Isolation,Identification And Biological Activities Of Secondary Metabolites Of Endophytic Actinomycetes From Neem

By taking the Actinomyces strain R6 separated from the neem as the research object,the taxonomic status,antibacterial activity of fermentation broth and optimization of fermentation medium and conditions were studied systematically,and the extraction and purification of the secondary metabolites of strain R6 were also studied.The antibacterial activity and safety of these metabolites,which structures were identified,were measured.The main results are as follows:1.Screening of endophytic actinomycetes R6 with high antibacterial activity from neem.114 endophytic fungus,26 bacteria and 85 actinomycetes were obtained from the leaves and nutlet of neem.Then the fungicidal activities of the 85 actinomycetes against 13 kinds of plant pathogenic fungus were tested by dual culture,which showed that 24 strains were good activity.The antibacterial activity and insecticidal activity of these 24 strains were also evaluated,and of which G20,G27,G49,G60 and R6 showed low toxicity activity.Finally,R6 with high bactericidal activity and rich substances was chosen as the experimental materials preliminarily by the antibacterial activity screening and chemical screening.2.R6 strain was Streptomyces hygroscopicus by traditional taxonomy and biology identification.The morphological characteristics and culture characteristics of actinomycete strain R6 were observed,and the physiological and biochemical characteristics were measured in this study.The 16 S r DNA sequence was analysed and compared.By sequence comparing,the results showed that R6 was most similar with S.pactum NBRC13433 and S.olivaceus B-3009,in which the homologies was 98.87% and 98.85%.The R6 strain was S.pactum,which was consistent with the characteristics of R6 strain,which was consistent with the characteristics of S.hygroscopicus.3.Optimization of Fermentation Process of Actinomycete R6 Strain.The optimization of fermentation medium and conditions were studied by single factor experiment and orthogonal test.The favorable fermentation condition was determined,which was the age of inoculum 3 d,fermentation time 7.0 d,fermentation temperature 28.0 oC,rotate speed 180 r/min,inoculation amount 8.0% and initial p H value of medium 8.0.The optimized formula of medium?g/100 m L?was as follow: soluble starch 20.0,glucose 10.0,peptone 5.0,yeast extract powder 5.0,NaCl 4.0,K₂HPO₄ 0.5,MgSO₄·7H₂O 0.5,CaCO₃ 2.0 and pH 8.0.4.Monomer compound Y₁,Y₂ and Y₃ were separated and purified from the R6 fermentation products.The fermentation products?11.58 g?of R6 strain was extracted by macroporous resin adsorption process and solvent extraction.The antimicrobial activity of fermentation products was carried out by bioassy-driven separation,which showed that these products were moderately active with good activity against fungus and bacteria.The toxicity activities of C₁,C₂,C₃,C₄,C₅ and C₆ were obtained by separation and purification of the active substances in the fermentation products by a column chromatography on silica gel.By determination of the activity of Botrytis cinerea and Erwinia cawtovora,C₁ and C₂ with strong antibacterial activity were isolated and purified.After several times silica gel column chromatography,gel column chromatography,thin layer chromatography and high performance liquid preparation and other methods,three pure compounds were obtained?Y₁ 48.2 mg,Y₂ 24.4 mg and Y₃ 81.6 mg?.5.Y₁ and Y₂ were polyketone new compounds by structures identification.Three monomer compounds were identified by IR,MS,NMR,optical rotation and other technological means,and the activity was detected by microdilution method.The results showed that both Y₁ and Y₂ were new compounds and polyketone compounds,Y₁(Lobophorin N,C₃₃H₄₄O₆)and Y₂(Lobophorin O,C₃₉H₅₄O₉);Y₃ was a known macrolides antibiotics?Divergolide C,C31H35NO8?.These three compounds showed good antibacterial activities,and the Y₁ was the best one.And the minimum inhibition concentration?MIC?of the compound Y₁ against Xanthomonas campstris pv,Pseudomonas syringae pv and E.cawtovora was 7.81 ?g/m L,15.63 ?g/m L and 3.91 ?g/m L,respectively.6.Compound Y₁ has high antibacterial activity against tomato gray mold and safe for tomato plants.The influence of compound Y₁ on the mycelium growth,spore germination,mycelial biomass,conidia and sclerotium of B.cinerea was tested by mycelium growth rate method,spore germination and mycelial dry weight test.With the increase of concentration,the inhibitation was steadily increasing.The biological activity and safety were tested by potculture method.The control effect of Y₁ was 85.44% at the concentration of 7.81 ?g/m L,and safe for tomato plants.