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一株土壤来源的贝莱斯芽孢杆菌抑制黄曲霉生长机理研究

摘要: 为了更加安全有效地防治黄曲霉及其毒素污染,从花生田地土壤中分离获得了一株对黄曲霉生长具有良好抑制作用的菌株,通过平板对峙试验,验证其拮抗黄曲霉的效果;通过16S rDNA序列分析、构建进化树以及生理生化指标明确菌株种属信息;通过发酵优化、孢子萌发试验,发现该菌株在葡萄糖培养基、蔗糖培养基、牛肉膏培养基中的发酵液均能抑制黄曲霉孢子萌发,其中在葡萄糖培养基中的发酵液抑制效果最强,并考察了其对黄曲霉菌丝生长及孢子产生的抑制作用;采用PI和DAPI染色探讨了发酵液抑制黄曲霉菌丝生长的机理。结果表明:分离获得的菌株在平板对峙试验中能够显著抑制黄曲霉菌落生长,该菌株为革兰氏阳性菌株贝莱斯芽孢杆菌;在葡萄糖培养基中发酵48 h获得的发酵液对黄曲霉孢子萌发抑制率最佳,达到79.35%;在固体PDA培养基上对黄曲霉菌落生长抑制率为29%,对孢子生成的抑制率为54%;染色结果显示,发酵液中的抑菌物质能够通过破坏黄曲霉细胞膜的通透性以及损伤DNA的方式抑制菌丝生长。该菌株及其发酵液对黄曲霉具有较好的防治作用,具有进一步开发为农产品防霉剂的潜力。

Abstract: In order to control Aspergillus flavus growth and aflatoxin contamination more safely and effectively, a strain with excellent inhibitory effect on the growth of A. flavus was isolated from the soil of peanut field. The antagonistic effect against A. flavus was confirmed by plate confrontation examination; 16S rDNA sequence alignment analysis, physiological and biochemical characterization were employed for strain identification. Furthermore, the optimal medium for producing antibacterial substance was evaluated by spore germination investigation, and the inhibition effect of fermentation broth on mycelial growth and spore production of A. flavus was evaluated. In addition, the inhibitory mechanism was elucidated through PI and DAPI staining. The results showed that the colony growth of A. flavus was inhibited by strain 41 in a noncontact mode, indicating that strain 41 could antagonize A. flavus via producing some antifungal compounds. The physiological and biochemical analysis showed that strain 41 was a gram-positive bacterium and could utilize glucose, maltose, fructose, sucrose, and xylose, as well as hydrolyze starch. In salt-tolerance experiments, strain 41 grew normally in 2% NaCl, 5% NaCl, and 7% NaCl, but not in media containing 10% NaCl. Furthermore, strain 41 could inhibit spore germination of A. flavus in glucose medium, sucrose medium and beef extract medium, among which glucose medium was the most effective, and the inhibitory effects of the fermentation broth on the mycelial growth and spore production of A. flavus were investigated under this condition. The inhibition rate of A. flavus spore germination was 79.35% by fermentation broth obtained in glucose medium of the isolated strain for 48 hours. On solid PDA medium, the inhibition rate of A. flavus colony growth and spore production was 29% and 54% respectively. The results of PI and DAPI staining on the mycelia treated with the fermentation broth showed that the antibacterial substances in the fermentation broth could inhibit the growth of A. flavus mycelia by destroying the permeability of cell membrane and damaging DNA respectively. This study demonstrated that the strain and its fermentation broth had an excellent inhibitory effect on A. flavus and provided a theoretical basis for development of the antifungal substance as a mildew inhibitor in agricultural field.

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