| Transposon mutagenesis has been become a very mature technology in the preparation of mutants. Transposon Tn917 was specially used for Gram-positive bacterium to generate mutant pools, which have been reported in many articles. We discussed the relationship between the yield of Cytokinin and the biocontrol ability of Bacillus megaterium using the technology of transposon Tn917-mediated insertional mutagenesis with Rhizoctonia cerealis of wheat as control object.A bacterium strain B1301 with biocontrol capability was isolated from field soils of ginger, and identified as Bacillus megaterium. It produces Cytokinin. In this study, plasmid pTV1::Tn917 from Bacillus subtilis PY143 was introduced into Bacillus megaterium B1301 through transformation by electroporation and protoplasts. A mutant library of Bacillus megaterium B1301 was constructed by transposon mutagenesis and removing plasmid at high temperature. About 98% mutants had the genetic stability by subculturing on selective and nonselective media alternately. The presence of Tn917 in the chomosomal DNA of the transformants was confirmed by PCR amplification.Cytokinin production of mutants were assayed with the radish cotyledons expansion test. Two mutants, B1301-22 and B1301-6, were got through Cytokinin biometrics. Compared with the B1301, the Cytokinin yield of two mutants have been changed. The relative adding weight ratio of B1301-22 to radish cotyledons was increased by 18.34%and B1301-6 was decreased by 9.30%, relative to the B1301 control. Southern blotting analysis showed Tn917 was randomly integrated into the genome of recipient strains, and contained two copy of Tn917. The phenotype of two mutants were the same with B1301.B1301 and its two mutants were dually cultured with nine pathogens respectively, including Rhizoctonia solani, Fusarium graminearum, Bipolaris sorokiniana,Fusarium moniliforme, Fusarium oxysporumf.sp.vasinfectum, Botrytis cinerea , Alternaria sp, Colletotrichum gossypii and Rhizoctonia cerealis. The inhibitory ability of this two mutants declined greatly or lost compared with the wild type strain B1301. It suggested that one copy of Tn917 had integrated into genes about inhibition ability and the expression of related gene weakened. The other copy of Tn917 integrated into gene related with Cytokinin and two effects were brought about: decline and improvement of the expression of Cytokinin encoding gene.B1301 and its two mutants were used to test their biological control on the wheat sheath blight caused by Rhizoctonia cerealis. The results showed that biological control ability of B1301-22 mutant which expressed more Cytokinin was increased by 11.84%, and on the contrary that of B1301-6 mutant which produced less Cytokinin was decreased by 40.34%. This conclusion showed that Cytokinin was tight correlation in biological control with B1301. Two mutants obviously declined in the inhibitory ability to nine pathogens. Hower, the ability of biological control from B1301-22 became high than B1301, B1301-6 became low. So, Cytokinin is of importance to B1301 in biological control.Effect of different carbon source, nitrogen source, initialize medium pH, temperature and rotate speed on the Cytokinin production of mutation strain B1301-22 was studied. Result showed that proper condition of producing cytokinin was 1% cornmeal as carbon source , 0.8% bean flour as nitrogen source, 28–30℃, initialize pH to 7.2, rotate speed 180–200r/min. The relative adding weight on radish cotyledons of B1301-22 increased by 21.60% under culture conditions as above mentioned than that in basic culture at 30℃, rotate speed 180r/min.
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