百合枯萎病拮抗细菌的筛选、鉴定及其抑菌物质研究

Isolation and identification of an antagonistic bacterium against Fusarium on lily and its antifungal substances

【目的】筛选对百合枯萎病具有抑菌活性的拮抗细菌,对其抑菌活性物质进行初步分离纯化分析。【方法】以强致病力的百合尖孢镰刀菌(Fusarium oxysporum)为靶菌,采用系列稀释法和平板对峙法初筛拮抗细菌,并通过产铁载体能力、水解酶活性、土壤定殖力等多种生防特性指标进行复筛,结合形态学特征、生理生化指标和16S r RNA基因序列比对鉴定其分类地位;利用百合尖孢镰刀菌作为靶菌进行活性追踪,结合酸沉淀、快速柱色谱、HPLC等分离纯化手段,对菌发酵液中的抑菌活性物质进行纯化分析。【结果】在64株百合根际细菌和386株海洋细菌中进行初筛,得到9株对百合镰刀菌具有较强拮抗活性的菌株,最后筛选了1株拮抗活性较强且产铁载体能力和水解酶活性、土壤定殖能力较高的菌株11B91,鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),其抑菌活性物质初步推测可能为Iturin和Fengycin脂肽类化合物。【结论】菌株11B91在百合枯萎病的生物防治中具有潜在的应用价值,证实海洋来源的微生物也具有防治陆地植物病原菌的潜力,为植物病害的防治拓宽了思路。

[Objective] The purpose of this study was to isolate and characterize an antagonistic bacterium against Fusarium on lily and to separate and identify its antifungal compounds. [Methods] Serial dilution method and dual-culture inhibition on agar plate were adopted for screening bacteria antagonistic to Fusarium oxysporum. The bacterial antagonists were rescreened by measuring siderophores-producing potential, hydrolytic enzymes activity and soil colonization. The previously isolated bacterial strain was identified using morphological, physiological and biochemical analyses as well as 16S rRNA gene sequencing. Antifungal substances produced by the antagonists were isolated, purified and analyzed based on bioactivity-guided method together with acid precipitation, flash chromatography and HPLC. [Results] A total of 64 bacterial isolates were obtained from the rhizosphere of filed-grown lily and 386 bacterial isolates were from marine organisms and silt. In a dual-culture inhibition test, 9 isolates displayed the antagonistic activity towards Fusarium oxysporum. Strain l lB91 was obtained by rescreening and identified as Bacillus amyloliquefaciens. Antifungal substances produced by strain llB91 were speculated to be lipopeptides iturin and fengycin. [Conclusion] Strain 11B91 deserved to be developed a kind of biocontrol agent. This is to certify that bacteria from ocean could have potential for the biological control of pathogens which reside in soil so as to broaden our horizon for the prevention and control of plant diseases.