Protein characterization reveals cell wall-degrading enzymes underlying antifungal activity of Bacillus velezensis against Botrytis cinerea and Ascosphaera apis

Bacterial biocontrol agents offer environmentally sustainable alternatives to chemical fungicides for managing plant and livestock pathogen diseases. This study screened twelve bacterial isolates for antifungal activity against Botrytis cinerea and Ascosphaera apis using antagonistic assays. Seven isolates demonstrated significant inhibitory effects, with Bacillus velezensis SSH60 showing overall robust biocontrol efficacy against B. cinerea and A. apis . Cell-free supernatant concentrates from SSH60 showed dose-dependent antifungal activity, confirming that bioactive compounds are secreted extracellularly. In vivo validation using tomato fruits demonstrated practical biocontrol potential, with SSH60 crude protein extract reducing B. cinerea lesion size by 90 %. To identify the active antifungal components, extracellular proteins were fractionated by ammonium sulfate and fast protein liquid chromatography (FPLC). The 25–50 % ammonium sulfate fraction of SSH60 supernatant exhibited the strongest antifungal activity against both pathogens. Protein characterization identified three major bioactive enzymes: subtilisin or β-1,3-1,4-glucanase (∼27 kDa), α-amylase (∼60 kDa), and β-mannosidase (∼40 kDa). These enzymes target different fungal cell wall components through proteolytic degradation, polysaccharide hydrolysis, and mannoprotein disruption, representing a multi-enzyme biocontrol mechanism. Phylogenetic analysis confirmed SSH60 as B. velezensis . The activity of SSH60 against both plant and insect fungal pathogens represents a new approach to broad-spectrum biocontrol. This study demonstrates that B. velezensis SSH60 produces antifungal enzymes with potential for sustainable, dual-purpose biocontrol applications against economically important fungal pathogens in integrated disease management systems where both crop protection and pollinator health are priorities. • Bacillus velezensis SSH60 demonstrated dual biocontrol efficacy against fungal diseases affecting crops and honeybees. • Three cell wall-degrading enzymes in culture filtrate identified: subtilisin/glucanase, α-amylase, β-mannosidase. • Multi-enzyme mechanism degrades fungal walls via disruption of proteins, polysaccharides, and mannoproteins. • In vivo validation in tomato fruits showed bacterial proteins reduced B. cinerea lesion area by 90 %. • Extracellular protein-based biocontrol offers sustainable alternative to chemical fungicides with defined mechanisms.