Distinct physiological strategies underpin antagonism of native Pichia yeasts against Botrytis cinerea

Botrytis cinerea causes severe losses in viticulture and postharvest systems, requiring biological control agents adapted to the grape environment. Here, two native epiphytic yeasts, Pichia kluyveri B(F8) and Pichia kudriavzevii C(A4), were assessed for antagonistic activity against B. cinerea strains B05.10 and 992 using dual-culture and volatile-mediated in vitro assays, followed by evaluation of antagonistic activity on wine and table grape berries. Both yeasts markedly inhibited mycelial growth and significantly reduced disease severity on fruit. Additional physiological insight was obtained through integrated volatilomic and extracellular proteome analyses using B. cinerea B05.10. GC-MS/SPME profiling revealed pronounced remodeling of yeast volatile profiles, in which esters, higher alcohols and organic acids represented the largest fractions of the detected compounds, with patterns strongly modulated by context, including substrate and interaction timing. Volatilome differences were most pronounced on grape berries under preventive conditions, particularly in P. kudriavzevii C(A4). Analysis of the extracellular proteome revealed sharply contrasting physiological responses between strains: P. kudriavzevii exhibited a broadly inductive profile involving energy metabolism, cell-wall remodeling and stress responses, whereas P. kluyveri displayed a predominantly repressive proteome with selective upregulation of ubiquitin-related proteins, consistent with a maintenance-oriented strategy. Together, these results provide integrated volatilomic and proteomic evidence that comparable antagonistic outcomes may be accompanied by distinct physiological programs, offering new insight into yeast-Botrytis interactions and supporting the potential of native Pichia strains for biological control of gray mold.