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MOLECULAR BIOLOGY and EPIDEMIOLOGY of GRAPEVINE LEAFROLL- ASSOCIATED VIRUSES by BHANU PRIYA DONDA a Dissertation Submitted in Pa

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MOLECULAR BIOLOGY and EPIDEMIOLOGY of GRAPEVINE LEAFROLL- ASSOCIATED VIRUSES by BHANU PRIYA DONDA a Dissertation Submitted in Pa MOLECULAR BIOLOGY AND EPIDEMIOLOGY OF GRAPEVINE LEAFROLL- ASSOCIATED VIRUSES By BHANU PRIYA DONDA A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSPHY WASHINGTON STATE UNIVERSITY Department of Plant Pathology MAY 2016 © Copyright by BHANU PRIYA DONDA, 2016 All Rights Reserved THANKS Bioengineering MAY 2014 © Copyright by BHANU PRIYA DONDA, 2016 All Rights Reserved To the Faculty of Washington State University: The members of the Committee appointed to examine the dissertation of BHANU PRIYA DONDA find it satisfactory and recommend that it be accepted. Naidu A. Rayapati, Ph.D., Chair Dennis A. Johnson, Ph.D. Duroy A. Navarre, Ph.D. George J. Vandemark, Ph.D. Siddarame Gowda, Ph.D. ii ACKNOWLEDGEMENT I would like to express my respect and deepest gratitude towards my advisor and mentor, Dr. Naidu Rayapati. I am truly appreciative of the opportunity to pursue my doctoral degree under his guidance at Washington State University (WSU), a challenging and rewarding experience that I will value the rest of my life. I am thankful to my doctoral committee members: Dr. Dennis Johnson, Dr. George Vandemark, Dr. Roy Navarre and Dr. Siddarame Gowda for helpful advice, encouragement and guidance. I would like to thank Dr. Sandya R Kesoju (USDA-IAREC, Prosser, WA) and Dr. Neil Mc Roberts (University of California, Davis) for their statistical expertise, suggestions and collaborative research on the epidemiology of grapevine leafroll disease. To Dr. Gopinath Kodetham (University of Hyderabad, Hyderabad, India), thank you for believing in me and encouraging me to go the extra mile. I thank Dr. Sridhar Jarugula (Ohio State University Agricultural Research and Development Center, Wooster, University of Ohio, Ohio, USA), Dr. Sudarsana Poojari (Agriculture and Agri-Food Canada, Canada) and Dr. Olfemi Alabi (Texas A&M University, Texas, USA) for help, guidance and motivation during the initial years of my Ph.D. at WSU. To all members of Dr. Rayapati, past and current, I enjoyed learning alongside you and am grateful for your assistance and support. I sincerely thank the faculty, staff and students in the WSU Department of Plant Pathology for their help and support. It is my pleasure to thank all the members of WSU-Irrigated Agriculture Research and Extension Centre for their help during my PhD. I wish to acknowledge the following for supporting my graduate research program: Department of Plant Pathology, WSU Agricultural Research Center, the Wine Advisory iii Committee, the Washington Wine Commission, Washington State Grape & Wine Research Program, Northwest Center for Small Fruits Research, and Altria - Chateau Ste. Michelle Wine Estates. I sincerely acknowledge the following for scholarships and travel awards: Dr. Walter J Clore scholarship from Washington Wine Industry Foundation, 2015-16 American Society for Enology and Viticulture scholarship and Student Travel Award to attend the American Society for Enology and Viticulture annual meeting, June 15-18, 2015, Portland, OR, and Raymond G. Grogan Student Travel Award from the American Phytopathological Society to attend the American Phytopathological Society annual meeting, August 1-5, 2015, Pasadena, CA. I am forever grateful to my parents and my brother for their unconditional love and support, which has always been a source of inspiration for me. Lastly, I would like to thank my husband, Kale Harrison, without his encouragement and support this degree would not have been possible. I thank you for being my anchor. iv MOLECULAR BIOLOGY AND EPIDEMIOLOGY OF GRAPEVINE LEAFROLL- ASSOCIATED VIRUSES Abstract by Bhanu Priya Donda, Ph.D. Washington State University May 2016 Chair: Naidu A. Rayapati Studies were conducted on molecular biology and epidemiology of grapevine leafroll- associated viruses infecting wine grape (Vitis vinifera) cultivars in Washington State. In the first objective, the complete genome sequence of two isolates of Grapevine leafroll-associated virus 1 (GLRaV-1, genus: Ampelovirus, family Closteroviridae) was determined to be 18,731 and 18,946 nucleotides. The genome of GLRaV-1 isolates contain nine open reading frames with long 5’ and 3’ non-translated regions (NTRs). The sequence differences in the 5’NTR was used to develop a restriction fragment length polymorphism assay for distinguishing GLRaV-1 variants in vineyards. Northern blot hybridization revealed the presence of three of the eight putative 3' co-terminal subgenomic (sg) RNAs at higher levels in virus infected grapevine samples. The 5’ termini of five sgRNAs were mapped and their leader sequences determined. The results provided a foundation to further