Smart Biotechnology: Recycling Grape Wastes for Sustainable Protection Against Fungal Pathogens in Berry Cultivation

Viticulture, a thriving industry focused on wine production, faces challenges such as resource-intensive practices and significant byproducts like grape pomace and pruning residuals. These byproducts, along with environmental and health concerns related to pesticide use, highlight the need for sustainable alternatives in terms of ecological management. To tackle these challenges, this project aims to optimize grape pomace as a growth medium for fungi producing double-stranded RNA (dsRNA), a promising tool for controlling grapevine diseases such as downy mildew and grey mold. Although in vitro production of dsRNA is feasible, it is costly. Additionally, the project seeks to induce plants to produce dsRNA. Furthermore, to confer resistance to fungal pathogens and introduce antifungal peptides, genome editing and knock-down techniques are employed. Indeed, also short peptides could be a sustainable treatment, reducing chemical pesticide dependency and preserving biodiversity. In this work, pomaces from Chardonnay grapes harvested in Franciacorta area were used. Pomaces were separated from stalks and treated with heat at different temperatures (50, 65, 75°C), followed by dehydration and reduction into fine dry particles. Microbiological analyses revealed microbial contamination below 10 cells/mL with both 65°C and 75°C treatments; while, enzymatic treatment with cellulase increased glucose concentration by about 25%. Incubation conditions were maintained for Saccharomyces cerevisiae EC1118 and Pichia pastoris GS115, with higher performance observed for the S. cerevisiae strain. A CRISPR/Cas9 approach was utilized to generate a S. cerevisiae EC1118 mutant for RNAseIII, enhancing the accumulation of specific dsRNA. Preliminary results of expression experiments verified the synthesis of dsRNA to silence VviLBDIf7, responsible for susceptibility to downy mildew in Vitis vinifera. Simultaneously, P. pastoris GS115 has been modified to produce small peptides with antifungal activities. In vivo tests will be performed. These efforts represent substantial advancements in sustainable viticulture and fruit crop management, fostering the principles of the circular economy.