Screening Of Stronger Antibiotic Activity Strain Based On SF-628 By N~+ Implantation And Optimizing Of The Cultural Conditions Using Response Surface Methodology

The biocontrol bacterium sf-628, isolated from plant rhizosphere could obviously inhibit the growth of Rice Sheath Blight, which was proved by the disease control experiment in green-house. After bacterizing rice seeds, the growth-promoting effects of sf-628 on rice seeding was tested in pot. The results indicated that the growth-promoting effect of sf-628 and other several antagonistic was significant.Biocontrol bacterium sf-628 was an important biocontrol factor in control of rape disease. In order to improve the growth rate and antagonistic ability of the strain, and to obtain high-efficiency strains,N⁺of different doses was implanted into the biocontrol bacterium sf-628. Of 500 strains mutation treated, 11 strains antagonisnic ability increased more than 12% that of sf-628, by the first and the second screening, and fix quantify screening. Which was 1.86% total screening strains, and three of 11 strains of antagonistic ability increased 35.7%⁴2.9% than sf-628 strain. Efficiency of controlling disease in the pot were measured. The results indicated that the optimum dose of ion implantation for the sf-628 was 100～150×5.3×10¹³N+/cm² ; the control effect of Sclerotinia sclerotiorum increased by 35. 7% -42. 9% over that of sf-628.Response surface methodology(RSM) based on a three-level three-factor Box-Behnken design of experiment was used to optimize the growth and the bacillus biomass in submerged cultivation of biocontrol bacterium sf-628.The critical factors selected for the investigation were temperature,time of cultivation and initial inoculation volume based on the results of previous Plackett-Burman design. By solving the regression equation and also by analyzing the response surface contour plots-the optimal process conditions were determined trader conditions of temperature:31.91℃,cultivation time:2.13 d and volume of inoculation 3.79%, the prediction of colony forming units(CFU) of sf-628 was 9.65×10⁹cfu/ml per milliliter of fermentation liquor. While cultivation temperature,time and volume of inoculation were 35.55℃,2.13 d and 2.86% respectively,the bacillus growth expressed as colony dimater(CD) was 25.15mm. In order to simultaneously obtain the maximum field of CFU and bacillus growth the above conditions would be located at 32.5℃,1.99d and 3%. In these conditions, the maximum predicted yield of CFU and CD were found to be 9.34×10⁹cfu/ml and 24.33mm respectively. These predicted values were also verified by validation experiment.Based on the results of previous Plackett-Burman design,the most important factors affecting the yield of CFU(colony-forming units) and bacillus growth of sf-628 in submerged cultivation were further investigated by response surface methodology. Three-factor,i.e.yeast extract,soybean meal and corn steep olquor, three-level Box-Behnken design was employed. By solving the quadratic model equation using appropriate statistic methods,the optimum concentrations were determined: yeast extract ,soybean meal and corn steep olquor were 0.96,2. Hand 0.76 g.L^-1,respectively. The predicted yield of bacillus biomass was 2.13×10¹⁰cfu/ml.When the above factors were 0.74,3 and 0.77 g.L^-1, respectively. The colony diameter was 29.79mm.In order to obtain the maximum yield of bacillus biomass and bacillus growth at the same time,the optimal concentrations of yeast extract,soybean meal and corn steep olquor would be 0.79,2.41and 0.77,respectively.In this situation ,2.01-×10¹⁰ cfu/ml of CFU(colony-fomting units) and 29.18mm bacillus growth were gained simultaneously.