苏云金芽孢杆菌对菜青虫的防治技术研究
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摘要
用选择培养基从四川成都平原的亚热带黄壤中成功的分离到了一株苏云金芽孢杆菌，定名为SC-1，并对其进行了鉴定。实验证明：该菌株在基础培养基上培养72h以后，可以产生大量的伴孢晶体，晶体的大小为0.5～1.0μm，形状为菱形。
用土壤中分离的苏云金芽孢杆菌SC-1以及库尔斯塔克亚种(血清型为H3abc)和肯尼亚亚种(血清型为H4a-4c)对鳞翅目昆虫菜青虫的幼虫进行了室内生物测定，结果表明：这三株菌都对菜青虫幼虫具有致病性，但毒力各不相同。在同一浓度下，库尔斯塔克亚种的毒力最强，肯尼亚亚种次之，而SC-1菌株较两者弱之。并且随着浓度的升高，其杀虫毒力也增强，二者的关系可用一元线性回归方程表示。由于不同的培养条件下，苏云金芽孢杆菌的杀虫毒力也呈现不同，所以，我们分别用LB培养基和改良培养基培养苏云金芽孢杆菌，然后对菜青虫进行攻毒处理，结果显示：同一菌株经改良培养基培养后，其杀虫毒力与经LB培养基培养的菌株相比，平均要高出1.3倍。
家蚕微孢子可以感染菜青虫，我们首次将苏云金芽孢杆菌与家蚕微孢子混合施用，结果表明：菜青虫的死亡率较单独使用二者大约高出1倍。
通过对三株苏云金芽孢杆菌伴孢晶体蛋白的提取和SDS—PAGE电泳分析，证明三株菌中库尔斯塔克亚种可以产生对鳞翅目昆虫有较强毒杀作用的130～140kD的伴孢晶体蛋白，肯尼亚亚种和从土壤中分离的菌株SC-1均可
产生对鳞翅目昆虫有毒杀作用的71kD的伴抱晶体蛋白。
本文的实验方法和实验结果对微生物农药的生产和应用具有一定的参考
价值。
Bacillus thuringiensis (Bt) was isolated successfully by selective medium from subtropics yellow soil in Chengdu plain of Sichuan. It was named SC-1 and identified. The results proved that: it can produce a lot of parasporal crystal after 72h in BP medium. The size of crystal is 0.5~1.0 u m. Its shape is rhombus.
Indoor bioassay was carried out about SC-1, kurstaki and kenyae against Lepidoptera insect-Pieris rapae larvae. Results of bioassay shows: Pieris papae larvae can be infected with 3 strains. But the toxicity is different. In the same concentration, the toxicity of kurstaki is the highest, kenyae is lower than kurstaki and SC-1 is the lowest. At the same time, the toxicity will be enhanced with the increase of concentration, whose rule of enhancement can be expressed by the linear regression equation. In different culture condition, the toxicity is not same. 3 strains of Bt were respectively cultured by LB and improved medium. Then bioassay proved: toxicity of the same strain cultured by improved medium is 1.3 times higher than the one cultured by LB medium.
Pieris rapae larvae can be infected with Nosema bombycis (Nb). The Bt and Nb were mixed and used. The results proved: the death rate of Pieris rapae larvae was obviously improved compared with separate use of them.
Crystal protein of 3 strains Bt was purified and analized with SDS-PAGE. The results demonstrated that: 130~140 kD protein was produced by kurstaki, which has
high toxicity against Lepidoptera pest. Kneyae and SC-1 can produce 71kD crystal protein, the kind of protein also has toxicity against Lepidoptera pest.
