B.t.与生物杀虫剂的联合作用及其毒理初探
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摘要
苏云金杆菌(Bacillus thuringiensis)是近缘于蜡状芽孢杆菌能产生晶体具芽孢，寄生于体内引起昆虫发病的杆菌。本文从昆虫神经毒性反应角度，通过对B．t．与其他新型胃毒性生物杀虫剂的联合作用机制进行研究，特别是与一些动、植物源的毒蛋白类物质如蝎毒、沙蚕毒素等的联合作用机制进行研究，以期指导B．t．等生物源胃毒杀虫剂的合理混用，并在此基础上提出B．t．与生物源杀虫物质的混配使用技术。本研究得出下列结果：
1 B．t．与转δ-内毒素基因棉(下称转基因棉)联合作用后使得棉铃虫对B．t．的敏感性增强，但多代取食转基因棉的试虫却对B．t．的敏感性下降。通过对试虫体内的酯酶比活力的测定发现，多代取食基因棉的试虫乙酰胆碱酯酶及羧酸酯酶比活力增强，两项指标比活力分别增强了1.69及1.78倍。
2 中毒症状调查结果发现，蝎毒素、沙蚕毒素、阿维菌素及楝素与B．t．混配处理后的试虫同时表现出明显的多种累加症状，且症状出现时间也明显提前。处理混剂后大部分试虫出现明显症状的时间要比B．t．提前24小时左右。
3 B．t．与蝎毒素、沙蚕毒素、阿维菌素、楝素及氰戊菊酯混配，利用共毒因子法，考虑到以B．t．为主，选出几种增效作用比较明显的混剂配方。其配比是B．t．与蝎毒素为4：1，B．t．与沙蚕毒素为10：1，B．t．与阿维菌素为16：1，B．t．与楝素为50：3，B．t．与氰戊菊酯为40：1的配比。室内测定结果显示各混剂对棉铃虫有较高增效作用，致死中浓度分别为75.405mg／L，116.830mg／L，20.110mg／L，80.593mg／L，27.992mg／L。共毒系数分别为217.9、254.8、1064.0、443.4、1082.6。
4 测定混剂对棉铃虫中肠神经肌肉动作电位结果表明，B．t．与蝎毒素、沙蚕毒素及阿维菌素混配均导致了试虫中肠神经肌肉动作电位的发放频率或振幅的加剧。B．t．和蝎毒素混剂处理平均自发放频率加药前为0.9±0.08次／秒，加药后1分钟为1.4±0.25次／秒，2分钟后为2.9±0.31次／秒。平均振幅加药前为0.12±0.029mv，加药1分钟后为0.18±0.053mv，2分钟后为0.31±0.032mv。以B．t．和沙蚕毒素混剂处理平均自发放频率加药前为0.8±0.10次／秒，加药后3分钟为1.3±0.14次／秒，6分钟后为2.8±0.31次／秒。平均振幅加药前为0.10±0.018mv，加药3分钟后为0.15±0.017mv，6分钟后为0.34±0.044mv。以B．t．和阿维菌素混
剂处理平均自发放频率加药前为 l．2 i 0二24次／秒，加药后 5分钟为 0．sic．09次／
秒，8分钟后为 2，2士 0．17次／秒。平均振幅加药前为 0．14土 0刀29my，加药 5分钟
后为0刀9士0刀17my，8分钟后为0。28上0．054my。
以上结果表明，Bt与转基因棉呈协同增效作用，其增效机理与酯酶及乙酸胆
碱酯酶有关；Bj．与蝎毒素、沙蚕毒素及阿维菌素及多种杀虫剂联合处理有较广泛
协同效应，其增效机理可能与B．t．的神经毒性作用协同相关。这种协同效应在抗
性治理和害虫防治，包括转基因作物利用中具有潜在的应用价值，值得进一步研
究。
Bacillus thuringiensis(B.t.) has been widely used because of its specificity for target insects, low development cost and environmental compatibility. This thesis aims at guiding a reasonable combination of B.t. and other biological insecticides and also introducing the practical combination technique of B.t. and other biological insecticides in the perspective of insect never toxication. The result is obtained by carrying out on the combination mechanism of B.t. and other newly developed biological insecticides, especially researches on the combinational functions of some animal and plant toxins, for example , Buthotoxin and Avermectin.The following is the main results of the research.
(1) The susceptibility of larvae of less army worm Helicoverpa armigera to B.t. was increased when the larvae were fed with transgenic cotton compared to those fed on the non-transgenic cotton, but decreased after 10-generation's feeding with the bt transgenic cotton plants. Biochemical analysis showed that the activities of acetylcholinesterase and carboxylesterase were obviously higher in those larvae fed on the transgenic cotton.
