AN ABSTRACT OF THE DISSERTATION OF Anne B. Haigren for the degree of Doctor of Philosophy in Botany and Plant Pathology, presented on January 24, 2006. Title: Characterization, Epidemiology, and Ecology of a Virus Associated with Black Raspberry Decline. Abstract .aprud: Redacted for privacy R. Martin The objective of this study was to characterize an unknown agent associated with decline in black raspberry (Rubus occidentalis) in Oregon.A virus was found consistently associated with decline symptoms of black raspberries and was named Black raspberry decline associated virus (BRDaV). Double stranded RNA extraction from BRDaV-infected black raspberry revealed the presence of two bands of approximately 8.5 and 7 kilobase pairs, which were cloned and sequenced. The complete nucleotide sequences of RNA 1 and RNA 2 are 7581 nt and 6364 nt, respectively, excludingthe 3' poly(A) tails.The genome structure was identical to that of Strawberry mottle virus (SMoV), with the putative polyproteins being less than 50% identical to that of SMoV and other related sequenced viruses. The final 189 amino acids of the RNA-dependent- RNA-polymerase (RdRp) reveal an unusual indel with homology to A1kB-like protein domains, suggesting a role in repair of alkylation damage. This is the first report of a virus outside the Flexiviridae and ampeloviruses of the Closteroviridae to contain these domains. An RT-PCR test was designed for the detection of BRDaV from Rubus tissue. BRDaV is vectored non-persistently by the large raspberry aphid Amphorophora agathonica, the green peach aphid Mvzus persicae, and likely nonspecifically by other aphid species.Phylogenetic analysis of conserved motifs of the RdRp, helicase, and protease regions indicate that BRDaV belongs to the Sadwavirus genus. To assess the rate of spread BRDaV, four newly planted fields of black raspberries (Rubusoccidentalis)in Oregon were studied for three years. In an effort to characterize the suspected complexity of synergistic interactions between BRDaV and other Rubus-infecting viruses, the prevalence of ten additional Rubus viruses was also monitored in the study fields.The timing of BRDaV infection as it relates to aphid populations and flights was also determined. Testing of nearby vegetation identified several symptomless Rubus hosts of BRDaV, as well as detection in multiple cultivars of black raspberry and several non-Rubus weeds.It was determined that BRDaV spreads rapidly with a low aphid threshold and consistently is associated with decline of black raspberries in Oregon. © Copyright by Anne B. Haigren January 24, 2006 All Rights Reserved Characterization, Epidemiology, and Ecology of a Virus Associated with Black Raspberry Decline by Anne B. Haigren A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented January 24, 2006 Commencement June 2006 Doctor of Philosophy dissertation of Anne B. Haigren presented on January 24. 2006. APPROVED: Redacted for privacy or Professor, representing Botany and Plant Pathology Redacted for privacy Chair of the Depftment çBotany and Plant Pathology Redacted for privacy Dean of tl* fIiadji'ate School I understand that my dissertation will becomepartof the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request. Redacted for privacy Anne B. Hahen. Author ACKNOWLEDGEMENTS I extend my utmost appreciation to my major professor, Dr. Bob Martin, for his patience, support, and enthusiasm, and for teaching me the values of perseverance and independent thinking. I thank my committee members Dr. Valerian Dolja, Dr. Tom Wolpert, Dr. Jennifer Parke, and Dr. Kate Field for their ideas, feedback, and genuine interest in my project. The Oregon black raspberry growers made this research possible by allowing me to collect data in their fields. The financial support of the Northwest Center for Small Fruits Research and the Oregon Raspberry and Blackberry Commission also made this work possible. Financial support was also provided by the USDA-ARS, an NSF GK-12 Rural Science Education Program Fellowship, and two graduate teaching assistantships. I thank the members of the Martin lab for supporting me so strongly. Special thanks go to Dr. loannis Tzanetakis, for sharing daily struggles and insights, and for challenging me to think more rigorously about the scope of my project, and Dr. Jennifer Kraus, who has extended to me her scientific wisdom and shown me the value of really listening. I extend a big thank you to the whole crew at the USDA ARS Horticultural Crops Research Lab for their daily support and humor, and to the BPP office staff for keeping my program running smoothly. I thank those who have made it possible for me to get to where I am: my professors at Bucknell University who encouraged my scientific development, my professors at Oregon State University for keeping the spark of excitement alive, my parents Nancy and Don for encouraging me to pursue higher education and supporting the decisions I have made, the BPP grad students and especially my good friend Rachael for being my life raft in the sea of grad school, my dogs for being unconditionally happy to see me every day, and my husband and greatest asset Jason McKerr for keeping me looking forward. TABLE OF CONTENTS gç Chapter 1. INTRODUCTION AND LITERATURE REVIEW..................... 1 Raspberry history.................................................................. 1 Black raspberry botany............................................................ 1 Black raspberry production systems in Oregon................................2 Black raspberry viruses............................................................3 Major aphid-transmitted viruses..................................................4 Minor aphid-transmitted viruses..................................................7 Nematode-transmitted viruses.....................................................8 European nematode-transmitted viruses................................8 North American nematode-transmitted viruses........................9 Pollen-borne viruses................................................................10 Major pollen-borne viruses...............................................11 Minor pollen-borne viruses...............................................12 Viruses with unknown vectors.....................................................13 Insects as vectors....................................................................17 Biology of Amphorop ho ra.........................................................19 Strategies forRubusvirus control.................................................20 Sadwavirus review..................................................................22 Overview of Dissertation ............................................................ 24 References............................................................................24 Chapter 2. IDENTIFICATION, CHARACTERIZATION, AND DETECTION OF A VIRUS ASSOCIATED WITH DECLINE SYMPTOMS IN BLACK RASPBERRY................................................................................31 ABSTRACT.........................................................................31 INTRODUCTION..................................................................32 MATERIALS AND METHODS.................................................33 Test plants and virus isolates.............................................33 Mechanical transmissions................................................34 TABLE OF CONTENTS (Continued) gç Aphid transmissions . 34 Virus purification..........................................................35 Isolation of dsRNA.........................................................36 cDNA synthesis and cloning.............................................37 RT-PCR detection.........................................................38 PCR and RACE............................................................40 Sequencing and genome analysis........................................40 Coat protein expression...................................................42 RESULTS............................................................................43 Hostrange...................................................................43 Aphid transmissions........................................................44 Properties of virions........................................................45 Protein purification.........................................................46 Cloning from dsRNA......................................................48 Nucleotide sequence and phylogenetic analysis.......................50 Non-coding regions..............................................50 RNA1.............................................................50 RNA2.............................................................58 DISCUSSION...................................................................... 61 ACKNOWLEDGEMENTS......................................................65 REFERENCES.....................................................................65 Chapter 3. EPIDEMIOLOGY AND ECOLOGY OF BLACK RASPBERRY DECLINE ASSOCIATED VIRUS.......................................................70 ABSTRACT........................................................................70 INTRODUCTION..................................................................70 MATERIALS AND METHODS.................................................75