Isolation, Screening and Identification of Native and New Bacillus subtilis with Strong Antifungal Compound against Fusarium oxysporum

The genus Fusarium causes a wide range of infections in human, animals and herbs. The purpose of this research was to investigate and identify the native strains of Bacillus subtilis playing an inhibitory role against Fusarium oxysporum by producing surfactin. B. subtilis was isolated from the soil of various parks in Tehran-Iran, and identified by biochemical tests. Growth inhibition zone, minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of B. subtilis were determined. After purification of surfactin, quantitative and qualitative analysis of surfactin conducted using high performance liquid chromatography (HPLC) . Finally, two selected native strains with the highest production rate of surfactin identified using PCR for 16S rRNA and phylogenetic tree was drawn. Sixty strains of B. subtilis were isolated from soil, after identification through phenotypical and biochemical tests, the antagonistic activity of 27 different strains against F. oxysporum by Agar well diffusion assay determined and the highest inhibition zone was 13.66 mm. Six strains showing the best inhibitory effect, were isolated and their metabolite were purified by methanol. MIC and MFC values of different strains were in the range of 0.5-1.6 and 1.6-2.6 mg/mL. Using HPLC, the purified surfactin content in B. subtilis was about 56.7 – 131.9 μg/mL. Based on the curves of the chromatogram, the preferred strains with the highest production of surfactin, by molecular identification, displayed high similarity to B. subtilis. We got a maximum amount of yellow and transparent surfactin from native strains. Furthermore, the selected bacteria can be good candidates for biological control of fungal pathogens.