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Blackberry Virosome: a Micro and Macro Approach a Thesis Submitted Blackberry Virosome: A Micro and Macro Approach A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Cell and Molecular Biology by Archana Khadgi Purbanchal University, SANN International College and Research Center Bachelor of Science in Biotechnology, 2010 December 2015 University of Arkansas This thesis is approved for recommendation to the Graduate Council. Dr. Ioannis E. Tzanetakis Thesis Director Dr. Craig Rothrock Dr. Byung-Whi Kong Committee Member Committee Member ProQuest Number: 1605391 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. ProQuest 1605391 Published by ProQuest LLC (2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, MI 48106 - 1346 Abstract Viruses pose a major concern for blackberry production around the world with more than 40 species known to infect the crop. Virus complexes have been identified recently as the major cause of plant decline with blackberry yellow vein disease (BYVD) being the most important disease of the crop in the Southern United States. The objective of this research was to study the blackberry virosome in both the macro and micro scale. The large scale approach involves identification of the major viruses known to be associated with BYVD in the Southern United States as well as the identification of other viruses whose prevalence is still unknown. RT-PCR was employed to detect the viruses present in wild, cultivated and sentinel blackberries from different states. In the micro approach, the virosome of a single field was studied using large scale sequencing. Understanding the virosome on a regional and local scale provides important information which could greatly enhance disease management. The ultimate goal of this research is to better understand virus distribution in nature and aid in the development of proper management strategies to control epidemics. Acknowledgements Foremost, I would like to express my sincere gratitude to my advisor Dr. Ioannis E. Tzanetakis for his continuous support and encouragement throughout my study duration. I have been amazingly fortunate to have an advisor who gave me the freedom to explore on my own and at the same time the guidance to recover when my steps faltered. His patience and support helped me overcome many crisis situations and finish this thesis. I sincerely thank my advisory committee members Dr. Craig Rothrock and Dr. Byung- Whi-Kong for their support, suggestions, and recommendations. I would like to thank Dr. Thien Ho for his insightful comments and constructive criticisms at different stages of my research. I am thankful to all my lab members, Mohamad Hassan, Muhammad Shafiq Shahid, Thekke-Veetil Thanuja, Joanna Gress, Terea Stetina, Marites Sales, Patrick Di Bello, and Jing Zhou for a great company and valuable assistance during the course of my study. I am also indebted to all my friends in the Department of Plant Pathology and Fayetteville for making my stay so wonderful. I would like to thank Sandeep Sharma, who as a good friend was always willing to help and give his best suggestions. Most importantly, none of this would have been possible without the love and patience of my family. My family, to whom this dissertation is dedicated to, has been a constant source of love, concern, support and strength all these years. I would like to express my heart-felt gratitude to my family. Table of Contents Chapter I. Introduction……………………………………………………….......................1 Abstract ......................................................................................................................2 Introduction................................................................................................................3 Blackberry virus distribution in the southern United States…………………….…..4 Field Virosome - Understanding the virus movement in the field scale…………....19 References .................................................................................................................22 Chapter II. Blackberry virus distribution in the Southern United States………………....41 Abstract ....................................................................................................................42 Introduction..............................................................................................................43 Materials and Methods…………………………………....………………….…….45 Results……………………………………………………………………….……..53 Discussion ................................................................................................................86 References ................................................................................................................91 Chapter III. Field Virosome- Understanding virus movement in the field scale…………………………………………………………………..…….94 Abstract...................................................................................................................95 Introduction............................................................................................................96 Materials and Methods…………………………………....………..............…….97 Results……………………………………………………………….……….…115 Discussion.............................................................................................................140 References ............................................................................................................143 Chapter IV. Conclusions…………………………………………………………..…..146 Abstract ...............................................................................................................147 Significance of studying Blackberry virus distribution in the Southern United States………………………………........................................................148 Significance of studying field virosome to understand virus movement in the field scale …………………………………….…....…………………..….150 References ............................................................................................................152 List of Tables Chapter II 2.1 List of samples used for study……………………………………………….…….….48 2.2 List of all the viruses detected……………………………………………………..….50 2.3 List of oligonucleotide primers used in the detection…………………………….…..51 2.4 Geographical incidence of blackberry yellow vein associated virus in plants showing virus like symptoms………………………………………..……..63 2.5 Geographical incidence of blackberry chlorotic ringspot virus in plants showing virus-like symptoms………………………………………..………64 2.6 Geographical incidence of beet pseudo-yellows virus in plants showing virus-like symptoms…………………………………………….........…….65 2.7 Geographical incidence of blackberry virus Y in plants showing virus-like symptoms…………………………………………………………….……66 2.8 Geographical incidence of blackberry virus S in plants showing virus-like symptoms……………………………………………………………….…67 2.9 Geographical incidence of blackberry virus E in plants showing virus-like symptoms………………………………………………………………....68 2.10 Geographical incidence of blackberry leaf mottle associated virus in plants showing virus-like symptoms……………………………………….69 2.11 Geographical incidence of impatiens necrotic spot virus in plants showing virus-like symptoms………………………………………………70 2.12 Geographical incidence of tobacco ringspot virus in plants showing virus-like symptoms………………………………………………………71 2.13 Geographical incidence of strawberry necrotic shock virus in plants showing virus like symptoms………………………………….…………72 2.14 Geographical incidence of grapevine syrah virus-1 in plants showing virus-like symptoms………………………………………………………73 2.15 Geographical incidence of raspberry bushy dwarf virus in plants showing virus-like symptoms……………………………………………………….74 2.16 Geographical incidence of raspberry leaf mottle virus in plants showing virus-like symptoms…………………………………...……………75 2.17 Geographical incidence of black raspberry necrosis virus in plants showing virus-like symptoms……………………………………...………76 2.18 Incidence of different viruses in sentinel plants for Arkansas and North Carolina between 2010 and 2012………………………………..………77 2.19 Number of viruses found every month in sentinel plants from Arkansas 2010……………………………………………………………….………78 2.20 Number of viruses found every month in sentinel plants from Arkansas 2011……………………………………………………….…………..….79 2.21 Number of viruses found every month in sentinel plants from Arkansas 2012………………………………………………………………….……80 2.22 Number of viruses found every month in sentinel plants from North Carolina 2010…………………………………………………………………81 2.23 Number of viruses found every month in sentinel plants from North Carolina 2011……………………………………………………………..…..82 2.24 Number of viruses found every month in sentinel plants from North Carolina 2012…………………………………………………………………83 Chapter III 3.1 List of primers used in DOP-RT PCR …………………………………………..….103 3.2 List of samples and primers used in the experiment………………………………...105 3.3 List of detection primers designed for the known virus hits………………………..108 3.4 List of detection primers designed for verification of potential new virus hits……………………………………………………………………………..109 .3.5 Number of raw reads in each set from LSS………………………………….………128 3.6 List
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