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Università Degli Studi Della Tuscia Di Viterbo UNIVERSITÀ DEGLI STUDI DELLA TUSCIA DI VITERBO DIPARTIMENTO DI SCIENZE E TECNOLOGIE PER L'AGRICOLTURA, LE FORESTE, LA NATURA E L'ENERGIA Corso di Dottorato di Ricerca in SCIENZE DELLE PRODUZIONI VEGETALI E ANIMALI - XXIX Ciclo Investigations on the transmission of the bacterium Xylella fastidiosa by insect vectors (Settore Scientifico-disciplinare AGR/12) Tesi di dottorato di: Dott. ISSAM EDDINE BEN MOUSSA Coordinatore del corso Tutore Prof. STEFANIA MASCI Prof. LEONARDO VARVARO Prof. ANNA MARIA D'ONGHIA Co-tutore Prof. Caroline Roper Dott. Franco Valentini Prof. Stefano Speranza A.A. 2016/17 Dedicace To my beloved parents, my darling sister and to Safsoufa ii AKNOWLEDGEMENT It is my pleasure to express my heartfelt thanks to all the people who helped me to accomplish my PhD thesis and those who had great contribution in my life. First of all, I would like to thank the coordinator of the PhD program, Pr. Stefania Masci and the coordinator of IAMB's IPM department, Dr. Anna Maria D‟ONGHIA for giving me the opportunity to work on this interesting subject, their direction and for their guidance. Furthermore, I would like to gratefully and sincerely thank my supervisor Pr. Leonardo Varvaro and my advisors Dr. Valerio Mazzoni, Dr. Franco Valentini and Dr. Stefano Speranza for their assistance, patience and their critical comments in reviewing my paper. Special thanks to my co-supervisors during my training at the university of California Riverside, Pr. Caroline Roper, Pr. Elaine Backus and Dr. Rodrigo krugner for their collaboration, for giving me valuable remarks and most importantly for their friendship. My gratitude for all my friends who helped me to achieve this end and supported me during critical moments. My deepest thanks to my beloved parents who raise me up, educated me and still continue to take care of me. iii Table of contents Introduction ................................................................................................................................................... 1 Chapter I: Literature review .......................................................................................................................... 3 I.1. Background on Xylella fastidiosa ....................................................................................................... 3 I.1.1. History .......................................................................................................................................... 3 I.1.2. Biology ......................................................................................................................................... 4 I.1.3. Taxonomy and classification ........................................................................................................ 6 I.1.4. Geographical distribution ............................................................................................................. 7 I.1.5. Symptoms ................................................................................................................................... 10 I.1.6. Vectors ....................................................................................................................................... 12 I.2. The Olive Quick decline syndrome ................................................................................................... 15 I.2.1. Current situation in Apulia ......................................................................................................... 15 I.2.2. Host plants .................................................................................................................................. 16 I.2.3. Vectors ....................................................................................................................................... 17 Chapter II: Identification of potential insect vectors of Xylella fastidiosa in Apulia region and study of their seasonal dynamics in relation with disease diffusion. ......................................................................... 18 II.1. Background ...................................................................................................................................... 18 II.2. Material and Methods ...................................................................................................................... 18 II.2.1. Capture and Identification of Insects ........................................................................................ 18 II.2.2. Extraction and amplification of X. fastidiosa DNA .................................................................. 20 II.2.3. Cloning and sequence analysis ................................................................................................. 20 II.2.4. Data Analysis ............................................................................................................................ 22 II.3. Results ............................................................................................................................................. 23 II.3.1. Identification and abundance of insects .................................................................................... 23 II.3.2. Detection and seasonal fluctuations of X. fastidiosa infection in captured insects ................... 25 II.3.3. Sequences analysis .................................................................................................................... 30 II.4. Discussion ........................................................................................................................................ 32 Chapter III: Evaluation of the possibility to utilize potential vectors as indicators of the presence of the bacterium in apparently uncontaminated areas so called "Spy Insect approach" ........................................ 35 III.1. Background .................................................................................................................................... 35 III.2. Material and methods ..................................................................................................................... 35 III.2.1. Insects capture and identification ............................................................................................ 35 III.2.2. Extraction of X. fastidiosa DNA from insects and PCR assays .............................................. 36 iv III.3. Results ............................................................................................................................................ 37 III.3.1. Insects capture and identification ............................................................................................ 37 III.3.2. PCR assays .............................................................................................................................. 38 III.4. Discussion ...................................................................................................................................... 41 Chapter IV: Effect of X. fastidiosa endoglucanases on the acquisition, retention of the bacteria by an efficient insect vector .................................................................................................................................. 42 IV.1. Background .................................................................................................................................... 42 IV.2. Material and methods ..................................................................................................................... 42 IV.2.1. Bacterial strains and growth conditions .................................................................................. 42 IV.2.2. Insect maintenance .................................................................................................................. 43 IV.2.3. Insect acquisition experiments ................................................................................................ 43 IV.2.4. Quantification of X. fastidiosa ................................................................................................ 44 IV.2.5. Scanning electron microscopy ................................................................................................ 45 IV.2.6.Data analysis ............................................................................................................................ 45 IV.3. Results ............................................................................................................................................ 45 IV.3.1. Quantitative comparison (qPCR) ............................................................................................ 45 IV.3.2. Qualitative comparison (SEM) ................................................................................................ 46 IV.4. Discussion ...................................................................................................................................... 49 Conclusions ................................................................................................................................................. 52 References ................................................................................................................................................... 54 v List of figures Figure 1: Scanning electron micrographs of tangential sections through grapevine and associated xylem vessels. (A) Water-conducting xylem vessels of a grapevine cross section serve as the habitat for X. fastidiosa cells. (B) View
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