草鱼病原菌AvX005的分离鉴定及其拮抗菌筛选研究

Isolation of Pathogenic Strain AvX005 and Screening of Its Antagonistic Bacteria in Grass Carp Ctenopharyngodon idellus

2016年4—7月,湖南望城乔口渔场爆发了较大范围的草鱼疾病,自爆发病的草鱼病变组织中进行细菌分离与培养,分离得到一株优势菌,回复感染试验测定,该菌株具有较强的致病性。经菌落形态观察、冷场扫描电镜观察、生理生化试验及16SrDNA基因序列同源性分析,鉴定为维氏气单胞菌,编号为AvX005(GenBank登录号:KU64116.1),在此基础上以维氏气单胞菌AvX005为指示菌,通过点种法和纸片扩散法筛选拮抗菌,拮抗试验发现新筛选的解淀粉芽孢杆菌对维氏气单胞菌AvX005有较好的拮抗效果,采用酸沉淀、C18反相柱色谱分离到抑菌活性物,LC-MS/MS鉴定分子量为1045.29。根据抑菌活性物的理化性质,初步确定解淀粉芽孢杆菌产生的抑菌活性物可能为脂肽类化合物。该研究不仅可为快捷、准确检测由维氏气单胞菌引起的鱼类疾病提供参考及填补国内维氏气单胞菌拮抗菌在分类地位、生物学特性等方面的不足,而且也为我国草鱼维氏气单胞菌病的生物防治提供科学资料。

There was an outbreak mortality of grass carp Ctenopharyngodon idellus in the farms of Hunan province from the April to July,2016.Lesion tissue of grass carp was collected from the outbreak of fish disease farm in Wangchengk,Hunan Province,and the dominant strains were isolated from diseased fish using routine bacterial isolation methods.Artificial infection test showed that the isolated strain possessed relative strong pathogenicity and was identified as a Aeromonas veronii named as AvX005（GenBank：KU64116.1）by the morphological observation,scanning electron microscopy,physiological and biochemical characteristic analysis and 16S rDNA gene sequence analysis.The bacteria,using AvX005 as the index bacteria we screened antagonistic bacteria against pathogenic A.veronii by using a spot diffusion assay and plate diffusion experiments.It was found that Bacillus amyloliquefaciens had the maxmal antibacterial activity among these antagonists.Antibacterial compounds from the identified strain,B.amyloliquefaciens,with molecular weight of 1045.29 analyzed by LC-MS/MS,was separated and purified by acid precipitation,and C18 reverse phase column chromatography.According to the physicochemical properties of antibacterial activity.It is preliminarily inferred that the antibacterial activity of compounds may be a lipopeptide compound.The findings provide a reference for fast and accurate detection of fish diseases caused by A.veronii and to fill the gaps in the classification status and biological characteristics of antagonistic bacteria as well as provide the scientific information for the biological control of A.veronii for grass carp culture.