Walnut is a significant economic tree species worldwide. Walnut anthracnose, caused by the pathogen Colletotrichum gloeosporioides, greatly reduces walnut production and economic benefits. Our study showed that Bacillus velezensis effectively halted the growth of C. gloeosporioides, inducing noticeable abnormalities such as hyphal breakage and distortion, thereby curtailing the pathogen’s virulence. A 50–100 times dilution of B. velezensis fermentation broth, applied every two to three days, served as an efficient protective layer for walnut leaves and fruits against C. gloeosporioides infection. Transcriptomic analysis of B. velezensis unveiled its dynamic response against C. gloeosporioides. On the second day, B. velezensis upregulated a significant number of differentially expressed genes related to the synthesis of metabolic products, amino acid biosynthesis, and motility. On the fourth day, continuous synthesis of metabolic products and amino acids, along with differential expression of spore-related genes, was observed. By the sixth day, the focus shifted towards environmental adaptation and carbon source utilization. Throughout the process, B. velezensis likely employed strategies such as the release of metabolic products, increased chemotaxis, and nutrient competition to exert its antagonistic effect on C. gloeosporioides. Fluorescence quantitative results showed that 15 primer pairs were up-regulated and 15 were down-regulated, with a 100% similarity rate to transcriptome sequencing results, confirming their authenticity. These findings provided a foundation for the widespread application of B. velezensis as a biocontrol agent in agriculture and forestry.

Chestnut blight, caused by Cryphonectria parasitica (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 Bacillus amyloliquefaciens based on morphological characteristics and the phylogenetic analysis of 16S rRNA and gyrA genes. The antifungal activity of strain D39 against C. parasitica 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 (srfAA, ituC, fenD, bmyB, and bacA), 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 B. amyloliquefaciens D39 has strong potential as a biocontrol agent against chestnut blight, offering an effective and environmentally friendly alternative for disease management.