AN ABSTRACT OF THE DISSERTATION OF Kelsey Liann Dunnell for the degree of Doctor of Philosophy in Botany and Plant Pathology presented on May 19, 2016. Title: Understanding Host-Pathogen Interactions in the Sphaerulina musiva- Populus Pathosystem Abstract approved: ______________________________________________________ Jared M. LeBoldus Host-parasite interactions between the pathogen, Sphaerulina musiva, and its host, Populus spp., were examined in a series of studies. In the first study, variation in resistance of Populus nigra from 7 locations in Europe was differentiated based on the parameters cankers per cm and disease severity score. The analysis evaluated if location, isolate, genotype, and their interactions significantly affected variation in these parameters. In addition, the correlation in disease severity score between single isolate and bulk isolate inoculations was examined. Location (72%; P < 0.001) explained the majority of variation in resistance for cankers per cm followed by genotype(location) (28%; P < 0.001). In contrast, genotype(location) (51%; P < 0.001) explained the majority of variation in disease severity score followed by location (26%; P = 0.025). A significant isolate effect (P = 0.034) and genotype(location)*isolate interaction (P = 0.004) were also present in the disease severity score model. When correlating single isolate to bulk isolate inoculations the correlation coefficients for disease severity score indicated a significant range of correlations (r = 0.871 to r = 0.952) with significant P-values (P < 0.05). The second study determined whether or not there is a correlation between resistance to stem infection and resistance to leaf infection. The results indicated no correlation between leaf spot severity and stem canker severity (r = 0.452; P = 0.068). Older leaves showed significantly lower disease severity compared to younger leaves (leaf 1 vs. leaf 5) when averaged across all genotypes in both experiments. The lack of a consistent correlation among leaves and stems may have implications for disease management and deployment of resistant genotypes and suggests that there may be different mechanisms controlling resistance in the stem compared to the leaves. The third study examined the genetic basis of the interaction between S. musiva and P. trichocarpa. RNA-seq was used to identify potential S. musiva effectors involved in stem canker formation at 2 and 3 weeks post-inoculation. The total reads at 2 weeks were 4,198,267; at 3 weeks reads increased to 4,915,453 compared to 3,125,004 for the control. Approximately 20% of the total reads were aligned to the S. musiva reference genome and the remaining reads were aligned to P. trichocarpa. Analysis of the fungal reads identified 70 genes at 2 weeks post-inoculation and 110 genes at 3 weeks post-inoculation that were differentially expressed between the inoculated trees and the control. The most highly expressed gene at 2 weeks and second at 3 weeks had homology to Extracellular protein 2 (Ecp2) produced by Cladosporium fulvum. Sphaerulina musiva Ecp2 (SmEcp2) was infiltrated into P. trichocarpa leaves and caused necrosis on some susceptible genotypes. ©Copyright by Kelsey Liann Dunnell May 19, 2016 All Rights Reserved Understanding Host-Pathogen Interactions in the Sphaerulina musiva-Populus Pathosystem by Kelsey Liann Dunnell A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented May 19, 2016 Commencement June 2016 Doctor of Philosophy dissertation of Kelsey Liann Dunnell presented on May 19, 2016 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. Kelsey Liann Dunnell, Author ACKNOWLEDGEMENTS I would first like to thank my advisor, Dr. Jared LeBoldus, for accepting me as his graduate student, hiring me as a technician, and allowing me to transfer to Oregon State University! I could not have completed my PhD without your superb mentorship. I feel very fortunate to have been able to come to Oregon to finish my degree and I am very grateful for your support and guidance through this endeavor. Thank you so much for everything. I express sincere thanks to my committee members at North Dakota State University: Dr. Jack Rasmussen, Dr. Julie Pasche, and Dr. Todd West and to my committee members at Oregon State University: Dr. Thomas Wolpert, Dr. Kenneth Johnson, Dr. David Shaw, and Dr. Daniel Luoma. This research could not have been accomplished without the help of numerous people at North Dakota State University including Jim Walla, Achala Nepal, Ruqian Qin, Kelsie Mettler, and Jon Richards. I want to especially thank Dr. Robert