University of São Paulo “Luiz de Queiroz” College of Agriculture Aetiology and epidemiology of grapevine anthracnose Ricardo Feliciano dos Santos Thesis presented to obtain the degree of Doctor in Science. Area: Plant Pathology Piracicaba 2017 Ricardo Feliciano dos Santos Agronomist Aetiology and epidemiology of grapevine anthracnose Advisor: Prof. Dr. MARCEL BELLATO SPÓSITO Thesis presented to obtain the degree of Doctor in Science. Area: Plant Pathology Piracicaba 2017 2 Dados Internacionais de Catalogação na Publicação DIVISÃO DE BIBLIOTECA – DIBD/ESALQ/USP Santos, Ricardo Feliciano dos Aetiology and epidemiology of grapevine anthracnose / Ricardo Feliciano dos Santos. - - Piracicaba, 2017. 119 p. Tese (Doutorado) - - USP / Escola Superior de Agricultura “Luiz de Queiroz”. 1. Vitis spp. 2. Elsinoë ampelina 3. Diversidade genética 4. Produção de conídios 5. Patometria 6. Progresso temporal e espacial I. Título 3 I dedicate with love to my parents Vicente and Kátia, my grandparents Diogo (in memoriam) and Adelina, my brother Rodrigo and my fiancée Amanda. 4 ACKNOWLEDGEMENTS I wish to express my sincere thanks to the following: God for guiding my steps. The “Luiz de Queiroz” College of Agriculture - University of São Paulo (ESALQ-USP) and the Graduate Program in Plant Pathology for my education during my doctoral study. The Brazilian National Council for Scientific and Technological Development (CNPq) for providing the scholarship from March 2014 to February 2015 (grant number: 140710/2014-0). The São Paulo Research Foundation for providing the scholarship in Brazil from March 2015 to October 2017 (grant number: 2014/24472-1) and in Australia from August 2016 to March 2017 (Research Internship Abroad – BEPE, grant number: 2016/01508-6). My advisor Dr. Marcel Bellato Spósito for his generosity, guidance, support, friendship and knowledge, which were essential during my doctoral study. The professor Dr. Lilian Amorim for her help and constructive suggestions. My supervisor Dr. Mark Sosnowski for his help, guidance, patience and encouragement during my stay at South Australian Research and Development Institute, Australia. The professor Dr. Nelson Sidnei Massola Júnior and Dr. Maisa Ciampi-Guillardi for their advice and support on molecular biology procedures. The professors and staff of the Department of Plant Pathology and Nematology for their support and extensive knowledge provided. The staff of the University of Adelaide and the South Australian Research and Development Institute for their valuable support. Thanks especially to Matthew Ayres for his friendship, help and interesting conversations about the Aussie life. The Australian and Brazilian grape growers for providing anthracnose samples. My colleagues from the Graduate Program in Plant Pathology at ESALQ. My colleagues and friends from the Epidemiology Laboratory for all their support, talks, friendship and scientific discussions. All my friends for their presence throughout this study. 5 All my family. Thanks especially to my parents, Vicente and Kátia, my brother Rodrigo and my grandmother Adelina for their continuous love, help and support. My fiancée Amanda for her unconditional support, patience, encouragement and love. Lastly, everyone who supported me throughout the course of this journey. Thank you all! 6 "A person who never made a mistake never tried anything new." Albert Einstein 7 CONTENTS RESUMO ................................................................................................................................. 10 ABSTRACT ............................................................................................................................ 11 1 GENERAL INTRODUCTION ..................................................................................... 13 References ............................................................................................................................ 14 2 AETIOLOGY OF ANTHRACNOSE ON GRAPEVINE SHOOTS IN BRAZIL .... 17 Abstract ................................................................................................................................. 17 2.1 Introduction ................................................................................................................ 17 2.2 Materials and methods ............................................................................................... 19 2.2.1 Fungal isolates .................................................................................................... 19 2.2.2 Molecular characterization ................................................................................. 20 2.2.3 Morphological characterization .......................................................................... 22 2.2.4 Pathogenicity ...................................................................................................... 23 2.3 Results ........................................................................................................................ 24 2.3.1 Molecular characterization ................................................................................. 24 2.3.2 Morphological characterization .......................................................................... 33 2.3.3 Pathogenicity ...................................................................................................... 35 2.4 Discussion .................................................................................................................. 37 References ............................................................................................................................ 41 Supporting information ........................................................................................................ 47 3 PHYLOGENY, MORPHOLOGY AND PATHOGENICITY OF ELSINOË AMPELINA, THE CAUSAL AGENT OF GRAPEVINE ANTHRACNOSE IN BRAZIL AND AUSTRALIA ................................................................................................................. 49 Abstract ................................................................................................................................. 49 3.1 Introduction ................................................................................................................ 49 3.2 Materials and methods ............................................................................................... 51 3.2.1 Sampling and fungal isolation ............................................................................ 51 3.2.2 DNA isolation, PCR amplification and sequencing ........................................... 53 3.2.3 Phylogenetic analyses ......................................................................................... 54 3.2.4 Intraspecific diversity ......................................................................................... 54 3.2.5 Morphological characterization .......................................................................... 54 3.2.6 Pathogenicity tests .............................................................................................. 55 8 3.3 Results ....................................................................................................................... 57 3.3.1 Phylogenetic analyses ........................................................................................ 57 3.3.2 Intraspecific diversity ......................................................................................... 61 3.3.3 Morphological characterization ......................................................................... 61 3.3.4 Pathogenicity ...................................................................................................... 62 3.4 Discussion ................................................................................................................. 64 References ............................................................................................................................ 67 Supporting information ........................................................................................................ 72 4 IN VITRO PRODUCTION OF CONIDIA OF ELSINOË AMPELINA, THE CAUSAL FUNGUS OF GRAPEVINE ANTHRACNOSE ................................................ 73 Abstract ................................................................................................................................ 73 4.1 Introduction ............................................................................................................... 73 4.2 Materials and methods............................................................................................... 74 4.2.1 Fungal isolates.................................................................................................... 74 4.2.2 In vitro production of conidia ............................................................................ 75 4.2.3 Conidia quantification and germination ............................................................. 76 4.2.4 Infectivity of conidia produced in vitro ............................................................. 77 4.2.5 Statistical analyses ............................................................................................. 78 4.3 Results ....................................................................................................................... 78 4.4 Discussion ................................................................................................................. 81 References ...........................................................................................................................