Distinct temporal dynamics of tomato defense induced by surfactin and Bacillus subtilis against gray mold

Botrytis cinerea , the causal agent of gray mold, represents a major constraint to tomato production worldwide, driving the need for sustainable alternatives to chemical control. Although microbial metabolites are often regarded as key determinants of the biocontrol activity exerted by beneficial microorganisms, the extent to which purified metabolites reproduce the defense responses induced by their producing strains remains largely unresolved. This study investigated the individual and combined effects of Bacillus subtilis GB2 and its biosurfactant surfactin on fungal suppression and the priming of plant defense responses in tomato. Antifungal activity against B. cinerea was first evaluated through in vitro plate and dual-culture assays. Subsequently, tomato plants were treated with surfactin (foliar application), GB2 (root inoculation), or their combination (ENT), and analyzed in two independent experiments under non-infected and pathogen-challenged conditions. In vitro assays showed that surfactin strongly inhibited B. cinerea growth (90% at 24 h; 60% at 7 days), while B. subtilis GB2 showed moderate antagonism (∼39%) and induced structural alterations in fungal hyphae. Plant growth was not negatively affected and was often enhanced, particularly under GB2 root-inoculation and combined treatments. Surfactin induced early responses, whereas GB2 triggered stronger and more sustained effects, especially under pathogen pressure. These responses were associated with enhanced antioxidant activity and differential modulation of marker genes associated with ethylene-, jasmonate-, and salicylic acid-dependent defense pathways. The combined treatment did not enhance defense responses beyond those induced by the individual treatments under the conditions evaluated. These findings demonstrate that the defense responses induced by a living microbial inoculant are not fully reproduced by its purified metabolite, highlighting distinct temporal patterns of defense activation. Overall, this study provides a mechanistic and temporal framework for the targeted use of microbial agents and their metabolites in more sustainable gray mold management.