Study On Preparation And Application Method Optimization Of Phage For Preventing And Controlling Soil-borne Bacterial Wilt Of Tomato

Bacterial wilt caused by Ralstonia solanacearum is one of the most serious soilborne bacterial diseases in the world,which seriously threatens the planting quality and economic output of tomato.Phages have ability to precisely target and efficiently lyze the pathogen bacterial blight,and their antimicrobial action mode is not affected by the mechanism of bacterial resistance to antibiotics.However,in the process of practical application of agriculture,there are still many factors that can not be ignored limit the extensive application of phage.For example,biological and abiotic factors restrict the prevention and control effect of phage,temperature,p H,resources and host polymorphism all affect the stability of phage therapy.This is an urgent problem to be solved in the future application of bacteriophage to control soil-borne diseases.Through literature review,this paper started from two aspects to explore the methods and materials that can improve the phage to better prevent and control tomato soil-borne bacterial wilt.On the first hand,NN-P42isolated and screened in the laboratory was used as the model strain,and different kinds of protective agents were added to provide external protection for phages.The potency of phages was investigated under the conditions of adding different concentrations of protective agents and different storage temperatures.In the second aspect,four strains of phages(NJ-p3,NB-p21,NC-p34,and NN-p42)from previous laboratory studies were combined into a phage cocktail,and three variables of inoculation frequency,concentration and method were set to explore the effects of different application methods on the inhibition of R.solanancearum invasion by phages and the effects on the microbial community in tomato rhizosphere soil.The main research results are as follows:1.Different types of protectant,concentration of protectant and storage temperature have significant influence on bacteriostatic effect of bacteriophage.At 4℃,the bacteriophage potency of all treatments was significantly higher than that at 30℃,and the bacteriophage activity was strong,which was not significantly different from that of the control treatment(only the bacteriophage suspension),and the bacteriostatic effect was maintained at a high level for a long time.On the contrary,Phage titers was low at 30℃.With the prolongation of time,the bacteriostatic effect of phage decreased significantly.The10%plant peptone,0.1%glycerol and 1%trehalose treatment as the protectant had better bacteriostatic effect.In the actual production application,the phages that need short-term application can be stored through the cold chain without adding protective agent,which can reduce the cost and labor to obtain higher economic benefits.Protectant can be added to maintain high phage activity for long-term preservation,and the protection effect is better when the concentration of plant peptone is 10%,glycerol is 0.1%and trehalose is 1%.2.At the same concentration,the bacteriophage cocktail formed by the combination of NJ-P3,NB-P21,NC-P34 and NN-P42 showed significantly better bacteriophage inhibition effect than that of single bacteriophage.On the basis of bacteriophage cocktail,different inoculation frequency(one inoculation,two inoculations)and inoculation concentration(10~8pfu/m L、10~7pfu/m L、10~6pfu/m L、10~4pfu/m L)were set.Two inoculations and the inoculation concentration of 10~8pfu/m L had the best bacteriostatic effect,and the bacteriostatic rate reached the highest 70%.In a pot experiment,the control effect of bacteriophage cocktail on tomato R.solanancearum was observed.In a certain range,the higher the concentration and frequency of application,the better the inhibition effect on the growth of R.solanancearum.The best control effect of bacterial wilt disease was the treatment of 108 pfu/g soil inoculation and three inoculation treatments.3.To explore the influence of bacteriophage cocktail on tomato rhizosphere soil and analyze its microbial community.Compared with each treatment inoculated with bacteriophage cocktail,the diversity of microbial community in the control treatment inoculated only with pathogenic R.solanancearum decreased significantly,The abundance of Proteobacteria and Blastomyces increased significantly,and the difference of community structure was significant.After inoculation with phages,the composition and diversity of rhizosphere soil microbial community could be restored to a certain extent,and the disturbance of R.solanancearum bacteria to rhizosphere soil microbial community could be reduced,but there was no significant difference between different phage treatments.