Abstract
Late blight, caused by Phytophthora infestans, is one of the most destructive oomycete diseases affecting global potato and tomato production, leading to annual economic losses exceeding $6 billion. Overreliance on synthetic fungicides has raised serious environmental and resistance-related concerns, highlighting the need for sustainable biological alternatives. This study aimed to isolate and characterize antagonistic rhizobacteria and evaluate their bioactive metabolites as potential inhibitors of the CRN8 effector protein of P. infestans using computational approaches. Six bacterial isolates were recovered from rhizospheric soil collected from the Bangladesh Rice Research Institute (BRRI), Rajshahi, Bangladesh. Among them, Isolate-2 exhibited the strongest antagonistic activity, causing approximately 54% mycelial growth inhibition against P. infestans in dual-culture assays, and was identified as Bacillus subtilis NJ01 based on morphological, biochemical, and 16S rRNA gene sequence analyses. The three-dimensional structure of the CRN8 protein was predicted using AlphaFold2 and validated using PROCHECK, ERRAT2, and Verify3D. Virtual screening of 76 reported B. subtilis-derived antimicrobial compounds identified three compounds: Androst-5-en-3-ol, 4,4-dimethyl-, (3beta)-, 7-O-Malonyl macrolactin A, and Simiarenol, with predicted binding affinities of -8.4, -8.1, and -8.0 kcal/mol, respectively. ADMET (absorption, distribution, metabolism, excretion, and toxicity) profiling indicated favorable drug-likeness and generally favorable predicted toxicity profiles among the selected compounds, although compound-specific differences were observed. POM analysis of five representative compounds (C1-C5) revealed distinct predicted bioactivity profiles across multiple target classes, providing additional insights into their potential molecular features and biological relevance. Furthermore, 100 ns molecular dynamics simulations supported stable protein-ligand interactions, as indicated by RMSD, RMSF, radius of gyration, SASA, principal component analysis (PCA), and free energy landscape analyses. Collectively, these findings suggest that B. subtilis NJ01 possesses promising biocontrol potential and that its reported metabolites warrant further experimental isolation and validation as potential inhibitors of the CRN8 effector protein for the sustainable management of late blight disease.