Isolation Of The Antagonist Streptomyces And Identification Of The Active Compound

The actinomycete strains with its high practical value in biocontrol can produce some kinds of secondary metabolites which are source for the development of new biological pesticide or precursor substances.In this context,isolation of antagonistic actinomycete has been considered as an important step towards the biological pesticide.In our study,firstly we isolated 320 strains of actinomycetes from soil samples,which collected from different geographical conditions such as farmland,forest,hot spring and river bed.Fusarium oxysporum f.sp.cucumerinum is the causal agent of cucumber fusarium wilt.In the experiment pathogenic fungus F.oxysporum f.sp.cucumerinum was used as indicator strain.Among the isolated strains,there are 77 strains showed antagonistic activity against F.oxysporum f.sp.cucumerinum,among which strain M527 showed the strongest bioactivity and broad spectrum antimicrobial activity.It was characterized as Streptomyces rimosus M527 by testing its morphology,physiology,biochemistry and 16 s rDNA homologous sequence analysis.The fermentation broth of S.rimosus M527 can completely inhibited the spore germination and hyphal growth of F.oxysporum f.sp.cucumerinum when it was deluted 8 times.According to the biocontrol experiment,fermentation broth of S.rimosus M527 promoted growing of cucumber plantlets and showed good effect inhibiting on cucumber wilt disease,with the disease index from 71.1 down to 20.0 and the treatment effect is 72.1 %.Then the fermentation broth of S.rimosus M527 was extracted with organic solvent and separated on silica gel column chromatography,then further separated by chromatography on Sephadex LH-20 and preparative HPLC,and during the separation process the F.oxysporum f.sp.cucumerinum was used as the indicator strain.The structure was determined on the basis of MS,1H-NMR and 13C-NMR spectroscopic analyses and comparison with the literatures.The active compound was identified as rimocidin and the HPLC condition was optimized by using ZORBAX SB-C18(150×4.6 mm,5 ?m)chromatographic column and 2998 PDA photodiode detector.Methanol as A mobile phase and water(0.1 % acetic)as B mobile phase,with 1ml/min flow rate and detection wavelength wa s 304 nm and injection volume was 5 ?L,the gradient elution program as follow: 0~15 min,5 %~70 % A;15~25 min70 % A;25~35 min 70 %~100 % A;35~45 min 100 %~5 % A.Further the rimocidin was diluted 17.56 mg/L;8.78 mg/L;4.39 mg/L;2.20 mg/L;1.10 mg/L;0.55 mg/L;0.27 mg/L;0.14 mg/L.The hyphal growth of F.oxysporum f.sp.cucumerinum can be inhibited by the rimocidin at the concentration of 8.78 mg/L,while the spore germination can be inhibited completely under 2.20 mg/L of rimocidin.The greenhouse cucumber potted plant experiment result showed that inhibitory effect of rimocidin was better than ethylicin.After treatment for 14 d and 21 d,the therapeutic effect of rimocidin against F.oxysporum f.sp.cucumerinum in potted cucumber seeding was 78.3 % and 70.6 %,while the 1000-fold diluted ethylicin(80 %)was 66.7 % and 42.7 %.At last,in order to study the expresson of the M527 resistance gene otrA effct on the secondary compunds of type strain Streptomyces coelicolor M145,otrA resistance gene was cloned from S.rimosus M527 genome DNA.The otr A gene was placed under the control of the constitutive promoter Perm E* to constructed pIB139-otrA,respectively.Recombinant strains M145-OA was constructed by conducting intergeneric conjugation.The sporulation ratio of recombinant strain M145-OA was more fast than the wild-type strains.The wild-type strain and the recombinant strain M145-OA was used for actinorhodin fermentation,the production of actinorhodin was higher in M145-OA comparing to those of original strain by 2 times.Furthermore,by conducting reverse transcription polymerase chain reaction(RT-PCR)analysis,the results indicated that the transcriptional level of ACT II-orf4 which was the regulatory gene in the actinorhodin biosynthesis was enhanced in recombinant strain M145-OA compared to corresponding values of S.coelicolor M145.So the production of actinorhodin was higher than wild-type strain.