ARDRA方法在荧光假单胞菌分离鉴定中的应用

Application of ARDRA Technology in Pseudomonas fluorescens Isolation and Identification

【目的】探索荧光假单胞菌的分类方法,分离筛选拮抗性较强的荧光假单胞菌。【方法】利用紫外下产生荧光的特性,采用梯度稀释及鞭毛染色的方法,对采自云南、海南、山东和新疆的488份植物根际土壤进行分离筛选,分离菌株进行ARDRA分析,采用室内平板对峙法筛选对植物病原真菌具有较强拮抗作用的菌株,并对其进行生理生化分析和分子鉴定。【结果】分离筛选到102株紫外下产生荧光的杆状单极生鞭毛菌株。ARDRA分析产生荧光假单胞菌类型、恶臭假单胞菌类型以及一个未知的谱带类型。以油菜立枯病菌作为靶标菌,筛选到25株具有拮抗作用的菌株,其中TC222产生的抑菌圈最大,进一步的生测结果表明该菌株对9种重要的植物病原真菌如番茄灰霉病菌等具有很强的拮抗活性。TC222的16SrDNA核苷酸序列与荧光假单胞菌PfO-1同源性达到99%,其最终鉴定为荧光假单胞菌。【结论】ARDRA方法在分子水平上为荧光假单胞菌的分离鉴定探索了一条新途径;TC222菌株具有拮抗多种植物病原真菌的能力,显示了良好的应用前景。

[ Objective ]The study isolated strongly antagonistic P. fluorescens. [ Method ] Fluorescence under UV and flagella staining were used to isolate fluorescent pseudomonas from 488 soil samples collected from Shandong, Hainan, Yunnan and Xinjiang Provinces. About 1.5 kb 16S rDNA fragments of these strains were amplified with 16S rDNA primer pairs of eubacterium.Amplified ribosomal DNA restriction analysis （ARDRA） was performed. Antagonistic activities to plant pathogens were tested on PDA plates. Nucleic acid BLAST searches were performed using the website http：//www.ncbi.nlm.nih.gov/blast. TC222 identification was performed according to 16S rDNA sequenced together with physiological and biochemical analysis. [Result] A total of 163 fluorescent bacterial strains were isolated, of which 102 were rod shaped and had monotrichous or lophotrichous flagella. All strains produced three digestion patterns according to ARDRA analysis.These were similar to the digestion patterns of P. fluorescens, P. putida. The data suggested that all isolated strains belonged to at least three species. 25 strains showed inhibitory to Rhizoctonia solani in dual-culture test on PDA plates, one of which showed the highest antagonistic activity was named TC222. Moreover, TC222 displayed inhibitory to other plant pathogens, e.g. Gaeumannomyces graminis, Magnaporthe grisea, Drechslera sorakiniana, Alternaria brassicae, Botrytis cinerea, Phomopsis asparagi, Fulvia fulva, Colletotrichum lindemuthianum Sacc. et Magn and Phytophthora parasitica vat nicotianae. 16S rDNA sequence analysis of TC222 showed 99% homology to P. fluorescens PfO- 1. TC222 was further confirmed to belong to P. fluorescens according to physiological and biochemical characteristics. [Conclusion ] ARDRA analysis provides a new way for P. fluorescens isolation and identification. TC222 showed a potential prospect in biocontrol for plant diseases.