Screening And Identification Of Antagonistic Bacteria And Detecting The Genes Related To Cyclic Lipopeptides Biosynthesis

As the demand on the food safety and improvement of ecological environment, the biological control got more and more attentions and became the hot area of research and development. In this study, several antagonistic bacteria were isolated and indentified, the antimicrobial spectrums were tested, and 4 genes related to cyclic lipopeptides biosynthesis were also detected. The main concludes of this paper are as follows.1.Twelve strains of bacteria were isolated from the soil samples collected from Arctic area. However, only strain X1-2 had the antagonist activity to the plant fungal pathogen Sphaeronaemella fragariae CMB-2 when studied in the way of co-culture on PDA plate, and the radius of the inhibition zone get to 17 mm.2、The strain X1-2 and other 8 strains of antagonistic bacteria kept in this lab were identified with 16Sr DNA, gyrB and gyrA gene sequences. The results showed that 5 strains (BJ-6, BJ-7, BJ-8,22# and 33#) were identified as Bacillus amyloliquefaciens; 2 strains (BJ-9 and X1-2) were B. subtilis; strain 1# was Stenotrophomonas maltophilia and strain BT was B. thuringiensis.3、In order to detect the antimicrobial activity of the above bacteria strains,28 kinds of plant pathogenic fungi and bacteria were tested with confrontation culture method. The results showed that the B. amyloliquefaciens strains (BJ-6, BJ-7, BJ-8,22#,33#) had the widest antimicrobial spectrums and highest antimicrobial activities. And the S. maltophilia strain 1# had the wide antimicrobial spectrum and highly antimicrobial activity either. B. subtilis strain BJ-9 and X1-2 and B. thuringiensis strain BT had low antimicrobial activity and a narrow antimicrobial spectrum.4、Four genes(fenD、bacD、ituA、sfp)related to cyclic lipopeptides:fengycin, iturin, surfactin and bacillomycin D biosynthesis were detected in 9 antagonistic bacteria strains. The results indicated that 4 genes (fenD、bacD、 ituA、sfp) had existed in the strains B. amyloliquefaciens BJ-6, BJ-8,22# and 33#; however B. amyloliquefaciens BJ-7 had lost the surfactin biosynthesis related gene sfp; The sfp and fenD gene which related to surfactin and fengycin biosynthesis have not found in the strain S. maltophilia 1#; and none of the four gene has been amplified in strain B. subtilis BJ-9 and X1-2.5、The overlap PCR was used for connecting the upstream and downstream fragments of the delete gene, and then connection fragment were cloned on the temperature-sensitive plasmid pKSV7 to finished the recombinant plasmid. The fenD gene deleted mutant was screened by homologous recombination method. However, because of the low rate of electroporation and genetic recombination, the fenD gene deleted mutant was not obtained in the end.