Isolation and Identification of Native <i>Bacillus subtilis</i> Strains from Soil for Biocontrol of <i>Fusarium solani</i> -Induced Fungal Diseases

Fusarium solani (F. solani), a pathogenic fungus in the Ascomycota division, poses significant risks to plants and humans, causing diseases like fusariosis and corneal infections. This study focused on isolating and identifying native Bacillus subtilis ( B. subtilis ) strains from soil to assess their potential as biocontrol agents against F. solani , leveraging their antifungal metabolite production. Soil samples were collected from various locations in Tehran, and B. subtilis strains were isolated using heat treatment and serial dilution methods. Morphological and biochemical tests were conducted to identify the strains. The antifungal activity of B. subtilis strains was assessed using the agar well diffusion assay, and the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined. Surfactin, a key antifungal metabolite produced by B. subtilis , was purified and analyzed using high-performance liquid chromatography (HPLC). Polymerase chain reaction (PCR) amplification of the 16S rRNA gene was carried out for molecular identification, and the PCR products were sequenced. Of the 60 B. subtilis strains isolated and confirmed through morphological and biochemical identification, 27 exhibited antifungal activity against F. solani . Among these, 6 strains demonstrated statistically significant inhibition, with inhibition zones ranging from 7.6 to 14.4 mm. The MIC values ranged from 0.5 to 2 mg/mL, while the MFC varied between 1 and 8 mg/mL. HPLC analysis identified two strains with the highest levels of surfactin production. PCR amplification of the 16S rRNA gene for these strains revealed a 99.7% similarity to B. subtilis sequences available in the NCBI database, a finding further corroborated by phylogenetic analysis. Native B. subtilis strains from soil demonstrated strong antifungal activity against F. solani , highlighting their potential as biocontrol agents. The production of surfactin by these strains further supports their application in sustainable agricultural practices.