To my dear father CONTENTS Chapter 1. General Introduction and Outline of Thesis........................................................................1 GENERAL INTRODUCTION.................................................................................................................. 2 ESCA DISEASES COMPLEX: STATE OF THE ART....................................................................2 Brown wood streaking disease .........................................................................................................3 Petri disease of grapevine .................................................................................................................3 Esca (young esca)....................................................................................................................... ....4 Symptoms inside trunk and main branches........................................................................ ..4 Symptoms on leaves........................................................................................................... ..4 Symptoms on shoots and branches........................................................................................6 Symptoms on berries and clusters.........................................................................................6 White rot..............................................................................................................................................7 Esca proper.........................................................................................................................................8 Apoplexy.............................................................................................................................................9 The genus Phaeoacremonium and Phaeomoniella: STATE OF THE ART ...................................10 Hosts ...............................................................................................................................................10 Epidemiology...................................................................................................................................12 Pathogenesis ....................................................................................................................................14 Molecular identification and detection ............................................................................................16 Disease management .......................................................................................................................17 OUTLINE AND SCOPE OF THE THESIS..............................................................................................19 Chapter 2. Development of a semi nested-multiplex PCR approach for the simultaneous detection of fungi associated with esca and related diseases of grapevine ...........................................................22 ABSTRACT ..................................................................................................................................................23 INTRODUCTION ........................................................................................................................................23 MATERIALS AND METHODS .................................................................................................................25 Isolates .............................................................................................................................................25 DNA Extraction ...............................................................................................................................27 Primer design ...................................................................................................................................28 Botryosphaeriaceae species ..................................................................................................28 Phaeoacremonium aleophilum .............................................................................................31 Non-nested species-specific PCR ....................................................................................................33 Nested species-specific PCR ...........................................................................................................34 Semi - nested species-specific PCR ................................................................................................34 Semi Nested Multiplex PCR using DNA extracted from fungal mycelium ...................................34 Semi Nested Multiplex PCR using DNA extracted from plant tissue ...........................................35 Sequencing ......................................................................................................................................35 RESULTS .....................................................................................................................................................36 PCR amplification for primer specificity ........................................................................................36 Nested species-specific PCR ...........................................................................................................36 Semi - nested species-specific PCR ................................................................................................37 Semi nested Multiplex PCR using DNA extracted from fungal mycelium ....................................37 Semi nested Multiplex PCR using DNA extracted from plant tissue ......................................... 38 DISCUSSION ...............................................................................................................................................39 Chapter 3. Genetic diversity among isolates of Phaeomoniella chlamydospora obtained from esca diseased grapevines ............................................................................................................................42 ABSTRACT ..................................................................................................................................................43 INTRODUCTION .......................................................................................................................................43 I MATERIALS & METHODS .....................................................................................................................45 Plant material ..................................................................................................................................45 Isolations .........................................................................................................................................47 Molecular analyses ..........................................................................................................................48 DNA isolation and amplification ....................................................................................................48 Sequence and phylogenetic analysis ...............................................................................................49 RESULTS .....................................................................................................................................................50 PCR results of the amplification of the Elongation factor EF 1-α gene .........................................50 PCR results of the amplification of ß-Tubulin gene .......................................................................50 Sequence analysis ...........................................................................................................................51 Phylogenetic analysis ......................................................................................................................51 DISCUSSION ...............................................................................................................................................54 Chapter 4. Molecular and phenotypic characterisation of novel Phaeoacremonium species isolated from esca diseased grapevines...................................................................................................................56 ABSTRACT .................................................................................................................................................57 INTRODUCTION .......................................................................................................................................57 MATERIAL AND METHODS ..................................................................................................................59 Fungal isolates ................................................................................................................................59 DNA isolation and amplification ....................................................................................................61 Phylogenetic analysis ......................................................................................................................65 Morphology ....................................................................................................................................68 RESULTS .....................................................................................................................................................69 Phylogenetic analyses .....................................................................................................................69 Taxonomy