The Identification Of The Soil Actinomycete 11-9-3 And Studies On The Insecticidal Active Ingredients Of Its Secondary Metabolites

The secondary metabolites of microorganisms are important natural product resources.The development of new drugs through structural modification or derivatization of secondary metabolites of microorganisms is one of the main directions of research and development of new pesticides and natural products.Actinomycetes are an important bioactive substance-producing bacterium.At present,about 70%of the more than 30,000 kinds of biologically active products discovered from microbial secondary metabolites are derived from actinomycetes,which are widely distributed and widely distributed.The resulting secondary metabolites are novel and diverse in structure,so the discovery of new active substances from the metabolites of actinomycetes provides the basis for the development of new pesticides and the development of new lead compounds.In this study,several insecticidal activities of soil actinomycetes with certain agricultural activities were isolated from the Qinghai-Tibet Plateau and Inner Mongolia region with complex climatic conditions and varied soil conditions,and a soil actinomycetes with significant insecticidal activity was screened.In order to further clarify the insecticidal active ingredient of strain 11-9-3,this strain was systematically studied in this study,and the metabolites of this strain were isolated and identified.The activity was evaluated and the strain was classified and identified.The main results are as follows:?1?Using the slide dipping method and the dip-leaching method,Plutella xylostella,Tetranychina harti,Mythimna separata,and Aphis citricola were used as test insects.Seven strains of soil actinomycete fermentation broth deposited in the laboratory were screened for insecticidal activity.The results showed that:11-9-3,11-9-49,12-9-10,11-12-1,11-12-2.It has obvious toxicity to Tetranychina harti,and the corrected mortality rate is higher than 60%at 24h,while 11-9-3 and 12-9-10 have the best toxic activity against Tetranychina harti,and the corrected mortality rate is up to 80%at 24h;11-9-3,11-9-49,12-9-10 have better toxicity to Aphis citricola,24h corrected mortality is higher than 50%,and 11-9-3 against Aphis citricola The toxicity was the best,and the corrected mortality rate reached 70%in 24h.The7 strains of actinomycete fermentation broth had low toxicity to the Mythimna separata,and no obvious poisoning activity.The 72h mortality rate was less than 20%;The toxicity of 7strains actinomycete to Plutella xylostella was significantly different.The corrected mortality rate of 12-9-26 and 12-9-27 at 72h was less than 20%,and the corrected mortality rate of 11-9-49 and 12-9-10 at 72h was above 50%,while 11-9-3 poisoned the Plutella xylostella of the most significant,the corrected mortality rate was above 90%at 72h.The fermentation broth of 11-9-3 showed significant insecticidal activity,and the insecticidal activity of the crude extracts of the four polar segments of the strain was further quantitatively determined.The results showed that the ethyl acetate segment and the chloroform segment were poisoned against the Plutella xylostella.The activity was the best,and the corrected mortality rate was higher than 90%at 72h,so it was confirmed that the active substance was concentrated in the chloroform segment and the ethyl acetate segment.?2?Using multi-phase taxonomy method,by analyzing the morphological characteristics and physiological and biochemical characteristics of strain 11-9-3,combining the 16S rDNA sequence of the strain with the characteristic strain and the construction of phylogenetic tree,it can be basically It was confirmed that the strain 11-9-3 belongs to the Streptomyces.Through sequence alignment,the actinomycete strain 11-9-3 and Streptomyces hygroscopicus were on the same phylogenetic branch with a similarity of 98%.The morphological characteristics and culture characteristics were also similar to Streptomyces hygroscopicus.But its physiological and biochemical characteristics are quite different from the typical Streptomyces hygroscopicus.Therefore,11-9-3 may be a new subspecies of Streptomyces hygroscopicus.The classification status needs to be determined by DNA-DNA hybridization and comparison of cell wall's chemical composition.?3?A large amount of fermentation was carried out on strain 11-9-3,and the supernatant was collected and concentrated to obtain a crude extract of chloroform and ethyl acetate.The activity was followed by silica gel column chromatography and gel column.Separation and extraction of the chloroform active fraction of the 11-9-3 strain and the crude extract of the ethyl acetate active fraction were carried out by various separation methods such as separation,thin layer chromatography and HPLC,respectively,and finally 8 compounds were obtained,using nuclear magnetic hydrogen carbon.Spectral isotherm analysis combined with literature analysis,identified six compounds,namely:4-Hydroxybenzoic acid?A?,cyclo-?Pro-Leu??B?,1-acetyl-4,5-dihydroxyanthracene-9,10-dione?C?,1,8-dihydroxy-6-methylanthracene-9,10-dione?E?,Convolvulol?I?,cyclo-?L-phenylalanyl-L-pro?dipeptide?D?,C and E are all terpenoids,B and D are all cyclic dipeptide compounds,and I is a benzofuran compound.?4?The preliminary insecticidal activity of 6 compounds was studied.The toxicity of Aphis citricola,Tetranychina harti,Plutella xylostella,Mythimna separata,antifeedant activity against Mythimna separata,and Plutella xylostella were determined.Growth inhibition,the results showed that two compounds C with a significant toxicity to Tetranychina harti,(the lethal LC₅₀ of 756.46?g/ml)and compound E(LC₅₀ of 389.79?g/ml)were screened,and The compound I had certain antifeedant activity,and the antifeedant rate was 44%.