Screening,Identification And Action Mechanism Of Active Compounds Of Antagonistic Streptomyces Against Rhizoctonia Solani Caused Tobacco Target Spot Disease

Rhizoctonia solani AG-3 is the causal agent of target spot disease on tobacco leaf.Streptomyces spp.produced several types of secondary metabolites;these are the potential source of agro-antibiotics and as well environment friendly.A systematical study was carried out to evaluate the most potent antagonistic Streptomyces strain with better properties in such aspects as isolation and screening,classification and identification,optimization of fermentation conditions and nutrient medium,extraction,purification and identification of antagonistic substance,action mechanism of active substance against the pathogen,which led to the inhibition of Rhizoctonia solani AG-3 and control of target spot.It is of great significance in disease management of several plants diseases associated to Rhizoctonia solani.The main results as followed:1.Twelve strains were isolated from the soil samples and screened against the R.solani,assessed by the living organism's inhibition effect.For secondary screening six strains were selected on the base of primary screening,by the fermented broth on Gause's synthetic medium.Stl-9,TA1123,9407,128,163 and 330 all have satisfied inhibitory effects,while the most potent Streptomyces antagonistic strain was TA1123.Streptomyces strain TA1123 was tested against other phytopathogens and showed inhibitive effects against each.On the base of inhibitive effects Streptomyces TA1123 was selected for further research.2.Classical taxonomy and molecular biological taxonomy were adopted in classifying and identifying strain TA1123.Strain TA1123 was identified as Streptomyces var.diastatochromogenes.Gene bank accession number KX852460 was assigned by NCBI.3.To enhance production of secondary metabolites,central composite design of response surface methodology was applied in submerged fermentation.The maximum metabolite production was using medium volume 55 ml in 250 ml flask,agitation speed of 165 rpm,incubation temperature 30c,initial pH 6.8 and inoculum size of 7%.Peanut meal,soluble starch,NaCl,yeast extract,and ammonium sulphate were identified the best ingredient for high antifungal activity of Streptomyces strain TA1123 against the R.solani AG-3.For the improved production of secondary metabolites,central composite design of response surface methodology was applied in submerged fermentation.The best concentrations of ingredients were peanut meal 4.88%,soluble starch 4.40%,NaCl 0.52%,and yeast extract 0.47%,and ammonium sulphate 0.0360%.Study of metabolism changes in the submerged fermentation process was analyzed.Level of the reducing sugar increased,as the total sugar was consumed.Amino nitrogen and total sugar tendency was decreased,which indicated the growth of bacteria in submerged fermentation batch.Production of secondary arid other metabolites influenced the pH of the fermentation batch.4.Stability of fermentation broth was determined at various temperatures,pH values and treatments under illuminated light and U.V light.Fermented broth was stable and had the potential to suppress the pathogen.Crude substance was produced by submerged fermentation process from Streptomyces strain TA1123.Various solvent was used to extract the culture filtrate.Among all,ethyl acetate extracted supernatant showed great potency against R.solani AG-3.The active fractions were purified by silica gel column chromato~graphy having 52 mm zone of inhibition against R.solani AG-3.Most active fraction was analyzed by Gas chromatography-Mass spectrometry(GC-MS)to identify the compounds.Twenty seven compounds were identified in the extract of ethyl acetate;.most of the compounds were the derivatives of aromatic compounds.Eicosane(C20H42)and dibutyl phthalate(C16H22O4)were found antifungal compounds in this study.While morphinan,7,8-didehydro-4,5-epoxy-17-methyl-3,6-bis[(trimethylsilyl)oxy]-,(5.Alpha.6.Alpha)-(C23H35NO3Si2),cyclononasiloxane,octadecamethyl-(C,8H54O9Si9)and benzoic acid,2,5-bis(trimethylsiloxy)(Ci6H30O4Si3)are the major compounds with highest peak number.Eicosane(C20H42)and dibutyl phthalate(C16H22O4)were found antifungal compounds in this study.Dibutyl phthalate was found in both extract,both compounds already reported as antifungal compounds.5.Antifungal action mechanism of ethyl acetate extracts of Streptomyces TA1123 against the tobacco target spot disease pathogen,Rhizoctonia solani AG-3 were evaluated.Bacterial extract had strong antifungal activity against the R.solani AG-3.Bacterial extract had 96%inhibition effect against the mycelium,formation of the sclerotia decreased from 71.73 to 0%as the concentration of the treatment increased from 1×104 mM to 7×10-4 mM and 88.22-97%inhibitive effect the disease lesion.Transmission electron microscopic(TEM)observation showed suppression of hyphae.Endogenous ROS generated and caused oxidative damaged evidenced by the CLSM images.According to our knowledge this is the first report to study action mechanism to encounter the basidiospores of R.solani AG-3.6.Ethyl acetate extracts of Streptomyces TA1123 could induce the defense response by activating the enzymes in tobacco plants to combat the ROS and extracellular metabolites in tobacco leaf,which might increase the immunity.Tobacco plants were treated with ethyl acetate extracts of Streptomyces TA1123.Changes in the activity of the enzymes were evaluated and exometabolomic study was carried to analyze the effects of Ethyl acetate extracts in the tobacco leaf metabolism by LC-MS/MS.Metabolic profile of the treated sample was compared with control(untreated).Activation of defense enzymes indicated that enzymes burst the exogenous ROS and guard the tobacco plant.It was observed in the exometabolomic study that in the treated sample amino acid(L-Cysteine,N,S-di(trifluoroacetyl)-,trimethylsilyl ester),mammalian steroid 5?-Androstane-3a,17?-diol,bis(pentafluoropropionate),flavonoids(Rutin),amines and amides were identified.Current study determined that ethyl acetate extract of Streptomyces TA1123 activated the defense enzymes to burst the exogenous ROS.Exometabolomic study suggested that ethyl acetate extract caused changes in the metabolic profile of tobacco leaf which could immune the tobacco plants against the microbes.