棉花黄萎病拮抗芽孢杆菌S12的筛选鉴定及拮抗机制的分析

[目的] 近年来,新疆棉田由于长期连作导致棉花黄萎病呈逐年加重的趋势,筛选适合新疆特殊地理环境的高效拮抗菌株对防治棉花黄萎病尤为重要。[方法] 采用平板对峙和琼脂平板扩散法从生长健康的棉花根际土壤中筛选拮抗细菌,利用常规PCR方法扩增拮抗细菌16S rRNA基因序列,应用BLAST进行同源性搜索,通过构建系统发育树并结合生理生化检测对拮抗细菌进行鉴定。用底物水解法检测其产生水解酶的能力,PCR方法检测其产生抗菌肽的潜力。[结果] 枯草芽孢杆菌S12对大丽轮枝菌(Verticillium dahliae Kleb.)的抑菌率为56%,抑菌圈直径为20 mm,其发酵液对病原菌孢子萌发抑制率为95.93%,表现出较好的抑菌能力;菌株S12能合成蛋白酶,含有srfAA、spaS、bacA抗菌肽合成基因,具有一定的生防潜力。[结论] 获得了可抑制大丽轮枝菌的芽孢杆菌菌株S12,并分析了其拮抗机制,为新疆棉花黄萎病的生物防治提供菌株资源和理论指导。
[Objectives] Cotton is the most important economical crop in Xinjiang, but the incidence of cotton Verticillium wilt had been increasing significantly under long-term cotton continuous cropping in recent years. It is particularly important for biological control of cotton Verticillium wilt to screen antagonistic strains from the special geographical environment in Xinjiang. [Methods] The antagonistic bacterium was isolated by the methods of the dual culture and agar diffusion. Antagonistic bacterial genomic DNA was extracted using the cetyl-trimethyl ammonium bromide protocol(CTAB). The 16S rRNA gene sequence of antagonistic bacterium was amplified from genomic DNA by a polymerase chain reaction(PCR)standard method using two universal primers:27F/1492R. A phylogenetic tree was established based on published 16S rRNA gene sequences of related strains from GenBank. Based on the physiological and the 16S rRNA gene sequence analysis, the strain S12 was identified as Bacillus subtilis. Activities of fungal cell wall degrading enzymes were tested by Substrate Hydrolysis methods. Genes responsible for the synthesis of antimicrobial peptides were identified in antagonistic bacteria. [Results] The results show that inhibiton rate of strain S12 on hypha growth was 56%, and inhibition zone of fermentation liquid on hypha growth was 20 mm. The inhibiton rate of fermentation liquid on conidial germination was 95.93% by concave slide method. The strain S12 can synthesize of protease. The genes of srfAA, spaS and bacA responsible for the synthesis of antibiotics surfactin, subtilin and bacylisin were identified in the strain S12. [Conclusions] This study provides a theoretical basis and strain resource for biological control of cotton Verticillium wilt