AN ABSTRACT OF THE DISSERTATION OF Diego F. Quito-Avila for the degree of Doctor of Philosophy in Botany and Plant Pathology presented on November 21, 2011 Title: Impact of Raspberry bushy dwarf virus, Raspberry leaf mottle virus, and Raspberry latent virus on Plant Growth and Fruit Crumbliness in Red Raspberry (Rubus idaeus L.) ‘Meeker’ Abstract approved: _____________________________________________________________________ Robert R. Martin The United States is the third-largest producer of raspberries in the world. Washington State leads the nation in red raspberry (Rubus idaeus L.) production. ‘Meeker’, the most grown red raspberry cultivar in the Pacific Northwest (Washington, Oregon and British Columbia, Canada) is highly susceptible to Raspberry crumbly fruit, a virus- induced disease that produces drupelet abortion and reduces fruit quality and yield. The disease has long been attributed to Raspberry bushy dwarf virus (RBDV), a pollen-and-seed transmitted virus found in most commercial raspberry fields around the world. In recent years, an increased severity of crumbly fruit was observed in areas where two additional viruses were common. One of these viruses, Raspberry leaf mottle virus (RLMV), was characterized recently and shown to be a novel closterovirus transmitted by the large raspberry aphid Amphorophora agathonica Hottes. The second virus, Raspberry latent virus (RpLV) was a tentative member of the family Reoviridae whose characterization remained to be completed. To investigate the role of these two new viruses in the crumbly fruit disorder, ‘Meeker’ raspberry infected with single or mixtures of the three viruses, in all possible combinations, were generated by graft inoculation. Eight treatments, including a virus- free control, were planted in the field at the Northwestern Research and Extension Center in Mt. Vernon, WA. Plant growth and fruit crumbliness were evaluated during establishment and the second year. Simultaneously, the characterization of RpLV, at the genetic and biological level, was completed. RpLV is a novel member of the plant Reoviridae composed of 26,128 nucleotides divided into 10 genomic dsRNA segments. Analysis of the RNA dependent RNA polymerase (RdRP) indicated that RpLV was related most closely to members of the genus Oryzavirus. However, the genomic terminal regions, conserved at the genus level in reoviruses, did not show homology to those of oryzaviruses, suggesting that RpLV may be a member of a new genus. It was found later, that RpLV was transmitted by A. agathonica. As all other plant reoviruses are transmitted by leaf- and planthoppers, these findings strongly support the creation of a new genus in the plant Reoviridae for the classification of RpLV. Real-time quantification of viral titers in single or mixed infections revealed that the titer of RBDV was enhanced ~400-fold when the virus was found in co- infections with RLMV. Interestingly, plants co-infected with these two viruses showed significant reduction in plant growth during the establishment and second year. Crumbly fruit was more severe in plants infected with the three viruses compared to the virus-free control. Taken together, this work presents valuable information about the interactions between three important raspberry viruses and their effect on plant growth and fruit crumbliness in ‘Meeker’, the most important red raspberry cultivar in the Pacific Northwest. Impact of Raspberry bushy dwarf virus, Raspberry leaf mottle virus, and Raspberry latent virus on Plant Growth and Fruit Crumbliness in Red Raspberry (Rubus idaeus L.) ‘Meeker’ by Diego F. Quito-Avila A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented November 21, 2011 Commencement June 2012 Doctor of Philosophy dissertation of Diego F. Quito-Avila presented on November 21, 2011. APPROVED: _______________________________________________________________ Major Professor, representing Botany and Plant Pathology _______________________________________________________________ Chair 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. _______________________________________________________________ Diego F. Quito-Avila, Author ACKNOWLEDGMENTS It would take me a document, perhaps longer than this dissertation itself, to list all the great people that have been influential in the completion of this work. First, I would like to thank Dr. Robert Martin for giving me the opportunity to undertake this important project; his unconditional support, guidance, and infinite optimism have made the execution of this project an ecxiting journey. I would also like to thank