关键词: 青枯雷尔氏菌, 噬菌体, 分离, 裂解性, 生物学特性

Abstract: 【Objective】In order to provide a new formulation of anti tobacco 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.

Key words: Ralstonia solanacearum, phage, isolation, lytic, biological properties