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

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 2216E 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 yellow greenish 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 10⁴-10⁸ cfu/mL,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,non-flagellum bacterium wit size of 0.6 μm×0.4 μm-1.0 μm×0.4 μm.It grew well at temperatures from 4 to 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-32E.Based on phylogenetic analysis of multilocus sequences (16S rRNA, rpoA,recA,yprH),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.