常用化学杀虫剂和抗生素对苏云金杆菌的影响
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摘要
在微生物农药中，苏云金杆菌(Bacillus thuringiensis简称Bt)因其杀虫活性高、杀虫谱较广、易于大规模生产、对人畜安全、无环境污染等特点，已被广泛应用于防治农、林和环卫害虫，并取得了显著的经济效益、社会效益和生态效益，在微生物防治害虫的实践中占有极其重要的地位。本论文以微生物学和分析化学相结合为出发点，在微生物学的基础上，辅以化学知识，着眼于苏云金杆菌杀虫剂在实际应用和制剂保存中存在的问题，重点研究了化学杀虫剂与苏云金杆菌制剂混配和抗生素类作为防腐剂时，二者对苏云金杆菌杀虫剂主要杀虫活性成分伴胞晶体蛋白及芽孢的影响。
研究明确了4类(菊酯类、有机磷类、氨基甲酸酯类和杀螨类)9种常用化学杀虫剂与苏云金杆菌制剂混用时对苏云金杆菌芽孢及主要活性成分伴胞晶体蛋白的作用和影响。十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表明，经菊酯类和有机磷类杀虫剂处理的伴胞晶体蛋白带中，均有一浓度可使伴胞晶体蛋白带颜色加深；经80％敌敌畏500倍稀释液处理的伴胞晶体的两条主要蛋白带较未处理对照相应色带明显变浅，说明该杀虫剂对伴胞晶体蛋白有明显影响。该杀虫剂导致伴胞晶体蛋白带变浅的可能原因：①敌敌畏有效成分分子结构中的P=O键具有一定的氧化作用；②80％敌敌畏制剂中的某种助剂对伴胞晶体蛋白有降解作用。上述两种原因均有可能导致电泳蛋白带变浅。经20％灭多威、20％三氯杀螨醇和10％哒螨酮处理的伴胞晶体蛋白电泳带未发生明显变化。生物测定结果表明，80％敌敌畏与Bt制剂混配一定时间后在某种程度上将造成Bt杀虫毒力的降低，而其余8种杀虫剂与Bt制剂混配后的一定时间内均有不同程度的增效作用。常规使用浓度下，25％敌杀死、48％乐斯本、20％三氯杀螨醇和10％哒螨酮
l－l。I 文 摘 要
乳油可与苏云余杆菌制剂配制成混剂；4．SO／O高效氯氟菊酯乳汕、Z．5％功大乳汕。
20％灭多威和 40％辛硫磷。IJ与书、云朵杆菌制剂现混现用；80％敌地畏不’1’I’与苏云
朵杆菌制剂混用。
针对苏云朵朴自悬浮剂剂型。卜存在f叫Z柏山高化及费力一门a两人问题，州大队
确了］4种常用医用抗生素作为防购利对苏云今杆菌制剂芽地价发的抑制作用和
对伴胞晶体的影响。结果在；刃，红有素、欢酸环丙沙星对＊H菌株芽他。的发有较
强的抑制作用，在 0．5 u g／ml低剂量下］－I在一个月内抑制芽地的Iw发；氧氟沙星
和麦迪霉素抑制作用稍差，在 5 u g叩l白剂量下抑制作用15ti 日问延长而减宕，96h
后基本丧失抑制作用。其它几种抗生素对苏云金杆菌芽他萌发的抑制终浓度均在
30vg／ml以上；试验研究了对芽泡萌发抑制作用较强的麦迪霉素、红霉素。氧氟
沙星和盐酸环丙沙星对CH菌株伴胞晶体的作用，SDS－PAGE检测结果表明，试
验浓度下，这4种抗生素对Cll菌株伴胞晶体无损伤作用。其中氧氟沙星45℃条
件下放置4d，发酵液毒力保持率为96二％。在苏云朵杆菌悬浮剂中加入适当的抗
生素作为防腐剂，既可防止其二次发酵，抑制某些杂菌的生长，又对苏云来杆菌
悬浮剂中的主要杀虫话性成分伴胞晶体蛋白无损伤作用，从而可维持。悬浮别的稳
定性，延长保存期。
As one of important branch of micro-pesticide. Bacillus thuringiensis (Bt) are widely used in controlling pests on agriculture , forest , sanitation and so on. And notable effects in economic society and zoology were obtained. In micro-controlling, B1 has showed its unsubstitute state. On the basis of microbiology, the research work is on the problems about conservation and. application of preparation of Bt. The effects of chemistry pesticides when blended with Bt and antibiotics as antisepsis on spore and parasporal crystal of Bt were studied.
