凡隆气单胞菌(Aeromonas veronii)拮抗菌4-1-3的筛选鉴定及其活性物质初探

Screening and Characterization of a Bacterium Named 4-1-3 Antagonizing Aeromonas veronii

关键词:  凡隆气单胞菌  拮抗菌  荧光假单胞菌  活性物质  胞外蛋白

Key words:  Aeromonas veronii  Antagonistic bacterium  Pseudomonas fluorescens  Active substance  Extracellular protein

摘要:

凡隆气单胞菌是泥鳅养殖中容易发生而且致死率较高的病原菌之一。为筛选凡隆气单胞菌拮抗菌、研发防治此病害的生物制剂，采集泥鳅养殖池底泥样品，通过滤纸抑菌圈法筛选凡隆气单胞菌拮抗细菌，结合形态观察、生理生化实验和16S rRNA基因序列分析进行菌株鉴定。采用硫酸铵沉淀和离子交换层析方法分离纯化抑菌物质，电泳检测其分子量。结果显示，从样品中分离到17株细菌，其中筛选出的拮抗菌4-1-3，能够高效抑制凡隆气单胞菌，抑菌圈直径达到15.2 mm。经菌株鉴定后命名为Pseudomonas fluorescens 4-1-3。该菌产生的抑菌物质为胞外蛋白，分子量约为100 kDa，推测该物质是荧光假单胞菌产生拮抗物质的一种新类型，有望进一步研制成防治水产病害的生物制剂。

Abstract:

Aeromonas veronii is a highly mortal pathogenic microorganism in the loach aquaculture. To screen the antagonistic bacteria and to develop microbial preparation against the pathogens, the samples were collected from loach breeding pond and the bacteria strains were isolated and screened for the ability to inhibit the size of the zone of the strains. The active substances were separated and purified with the ammonium sulfate precipitation and ion exchange chromatography, and the molecular weights were revealed with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A strain named 4-1-3 was isolated and screened from the samples collected from the bottom sediments of a loach breeding pond in the Lianyungang city, and it identified as Pseudomonas fluorescens based on its morphology and the analysis of 16S rRNA gene sequence, designated as P. fluorescens 4-1-3. The strain strongly inhibited the growth of A. veronii, with 15.2 mm inhibition zone diameter. The antibacterial substance produced by the strain P. fluorescens 4-1-3 is an extracellular protein which can be precipitated using 60% ammonium sulfate and eluted by 600 mmol/L NaCl through Sepharose DEAE Fast Flow. The molecular weight of P. fluorescens 4-1-3 is around 100 kDa determined by SDS-PAGE. In this study, we obtained a strain that can inhibit the growth of A. veronii and we found a new bacteria antagonistic substance produced by P. fluorescens. The present study provides evidence to the improve disease control in loach breeding and other aquaculture.