Isolation,Identification And Genomic Analysis Of Bacteriophage Of Ralstonia Solanacearum

Bacterial wilt is a kind of soil borne bacterial disease,which has the characteristics of rapid diffusion and great destruction.It has devastating damage to the growth of mulberry,and seriously limited the healthy and sustainable development of sericulture industry in China.Bacterial wilt has become one of the most serious plant diseases in the world because of its wide incidence,great harm and strong destructive power.As the pathogen of bacterial wilt has many pathogenic varieties and a wide range of hosts,it is difficult to cure the bacterial wilt once it occurs.At present,the method for the controlling of Mulberry Bacterial wilt is limited.As the virus of bacteria,phage has great potential in controlling bacterial diseases because of its natural bactericidal ability,host specificity and environmental friendliness.It has become one of the effective strategies to replace antibiotics and chemical fungicides.In recent years,with the successful application of phage therapy in medical,food and other industries,phages have been gradually applied to the prevention and control of agricultural bacterial diseases,which provides ideas for the prevention and control of plant bacterial wilt.In this study,159 strains of were isolated from diseased mulberry plants in Guangxi.According to the 16 S identification,it is preliminarily considered that the bacteria causing Mulberry Bacterial Wilt in Guangxi are mainly Enterobacter and Ralstonia solanacearum.We collected 45 soil samples from Province Guangxi,Guangdong,Hubei,Sichuan and other places.Taking the isolated bacterial wilt strains as indicator bacteria,we isolated five bacteriophages from soil,and completed the purification and basic characteristics of two bacteriophages(Mul Y4 and Mulvp2),including plaque morphology,host spectrum,temperature and ph stability,one-step growth curve and inhibition effect on bacteria under medium conditions.The whole genome sequencing and annotation analysis of two phages were completed.The plaque formed by phage Mul Y4 is small and clear,with neat edges.The plaque formed by phage Mulvp2 has a translucent halo around the central clear plaque,and the halo becomes larger and larger with the extension of culture time,which indicates that Mulvp2 has the function of degrading extracellular polysaccharides.The results of host range detection showed that the both phages could infect Ralstonia solanacearum and Enterobacter.The detection of temperature and ph stability showed that the phage could exist relatively stably under the temperature below 37℃ and neutral PH environment.And the stability of different phages to temperature was different.The one-step growth curve showed that the cleavage amounts of Mul Y4 and Mulvp2 were 189 PFU/cell and571 PFU/cell,respectively.The bacteriostatic test under the condition of culture medium showed that Mul Y4 inhibited the growth of pathogen population,and Mulvp2 could reduce the host bacterial population to a very low level.The whole genome of the two phages was sequenced through high-throughput sequencing technology,and the genome comparison and annotation analysis were completed.The total length of phage Mul Y4 is 52,669 bp and the content of G+C is49.43%.There is a 166 bp forward repeat at the end of the Mul Y4 genome.The Mul Y4 genome contains 67 coding sequences(CDSs),of which 32 have possible functions and the rest are unknown functional proteins.The nucleotide sequence similarity between Mul Y4 and the reported Escherichia phage phb10 was 58.49%(coverage 62%,consistency 94.35%).The phage Mulvp2 has a total length of 58604 bp and a G+C content of 50.24%.Its genome contains 72 coding sequences,while the annotation and conserved domain analysis results showed that there are only 14 possible functions.The comparative analysis shows that the genome of Mulvp2 has no similarity with the reported phages,indicating that Mulvp2 is a new phage.We have further completed the gene clone of phage Mul Y4 lysozyme and the over-expression of protein.In the follow-up,we will carry out the functional research of phage enzymes for lysis bacteria.This study enriched the bacteriophage resources of bacteria caused Bacterial Wilt and provided resources and theoretical basis for the application of bacteriophages and their enzymes for lysis bacteria.