Integrative GC–MS and In Silico Analysis of Bacillus subtilis Metabolites Reveals Protein Focused Antifungal Action Against Virulence Proteins of Fusarium oxysporum

The exploration of Bacillus spp. as eco-compatible bioresources offers a promising frontier in antifungal biotechnology. In the present study, Bacillus subtilis Bs-06, isolated from the rhizosphere of healthy tomato plants, demonstrated robust antagonistic potential through the secretion of diverse bioactive secondary metabolites. GC-MS analysis of its culture extract revealed ten key compounds, notably phenol, 3,5-bis(1,1-dimethylethyl)-, palmitic acid, oleic acid, and octadecanoic acid, all previously reported for their antimicrobial properties. To elucidate the molecular mechanism underlying this antagonism, a structure-based in silico approach was employed, targeting essential virulence proteins Cutinase (5AJH), Avr1/SIX4 (7T6A), and Avr3/SIX1 (7T69). Molecular docking simulations revealed high binding affinities between these Bacillus derived ligands and the target proteins, with phenol, 3,5-bis(1,1-dimethylethyl)- exhibiting the strongest interaction (–11.53 kcal/mol) toward Avr3. Hydrogen bonding and hydrophobic interactions stabilized these ligand–protein complexes, indicating potential inhibition of pathogenic functions. Further, normal mode analysis (NMA) using the iMODS platform evaluated the dynamic behavior and structural rigidity of ligand-bound complexes. Avr1 exhibited the highest flexibility, while Cutinase demonstrated the most structural rigidity, as reflected in eigenvalues and deformability profiles. These findings position B. subtilis Bs-06 as a reservoir of antifungal compounds capable of targeting and destabilizing virulence-associated proteins at the molecular level. The integration of GC-MS profiling, docking studies, and structural simulations presents a powerful framework for discovering and characterizing microbial biocontrol agents.