UNIVERSITÀ DEGLI STUDI DI UDINE Sede convenzionata: Fondazione Edmund Mach Corso di Dottorato di Ricerca in Scienze e Biotecnologie Agrarie Ciclo XXIX TESI DI DOTTORATO DI RICERCA A comparative study of plant-pathogen interaction in different genotypes and organs of grapevine (Vitis spp.), based on optimized and new screening methods for resistance to downy mildew (Plasmopara viticola - Berk. & Curt.) Coordinator: Prof. Giuseppe Firrao Supervisor: Dr. Riccardo Velasco Co-supervisors: Dr. Silvia Vezzulli PhD student: Prof. Rita Musetti Daniele Buonassisi ANNO ACCADEMICO 2016/2017 Ho domandato, allora: "Signore, Tu avevi detto che saresti stato con me in tutti i giorni della mia vita, ed io ho accettato di vivere con te, perché mi hai lasciato solo proprio nei momenti più difficili?" Ed il Signore rispose: "Figlio mio, Io ti amo e ti dissi che sarei stato con te e che non ti avrei lasciato solo neppure per un attimo: i giorni in cui tu hai visto solo un'orma sulla sabbia, sono stati i giorni in cui ti ho portato in braccio". (L’orma sulla sabbia) Index i) INDEX i) INDEX ....................................................................................................................................................... 3 ii) ABSTRACT ............................................................................................................................................. 6 CHAPTER 1 .................................................................................................................................................... 8 GENERAL INTRODUCTION ........................................................................................................................ 8 Summary ..................................................................................................................................................... 8 1.1. GRAPEVINE ........................................................................................................................................ 9 1.1.1. Grapevine taxonomy ................................................................................................................... 9 1.1.2. Viticulture and its economic importance ................................................................................. 11 1.1.3. Grapevine pathogenic diseases .............................................................................................. 12 1.1.3.1. Gray mould ........................................................................................................................ 12 1.1.3.2. Powdery mildew ............................................................................................................... 13 1.1.3.3. Black rot .............................................................................................................................. 14 1.1.3.4. Eutypa dieback ................................................................................................................. 15 1.1.3.5. Esca ..................................................................................................................................... 15 1.1.3.6. Crown gall disease........................................................................................................... 16 1.1.3.7. Pierce’s disease ................................................................................................................ 17 1.1.3.8. Fanleaf virus (GFLV) ........................................................................................................ 17 1.2. GRAPEVINE DOWNY MILDEW .................................................................................................... 19 1.2.1. Grapevine downy mildew life cycle ......................................................................................... 19 1.2.2. Grapevine downy mildew symptoms ...................................................................................... 20 1.2.3. Grapevine downy mildew disease control ............................................................................. 23 1.2.4. Grapevine breeding for disease control ................................................................................. 24 1.3. PLANT-PATHOGEN INTERACTION ............................................................................................ 25 1.3.1. The plant immune system ........................................................................................................ 25 1.3.2. Hormone signalling .................................................................................................................... 27 1.3.2.1. Systemic immunity .......................................................................................................... 27 1.4. “OMICS” APPROACHES TO STUDY THE GRAPEVINE-DOWNY MILDEW INTERACTION .......................................................................................................................................... 29 1.4.1. Phenomics .................................................................................................................................. 29 1.4.1.1. Macroscopic and microscopic inspection ................................................................ 29 1.4.1.2. Image-based, staining and ultrastructural analyses .............................................. 31 1.4.1.3. Metabolic profiling ........................................................................................................... 32 1.4.1.4. Genetics coupled with Genomics ................................................................................ 33 3 Index 1.4.1.4.1. Grapevine .................................................................................................................... 33 1.4.1.4.2. Downy mildew ............................................................................................................ 39 1.4.2. Transcriptomics and Proteomics ............................................................................................. 40 1.4.2.1. Grapevine ........................................................................................................................... 40 1.4.2.2. Downy mildew ................................................................................................................... 44 1.5. REFERENCES: ................................................................................................................................. 46 CHAPTER 2 .................................................................................................................................................. 57 Development of a novel phenotyping method to assess downy mildew symptoms on grapevine inflorescences ......................................................................................................................... 57 Abstract ..................................................................................................................................................... 57 2.1 INTRODUCTION ................................................................................................................................ 58 2.2. MATERIALS AND METHODS ....................................................................................................... 60 2.2.1. Plant material .............................................................................................................................. 60 2.2.2. Fruiting cutting production ........................................................................................................ 62 2.2.3. Leaf disc in vitro bioassay upon downy mildew infection .................................................... 62 2.2.4. Inflorescence in vitro bioassay upon downy mildew infection ............................................ 64 2.2.5. Statistical analysis ..................................................................................................................... 65 2.2. RESULTS ........................................................................................................................................... 66 2.3.1. Field-derived leaf disc downy mildew resistance assessment ........................................... 66 2.3.2. Field-derived inflorescence downy mildew resistance assessment................................... 71 2.3.3. Comparison between field-derived leaf disc and inflorescence disease response ......... 75 2.3.4. Phytotron-derived leaf disc and inflorescence downy mildew assessment ...................... 75 2.4. DISCUSSION .................................................................................................................................... 78 2.4.1. The in vitro foliage disease response variability ................................................................... 78 2.4.2. The new in vitro inflorescence phenotyping method ............................................................ 79 2.4.3. Phenotype predictability based on a single organ evaluation ............................................. 81 2.5. ACKNOWLEDGEMENTS ............................................................................................................... 83 2.6. APPENDIX 1 ...................................................................................................................................... 84 2.7. REFERENCES .................................................................................................................................. 90 CHAPTER 3 .................................................................................................................................................