Isolation And Identification Of Streptomyces Sp.ⅠT2 And ⅡW1 With Antifungal Activity To Pyricularia Oryzae And Construction The Method Of Conjugation Transfer

In order to find new microbial resources against the Pyricularia oryzae,the bacteria was isolated from the soil collected from Shenlongjia in Hubei Province with Pyricularia oryzae NO-1 strain and Pyricularia oryzae 168 strain as indicator.And the bacterial strains are fermenting under different fermentation conditions,and detecting the antifungal activity of the strains to Pyricularia oryzae and the stability of the fermentation broth.At the same time,cloning and analysing the attB sequence of the strains and construction of conjugation transfer method with E.coli.Two strains of IT2 and IIW1 with antifungal activity to Pyricularia oryzae were isolated with Pyricularia oryzae NO-1 strain and Pyricularia oryzae 168 strain as indicator.The inhibition zone diameter of IT2 strain agar block against Pyricularia oryzae NO-1 and Magnapoutforthe oryzae 168 were 29.7mm and 27.4mm respectively,and the inhibition zone diameter of IIW1 strain agar block against Pyricularia oryzae NO-1 and Pyricularia oryzae 168 were 30.4mm and 32.6mm respectively.Base on the colony morphology,physiological and biochemical characteristics of IT2 and IIW1 strain and phylogenetic analysis of 16S rDNA,the IT2 and IIW1 strain were initially identified as Streptomyces.The effects of different culture conditions on the antifungal activity of Streptormyces sp.IT2 fermentation broth to Pyricularia oryzae were detected,it was preliminarily determined that the suitable fermentation conditions were:The Streptomyces sp.IT2 was cultured with sucrose-beef cream fermentation medium at 26.5-30℃,150rpm for 48h.Under this condition,the inhibition zone diameter of the fermentation broth of Streptomyces sp.IT2 to Pyricularia oryzae NO-1 was 42.2mm,and the inhibition zone diameter to Pyricularia oryzae 168 was 45.4mm.The stability of the fermentation broth of Streptomyces sp.IT2 was detected.When the temperature was below 55 C,the fermentation broth of Streptomyces sp.IT2 with stable antifungal activity,and dealed it at 80℃ for one hour,the antifungal activity was decreased more than 40%,and dealed it at 100℃ for one hour,the antifungal activity was compeletly lost.And at pH 1.0,the antifungal activity of fermentation broth of Streptomyces sp.IT2 decreased more than 50%,at the range of pH 2.0-7.7,the antifungal activity was stable,at the range of pH7.7-11.0,the antifungal activity was decreased as the pH rises,at pH 11.0,the antifungal activity was compeletly lost.The attB sequence of Streptomyces sp.IT2 and IIW1 was obtained by PCR amplification.According to the bioinformatics analysis,the attB sequence of Streptomyces sp.IT2 and IIW1 contains the essential 5’-TT sequence for site-specific recombination.The apramycin as the resistance marker to conjugation transfer of Streptomyces sp.IT2 and IIW1 strain,and constructed the Streptomyces sp.IT2 and IIW1-E.coli conjugation transfer method by the integrative plasmid pSET152,and obtained apomycin-resistant conjugation transfer of Streptomyces sp.IT2 and IIW1 strain.In order to construct the gene replacement vector for homologous recombination,two non-contiguous DNA fragments on a potential antibiotic synthesis gene cluster of Streptomyces ⅡR21 strain were cloned into the plasmid pOJ260,and obtained two recombinant plasmids pSCU215 and pSCU216.