Dottorato di Ricerca in Scienze Agrarie – Indirizzo “Gestione Fitosanitaria Ecocompatibile in Ambienti Agro-Forestali e Urbani” Dipartimento di Agraria – Università Mediterranea di Reggio Calabria (sede consorziata) Agr/12 – Patologia Vegetale Molecular analysis of the fungal community associated with phyllosphere and carposphere of fruit crops IL DOTTORE IL COORDINATORE Ahmed ABDELFATTAH Prof. Stefano COLAZZA IL TUTOR CO-TUTOR Prof. Leonardo SCHENA Dr. Anna Maria D'ONGHIA Dr. Michael WISNIEWSKI CICLO XXVI 2016 ACKNOWLEDGEMENTS I appreciate everyone’s contributions of time, ideas, and funding to make my PhD possible. First I want to thank my advisor Leonardo Schena. He has taught me so many things in science and in life. It was a great honor to work with him during this PhD. It has been an amazing experience, not only for his tremendous academic support, but also for giving me so many wonderful opportunities. I am greatly thankful to Dr. Michael Wisniewski for giving the opportunity to work in his lab at the USDA in West Virginia. Michael was a great advisor, I learnt so many thing from working with him and it was a great honor to know him and his amazing family. I am grateful to the University of Reggio Calabria for giving me the chance to work in their laboratories during my PhD. I’m sincerely grateful to the past and present group members of the Reggio Calabria University that I have had the pleasure to work with or alongside: Demetrio Serra, Antonio Biasi, Marisabel Prigigallo, Sonia Pangallo, David Ruano Rosa, Antonino Malacrinò, Orlando Campolo, Vincenzo Palmeri, and especially Saveria Mosca for being a great coworker and wonderful friend. Thanks to them, I had an incredible time in their amazing city. I would like also to thank the University of Palermo, and the coordinator of the PhD program, Prof. Stefano Colazza, for giving me this wonderful opportunity. Thanks also goes to the Mediterranean Agronomic Institute of Bari (MAIB) especially Anna Maria D'Onghia and Thaer Yaseen for their support. I will be always thankful to the people in Shepherdstown for their hospitality, especially Katie Allen, Caitlin McAteer, Bob Keel and Richard Womeldorf for giving such a wonderful time during my visit to their beautiful town. I would like to end this acknowledgment with thanking the most especial people in my life, my family, for their limitless love and support all my life, keeping me full of enthusiasm and happiness. Same goes to a wonderful and beautiful person, Melissa Si Ammour, thank you for your support and encouragement. Table of Contents Table of Contents Chapter I. Introduction ................................................................................................... 4 Metabarcoding ................................................................................................................ 4 1. ITS as a barcode gene for fungi ........................................................................ 6 2. Challenges with the ITS as a DNA barcode ..................................................... 7 Metabarcoding Workflow ............................................................................................... 8 1. Sam pling ........................................................................................................... 8 2. DNA extraction and library preparations ......................................................... 9 3. Sequencing technologies ................................................................................ 10 4. Quality and chimera filtering ......................................................................... 13 5. Operational Taxonomic Units (OTUs) ........................................................... 13 6. Normalizations ............................................................................................... 15 7. Taxonomy assignment .................................................................................... 16 8. Methods in calculating microbial diversity .................................................... 17 9. Statistical comparisons ................................................................................... 20 Scope of the thesis......................................................................................................... 20 References ..................................................................................................................... 21 Chapter II. Metabarcoding analysis of fungal diversity in the phyllosphere and carposphere of olive (Olea europaea) .............................................................................. 28 Abstract ......................................................................................................................... 28 Introduction ................................................................................................................... 28 Materials and Methods .................................................................................................. 30 Sampling and DNA extractions ................................................................................ 30 Fungal DNA amplification ....................................................................................... 31 Data analysis and statistics ....................................................................................... 32 Identification of fungal species ................................................................................. 33 Results ........................................................................................................................... 34 1 Table of Contents Fungal diversity and richness ................................................................................... 34 Olive fungal community structure ............................................................................ 37 Identification of fungal species ................................................................................. 40 Discussion ..................................................................................................................... 42 Conclusion .................................................................................................................... 48 References ..................................................................................................................... 49 Chapter III. Metagenomic analysis of fungal diversity on strawberry plants and effect of management practices on the fungal community structure of aerial organs .................... 54 Abstract ......................................................................................................................... 54 Introduction ................................................................................................................... 54 Materials and Methods .................................................................................................. 56 Sampling and DNA extractions ................................................................................ 56 Fungal DNA amplification and amplicon library preparation .................................. 57 Data analysis and statistics ....................................................................................... 58 Identification of fungal taxa ...................................................................................... 59 Results ........................................................................................................................... 59 General sequencing analysis ..................................................................................... 59 Alpha diversity and richness ..................................................................................... 60 Beta diversity analysis .............................................................................................. 61 Strawberry fungal community structure and identified taxa .................................... 63 Discussion ..................................................................................................................... 69 Conclusion .................................................................................................................... 72 References ..................................................................................................................... 73 Chapter IV. Analysis of fungal diversity in orange (Citrus sinensis) leaves with and without greasy spot disease symptoms ............................................................................. 77 Abstract ......................................................................................................................... 77 Introduction ................................................................................................................... 77 Materials and Methods .................................................................................................. 80 Data analysis and statistics ....................................................................................... 81 2 Table of Contents Identification of fungal taxa ...................................................................................... 82 Results and Discussion.................................................................................................. 83 Conclusion .................................................................................................................... 93 References ....................................................................................................................