Screening, Identification And Optimization Of Fermentation Conditions For Microorganisms Against Plant Pathogens And Preliminary Study Of PKS Gene Cluster

Plant pathogens, seriously harmful to the growth of crops, were one of the main reasonsfor agricultural losses. Actinomycete can produce active substances to inhibit plant pathogens,and was an important source of biologically active substances.In this study, Pyricularia oryzae, Rhizotonia solanikuhn, Verticilium dahllae, Fusariumgramineariumschwabe and Botrytis cinerea Per-soon were used as indicator fungal,15kindsof actinomycetes with antifungal activity were isolated and screened out from soil microbialculture bank in Shenlongjia area. One antifungal strain IIB21was chosen with the inhibitionzone diameter up to48mm for Pyricularia oryzae. Based on morphology characteristics,cultural characteristics, physiological, biochemical characteristics and16S rDNA sequencephylogenetic analysis, strain IIB21was identified as Streptomyces.The16S rDNA sequencewas submitted to NCBI with the obtained accession numbers JQ950680.Based on orthogonal test, the optimization of fermentation conditions were as follows:sucrose as carbon source, beef extract as nitrogen source, initial pH7.0, incubation for4dayswith temperature at30℃and rotation speed180rpm.Fermentation broth of strain IIB21was up to the best antifungal activity at30℃, theinhibition diameter was41.16mm,and was fairly stable at4℃~60℃, the inhibition diameterswere above72.10%of the best antifungal activity; And was fairly stable at pH3.0~10.0, theinhibition diameters were above74.95%of the best antifungal activity; antifungal activitysubstance of the fermentation broth was fairly stable at UV.Extracted by ethyl acetate, the bioactive substances in fermentation broth of strainIIB21was concentrated about5-fold. The best ratio of the TLC expand agent was V(petroleum ether): V (ethyl acetate)=7:3; two kinds of antifungal active substances wereseparated by silica gel column chromatography preliminarily. The average inhibition zonediameter of component I was up to21.06mm and the average inhibition zone diameter ofcomponent II was up to25.31mm.The conserved sequence of KS region in PKS gene cluster was used to design primerand amplified15strains of actinomycetes by PCR. The results showed that total DNA in sixkinds of actinomycetes obtained the expected fragment of750bp.The amplified sequences ofstrain IIID1and strain IIR21were cloned, sequenced and submitted to NCBI, the accessionnumber for PKS gene of strain III D1was JQ950678, and accession number for PKS geneof strain IIR21was JQ950679. Bioinformatics analysis indicated that: Strain III D1and strainIIR21contained PKS gene cluster in the genome.