Evaluation of Biocontrol Efficacy by Competing Yeasts

Botrytis cinerea causes gray mold across a wide range of crops and postharvest contexts. While integrated agronomic practices help, chemical fungicides are still often required, raising concerns about resistance development and environmental impact. Biological control agents (BCAs), particularly non-Saccharomyces yeasts, offer advantages such as simple nutritional needs, surface colonization potential, and lower risk of mycotoxin production. A robust, rapid, and cost-effective in vitro screening workflow is essential to identify promising BCAs that inhibit B. cinerea through fungistatic diffusible substances or volatile organic compounds (VOCs). This manuscript presents two complementary assays-the Cellophane Agar Layer technique (CALt) and the Sandwich Dual Culture technique (SDCt)-as practical tools for high-throughput screening and preliminary mechanism characterization. Methods are described in detail. CALt assesses the production of diffusible antifungal substances by yeasts by placing a cellophane layer over YPDA, applying yeast suspensions, and subsequently introducing B. cinerea to measure changes in fungal colony extension. SDCt evaluates the influence of yeast-emitted VOCs by sandwiching a B. cinerea mycelial disk between two plates containing 48-h-old yeasts and sealed to permit VOC diffusion. In both assays, daily mycelial growth of B. cinerea is recorded and analyzed as colony diameter growth over time, with results presented as mean ± standard deviation. The materials, media compositions (YPD and YPDA), and step-by-step protocols for yeast preparation, plate setup, inoculation, incubation conditions, and outcome calculations are provided to enable rapid replication and adoption in screening programs. Together, CALt and SDCt offer a streamlined framework to identify yeast BCAs with strong antagonistic activity and to gain preliminary insights into their mechanisms of action, informing subsequent validation under commercial conditions.