(2)Observation on the poisoning symptom showed that the poisoning of the insects treated with B.t.+ Buthotoxin, B.t.+ Avermectin, B.t.+ Nereistoxin and B.t.+ Azadirachtin mixture were enhanced and accelerated for about 24hrs..
(3) Greenhouse tests indicated that joint treatment with B.t. and Nereistoxin, Avermectin, Buthotoxin and Azadirachtin presented better control effects than any single toxicant. The LC50 value of each mixture is 75.405mg/L,116.830 mg/L,20.110 mg/L,80.593mg/L5 27.992 mg/L respectively, and the C.T.C values of LC50 is 217.9, 254.8,1064.0, 443.4 and 1082.6 respectively.
(4)Electrophysiological effects of the mixtures on the midgut never-muscle system
showed synergistic symptoms. The frequency and amplitude of spontaneous action potentials was increased obviously. The average frequency and amplitude value caused by the joint treatment with B.t, and Buthotoxin is as follow: 0.9±0.08 times/second and 0.12±0.029mv before the treatment, 1.4±0.25 times/second and 0.18?.053mv at 1minute, 2.9±0.31 times/second and 0.31±0.032mv in 2 minutes . The value for the joint treatment B.t. and Avermectin is 0.8±0.10 times/second and 0.10±0.018mv before the treatment, 1.3±0.14 times/second and 0.15±0.017mv in 3minutes , 2.8± 0.31 times/second and 0.34±0.044mv in 6minutes . The value for the Joint treatment B.t. and Nereistoxin,. is 1.2±0.24 times/second and 0.14±0.029mv before the treatment , 0.8±0.09 times/second and 0.09±0.017mv in 5minutes , 2.2±0.17 times/second and 0.28±0.054mv in 8 minutes .
For conclusion, the author believed that the synergistic mechanism of B.t and Bt-transgenic cotton relate to the acetylcholinesterase and carboxylesterase, and there existed different kinds of joint action between B.t. and many other toxicants, and which is useful in insect pest management, or resistant management of the transgenic cotton plants.
1 B．t．与转δ-内毒素基因棉(下称转基因棉)联合作用后使得棉铃虫对B．t．的敏感性增强，但多代取食转基因棉的试虫却对B．t．的敏感性下降。通过对试虫体内的酯酶比活力的测定发现，多代取食基因棉的试虫乙酰胆碱酯酶及羧酸酯酶比活力增强，两项指标比活力分别增强了1.69及1.78倍。
2 中毒症状调查结果发现，蝎毒素、沙蚕毒素、阿维菌素及楝素与B．t．混配处理后的试虫同时表现出明显的多种累加症状，且症状出现时间也明显提前。处理混剂后大部分试虫出现明显症状的时间要比B．t．提前24小时左右。
3 B．t．与蝎毒素、沙蚕毒素、阿维菌素、楝素及氰戊菊酯混配，利用共毒因子法，考虑到以B．t．为主，选出几种增效作用比较明显的混剂配方。其配比是B．t．与蝎毒素为4：1，B．t．与沙蚕毒素为10：1，B．t．与阿维菌素为16：1，B．t．与楝素为50：3，B．t．与氰戊菊酯为40：1的配比。室内测定结果显示各混剂对棉铃虫有较高增效作用，致死中浓度分别为75.405mg／L，116.830mg／L，20.110mg／L，80.593mg／L，27.992mg／L。共毒系数分别为217.9、254.8、1064.0、443.4、1082.6。
4 测定混剂对棉铃虫中肠神经肌肉动作电位结果表明，B．t．与蝎毒素、沙蚕毒素及阿维菌素混配均导致了试虫中肠神经肌肉动作电位的发放频率或振幅的加剧。B．t．和蝎毒素混剂处理平均自发放频率加药前为0.9±0.08次／秒，加药后1分钟为1.4±0.25次／秒，2分钟后为2.9±0.31次／秒。平均振幅加药前为0.12±0.029mv，加药1分钟后为0.18±0.053mv，2分钟后为0.31±0.032mv。以B．t．和沙蚕毒素混剂处理平均自发放频率加药前为0.8±0.10次／秒，加药后3分钟为1.3±0.14次／秒，6分钟后为2.8±0.31次／秒。平均振幅加药前为0.10±0.018mv，加药3分钟后为0.15±0.017mv，6分钟后为0.34±0.044mv。以B．t．和阿维菌素混
剂处理平均自发放频率加药前为 l．2 i 0二24次／秒，加药后 5分钟为 0．sic．09次／
秒，8分钟后为 2，2士 0．17次／秒。平均振幅加药前为 0．14土 0刀29my，加药 5分钟
后为0刀9士0刀17my，8分钟后为0。28上0．054my。
以上结果表明，Bt与转基因棉呈协同增效作用，其增效机理与酯酶及乙酸胆
碱酯酶有关；Bj．与蝎毒素、沙蚕毒素及阿维菌素及多种杀虫剂联合处理有较广泛
协同效应，其增效机理可能与B．t．的神经毒性作用协同相关。这种协同效应在抗
性治理和害虫防治，包括转基因作物利用中具有潜在的应用价值，值得进一步研
究。
Bacillus thuringiensis(B.t.) has been widely used because of its specificity for target insects, low development cost and environmental compatibility. This thesis aims at guiding a reasonable combination of B.t. and other biological insecticides and also introducing the practical combination technique of B.t. and other biological insecticides in the perspective of insect never toxication. The result is obtained by carrying out on the combination mechanism of B.t. and other newly developed biological insecticides, especially researches on the combinational functions of some animal and plant toxins, for example , Buthotoxin and Avermectin.The following is the main results of the research.
