AN ABSTRACT OF THE THESIS OF Hazel A. Daniels for the degree of Master of Science in Sustainable Forest Management presented on September 19, 2017. Title: Phylogenetic Identification of Pathogenic and Endophytic Fungal Populations in West Coast Douglas-fir (Pseudotsuga menziesii) Foliage. Abstract approved: _____________________________________________________ James D. Kiser Douglas-fir provides social, economic, and ecological benefits in the Pacific Northwest (PNW). In addition to timber, forests support abundant plant and animal biodiversity and provide socioeconomic viability for many rural communities. Products derived from Douglas- fir account for approximately 17% of the U.S. lumber output with an estimated value of $1.9 billion dollars. Employment related to wood production accounts for approximately 61,000 jobs in Oregon. Timberland also supports water resources, recreation, and wildlife habitat. Minor defoliation has previously been linked to Swiss Needle Cast, associated with the fungus Phaeocryptopus gaeumannii, however, unprecedented large-scale defoliation began in the 1990s and has increased since, leading to decreased growth and yield. Areas affected areas by SNC exceed 500,000 acres in Oregon. Defoliation symptoms are inconsistent with predicted effects of P. gaeumannii, and targeted chemical control has had mixed results. While the microbiome community of conifer needles is poorly described to date, we hypothesize the full interstitial microbiome complex is involved in disease response in conifers. There are at least three known pathogenic endophytes of Douglas-fir, in addition to a large number of endophytes with undetermined host relationships. In order to understand the mechanistic dynamics of needle cast, it is necessary to uncover the underlying cause of the symptoms. The first step is establishing a baseline inventory of endophytes in PNW Douglas-fir needles. Two-year old needles were collected from two sites each in Oregon and Washington, for a total of four sites. Study sites varied in elevation (185 – 860m) and mean annual precipitation (250 – 1,575 mm/year). DNA extraction and sequencing revealed 46 unique isolates from 39 taxa in Douglas-fir needles gathered from four test sites in Oregon and Washington. Rates of infection for all needles was 39%, with infection ranging from 5% to 60% among seed sources. The results suggest that the probability of endophyte occurrence at the cool, wet site, was 2.8 (p < 0.0001) times higher than the warm, dry site, and 2.2 (p = 0.0007) times higher than the warm, wet site. Non-local needles at the warm, dry site were 13.6 (p < 0.0001) times more likely to be infected than needles from the local seed source. The environmental variable that seemed most positively correlated with endophyte presence was winter relative humidity. There was strong evidence for the influence of seed source type on the relationship between continentality (the difference between mean warmest month and mean coldest month temperatures) and mean average number of endophytes. Long-term silvicultural management of PNW forests will benefit as a result of this study. It is the first study to use modern molecular techniques to uncover the fungal endophytic communities of West-Coast Douglas-fir foliage. It is vital to understand the complete etiology of the needle cast disease affecting Douglas-fir, and whether to focus on mitigating a single-species pathogen, or a number of different pathogens. Because forests are a long-term strategy, it is critical to understand the ecological implications of disease in this important tree species. ©Copyright by Hazel A. Daniels September 19, 2017 All Rights Reserved Phylogenetic Identification of Pathogenic and Endophytic Fungal Populations in West Coast Douglas-fir (Pseudotsuga menziesii) Foliage. by Hazel A. Daniels A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented September 19, 2017 Commencement June 2018 Master of Science thesis of Hazel A. Daniels presented on September 19, 2017 APPROVED: Major Professor, representing Sustainable Forest Management Head of the Department of Forest Engineering, Resources & Management Dean of the Graduate School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Hazel A. Daniels, Author ACKNOWLEDGEMENTS There are probably a thousand people who have helped me reach this point in my life; I know I haven’t walked this road alone. Chief among those is Jim Kiser, my advisor and advocate, who has empowered me to think big (and small) and aim high. A good mentor, one whose core values align with your own, and with whom you can be honest in the face of despair, is priceless. For this reason and others, I feel especially fortunate to have Jim on my side. I am also fortunate to have Jeff Morrell, Dave Shaw, and Jay Pscheidt as committee members. Their collective experience, guidance, and helpful questions have made me a better researcher (I hope!). My thanks to Connie Harrington, USFS, for granting me access to the Seed Source Movement Trial sites in my study, and helping me stay warm at the Washington sites. Thanks also to Starker Forests, Inc. for allowing me to access one of these sites on their property, and Chris Poklemba, USFS, for accompanying me to Southern Oregon. Thanks to Jed Cappalazzi for his tireless enthusiasm and goodwill, despite my constant barrage of questions and various spills in the lab. He welcomed me into the Morrell lab, and helped me learn new techniques and keep better records. My sincere appreciation to Amy Klocko, Steve Strauss, Anna Magnusen, and Michael Freitag for allowing me the use of their lab space and hardware. Thanks also to Sarath Vega Gutiérrez, Daniel Way, Daniel Trebelhorn for assistance in DNA extraction, to Lisa Ganio and Ariel Muldoon for statistical advising, to Dave Shaw and Glenn Howe for support and advice, and to Jared LeBoldus and his entire lab group for being so welcoming after I lost my office. I’d especially like to thank the College of Forestry, Department of Forest Engineering, Resources and Management for their support of my research, and Madison Dudley, Erika Hanna, and Chelsea Durling for being absolute rock stars. I thank you for the often- unrecognized administrative and care work that keeps our department running. I am grateful to Jennifer Franklin, Ðenita Hadžiabdić Guerry, and Mark Windham and the University of Tennessee, for seeing and fostering my inner forest pathologist. I dedicate this thesis in part to the memory of Lisa Vito, who kept me company in the lab, taught me how to hold a pipette, and made every day full of laughter. I also dedicate this thesis in part to my mother, Beth Daniels, and aunt, Dianne Muncey. Your love and encouragement have always made me feel invincible. Thanks to my patient and forgiving partner, Josh Goben, who always follows me when I wander into the woods, and to Ashnod and Jem for comforting me in the way only cats can do. I also thank my CGE family, who help keep my spirits high with solidarity, potlucks, and radical conversation. Finally, I gratefully recognize and give thanks to the Kalapuya people, on whose land this university sits. CONTRIBUTION OF AUTHORS Jim Kiser and Hazel Daniels conceived and designed the study. Ariel Muldoon and Connie Harrington helped with some aspects of study design. Hazel Daniels collected, analyzed, and interpreted data, with the assistance of Jim Kiser. Hazel Daniels wrote this thesis, with critical revisions by Jim Kiser. TABLE OF CONTENTS Page 1 General introduction ................................................................................................................ 1 1.1 What are endophytes ............................................................................................... 4 1.2 Endophyte classifications ........................................................................................ 5 1.3 Endophyte infections ............................................................................................... 7 1.4 Other roles of endophytes ....................................................................................... 9 1.5 Endophytes as pathogens ..................................................................................... 12 1.6 Endophytic pathogens of Douglas-fir ................................................................. 13 1.7 Literature cited ........................................................................................................ 23 2 Phylogenetic identification of pathogenic and endophytic fungal populations in West Coast Douglas-fir (Pseudotsuga menziesii) foliage .................................................... 32 2.1 Introduction ............................................................................................................ 32 2.2 Objectives .................................................................................................................. 36 2.3 Methods ................................................................................................................... 37 2.3.1 Traditional plate culturing .................................................................... 41 2.3.2 DNA extraction ...................................................................................... 43 2.3.3 Data Analysis ...........................................................................................