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Botrytis cinerea poses a recurring threat to viticulture, causing significant yield losses each year. The study explored the biocontrol capabilities of commercially used winemaking yeasts as a strategy to manage B. cinerea in grape berries. The winemaking yeast strains— Saccharomyces cerevisiae ES181, Saccharomyces pastorianus KBG6, S. cerevisiae BCS103, Lachancea thermotolerans Omega, and Torulaspora delbrueckii TD291-reduced B. cinerea growth and conidiation in vitro. Furthermore, they demonstrated a decreased disease severity and number of conidia in grape berries. Among these strains, S. cerevisiae BCS103 was the most effective, inducing the expression of the defense-related gene PR4 in berries. Its diffusible compounds and volatile organic compounds also reduced the expression of BcLTF2 , a positive regulator of B. cinerea conidiogenesis. The examined winemaking yeast strains, especially S. cerevisiae BCS103, demonstrated effective inhibition of B. cinerea in vitro and in grape berries, influencing key defense genes and reducing BcLTF2 expression, offering potential solutions for disease management in viticulture. The study underscores the promise of commercially available winemaking yeast strains as eco-friendly tools against B. cinerea in viticulture. Leveraging their safety and existing use in winemaking offers a potential avenue for sustainable disease management. [ABSTRACT FROM AUTHOR]