(1) The susceptibility of larvae of less army worm Helicoverpa armigera to B.t. was increased when the larvae were fed with transgenic cotton compared to those fed on the non-transgenic cotton, but decreased after 10-generation's feeding with the bt transgenic cotton plants. Biochemical analysis showed that the activities of acetylcholinesterase and carboxylesterase were obviously higher in those larvae fed on the transgenic cotton.
(2)Observation on the poisoning symptom showed that the poisoning of the insects treated with B.t.+ Buthotoxin, B.t.+ Avermectin, B.t.+ Nereistoxin and B.t.+ Azadirachtin mixture were enhanced and accelerated for about 24hrs..
(3) Greenhouse tests indicated that joint treatment with B.t. and Nereistoxin, Avermectin, Buthotoxin and Azadirachtin presented better control effects than any single toxicant. The LC50 value of each mixture is 75.405mg/L,116.830 mg/L,20.110 mg/L,80.593mg/L5 27.992 mg/L respectively, and the C.T.C values of LC50 is 217.9, 254.8,1064.0, 443.4 and 1082.6 respectively.
(4)Electrophysiological effects of the mixtures on the midgut never-muscle system
showed synergistic symptoms. The frequency and amplitude of spontaneous action potentials was increased obviously. The average frequency and amplitude value caused by the joint treatment with B.t, and Buthotoxin is as follow: 0.9±0.08 times/second and 0.12±0.029mv before the treatment, 1.4±0.25 times/second and 0.18?.053mv at 1minute, 2.9±0.31 times/second and 0.31±0.032mv in 2 minutes . The value for the joint treatment B.t. and Avermectin is 0.8±0.10 times/second and 0.10±0.018mv before the treatment, 1.3±0.14 times/second and 0.15±0.017mv in 3minutes , 2.8± 0.31 times/second and 0.34±0.044mv in 6minutes . The value for the Joint treatment B.t. and Nereistoxin,. is 1.2±0.24 times/second and 0.14±0.029mv before the treatment , 0.8±0.09 times/second and 0.09±0.017mv in 5minutes , 2.2±0.17 times/second and 0.28±0.054mv in 8 minutes .
For conclusion, the author believed that the synergistic mechanism of B.t and Bt-transgenic cotton relate to the acetylcholinesterase and carboxylesterase, and there existed different kinds of joint action between B.t. and many other toxicants, and which is useful in insect pest management, or resistant management of the transgenic cotton plants.
引文
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85．张宗炳．昆虫神经生理与神经毒剂．北京：科学出版社．1986，104-135．
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57．冯夏等，广东小菜蛾对苏云金杆菌的抗药性研究昆虫学报，1996，14(3)：238-245．
58．张兴 编 昆虫毒理学实验指导书 西北农业大学植保系 1994：11-12．
59．徐健 蔡扬生 等．苏云金杆菌与化学杀虫剂的联合作用测定．2001(2)：45-51．
60．陈立 徐汉虹 等．农药复配最佳增效配方筛选方法的探讨．植物保护学报，2000，27(4)：349-354．
61．赵小公 烟碱乳油及其增效配方研究和增效机理初探．1997：16．
62. Sun,Y.P.& E.R.Johnson Analysis of joint action of insecticides abainst house flies. J. Econ. Entomol. 1960,53:887-891.
63．郝小草 等 防治棉铃虫的药剂田间筛选试验研究初报 植物保护学报 1995，22(2)：181-186．
64．刘安西，陈守同．昆虫电生理学实验研究方法．北京：科学出版社．1990，67-72．
65．赵勇 刘安西 李军 等．神经敏感性降低是棉铃虫对拟除虫菊酯抗药性的重要机制．昆虫学报．1996，39(4)：393-398．
66. Bliss.The toxicity of poisons applied jointly .Annl. Appl. Biol., 1939, 26:585-615.
67. Sakai S, Sato M, Kojima K. Insect toxicological studies in the joint action of insecticides. Ⅱ.Joint action between contact insecticides. Botyu Kagaku Kagaku, 1951, 16: 130-140.
