Solanum lycopersicum, a prominent vegetable crop, suffers major yield losses from early blight disease driven by Alternaria solani. Sustainable alternatives to conventional chemical fungicides are urgently needed to mitigate these losses while minimizing environmental impact. This study investigates the application of the microbial consortium derived from Gir cow dung for enhancing tomato resistance against early blight. Metagenomic analysis of Gir cow dung revealed a rich diversity of beneficial microbes, with Bacillus species being one of the prominent. Three key strains Bacillus subtilis, Bacillus tequilensis, and Bacillus licheniformis were isolated, characterized, and tested for their antagonistic activity against A. solani. The microbial consortium exhibited significant antifungal properties, and the application of the consortium on tomato plants led to a marked reduction in disease progression. Histochemical analyses of treated plants revealed a pronounced increase of hydrogen peroxide, phenolic compounds, and superoxide dismutase, suggesting enhanced activation of plant defense pathways. Comparative gene expression profiling showed the upregulation of key defense-related genes, including PR2b, Chi3, and Pto kinase, in Bo-treated plants, indicating a systemic activation of host defense mechanisms. Untargeted metabolomic analysis using LC-MS/MS further demonstrated significant metabolic reprogramming in microbial consortium-treated plants. Pathway enrichment analysis revealed enhanced accumulation of secondary metabolites, including phenolics and terpenoids, which are known to play vital roles in pathogen defense. Additionally, hierarchical clustering of metabolite profiles highlighted distinct metabolic signatures between Bo-treated and control plants, underscoring the broad biochemical impact of the microbial treatment. Collectively, our findings demonstrate that the microbial consortium derived from Gir cow dung significantly enhances tomato plant resistance to A. solani through multifaceted biochemical, molecular, and metabolic mechanisms. This study provides a sustainable biocontrol strategy that could effectively replace chemical fungicides, contributing to resilient tomato production and advancing the field of agroecological plant disease management.
