Screening Of Endospore Variation Mutant For Endophytic Biocontrol Strain Bacillus Cereus0-9

Bacillus cereus0-9, an endophytic bacteria isolated from roots of healthy wheat in previous study,could colonize inner roots and rhizosphere of wheat persistently, form biofilm and had potential tobiological control against sharp eyespot of wheat under room and field conditions. Endospore formation isusually triggered by a lack of nutrients and endospores can survive environmental assaults that wouldnormally kill the bacterium. These stresses include high temperature, high UV irradiation, desiccation,chemical damage and enzymatic destruction. This feature of endospore contributes it to formulationprocessing for biological control agents. Therefore, the purpose of this study was to isolated endosporeformation variance mutants of B.cereus0-9so as to elucidate the regulation of endospore differentiationprocess and analyse the relationship between endospore differentiation and biological control against plantdisease of biological control strain0-9.Plasmid pMarB,containing the replicon repG, and transpon TnYLB-1and existed in0-9strain wasinduced under high temperature. Transposon TnYLB-1inserted into the genome of0-9strain. transposoninsertion library which contained1163strains was contructed. In addition, according to the differenthigh-temperature-induced transposition conditions, we confirm to the best transposition condition:46°C for10h under this condition, the transposition efficiency up to90.38%.Endospore staining and counts technique were applied to determine the B.cereus0-9endosporeformation curve. The results revealed that26hours after incubation is optimal time for analyzing endosporeformation under the condition of37℃culture,1/5times liquid LB culture.From the mutant library of1163mutants by spore staining and flat contrast, we screened four sporulation significant differences in thestrains compare to wild-type strain0-9which designate as B2,B117,B695and B1108.Sporulation mutationfrequency is0.43%。Inverse PCR was used to determine and analyze the transposon insertion site flanking sequences of B2mutant. After sequencing,homology comparison with genome sequence of B.cereus in Genebank show thatthe gene encoded for NADH dehydrogenase was disruption by insertion transposon,which suggested the gene of NADH dehydrogenase involved in the formation of spores.Result of this research have not beenreported previously.