小麦赤霉病原菌拮抗菌Bacillus amyloliquefaciens 7M1产抗菌素的研究

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

摘要:

【目的】从小麦根际土壤分离鉴定一株赤霉病拮抗菌，对该菌产的抗菌素进行生物学性质研究、种类鉴定和抑菌实验。【方法】利用牛津杯法和光照培养箱实验对其抑菌活性进行测定，通过16S rRNA基因序列分析对目标菌株的种属进行初步鉴定，根据抗菌素相关基因进行PCR扩增和测序，利用在线软件Pro Param tool和TMHMM对编码蛋白进行生物信息学分析。【结果】7M1菌体和抗菌素对禾谷镰刀菌的抑菌圈直径分别为16.33±0.13 mm和15.43±0.21 mm，16S rRNA基因序列分析结果显示其为芽孢杆菌，并与解淀粉芽孢杆菌具有较近的亲缘关系，菌株7M1抗菌素对小麦赤霉病的温室防治效果为76.41%，而且热稳定性好，可被蛋白酶K、胰蛋白酶、胃蛋白酶降解，在pH 5.0?10.0有较好的抑菌活性，但是紫外线辐射会降低其抑菌活性。菌株7M1含有bacAB、ituC、bamD 3种基因，通过与GenBank中相关的抗菌素基因进行比对，发现其编码的氨基酸序列与GenBank库中的芽孢杆菌溶素、伊枯草菌素和杆菌抗霉素D等抗菌素的相似性达到99%。bacAB编码蛋白和ituC编码蛋白是稳定蛋白，bamD编码蛋白是不稳定蛋白，此外，3种基因的编码产物不具有明显的跨膜结构。【结论】从该菌发酵液提取的抗菌素有很好的抗禾谷镰刀菌活性而且性质稳定，因而在小麦赤霉病的生物防治中具有潜在的应用价值。

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.