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ABSTRACT THOMAS, ANNA. Biology, Epidemiology And ABSTRACT THOMAS, ANNA. Biology, Epidemiology and Population Genomics of Pseudoperonospora cubensis, the Causal Agent of Cucurbit Downy Mildew. (Under the direction of Dr. Peter S. Ojiambo and Dr. Ignazio Carbone). Cucurbit Downy Mildew (CDM) caused by the obligate oomycete, Pseudoperonospora cubensis, is economically the most important disease of cucurbits worldwide. During the past two decades, a resurgence of CDM has occurred around the world resulting in severe disease epidemics. In the United States, resurgence of the disease occurred in 2004 and resulted in significant crop losses. Introduction of a new lineage, a new pathotype or new genetic recombinants of the pathogen have been suggested as potential reasons for this resurgence. The potential for sexual reproduction, the current pathotype structure of P. cubensis in the United States, and the role different cucurbit host types and geography play in the shaping the genetic structure of P. cubensis were investigated. Pairing assays involving known mating type tester strains and unknown isolates revealed, for the first time, the presence of both A1 and A2 mating types in the United States. Mating type was found to be significantly (P < 0.0001) associated with host type with isolates collected from cucumber being primarily of the A1 mating type, while those from squash and watermelon being of the A2 mating type. Similarly, a significant (P = 0.0287) association of mating type with geography was also observed. Isolates collected from northern tier states of Michigan, New Jersey, New York and Ohio were all A1, while isolates belonging to either the A1 or A2 mating type were present in equal proportions in southern- tier states of Alabama, Florida, Georgia, North Carolina, South Carolina and Texas. Plasmolysis test showed that about 40% of the oospores produced were viable and this suggests that oospores may play a role in the epidemiology of CDM in the United States. Compatibility assays were conducted using 22 isolates and fifteen host differentials to classify the isolates into pathotypes. Based on the compatibility with the differential host set, five pathotypes (1, 3, 4, 5 and 6) were identified among the 22 isolates. Pathotypes 1 and 3 had not been previously described in the United States and isolates of these two new pathotypes were also compatible with Poinsett 76, a cultivar of C. sativus known to be resistant to CDM prior to 2004. Virulence within the pathogen population was expressed based on virulence factors, virulence phenotypes and virulence complexity. The number of virulence factors ranged from 2 to 8 indicating a complex virulence structure with 77% of the isolates having 5 to 8 virulence factors. Thirteen virulence phenotypes were identified and the mean number of virulence factors per isolate and mean number of virulence factors per virulence phenotype was 5.05 and 5.23, respectively, indicating that complex isolates and phenotypes contributed equally to the complex virulence structure of P. cubensis. Gleason and Shannon indices of diversity were 3.88 and 2.32, respectively and indicated a diverse virulence structure of P. cubensis population within the United States. Comparative whole genomic analysis of nine isolates collected from diverse cucurbit hosts revealed the presence of two distinct evolutionary lineages. In addition, phylogenetic analysis showed that P. cubensis and its sister species, P. humuli, shared a recent common ancestor. A reconstruction of ancestral recombination suggested a hybrid origin of lineage II of P. cubensis, with P. humuli and lineage I of P. cubensis as putative parents. Subsequently, the role of different cucurbit host types in shaping the structure of P. cubensis population was investigated using 93 isolates with the double digest Restriction Associated DNA Sequencing technology. Many fixed polymorphisms throughout the genome separated lineage II that included isolates mainly from Cucumis spp. from lineage I that comprised isolates from Cucurbita spp. and Citrullus lanatus. A distinct genetic cluster within lineage I associated mainly with C. lanatus was also identified. The association of lineage II with new pathotypes and the lack of genetic differentiation among lineage II isolates suggest that lineage II might have been responsible for the resurgence of CDM in the United States. A strong association of mating types with lineages was also observed with all A1 mating type isolates belonging to lineage II and all A2 mating type isolates belonging to lineage I, irrespective of the host of origin. However, the structuring of the pathogen population by geography was not evident and hence, the direction of seasonal migration of