Efficient Breeding and Metabolomic Characterization of Bacillus safensis Mutants with High-Antagonistic Activity Based on Droplet Microfluidics Technology

The limited antimicrobial efficacy of biocontrol microorganisms often constrains their practical implementation in crop disease management. To address this challenge, we developed an integrated high-throughput screening platform combining atmospheric and room temperature plasma (ARTP) mutagenesis with droplet microfluidics, which efficiently selected Bacillus safensis mutants with high-antagonistic activity from ∼10 6 variants by low fluorescence screening of GFP-labeled Erwinia carotovora Ecc15 (GFP-Ecc15) in microdroplets. The optimal mutant R1 demonstrated enhanced broad-spectrum antagonistic activity against Ecc15 and nine phytopathogens. Untargeted metabolomic analysis identified quinolinic acid and clindamycin as key synergistic antimicrobial metabolites in R1. HPLC and in vitro assays confirmed their biosynthesis and synergistic efficacy, contributing to R1’s improved antimicrobial performance. This study represents the first report of microbial-derived clindamycin biosynthesis and provides systematic characterization of quinolinic acid’s antimicrobial properties. Our work provides a feasible strategy for breeding high-performance biocontrol strains while offering mechanistic insights into metabolite-mediated antagonism, advancing the design of next-generation microbial pesticides.