Isolation of Bacillus amyloliquefaciens D39 and Identification of Its Antimicrobial Proteins Active Against Chestnut Blight

Chestnut blight, caused by <i>Cryphonectria parasitica</i> (Murrill) M.E. Bar, is a destructive fungal disease threatening chestnut cultivation and production. In response to the limitations of chemical control, biological control using antagonistic microbes has gained increasing attention. A rhizosphere-derived bacterium, strain D39, was isolated from healthy chestnut trees and identified as <i>Bacillus amyloliquefaciens</i> based on morphological characteristics and the phylogenetic analysis of 16S rRNA and <i>gyrA</i> genes. The antifungal activity of strain D39 against <i>C. parasitica</i> was evaluated using dual-culture and double-layer Oxford cup assays. The strain exhibited broad-spectrum and stable antagonistic effects and harbored five key genes associated with antimicrobial compound biosynthesis (<i>srfAA</i>, <i>ituC</i>, <i>fenD</i>, <i>bmyB</i>, and <i>bacA</i>), as confirmed by PCR. A 145 kDa extracellular protein with strong antifungal activity was extracted and purified by ammonium sulfate precipitation, DEAE ion-exchange chromatography, and Sephadex G-75 gel filtration. LC-MS analysis identified the protein as a serine peptidase belonging to the S8 family, and its structure was predicted using multiple bioinformatic tools. In pot experiments, treatment with the strain D39 significantly reduced disease severity, achieving control efficiencies of 66.07% and 70.89% at 10 and 20 days post-inoculation, respectively. These results demonstrate that <i>B. amyloliquefaciens</i> D39 has strong potential as a biocontrol agent against chestnut blight, offering an effective and environmentally friendly alternative for disease management.