Integrated chemical and genomic analysis of lipopeptides produced by Bacillus velezensis CMRP4489 with antifungal activity

Background Bacillus velezensis CMRP4489 shows promise as a biological control agent thanks to its production of secondary metabolites with antimicrobial properties. Nevertheless, the specific compounds contributing to its antifungal effects are not fully understood. This study aimed to identify and characterise bioactive compounds from B. velezensis CMRP4489 that exhibit antifungal activity against Sclerotinia sclerotiorum , a major phytopathogen affecting various crops. Results We utilised vacuum liquid chromatography (VLC) and thin-layer chromatography (TLC) to separate and identify antifungal compounds present in the cell-free supernatant of B. velezensis CMRP4489. Initial antifungal activity was detected in fraction F2, but further purification led to the identification of fraction F6F, which displayed significantly enhanced activity, evident from inhibition halos measuring 26.25 ± 1.8 mm in disk diffusion assays. High-performance liquid chromatography (HPLC) revealed four subfractions, with F6F.1 demonstrating the highest activity, forming inhibition halos of 27.5 ± 2.0 mm. Through spectroscopic and spectrometric analysis, we identified F6F.1 as Bacillopeptin A and B, structurally similar lipopeptides. Electron microscopy confirmed that Bacillopeptins disrupt the hyphal structure of S. sclerotiorum , while minimal inhibitory concentration (MIC) assays showed that as low as 25 µg/mL could be effective. Additionally, genomic analysis using antiSMASH showed the presence of biosynthetic gene clusters (BGCs) in B. velezensis CMRP4489 linked to lipopeptide production, particularly those related to bacillopeptins, iturins, and fengycins, reinforcing the strain's potential for the production of diverse bioactive compounds. Conclusions This research is the first to illustrate the effectiveness of Bacillopeptins A and B from B. velezensis CMRP4489 against S. sclerotiorum , highlighting their strong antifungal properties and potential as biocontrol agents. The identification of pertinent BGCs suggests that B. velezensis CMRP4489 can be optimised for increased production of secondary metabolites. This study highlights the potential of Bacillopeptins A and B as alternatives to chemical fungicides, warranting further investigation in greenhouse and field trials to evaluate their effectiveness in real-world applications. Our findings provide important insights into the antimicrobial potential of B. velezensis CMRP4489 and emphasise its promise in sustainable crop protection strategies.