用土壤中分离的苏云金芽孢杆菌SC-1以及库尔斯塔克亚种(血清型为H3abc)和肯尼亚亚种(血清型为H4a-4c)对鳞翅目昆虫菜青虫的幼虫进行了室内生物测定，结果表明：这三株菌都对菜青虫幼虫具有致病性，但毒力各不相同。在同一浓度下，库尔斯塔克亚种的毒力最强，肯尼亚亚种次之，而SC-1菌株较两者弱之。并且随着浓度的升高，其杀虫毒力也增强，二者的关系可用一元线性回归方程表示。由于不同的培养条件下，苏云金芽孢杆菌的杀虫毒力也呈现不同，所以，我们分别用LB培养基和改良培养基培养苏云金芽孢杆菌，然后对菜青虫进行攻毒处理，结果显示：同一菌株经改良培养基培养后，其杀虫毒力与经LB培养基培养的菌株相比，平均要高出1.3倍。
家蚕微孢子可以感染菜青虫，我们首次将苏云金芽孢杆菌与家蚕微孢子混合施用，结果表明：菜青虫的死亡率较单独使用二者大约高出1倍。
通过对三株苏云金芽孢杆菌伴孢晶体蛋白的提取和SDS—PAGE电泳分析，证明三株菌中库尔斯塔克亚种可以产生对鳞翅目昆虫有较强毒杀作用的130～140kD的伴孢晶体蛋白，肯尼亚亚种和从土壤中分离的菌株SC-1均可
产生对鳞翅目昆虫有毒杀作用的71kD的伴抱晶体蛋白。
本文的实验方法和实验结果对微生物农药的生产和应用具有一定的参考
价值。
Bacillus thuringiensis (Bt) was isolated successfully by selective medium from subtropics yellow soil in Chengdu plain of Sichuan. It was named SC-1 and identified. The results proved that: it can produce a lot of parasporal crystal after 72h in BP medium. The size of crystal is 0.5~1.0 u m. Its shape is rhombus.
Indoor bioassay was carried out about SC-1, kurstaki and kenyae against Lepidoptera insect-Pieris rapae larvae. Results of bioassay shows: Pieris papae larvae can be infected with 3 strains. But the toxicity is different. In the same concentration, the toxicity of kurstaki is the highest, kenyae is lower than kurstaki and SC-1 is the lowest. At the same time, the toxicity will be enhanced with the increase of concentration, whose rule of enhancement can be expressed by the linear regression equation. In different culture condition, the toxicity is not same. 3 strains of Bt were respectively cultured by LB and improved medium. Then bioassay proved: toxicity of the same strain cultured by improved medium is 1.3 times higher than the one cultured by LB medium.
Pieris rapae larvae can be infected with Nosema bombycis (Nb). The Bt and Nb were mixed and used. The results proved: the death rate of Pieris rapae larvae was obviously improved compared with separate use of them.
Crystal protein of 3 strains Bt was purified and analized with SDS-PAGE. The results demonstrated that: 130~140 kD protein was produced by kurstaki, which has
high toxicity against Lepidoptera pest. Kneyae and SC-1 can produce 71kD crystal protein, the kind of protein also has toxicity against Lepidoptera pest.
引文
[1] 周传恩．我国杀虫微生物的应用研究进展及发展前景．农药，2001，40(7)：8～10
[2] 张敏恒．国外农药开发的一些新特点．农药，1999,38(10)：27～30
[3] 洪华珠，杨红．杀虫微生物学纲要．武汉：华中师范大学出版社，1997．231
[4] 雷国明．生物农药的龙头老大—苏云金芽孢杆菌百年之路．农药快讯，2002，5：14～17
[5] 戴莲韵，王学聘．苏云金芽孢杆菌研究进展．北京：科学出版社，1997．2～3
[6] 刘银泉，冯明光，刘树生．球孢自僵菌对桃蚜的毒力测定．植物保护学报，1999，26：30～34
[7] 殷向东．生物源杀虫剂研究应用进展及其在我国的发展思路．农药，1999，38(11)：45～46
[8] 洪华珠，杨红．杀虫微生物学纲要．武汉：华中师范大学出版社,1997．81～85
[9] Berliner E. Uber die schlaffsuchi der mehlmottenraupo. (Ephestia kuhuiella zell.) and ihren erreger Bacillus thuringiensis n.sp. Zeitschrift fur Angewandte Entonologie. 1911, (2): 29～56
[10] Hannay C L. Crystalline inclusions in aerabic spore forming bacteria. Nature, 1953, 172: 1004
[11] Hannay C L, and Fitz-james P C. The protein crystals of Bacillus thringiensis Berliner. Can. J. Microbiol. 1995, (1): 694～710
