Isolation And Screening Of Streptomyces Corchorusii AUH-1 And Study On Its Activity Evaluation And Antagonistic Mechanism Against Phytopathogenic Fungi

Soil microorganism is an important basic resource of soil ecological environment and plays a significant role in biological control of plant diseases.Actinomyces is one of the most influential groups of soil microorganisms,and it can produce many metabolites with novel structure and significant biological activity,and become a valuable biological control resource.In this paper,an actinomycetes AUH-1 with strong antagonistic activity against phytopathogenic fungi was isolated from 26 different types of soil samples collected in Jiangxi,Fujian and Hebei,etc..On the basis of strain identification and antibacterial activity determination of the antagonistic actinomycetes AUH-1,we focused on the fermentation medium optimization,metabolic regulation analysis,antibacterial activity evaluation,antibacterial mechanism and the preliminary separation and identification of the active products.In order to provide theoretical guidance and direct basis for biocontrol application.1.We isolated and screened 15 antagonistic actinomycetes from collected 26 soil samples by using dual culture,and the indicator strain was F.oxysporum f.sp.niveum.The15 strains were fermented,and the fermentation broth was measured antibacterial activity by using cylinder-plate method,such as bacteria,fungi and plant pathogens,and further rescreening to obtain an antagonistic actinomycetes AUH-1 which had a best inhibitory effect on Rhizoctonia solani and F.oxysporum f.sp.niveum.We had determined the taxonomical status of Actinomycetes AUH-1 through the used of multiphase classification and identification method.Based on the comprehensive results,the strain AUH-1 was identified as Streptomyces corchorusii.2.We studied preliminarily for fermentation medium formula,antifungal activity and stability of S.corchorusii AUH-1.After optimization with response surface,the composition mass concentration of S.corchorusii AUH-1 culture medium was showed as follows:sucrose 3.1%,corn starch 0.25%,corn steep liquor 4.5%and?NH₄?₂SO₄ 0.375%.By using the method of inhibition rate of mycelial growth,we determined the antifungal activity of the S.corchorusii AUH-1.The results showed that strain AUH-1 had tested eight kinds of phytopathogenic fungi?F.oxysporum f.sp.niveum,Phytophthora parasitica var.nicotianae,R.solani,Phytophthora capsica,Botryosphaeria dothidea,Fusarium oxysporum f.sp.vasinfectum,Verticillium dahliae,and Fusarium oxysporum f.sp.cucumerinum?and inhibition rates were 81.81%,78.99%,68.25%,67.23%,29.66%,28.25%,24.67%and37.05%,respectively.It suggests that S.corchorusii AUH-1 have a broad spectrum in suppressing the growth of various fungal plant pathogens.The stability test showed that the antifungal active substances produced by this bacterium had favorable temperature stability and UV stability and have promising prospects for the development of microbial pesticides.3.The characteristic curves of fermentation metabolism of S.corchorusii AUH-1indicated that its antifungal active substances began to be synthesized in large quantities only when the concentration of bacteria reached a certain stage.Furthermore,the models and parameters which can be used in biomass growth,product formation and substrate consumption were obtained by simulation of fermentation kinetics model.The fitting degree of each model was 96.329%,95.954%and 96.227%,respectively.The relative error was less than 5%.The fitting effect was better,which provides a kinetic theoretical reference for the further optimization of the fermentation process of the strain.4.The inhibition mechanism of S.corchorusii AUH-1 was studied by using R.solani and F.oxysporum f.sp.niveum as indicator strains.The results showed that S.corchorusii AUH-1could inhibit the germination of pathogenic sclerotia and spores,inhibit the mycelial growth,increase the cell membrane permeability,accelerate K⁺,Na⁺exosmosis of protoplasm in mycelium,and inhibit the synthesis of ergosterol in the cell membrane.The degree of lipid peroxidation of the cell membrane was aggravated and synthesis and metabolism system of the hypha protein was destroyed so as to affect the normal growth and development of mycelium and achieve the antagonistic effect.It can be seen that the active substances produced by S.corchorusii AUH-1 is mainly applied to the cell membrane of plant pathogenic fungi,further interfered and destroyed the structure and function of the membrane to achieve the bacteriostatic effect.5.The results of the determination of the active substances in the fermentation supernatant and intracellular showed that active substance of the S.corchorusii AUH-1 was mainly in the cell.The results of solvent extraction showed that the extraction efficiency of n-butanol was the best.According to principle of similar compatibility,it was preliminarily determined to be a medium-polarity substance.The results of Doskochilova solvent system indicated that the chromatographic profile of AUH-1 was the closest to the macrolide antibiotics,but whether the main antibacterial active components have the properties of macrolide antibiotics remains to be further studied.The TLC results showed that five components were separated on the chromatoplate,wherein the component 5 had the highest antibacterial activity,the component 1 and the component 3 also had antibacterial activity,and the other components were not.The UV absorption spectrum showed that there are three maximum absorption peaks,which was 197 nm,202 nm and 256 nm,respectively.The HPLC analysisi found that when the methanol concentration was 60%isocratic elution,the sample active substances was up to 17 species,and the maximum peak time was 10.798 min.The Infrared Spectrum showed that O-H,-CH₂,C=O,C=C,C-N,C-H and C-O are present in the sample.The SEM found that its surface was smooth,viscoelastic and adsorptive.The LC-MS results showed that the molecular weight of AUH-1 was about 301 and 318.The above results suggest that AUH-1 antifungal active substances may be a cyclic polyhydroxy carboxylic acid compounds.However,its specific structure still needs further identification.