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Characterization of Moroccan Citrus Tristeza Virus (CTV) Isolates and Study of Their Genomic Variability After Aphid Transmission

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Characterization of Moroccan Citrus Tristeza Virus (CTV) Isolates and Study of Their Genomic Variability After Aphid Transmission UNIVERSITY OF CATANIA DEPARTMENT OF AGRI-FOOD AND ENVIRONMENTAL SYSTEMS MANAGEMENT SECTION OF PLANT PATHOLOGY INTERNATIONAL Ph. D. PROGRAM IN PLANT HEALTH TECHNOLOGIES AND PROTECTION OF AGRO-ECOSYSTEMS XXV-Cycle 2009-2012 Characterization of Moroccan Citrus Tristeza Virus (CTV) isolates and study of their genomic variability after aphid transmission This thesis is presented for the degree of Doctor of Philosophy By Mohamed AFECHTAL COORDINATOR: SUPERVISORS: Prof. Carmelo RAPISARDA Prof. Giuseppe COCUZZA Dr. Anna Maria D’ONGHIA Dr. Khaled DJELOUAH Academic years 2009-2012 BIOGRAPHY Mohamed AFECHTAL was born in Ouaouizerth, Morocco, on February 2nd 1983. He attended Ouaouizerth High School and graduated in October 2007 at the Hassan II Institute of Agronomy and Veterinary Medicine (IAV-Hassan II), Rabat, Morocco, as an Agronomic Engineer in Plant Pathology. In 2005, he got a scholarship from CIHEAM to pursue a Master of Science degree in Integrated Pest Management at the Mediterranean Agronomic Institute of Bari (MAI-Bari), Italy - in collaboration with IAV-Hassan II - which he completed in October 2007. Two months later, he became a researcher in virology of citrus and fruit trees at the National Institute of Agricultural Research (INRA-Morocco). In the same year, 2008, he reported the largest outbreak ever reported of Citrus tristeza virus in Morocco, which led him to perform his Ph.D. thesis on the same topic at the University of Catania, Italy - in collaboration with the MAI-Bari - under the supervision of Prof. G. Cocuzza, Dr. A.M. D’Onghia and Dr. K. Djelouah. i ACKNOWLEDGEMENTS It is with great pleasure that I write these words to express my deep gratitude to everyone who helped, guided and contributed directly or indirectly for completing this Ph.D. thesis. INRA-Morocco Well, this work was only possible because of Prof. M. Badraoui, the Director of INRA- Morocco, and I will always be indebted to him for giving me this chance to make my Ph.D. thesis. It is my utmost pleasure to express my deep sense of heartfelt gratitude towards Mr. A. F. Andaloussi, the Chief of the Plant Protection Department in INRA; Mr. H. Benaouda, the Chief of CRRA-Kénitra; and Dr. M. El Guilli, the Coordinator of the Plant Protection Unit in CRRA-Kénitra for their help and encouragement. UNICT Sincere gratitude goes to my supervisor Prof. G. Cocuzza, for blessing me with his kind supervision, guidance and encouragement. I am very much thankful to our kind coordinator Prof. C. Rapisarda, for his friendly guidance and coordination. I would also like to take the opportunity to thank Prof. G. Cirvilleri for her valuable advice and help. CIHEAM / MAI-Bari My deep gratitude to all the staff of the Mediterranean Agronomic Institute of Bari, represented by the Director Prof. C. Lacirignola, for their support, hospitality and for permitting me to use all available facilities in the Institute. My deepest thanks and appreciation go to my supervisor Dr. A.M. D’Onghia, Head of IPM department, for blessing me with her kind guidance and for her enlightening knowledge which helped me to complete my project and allowed me to achieve this work. I enjoyed being here and am enriched by many dimensions being a student of the department. But also, my appreciation and my deep gratitude to my kind supervisor Dr. K. Djelouah, who always helped me with promptness and kindness and for all the scientific information he provided me. Since my working with him, not a single day has been spent without exploring and experiencing his new aspects of expertise in teaching, research, maintaining human relations and resources management. Without him, this work would not have been accomplished. ii My special thanks to all the staff of IPM department, Dr. M. Digiaro, Dr. T. Elbeaino, Dr. T. Yaseen, Dr. D. Frasheri, Dr. F. Valentini, Mme. S. Santoro, Mr. F. Santoro, Mr. G. Santoro and Mr. G. Cavallo for their kind support and their scientific information shared with me. I have a great pleasure in extending my sincere thanks to my lab-mates, some of them left earlier and some are still around, for their help and kind co-operation during all these years. Special and cordial thanks to my friends Dr. H. El Bilali, Dorsaf Yahyaoui and Yosra Ahmed for their help and for the moments we shared together. At last but not least I would like to thank my parents and my brothers Ahmed and Ali. To them I dedicate this thesis. Above all, thanks to the ALMIGHTY GOD for his blessings. Mohamed AFECHTAL iii TABLE OF CONTENTS BIOGRAPHY .........................................................................................................................................................i ACKNOWLEDGEMENTS ................................................................................................................................ ii TABLE OF CONTENTS ................................................................................................................................... iv ABSTRACT ........................................................................................................................................................ vii RESUME .............................................................................................................................................................. ix RIASSUNTO ...................................................................................................................................................... xi LIST OF TABLES ............................................................................................................................................. xiii LIST OF FIGURES ........................................................................................................................................... xiv LIST OF ANNEXES ........................................................................................................................................ xvii LIST OF ABBREVIATIONS ........................................................................................................................ xviii Chapter I. INTRODUCTION ............................................................................................................................ 1 Chapter II. BIBLIOGRAPHIC REVIEW .......................................................................................................... 3 2.1. Origins of the tristeza disease ................................................................................................................ 3 2.2. Brief history of Citrus tristeza virus in Morocco ................................................................................ 4 2.3. Causal agent ............................................................................................................................................... 6 2.4. Host-virus relationships .......................................................................................................................... 7 2.5. Symptoms of CTV .................................................................................................................................... 8 2.5.1. Quick decline (QD) syndrome of trees on sour orange rootstock ............................................ 9 2.5.2. Stem pitting (SP) syndrome .......................................................................................................... 11 2.5.3. Seedling yellows (SY) syndrome .................................................................................................. 12 2.6. The CTV genome .................................................................................................................................... 13 2.7. Expression, replication strategies and translation of CTV ............................................................. 15 2.8. Detection and strain characterization of CTV ................................................................................... 17 2.8.1. Biological techniques ..................................................................................................................... 17 2.8.2. Serological techniques ................................................................................................................... 18 2.8.3. Molecular techniques ..................................................................................................................... 19 2.9. CTV genetic diversity ............................................................................................................................ 20 2.10. Transmission and epidemiology of CTV ......................................................................................... 22 2.10.1. CTV transmission .......................................................................................................................... 22 2.10.1.1. The brown citrus aphid Toxoptera citricidus (Kirkaldy) ................................................ 23 2.10.1.2. The cotton or melon aphid Aphis gossypii (Glover) ........................................................ 25 2.10.1.3. The green citrus or spirea aphid Aphis spiraecola (Patch) ............................................. 26 2.10.1.4. The black citrus aphid Toxoptera aurantii (Boyer de Fonscolombe) ........................... 27 2.10.2. CTV epidemiology ........................................................................................................................ 28 2.10.3. Genomic alteration of viral CTV population by aphid transmission ................................
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