Abstract: This study developed a composite freeze-dried biocontrol agent for controlling postharvest gray mold on strawberry by co-culturing three antagonistic yeasts (Pichia guilliermondii, Meyerozyma caribbica, and Pichia rarassimilans). The nutritional conditions for mixed culture and the formulation of cryoprotectants were optimized using dual-response surface methodologies to enhance the biocontrol efficacy and storage stability of the agent. The results showed that the optimal biomass production was achieved when the yeasts were cultured at a ratio of 3:1:2 (v/v/v) in a medium containing sucrose (15.49 g/L), yeast extract (16.73 g/L), MgSO? (4.74 g/L), and KH?PO? (3.34 g/L), under conditions of pH 6.0, 2% inoculation volume, at 24 °C for 60 h. The composite agent, protected with 9.63% skim milk powder, 14.61% xylooligosaccharides, and 21.82% inulin, retained a high survival rate of 64% after freeze-drying. Scanning electron microscopy observations indicated that this protective formulation effectively maintained the integrity of yeast cell morphology, minimizing damage caused by freeze-drying. The agent maintained high biological activity (4.07×10? CFU/mL) after 5 months of storage at ?20 °C. Application experiments confirmed that the composite freeze-dried agent could rapidly colonize strawberry wounds, effectively suppress infection by Botrytis cinerea, and significantly delay spore germination and mycelial growth of the pathogen. This study provides an efficient and stable microbial preparation strategy for the green control of postharvest gray mold in strawberry.

Key words: Composite yeast agent, Strawberry gray mold, Biocontrol, Dual-response surface optimization