Identification of antagonistic bacterium strain Bacillus amyloliquefaciens 7M1 against Fusarium graminearum and characterization of the antibiotics from the target strain

Abstract:

[Objective] Isolation and identification of a strain with antagonistic activity against Fusarium graminearum, and the antibiotics from the target strain were identified and their bio-characteristics, antifungal activity was determined. [Methods] The inhibition activity of antibiotics was analyzed using oxford cup method and light growth chamber assay. Phylogenetic tree was constructed using the Neighbor-Joining method based on the 16S rRNA gene sequences, the antibiotics related genes were amplified by PCR, then the bioinformatics were determined using online software (Pro Param tool, TMHMM). [Results] The inhibitory zone diameter of 7M1 strain and antibiotics were 16.33±0.13 mm and 15.43±0.21 mm, light growth chamber assay showed that the control efficacy of wheat scab by 7M1 antibiotics was 76.41%. The 7M1 strain was found closely related to Bacillus amyloliquefaciens HQ840415.1 (98% similarity) based on the 16S rRNA gene sequence analysis. The antibiotics showed strong resistance to high temperature and a clear inhibition zone (5.33±0.32 mm) was still obtained after intense heating treatment of 90 °C for 30 min. The inhibitory activity was partially sensitive to proteinase K, pepsin and trysin. The antifungal activity of antibiotics was sustained throughout the pH range from 5.0 to 10.0. Along with the time of ultraviolet irradiation extending, the inhibitory activity was reduced. The gene essential for bacilysin, iturin and bacillomycin D synthetase was present in strain 7M1, which was naturally clustered from Bacillus sp. in GenBank, the amino acid sequence of the encoded product of the bacilysin, iturin and bacillomycin D synthetase gene exhibited high similarity up to 99% to that of iturin, bacilysin and bacillomycin D. bacAB encoded protein and ituC encoded protein were stable protein, bamD encoded protein were unstable protein. In addition, there was no obvious transmembrane structure present in the encoded product. [Conclusion] The antibiotics extracted from 7M1 had an obvious inhibiting effect against Fusarium graminearum and the antagonistic activity was resistant to some extent against temperature, pH proteinase and ultraviolet irradiation. These results suggested that 7M1 antibiotics were a promising material to control wheat scab.

Key words:Bacilli, Antibiotics, Genes, Antimicrobial activity, Wheat scab