The effects of 9 kinds of 4 species (pyrethroid compounds, organic phosporus compounds, carbamate compounds and killing-mite compounds) of chemical pesticides on spore and parasporal crystal of CH strain of Bacillus thuringiensis were studied when the two kind of pesticides were blended to control pests. The results of the present study showed that these chemical insecticides had different effects on Bt spore and insecticidal crystal protein (ICP). The analysis of SDS-PAGE showed that pyrethroid compounds and organic phosporus compounds could do good to ICP of Bt. Dichlorvos was very harmful to parasporal crystals. The reason of the latter may be: (1)the P=O configuration of the efficient content of dichlorvos may oxygenize ICP so that the activity of ICP was destroyed: (2)some reducing agent in the dichlorvos preparation would do great harm to ICP. Methomyl, dicofol and pyridaben hadn't obvious effects on ICP. Among the nine insecticides studied, deltamethrin. chlorpyrifos. dicofol and pyridaben had no harm
ful effects and they may be mixed with Bt as composite preparation; Beta-cypermethrin. cyhalothrin. methomyl and phoxim may be temporarily mixed with Bt for use. Dichlorvos can't be mixed with Bt as composite preparation or temporarily mixed with Bt for use.
On the contagion of preparation and the decrease of toxin in Suspension Concentrate (SC), fourteen kinds of medical antibiotics were studied about their effects on prohibiting the revegetation of spores and parasporal crystals as antisepsis. The
results showed that these antibiotics had different effects on Bt spore and ICP. The optimal antibiotics for controlling spore revegetation were ncrythromycin and ciprofloxacin hydrochloride, both at 0.5 u g/ml. Oflixacin and ofloxacin needed higher doses, at 5 u g/ml in liquid which could control spores growth for 96h. The others were above 30 u g/ml to take effect. The spores treated with these antibiotics could bourgeon when the antibiotic was diluted or eliminated. SDS-PAGE and bioassay showed that Oflixacin , erythromycin , ofloxacin and ciprofloxacin hydrochloride did not destroyed parasporal crystal. Ofloxacin had better preservation among the four antibiotics. After the fermented liquid added with ofloxacin was stored at 45℃ for four days, the percent of the original activity remained was 96.2%. According to the laboratory research, the author thought that some kinds of antibiotics could prohibit the growth of spore and some other saprophyte when antibiotic was used as antisepsis in SC. Thus, some an
tibiotics can replace chemical-synthesizing antisepsis which may contaminate air, water in great extent. Meanwhile. these antibiotics can also retain the stabilization of SC better.
研究明确了4类(菊酯类、有机磷类、氨基甲酸酯类和杀螨类)9种常用化学杀虫剂与苏云金杆菌制剂混用时对苏云金杆菌芽孢及主要活性成分伴胞晶体蛋白的作用和影响。十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表明，经菊酯类和有机磷类杀虫剂处理的伴胞晶体蛋白带中，均有一浓度可使伴胞晶体蛋白带颜色加深；经80％敌敌畏500倍稀释液处理的伴胞晶体的两条主要蛋白带较未处理对照相应色带明显变浅，说明该杀虫剂对伴胞晶体蛋白有明显影响。该杀虫剂导致伴胞晶体蛋白带变浅的可能原因：①敌敌畏有效成分分子结构中的P=O键具有一定的氧化作用；②80％敌敌畏制剂中的某种助剂对伴胞晶体蛋白有降解作用。上述两种原因均有可能导致电泳蛋白带变浅。经20％灭多威、20％三氯杀螨醇和10％哒螨酮处理的伴胞晶体蛋白电泳带未发生明显变化。生物测定结果表明，80％敌敌畏与Bt制剂混配一定时间后在某种程度上将造成Bt杀虫毒力的降低，而其余8种杀虫剂与Bt制剂混配后的一定时间内均有不同程度的增效作用。常规使用浓度下，25％敌杀死、48％乐斯本、20％三氯杀螨醇和10％哒螨酮
l－l。I 文 摘 要
乳油可与苏云余杆菌制剂配制成混剂；4．SO／O高效氯氟菊酯乳汕、Z．5％功大乳汕。
20％灭多威和 40％辛硫磷。IJ与书、云朵杆菌制剂现混现用；80％敌地畏不’1’I’与苏云
朵杆菌制剂混用。
针对苏云朵朴自悬浮剂剂型。卜存在f叫Z柏山高化及费力一门a两人问题，州大队
确了］4种常用医用抗生素作为防购利对苏云今杆菌制剂芽地价发的抑制作用和
对伴胞晶体的影响。结果在；刃，红有素、欢酸环丙沙星对＊H菌株芽他。的发有较
强的抑制作用，在 0．5 u g／ml低剂量下］－I在一个月内抑制芽地的Iw发；氧氟沙星
和麦迪霉素抑制作用稍差，在 5 u g叩l白剂量下抑制作用15ti 日问延长而减宕，96h
后基本丧失抑制作用。其它几种抗生素对苏云金杆菌芽他萌发的抑制终浓度均在
