Screening Of Antagonistic Actinomycetes In Populus Euphratica Wetland And Primary Investigation Of Their Secondary Metabolites

Populus euphratica wetland is a typical wetland environment formed by the convergence of the Tarim River Basin and the desert in Xinjiang,and it is an ideal place for excavating actinomycete resources.This study is based on the early isolation of 140 strains of actinomycetes from the soil samples of Populus euphratica in Alar,Xinjiang.Alternaria tenuissima,Fusarium oxysporum and Verticillium dahliae were used as target bacteria for the initial screening and rescreening of antagonistic activity.Strains with better antibacterial activity were selected and fermented to obtain crude extracts.The secondary metabolites were separated and identified by thin-layer chromatography,high-performance liquid chromatography and nuclear magnetic resonance.In the end,two strains were new species of the genus Streptomyces were identified to determine their taxonomic status.Afterwards,The research results are as follows:A total of 60 strains with bacteriostatic activity were screened for 140 strains of actinomycetes by plate confrontation method.and then rescreened by agar punching method,a total of 40 strains of actinomycetes with antibacterial activity were obtained.Among them,12 strains were screened for antibacterial activity against Alternaria tenuissima,TRM68008,TRM68068,TRM68080,TRM680120,TRM68124 and TRM68139 had the most significant antibacterial activity.6 strains had antibacterial activity against Fusarium oxysporum,TRM68014 had the most significant antibacterial activity.12 strains with Verticillium dahliae had antibacterial activity,TRM68051 had the most significant antibacterial activity.20 strains had antibacterial activity against Staphylococcus aureus,TRM68023,TRM68028 and TRM68037 had the most significant antibacterial activity.7 strains were screened for Candida albicans,and TRM68022,TRM68059,TRM68068,TRM68080 and TRM68120 had the most significant antibacteri-al activity.There were 9 strains with antibacterial activity against the three pathogenic bacteria,and 4strains with better antibacterial activity were TRM68014,TRM68022,TRM68068 and TRM68080.Separation of secondary metabolites by the two strains of TRM68068 and TRM68080 with broad-spectrum antibacterial activity using silica gel column chromatography,gel column chromatography and preparative chromatography,etc.,and according to physical and chemical properties and their Spectral analysis obtained a total of 3 compounds,of which two compounds 068-1 and 068-2 were isolated from TRM68068,which were identified as genistein and daidzein;one compound 080-1 was isolated from TRM68080-1,which were identified as N-acetyltyramide.The multi-phase classification method was used to classify the strains TRM 68005T and TRM68085T isolated from the soil of Populus euphratica wetland and found that these two strains had the same phylogenetic and chemical taxonomic characteristics as Streptomyces.The optimal growth condition for strain TRM6005^T is Gause's synthetic agar medium with a salt concentration of 1%,pH 7.0 and a growth temperature of 37°C.It has the ability to produce lipase,oxidase,starch hydrolase,urease and melanin,and has the ability The ability of nitrate,catalase and gelatin to liquefy.Phylogenetic analysis of the 16S rRNA gene sequence of TRM 68005^T showed that it had high similarity with S.chromofuscus NBRC12851^T?98.34%?and S.ferrugineus HV38^T?98.25%?,and formed obvious branches Through morphological observation of strain TRM68005^T and relative strains,it was found that there was a significant difference,followed by chemical taxonomy identification results showed that the strain TRM 68005^T whole cell hydrolyzed sugars including ribose,xylose,glucose and galactose Amino acids are mainly L,L-DAP,menaquinone is mainly MK-9?H₆??51.04%?,MK-9?H₈??30.78%?and MK-9?H4??10.16%?as the Lord.According to these genes and physiological and biochemical characteristics,TRM 68005^T strain is a new species of Streptomyces,and the species is named Streptomyces lasuensis TRM 68005^T.The optimal growth condition of strain TRM68085 is Gause's synthetic agar medium with salt concentration of 0%,pH 7.0 and growth temperature of 28°C.TRM68085^T has the ability to produce lipase,oxidase and starch hydrolase.Chemical taxonomic results show that the whole cell hydrolyzed sugar of strain TRM 68085^T mainly includes ribose,xylose,glucose and galactose,fatty acids are mainly C16:0?25.4%?and iso-C16:0?18.3%?,methylnaphthalene The quinones are mainly MK-9?H₆?and MK-9?H₈?,and the whole cell amino acids are mainly L,L-DAP.By comparing the 16S rRNA gene sequence of strain TRM 68085^T on Ezbiocloud,the most similar strain is S.capitiformicae 1H-SSA4^T,and the similarity is98.54%.Through phylogenetic analysis of three evolutionary trees,TRM68085^T and Streptomyces S.capitiformicae 1H-SSA4^T?98.6%?formed a stable independent branch,in addition,the DDH value analysis of the whole genome sequence of strain TRM68085^T and related strain S.capitiformicae1H-SSA4^T,its value is far below 70%,according to these genes and physiological and biochemical The characteristics show that TRM 68085^T is a new species of Streptomyces,and the species is named Streptomyces arboris TRM 68085^T.