[12] Dulmage HT. Insecticidal activity of HD-1, a new isolate of Bacillus thringiensis. J. Incertebr, Pathol. 1970a, (15): 232～239
[13] Intemet http://www.china-agri.com
[14] Intemet http://www.Pesticideinfo.com
[15] 李钟华，王龙根，张新民．创制新农药是我国“十五”农药科技攻关的主旋律．农药，2001，40(4)：1～2
[16] 朱星华，潘舟，郝国防．在WID／TBT规范下Bt技术标准的制定及其对贸易的影响．科技进步与对策，2001，12：35～36
[17] 晓岚．农用生物农药展望．农药译丛，1994，16(2)：7～17
[18] Salama H S, Morris O N, Rached E. The biopesticide Bacillus thuringiensis and its applications in developing countries. Egypt, Cairo: NRC, 1993, 267～281
[19] 喻子牛．苏云金芽孢杆菌杀虫晶体蛋白及其基因的研究与应用．见：李阜棣，李学恒，刘武定，喻子牛编．生命科学和土壤学中几个领域研究的进展．北京：农业出版
社, 1993. 170～179
[20] Gelemter W D, Schwab G E. Transgenic bacteria, viruses, algae and other microorganisms as Bacillus thuringiensis toxin delivery systems. In: Entwist E F, Cory J S, Bailey M J, et al eds. Bacillus thuringiensis, an environmental biopesticide: theory and practice. John Wiley & Sons Ltd: 1993, 89～104
[21] 黄大昉主编，林敏副主编．农业微生物基因工程．北京：科学出版社，2001．467～479
[22] 福建农学院主编．害虫生物防治．北京：农业出版社，1982．295
[23] 梁文平，郑斐能，王仪等．21世纪农药发展的趋势：绿色农药与绿色农药制剂．农药，1999，38(9)：1-2
[2] 张敏恒．国外农药开发的一些新特点．农药，1999,38(10)：27～30
[3] 洪华珠，杨红．杀虫微生物学纲要．武汉：华中师范大学出版社，1997．231
[4] 雷国明．生物农药的龙头老大—苏云金芽孢杆菌百年之路．农药快讯，2002，5：14～17
[5] 戴莲韵，王学聘．苏云金芽孢杆菌研究进展．北京：科学出版社，1997．2～3
[6] 刘银泉，冯明光，刘树生．球孢自僵菌对桃蚜的毒力测定．植物保护学报，1999，26：30～34
[7] 殷向东．生物源杀虫剂研究应用进展及其在我国的发展思路．农药，1999，38(11)：45～46
[8] 洪华珠，杨红．杀虫微生物学纲要．武汉：华中师范大学出版社,1997．81～85
[9] Berliner E. Uber die schlaffsuchi der mehlmottenraupo. (Ephestia kuhuiella zell.) and ihren erreger Bacillus thuringiensis n.sp. Zeitschrift fur Angewandte Entonologie. 1911, (2): 29～56
[10] Hannay C L. Crystalline inclusions in aerabic spore forming bacteria. Nature, 1953, 172: 1004
[11] Hannay C L, and Fitz-james P C. The protein crystals of Bacillus thringiensis Berliner. Can. J. Microbiol. 1995, (1): 694～710
[12] Dulmage HT. Insecticidal activity of HD-1, a new isolate of Bacillus thringiensis. J. Incertebr, Pathol. 1970a, (15): 232～239
[13] Intemet http://www.china-agri.com
[14] Intemet http://www.Pesticideinfo.com
[15] 李钟华，王龙根，张新民．创制新农药是我国“十五”农药科技攻关的主旋律．农药，2001，40(4)：1～2
[16] 朱星华，潘舟，郝国防．在WID／TBT规范下Bt技术标准的制定及其对贸易的影响．科技进步与对策，2001，12：35～36
[17] 晓岚．农用生物农药展望．农药译丛，1994，16(2)：7～17
[18] Salama H S, Morris O N, Rached E. The biopesticide Bacillus thuringiensis and its applications in developing countries. Egypt, Cairo: NRC, 1993, 267～281
[19] 喻子牛．苏云金芽孢杆菌杀虫晶体蛋白及其基因的研究与应用．见：李阜棣，李学恒，刘武定，喻子牛编．生命科学和土壤学中几个领域研究的进展．北京：农业出版
社, 1993. 170～179
[20] Gelemter W D, Schwab G E. Transgenic bacteria, viruses, algae and other microorganisms as Bacillus thuringiensis toxin delivery systems. In: Entwist E F, Cory J S, Bailey M J, et al eds. Bacillus thuringiensis, an environmental biopesticide: theory and practice. John Wiley & Sons Ltd: 1993, 89～104
[21] 黄大昉主编，林敏副主编．农业微生物基因工程．北京：科学出版社，2001．467～479
[22] 福建农学院主编．害虫生物防治．北京：农业出版社，1982．295
[23] 梁文平，郑斐能，王仪等．21世纪农药发展的趋势：绿色农药与绿色农药制剂．农药，1999，38(9)：1-2