elucidate the comparative molecular biology of grapevine-infecting members of the family Closteroviridae. In the second objective, the spread of grapevine leafroll disease (GLD) was monitored for several seasons in vineyard blocks planted with three red-berried wine grape cultivars. v Grapevines exhibiting GLD symptoms in these blocks were tested positive for Grapevine leafroll-associated virus 3 (GLRaV-3, genus: Ampelovirus, family Closteroviridae). The temporal spread of GLD indicated higher number of symptomatic vines in each season compared to previous seasons, suggesting increased incidence of the disease during successive seasons. The spatial distribution of symptomatic vines in all three blocks indicated a disease gradient in which the highest percentage of symptomatic vines were present in rows closest to old vineyard blocks showing GLD symptoms. Spatial autocorrelation (dependence) analysis indicated random distribution of symptomatic vines during initial years of post-planting suggesting primary spread and clustering of symptomatic vines during subsequent years suggesting secondary spread of GLD. Sequence analysis of a portion of the heat-shock protein 70 homolog gene encoded by GLRaV-3 revealed predominance of one of the several genetic variants of the virus in the three vineyard blocks. These results provided for the first time science-based knowledge on nature of the spread of GLD in young vineyards to pursue site-specific disease management strategies under conditions prevailing in Washington State. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENT ............................................................................................................. iii ABSTRACT….. .............................................................................................................................. v LIST OF TABLES ......................................................................................................................... ix LIST OF FIGURES ........................................................................................................................ x CHAPTER 1. GENERAL INTRODUCTION.......................................................................................... 1 REFERENCES ................................................................................................................ 18 2. SEQUENCE ANALYSIS OF GRAPEVINE LEAFROLL-ASSOCIATED VIRUS 1 ISOLATES FROM WASHINGTON VINEYARDS ...................................................... 34 ABSTRACT ..................................................................................................................... 34 INTRODUCTION ........................................................................................................... 35 MATERIALS AND METHODS ..................................................................................... 38 RESULTS ........................................................................................................................ 48 DISCUSSION .................................................................................................................. 70 ACKNOWLEDGEMENTS ............................................................................................. 77 AFFLIATION OF CO-AUTHORS ................................................................................. 77 AUTHORS' CONTRIBUTIONS ..................................................................................... 77 REFERENCES ................................................................................................................ 78 2. SPATIO-TEMPORAL SPREAD OF GRAPEVINE LEAFROLL-ASSOCIATED VIRUS 3 IN WASHINGTON VINEYARDS ................................................................. 87 ABSTRACT ..................................................................................................................... 87 INTRODUCTION ........................................................................................................... 88 vii MATERIALS AND METHODS ..................................................................................... 91 RESULTS ........................................................................................................................ 97 DISCUSSION ................................................................................................................ 116 ACKNOWLEDGEMENTS ........................................................................................... 121 AFFLIATIONS OF CO-AUTHORS ............................................................................. 121 AUTHORS' CONTRIBUTIONS ................................................................................... 122 REFERENCES .............................................................................................................. 123 3. SUMMARY AND CONCLUSIONS ...........................................................................
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