GRAPEVINE-ASSOCIATED MICROORGANISMS AS BIOCONTROL AGENTS AGAINST THE PROLIFERATION OF PATHOGENIC FUNGI

This PhD project aim at selecting potential biocontrol agents able to prevent, or limit, the infection of grapevine and grape (table and wine) by pathogenic fungi, in pre- harvest periods, in order to set an eco-friendly plant protection strategy as alternative to reduce both the use of chemicals fungicides in vineyard and possible residues in wine. The project will investigate the putative mode of actions of selected endophytic species to counteract the pathogen proliferation and will evaluate how to exploit these metabolic traits to optimize the antagonistic effect under different plant growth conditions and grape management. Chapter 1 focuses on developing techniques for isolating grapevine endophytes, both cultivable and non-cultivable. A total of 148 endophytes were isolated and cultivated, including 42 fungi and 106 bacteria. High-frequency isolates included Ralstonia pickettii and Cladosporium allicinum. Metabarcoding analysis revealed 78 bacterial and 72 fungal species, highlighting genera such as Sphingomonas and Cladosporium. Biodiversity analysis showed variability in microbial communities related to grapevine species, vineyard management, and geography, but no significant differences were found. Chapter 2 assesses the biocontrol efficacy of the isolated endophytes against three pathogenic fungi: Penicillium glabrum, Aspergillus carbonarius, and Botrytis cinerea. The study found that A. pullulans strains ED203, ED206, ED217, ED221 and B. velezensis ED163 effectively antagonized all three pathogens, with this least showing the highest inhibition rates. Tolerance tests showed that these BCAs can survive in presence of copper (up to 100 mg/L) and commercial fungicide SWITCH (up to 1 g/L), although ED163 was inhibited by both. All BCAs were sensitive to an oenological dose of sulphur dioxide (40 mg/L). VOCs analysis revealing the production of compounds with antifungal properties as alcohols, esters and terpenes and significant biofilm production and enzymatic activities (glucanase and chitinase) enhanced antagonistic effects of candidates. Chapter 3 investigates BCA-plant interactions using plant cell cultures and in vivo experiments. The ability of BCAs to limit B. cinerea infection was tested on V. vinifera berries, leaves, and pot plants. In vivo trials revealed that ED163 completely inhibited pathogen growth on grape leaves, while ED221 provided partial inhibition. No significant differences in plant health or growth were observed between plants treated with BCAs and those treated with sterile water. Callus cultures from grape leaves were used to study interactions between ED163, host, and pathogen. Scanning electron microscopy observation showed an interaction between leaves cells and ED163 which formed a patina on cells surfaces, while B. cinerea spores adhered in clumps. VOCs analysis showed variations in compound profiles over time, with increased production of compounds like octanoic acid and phenacyl thiocyanate by ED163 in co-culture with plant cells and B. cinerea. Gene expression analysis of the phenylpropanoid pathway indicated increased expression of the resveratrol transporter gene in grapevine leaves inoculated with both B. cinerea and B. velezensis, suggesting enhanced plant defensive responses. In conclusion, B. velezensis ED163 is a valid candidate for further investigation in vivo applications, as it shows significant promise for adopting a sustainable biological approach to reduce the use of chemical pesticides.