my three “foster moms” Karen Keller, Nola Mosier and Ruth Price, for being always there for me. Karen’s discipline, Nola’s kindness and Ruth’s sweetness have been key ingredients for my endurance during these four years away from home. I also want to express my appreciation to all the undergraduates in the Martin’ lab that one way or another contributed with the development of this project. My especial gratitude goes to my good friend and collegue Danielle Lightle for all her support, charisma, and kindness. Dani’s awesomeness made every trip to Washignton an exciting and fun adventure. I would also like to thank Ioannis Tzanetakis for guidance and support during these four years. His remarkable achievements kept me motivated and enthusiastic during my entire graduate program. I would like to express my gratitude to all the folks at the USDA-ARS, Horticultiral Crops Research Lab, especially Chad Finn, Michael Dossett, Nik Grunwald, Joyce Loper, Walt Mahaffee, Jana Lee, Paul Charron, Pete Moppin, Kenny, Ted Mackey, Mary Peterson, Brenda Shaffer, Sierra Hartney, Stefani Morgan, Maryann Resendes, Ruth Hamlyn, and my friend Phillip Ortiz (Phil) whose fun stories (told during his breaks) would keep me from falling asleep on my lab bench after midnight. I would like to thank Dr. Valerian Dolja, Jay Psheidt, Walt Ream and Chad Finn, for their valuable time and willingness to be part of my committee. I could not forget the folks at the Northwestern Research and Extension Center in Mt. Vernon Washington, particularly Gary Moulton and Tom Walters for their unconditional support in the execution of the field work. I would also like to thank my dear friends Jacob de Soto and James Ruff. Without Jacob’s decision to give me the opportunity to come to the United States for the first time, I would not be writing these lines now. Jim’s continuous support as friend and English tutor, allowed me to obtain those high TOEFL and GRE scores I needed to be accepted in Graduate School. Thanks to all my friends from the Association of Latin American Students, of OSU; their company, encouragement and support were essential during these last years. I wish I had the space to list all other people whose support was vital at some point during this stage of my life. Please forgive me if did not mention you. Last but not least, my especial gratitude to my brothers and sisters for their fundamental support, encouragement and, most importantly, for taking care of mom during my absence. CONTRIBUTION OF AUTHORS Dr. Robert Martin supervised and assisted with the overall project design, planning and execution. Drs. Ioannis Tzanetakis, Wilhem Jelkmann, and Karen Keller oversaw and assisted in the genetic characterization of RpLV (Chapter 2). Danielle Lightle performed the EPG experiments (Chapter 4) and assisted in the collection and statistical analysis of field data (Chapter 5). Dr. Tzanetakis helped editing and proofreading Chapters 3 and 4. All co-authors helped to edit and have approved the final version of the chapters with which they are associated. TABLE OF CONTENTS Page Chapter 1: Introduction …………………………..……….…………………………...1 Chapter 2: Complete Sequence and Genetic Characterization of Raspberry latent virus, a Novel Member of the Family Reoviridae …...…...…………………………..6 Abstract ……..……………...…………………………………………..………7 Introduction .. ………..…………………………………………………………8 Materials and Methods .…………………...…………………………………....9 Type Isolate Source .… ..……………………….………………9 Detection …………………………………….………………. 10 Double-stranded RNA Extraction ………………………..……10 Electrophoretic Analysis ……………………….………......….11 Genome Assembly……………………….………………….…12 Sequence Comparison and Phylogeny ……………………...…13 Results ………………...……………………………………………...13 Discussion ………………………………….…………………………...…..18 References ……………….………………………………………………….23 Chapter 3: Real-time RT-PCR for Detection of Raspberry bushy dwarf virus, Raspberry leaf mottle virus and Characterizing Synergistic Interactions in Mixed Infections……………...………………………………………………………………37 Abstract ………………………………………………….…………...……. 38 Introduction …………………………….………………….…………….….38 TABLE OF CONTENTS (Continued) Page Materials and Methods ……………….……………………………………..41 Virus Isolates and Plant Material………………………………….…….41 Primers and TaqMan Probes ………………………………………….42 Sensitivity and Efficiency Assays ……..……………………………….46 Comparison of Assays with Conventional Detection Methods…………45 Results………………………………………………………………………..46 Discussion ……………………………………………………………...…....58 References …...….…………………………………………………………...50 Chapter 4: Transmission Biology of Raspberry latent virus, a Plant Reovirus