一株条斑紫菜丝状体黄斑病致病菌病原学研究#br#

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10³~10⁸CFU·mL^-1的PZ201809121102菌悬液浸泡感染健康条斑紫菜丝状体。在13 d时,10⁸CFU·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10⁸CFU·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,以为今后对条斑紫菜育苗期丝状体黄斑病的防治提供参考。

On etiology of Pyropia yezoensis filament macular disease

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,10³-10⁸CFU·mL^-1of PZ201809121102 bacterial suspension was used to soak and infect healthy P.yezoensis filaments.After 13 days,10⁸CFU·mL^-1P.yezoensis filaments appeared in different sizes in the experimental group.The yellow spots were consistent with the symptoms of naturally-occurring filaments macular disease,and the bacteria was isolated again to obtain the same domain isolate,which proved that PZ201809121102 was a pathogen of P.yezoensis filaments macular disease.However in treatments of P.yezoensis suspension concentrations lower than 10⁸CFU·mL^-1,there was no obvious symptom of macular disease.5)Results of drug sensitivity experiments showed that PZ201809121102 was highly sensitive to two drugs,romefloxacin and fleroxacin,but not sensitive to cefazolin,cephalexin,and cefraditin.In this study,a species of P.yezoensis filamentous macular disease caused by P.luteola in seedling stage was discovered,providing a useful reference for the prevention and control of Pyropia yezoensis disease.