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Fusarium Species Responsible for Mycotoxin Production in Wheat Crop: Involvement in Food Safety

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Fusarium Species Responsible for Mycotoxin Production in Wheat Crop: Involvement in Food Safety Alma Mater Studiorum – Università di Bologna DOTTORATO DI RICERCA IN Scienze Agroambientali Ciclo XXIV Settore Concorsuale di afferenza: 07/B1 Settore Scientifico disciplinare: AGR/02 Fusarium species responsible for mycotoxin production in wheat crop: involvement in food safety Presentata da: DIMA ALKADRI Coordinatore Dottorato Relatore Prof. Giovanni Dinelli Prof. Alberto Vicari _______________________ _________________________ Esame finale anno 2012 I Alma Mater Studiorum Bologna University Faculty of Agriculture Department of Agroenvironmental Sciences and Technologies PhD in Agroenvironmental Science Fusarium species responsible for mycotoxin production in wheat crop: involvement in food safety PhD thesis of Dima Alkadri Supervisor: Co- Supervisors: Prof. Alberto Vicari Dr. Annamaria Pisi Dr. Antonio Prodi Coordinator: Prof. Giovanni Dinelli Academic year 2011- 2012 II I would like to dedicate this thesis to my family especially to my father, Hassan Alkadri and mother Ibtehal Alkadri, whom I didn't see for the past few years while I was busy in carrying out this scientific work. Their support, positive encouragement, constructive criticism and prayers still ring deep in my ears. No words could well thank you for everything you have done for me over the years, of all icons you are the most precious. To my brother Orwah Alkadri and his wife Doaa Rikab, their sons Dima and Hassan, who have been always a great source of motivation and inspiration. I am grateful to all of you. To my Damascene poem Shaam and Abed, my small family, I have been justly questioned by you, whether there would ever be a completion date for this project?, now that I have come this far I would like to thank You for being with me all along and in every step, I want to write different words for you, to invent a language for you alone to fit the size of my love. I dedicate this thesis to my beloved grandmothers and all my relatives, your words of support enlightened my way . In a word, I know I always have an amazing family to count on when times are rough. I also dedicate my thesis to the souls of my grandfathers, Ahmed Alkadri and Muhammad Munier Alkadri, which I am very proud to be their granddaughter. Special dedication is to my beloved father in law Yasein Aljahmany. I would like to dedicate this dissertation to the souls of my professors who died during my stay here, in Italy pursuing my studies, Prof. Jamal Hasnah, Prof. Ghassan Khayyat, Prof. Abdou Qasem and Prof. Ahmad Younes, God bless their souls and may they rest in peace and may almighty give their families all strength they need to overcome this great loss. To my freinds and colleagues in Syria and all over the world and to everybody who encouraged me during my study. III Last but not least, and in view of the tragic events that are engulfing those days my beloved Syria, I wish to start the dedication to her, my wounded beloved homeland I want you as always strong and like the old Syrian mythological phoenix rising from its own ashes, you will overcome all challenges and revive stronger and more determined. I am your daughter, back after three years of hard and continuous efforts. My research is devoted to you and through you to all human beings. I wish that you accept this, as a sign of my love and affection for you My Syria, My Damascus. IV Abbreviations Ac-DON acetyldeoxynivalenol AF aflatoxins ANOVA analysis of variance APindex Petri-dish aggressiveness index AUDPC standard area under disease progress curve standard AUHPC area under healthy progress BEA beauvericin bp base pair CE collision energy CEP collision cell entrance potential CID collision-induced dissociation CLA carnation leaf piece agar Clr coleoptile length reduction CTAB hexadecyl-trimethyl-ammonium bromide cv cultivar CXP collision cell exit potential DAI days after inoculation DI disease incidence DNA deoxyribonucleic acid DON deoxynivalenol DP declustering potential DS disease severity ELISA enzyme-linked immunosorbent assay ENN cyclohesadepsipeptide enniatin FB fumonisin B EP entrance potential FDK Fusarium damaged kernels FHB Fusarium head blight FUS-X fusarenon-X V GFC Gibberella fujikuroi complex Gr germination rate reduction HPLC-MS/MS high-performance liquid chromatography combined with tandem mass spectrometry HW hectolitric weight IDA information dependent acquisition KW kernel weight LOD limits of detection LOQ limits of quantification MSPD matrix solid-phase dispersion NIV nivalenol OTA ochratoxins PCR polymerase chain reaction PDA potato dextrose agar PPM part per million QqQ triple quadrupole QTL quantitative trait loci TDI Tolerable Daily Intake TRI trichodiene synthase ZEN zearalenone Key words wheat, FHB, Fusarium, PCR, chemotype, mycotoxins, HPLC-MS/MS, aggressiveness, Petri-dish test, inoculation, cultivars, resistance. VI Table of contents Pages SUMMARY .................................................................................................................................... 1 GENERAL INTRODUCTION ....................................................................................................... 4 Wheat............................................................................................................................................... 5 Fusarium spp. .................................................................................................................................. 6 Fusarium head blight in wheat ........................................................................................................ 9 Epidemiology ................................................................................................................................ 10 Conditions for Infection and Colonization .................................................................................... 11 Fusarium head blight and food quality.......................................................................................... 12 Mycotoxins.................................................................................................................................... 13 Control........................................................................................................................................... 15 FHB resistance............................................................................................................................... 16 OBJECTIVES................................................................................................................................ 18 CHAPTER I................................................................................................................................... 20 Mycoflora isolated from Syrian wheat kernels and characterization of Fusarium species in two Mediterranean countries, Syria and Italy....................................................................................... 20 ABSTRACT .................................................................................................................................. 21 INTRODUCTION......................................................................................................................... 22 MATERIALS AND METHODS .................................................................................................. 24 Fungal isolation.......................................................................................................................... 24 DNA extraction.......................................................................................................................... 24 Chemotype identification........................................................................................................... 25 RESULTS...................................................................................................................................... 26 Fungal colonization of Syrian wheat kernels............................................................................. 26 Morphological and molecular identification of Syrian Fusarium species................................. 27 Morphological and molecular identification of Italian Fusarium species................................. 27 Chemotype identification........................................................................................................... 28 Syrian strains.......................................................................................................................... 28 Italian strains .......................................................................................................................... 28 DISCUSSION................................................................................................................................ 28 FIGURES AND TABLES............................................................................................................. 32 VII CHAPTER II ................................................................................................................................. 37 Screening and quantification of mycotoxin produced by Syrian Fusarium species ..................... 37 ABSTRACT .................................................................................................................................. 38 INTRODUCTION......................................................................................................................... 38 MATERIALS AND METHODS .................................................................................................. 41 Fusarium strains ........................................................................................................................41
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