高通量测序分析鉴定东星斑溃烂病主要致病菌  被引量:5

High-throughput Sequencing Analysis of the Main Pathogens of Ulcer Diseased Plectropomus leopardus

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作  者:王磊[2] 张天时[2] 刘洋[2] 张子威 李开敏 朱春华 陈松林[2] WANG Lei;ZHANG Tian-Shi;LIU Yang;ZHANG Zi-Wei;LI Kai-Min;ZHU Chun-Hua;CHEN Song-Lin(Southern Marine Science and Engineering Guangdong Laboratory(Zhanjiang)/Guangdong Research Centre on Reproductive Control and Breeding Technology of Indigenous Valuable Fish Species,Zhanjiang 524088,China;Yellow Sea Fisheries Research Institute/Key Laboratory for Sustainable Development of Marine Fisheries,Ministry of Agriculture and Rural Affairs/Qingdao National Laboratory for Marine Science and Technology/Laboratory for Marine Fisheries Science and Food Production Processes,Chinese Academy of Fishery Sciences(CAFS),Qingdao 266071,China)

机构地区:[1]南方海洋科学与工程广东省实验室(湛江)/广东省名特优鱼类生殖调控与繁育技术工程中心,湛江524088 [2]中国水产科学研究院黄海水产研究所/农业农村部海洋渔业可持续发展重点实验室/青岛海洋科学与技术试点国家实验室/海洋渔业科学与食物产出过程功能实验室,青岛266071

出  处:《农业生物技术学报》2023年第3期617-628,共12页Journal of Agricultural Biotechnology

基  金:南方海洋科学与工程广东省实验室(湛江)资助项目(ZJW-2019-06);山东省重点研发计划项目(2021LZGC028);山东省泰山学者攀登计划专家项目;中国水产科学研究院基本科研业务费(2020TD20)。

摘  要:东星斑(Plectropomus leopardus)是一种新兴的经济型养殖鱼类,近年来溃烂症已成为其主要病害,严重影响幼鱼和成鱼,造成极大的经济损失,分离鉴定溃烂症主要致病菌是开展抗病选育的前提。本研究利用PacBio三代高通量测序方法对东星斑病鱼溃烂部位和正常鱼皮肤进行了微生物测序和聚类分析。结果显示,正常组中特有的OTUs(operational taxonomic units)为403个,而病鱼组有72个特有的OTUs,两组共有OTUs数是74个。正常组平均识别出16个门、30个纲、63个目、96个科、145个属、168个种,然而在病鱼皮肤平均识别出9个门、12个纲、27个目、38个科、55个属、67个种。这种差异说明致病菌抑制了正常菌群的生长,导致病鱼皮肤中的菌群结构多样性明显降低。正常组和病鱼组优势菌群组成存在较大差异,正常鱼中以变形菌门(Proteobacteria)和厚壁菌门(Firmicutes)为主;病鱼中以拟杆菌门(Bacteroidetes)和ε变形菌门(Epsilonbacteraeota)为主。ANOVA方差分析显示病鱼皮肤中弧菌属(Vibrio)的含量显著升高,通过KRONA分析注释出哈维氏弧菌(V.harveyi)、塔式弧菌(V.tubiashii)和不可培养弧菌(uncultured-Vibrio)。经细菌分离实验鉴定1株哈维氏弧菌,命名为V.harveyi stain Dx21。通过腹腔注射法进行回归感染实验,发现V.harveyi stain Dx21能够导致东星斑幼鱼体表溃烂,随后导致死亡,症状与自然感染相似。该菌株对体重为100 g东星斑鱼苗的半致死浓度为1.0×105 cfu/mL。综上所述,本研究揭示了东星斑病鱼皮肤的菌群特征,提示哈维氏弧菌是主要致病菌之一。本研究对于东星斑溃烂病的病害防控以及疫苗的研制具有一定的指导意义。Plectropomus leopardus is a kind of newly farmed fish with high economic value.In recent years,skin ulcer disease occurs in both juvenile and adult fish,resulting in a large number of deaths and great economic losses.It is important to identify the main pathogenic bacteria for disease-resistant breeding.In this study,the diseased fish and normal fish were collected from Hainan,and the skin microbial sequencing and analysis were carried out on 3 generation high-throughput sequencing method.The results showed that there were 403 unique operational taxonomic units(OTUs)in the normal group and 72 unique OTUs in the ulcerated group.The number of OTUs shared by the two groups was 74.In the normal group,there were 16 phyla,30 classes,63 orders,96 families,145 genera and 168 species were identified on average,while 9 phyla,12 classes,27 orders,38 families,55 genera and 67 species were identified on average in the diseased fish skin.The difference between two groups indicated that pathogenic bacteria inhibited the growth of normal bacteria,resulting in a significant decrease in the diversity of bacteria structure.There were great differences in dominant bacterium composition between normal group and diseased group.Proteobacteria and Firmicutes were dominant phyla in normal group,while Bacteroidetes and Epsilonbacteraeota were dominant phyla in the diseased fish.ANOVA analysis of variance showed that the content of Vibrio in diseased fish skin was significantly increased,and Vibrio harveyi,V.tubiashii and a specie of uncultured Vibrio were annotated in KRONA analysis.Based on above research,a strain of V.Harveyi was isolated and identified from the ulcer skin of P.leopardus,named as V.harveyi stain Dx21.After the artificial infection with the intraperitoneal injection,the result showed that the V.harveyi stain Dx21 could cause surface ulceration and death in juvenile starfish,which was similar to natural infection.The half-lethal dose(LD50)was 1.0×105 cfu/mL for P.leopardus fries weighing 100 g.This study revealed the flo

关 键 词:东星斑 溃烂病 16S rRNA 高通量测序 哈维氏弧菌 

分 类 号:S943[农业科学—水产养殖]

 

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