一株条斑紫菜丝状体黄斑病致病菌病原学研究  

On etiology of Pyropia yezoensis filament macular disease

作　　者：尚晓金 荣小军[2,3] 王印庚 汪文俊[2,3] 刘福利 廖梅杰[2,3] 张正 李彬[2,3] 于永翔 SHANG Xiaojin;RONG Xiaojun;WANG Yingeng;WANG Wenjun;LIU Fuli;LIAO Meijie;ZHANG Zheng;LI Bin;YU Yongxiang(College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China;Key Laboratory of Marine Aquaculture Disease Control,Ministry of Agriculture and Rural Science,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao Shandong 266071,China;Qingdao National Laboratory for Marine Science and Technology,Laboratory of Marine Fisheries Science and Food Production Process,Qingdao Shandong 266237,China)

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]中国水产科学研究院黄海水产研究所,农业农村部海水养殖病害防治重点实验室,山东青岛266071 [3]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,山东青岛266237

出　　处：《海洋渔业》2021年第2期219-229,共11页Marine Fisheries

基　　金：现代农业产业技术体系(CARS-50)。

摘　　要：采集山东省日照市岚山区藻类养殖合作社某紫菜育苗场的患有黄斑病的条斑紫菜(Pyropia yezoensis)丝状体,经2216E、TSB和TCBS 3种培养基体外培养,筛选获得优势菌株PZ201809121102,通过革兰氏染色、电镜负染观察、16S rDNA鉴定、生理生化分析、药物敏感性等技术方法对菌株进行鉴定分析,并结合回归感染实验确认菌株PZ201809121102的致病能力。结果表明:1)分离纯化后的优势菌株PZ201809121102在2216E海水培养基上的菌落形态呈细小圆形、中间突起、表面光滑呈亮黄色、匍匐镶嵌生长、属革兰氏阴性菌、短杆状、细胞大小(0.5~1.0)μm×(1.5~5.0)μm,透射电镜下观察到该菌株孢外具有单端生鞭毛;2)16S rDNA测序分析表明,该菌在所建16S rDNA系统发育树中与假单胞菌属聚为一支,与浅黄假单胞菌(Pseudomonas luteola)同源性最高,为100%;3)生理生化显示,该菌株在6%NaCl胰胨水、10%NaCl胰胨水中均生长,氧化酶反应阳性,鸟氨酸脱羧酶、赖氨酸脱羧酶、精氨酸双水解酶反应阳性;3%NaCl ONPG反应阳性。能同化利用葡萄糖,不能利用蔗糖、乳糖、密二糖、山梨醇等多糖;4)进行回归感染实验时,用10^(3)~10^(8)CFU·mL^(-1)的PZ201809121102菌悬液浸泡感染健康条斑紫菜丝状体。在13 d时,10^(8)CFU·mL^(-1)感染试验组的条斑紫菜丝状体出现大小不一的黄色斑点,与自然发病的丝状体黄斑病症状一致,且可再次分离得到与自然发病丝状体中相同优势分离株,证明PZ201809121102有致病性,是引发条斑紫菜丝状体黄斑病的一种病原,菌悬液浓度小于10^(8)CFU·mL^(-1)的各试验组丝状体中无明显黄斑病患病症状。5)药敏实验结果显示,PZ201809121102对罗美沙星和氟罗沙星两种药物高度敏感,对头孢唑啉、头孢氨苄、头孢拉定不敏感。本研究发现了导致条斑紫菜育苗期丝状体黄斑病的一株浅黄假单胞菌(P.luteola),可丰富对紫菜丝状体黄斑病的认识,以The experiment carried out an pathogenic analysis of Pyropia yezoensis filaments from a laver nursery in the Lanshan alage cultivation cooperative in Rizhao City of Shandong Province.We collected P.yezoensis filaments with typical symptoms of macular disease,cultured on three mediums of 2216E,TSB and TCBS.The dominant strain PZ201809121102 was isolated and purified from the focus of P.yezoensis filaments body.The strain PZ201809121102 was identified and obtained.Through Gram staining,electron microscope scanning,negative staining observation,16S rDNA identification,physiological and biochemical analysis and drug sensitivity test,we identified and analyzed the strains and combined with regression infection experiments to confirm the pathogenicity of the strain PZ201809121102.Results showed that:1)The colony shape of the predominant strain PZ201809121102 after separation and purification on 2216E medium was small and round,with intermediate impurities,smooth and bright yellow surface,growing in creeping status,belonging to Gram-negative bacteria,short rot-shape and with the cell size of(0.5-1.0)μm×(1.5-5.0)μm.Under transmission electron microscope,it was observed that there was a single flagellum outside the spore.2)16S rDNA sequencing analysis showed that the bacterium clustered with Pseudomonas in the 16S rDNA phylogenetic tree,and had the highest homology with Pseudomonas luteola,which was 100%.3)Physiological and biochemical results showed that the strain PZ201809121102 could grow in 6%NaCl tryptone water and 10%NaCl tryptone water.Oxidase reaction was positive.Ornithine decarboxylase,lysine decarboxylase,arginine dihydrolase reaction was positive.3%NaCl ONPG reaction was positive.It could assimilate and utilize glucose,but not polysaccharides as sucrose,lactose,denbiose and sorbitol.4)In regression infection experiment,10^(3)-10^(8)CFU·mL^(-1)of PZ201809121102 bacterial suspension was used to soak and infect healthy P.yezoensis filaments.After 13 days,10^(8)CFU·mL^(-1)P.yezoensis filaments appeared in dif

关 键 词：条斑紫菜 黄斑病 浅黄假单胞菌 病原学 

分 类 号：S946.2[农业科学—水产养殖]