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Citrus Tristeza Virus: Characterization of Texas View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Texas A&M University CITRUS TRISTEZA VIRUS: CHARACTERIZATION OF TEXAS ISOLATES, STUDIES ON APHID TRANSMISSION AND PATHOGEN-DERIVED CONTROL STRATEGIES A Dissertation by CAROLINE MARY HERRON Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY August 2003 Major Subject: Plant Pathology CITRUS TRISTEZA VIRUS: CHARACTERIZATION OF TEXAS ISOLATES, STUDIES ON APHID TRANSMISSION AND PATHOGEN-DERIVED CONTROL STRATEGIES A Dissertation by CAROLINE MARY HERRON Submitted to Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved as to style and content by: ____________________________ ____________________________ John V. da Graça Herman B. Scholthof (Co-Chair of Committee) (Co-Chair of Committee) ____________________________ ____________________________ T. Erik Mirkov Karen-Beth G. Scholthof (Member) (Member) ____________________________ ___________________________ Richard F. Lee Dennis C. Gross (Member) (Head of Department) August 2003 Major Subject: Plant Pathology iii ABSTRACT Citrus tristeza virus: Characterization of Texas Isolates, Studies on Aphid Transmission and Pathogen-Derived Control Strategies. (August 2003) Caroline Mary Herron, B.Sc. (Hons.), University of Nottingham, UK; M.Sc., University of Bristol, UK Co-Chairs of Advisory Committee: Dr. John V. da Graça Dr. Herman B. Scholthof Citrus tristeza virus (CTV), an economically important graft-transmissible pathogen of citrus, causes major global declines in citrus production. In the commercial citrus region of the Lower Rio Grande Valley of Texas (LRGV), where red grapefruit on tristeza-decline sensitive sour orange rootstocks predominates, incidence of CTV is low. The efficient CTV vector, the brown citrus aphid (BrCA, Toxoptera citricida Kirkaldy) is now established in Mexico and Florida, thus information is needed on the severity of CTV, CTV aphid transmission and the performance of transformed citrus towards CTV before T. citricida arrives in Texas so that appropriate management strategies can be selected. Biological indexing and molecular typing were performed on fifteen Texas CTV isolates. The majority of the CTV isolates tested contained the most severe CTV types known. In Florida, T. citricida were fed on crude CTV preparations in vitro and could transmit CTV to virus-free receptor plants with two CTV isolates, whereas a more iv highly purified CTV preparation from one CTV isolate was not transmitted by T. citricida. There were no differences in the majority of treatments in infectivity neutralizations using three CTV-derived antibodies (p25, p27 and p20). CTV p20 antibodies significantly enhanced the occurrence of CTV transmission in one test. The CTV genome of isolate H33 was sequenced using ‘shot gun’ methods. The H33 major component and H33 minor components were phylogenetically compared to six other full-length CTV sequences. An untranslatable CTV coat protein gene was genetically transformed into the genome of the Texas commercial Rio Red grapefruit variety, and fifty-two independent transgenic lines were produced. CTV challenge responses by the transgenic lines were variable. Individual plants could be identified which had low virus titers by ELISA detection, a temporal decrease in virus titer, or a delay in virus titer accumulation. Comparing all wild types to all transgenic lines over every assessment revealed significant decreases in virus titer in the transgenic lines compared to that of the wild type. An RNA entity with similarities to marafiviruses was identified in a CTV infected plant. The entity appears non-graft transmissible to citrus, and non- mechanically transmissible to a range of herbaceous species. v This work is dedicated to my parents John James Herron and Kathleen Nuvart Herron vi ACKNOWLEDGEMENTS I would like to thank my extraordinary committee members, all of whom have contributed to my personal development. Thanks to my brave supervisor, Dr. John da Graça, for taking up the position half way through the program, and for his constant belief in my abilities. Thank you to my steadfast molecular virology triumvirate who have all generously given knowledge, time, and space for the learning process to occur; Dr. T. Erik Mirkov, within whose laboratory most of the project was executed, co-chair Dr. Herman B. Scholthof who has helped me through the administrative and logistical ups and downs, and Dr. Karen-Beth G. Scholthof, who together with Herman, gave me early laboratory experience and the best plant virology course ever. I will always be indebted to Dr. Gary McBride, former interim assistant director of the Citrus Center for sorting out a visa problem, and