Viticulture is a multibillion-dollar market based on grapes, one of the most valuable fruits in agriculture, but it is also characterized by the highest uses of fungicides per hectare for disease management. In fact, despite the availability of genetic resources resistant to the most diffuse fungal pathogens, those cover less than a fifth of the worldwide vine area. The remaining 80% is represented mainly by susceptible Vitis vinifera L. varieties. Instead, re-sistance traits are known to derive from other Vitis species, due to their coevolution with the pathogens, and their exploitation in breeding is a valuable strategy to reduce pesticide treat-ments. Black rot (BR) is a destructive disease caused by the ascomycete, Phyllosticta ampeli-cida, whose telomorphic form was known as Guignardia bidwellii. In the European continental area in the last two decades its pressure increased due to the concomitant decreased usage of chemicals and the occurrence of mild-rainy summer because of the climate change. This work was established to fight the spread of BR by means of the introduction of BR resistance in susceptible genetic background. The study consisted of (i) the reconstruction of BR resistant donor pedigree based on the collection of historical phenotypic data, (ii) the eval-uation of parental lines and selections of the breeding program of the Edmund Mach Founda-tion (Italy), (iii) the microscopic inspection of disease progression, and (iv) QTL analysis of the segregating population (N=146) ‘Merzling’ (hybrid, resistant) × ‘Teroldego’ (V. vinifera, suscep-tible). The outcomes revealed a large unexplored pool of disease resistance donors of 148 va-rieties belonging to 14 different species, that permitted to evaluate and identify five new promising genotypes readily exploited for breeding. Concurrently, a new inoculation method based on spore production from fresh infected leaves was developed. Ex vivo (detached leaves) inoculation did not reveal significant differences among the assessed genotypes and highlight-ed the tendency of spores to accumulate and germinate near leaf hairs, while disease progres-sion did not occur. The screening of the segregating population, both under greenhouse and field conditions, allowed the discrimination on chromosome 14 between two distinct QTLs as-sociated with leaf/shoot and bunch resistance. The first QTL confirmed the Resistance to Gui-gnardia bidwellii (Rgb)1 locus previously identified by three studies and the high-density link-age mapping allowed to reduce it from 2.4 to 0.7 Mb along the PN40024.v4 reference genome. The region resulted enriched in genes belonging to phloem dynamics and mitochondrial pro-ton transfer. The second QTL associated with bunch resistance was designated Rgb3. Located at 9 cM (6 Mb) upstream Rgb1, it was characterized by a cluster of Germin-like protein 3 genes and lipid transfer and localization, notably known to be linked to broad spectrum disease re-sistance and stress response. No resistance (R)-genes have been annotated in the region under-lying the QTL in the PN40024 reference genome. In conclusion, this work provided new insights for grapevine breeding programs by the identification of previously unknown BR resistance donors, the development of protocols and good practices towards large-scale resistance screening as well as the discovery of a novel QTL associated with bunch resistance. Marker validation in different genetic backgrounds is ongoing for its routinary implementation in marker-assisted breeding and further studies are planned to dissect BR resistance mechanism by the sequencing of the genomic regions, the study of the Germin-like 3 gene cluster and the cell wall characterization.

Grapevine (Vitis spp.) is one of the most widely cultivated perennial fruit crops in the world and its economic relevance is mainly related to wine production. In recent years, the increased frequency of extreme phenomena such as heat waves has been acknowledged as one of the most significant climate variables negatively affecting grape yield and berry composition, with consequences also on wine quality. Thus, studying the physiological, metabolic and genetic factors that are involved in grapevine response to high temperatures is essential to improve the knowledge of mechanisms underlying thermotolerance, aiming to support plant breeding innovation and the development of new management strategies in viticulture. In this work, a segregating population obtained from the crossing of ‘Rhine Riesling’ and ‘Cabernet Sauvignon’ was studied in the field with a multidisciplinary approach. The progeny (around 120 genotypes) was evaluated for phenological traits affected by changing temperatures, in particular bud burst, flowering and véraison, while physiological response to heat stress was assessed in various hot summer days by measuring chlorophyll fluorescence kinetics and stomatal conductance. Measures were collected in the early morning as control and in the afternoon during hot hours. Phenotypic data were then used in combination with a high-density linkage map (average distance between adjacent markers 0.78 cM), previously developed using genotypic information from 139 individuals, to perform QTL analysis. Based on physiological responses to high temperatures, selected individuals showing contrasting behaviour, together with parental lines, were further studied in controlled conditions. In the field, in fact, plants may be subjected to combined stresses and changes in environmental conditions may heavily influence plants response. With the experiment in controlled condition, on the other hand, plants were stressed at higher temperatures, compared to the ones registered in the field, by maintaining all the other sources of variability constant. In the growth chamber plants were studied for their physiological response to heat stress by using the same approach adopted in the field. To better understand mechanisms involved in grapevine adaptation to heat stress conditions, individuals with contrasting behaviour were studied also for their metabolome modifications, both in the field and in controlled conditions. Volatile organic compounds (VOCs) were investigated with an untargeted approach applying conventional methods of analysis. Accumulation of VOCs in grapevine leaves was analysed using gas chromatography coupled with mass spectrometry (GC-MS) after a pre-concentration with a solid-phase micro-extraction (SPME) approach. On the other hand, VOCs emission during stress was investigated in controlled conditions thanks to the use of the Closed-Loop Stripping Analysis (CLSA) which allows the collection of VOCs directly emitted by plants. Analysis was then performed with GC-MS. Metabolic alterations of non-volatile compounds were examined with an untargeted analysis using high-performance liquid chromatography coupled with a high-resolution mass spectrometer equipped with an electrospray soft ionization (HPLC-HR-ESI-MS). In this work a metabolomic workflow was developed, starting from sample collection and extraction to sample analysis and data interpretation. The analytical method developed allowed the preliminary evaluation of leaf metabolome alterations due to stress factors. In fact, the use of a weak cation-exchange mixed mode column, in combination with a data dependent acquisition mode, allowed a first wide screening of both primary and secondary metabolites resulting in a good compromise for metabolic fingerprinting. QTL analysis on the segregating population allowed the identification of several QTLs, related to both phenological and physiological traits, with the discovery of interesting putative candidate genes for grapevine resilience to changing temperatures. This is the first time that a similar approach has been applied to a perennial fruit crop by analysing chlorophyll fluorescence and leaf transpiration traits related to heat stress. On the other hand, the multidisciplinary approach allowed the fine characterization of Rhine Riesling and Cabernet Sauvignon response to high temperatures, both in controlled and field conditions, a tentative classification of ‘tolerant’ and ‘susceptible’ progeny individuals and the identification of metabolic pathways altered during heat stress in the susceptible plants. Together with the implementation of a novel metabolomic workflow based on HPLC-HR-ESI-MS, this work represents a novelty in studies on grapevine response to changing temperatures, as it considered not only the berry metabolism but the resilience of the plants itself, paving the way for future studies on thermotolerance.