Isolation And Identification Of Antimicrobia Ingredients From Xenorhabdus YL001 Secondary Metabolites And Its Biological Activities

Till now,finding new natural products with bioactivities from metabolites is an effective ways to create new pesticides.X.nematophila,a mutualistic symbiont of the soil-dwelling nematode,is a potent producer of natural bioactive compounds.In X.nematophila ATCC19061,7.5%of the genomic genes encode the proteins involving in secondary metabolism.While less than 5%of the genes involved in biosynthesis of secondary metabolites in Streptomycetes which the main bacteria produced antibiotic.X.nematophila YL001 was isolated from its nematode symbiont,Steinernema sp.YL001 obtained from the soil from Yangling,Shannxi,and had been identified according to its morphological and molecular characteristics.Previous research found that X.nematophila could be produce several secondary metabolites with antimicrobial activity.In order to determine the main metabolites of the X.nematophila YL001,we isolated the lipid-solubility abstraction based on tracking method and structure elucidation of the bioactive metabolites by the methods of NMR and HPLC.The main results and conclusions of the thesis are as follows:1.Seven compounds(A2Petroleum ether,A3Petroleum ether,A4Petroleum ether,C9Chloroform,D3Chloroform,B4_{Ethyl acetate} and B7_{Ethyl acetate})were isolated from the fermentation broth of the Xenorhabdus nematophila YL001 by using solvent extraction and silica gel column chromatography.And six1kinds2of2compounds4were3identified,4namely:A2_{Petroleum1ether1}was13-ethyl1（3’-methyl-2’-keto）-pentane1（Nematophin）;1A3_{Petroleum1ether1}was16-benzyl-3-isopropyl-1-methylpiperazin-2,5-dione,1（piperazine1derivatives）;1C9_Chloroform1was12-acetylamino-N-[5-（6-aminopurine-9-oxy）-4-Hydroxy-2hydroxymethyl-tetrahydrofuran-3oxy]-3-phenyl-propionamide,1amides;1D3_Chloroform1was1phenazine-1-carboxylic1acid1（Ketrazin）;1B4_{Ethyl1acetate1}was1hexahydropyrrole-1,4-dione,（Pyrrole）;B7 Ethyl acetate was Genistein.Among them,C9Chloroform and D3Chloroform were isolated from Xenorhabdus nematophila YL001 for the first time.2.Created a simple and fast method to separate nematophin.The fermentation broth of X.nematophila YL001was extracted with resin for 6 h at room temperature.Resin was extracted four times with petroleum ether.Petroleum ether extracts further tested by HPLC to afford nematophin.The purity of nematophin is over 90%.3.Evaluated the antifungal activity of C9 _Chloroform against Exserohilum turcicum in vitro.Compounds C9 _Chloroform showed high effect in the mycelial inhibition assay,with the EC50values estimated to be 21.134μg/m L.4.Evaluated the antifungal activity of nematophin against Rhizoctonia solani.The results showed that nematophin possessed significant antifungal activities against R.solani(EC₅₀ 42.144μg/m L).As the nematophin concentration increased,a reduction in the percentage of mycelial growth was observed.Furthermore,the nematophin showed pronounced reduction in sclerotia formation for R.solani.Effects of the NEP on the hyphal morphology of R.solani was observed by SEM and TEM after 12 h treatment.The control sample of SEM presented no morphological changes,which exhibited a normal morphology with uniform,smooth,uniseriate,and robust hyphae with plump growing points.However,the NEP treated sample,showed altered morphology characterized by irregular hyphae at the growing points,twisted,shrivelled and with distorted morphology.These results indicated that NEP may disrupted the intracellular structure of the pathogenic fungi.Observation under the TEM revealed that the cell wall and septa of R.solani hyphae were uniform in the control sample.The most obvious change of R.solani hyphae was the deformation of cells with an increase in vacuolization,mitochondria decreased significantly and the cytoplasmic matrix was obscured.Disease control efficacy of the NEP was evaluated against broad bean under growth chamber conditions.5.Nematophin possessed significant antifungal activities against V.dahliae,with the EC₅₀ value estimated to be 37.68μg/m L.Without treatment by Nematophin to Fusarium oxysporum,the mycelium is thick and the growth point is round,with 37.68μg/m L Nematophin to V.dahliae,hypha surface roughnes,mycelium deformation shrinkage,and the growth point cracking.6.Nematophin possessed significant antifungal activities against Phytophthora infestans,with the EC₅₀ value estimated to be 51.247μg/m L.Effects of the NEP on the hyphal morphology of Phytophthora infestans was observed by SEM,the NEP treated sample,showed irregular hyphae,twisted,rings on the mycelium surface and shrinked at the surface of mycelium.Disease control efficacy results showed that NEP possessed significant preventive efficacy which was lower than that of referenced mancozeb,with control efficacies of 15.58 and 94.8%,respectively.However,it exhibited high curative activity,with control efficacy was 56.41%.