An Abstract of the Dissertation Of
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AN ABSTRACT OF THE DISSERTATION OF Edward Gilman Barge for the degree of Doctor of Philosophy in Botany and Plant Pathology presented on December 13, 2019. Title: Structure and Function of Foliar Fungal Communities of Populus trichocarpa Across its Native Range, Pacific Northwest, USA. Abstract approved: ______________________________________________________ Posy E. Busby Foliar fungi – pathogens, endophytes, epiphytes – form taxonomically diverse communities that affect plant health and productivity. The composition of foliar fungal communities is variable at spatial scales both small (e.g., individual plants) and large (e.g., continents). However, few studies have focused on how environmental factors and host plant traits influence the composition and temporal variability of these communities. Moreover, predicting how nonpathogenic members of these communities affect the plant host remains a challenge. In Chapter two we used ITS metabarcoding to characterize foliar fungal communities of Populus trichocarpa in two consecutive years at the same sites located across its native range in the Pacific Northwest of North America. We used multivariate analyses to test for and differentiate spatial and environmental factors affecting community composition, and tested whether the magnitude of year-to-year variation in community composition varied among environments. We found that climate explained more variation in community composition than geographic distance, although the majority of variation was shared, and that the year-to-year variability of communities depended on the environmental context, with greater variability in the drier sites located east of the Cascade Range. In Chapter three we used ITS metabarcoding and multivariate analyses to test whether the influence of intraspecific host genetic variation on the foliar fungal community diminished over the course of one growing season. We utilized 12 P. trichocarpa genotypes that vary in two functional traits: phenology of bud-burst (early vs. late) and Sphaerulina leaf spot resistance (resistant vs. susceptible). We found that both of these traits drove differences in community composition among trees, but that the strength of the effect diminished through time as the communities converged. Our results suggest that for the leaves of deciduous plants, intraspecific host genetic variability may have its strongest impact on microbial community composition early in assembly. In Chapter four we tested whether Cladosporium endophyte phylogeny can be used to predict endophyte effects on P. trichocarpa leaf rust disease severity caused by Melampsora × columbiana. We used multilocus sequence typing to infer phylogenetic relationships among 96 Cladosporium endophyte isolates collected from wild P. trichocarpa trees throughout its native range. We then conducted a double- inoculation leaf-disk assay (endophyte inoculated first, then rust pathogen) for a subset of 50 Cladosporium isolates to characterize disease modification for the endophyte isolates; data on endophytes parasitizing rust was collected simultaneously for each isolate. We found that Cladosporium phylogeny was a significant predictor of rust disease severity and was also correlated with rust mycoparasitism, demonstrating that fungal endophyte phylogenetic relatedness can help predict differences in endophyte function. ©Copyright by Edward Gilman Barge December 13, 2019 All Rights Reserved Structure and Function of Foliar Fungal Communities of Populus trichocarpa Across its Native Range, Pacific Northwest, USA by Edward Gilman Barge A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented December 13, 2019 Commencement June 2020 Doctor of Philosophy dissertation of Edward Gilman Barge presented on December 13, 2019 APPROVED: Major Professor, representing Botany and Plant Pathology Head of the Department of Botany and Plant Pathology Dean of the Graduate School I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request. Edward Gilman Barge, Author ACKNOWLEDGEMENTS I am indebted to too many people, things, ideas, places to name them all. But, here’s a start. I would like to thank the Botany and Plant Pathology department as a whole for providing a warm, welcoming, and stimulating environment for my dissertation. I would like to thank everyone in both the Busby and Spatafora labs. Thank you Posy Busby for being an excellent advisor and mentor. You were very gracious and helped me grow in so many ways. Shawn Brown helped kick things off. Devin Leopold, Kyle Gervers, Gillian Bergmann and Sabrina Heitmann were always there to chat and trouble-shoot and push me