Metabolomics of Quercus spp. to understand and predict resistance to Phytophthora ramorum DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Anna Olivia Conrad Graduate Program in Plant Pathology The Ohio State University 2015 Dissertation Committee: Pierluigi Bonello, Advisor Laurence V. Madden P. Larry Phelan Thomas K. Mitchell 1 Copyrighted by Anna Olivia Conrad 2015 2 ABSTRACT Sudden oak death (SOD) is a devastating disease of oak and tanoak in the western United States, caused by the non-native, generalist pathogen Phytophthora ramorum Werres et al. Quercus agrifolia Née (CLO—coast live oak) is a highly susceptible host of P. ramorum in California forests and Quercus rubra L. (NRO—northern red oak) is an important Eastern forest species that is also susceptible to P. ramorum. CLO naturally resistant to P. ramorum have been observed and include trees that fail to develop symptoms of infection or appear to recover after symptom development. Variability in CLO susceptibility has been associated with variation in the concentration of certain phenolic compounds produced in CLO phloem tissue, and putative phenolic biomarkers of resistance were identified from trees already infected with P. ramorum. However, the association between constitutive (i.e. pre-infection) levels of phenolics in naïve CLO and variation in host susceptibility has not been examined, and little is known about the relationship between NRO susceptibility and variation in phenolic levels. This research aimed to elucidate chemical mechanisms of resistance and identify factors that may affect the production of phenolic defenses in susceptible Quercus spp., including CLO and NRO, before and after infection with P. ramorum. Time was a significant explanatory factor for the variability of certain phenolics, both within and between years, though overall variation was low and did not appear to be related to ii variability in CLO susceptibility to P. ramorum. NRO treated with a known, phosphonate-based elicitor of host defenses, were more resistant to P. ramorum, and both constitutive and induced levels of certain phenolics were also significantly impacted. Additionally, new approaches for identifying naturally resistant CLO based solely on constitutive concentrations of phenolics were tested. Four phenolic biomarkers of resistance were partially identified in CLO phloem collected before P. ramorum infection and were used to estimate the probability of CLO resistance and survival following infection with P. ramorum. Finally, this research demonstrated that Fourier-transform infrared (FT-IR) spectroscopy, combined with multivariate statistical analysis, can be used to distinguish between resistant and susceptible CLO prior to infection. The ability to identify resistant trees within forest stands could be a valuable tool for conserving and breeding resistant germplasm and identifying areas at risk of SOD. Moreover, the approaches utilized in these studies to identify resistant trees may be useful in other systems where forest pests and pathogens are of concern. iii DEDICATION In memory of my mother, Marcia Rees Conrad, whose unconditional love and endless support made everything possible. iv ACKNOWLEDGEMENTS First and foremost, I would like to thank my advisor Pierluigi (Enrico) Bonello for his encouragement and guidance throughout my Ph.D. program. He challenged me to think critically and gave me the freedom to pursue topics that interested me, and without his support I would have been unable to participate in many unique educational and professional experiences. I would also like to thank the remaining members of my advisory committee: Larry Madden, Tom Mitchell, Larry Phelan, and former member Terry Graham. They provided constructive feedback during critical stages of my research, and each provided me with specific knowledge related to their specialties. Many thanks those who collaborated with me including: Brice McPherson and David Wood from UC Berkeley; Luis Rodriguez-Saona from the Department of Food Science and Technology; Karen Suslow, Wolfgang Schweigkofler, and Vernon Huffman from the National Ornamental Research Site at Dominican University of California, and Sylvia Mori (USDA Forest Service) and Stephen Opiyo (MCIC) who advised me on statistics. A special thanks to Brice and David who made me feel at home during my many trips to California. They were also instrumental in the establishment of the Briones site, and collected hundreds of oak phloem samples for my research over the last five years. v Special thanks also to members of the Bonello Lab: Amy Hill was instrumental in processing samples from NORS-DUC and Briones studies; Patrick Sherwood, along with former members Sourav Chakraborty and Justin Whitehill, taught me the basics of liquid chromatography and mass spectrometry, and David Showalter and Caterina Villari provided helpful insights on various topics. Finally, none of this would have been possible without the support of my family and friends, in particular my sister, Rachel Conrad, and my boyfriend, Jong Hyun Park. vi VITA 1988................................................................Born, Columbus, OH 2006 ...............................................................Graduated Thomas Worthington High School, Worthington, OH 2010 ...............................................................B.S. Environmental Biology, SUNY College of Environmental Science and Forestry, Syracuse, NY 2010 to present ..............................................Graduate Research Associate, Department of Plant Pathology, The Ohio State University 2013 ...............................................................M.S. (non-thesis), Department of Plant Pathology, The Ohio State University 2013 to present ..............................................Ph.D. Candidate, Department of Plant Pathology, The Ohio State University PUBLICATIONS Conrad, A.O., Rodriguez-Saona, L.E., McPherson, B.A., Wood, D.L., Bonello, P., 2014. Identification of Quercus agrifolia (coast live oak) resistant to the invasive pathogen Phytophthora ramorum in native stands using Fourier-transform infrared (FT-IR) spectroscopy. Front. Plant Sci. 5, 521. vii Conrad, A.O., Segraves, K.A., 2013. Mycorrhizal colonization of Palafoxia feayi (Asteraceae) in a pyrogenic ecosystem. Mycorrhiza 23, 243–249. McPherson, B.A., Mori, S.R., Opiyo, S.O., Conrad, A.O., Wood, D.L., Bonello, P., 2014. Association between resistance to an introduced invasive pathogen and phenolic compounds that may serve as biomarkers in native oaks. For. Ecol. Manage. 312, 154–160. FIELDS OF STUDY Major Field: Plant Pathology viii TABLE OF CONTENTS Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgements ............................................................................................................. v Vita .................................................................................................................................... vii List of Tables .................................................................................................................... xv List of Figures .................................................................................................................. xix Chapter 1: Literature review ............................................................................................... 1 Generalized defense mechanisms in trees ........................................................................... 1 Physical defenses ......................................................................................................... 2 Chemical defenses ....................................................................................................... 4 Defense related proteins and enzymes .................................................................... 5 Plant specialized metabolites (PSMs) ..................................................................... 7 Phenolics in tree defense ................................................................................................... 10 Phenolics in Quercus spp. defense ............................................................................ 12 Sudden oak death .............................................................................................................. 17 Hypothesized origin of P. ramorum .......................................................................... 18 ix Phytophthora ramorum ............................................................................................. 19 Host and associated species of P. ramorum .............................................................. 21 SOD in Q. agrifolia and other true oaks .................................................................... 23 Q. agrifolia susceptibility .......................................................................................... 24 SOD management ...................................................................................................... 26 New approaches to identify resistant trees for use in disease management .............. 28 Research goals and objectives .......................................................................................... 30 Chapter