Isolation And Identification Of Secondary Metabolites From Photorhabdus Temperata SN259 And Their Antifungal Activities Against Phytopathogenic Fungi

The symbiotic bacteria associated with insect pathogenic nematodes are belonging to the family Enterobacteriaceae.Recent studies have shown that Photorhabdus.spp can produce a wide variety of secondary metabolites,these metabolites have diverse chemical structures and extensive bioactivities of medicinal and agricultural significance,such as antibacterial,antifungal,insecticidal,nematicide,antiviral and anticancer,and have a new type biologic resource with the potential of exploitation and the prospect of application.In this paper,50 strains of entomopathogenic bacteria are isolated from the soil samples collected in June,2015,from Fengcheng City of Liaoning Province,Zhalantun City of Inner Mongolia,Changbai Mountains of Jilin Province,China,by an entrapment method using Galleria melonella larvae.The optimal strain is determined by chemical screening and bioactive screening and identified as Photorhabdus temperata.The isolation,identification and bioactivities of the secondary metabolites of the strains are also studied,the main results are as follows:1.Screening of dominant strain:The crude extractions of the 57 bacteria are obtained by using macroporous resin adsorption method and screen by using TLC detection technology,the results show that the crude extract of SN259 is rich in secondary metabolites.The activities of the crude extractions are evaluated against phytopathogenic fungi using mycelium growth rate method.The results show that the crude extract of SN259 demonstrated obviously inhibition against mycelial growth of Pythium aphaniderrmatum,Rhizoctonia solani Kuhn,Exserohilum turcicum,and Fusarium oxysporum,when the concentration of crude extract is 50 ?g/mL,the inhabition rates of the four fungi are 72%,51%,41%,38%,respectively.So the strain of SN259 is selected as the optimal strain.2.Identification of SN259 strain:The SN259 strain is identified by 16S rRNA genes sequence analysis.The 16S rRNA genes of SN259 strain is amplified by PCR,the homology comparison and clustering analysis of the 16S rRNA sequence of SN259 strain are conducted by constructing phylogenetic tree.In the constructed phylogenetic tree,the sequence of SN259 strain is most similar(100)to the sequence of Photorhabdus temperata(GenBank accession number EU136626)and is named Photorhabdus temperata SN259(GenBank accession number KU240002).3.Isolation,purification and identification of secondary metabolites from SN259 strain:SN259 strain is large-scale cultured in liquid fermentation and the crude extract is obtained by extract technology.Seven stilbene derivatives are isolated from secondary metabolites of Photorhabdus temperata SN259 by column chromatography and semi-preparative high-performance liquid chromatography.Their structures are identified as 3-hydroxy-2-isopropyl-5-Phenethylphenyl carbamate(1),2-(1-hy droxypropan-2-yl)-5-[2-phenylethyl]benzene-1,3-diol(2),2-(1-hydroxypropan-2-yl)-5-[(E)-2-phenylethenyl]benzene-1,3-diol(3),2-ethyl-5-(2-phenylethyl)benzene-1,3-diol(4),2-isopropyl-5-[2-phenylethyl]benzene-1,3-diol(5),2-ethyl-5-[(E)-2-phenylethenyl]benzene-1,3-diol(6),and2-isopropyl-5-[(E)-2-phenylethenyl]benzene-1,3-diol(7),by 1H and 13C NMR and HRESIMS,as well as the published literatures.In which 1-3 are new compounds,4 is novel natural product,5-7 are three known compounds.4.Bioactivity assay and structure-activity analysis:Compounds 1-7 are evaluated for their antifungal activities against four phytopathogenic fungi by using the agar medium assay and the 96-well microdilution broth assay.Compounds 1 and 7 demonstrated strong inhibition against mycelial growth of P.aphanidermatum with the IC50 values estimated to be 2.8 and 2.7 ?g/mL,respectively,which is comparable to that of the positive control.The two compounds also exhibit significant antifungal properties in the 96-well microdilution broth assay against the P.aphanidermatum,with IC50 values of 2.4 and 1.9 ?g/mL,respectively.Although these compounds bioassays reveal different which provid information that the antifungal activities of these compounds are certainly influenced by the of and the isopropyl group.