| Tobacco bacterial wilt,one of a devastating bacterial soil-borne disease caused by Ralstonia solanacea,can cause the death of the whole tobacco plant,which seriously restrict the development of tobacco.Although the economic losses caused by the disease was reduced to some extent by integrated control measures such as biological control,chemical control,planting resistant varieties,adjusting the transplanting period or reasonable rotation to protect the tobacco plant from this disease,we still cannot effectively control the harm caused by this disease due to the high diversity,wide host range and the high survival ability of Ralstonia solanacearum in the soil.Thus,a novel and effective biological control method-phage therapy was developed,which refers to controlling the Ralstonia solanacea with phages.Therefore,the detection and separation of a new lytic phage will benefit to the phage therapy.In this study,lytic phages of Ralstonia solanacearum were isolated from the bulk soil planted with tobacco with the host of TB15-14 and TB15-15 by using double-layer plate method.The morphological and biological characteristics of phages were observed by transmission electron microscopy(TEM)and double-layer plate method,respectively.The genetic characteristics were studied by RAPD-PCR cloning and sequencing,high-throughput sequencing of the whole genome,genomic structure and comparative genomics analysis.This study not only provide technical support for phage control of tobacco bacterial wilt,but also provide data support for the production of bacteriophage preparations.The main results are as follows:1.According to the phage plaque size,three phages were isolated from TB15-14 and another three phages were isolated from TB15-15.The results of morphological characteristics showed that these six phages belonging to the order of Caudovirales and the family of Podoviridae.In addition,the phages from TB15-14 were named as RS-PI-1,RS-PI-2,RS-PI-3,respectively,and the phages from TB15-15 were named as RS-PII-1,RS-PII-2,RS-PII-3,respectively.2.The results of biological characteristics showed that there were no significant differences in the optimal multiplicity of infection among the six phages.The optimal multiplicity of infection in all phages were 1 except in one phage was 0.1.The one step growth curve indicated that the incubation period,outbreak period and burst size of the phages from the host TB15-15 were larger than those from TB15-14.3.High temperature environment is not suitable for six phages,and the best effect temperature was between 28 oC to 40 oC.pH has a greater impact on the phages from TB15-15 when it is higher than 8.In general,the alkaline environment was not suitable for these sixphages to live,and the optimum pH ranged from 3 to 8.4.RAPD analysis showed that the phage fragment of RS-PI-3-1 has a close relationship with Ralstonia phage RS-PI-1 and the similarity reached 99%.The comparative protein of Ralstonia phage RS-PI-1 is a hypothetical protein,thus we assumed that the phage fragment RS-PI-3-1 is also a hypothetical protein.Based on the intergrated biological characteristics analysis,we assumed that the phage fragment of RS-PI-3-1 was a new Ralstonia solanacearum phage.The similarity between phage fragment of RS-PII-2-1 and Ralstonia phage RS-PII-1 was 94%.The comparative protein of Rslatonia phage RS-PII-1 is a DNA helicase,thus we assumed that the phage fragment of RS-PII-2-1 is a DNA helicase.Phylogenetic tree analysis revealed that the bootstrap value of phage fragment of RS-PII-2-1 and Ralstonia phage RS-PII-1 was only 78%,therefore we assumed the phage fragment of RS-PII-2-1 is a new Ralstonia solanacearum phage.5.The results of the whole genome sequencing showed that RS-PI-1 and RS-PII-1 were belonging to double-stranded closed circular phages.The dsDNA sequence of phage RS-PI-1had length of 43211 bp with a G + C content of 61.5% and 48 open reading frames.The dsDNA sequence of phage RS-PII-1 had length of 42042 bp with a G + C content of 63.2% and 46 open reading frames.By comparing the whole amino acid of the whole genome sequences and constructing the phylogenetic tree based on the conserved regions,the coverage of these two phages was only 10% and the similarity was 78%,which are different.From other Ralstonia solanacearum phages.Therefore,we assumed that the phage RS-PI-1 and RS-PII-1 are new lytic phages of Ralstonia solanacearum. |
