From genome to field: Bacillus halotolerans BJ-3 as a novel biocontrol solution for grapevine Botryosphaeria dieback

Grapevine trunk diseases (GTD), particularly Botryosphaeria dieback caused by Lasiodiplodia theobromae , pose a significant threat to global viticulture. Current study aimed to develop a biocontrol solution using the locally isolated BJ-3 bacterial strain. We assessed the genomic biocontrol potential of BJ-3 through genome mining, evaluated its efficacy against 19 fungal pathogenic strains in vitro , and validated its efficiency against L. theobromae through spore inhibition assay, grapevine shoot inoculation and field experiments. Based on morphological characteristics and phylogenomic analysis, BJ-3 was identified as Bacillus halotolerans . Genome mining revealed eleven antimicrobial biosynthesis gene clusters, encoding secondary metabolites including bacillibactin, bacilysin, bacillaene, fengycin, subsilon, aurantinin, thailanstatin, and laterocidine; many of which are associated with biocontrol activities. In vitro assays demonstrated BJ-3′s broad-spectrum antagonistic capacity, with inhibition rates ranging from 54.74% to 91.62% against economically important GTD pathogens, including Botryosphaeria dothidea (76.18%), Neofusicoccum parvum (70.59%), Diaporthe sojae (91.62%), D. eres (81.31%), and L. theobromae (69.89%). In the spore germination assay, BJ-3 achieved 98.47% inhibition at 4×10 7 CFU/mL after 10-12 hours of incubation. Preventive treatments with BJ-3 at a concentration of 1.08×10 8 CFU/mL resulted in 74.39% control efficiency against L. theobromae . In the field experiment, BJ-3 effectively controlled Botryosphaeria dieback disease, with an inhibition rate of 71.94 %. These findings highlight the potential of the B. halotolerans BJ-3 strain as a biofungicide for managing Botryosphaeria dieback disease in grapevine, providing a foundation for developing B. halotolerans BJ-3-based antimicrobial secondary metabolites in future studies. (248 words)