Screening Of Endophytic Bacteria From Sophora Flavescens And Identificaton Of Its Antagonistic Substances

An endophytic bacterium with antagonistic activity was isolated from Sophora which belonged to traditional Chinese medicines and its classification,ferment conditions, colonizing ability,and its extraction method and antifungal mechanism to pathogen of its antagonistic substances were studied.Strain B₃₆ was abtained which was Bacillus subtilis and it could colonize effectively on Figs.Two antagonistic substances of endophytic bacteria from Sophora were the first time obtained,and it belonged to cyclic lipopeptide,which destroyed the structure of plasma membrane and expressed obvious inhibition on spores germination and grown on hyphae of Fulvia fulva.Strain B₃₆ was obtained from Sophora flavescens Ait root.148 bacteria strains were isolated by dilution- Fig method from roots of Sophora flavescens Ait were sampled at Tianzhu mountain in Shenyang City Liaoning Province.29bacteria strains showed antagonistic activity though confront culture method against pathogens on Fig,then the aimed strain B₃₆ which sterile-filtered supernatant had the strongest antagonistic activity against pathogens in Fig experiment as well as in pot culture trails was screened.Strain B₃₆ had antagonistic effect on Helminthosporium sativu Pammel.el al.,Curvularia lunata (Wakker) Boed,Colletotrichum lindemuthianum,Botrytis cinerea Pers.,and Fulvia fulva (Cooke) Cifferris et al.,the inhibition zone diameters were all arrived at 25.0mm.As well as strain B36 showed biological control activity against Wheat powdery mildew of experiment in vitro and in pot and against tomato gray mold with control effect were 95.71%,83.20% and 71.49%respectively,so Strain B₃₆ had great capacity of research and application.According to the characteristics of morphological feature,physiology,biochemistry tests and the comparison of 16SrRNA sequence the classification of strain B₃₆ was established. The results indicated that it was positive of gram,germ staining and acid production from glucose,and utilized glucose,sucrose,mannitol,fructose as carbon source,it could reduce nitrate,hydrolysis starch and gelatin.The16S rDNA sequence of B₃₆ Strain was 1463bp, according to the phylogenetic tree,closest relative strain to B₃₆ Strain was Bacillus subtilis B432,as the strain B₃₆ was Bacillus subtilis.The culture conditions and the compositions of cultural medium of Endophytic B36 strain which was isolated from Sophora with Antifungal Activity were studied by using the single factor and orthogonal design method.According to the antagonistic activity of sterile-filtered B36 and its grown.The optimal fermentation parameters were 28-33℃,150 r·min-1,pH 9.0,20%inoculation volume,and 50mL culture in 250mL flask for 36h,which had the strongest antagonistic activity.The fermentation medium was modified and the optimized medium was composed of sucrose 2.0%,yeast 0.5%,soybean flour 0.5%, NH₄NO₃0.5%and CaCO₃1.0%.Under this optimized condition,the inhibition zone diameter increased 13.3%to the initial condition.Colonizing dynamics of t he strain B₃₆ in tomato were studied by labeling method with double antibiotics-resistance.The results showed that strain B₃₆ marked with double antibiotics-resistance could colonize the root of tomato and deliver into the issues which were influenced significantly by inoculation methods.The density of marked strain in the tomato after inoculated by pruned root and soaking seed were munch more then by soil drench,there were no marked strain in root of tomato by spraying foliar and daubing leaf method.After inoculated with pruned root,the marked strain B₃₆ could colonize the root of tomato that could enhance the biocontrol effect to pantheons,and the strain B₃₆ had stronger colonize ability around the roots then in the surface of the roots.Then the marked strain could deliver into internal stem and leaf tissues,and its density decreased in root gradually while increased in stem and foliage then decreased as the plant growing.The antifungal substances of Baciliius subtilis B₃₆ strain were extracted by organic solvents precipitates,solvents extraction and macroporous adsorption resin methods.The extraction effect of macroporous adsorption resin method had showed better effection than the other methods.So macroporous resin HP20 was adopted to adsorb the antifungal substances of Baciliius subtilis B₃₆ and 75%ethanol was selected for desorption the antifungal substances that with dynamic experiment.A component had greatest antagonistic activity and content was obtained by three times separated with silica gel column,then analyzed by HPLC-MS negative ion.Two components with elution time were 5.7min and 6.9min respectively were obtained,and their composition were determined by electro spray ionization mass spectrometry.The results showed that component A(with a molecular weight of 1042.5Da) had a primary structure of seven cyclic lipopetides link to C₁₄:Cys-Asp-Tyr-Gln-Fatty acid-Pro-Asn-Gln,and component B(with a molecular weight of 1056.5Da) had a primary structure of ten cyclic link to C₁₇:Fatty acid-Asn -Asp-Met-Gln-Tyr-Asn-Gln-Asn-Pro-Ser. Antifungal effect of active substances extracted by macroporous adsorption resin method from endophytic bacteria B₃₆ to Fulvia fulva(Cooke)Cif.were studied.The results showed that the pathogenic hyphae grew in abnormal shape and produced sac,and the inhibition zone diameter was 20.5mm after treated with the crude extracts of antifungal substances.The antifungal substances also expressed obvious inhibition effect on spores germination.The inhibition rate was 86.56%and the activity of PMG and Cx were 0.5911 U·mL^-1 and 0.01757 U·mL^-1 which were both lower than the control significantly when the concentration of antifungal substances arrived at 1.06×10³Î¼g·mL^-1.So it was concluded that the antifungal substances could inhibit the activity of PMG and Cx and destroy the structure of plasma membrane,but could not decompose the chitin or inhibit the content of proteins.