Screening Of Antagonistic Actinomycetes FX-H-51 And Study Of Its Metabolites With Bioactivity

This research aimed at isolating and screening of antagonistic actinomycetes with high activity to plant pathogens,obtaining a strain FX-H-51 of Rhizoctonia cerealis has efficient antagonistic activity.It is initially identified as Streptomyces spectabilis through a series of classification methods.The following is optimization of fermentation conditions.Antimicrobial active substances is separated,purified and characterized by various detective methods.The detailed studies as follows:1.Isolation of actinomyces.Selected fifty Apple rhizosphere soil samples,which came from Shanxi Province,Shanxi Province,Gansu Province and Shandong Province,the natural drying pretreatment of soil samples indoors and then isolating strains using plate dilution method with selective isolation medium GA medium wih 80mg/L Potassium dichromate for the selective isolation of actinomycetes,a total of 217 strains were isolated from soil.2.Screening of activity strains.Using three kinds of pathogenic fungi as targets,screening of antagonistic actinomycetes by plate confrontation method and gray related degree analysis method.The results show that using Rhizoctonia cerealis,Bipolaris sorokinian,Fusarium culmorum as indicator fungi,screening out 4 strains actinomycetes FX-H-51,FX-H-71,FX-S-114,FX-S-178 that have efficient antagonistic activities by plate confrontation method.Gray related degree analysis showed that the strain of FX-H-51 not only has good inhibition of pathogenic fungi,but has good tolerance to adverse growing conditions.3.The taxonomy of actinomyces.The strain FX-H-51 is preliminarily identified by the morphological characteristics,physiological and biochemical.The results show that the strain FX-H-51 is identified as the Streptomyces spectabilis.16 S rDNA sequence of strain FX-H-51 is 1447 bp.The 16 S r DNA sequence homology of the strain FX-H-51 and Streptomyces spectabilis can come up to 99% by comparing the 16 S rDNA sequence of strain FX-H-51 with relevant sequence of GenBank database,and their relationships are the most recent.So the strain FX-H-51 is preliminarily identified as Streptomyces spectabilis.4.Optimization of fermentation condition.The single factor experiment and the orthogonal method are used to optimize fermentation conditions.The nutritional content of optimal media are maltose 50.3g,tryptone 9.6g,Na Cl 2.5g,Ca CO₃ 2.0g?1000ml?.Optimal culture conditions are 100 mL liquid medium volume,200r/min,pH8.0,28?,and 7d.5.Stability test of fermentation product.The physical and chemical properties are understood by the stability test.The results show that antimicrobial substance is better stabilization at room temperature and low temperature.Antimicrobial substance is better stabilization when pH3.0¹1.0.The fermentation product still has a higher antibacterial activity after UV processing 45 min.6.Fermentation of crude extracts of fungi inhibition.Antimicrobial substance from fermentation liquid of strain FX-H-51 are separated by solvent extraction method.The results show that bacteriostatic rate of yellow brown viscous crude extracts which are gained under the condition of ethyl acetate can come up to over 58.8% against Rhizoctonia cerealis.7.Isolate and purify fermentation extracts.Powdery antimicrobial substance is purified by thin-layer and column chromatography methods.The results show that the purit y of fermentation product can be detected with iodine fumigation.The compound exhibited high antimicrobial activity against Rhizoctonia cerealis and inhibition rate is 76.47%.