| Bacillus subtilis strain 916 (Bs-916) is characteristic with several advantages over some other antagonistic bacteria, such as its broad antagonistic spectrum, quick growth, strong resistance to diverse environment conditions and stabilized populations on plants, and strong activity against Rhizoctonia sotani. It was served as an important biocontrol agent in rice diseases by its ability to promote plant growth and suppress plant pathogenic organisms. Ion implantation is a mutation resource with some advantages, such as high mutation efficiency, wide mutation spectrum. Ion implantation being as an synthesis mutation method is more efficiency than the single mutation method.Improving the antagonisnic ability is a critical task for the Bs-916 strain applying in agricultural industry. In this paper, low-energy ion beam implantation was applied in Bs-916 by three times mutant treatment.The first time, six doses were selected to apply in Bs-916 from 30×2.6×1013 N+/cm2 to 250×2.6×1013 N+/cm2, such as 30,60,90,120,150 and 250×2.6×1013 N+/cm2. On basis of the results from the first treatment, eight doses were selected to apply from 90×2.6×1013 N+/cm2 to 800×2.6×1013 N+/cm2, such as 90,120,150,200,250,400,500和800×2.6×1013 N+/cm2. Afterward, two doses as 150×2.6×1013 N+/cm2 and 250×2.6×1013N+ /cm2 were selected to apply in Bs-916 in the fixed screeing treatment. From1300 strains treated by mutation, antagonisnic abilities of 31 strains were increased 8％more than that of Bs-916, and antagonisnic abilities of 11 strains were increased 10％more than that of parental strain by the first and the second screening, and fixed quantify screening. The results indicated that the optimum dose of ion implantation for the Bs-916 was 150×2.6×1013 N+/cm2 to 250×2.6×1013 N+/cm2. The high-yielding mutant strains were screened by the doses in range from 150×2.6×1013 N+/cm2 to 250×2.6×1013 N+/cm2. The conditions for the mutant were stable and the mutant strains acquired high positive mutation.The inhibition zone of the mutant strains screened by againsting indicator organism R. solani was increased when compared to those of the parental strains, and their antagonistic abilities against different fungi were increased from 4.3％to 7.9％. This enhanced stability was tested for ten generations. The inhibition to pathogens in vitro was measured and the control of rice sheath blight was tested in the pot and field by the screened strains. The inhibition zone of the screened strains against R. solani were increased by 4.3％～30.％% in vitro.The results indicated that the control effect of rice sheath blight in pot tests were 84％～87.4％, and superior to those from the parental strains (72.4％) The strains, K33,H74,J71 and E73, exhibitied high antagonisnic abilities. The control effect of rice sheath blight in field tests were 80.55％～82.87％and increased 3.2％～19.1％over that of Bs-916.The antagonistic substances produced by Bs-916 and the high-yielding mutant strains were extracted and analysed. TLC and HPLC were used to determine the natures and fixed quantify analysis.Lipopeptides production of Bs- 916 and the mutant strains appeared as a single elongated spot on the TLC plates at Rf values of 0.52. The fact that the lipopeptides had the same Rf value as the standard surfactin on TLC, which suggested that the lipopeptides produced by three strains were belonged to surfactin family. Surfactins produced in liquid cultures of Bs-916 and the mutant strains were analyzed by HPLC.The typical chromatogram showed the commercially purchased standard surfactin produced two different peaks depicting different isomers of surfactin through calibration of the quantify of surfactin production. Bs-916 and the mutant strains produced the substances with a profile similar to that of standard surfactin. The retention time of the peaks of substances produced by the mutant and original strains showd they were corresponding to the standard surfactin.It was concluded that the produced lipopeptide was belonged to surfactin family. The rates of surfactin production by Bs-H74 and Bs-J71 were significantly greater than that of the wild-type strains. The concentrations of surfactin were 3.332 mg/ml and 2.115 mg/ml, respectively, whereas the concentration of the wild-type strain was 0.973mg/ml and the Bs-M49 exhibited impaired capacity of surfactin production (0.311mg/ml). The mutant strain Bs-H74 produced 3.0-fold surfactin compared to that of the parental strains. The regulatory region of promoter gene of suafactin synthesis from Bs-H74 was located upstream of the srfA operon in B. subtilis. Sequences results showed that some nucleotides of the romoter gene of suafactin synthesis from Bs- H74 were different from that of original strain Bs-916, which indicated that the changes of promoter genes induced by N+ implantation may enhanced the surfactin production.In order to determine the roles of surfactin in biocontrol by Bs-916, another mutant strain, Bs-M49; which produced lower levels of surfactin than that of Bs-916, was tested. It was found that Bs-M49 had no obvious effects as a biocontrol agent against R. solani. These results suggested that the surfactin family produced by Bs-916 and the mutant strains may play an important role in the suppression of rice sheath blight caused by R. sotani.The surfactin inhibited the growth of mycelia and caused the damages to vesicle structures in the tips of hyphae and leaded to the cell wall broken and the protoplasm leaked out on the screening plate. The enzymes of POD, PPO and SOD as indicators of resistance to plant disease were selected and compared. The activities of three enzymes were various after the rice inoculated with Bs-916 and high-yield mutant strains of R subtilis.The results showed that the effection to three enzymes by inoculating antistrains and Rhizoctonia solani simutanously was higher than that of inoculating single R solani or antistrains. The effects to enzyme activities by four high effective mutant strains were higher than that of Bs-916.
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