Multi-marker analysis elucidates genetic diversity and antifungal mechanisms of native Bacillus thuringiensis strains against Agroathelia rolfsi

Abstract Bacillus thuringiensis is a ubiquitous, widely studied biocontrol agent primarily recognized for its insecticidal properties; however, its antifungal potential remains largely underexplored. Additionally, differentiating between its strains remains challenging, despite the availability of several genetic fingerprinting techniques. In this study, 50 native B. thuringiensis isolates were obtained from the Western Ghats and the North Eastern Region of India. These isolates exhibited bipyramidal (100%), spherical (76%), and cubical (12%) crystal morphologies. Genetic characterization using repetitive element polymorphism fingerprinting revealed diverse profiles. Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR produced a distinct 100–200 bp amplicon in all isolates, while BOX-A1R-based repetitive extragenic palindrome (BOX) displayed the highest diversity with 42 electrophoretic patterns, followed by Repetitive Extragenic Palindromes (REP) (23) and ERIC (17). Combined cluster analysis at a 48% similarity threshold grouped the strains into 10 clusters, with 70% clustering based on geographic origin, further supported by Principal Coordinate Analysis. The cophenetic correlation coefficient of 0.64 indicated a reliable fit between clustering and data matrices. Siderophore production and chitinase activity were detected in a total of 15 and 23 strains, respectively. Additionally, all strains tested positive in the oil spreading assay, confirming lipopeptide production. In vitro antifungal assays demonstrated complete inhibition of the mycelial growth of Agroathelia rolfsii ranging from lipopeptide concentration of 50.00–87.5 µL/mL. Overall, this study establishes a comprehensive genetic and functional characterisation of native B. thuringiensis isolates, identifies strains with significant antifungal potential against A. rolfsii , and provides a valuable foundation for future bioformulation development and integrated biological control strategies.