68. Finney D J. Probit analysis. London : Cambridge University Press, 1947.
69. Sun Yun-Pei, Johson E R. Analysis of joint action of insecticides against houseflies. J, Econ. Entomol., 1960, 53: 887-892.
70. Mansour N A, Eldefrawi M E, Toppozada A et al. Toxicological studies on the Egyptian cotton leafworm, Prodenia litura.Ⅵ.Potentiation and antagonism of organophosphorus and carbamate insecticides .J.Econ Entomol., 1966, 59:307-311.
71. Harris H A, Chamber H W. Interaction of insecticidal mixtures in tobacco budworm. J.Econ. Entomol., 1973, 66: 517-518.
72. Crafts A S, Cleay C W. Toxicity of arsenic, borax, chlorate and their combinations in three California soils. Hilgardia, 1936, 10: 401-412.
73．韩丽娟 顾中言 王强 等．农药复配与复配农药 江苏科学技术出版社 1994，45-52．
74．张兴 植物性杀虫剂川楝素的开发研究 西北农业大学学报 1993，21(4)：1-5．
75．赵善欢 张兴 从植物与昆虫的关系谈害虫防除 百科知识 1986，8：54-57．
76. Wu,S.J.et al Bacillus thuringensis Bioteachnology and Environmental Benefits, 1995,1:51.
77. Chen, X.J.et al The thermostable exotoxin of Bacillus thuringiensis.In "Microbial Control of Insects and Mites.Proce. Natl.Acad. USA, 1993,90,904.
78．顾中言 韩丽娟 钟定亮 等农药复配剂增效作用的定性定量分析．江苏农业科学，1990，(3)：31-34．
79．欧晓明 林雪梅 盛书祥等 评价杀虫剂混用联合作用的六种方法比较 农药科学与管理 1997，1：20-23．
80．欧晓明 王跃龙 林雪梅 等 灭多威与杀螟硫磷的筛选及药效评价 植物保护
1997，23(2)：26-29．
81．瞿启慧．昆虫分子生物学的一些进展：神经递质和离子通道．昆虫学报．1995，38(3)：379-379．
82. Kumar, N.S., G.Ventateswerdu Molecular Biol. Intl. 1998, 45 (4):769-774.
83. Shao.Z., et al Transmission of peanut bud necrosis virus J.Invertbr. Pathol. 1998,72:73-81.
84．张宗炳．杀虫药剂的分子毒理学．北京：农业出版社．1987，18-124．
85．张宗炳．昆虫神经生理与神经毒剂．北京：科学出版社．1986，104-135．
86. Bloomquist J R. Ion channels as targets for insecticides. Annu. Rev. Entomol. 1996, 41: 163-190.
87. Neher E, Sakmann B. Single channel currents recorded from membrance of denervated frog muscle fibes. Nature. 1976, 260: 779-820.
88. Hamill O P, Marty A, Nether E. Improved patch clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflugers Arch. Eur. J. Physiol. 1981, 391: 85-100.
89. Bloomquist J R. Ion channels as targets for insecticides. Annu. Rev. Entomol. 1996, 41: 163-190.
90．瞿启慧．昆虫分子生物学的一些进展：神经递质和离子通道．昆虫学报．1995，5(3)：59-61．
91．刘惠霞 吴文君 李新岗．昆虫生物化学．西安：陕西科学技术出版社．1998，45-52．
92. Neher E, Sakmann B. The patch clamp technique. Scientific A mencan. 1992, 266: 44-52.
93. Chow R H, Klingauf J, Neher E. Time course of Ca~(2+) concentration triggering exocytosis in neuroendocrine cells. Proc. Natl. Acacl. Sci. USA. 1994, 91: 12765-12769.
94. Chow R H, Ruden I, Neher E. Delay in vesicle fusion revcealed by electroche monitoring of single secretary events in adrenal chromaffin cells. Nature. 1992, 356: 60-63.
95. Chow R H, Klingauf J, Neher E. Time course of Ca~(2+) concentration triggering
exocytosis in neuroendocrine cells. Proc. Natl. Acacl. Sci. USA. 1994, 91: 12478-12489.
96. Chow R H, Ruden I, Neher E. Delay in vesicle fusion revcealed by electroche monitoring of single secretary events in adrenal chromaffin cells. Nature. 1992, 356: 76-79.