Isolation and identification of antimicrobial compounds produced by Bacillus nakamurai CBAS-959 and their activity against phytopathogenic bacteria

The application of beneficial microorganisms is an effective solution for the control of crop diseases caused by phytopathogenic bacteria. Bacillus sp. plays a significant role as a biofactory of antimicrobial compounds, and bioformulations containing species of the Bacillus subtilis complex are commercialized. However, there is limited information on the chemical structural diversity of antimicrobial compounds produced by Bacillus nakamurai and its own potential use in agriculture as a suppressor agent of phytopathogenic bacteria. In this study, the bacterium strain CBAS-959, which produces diffusible black pigmentation in the growth medium, was identified as Bacillus nakamurai through biochemical characterization and the analysis of the 16S rRNA and gyrA genes. Co-culture assays confirmed a strong antagonistic effect against Ralstonia solanacearum and Pectobacterium carotovorum, producing inhibition halos of 23.9 ± 1.8 mm and 23.2 ± 1.8 mm, respectively. The crude extract and pure fractions analyzed by MS/MS mass spectrometry and evaluated by disk diffusion assays demonstrated that B. nakamurai CBAS-959 produces iturin A, bacillibactin, bacillaene, bacillaene B, and dihydrobacillaene, and five isoforms with different surfactin homologs. Extracts obtained from PDB and M1 media showed zones of inhibition of phytopathogenic bacteria between 9.5 ± 0.5 and 23.5 ± 0.5 mm, while fractions of purified compounds between 13.2 ± 0.8 and 21.0 ± 2.6 mm. Bacillibactin showed a MIC of 31.25 µg/mL against R. solanacearum and P. carotovorum but did not inhibit X. citri. PDB-2 L extract was the most active against X. citri, with an MIC of 62.5 µg/mL. This is the first study that purified and demonstrated that bacillibactin has a direct antibacterial effect against R. solanacearum and P. carotovorum. In addition, this work reports that B. nakamurai CBAS-959 produces bacillaene B, five isoforms of surfactins, inhibits the growth of R. solanacearum on co-culture, and the extracts obtained in PDB and M1 inhibit the growth of X. citri. Therefore, this research provides new information on the metabolic diversity and antimicrobial activity of B. nakamurai CBAS-959 and its potential use for future studies to explore new alternatives for the control of phytopathogens.