Isolation,Identification And Application Research Of A Lytic Phage Infecting Ralstonia Solanacearum

Tobacco bacterial wilt which caused by Ralstonia solanacearum is one of the major diseases on tobacco and there is still no effective means to control it. From the aspect of biological control there was a phage of Ralstonia solanacearum isolated from rhizosphere soil of normal tobacco which growth in disease nursery of tobacco bacterial wilt. The taxonomic status of the phage was determined by virus morphology, conventional biological assays and molecular biology sequencing technology. The control effect of this phage was measured by pot experiment. The impact of the phage to the bacterial populations in soil was measured by soil bacterial metagenomics. The results were as follows:1. A strain of lytic Ralstonia solanacearum phage which was named ∈RS-1 was isolated and purified. Its plaques are circular, clear, transparent and have smooth edges, its diameter is between 1 mm~2 mm. The phage ∈RS-1 is tadpole-like and has icosahedra head with 94 nm in diameter, has a long flexible tail about 27 nm × 100 nm. There were more than 25 protein bands showing in the result of SDS-PAGE, meaning the phage has at least 25 structural proteins.2. Sequence analysis showed that the phage ∈RS-1 has high homology with Ralstonia phage RS603(AB937974.1), they are both Ralstonia phage which belong to Caudovirales, Myoviridae, P2-like viruses.3. Measurement of biological properties showed that the multiplicity of infection(MOI) of the R. solanacearum phage ∈RS-1 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 min-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.4. The pot experiment which determinate the control effect of the phage ∈RS-1 to tobacco bacterial wilt showed that the control effect were both significant when the phage ∈RS-1 was used to pouring and soaking tobacco root before the R. Solanacearum infect. Especially when pouing root advance, the tobacco bacterial wilt was significantly suppressed and the relative control efficiency reached 94.87%.5. With the method of soil bacterial metagenomics, after comparing the difference of soil bacterial community diversity between the soil without phage ∈RS-1 and the soil which add the phage ∈RS-1, the result showed that the inhibitory effect of phage ∈RS-1 to R. solanacearum was significant when compared with the soil without adding phage ∈RS-1, and its impact to other soil bacterial wasn’t very significant. All of this showed that when control the tobacco bacterial wilt with phage ∈RS-1, the effect to soil microbial populations is limited. The cleavage of phage ∈RS-1 to R. solanacearum was specificity, and it had high security to soil microflora.