一株引起坛紫菜绿斑病病原的分离鉴定及致病性研究

Identification and pathogenecity of a novel pathogen causing green rot disease in Porphyra haitanensis

为了明确引起坛紫菜绿斑病的病原,对2012年发生在福建省莆田养殖海区坛紫菜叶状体绿斑病开展了病原的分离鉴定和致病性研究。利用2216E海水培养基平板,从患病藻体分离得到一株优势细菌X5;在实验室条件下将X5进行回接感染实验,在显微镜下观察到坛紫菜叶状体发生组织病理变化,出现绿斑病症状:感染初期出现黄绿色小斑点,病斑逐渐扩大出现空洞,最后导致紫菜叶片流失;用浓度为104~108cfu/m L的X5感染坛紫菜时,在7 d内引起紫菜出现10%~100%病斑面积,这些结果表明X5为坛紫菜绿斑病的病原。利用生理生化和多基因序列分析方法对X5进行鉴定,结果显示该菌为革兰氏阴性菌,大小为0.6μm×0.4μm^1.0μm×0.4μm,无鞭毛,在温度4~42℃、盐度50~150、p H 6~10范围内生长,最适生长温度为16℃,最适生长盐度为50,最适p H为7,对甲硝唑、林可霉素和青霉素耐药,对25种抗生素敏感,Biolog和API ID-32E细菌鉴定系统的结果显示X5为弧菌属细菌(Vibrio sp.);基于多基因序列(16S rRNA,rpo A,recA,pyr H)构建的系统进化树分析显示,X5与V.casei、V.litoralis、V.rumoiensis聚为一支,进化距离为0.095~0.108,这些结果说明X5为弧菌属的一个新种或是V.casei、V.litoralis、V.rumoiensis之一的一个新亚种。本研究报道了弧菌X5能够引起坛紫菜绿斑病,为紫菜流行病学及疾病防治提供了理论支撑。

In 2012,a green rot disease incidence occurred in a Pyropia haitanensis culture farm located in Putian,Fujian Province. The present study was carried out to investigate the pathogenic agent of green rot disease. The combination approach of biochemical tests and mutilocus sequences analysis was used to identify the bacterial isolate,and the artificial infection experiment and histopathological observation were performed to determine the pathogenecity of the isolate. A dominant bacterial strain assigned as X5 was isolated from the diseased blades using 2216 E medium plate. In artificial infection experiment on the P. haitanensis blades,X5 was able to cause the same characteristic histopathology of green rot disease seen in natural infections：small yellowgreenish spots appeared over the blades,then gradually developed into large green holes,and finally leading to collapse of the whole blade. By infection with X5 at 10^4-10^8 cfu / m L,the infected P.haitanensis blades were found to have lesion area rates from 10% to 100%. These results indicated that X5 was the etiological agent of green spot disease of P. haitanensis. X5 was gram-negative,short rod,nonflagellum bacterium wit size of 0. 6 μm × 0. 4 μm-1. 0 μm × 0. 4 μm. It grewwell at temperatures from 4to 42 ℃,salinities from 50 to 150 and pH from 6 to 10. The optimum growth condition was 16 ℃,50 salinity and pH 7. X5 was resistant to metronidazole,clindamycin and penicillin,while susceptible to 25 antibiotics. It was identified as a member of the genus Vibrio by Biolog and API ID-32 E. Based on phylogenetic analysis of multilocus sequences（ 16 S rRNA,rpo A,rec A,ypr H）,X5 was showed to be highly related with V. casei,V. litoralis and V. rumoiensis,with the evolutionary distance of 0. 095-0. 108. These results indicate that X5 might be a novel species of the genus Vibrio or a novel subspecies of V. casei,V. litoralis or V. rumoiensis.The present study indicates that a Vibrio sp. strain can cause green rot disease in P. haitanensis,which will support the study on P. haitanensis epidemiology and disease control of P. haitanensis.