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.)

The first target of my research project has been to study the organ-specific interaction between grapevine and downy mildew (DM), caused by the biotrofic pathogen Plasmopara viticola (Berk. & M.A. Curtis) Berl. & De Toni. The studied genotypes have been chosen based on their susceptibility, tolerance and resistance to DM according to previous field observations based on the OIV452 and OIV453 descriptors for leaves and inflorescences, respectively. These two organs have been collected from 9 Vitis hybrids and a Vitis vinifera L. variety grown in an untreated experimental field at Edmund Mach Foundation (FEM). In particular, inflorescences have been harvested at three phenological stages (17, 25 and 29) of the Eichhorn-Lorenz (E-L) scale. Firstly, I have improved the annotation procedure of foliar resistance/susceptibility under controlled conditions (optimized and updated OIV descriptor 452-1). The in vitro leaf disc bioassay resulted significantly associated to the infield DM response. Secondly, I have developed a new in vitro phenotyping method - from infection to symptom evaluation - for DM resistance assessment on grapevine inflorescence (developed and proposed OIV descriptor 453-1). The 17 E-L phenological stage of inflorescence upon in vitro assay resulted significantly correlated to the in-field DM response. Based on the latter DM response assessment, genotypes have been assigned to classes following the four thresholds set for leaf discs. Linear correlations between organs within the same genotype have confirmed that the E-L 17 stage is the most responsive to the DM attack. Genotypes classified as mid-resistant or resistant at leaf level have also resistant inflorescences; genotypes with mid-susceptible leaves present susceptible inflorescences except for Cabernet Cortis (CC) (Cabernet Sauvignon x Solaris), carrying mid-resistant leaves and mid-susceptible inflorescences. To verify that the CC had a significantly different response between organs under controlled conditions, I have carried out some experiments detaching organs from fruiting cuttings grown in phytotron. Thus, CC organs were evaluated upon mock- and P. viticolainoculation, using the susceptible Pinot Noir organs as a reference. Unlike the latter, CC presented a significant difference in DM response; this represented an interesting result and corroborates the diverse DM epidemiological behaviour at organ level. In the second part of my research project, I have used CC as a model to study divergent dual epidemics, compared to its parent Cabernet Sauvignon (CS) displaying out susceptible organs. Leaves and inflorescences – at the 17 E-L phenological stage – obtained from fruiting cuttings have been used. To assess the response of different grapevine organs to P. viticola attack, the disease development has been evaluated with different methods, from the macroscopic/microscopic to the ultrastructural point of view. Upon P. viticola inoculation, the CC and CS organs showed different levels of resistance or susceptibility, according to previous phenotypic observations and organ classification. Indeed, CC leaves were characterized by some limited attack areas with sparse sporulation and necrotic dots, while the CC inflorescences by abundant and localized sporulation. Moreover, CS organs were both characterized by an abundant and widespread sporulation. Afterward, H2O2 production was histochemically evaluated by DAB staining. Its accumulation was clearly visible only on the surface of the CC leaves, suggesting that only the mid-resistant organ could be able to activate H2O2 production that interferes with pathogen growth and diffusion. There results have led me to investigate the different interaction between P. viticola and two grapevine organs at ultrastructural level. Interestingly, three zones showing different response to P. viticola-inoculation in CC leaf tissues were found: the leaf areas in correspondence to pathogen sporulation, the leaf areas corresponding to the border between green and brown spots, and the necrosis zones. CC inflorescence tissues presented two types of pathogen-response zones: areas in which pathogen structures were deformed and the necrosis areas. Conversely, P. viticola was typically structured in both CS tissues. Encouraged by the preliminary results and since the divergent dual epidemics are poorly studied on a molecular point of view, I have decided to perform a trascriptomics study to identify the differential expressed genes (DEGs) in CC organs both at baseline and upon P. viticola-inoculation, using CS as a reference. DEGs were grouped in 14 clusters based on their expression profiles. Interestingly, in the mid-resistant leaves of CC, there was an up-regulation of genes related to secondary metabolism, in particular genes belonging to signal transduction, phytoalexin biosynthesis and oxidative stress response. Meanwhile the genes involved in the protein modification and secondary metabolic process were upregulated in the susceptible leaves of CS. Genes implicated in the secondary metabolism, response to stimuli, protein modification, carbohydrate metabolism and reproduction were up-regulated in the mid-susceptible inflorescences of CC, while only a weak response involving genes of secondary metabolism and response to stimuli was found in the susceptible inflorescences of CS. Among genes down-regulated upon P. viticolainoculation, only in CC inflorescence the biological processes functional were significantly enriched. This cluster presented a down-regulation of genes involved in photosynthesis, generation of precursor metabolites and energy, and carbohydrate metabolic process suggesting a broad reduction of the photosynthetic process. Finally, to validate the organ-specific response to P. viticola-inoculation, the disease development and the expression level of some DEGs have been evaluated on an independent experiment, using the two studied genotypes, the reference V. vinifera variety Pinot noir and the CC-related Vitis hybrid Muscaris