USFS) for their collaboration, judicious guidance, and thoughtful feedback throughout. This research could never have been completed without the collaboration, selflessness, physical labor, mentorship and kinship of my labmates at WSU Puyallup Research and Extension Center, Katie Coats, Andree DeBauw, Gil Dermott, Dr. Marianne Elliott, Dr. Andrea Garfinkel, Dr. Anna Leon, David McLoughlin, Andy McReynolds, Kathy Riley, Lucy Rollins, Don Sherry, and Carly Thompson. Finally, thank you to Jordan Bowerman, my husband and the most extraordinarily capable, meticulous, and resourceful person I’ve ever met – the logistic escapades that were encountered in the course of this research were conceivable and achievable through the dedication and ingenuity of this man. You merit credit for this degree too. iii ASSESSING STATUS OF AND RESISTANCE TO PHYTOPHTHORA ROOT ROT ON TRUE FIR (ABIES SPP.) Abstract by Kathleen Mary McKeever, Ph.D. Washington State University December 2016 Chair: Gary A Chastagner Many fir species (Abies) are susceptible to Phytophthora Root Rot (PRR). Poor drainage and standing water facilitate pathogen survival, proliferation, and spore dispersal. Disease is caused by numerous Phytophthora spp. that constitute regionally-adapted communities. Survey results of diseased Abies in U.S. Christmas tree farms demonstrated a nearly uniform community profile dominated by P. cambivora on noble fir in the Pacific Northwest, and a more diverse array of Phytophthora species collected from Fraser, Turkish, balsam, and Canaan firs in the Great Lakes and Eastern states. It was concluded that Phytophthora community compositions vary in response to host availability, environment, and anthropogenic distribution of infested host material. Aside from short-term chemical and cultural individual-tree protection, there are few feasible methods for altering the environment to provide perennial disease abatement. An alternative approach is to reform host conduciveness to disease by identifying resistant members of a population through the application of molecular tools. As a precursor to molecular marker iv development, a multifactorial study was performed to characterize host phenotypes in response to multiple species of Phytophthora under variable environmental conditions. It was demonstrated that P. cinnamomi and P. taxon kelmania cause greater root damage and more mortality than P. pini or P. cambivora and that disease is more severe at warmer temperatures. Furthermore, it was established that there is a spectrum of resistances among the various species of Abies, ranging from highly susceptible noble and Fraser firs to more resistant Turkish and Nordmann firs. Previous observations of PRR-resistant Turkish fir plantings have indicated that the ability to resist disease is not uniform among seed sources. To test variability among Turkish fir families, a collection of 36 seed sources was inoculated with five Phytophthora species under saturated field conditions. Chi square analysis indicated that four seed sources were significantly more susceptible; however, mortality of less than 10% in the most susceptible Turkish fir families in comparison to 100% mortality in Fraser and noble fir suggested that Turkish fir is a PRR-resistant Abies species. Significantly more disease caused by P. cryptogea in this field study confirmed previous observations of variability in Phytophthora species aggressiveness. v TABLE OF CONTENTS Page ACKNOWLEDGEMENT ............................................................................................................. iii ABSTRACT ................................................................................................................................... iv LIST OF FIGURES ................................................................................................................................. viii LIST OF TABLES ...........................................................................................................................x INTRODUCTION ...........................................................................................................................1 Literature Cited ................................................................................................................................5 CHAPTER ONE: A SURVEY OF PHYTOPHTHORA SPP. ASSOCIATED WITH ABIES IN U.S. CHRISTMAS TREE FARMS .................................................................................................8 Literature Cited ..............................................................................................................................25 CHAPTER TWO: INTERACTIONS BETWEEN ROOT-ROTTING PHYTOPHTHORA, ABIES, AND ENVIRONMENT.................................................................................................................32 Literature Cited ..............................................................................................................................53 CHAPTER THREE: FIELD ASSESSMENT OF TURKISH FIR (ABIES BORNMUELLERIANA) RESISTANCE TO FIVE ROOT-ROTTING PHYTOPHTHORA SPECIES ........................................................................................................................................57 Literature Cited ..............................................................................................................................77 APPENDIX CHAPTER 1 FIGURES AND TABLES .......................................................................................83 vi CHAPTER 2 FIGURES AND TABLES .......................................................................................87 CHAPTER 3 FIGURES AND TABLES .....................................................................................100 vii LIST OF FIGURES Page CHAPTER ONE Figure 1. Maximum Likelihood tree using rDNA ITS sequences showing phylogenetic relationships between Phytophthora isolates collected during this survey and reference Phytophthora sequences obtained from GenBank .........................................................................83 CHAPTER TWO Figure 1. Mean lesion sizes produced on noble fir seedling stems seven days post-inoculation with various isolates of four Phytophthora species LIST OF TABLES .......................................87 Figure 2. Relationship between log-transformed area under the disease progress curve (AUDPC) values and root rot rating (expressed as the midpoint value of the percentage range) for each host species. ..........................................................................................................................................88 Figure 3. Relationship between log-transformed area under the disease progress curve (AUDPC) values and seedling dry weights (expressed as the percentage of the control seedlings) for each host species. ...................................................................................................................................89 Figure 4. Seedling root system dry weights (expressed as the percentage of the control seedling weights) among host species for each Phytophthora species and isolate treatment. ....................90 Figure 5. Seedling root system dry weights (expressed as the percentage of the control seedling weights) among Phytophthora species for each host species. ......................................................91 Figure 6. Average root rot severity (expressed as the midpoint values of the percentage range) among Phytophthora species for each host species. ......................................................................92 viii Figure 7. Average root rot severity (expressed as the midpoint value of the percentage range) among host species for each Phytophthora species and isolate treatment. ...................................93 Figure 8. Disease progress curves displaying mortality over 16 weeks for seven Abies species exposed to three isolates each of four Phytophthora species at two temperatures. .......................94 Figure 9. In vitro radial growth (mm/day) of three isolates of each of four species of Phytophthora at the two temperatures employed in the greenhouse study ..................................95