to think about things in different ways. I would like to thank the Stuntz Foundation, Oregon Mycological Society, Cascade Mycological Society, and the Ben Woo Research Grant for funding. I would like to thank Richard Tehan for being a great friend and partner in crime (mushroom hunting). I would like to acknowledge and thank the beaches, forests, mushrooms, and beer of Oregon. Truly a wonderful state. I would like to thank Ekhart Tolle. And last but not least, I would like to thank my family, my mother, father, brother aunt Patty, and my wife, Charissa Bujak for providing unconditional love and support throughout. One Love, Edward G. Barge CONTRIBUTION OF AUTHORS Chapter 2: Posy E. Busby and George Newcombe conceived the idea for the study. Posy E. Busby collected and processed the samples with assistance from Kabir Peay. Devin R. Leopold assisted with data analysis. Posy E. Busby, George Newcombe, Kabir Peay, and Devin R. Leopold advised and edited the manuscript. Chapter 3: Posy E. Busby and Sean P. Brown conceived the idea for the study, assisted with data analysis and edited the manuscript. Chapter 4: Posy E. Busby and Rytas Vilgalys assisted in the design of the study. Alejandro Rojas conducted a portion of the multi-locus sequence typing. Devin R. Leopold assisted with data analysis. Posy E. Busby, Alejandro Rojas, and Devin R. Leopold edited the manuscript. TABLE OF CONTENTS Page Chapter 1: Introduction...……………………………………………………………...1 References……………………………………………………………………..5 Chapter 2: Differentiating spatial from environmental effects on foliar fungal communities of Populus trichocarpa...………………………………………….…… 7 Abstract………………………………………………………………………..8 Introduction………………………………………………………………...….9 Materials and Methods…………………………………………………….…12 Results………………………………………………………………………..18 Discussion……………………………………………………………...…….20 References……………………………………………………………………25 Chapter 3: Intraspecific host genetic effects on the foliar fungal microbiome of Populus trichocarpa diminish through the growing season….....………………...…………...47 Abstract………………………………………………………………………48 Introduction………………………………………….……………………….49 Materials and Methods……………………………………………………….52 Results………………………………………………………………………..57 Discussion……………………………………………………...…………….59 References……………………………………………………………………65 Chapter 4: Phylogenetic relatedness among Cladosporium leaf endophytes predicts their ability to reduce the severity of a poplar leaf rust disease …………....78 Abstract………………………………………………………………………79 TABLE OF CONTENTS (Continued) Page Introduction……..…………………………………………..………………..80 Materials and Methods……………………………………………………….82 Results………………………………………………………………………..90 Discussion……………………………………………………………………92 References……………………………………………………………………99 Chapter 5: Conclusion………………………………………………………………128 LIST OF FIGURES Figure Page Figure 2.1 Map of study sites………………………………….…………..…………29 Figure 2.2 Taxonomic distribution of the 200 most abundant foliar fungal OTUs….30 Figure 2.3 NMDS ordination of foliar fungal community composition and scatterplots of site-level Bray-Curtis dissimilarity vs. geographic distance...…..…...31 Figure 2.4 Associations between OTUs and sites over the two years……………….32 Figure 2.5 Box plots of foliar fungal Shannon diversity and Chao1 richness.......…..33 Figure 2.6 Venn diagram of variation in community composition explained by spatial vs. climatic factors………………………………………….……..………….34 Figure 2.7 Bar graph showing the proportion of variation in community composition explained by year at each site…………………………………………..35 Figure 2.S1 Monthly precipitation and mean temperature at sites in 2013 and 2014……………………………………………………………………………...36 Figure 2.S2 Variation in sequencing depth by year and region…………………...…37 Figure 2.S3 Within-site and within-region betadispersion………………..…………38 Figure 2.S4 PCA plot of abiotic environmental variation among sites…………...…39 Figure 3.1 Verification of leaf phenology and disease resistance phenotypes…………………………………………………………………………...69 Figure 3.2 Convergence in community composition (mean pairwise Bray-Curtis dissimilarity) between disease resistant vs. susceptible trees and between early vs. late phenology trees…………………………………………..70 Figure 3.3 Convergence in community composition (PCoA axes) over time……………………………………………………………………..………71 Figure 3.S1 Map of study sites and genotypes and their associated phenology and disease resistance phenotypes…………………………………………………...72 Figure 3.S2 Variation in community composition (PCoA axes 1–4) over time and between early vs. late phenology trees and between disease resistant vs. susceptible trees……………………………………………………...…73 LIST OF FIGURES (Continued) Figure Page Figure 3.S3 Variation in richness and Shannon diversity over time and between