Alma Mater Studiorum – Università degli Studi di Bologna FACOLTÁ DI AGRARIA Dipartimento di Scienze Agrarie (DipSA) DOTTORATO DI RICERCA IN ECOLOGIA MICROBICA E PATOLOGIA VEGETALE Ciclo XXV° Settore/i scientifico-disciplinare/i di afferenza: AGR/12 PATOLOGIA VEGETALE TITOLO TESI: Epidemiology and population genetics of Podosphaera fusca and Golovinomyces orontii, causal agents of cucurbit powdery mildew Presentata da: Alessandro Pirondi Coordinatore dottorato Tutor e relatore Prof. Paolo Bertolini Prof. Agostino Brunelli Correlatore Prof. Alejandro Pérez-García Esame finale anno 2013 2 to Beatrice and Nerina 3 4 “I tell young people: Do not think of yourself, think of others. Think of the future that awaits you, think about what you can do and do not fear anything.” Rita Levi Montalcini 5 6 TABLE OF CONTENTS GENERAL INTRODUCTION .......................................................................................... 9 1. Importance and origin of cultivated cucurbits ........................................................... 9 2. Cucurbit powdery mildew disease ........................................................................... 10 2.1 Taxonomy and host range ................................................................................. 12 2.2 Biology ............................................................................................................. 13 2.3 Life cycle and epidemiology ............................................................................. 18 2.4 Ecological requirements .................................................................................... 20 2.5 Races and pathotypes ........................................................................................ 20 2.6 Control ............................................................................................................. 22 3. Population genetics in plant pathogens and evolutionary potential ......................... 25 3.1. Risk assessment: Evolutionary potential of powdery mildew fungi .................. 27 3.2. Tools and techniques of population genetics .................................................... 28 3.2.1. Amplified Fragment Length Polymorphism (AFLP).................................. 30 3.2.2. Multilocus Sequence Typing (MLST) ....................................................... 32 4. Aim of the thesis ..................................................................................................... 33 PART ONE: EPIDEMIOLOGY ..................................................................................... 35 SPECIES REPLACEMENT OF CUCURBIT POWDERY MILDEW-CAUSING FUNGI IN NORTHERN ITALY ............................................................................................................................. 35 1. Introduction ............................................................................................................ 35 2. Material and methods ............................................................................................. 37 2.1. Sampling ......................................................................................................... 37 2.2. Isolation of chasmothecia ................................................................................. 38 2.3. Species identification ....................................................................................... 39 2.3.1. Morphological identification ..................................................................... 40 2.3.2. Molecular identification ............................................................................ 41 2.4. Mating type identification ................................................................................ 42 2.5. Statistical analysis ............................................................................................ 43 2.6. Collection of powdery mildew fungi from weeds, spontaneous plants and non- cucurbitaceous crops............................................................................................... 43 2.7. Climate data ..................................................................................................... 44 3. Results .................................................................................................................... 44 3.1. Occurrence and distribution of cucurbit powdery mildew species..................... 44 3.1.1. Morphological identification ..................................................................... 44 Bologna .................................................................................................................. 45 Mantua ................................................................................................................... 48 3.1.2. Molecular identification ............................................................................ 51 3.2. Chasmothecia collection and mating type identification ................................... 53 3.3. Powdery mildew species in alternative hosts .................................................... 56 3.4. Climate data ..................................................................................................... 57 4. Discussion .............................................................................................................. 59 7 PART TWO: POPULATION GENETICS ..................................................................... 65 GENETIC DIVERSITY ANALYSIS OF THE CUCURBIT POWDERY MILDEW FUNGUS PODOSPHAERA FUSCA SUGGESTS A CLONAL POPULATION STRUCTURE ...................................................... 65 1. Introduction ............................................................................................................ 65 2. Material and methods ............................................................................................. 67 2.1. Fungal collection, maintenance and conservation ............................................. 67 2.2 Morphological identification ............................................................................. 71 2.3. DNA isolation .................................................................................................. 72 2.4 Primers design for Multilocus Sequence Typing (MLST) analysis .................... 72 2.5. Mating type identification ................................................................................ 74 2.6. AFLP (Amplified Fragment Length Polymorphism) analysis ........................... 75 2.7. Cloning of AFLP polymorphic bands ............................................................... 78 3. Results .................................................................................................................... 79 3.1. Isolation of housekeeping gene fragments ........................................................ 79 3.2. Genetic diversity analysis of P. fusca populations by MLST method ............... 83 3.3. Frequencies of P. fusca mating types ............................................................... 83 3.3. AFLP fingerprinting ........................................................................................ 84 3.4. Cloning of polymorphic bands ......................................................................... 88 4. Discussion .............................................................................................................. 90 GENERAL DISCUSSION ............................................................................................... 97 CONCLUSIONS ............................................................................................................ 109 REFERENCES .............................................................................................................. 111 ANNEXES ...................................................................................................................... 123 ACKOWLEDGMENTS ................................................................................................ 129 8 General introduction 1. Importance and origin of cultivated cucurbits Cucurbits belong to the family Cucurbitaceae and consist of about 118 genera and 825 species, according to the last taxonomic treatment of Jeffrey (1990). Cucurbits are present in both the New and Old World and are among the most important plant families that supply human with edible products and useful fibers. Cucurbits are divided into five sub-families: Fevilleae, Melothrieae, Cucurbitaceae, Sicyoideae, and Cyclanthereae. The most important cultivated genera are Cucurbita L., Cucumis L., Citrullus L., Lagenaria L., and Luffa L., found in the sub-family Cucurbitaceae, and Sechium L., found in the sub-family Sicyoideae (Whitaker and Davis, 1962). Among the cucurbits, watermelon is the most popular in the world. The United Nations' Food and Agriculture Organization (FAO) estimated an average annual area of cultivation of 2.5 million ha and an annual production of 46.6 million tons of watermelon fruits between 1996 and 1998. Next in total world production were cucumber, melon, squash and pumpkins. In terms of countries, China is the leading producer of major cucurbit crops followed by Turkey, Iran and Ukraine. In the Americas, Argentina is an important producer of squash and pumpkins and the United States is an important producer of cucumber, melon and watermelon (FAO, 1998). The most important cucurbits in Brazil are squash, watermelon and melon, whose total production in 1995 was 535 million fruits harvested from an area of 206,000 ha (IBGE, 1996). Although cultivated cucurbits are very