Brueggeman, who helped me through much of my RNA-seq project, and Danielle Holmes, who provided guidance through the protein expression part of my project. This research could not have been completed without funding from the United States Department of Agriculture National Institute of Food and Agriculture USDA- NIFA-RIPM 2012-34103-19771 grant and the United State Department of Energy, Sun Grant Initiative Regional Biomass Feedstock Partnership through a subcontract from South Dakota State University. A Graduate Research Assistantship was provided from North Dakota State University-Agriculture Experiment Station and from Oregon State University. Finally, I would like to thank my husband, Josh, for providing support through this crazy endeavor and for agreeing to move to Oregon with me! TABLE OF CONTENTS Page Chapter 1 Introduction ..…………...........…………………………………………… 1 The Genus Populus…………………………………………………………... 1 Hybrid Poplar ………………………………………….…………………...…2 Diseases of Poplar……………………………………………………………..2 Sphaerulina musiva…………………………………………………………....3 History of Sphaerulina musiva…………………………………...…...3 Symptoms and Signs…………………………………………………..4 Disease Cycle……………………………………………………….....5 Phylogeny…………………………………………………………..…5 Poplar Production Impacts……………………………………………………6 Managing Septoria Canker……………………………………………………7 Host-Parasite Interactions..................................................................................8 Research Objectives………………………………………………….………..9 References ………………………………………………………….……......11 Chapter 2. Variation in resistance of Populus nigra to Sphaerulina musiva in the north central US…………………………………………………………………...…17 Abstract……………………………………………………………………....18 Introduction…………………………………………………………………..18 Materials and Methods ………………………………………………………20 Experiment 1………………………………………………………....20 Poplar Culture …………………………………………...…...20 TABLE OF CONTENTS (Continued) Page Pathogen Culture……………………………………….…….21 Inoculation…………………………………………………...22 Experimental Design…………………………………………22 Data Analysis……………………………………………..….22 Experiment 2 ……………………………………………………...…23 Results………………………………………………………………………..24 Experiment 1…………………………………………………………24 Cankers per cm………………………………………………24 Disease Severity Score………………………………………24 Experiment 2…………………………………………………………25 Discussion……………………………………………………………………25 References……………………………………………………………………28 Chapter 3. The Correlation between Leaf Spot Resistance and Stem Canker Resistance……………………………………………………………………...…….37 Abstract…………………………………………………………………........37 Introduction…………………………………………………………..............37 Materials and Methods……………………………………………………….39 Pathogen Propagation………………………………………………..39 Host Propagation……………………………………………………..39 Inoculation…………………………………………………………...40 Experimental Design…………………………………………………41 Data Analysis………………………………………………………...41 TABLE OF CONTENTS (Continued) Page Results…………………………………………………………………...…..42 Discussion...……………………………………………………….……..…..43 References…………………………………………………………….….…..47 Chapter 4. Identifying a putative necrotrophic effector in the Sphaerulina musiva- Populus trichocarpa pathosystem using RNA-seq, in vitro expression, and infiltration……………………………………………………………………………55 Abstract………………………………………………………….…….……..55 Introduction……………………………………..……………………………55 Materials and Methods…………………………..…………………………..56 Pathogen Culture…………………………………………………….56 Host Propagation…………………………………………………….57 Inoculations, mRNA Isolation, and Library Preparation for RNA- seq……………………………………………………………………57 Data Analysis……………………………………………………..….58 Transformation and Protein Expression………………………….….59 Infiltration Experiments………………………………………….…..60 Results………………………………………………………………….…….60 Transcriptome Analysis……………………………...………………60 Infiltrations…………………………………………...………………61 Discussion……………………………………………………………………61 References………………………………………………………….………...65 Chapter 5. Conclusion……………………………………………………………….76 Summary……………………………………..………………………..……..76 TABLE OF CONTENTS (Continued) Page Implications for Management……………………………………...………...77 Future Research……………………………………...……………………....78 Bibliography………………………………………………………………………....80 Appendix… ………………………………………………………………………….88 LIST OF FIGURES Figure Page 1.1 Leaf spots caused by Sphaerulina musiva on a Populus nigra leaf with a close-up of a leaf spot containing pycnidia…..……………..…………………………..…15 1.2 Stem canker containing pycnidia caused by Sphaerulina musiva on a Populus nigra stem.…….………………………………………………………………....15 1.3 Sphaerulina musiva in culture on KV-8 medium. The pink spore tendrils of conidia are oozing out of the pycnidia. ……………………………..............…...16 2.1 Map of open pollinated Populus