中国农业科学 ›› 2007, Vol. 40 ›› Issue (1): 92-98 .

• 植物保护 • 上一篇    下一篇

ARDRA方法在荧光假单胞菌分离鉴定中的应用

年洪娟,张杰,樊利强,刘朔,宋福平,黄大昉   

  1. 中国农业科学院植物保护研究所
  • 收稿日期:2006-05-10 修回日期:1900-01-01 出版日期:2007-01-10 发布日期:2007-01-10
  • 通讯作者: 黄大昉

Application of ARDRA Technology to Isolation and Identification of Pseudomonas fluorescens

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  1. 中国农业科学院植物保护研究所
  • Received:2006-05-10 Revised:1900-01-01 Online:2007-01-10 Published:2007-01-10

摘要: 【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 16S rDNA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。

关键词: 荧光假单胞菌, 分离鉴定, ARDRA分析, 拮抗作用

Abstract: A total of one hundred and sixty-three isolates of fluorescent Pseudomonas spp were isolated from 488 plant rhizospheric soil samples collected from Shandong, Hainan, Yunnan and Xinjiang Province. One hundred and two were detected to be rod and had monopolar one or more flagellum observed under light microscope. About 1.5 kb 16S rDNA fragment of these strains were amplified with the conservative 16S rDNA primer pairs of the eubacterium. The PCR products were purified and digested with the restriction enzyme HeaIII. Amplified ribosomal DNA restriction analysis (ARDRA) was performed. All strains produce three digestion patterns, which were similar with the digestion patterns of Pseudomonas fluorescens, Pseudomonas putida and unknown type. ARDRA analysis showed that all isolated strain belonged to at least three species. All the One hundred and two isolated strains were inhibitory ability to plant pathogenic fungi. Twenty-five of them showed inhibitory to Rhizoctonia solani in dual-culture test on PDA plates, one of them shown the highest antagonistic activity to Rhizoctonia solani was named TC222. Moreover, TC222 displayed inhibitory to various 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 var nicotianae. 16S rDNA sequence analysis of TC222 showed 99% homology to Pseudomonas fluorescens. TC222 was further confirmed to belong to Pseudomonas fluorescens according to physiological and biochemical characteristics.

Key words: Pseudomonas fluorescens, Isolation, ARDRA analysis, Antagonism