P. cubensis could not be inferred. © Copyright 2016 Anna Thomas All Rights Reserved Biology, Epidemiology and Population Genomics of Pseudoperonospora cubensis, the Causal Agent of Cucurbit Downy Mildew by Anna Thomas A Dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Plant Pathology Raleigh, North Carolina 2016 APPROVED BY: _______________________________ _______________________________ Peter Ojiambo Ignazio Carbone Committee Co-Chair Committee Co-Chair _______________________________ _______________________________ Howard Shew Lina Quesada DEDICATION To my family ii BIOGRAPHY Anna Thomas was born and raised in a small village named Marady in the coastal state of Kerala, India. After finishing her high school, she joined the University of Agricultural Sciences, Bangalore, and studied agriculture. Having been brought up by parents who loved farming, Anna soon realized her passion for agriculture. She thoroughly enjoyed school and towards the end of her undergraduate studies developed a strong interest in Plant Pathology. Anna joined the Department of Plant Pathology at University of Georgia for her Masters under the guidance of Drs. Katherine Stevenson and David B. Langston, Jr. and worked on fungicide resistance management of the gummy stem blight pathogen, Didymella bryoniae. Upon completion of her Master’s program in 2011, Anna joined the Department of Plant Pathology at North Carolina State University in fall 2011 to study the population biology of Pseudoperonospora cubensis under the direction of Dr. Peter Ojiambo and Dr. Ignazio Carbone. Her project focused mainly on understanding the possible causes for the resurgence of the disease in the United States in an effort to device better disease management strategies. iii ACKNOWLEDGMENTS I am extremely thankful to many people who have contributed to the completion of my doctoral program. I would like to express my sincere gratitude to my co-advisors Drs. Peter Ojiambo and Ignazio Carbone for their guidance, support and kindness during my graduate education. I also would like to thank my advisory committee members, Dr. David Shew and Dr. Lina Quesada-Ocampo, for their valuable insights and assistance they availed to me during my graduate work. The past and present members of the Ojiambo lab, Lucky Mehra, Wendy Britton, Katie Neufeld, Barbara Stevens, Taylor Ghiloni, Mary Lewis, Mike Adams, Thomas Keever, and Jane Marian Luis, have been great co-workers and friends and would like to thank them for their friendship. I also would like to thank Rakhi Singh for her guidance and help in learning the molecular techniques. I also would like to thank all the collaborators of CDM ipmPIPE program for sending me infected samples without which my project would not have been possible: Drs. Amanda Gevens, David Langston, Anthony Keinath, Steve Rideout, Kate Everts, Andy Wyendant, Margaret McGrath, Mary Hausbeck, Melanie Ivey, Thomas Isakeit, Kenneth Seebold, Dan Egel, Mohammed Babadoost, Sally Miller, Beth Gugino, Steve Bost, Edward Sikora, Shouan Zhang, Gary Vallad, Elaine Roddy, Mahfuzur Rahman, Nathan Kleczewski, Bhabesh Dutta and Emily Pfeufer. Thanks are also due to Drs. Gerald Holmes and Anthony Keinath for very helpful discussions on cucurbit downy mildew. The faculty, staff and fellow graduate students in the department have always been a pleasure to interact with and I would like to thank them all for making my time at NCSU a very memorable one. For their constant encouragement and support, I would like to thank my dear parents and siblings. And I am indebted to my friends Sara Thomas and Nadia Chacko for being there at the other end of the phone whenever I needed them. Last but not iv the least, I would like to thank my husband Eldhose Kilithattil for his everlasting patience, love and support and our little Sarah for brightening my days with her sweetest smile. v TABLE OF CONTENTS LIST OF TABLES ............................................................................................................... viii LIST OF FIGURES ................................................................................................................ x CHAPTER 1 ............................................................................................................................ 1 Literature Review ............................................................................................................... 1 1.0 Importance of cucurbits ........................................................................................... 1 1.1 Cucurbit Downy Mildew .......................................................................................... 2 1.2 History and re-surgence of Cucurbit Downy Mildew ..........................................
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