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

【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20 μL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、pH、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dsDNA；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 kD，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菌时的潜伏期约为30 min，爆发期约为80 min，裂解量约为156；该噬菌体的裂解活性在28℃时最高，在28-50℃均较强，但在温度超过60℃后活性基本丧失；其对酸碱的耐受力较强，在pH 3-8的范围内均有较强的裂解活性，当pH值超过9后活性开始降低；其对紫外线有一定的耐受能力，经紫外线照射0-9 min后裂解活性依然较强，12 min后活性开始下降，21 min后活性基本丧失；其对氯仿不敏感，5%浓度的氯仿对其活性基本没有影响。【结论】分离到了裂解性的青枯雷尔氏菌噬菌体，属于有尾噬菌体目，肌尾噬菌体科，经过测定其各项生物学特性可知其潜伏期较短，裂解能力较强，具有很好的杀菌效果，且其裂解活性持续时间长，并能在不同温度、不同酸碱性的环境内有较强的适应能力，具有开发为抗青枯雷尔氏菌菌剂的潜力。

Isolation and Biological Properties of a Lytic Phage Infecting Ralstonia solanacearum

【Objective】In order to provide a new formulation ofantitobacco bacterial wilt, a lytic phageof 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×100 nm. According to the classification standards of the international commission on virus classification, it is a lytic phage of Myoviridae in Caudovirales, and its nucleic acid is dsDNA. There were 25 protein bands showing relative molecular mass of 10-100 kD in the result of SDS-PAGE, meaning the phage had at least 25 structural proteins. The agarose gel electrophoresis showed that its genome size was greater than 48 kb, fitting to the characteristics of Myoviridae (31-317 kb). Measurement of biological properties showed that the multiplicity of infection (MOI) of the R. solanacearum phage was 0.01. Its incubation period was 30 min, burst phase time was 80 min, and the burst size was 156 when infecting and locating on the R. solanacearum. The activity of phage ∈RS-1 was strong from 28℃ to 50℃, reaching a supreme at 28℃, however showing inactivation at 60℃. And it had wide susceptibility to pH changes, but its activity was decreased when the pH was above 9. The phage ∈RS-1 was also insensitive to ultraviolet ray, its activity was stable when exposed to ultraviolet ray for 0-9 min and began to decline after 21 min, 24 min later it was inactivated. It was insensitive to chloroform, 5% concentration of chloroform had no effect on its activity. 【Conclusion】In this experiment, a lytic R. solanacearum’s phage was isolated belonging to Myoviridae in Caudovirales. With some good biological properties including short incubation period, strong sterilization ability and stable in wide temperatures and pH, it has a potential to be developed as a new preparation for control the R. solanacearum bacterial disease.