giving me good advice. I thank the members of the first Citrus tristeza virus (CTV)/Brown citrus aphid workshop in Central America and the Caribbean for their inspiration; Drs. Richard Lee, Mario Rocha-Peña, Chuck Niblett, Steve Garnsey, David Gumpf, Chet Roistacher, Tim Gottwald, Ray Yokomi and Ramón Lastra, many of whom I have had the honor and privilege to talk and work with again. I acknowledge the financial support for this work from the USDA-CREES Special Program Citrus Tristeza Research and the Texas A&M University-Kingsville Citrus Center. I could not have done the citrus indexing without my co-workers Nora- Solís Gracia and Craig Kahlke; we were a great team! vii I thank Dr. Chuck Niblett for allowing me to work in his laboratory at the University of Florida, Gainesville, with appreciation to Dr. Richard Lee, Dr. K. L. Manjunath and Marty Dekkers for technical support whilst I was there. I thank Dr. Richard Lee, Dr. R. Brlansky, Debbie Howd and Fran Funk for their support in my visits to the University of Florida Citrus Research and Education Center, Lake Alfred, Florida. I am grateful to Dr. Michael Edwards, USDA-ARS, Northern Crop Science Laboratory, Fargo, North Dakota, Professor Giovanni Martelli, Departmento di Protezione delle Piante e Microbiologia Applicata, Universitá delgi Studi and Centro di Studio del CNR sui Virus e le Virosi delle Colture Mediterranee, Bari, Italy, and Agritest s. r. i., Technopolis, Valenzano, Italy for the gifts of antibodies used in Chapter VI. Many thanks also to Drs. Vincente Febres, University of Florida, Gainesville, Florida, Sidderame Gowda and Richard Lee, Citrus Research and Extension Center, University of Florida, Lake Alfred, Florida for the gifts of CTV antibodies. Thank you to Drs. Wengping Qui and Laszlo Kovaks, for useful discussions and the grapevine indicator plants. Great appreciation goes to Dr. Robert Ferry Sr. and Wilma Ferry for providing many needed distractions from work throughout my time in Texas. I am grateful to all the members of the McAllen International Orchid Society for their friendship whilst I have been in the Rio Grande Valley. To my nearest ‘family’ Dave, Annette, Oliver, Madeline and Francis Noble in Houston-it was great to have you so close. To my friends, teachers and workmates who have all made my time a sheer joy whilst I have been here; Nora Solís-Gracia, Carlos Gracia, Craig Kahlke, Kristen Kahlke, Joe Molina, viii Manuel Peña, Josephina Bustamante, Euphemia Bustamante, Jon Wong Park, Bénédicte Desvoyes, Tricia McCabe, Amrita Macguire, Ivan Ingelbrecht, Francis Moonan, Zhong Nan Yang, Abdourhamane Issoufou Kollo, Massimo Turina, Mamoudou Sétamou, Roberto Saldaña, Tichaona Mangwende, and Martin Yassi. I am thankful for the many friends who have kept me going throughout the years; Jacky Israel (nee Wallace), Richard Israel, June and John Wallace, Susan Mann, Penny Astridge, Di Ballantine (nee Schietzel), Angus Ballantine, Harry Sabal, the Sabal family and of course my two ‘brothers’, Peter Allen and Steve Riches. I owe much to the small holder citrus growers of Belize whose struggles against adversity are many but whose generosity, friendship and laughter will always remain with me. I also blame all of this on Dr. Richard Lee, whose unselfish and unpopular support of a plant pathologist in Belize twelve years ago and continuous support and advice cultivated my interest in CTV. ix TABLE OF CONTENTS Page ABSTRACT ........................................................................................................... iii DEDICATION ....................................................................................................... v ACKNOWLEDGEMENTS ................................................................................... vi TABLE OF CONTENTS ....................................................................................... ix LIST OF FIGURES................................................................................................ xii LIST OF TABLES ................................................................................................. xiv CHAPTER I INTRODUCTION AND LITERATURE REVIEW ............................. 1 1.1 Summary ............................................................................ 1 1.2 Introduction........................................................................ 2 1.3 Citrus tristeza virus Characterization................................. 22 1.4 Aphid Transmission of Plant Viruses................................. 37 1.5 Pathogen-Derived Resistance in Plants.............................. 46 1.6 The Aims of this Study....................................................... 53 II SEVERITY OF CITRUS TRISTEZA
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