石蒜根际拮抗细菌HH1的筛选鉴定及其抑菌活性研究

Screening and identification of antagonistic bacteria HH1 from rhizosphere soil of Lycoris radiata( L'Her.) Herb and its antifungal activity

[目的]以小麦赤霉病菌(Fusarium gramiuearum)为筛选靶标,分离石蒜根部土样中的拮抗细菌并研究其抑菌特性及机制。[方法]通过抑菌活性筛选拮抗菌株,根据菌落及细胞形态观察、生理生化试验结果和16S rRNA基因序列分析进行菌种鉴定,并对拮抗菌株产活性抗真菌成分的最佳培养基和培养条件进行优化。通过显微观察拮抗菌发酵上清液对病原菌生长的影响,对活性物质的热稳定性、酸碱稳定性及对蛋白酶K敏感性进行测定。[结果]共获得有稳定拮抗效果的菌株7株。以其中1株对小麦赤霉病菌具有较好拮抗作用的细菌HH1作为进一步研究的对象,鉴定其为短小芽孢杆菌(Bacillus pumilus)。抑菌试验发现菌株HH1能够分泌抑制小麦赤霉病菌孢子和菌丝生长的物质。沉淀菌株HH1发酵上清液中活性物质的最适硫酸铵饱和度为60%。优化了菌株HH1产活性抗真菌成分的培养基和培养条件。对活性物质热稳定性、酸碱稳定性及对蛋白酶K敏感性的测定结果显示:拮抗物质在p H中性和低于70℃的条件下较稳定,70℃以上的温度和蛋白酶K处理抗菌物质,其抑菌活性显著下降。[结论]初步推测菌株HH1所产拮抗物质可能是蛋白质类。

[ Objectives] Strains against Fusarium gramiuearum were screened from the root soil of Lycoris radiata（ L＇Her. ） Herb. For further application,the antagonistic mechanism of strain HH1 was also studied. [ Methods] Taking F. gramiuearum as target pathogen, rhizosphere bacteria were screened for their antifungal ability. Strain HH1 with remarkable antifungal effect was characterized by traditional and molecular techniques. The effect of the antagonistic substance produced by strain HH1 on the pathogen spores and mycelium was microscopically observed. The effects of temperature, pH and proteinase K on the inhibition activity of antagonistic substance were then studied. [ Results ] 7 strains with inhibitory activity against F. graminearum were isolated from rhizosphere soil of L. radiata. The antagonistic strain HH1 with strong inhibitory activity was identified as Bacillus pumilus. It was found that different concentrations of strain HH1 cell supernatant could inhibit the germination of spores and hyphae growth of F. graminearum, and the inhibition activity increased when the concentration was higher. 60% saturation of ammonium sulfate made a complete precipitation of the antagonistic substance in culture broth. The culture medium and culture conditions were then optimized. The antagonistic substance was stable under neutral pH and temperature lower than 70℃ .The inhibition activity of the antagonistic substance of strain HH1 significantly declined after treated with proteinase K for 1 h in 37℃. [ Conclusions] According to these characterizations, the antagonistic substance produced by strain HH1 might be identified as protein.