DNA Polymorphism Analysis Of Streptomyces Roseoflavus Men-myco-93-63 With Its Antibiotic Blocked Mutant Strains

Streptomyces roseoflavus Men-myco-93-63 isolated from potato scab decline soil is an antagonistic strain.The strain and its fermentation can inhibit many pathogenic fungi and control the related important plant disease such as Verticillium wilt of cotton,cumber powdery mildew,and so on.After being treated by microwave irradiation and diethyl sulfate(DES),the spores of S.roseoflavus Men-myco-93-63 were mutated.Eight blocked mutant strains named respectively as DE01,DE02,MV11,MV18,MV21,MV22,MV35 and MV40 were screened from its mutant strains.These mutants had no antifungal ability to Verticillium dahliae,Streptomyces scabies,Botrytis cinerea and many other pathogens, and were stable after ten subcultures.The genetic polymorphism of Men-myco-93-63 with its eight antibiotic blocked mutants was analyzed using amplified fragment length polymorphism(AFLP)and 3142 AFLP bands were amplified using 38 primer pairs,2362(75.2%)bands of them were polymorphic.Cluster analysis showed that the genetic similarity coefficient of S. roseoflavus Men-myco-93-63 and its eight antibiotic blocked mutants ranged from 0.52 to 0.88,which indicated that the relationship of Men-myco-93-63 and its eight antibiotic blocked mutants was close.All the nine strains were classified into two groups. Men-myco-93-63,DE01,MV11,MV22 and MV35 were in one group.MV21,MV40, MV18 and DE02 were in another group.The similarity between Men-myco-93-63 and DE02 was lowest than others.Three of the ninety-six pairs of primers were used for AFLP analysis,such as E-CA/M-GA,E-GA/M-AC and E-GA/M-AG.The bands with 254 base pairs(bp),312 bp,and 209bp amplified respectively by three pairs of primers,were present only in Men-myco-93-63,and named as EM254,EM312,and EM209,respectively.Southern hybridization results showed that EM254,EM312 and EM209 had a single copy in the genome of Men-myco-93-63,and they were present only in Men-myco-93-63. The three specific AFLP fragments were also respectively converted into single locus PCR markers of the sequence-characterized amplified region(SCARs),which provide a simple molecular detection method for identifying of Men-myco-93-63 from mutants.