30vg／ml以上；试验研究了对芽泡萌发抑制作用较强的麦迪霉素、红霉素。氧氟
沙星和盐酸环丙沙星对CH菌株伴胞晶体的作用，SDS－PAGE检测结果表明，试
验浓度下，这4种抗生素对Cll菌株伴胞晶体无损伤作用。其中氧氟沙星45℃条
件下放置4d，发酵液毒力保持率为96二％。在苏云朵杆菌悬浮剂中加入适当的抗
生素作为防腐剂，既可防止其二次发酵，抑制某些杂菌的生长，又对苏云来杆菌
悬浮剂中的主要杀虫话性成分伴胞晶体蛋白无损伤作用，从而可维持。悬浮别的稳
定性，延长保存期。
As one of important branch of micro-pesticide. Bacillus thuringiensis (Bt) are widely used in controlling pests on agriculture , forest , sanitation and so on. And notable effects in economic society and zoology were obtained. In micro-controlling, B1 has showed its unsubstitute state. On the basis of microbiology, the research work is on the problems about conservation and. application of preparation of Bt. The effects of chemistry pesticides when blended with Bt and antibiotics as antisepsis on spore and parasporal crystal of Bt were studied.
The effects of 9 kinds of 4 species (pyrethroid compounds, organic phosporus compounds, carbamate compounds and killing-mite compounds) of chemical pesticides on spore and parasporal crystal of CH strain of Bacillus thuringiensis were studied when the two kind of pesticides were blended to control pests. The results of the present study showed that these chemical insecticides had different effects on Bt spore and insecticidal crystal protein (ICP). The analysis of SDS-PAGE showed that pyrethroid compounds and organic phosporus compounds could do good to ICP of Bt. Dichlorvos was very harmful to parasporal crystals. The reason of the latter may be: (1)the P=O configuration of the efficient content of dichlorvos may oxygenize ICP so that the activity of ICP was destroyed: (2)some reducing agent in the dichlorvos preparation would do great harm to ICP. Methomyl, dicofol and pyridaben hadn't obvious effects on ICP. Among the nine insecticides studied, deltamethrin. chlorpyrifos. dicofol and pyridaben had no harm
ful effects and they may be mixed with Bt as composite preparation; Beta-cypermethrin. cyhalothrin. methomyl and phoxim may be temporarily mixed with Bt for use. Dichlorvos can't be mixed with Bt as composite preparation or temporarily mixed with Bt for use.
On the contagion of preparation and the decrease of toxin in Suspension Concentrate (SC), fourteen kinds of medical antibiotics were studied about their effects on prohibiting the revegetation of spores and parasporal crystals as antisepsis. The
results showed that these antibiotics had different effects on Bt spore and ICP. The optimal antibiotics for controlling spore revegetation were ncrythromycin and ciprofloxacin hydrochloride, both at 0.5 u g/ml. Oflixacin and ofloxacin needed higher doses, at 5 u g/ml in liquid which could control spores growth for 96h. The others were above 30 u g/ml to take effect. The spores treated with these antibiotics could bourgeon when the antibiotic was diluted or eliminated. SDS-PAGE and bioassay showed that Oflixacin , erythromycin , ofloxacin and ciprofloxacin hydrochloride did not destroyed parasporal crystal. Ofloxacin had better preservation among the four antibiotics. After the fermented liquid added with ofloxacin was stored at 45℃ for four days, the percent of the original activity remained was 96.2%. According to the laboratory research, the author thought that some kinds of antibiotics could prohibit the growth of spore and some other saprophyte when antibiotic was used as antisepsis in SC. Thus, some an
tibiotics can replace chemical-synthesizing antisepsis which may contaminate air, water in great extent. Meanwhile. these antibiotics can also retain the stabilization of SC better.
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