一株裂解性青枯雷尔氏菌噬菌体的分离及生物学特性分析  

Isolation and Biological Properties of a Lytic Phage Infecting Ralstonia solanacearum

作　　者：高苗[1] 杨金广[1] 刘旭[1] 刘伟[1] 孙航军 申莉莉[1] 钱玉梅[1] 杨清林[3] 余广宏 李锡宏 王凤龙[1] 

机构地区：[1]中国农业科学院烟草研究所/烟草行业病虫害监测与综合治理重点开放实验室,山东青岛266101 [2]黑龙江八一农垦大学农学院,黑龙江大庆163319 [3]山东中烟工业有限责任公司,济南250014 [4]云南烟草保山香料烟有限责任公司,云南保山678000 [5]湖北省烟草科研所,武汉430030

出　　处：《中国农业科学》2015年第7期1330-1338,共9页Scientia Agricultura Sinica

基　　金：国家烟草专卖局科技项目(110200902065);云南省烟草公司科技项目(2013YN37);湖北省烟草公司科技项目(027Y2013-006)

摘　　要：【目的】分离并纯化出一株裂解性青枯雷尔氏菌(Ralstonia solanacearum)噬菌体,并测定其各项生物学特性,为开发新的抗烟草青枯病制剂提供依据。【方法】取烟草青枯病重病田中健康烟株的根际土壤制成土壤悬浮液,并通过在青枯雷尔氏菌菌液中加入过滤后的土壤悬浮液富集噬菌体,用双层平板法验证噬菌体的存在后挑取单个最大噬菌斑进行反复纯化,直到得到单一清晰的噬菌斑。纯化后的单个噬菌斑加入对数早期的青枯雷尔氏菌菌液中进行增殖培养,将增殖液按常规方法进行噬菌体颗粒浓缩后,取20μL浓缩液用磷钨酸染色并通过电子显微镜观察噬菌体的形态特征;同时将浓缩液进行SDS-PAGE电泳,观察蛋白条带大小和数量;用λ噬菌体DNA提取试剂盒提取噬菌体增殖液中的噬菌体核酸进行琼脂糖凝胶电泳,确定其基因组片段大小;最后用常规方法测定噬菌体的滴度、最佳感染复数、一步生长曲线,并通过比较加入噬菌体液前后青枯雷尔氏菌菌液的OD600值变化测定其对温度、p H、紫外线、氯仿的敏感性。【结果】分离并纯化出了一株裂解性青枯雷尔氏菌噬菌体,命名为∈RS-1,噬菌斑为圆形,清晰透明,边缘光滑,直径1—2 mm,经电镜观察其形态为蝌蚪状,头部为二十面体的立体对称,直径约为94 nm,并有一带伸缩尾鞘的长尾大约为27 nm×100 nm,按照国际病毒分类委员会分类标准,其属于有尾噬菌体目(Caudovirales),肌尾噬菌体科(Myoviridae)的裂解性噬菌体,核酸性质为ds DNA;噬菌体浓缩液经SDS-PAGE分析至少可以观察到25条蛋白条带,相对分子质量在10—100 k D,说明其蛋白外壳至少含有25个结构蛋白;将提取的DNA进行琼脂糖凝胶电泳显示其条带大于48 kb,符合肌尾噬菌体科基因组大小范围(31—317 kb);生物学特性的测定显示该噬菌体对青枯雷尔氏菌的最佳感染复数为0.01;其吸附和感染青枯雷尔氏Objective]In order to provide a new formulation of anti tobacco bacterial wilt, a lytic phage of Ralstonia solanacearum was isolated and purified from natural environments, and its biological characteristics were determined.[Method]Rhizosphere soil samples were taken from healthy tobacco plants growing in the disease field of tobacco bacterial wilt. Soil suspension after filtration was added into the R. solanacearum bacterial liquid to enrich the phage by co-cultivating. The existence of phage was detected using the way of two-layer plating method and a single clear of plague was obtained through purifying the single maximum phage repeatedly. The purified single phage was added into R. solanacearum bacterial liquid at logarithmic phase to propagate the phage culture. 20 μL concentrate of the phage particles by the conventional method was stained with phosphotungstic acid to observe their morphological characteristics by electron microscope. Meanwhile, the concentrate of phage’s fluid was used to detect the number and size of phage’s structure protein by SDS-PAGE electrophoresis, and confirm the size of genome fragment by agarose gel electrophoresis after extracting the total nucleic acid of the phage using the phage DNA extraction kit. The other biological properties include titer, MOI, one-step growth curve were measured by the conventional method, and the sensitivity to temperature, pH, ultraviolet ray and chloroform was tested by comparing the change in the OD600 of the R. solanacearum bacterial liquid before and after adding the phage.[Result]A strain of lytic R. solanacearum phage named as ∈RS-1 was isolated and purified. Its plaques are circular, clear and transparent with smooth edge, showing 1-2 mm of diameter. The electron microscope result showed that the phage particles like tadpoles with an icosahedra head of 94 nm diameter, and a long flexible tail about 27 nm＆#215;100 nm. According to the classification standards of the international commission on virus classification, it is a lytic phage o

关 键 词：青枯雷尔氏菌 噬菌体 分离 裂解性 生物学特性 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治] S476[农业科学—植物保护]