METABOLIC RESPONSES OF CITRUS PLANTS TO THE BACTERIAL PATHOGEN Candidatus Liberibacter asiaticus AND ITS INSECT VECTOR Diaphorina citri By YASSER SOBHY AHMED NEHELA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2018 © 2018 Yasser Sobhy Ahmed Nehela To the memory of my mam and my brother Muhammed, who I will never ever forget them, To my beloved wife Doaa for always being there for me with unconditional love, To my children, Lojain, Malek, and Lana whose smiles give me a reason to wake up every day, To my parents and family for their love, endless support, encouragement and sacrifices. ACKNOWLEDGMENTS First and foremost, thanks to Allah “Subhanahu Wa Ta’ala” Completion of this work would not have been successful without the support and guidance of many people who always believed in me and helped me in many ways. I would like to thank my graduate committee who lead me through my graduate program. First of all, I would like to express my deep and sincere gratitude to my advisor and committee chair, Dr. Nabil Killiny, associate professor, department of plant pathology, CREC- IFAS-UF, for supporting my academic career since my first steps as a research scholar in UF. I would like to thank him for his perpetual patience and empathy, support, encouragement, scientific guidance, and thoughtful advice toward both personal and professional development. I am highly indebted to his believing in me as a scientist, have confidence in my abilities, and for permitting me to become a member of his team, where I cultivated a wealth of technical skills and a solid research infrastructure to build my academic career. His high ethical standards and respectful views for the others will never be forgotten. without your trust, scientific inputs and personal mentorship, I would not be able to conclude this step. In addition, I would like to convey my appreciation to my supervisory committee members who contributed to my personal advancement and aided in developing my research program. I would like to thank Dr. Jeff B. Jones - Professor, Department of Plant Pathology, UF for his valuable guidance and support throughout my doctorate. I would like to thank Dr. Bill Dawson - Eminent Scholar, Department of Plant Pathology, CREC-IFAS-UF for being a great mentor and taking time out of his exceptionally busy schedules to serve on my Ph.D. Additionally, my sincere thanks go to Dr. Larry W. Duncan - Professor, Department of Entomology and Nematology, CREC-IFAS-UF for his suggestions, and guidance in composing my research and dissertation. 4 The conclusion of this step would never be possible without the support of special people. I would like to thank all supportive Killiny’s lab members that I had the privilege to interact and work with them throughout my doctorate. I would like to express my gratitude to my soulmate friend Dr. Faraj Hijaz for technical assistance with lab equipment and methods and for helping me in many personal and family issues since my first day in Florida and during these years abroad. Additionally, I would also like to thank Mrs. Shelley E. Jones for her help throughout my research, showing me the ropes around the lab, sharing her invaluable expertise, and always leading by example. I would also like to thank Floyd Butz and Lorraine Jones for the technical assistance, maintaining the trees in the greenhouse, maintaining the psyllid colonies and making the Killiny’s lab a fun and friendly place to work. I would like to express my gratitude to my previous supervisors Dr. Hassan El-Zahaby and Dr. Abdelnaser Elzaawely – Department of Agricultural Botany, Faculty of Agriculture, Tanta University, Egypt for their collaboration, helpful discussions, and support throughout the first steps of my program. In addition, I would like to thank the Cultural Affairs and Missions Sector, Ministry of Higher Education, Egypt for funding my first two years in the USA as a research scholar. Thanks also are extended to the Egyptian cultural and educational bureau – Washington DC for their overall concern with me particularly, and other fellows in general. Lastly, my beloved family deserves the most thanks. They stood with me through the best and worst times. I would like to thank my brother Ramadan who helped me in many ways. My deep thank and love go to my wife, Doaa, who always encouraged me to follow my dream, and without her love and support, I would never be who I am today. Finally, I would like to express my sincere appreciation for everybody who contributes directly or indirectly in this study. Thanks for all. 5 TABLE OF CONTENTS Page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES .........................................................................................................................10 LIST OF FIGURES .......................................................................................................................12 LIST OF ABBREVIATIONS ........................................................................................................16 ABSTRACT ...................................................................................................................................27 CHAPTER 1 REVIEW OF LITERATURE .................................................................................................29 Introduction .............................................................................................................................29 The Genus Liberibacter and its Associated Species ...............................................................30 History of Huanglongbing (HLB) ..........................................................................................32 HLB Pathosystem ...................................................................................................................33 The Causal Agents (Pathogens) .......................................................................................34 The Rutaceous Host Plant(s) ...........................................................................................36 The Insect Vector(s) ........................................................................................................38 Geographical Distribution of HLB .........................................................................................40 The Geographical Distribution of the Bacterial Pathogens .............................................40 The Geographical Distribution the Insect Vectors ..........................................................42 Proposed Objectives and Hypotheses of this Study ...............................................................43 Objective 1: To Understand the Role of Phytohormonal Cross-Talk and its Role in Citrus Response to HLB ..............................................................................................43 Sub-objective 1.1: To develop a GC/MS-based method for phytohormone profiling of Citrus sinensis (L.) tissues .................................................................43 Sub-objective 1.2: To study the phytohormonal cross-talk that mediates the citrus responses to Ca. Liberibacter asiaticus and its vector D. citri ....................43 Objective 2: To Study the Role of Citrus Leaf Pigments in Citrus Response to HLB and Disease Symptoms Development ..........................................................................44 Objective 3: To Investigate the Role of Carboxylic Compounds in the Citrus Response to HLB .........................................................................................................44 Objective 4: To Study the Effect of CLas-Infection and/or D. citri-Infestation on the TCA Cycle of its Host Plant ..................................................................................44 2 PHYTOHORMONE PROFILING OF THE SWEET ORANGE (Citrus sinensis (L.) OSBECK) LEAVES AND ROOTS USING GC-MS-BASED METHOD ............................51 Introduction .............................................................................................................................51 Materials and Methods ...........................................................................................................52 Plant Materials .................................................................................................................52 6 Extraction of Phytohormones ..........................................................................................52 Derivatization of Phytohormones ....................................................................................53 Phytohormones Standards Curves ...................................................................................53 Method Evaluation ..........................................................................................................53 Extraction recovery ..................................................................................................53 Limit of Detection and Limit of Quantification .......................................................54 Reproducibility .........................................................................................................54 GC-MS Analyses .............................................................................................................54 Statistical Analysis ..........................................................................................................54