Optimization Of Solid State Fermention Conditions Of Streptomyces Sp.NEAU-174 And Its Control Effect On Ralstonia Solanacearum

Tomato bacterial wilt is a soil-borne plant disease caused by Ralstonia solanacearum,which infects a wide range of hosts.Tomato,as one of the most important vegetable crops in the world,has a serious decline in output due to the occurrence of bacterial wilt every year.So it is urgent to find effective control methods.At present,the main prevention and control means is chemical control,but it can produce drug-resistant pathogens and seriously pollute the environment.Biological control has been widely concerned because of its high efficiency and pollution-free.So,the study of antagonistic microorganisms became the focus.In this study,the actinomycetes were isolated and purified from root soil of catalpa ovata.A strain,NEAU-174,with strong antibacterial activity was screened out by in vitro screening technology.The effects of this strain on the control of tomato bacterial wilt were studied through tomato seedling disease resistance tests,in vitro leaf experiments and pot experiments.Therefore,response surface analysis（RSM）was used to optimize the culture conditions for solid fermentation of this strain.In order to better apply the strain in the field,natural soil was used for pot experiment to verify its control effect.The supernatant and precipitates of strain NEAU-174fermentation broth had good activity against tomato bacterial wilt and had the value of mining its active primary metabolites.Therefore,the stability test of fermentation broth was carried out and the active secondary metabolites were separated by activity tracking method.In addition,a preliminary identification test was conducted on the strain.The main experimental results are as follows:（1）Five kinds of isolation media（CPA,DPA,SSA,HV,GS）were used to isolate actinomycetes in root soil of catalpa ovata.After morphological and molecular analysis,they were combined to obtain 175 strains of actinomycetes,including Streptomyces,Nocardia,Micromonospora,Microbacteria,Actinomadura and Glycomyces.Streptomyces is the dominant genus,accounting for 85.1%.（2）The activity of 175 actinomycetes against R.solanacearum was tested.A total of 18strains of spore extracts had a bacteriostatic diameter greater than 10 mm,of which the strain NEAU-174 had the largest diameter of 34.6 mm.（3）The active strain NEAU-174 was identified by through 16S r RNA gene sequence alignment,physiological and biochemical characteristics and culture characteristics of different media analysis,the strain was determined to be a Streptomyces and the strain conformed to the characteristics of the genus Streptomyces.（4）Pot experiments showed that both the spore suspension of strain NEAU-174(10⁶,10⁷,10⁸CFU·g^-1)can effectively control tomato R.solanacearum,with control effects of 68.8%,74.5%and 83.2%,respectively.（5）The best formulation for culture medium of strain NEAU-174 was vermicompost and wheat bran with the ratio was 3:1.The solid state fermentation conditions of strain NEAU-174were optimized by response surface methodology.The inoculation amount was 10%,the water content was 50%and the temperature was 27℃.On the 7 day,the spore amount of strain NEAU-174 reached the maximum,which was 4.47×10¹¹CFU·g^-1.（6）Potted tomato experiments showed that the solid fermentation biologics of strain NEAU-174(10⁶,10⁷,10⁸CFU·g^-1)could effectively control tomato bacterial wilt,with control effects of 68.0%,74.9%and 81.6%,respectively.（7）When the strain NEAU-174 was fermented,the bacteriostatic diameter of the supernatant was 28.5 mm,and the bacteriostatic activity of the methanol extract of cell pellet was 32.3 mm.The active metabolites were separated by activity tracking method.The main active compounds was separated by silica gel plate separation method.According to the TLC chromatography characteristics,silica gel column separation characteristics and the activity of fermentation liquid,it was speculated polyether antibiotics.