一株病原发光杆菌的分离与鉴定

从三疣梭子蟹育苗场发病且大量死亡的大眼幼体中分离到优势生长的菌株SX1,人工感染试验证明,SX1对健康三疣梭子蟹大眼幼体及Ⅲ期幼蟹有较强的致病性;对菌株SX1进行了形态特征、理化特性等表型生物学特性检验,并进行了菌株SX1的16S rRNA、gyrB和rpoA 3种基因的同源性检索与系统发育学分析。结果显示,菌株SX1具有发光杆菌属的特征,且16S rRNA、gyrB和rpoA 3种基因序列均与发光杆菌具有较高的同源性,在系统发育树中与Photobacteriumganghwense聚为一个分支,自举数据值达100%。综合菌株SX1的形态特征、理化特性及3种基因的同源性检索与系统发育学分析,研究表明菌株SX1与P.ganghwense亲缘关系更近,鉴定SX1为P.ganghwense且为本次引起三疣梭子蟹大眼幼体大量死亡的病原菌。

Isolation and identification of pathogenic Photobacterium sp.

Dominant bacteria strain(SX1)was isolated from diseased and dead megalopas Portunus trituberculatus,and have pathogenicity to healthy megalopa and juvenile(Ⅲ)crab P. trituberculatus by artificial challenge.The phenotypic characteristics of the isolates including morphological,physiological and biochemical characteristics were determined.The 16S rRNA and two housekeeping genes,RNA polymerase a-chain(rpoA)and DNA gyrase B subunit(gyrB)were sequenced,and the phylogenetic trees based on 16S rRNA and gyrB sequences were constructed as an aid to identification.The sequenced 16S rRNA gene of strain SX1(GenBank accession No.JQ394838)was 1 454 bp,the gyrB gene of strain SX1(GenBank accession No.JQ394839)was 1 077 bp,the rpoA gene(GenBank accession No.JQ394840)was 913 bp.The 16S rRNA,gyrB and rpoA genes were compared with sequences deposited in databases,and molecular phylogenetic analyses were conducted.The results showed that the characteristics of identified strain(SX1)consist with genus of Photobacterium,and the 16S rRNA,gyrB and rpoA genes of the identified strain(SX1) exhibited high similarity with Photobacterium sp..The phylogenetic trees constructed using the neighbor joining method based on 16S rRNA and gyrB genes,strain SX1 clustered with the Photobacterium ganghwense,and was supported by a higher bootstrap value.Therefore,strain SX1 was similar to P. ganghwense.The results confirmed that the diseased megalopas were infected by P. ganghwense.To our knowledge,this is the first report of P. ganghwense as pathogenic bacteria of crab.The study showed the diversity and complexity of pathogenic bacteria from aquatic animals.