A Preliminary Study On The Production Of Resistant Secondary Metabolites By Pseudomonas Fluorescens LMA-2, SXM-13, SXM-19 And EG-42

The application of chemical pesticides leads to serious environmental and social problems.Biological control is an effective means of prevention and control of plant fungal disease,because it is the effective protection of the ecological environment and potential control,so it has been recognized by both at home and abroad in recent years,Pseudomonas spp.get more attention as bio-control strains.Pseudomonas spp.can produce a variety of secondary metabolites,such as pyoluteorin?plt?,2,4-diacetylphloroglucinol?2,4-DAPG.,phl?,pyrrolnitrin?pm?,phenazine?pca?and oomycetesA?Oomycin?,pseudomonas acid,biosurfactant siderophores,hydrogen cyanide?HCN?,which inhibit the growth of plant pathogenic microorganisms,plt,phl,prn,pca has been widely studied.The use of antibiotics has led to a large number of resistant bacteria.Pseudomonas peptide ring of fat because of its chemical structure and action mechanism different from existing all kinds of antibiotics,and its inhibition of many kinds of drug-resistant bacteria and the activity of killing tumor cells,make it become a hot spot in today's new antibiotics screening.Four strains fluorescent pseudomonas producing antibiosis of secondary metabolites as material.we research include the function of strain identification,genetic molecular detection and identification of metabolites.The main research contents and results are as follows:1.199 fluorescent pseudomonas strains is from rhizosphere soil of corn,eggplant,pepper,ARDRA analysis shows that there are four OTU.Experiments show that the four strains is antagonistic fungi and inhibits bacteria.LMA-2 is antagonistic against Exserohilumturcicum LeonardetSuggs and Cochliobolus sati-vu Drechsl.SXM-13 is antagonistic against Sclerotinia sclerotiorum?Lib.?de Bary,Valsa mali Miyabe et Yamada and Rhizoctonia solani Kuhn.SXM-19 is antagonistic against to Cochliobolus sati-vu Drechsl and Valsa mali Miyabe et Yamada.EG-42 is antagonistic Cochliobolus sati-vu Drechsl.2.Using 16s rDNA sequencing,phosphorus fatty acid composition analysis and morphology observation methods of environmental scanning electron microscopy identify the above four strains.The SXM-19 is the pseudomonas putida,the rest is of pseudomonas spp.3.Detect antibiosis of secondary metabolites synthesis related genes of LMA-2,the SXM-13,SXM-19 and EG-42 in the method of Polymerase chain reaction,and the results show that the SXM-19 with related genes of phl and cyclic lipopeptid,EG-42 with related genes of phl.Chemical methods tests show LMA-2,SXM-13,SXM-19,EG-42 don't produce HCN and chitinase.4.Through the discharge of oil ring experiment and surface tensiometer test show that fermentation of SXM-19 exists surfactant,TLC results shows SXM-19 can produce cyclic lipopeptide,separation and purification of SXM₁9 metabolites using SephadexG-25,Electrospray Mass spectrometry shows the molecular weight is respectively 1207 and 1168,which is the same as pseudomycinB and masstolideC/pseudophomin.Found through the literature,the stench pseudomonas SXM-19 is firstly known that can produce two cyclic lipidpeptide.5.The SXM-19 mixture of cyclic lipidpeptide is determined by MTT experiment,we find that it has kill activity on HT1080,Hela,and HEK293 cells.Through this experiment,we obtain the LMA-2,the SXM-13,SXM-19 and EG-42 is antagonistic fungi and inhibits bacteria.Determine the SXM-19 with related genes of cyclic lipopeptide and phl in molecular level,EG-42 with related genes of phl.Secondary metabolites of LMA-2,SXM-13 need further research.Isolate and purify secondary metabolites of SXM-19,which is preliminarily determined as cyclic lipopeptide.