Study On The Control Effect Of Streptomyces Sp.NEAU-HV9 On Ralstonia Solanacearum And Active Metabolites

Tomato bacterial wilt is an important soil-borne bacterial plant pathogen caused by Ralstonia solanacearum,which is distributed all over the world.The host range of it is unusually wide.It can infect more than 450 monocotyledonous and dicotyledonous plants,and is devastating to many hosts.Therefore,it is ranked as the second most destructive bacterial pathogen in the world.As one of the most important vegetable crops in the world,tomato production is severely reduced every year due to bacterial wilt,and it is urgent to find effective control methods.At present,chemical control is the main control method,but it not only causes the emergence of drug-resistant pathogens,but also directly causes environmental pollution.Biological control has attracted widespread attention due to its advantages such as high efficiency and no pollution to the environment.Antagonistic microorganisms and their secondary metabolites have become the focus of attention.In this research,the actinomycetes were isolated and purified from the riverside soil in Bama yao Autonomous County,Hechi City,Guangxi zhuang Autonomous Region.A strain with strong antibacterial activity was screened out by in vitro screening technology,and the secondary metabolites with antibacterial activity were separated and purified.The effects of this strain and its secondary metabolites on the control of tomato bacterial wilt were studied through tomato seedling disease resistance tests,in vitro leaf experiments,and pot experiments.In addition,the strain was identified and its whole genome was analyzed,its gene function was annotated and secondary metabolites were predicted.The main experimental results are as follows:?1?Five kinds of isolation media?CPA,DPA,SSA,HV,GS?were used to isolate actinomycetes in soil.After morphological and molecular analysis,they were combined to obtain101 strains of actinomycetes.Including Streptomyces,Nocardia,Kitasuria,Micromonospora,Microbacteria and Tsukamura,Streptomyces is the dominant genus,accounting for 79.4%.?2?The activity of 101 actinomycetes against R.solanacearum was tested.A total of 11strains of spore extracts had a bacteriostatic diameter greater than 10 mm,of which the strains NEAU-HV9 and NEAU-HV72 had a diameter of greater than 25 mm,and the strain NEAU-HV9had the largest diameter of 27.74 mm.When the strain NEAU-HV9 was fermented,the bacteriostatic diameter of the supernatant was 32.8 mm,and the bacteriostatic activity of the methanol extract of cell pellet was 30.5 mm.?3?The strain NEAU-HV9 was fermented and the activity-tracking method was used to isolate the active secondary metabolites.After mass spectrometry,¹H-NMR,¹³C-NMR analysis,the compound was initially determined to be actinomycin D.By comparing the antibacterial activity and the liquid phase retention time with commercial actinomycin D,it was finally determined to be actinomycin D.The minimum inhibitory concentration of R.solanacearum was0.6 mg/L.?4?The tomato disease resistance test preliminary showed that the strain NEAU-HV9 spore suspension?10⁹cfu/m L?and actinomycin D had good control effects,and the control effect was close to 100%.In the in vitro test,when the spore concentration was 10⁹cfu/m L,it could effectively control R.solanacearum.Pot experiments showed that both the spore suspension and actinomycin D of strain NEAU-HV9 can effectively control tomato R.solanacearum,with control effects of 82%and 100%,respectively.?5?The whole genome of strain NEAU-HV9 was sequenced.The strain had a genome size of10,797,268 bp,DNA G+C content was 69.1 mol%and contained 9727 coding genes.Gene function annotation of strain NEAU-HV9 was performed using GO,KEGG and COG functional databases.The results indicate that functional proteins of strain NEAU-HV9 are mainly involved in biological and metabolic processes.The genomic sequence of strain NEAU-HV9 was submitted to the anti SMASH program for analysis of secondary metabolite gene clusters.This strain has a total of 23 secondary metabolite synthetic gene clusters,of which the number of NRPS gene clusters is the largest,with 11 and includes gene clusters with 86%similarity to synthetic gene clusters of actinomycin D.?6?Polymorphic taxonomy identification of strain NEAU-HV9.The 16S gene r RNA sequence comparison was roughly determined as Streptomyces,and the physiological and biochemical characteristics,the culture characteristics of different media,the micro-morphological characteristics and chemotaxonomic characteristics were analyzed to determine the strain as a potential new species in Streptomyces.