Abstract: Canker disease of Carya cathayensis caused by Botryosphaeria dothidea is one of the most serious diseases affecting hickory plant in China. To effectively prevent and control the occurrence and development of this disease, we previously isolated an endophytic bacterium (strain ZL34) with strong antimicrobial activity against B. dothidea from the root of C. cathayensis. Taxonomic classification, antimicrobial characteristics of fermentation products, and optimization of fermentation medium of the strain ZL34 were investigated in this study. Firstly, based on its morphological and physiological characteristics combined with molecular biological analysis, the strain ZL34 was identified as Bacillus velezensis. Antimicrobial spectrum analysis revealed that, in addition to exhibiting strong antimicrobial activity against the canker disease pathogen B. dothidea, the fermentation supernatant of ZL34 also had significant antimicrobial effects against various pathogenic fungi and bacteria, including Fusarium oxysporum and Staphylococcus aureus. The fermentation supernatant of ZL34 significantly inhibited the growth of B. dothidea mycelia, with a 5-fold dilution inhibiting mycelial growth by 96.48%, although it had no noticeable effect on spore germination. The inhibited B. dothidea mycelia accumulated pigments markedly, showed abnormal hyphal protrusions and swelling, and a large number of conidia were produced from the substrate mycelia. Finally, we investigated the effect of medium components on the sporulation and active production of ZL34 through orthogonal design experiments, obtaining the optimal medium formulation. It was composed of 40 g/L soybean powder, 40 g/L corn flour, 5.0 g/L CO(NH₂)₂, 6.0 g/L(NH₄)₂HPO₄, 3.5 g/L CaCO₃ and pH 7.0. In the optimized medium, the viable bacteria count in the fermentation broth reached 6.97×10⁹ CFU/mL, a 44.79% increase compared to the medium before optimization. The 20-fold diluted supernatant had an antimicrobial rate of 87.16% against B. dothidea, a 45.41% increase compared to the supernatant before optimization. The above results indicated that B. velezensis ZL34 had strong inhibition activity against B. dothidea, and the medium formula of the strain was optimized with higher endospore production and antimicrobial activity. This research lays a solid foundation for the successful development of B. velezensis ZL34 into biological control agents against canker disease.

Key words: Botryosphaeria dothidea, Bacillus velezensis, identification, antimicrobial activity, Carya cathayensis