Purification Of Xenorhabdusbovienii SN269 Secondary Metabolitesand Analysis Of Inhibition Activityof Madumycin ? Against Fungi Pathogens

Entomophthogenic bacteria parasites in the intestinal tract of entomopathogenic nematode,is a Gram-negative bacterium and belongs to Enterobacteriaceae,which including Phophrabdus,Heterothabditidae and Serratia.Phothrabdus and He terothabdtidae,Xenohrabdus and steinernematidae,Serratia and Heterohitidoides chongmingensis are mutualistic,respectively.Entomophogenic bacteria is commonly used as a new biological pesticide now since it has a wide range of insecticidal and antifungal function,and it is playing an important role in biocontrol of plant diseases and insect pests.Especially the symbiotic bacteria,which play the key pathogenic role,could produce and secret many secondary metabolites during fermentation and have good bioactivity,if it could be isolated,purified and structure identified,it may have good potential in agricultural area and lay a foundation for the development of new biological pesticide.the main results are as follows:1.The isolation of bioactivity entomopathogenic bacteria:In this study,the soil samples are get from different counties in Liaoning Province,including Fushun,Fengcheng county in Dandong,Longshoushan county,Xifeng county in Tieling,and etc.,and some neighboring area close to Liaoning Province(Boketu county in Yakeshi,Zhalantun in Hulunbeier,Inner Mongolia;Changbaishan in Baishan,Jilin Province,and etc..),42 bacterial strains are get from the102 soil samples,including 38 primary types and 4 secondary types by using white-trap method.2.Screening of dominant strain:The crude extractions of the 42 bacteria are obtained by using macroporous resin adsorption method and screen by using TLC detection technology,the results show that the crude extract of SN231 has components different from all other crude extracts.By using the plate confrontation method and block displacement method to measure the crude extractions of the four symbiosis and bacteria on the 8 phytopathogenic fungi.The results show that the crude extract SN231 and bacteria also have better bacteriostasis on Phytophthora capsici.Therefore,SN231 are chosen as the target bacterial strain,renamed as SN269.3.Identification of SN269 strain:The SN269 strain is identified by 16S rRNA genes sequence analysis.The 16S rRNA genes of SN269 strain was amplified by PCR.The multi sequence homology was analyzed by clustal and Mega 6.0 software.The phylogenetic tree obtained by the neighbor-joining method showed that SN269 strain clustered together with Xenorhabdus bovienii,it can come up to 99%by comparing the 16S rRNA sequence of homology of the strain SN269.So SN269 is named Xenorhabdus bovienii SN269.4.Isolation,purification of secondary metabolites from SN269 strain:SN269 strain is large-scale cultured in liquid fermentation and the crude extract is obtained by extract technology.D3 stilbene derivative is isolated from secondary metabolites of Xenorhabdus bovienii SN269 by column chromatography and semi-preparative high-performance liquid chromatography.5.Identification and bioactivity assay of D3 stilbene derivative:Compound D3 is evaluated for its antifungal and inhibition of bacterial activities against phytopathogenic fungi and pathogenic bacteria by using the agar medium assay and the 96-well microdilution broth assay.The results show that D3 has a significant inhibiting effect on Phytophthora capsici and Botrytis cinerea,the EC50 values are 35.32 and 35.40 ?g/mL and has better inhibiting effect on Staphylococcus aureus,the IC50 valu is 0.13±0.02?g/mL.