生防细菌sf-628离子注入诱变育种及发酵条件的响应曲面法优化研究
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摘要
从土壤中分离获得了一株对水稻纹枯病菌有明显抑制作用的生防菌sf-628,室内控病试验结果表明,生防菌sf-628能显著降低水稻纹枯病的发病率,控病率达到51.5%;它同时能降低西瓜枯萎和油菜菌核病的发病率,控病率分别达到80%和78.5%。将其用于水稻种子处理,测定其对水稻幼苗的促生作用,在菌株发酵液1:100的处理浓度下,水稻苗根长和株高比对照分别增长了20.55%和10.84%。结果表明,生防细菌sf-628对水稻幼苗有明显的促生效应。
生防菌sf-628对油菜菌核病具有很好的防治效果。为进一步提高其拮抗性能,获得生防效果更好的高效菌种,利用不同剂量的N~+对sf-628进行离子注入诱变处理,在500多个诱变处理的菌株中,经过初筛、复筛和定量筛选,筛选到11株拮抗性能比出发菌sf-628提高12%以上的菌株,占筛选菌株总数的2.16%;其中有3个突变菌株对油菜菌核病的拮抗能力提高了35.7%～42.9%。对筛选出的高效突变菌株进行盆栽防病测定。结果表明,离子注入生防菌sf-628的最适剂量为100～150×5.3×10~(13)N~+/cm~2:防病效果比sf-628提高1.14%～14.80%。
在Plackett-Burman实验设计基础上,采用Box-Behnken响应曲面法(responsesurface methodology,RSM)对影响生防菌sf-628发酵活菌数与菌落直径的关键培养条件(培养温度、时间和接种量)进行了探讨。通过对发酵活菌数曲面方程以及细菌生长量二次多项回归方程求解得知,在自变量培养温度、时间和接种量分别为31.91℃、1.93d和3.79%时,生防菌发酵活菌数的最大预测值为96.53×10~8cfu/ml;35.55℃、2.13d和2.86%的时,该菌菌落直径可达25.15mm;而在32.5℃、1.99d和3%的工艺条件下,发酵结果可同时获得9.34×10~9cfu/ml的发酵活菌数和24.33mm(菌落直径)的细菌生长量。
在原有的plackett-Burman设计与实验结果基础上,采用响应曲面法(RSM)研究了影响生防菌sf-628生长与发酵活菌数的培养基关键成分的最佳水平。实验结果表明,当酵母膏、大豆粉和玉米桨分别为0.96g.L~(-1)、2.11g.L~(-1)和0.76 g.L~(-1)时,发酵活菌数最大预测值为2.13×10~(10)cfu/ml;当上述自变量为0.74 g.L~(-1)、3 g.L~(-1)和0.77 g.L~(-1)时,细菌最大生长量为29.79mm(菌落直径)。欲同时获得最大量的菌落直径和活菌数,则酵母膏、大豆粉和玉米浆须为0.79 g.L~(-1)、2.41 g.L~(-1)和0.77g.L~(-1),在此条件下,每毫升发酵液可同时获得2.01×10~(10)cfu/ml的活菌数和菌落直径为29.18mm的细菌生长量。
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 100～150×5.3×10~(13)N+/cm~2 ; 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 9.65×10~9cfu/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 9.34×10~9cfu/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 2.13×10~(10)cfu/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-×10~(10) cfu/ml of CFU(colony-fomting units) and 29.18mm bacillus growth were gained simultaneously.
生防菌sf-628对油菜菌核病具有很好的防治效果。为进一步提高其拮抗性能,获得生防效果更好的高效菌种,利用不同剂量的N~+对sf-628进行离子注入诱变处理,在500多个诱变处理的菌株中,经过初筛、复筛和定量筛选,筛选到11株拮抗性能比出发菌sf-628提高12%以上的菌株,占筛选菌株总数的2.16%;其中有3个突变菌株对油菜菌核病的拮抗能力提高了35.7%～42.9%。对筛选出的高效突变菌株进行盆栽防病测定。结果表明,离子注入生防菌sf-628的最适剂量为100～150×5.3×10~(13)N~+/cm~2:防病效果比sf-628提高1.14%～14.80%。
在Plackett-Burman实验设计基础上,采用Box-Behnken响应曲面法(responsesurface methodology,RSM)对影响生防菌sf-628发酵活菌数与菌落直径的关键培养条件(培养温度、时间和接种量)进行了探讨。通过对发酵活菌数曲面方程以及细菌生长量二次多项回归方程求解得知,在自变量培养温度、时间和接种量分别为31.91℃、1.93d和3.79%时,生防菌发酵活菌数的最大预测值为96.53×10~8cfu/ml;35.55℃、2.13d和2.86%的时,该菌菌落直径可达25.15mm;而在32.5℃、1.99d和3%的工艺条件下,发酵结果可同时获得9.34×10~9cfu/ml的发酵活菌数和24.33mm(菌落直径)的细菌生长量。
在原有的plackett-Burman设计与实验结果基础上,采用响应曲面法(RSM)研究了影响生防菌sf-628生长与发酵活菌数的培养基关键成分的最佳水平。实验结果表明,当酵母膏、大豆粉和玉米桨分别为0.96g.L~(-1)、2.11g.L~(-1)和0.76 g.L~(-1)时,发酵活菌数最大预测值为2.13×10~(10)cfu/ml;当上述自变量为0.74 g.L~(-1)、3 g.L~(-1)和0.77 g.L~(-1)时,细菌最大生长量为29.79mm(菌落直径)。欲同时获得最大量的菌落直径和活菌数,则酵母膏、大豆粉和玉米浆须为0.79 g.L~(-1)、2.41 g.L~(-1)和0.77g.L~(-1),在此条件下,每毫升发酵液可同时获得2.01×10~(10)cfu/ml的活菌数和菌落直径为29.18mm的细菌生长量。
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 100～150×5.3×10~(13)N+/cm~2 ; 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 9.65×10~9cfu/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 9.34×10~9cfu/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 2.13×10~(10)cfu/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-×10~(10) cfu/ml of CFU(colony-fomting units) and 29.18mm bacillus growth were gained simultaneously.
引文
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