Selection Of Ralstonia Solanacearum-specific Bacteriophages And Their Effects On Control Of Tomato Bacterial Wilt

"Bacteriophage therapy" has raisedwidely attention from scientists both domestic and overseas due to its high bacterial-lysis efficiency and host specificity.It has a wide application perspective on preventing pathogens of human,animals and plants.However,intensive studies are necessary for a large-scale and stable application in future.With the development of research on phages,its importance on controling plant diseases has been well-recogonized and investigated.In this study,four phages(named NJ-P3,NB-P21,NC-P34 and NN-P42)were isolated from tomato rhizosphere where suffered severe bacterial wilt in four provinces(Jiangsu,Zhejiang,Jiangxi and Guangxi).Ralstonia solanacearum,causing devastating bacterial wilt,was used as model soil-borne pathogen.Morphological structure and measured biocontrol efficiency of four phages were observed in vitro.We then analyzed their phylogenetic distance based on genomic data.In order to improve phage application in biocontrol of soil-borne diseases,optimal conditions for phage storage were tested.In the end,biocontrol efficiency of four phages was verified by greenhouse experiment and field experiment.The main results obtained were shown below:1.Four phages can form circular clear plaques with approximate 1 mm?2 mm diameter via two layer plating method.Four phages all had hexahedron head,which belonged to Caudovirales,Podoviridae.2.The whole genomes of four phages were sequenced through Illumina Hiseq sequencing platform.Genome analysis indicated that the length of NJ-P3,NB-P21,NC-P34 and NN-P42 genome were 42528 bp,41194 bp,41943 bp,42278 bp and the contents of G+C were 62.22%,62.22%,61.99%,62.10%,respectively.Four phages shared high genetic closest relationship,and some gene areas occurred rearrangement.They were most related to the phage DU_RP_? to Podoviridae.Four phages have 50 proteins in total respectively and 48 homologous proteins thereinto.The ratio of homologous proteins are above 96%of all proteins,indicating that they had relatively high homology.3.Measurement on biological properties of phages showed that four phages performed high lysis activity address to R.solanacearum from different hosts,can't lysis Escherichia coli and Pseudomonas protegens.The population of four phages were about 1011 PFU/mL.The multiplicity of infection(MOI)of four phages were between 0.01-0.1.During infection,incubation period of four phages was 30 min,and burst phase time was 90 min.NJ-P3 had the smallest lysis activity of which its burst size was 254.Four phages had high thermal stabilities,ranging from 20? to 40 ?.However,phages were inactivated when temperature reached to 60 ?.Meanwhile,they had wide resistance to pH changes,from pH 6 to 7.But their activities decreased when the pH was above 10 or below 4.Four phages were also insensitive to ultraviolet ray for 80 min,while their activity alleviated when exposed to ultraviolet ray for 90 min.4.Considering the feasibility of long-distance transportation,we tested optimal conditions for short-term storage of four phages.The results showed that phage NJ-P3,NB-P21,NC-P34 and NN-P42 could be stored at room temperature(25?).NJ-P3 and NB-P21 could maitain highest lysis ability in sterile water,while NC-P34 and NN-P42 had highest lysis ability in SM buffer solution and 30%glycerol,seperately.In addition,NJ-P3 and NC-P34 phage had stronger salt tolerance compared with NB-P21 and NN-P42.5.All four phages can significantly decrease tomato bacterial wilt with biocontrol efficiency above 50%in both field and greenhouse experiment.Soil-drenching and stem injection methods to inoculate phage both had high biocontrol efficacy of tomato bacterial wilt in field and greenhouse experiment.Phage NJ-P3 had the highest biocontrol efficiency(85%).While,phages had no significant effects on pathogen density in the rhizosphere.We also found that the reduction of disease incidence were not due to phage densities.In conclusion,four phages had high homology and could be used to control tomato bacterial wilt in the field.Based on optimal storage conditions,these four phages have potential of long-distance transportation and application.