Bacillus species compatibility enhances VOC-mediated systemic resistance against Botrytis cinerea

Botrytis cinerea, the causal agent of gray mold, poses a significant threat to global tomato production. This study explores the role of volatile organic compounds (VOCs) from three Bacillus strains in eliciting systemic resistance in tomato plants against gray mold. We tested VOCs from individual strains (LNXM12, GBAC46, LLTC93) as well as from both compatible and incompatible combinations of these bacteria. The result showed that compatible strains exhibited mutual growth, while incompatible strains displayed growth antagonism. Our findings further revealed that VOCs from both compatible and incompatible Bacillus spp. inhibited fungal mycelial growth, with significantly stronger suppression observed in compatible Bacillus spp. (54.05%) compared to incompatible Bacillus spp. (42.10%). Scanning electron microscopy (SEM) revealed that VOCs were produced by Bacillus spp. induced clear structural alterations in B. cinerea hyphae, with compatible strains causing more severe damage, including shrinking, twisting, and curling. Fungal hyphae exposed to VOCs from compatible Bacillus spp. also showed higher levels of reactive oxygen species (ROS) accumulation. In detached leaf assays, VOCs from both compatible and incompatible strains significantly reduced gray mold severity, with compatible Bacillus spp. providing stronger protection. Moreover, exposure to VOCs from incompatible and compatible strains resulted in a marked reduction in the oxidative stress marker malondialdehyde (MDA) (31.54% and 27.61%, respectively), along with decreased hydrogen peroxide (H₂O₂) levels (33.34% and 53.12%, respectively). Gene expression analysis demonstrated that VOCs from compatible Bacillus spp. upregulated all six tested defense-related genes (PR1, PR3, MPK6, LOX1, EF1, and PAL) in tomato plants challenged with B. cinerea, whereas VOCs from incompatible strains only induced PR1, PR3, and PAL. VOC profiling further revealed that compatible strains produced a broader spectrum of compounds, including alcohols, alkanes, and ketones, while incompatible strains primarily released alkanes and alkenes. Collectively, these findings highlight that strain compatibility plays a pivotal role in VOC-mediated antifungal activity and the induction of systemic resistance (ISR) in tomato plants. Thus, compatible Bacillus spp. represent promising eco-friendly biocontrol agents against gray mold.