Broad-spectrum Antibacterial Activity Evaluation And Active Product Identification Of Two Endophytic Streptomyces Isolated From Perilla Frutescens

Plant diseases,especially root rot,are caused by multiple pathogen infections.A single chemical agent can hardly achieve broad-spectrum disease resistance,and excessive use of chemical agents will threaten the environment and human health.In response to the green prevention and control policy proposed in the 14th Five-year Plan of China,the purpose of this study was to screen out microorganisms with broad-spectrum antibacterial ability to be used as microbial fertilizer,so as to achieve comprehensive protection of plants and reduce the use of chemical pesticides.In this study,endophytic actinomycetes from the leaves,stems and roots of Perilla frutescens were isolated and purified.Two strains ZSY13 and ZSG18 with broad-spectrum antagonistic activity were screened out by plate antagonism method.Then,their application potential was evaluated by in vivo pot experiment,solid fermentation condition optimization and biologic fertilizer pot experiment.Finally,their active products were isolated and identified.The main results are as follows:(1)Six screening media were used to isolate and purify endophytic actinomycetes from Perilla frutescens 24,17 and 35 strains of actinomycetes were obtained from leaves,stems and roots,respectively.(2)Strain activity test.Nineteen kinds of pathogenic fungi and three kinds of pathogenic bacteria were used to test the activity of 76 actinomycetes,and two strains ZSY13 and ZSG18 with broad-spectrum antagonistic activity were screened out.The inhibition rates of ZSY13 against 19fungal species were more than 50%,and the inhibition rates against Bipolaris sorokiniana,Bipolaris maydis and Alternaria Nees reached more than 80%;The inhibition rates of ZSG18 against 17pathogenic fungi were more than 50%,among which the inhibition rates against Bipolaris sorokiniana and Exserohilum turcicum were more than 80%.ZSY13 could inhibit Ralstonia solanacearum,Escherichia coli and Staphylococcus aureus,among which the inhibition effect on E.coli was the best.The diameter of inhibition zone was 24.3 mm.ZSG18 also had good inhibition effect on the three kinds of bacteria,the diameter of inhibition zone was more than 20 mm and the inhibition effect on Ralstonia solanacearum was the best,the diameter of inhibition zone was 28.1mm.(3)In vivo pot test.With Bipolaris sorokiniana as the tested fungus and Ralstonia solanacearum as the tested bacteria,the spore concentration was adjusted to 10~6CFU/g,10~7CFU/g and 10~8CFU/g by spore mixing with soil.The results showed that ZSY13 had the best control effect on wheat root rot and tomato bacterial wilt at 10~8CFU/g,which were 73.4%and 70.2%,respectively.The control effect of ZSG18 was the best when the conidium concentration was 10~8CFU/g,and the control effect was 70.6%and 78.2%,respectively.(4)Optimization of solid fermentation conditions.Wheat bran and earthworm dung were selected as fermentation substrates.The mass ratio of wheat bran to earthworm dung is 1:2,which was the optimal matrix ratio of ZSY13.Temperature,inoculum amount and water content were selected as the three factors affecting sporulation quantity.Box-behnken was used to design the experiment and response surface was used to optimize the conditions.The results showed that the optimum conditions of ZSY13 were temperature 28℃,inoculation amount 20%and water content60%.After 7 days of fermentation,the spore production was 3.09×10~9 CFU/g.The optimum substrate ratio of ZSG18 was wheat bran and earthworm manure with a mass ratio of 1:1.The optimum conditions were temperature 28℃,inoculation amount 15%,water content 50%.After 7days of fermentation,the maximum spore yield was 6.33×10~9 CFU/g.(5)Pot test of control effect of bacterial fertilizer.When solid fermentation products were used as bacterial fertilizer,the control effect was the best when the maximum applicable spore concentration(10~8CFU/g)was used.The control effects of ZSY13 and ZSG18 on wheat root rot were 78.3%and 72.2%,respectively,and on tomato bacterial wilt were 72.2%and 81.2%,respectively.(6)Strain identification.The 16S rRNA similarity between ZSY13 and Streptomyces melanosporofaciens DSM 40318 is the highest,with a similarity of 100%,and forms a stable branch with the strain in the phylogenetic tree.Therefore,it is preliminarily determined that ZSY13 is a subspecies of S.melanosporofaciens.ZSG18 has the highest similarity with 16S rRNA of Streptomyces antimycoticus NBRC 12839,with a similarity of 99.86%,and forms a stable branch with the strain in the phylogenetic tree.Therefore,it is preliminarily determined that ZSG18 is a subspecies of S.antimycoticus.(7)Identification of active secondary metabolites.The active compounds were isolated by silica gel column chromatography,gel column chromatography and semi-preparative high performance liquid chromatography combined with activity tracing method,and identified by NMR and MS.The active compounds in ZSY13 were niphenycin A and niphenycin C.The EC50 of niphenycin A and niphenycin C against Bipolaris sorokiniana were 3.4μg/mL and 3.9μg/mL,respectively.The lowest inhibitory concentrations against Ralstonia solanacearum were 8μg/mL,respectively.Guanidylfungins A was synthesized from ZSG18.The EC50 of guanidylfungins A against Bipolaris sorokiniana was 3.9μg/mL,and the minimum inhibitory concentration of guanidylfungins A against Ralstonia solanacearum was 8μg/mL.