Isolation Of The Antagonistic Actinomycetes Against Phytopathogenic Fungi And The Investigation On Its Active Substance

Agricultural microbiology resources is basic resources that the maintenance for sustainable development of agricultural ecological environment.Among them,the Actinomycetes produces various active secondary metabolites and become the main source of the biological pesticide.we collected 18 soil samples from Yunnan,Guangdong,Anhui,Hubei,Jiangxi and other places,then isolated and screened three strains of Actinomycetes which have strong antagonistic activity for plant pathogenic fungi（numbered C2,N2,E7 and D8,respectively）.Firtly,the four antagonistic Actinomycetes was conducted for strain identification,and antimicrobial activity was measured.Then we focused on the fermentation medium optimization,activity evaluation,antibactarial mechanism of the strain C2 and N2,and the separation of active metabolism,purfication and identification.In order to provide theoretical basis and direct criterion for biocotrol appliaction.1.We collected 18 soil samples,isolated and screened 169 actinomycetes from them.We screend 169 Actinomycetes by using flat confrontation method,the tested strains was the rice shealth blight strains.Which has screened 13 strains that have antibacterial effects.The 13 strains was for fermentation,the fermentation broth was measured antibacterial activity by using beidie method,and futher rescreening four better effect of antabgonistic Actinomycetes.2.We have determined the taxonomical status of Anctinomycetes C2,N2,E7 and D8 through the use of multiphase classification and indentification method.Based on the comprehensive results,the strains C2,N2,E7 and D8 were identified as Streptomyces sp.WPCB 129,Streptomyces prasinopilosus,Streptomyces corchorusii,and Streptomyces sp.A012304,respectively.3.We studied for fermentation medium formula,antibacterial activity of acive subtance,stability,purification and separation of Streptomyces.sp C2.Using Single factor experimental design,the fermentation medium as optimized,which was showed as follows（g/L）:sucrose 30,corn starch 10,corn syrup 20,（NH₄）₂HPO₄ 2,K₂HPO₄ 1,MgSO₄ 1,MnSO₄ 0.01,ZnSO₄ 0.01.Measured the antibacterial activity of Streptomyces.sp C2 thourgh growth rate method.The results show that the concentration10%of the fermentation broth have tested five kinds of plant pathogenic fungi（rice sheath blight strains,rice blast fungus,Penicillium orange,grape anthrax bacteria and Fusarium oxysporum）and inhibition rates were 49.78%,56.62%,94.10%,80.96%and18.59%respectively,it indicating that Streptomyces.sp C2 have a broad spectrum properties that antagonistic plant pathogenic fungi（rice sheath blight strains,rice blast fungus,Penicillium orange,grape anthrax bacteria and Fusarium oxysporum）.Stability experiments results showed that the fermentation broth of Streptomyces C2 was treated30 min on temperature 30℃and UV treated 60 min,still maintain a certain antibacterial activity,which shows that the active substance was produced by Streptomyces sp.C2 has good thermal stability and UV stability.The results of pH paper chromatography and Doskochilova solvent system indicated that the active substance was produced by Streptomyces C2 is a non-water-solubleⅡneutral type antibiotics.The active coarse fraction was obtained by further column chromatography pre-separstion that was subjected to LC-MS test,it was found that it obtained three components of retention time 29.9、31.1and 38.4min,which the active ingredient content of 29.9 min accounted for 54.47%,which the mass spectrum further shows the molecular weight of the component is 472.4.We studied for Streptomyces sp.N2 about optimization of fermentation mediud,the active substance evaluation,antibacterial mechanism and structure identification.The optimization of fermentation medium by quadratic rotation design showed that the ratio of the available nitrogen ammonium sulfate and slow-acting nitrogen soybean meal would have a significant impact on the synthesis of active substance was produced by Streptomyces N2.Among them,ammonium sulfate has significant N source repressor effect,while soybean meal is beneficial to the synthesis,which is most Actinomycetes N source demand characteristics are consistent.Antibacterial activity results showed that the concentration 10%of the fementation broth tested all have inhition effect.The inhibition rate reached 78.07%,71.59%,88.4%,38.31%,88.60%and 19.45%respectively,which indicates that the Streptomyces sp.N2 also has broad-spectrum antifungal activity（ice blast fungus,Rhizoctonia solani,Fusarium oxysporum,Fusarium,Italy Penicillium,orange Penicillium）.The inhitory mechanism of Streptomyces sp.N2 showed that the active substances were produced by them will cause progressively deformed,folding serious,damage oranelles and lead cell repture.It can be seen that Streptomyces sp.N2 had effect on membranes and organelles of plant pathogenic fungi,so as to achieve antibacterial effect.The experimental results of Streptomyces sp.N2 have good biological control effect on kiwi and grapes infected by Botryosphaeria bacterial.When the concentration of the liquid was 500μg/ml,fruit tissue has no ulceration and disease spot,it control effect of100%.Further purified by preparative liquid phase by solvent extraction,macroporous resin and silica gel column chromatography and pre-separation of the coarse active ingredient,and with UV,IR,mass spectrometry and nuclear magnetic resonance spectroscopy and other techniques for the eligible active monomer structure identification and analysis,identification of single structure:the solution of the compounds is3-methyl-3,5-two amino-4-ene-pyran-2-ketones（molecular formula:C₆H₇O₂N）,it belongs to amide compounds.It has an active four membered ring with antibacterial activity.Novelty search was by means of chemical authoritative scifind software,the results showed that no similarity structure,it illustrates that agricultural antibiotic N2 is new compound.