球孢白僵菌孢子悬乳剂与亚致死浓度吡虫啉对几种刺吸式口器害虫的室内与田间协同防效评价
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摘要
球孢白僵菌Beauveria bassiana作为一种重要的昆虫病原真菌，在害虫生物防治中已获得广泛研究和应用，但一直存在菌剂常温贮存难、杀虫效果滞后及不稳定等问题。本研究在本实验室关于球孢白僵菌SG8702菌株对付蚜虫等刺吸式口器害虫的一系列工作基础之上，系统测定了该菌孢子悬乳剂的常温贺架期并筛选到一种更为有效的乳化剂，并就孢子悬乳剂及其与亚致死用量吡虫啉混用对蚜虫、飞虱、叶蝉等刺吸式口器害虫的协同增效作用进行了较深入的室内测定和田间药效评价，以期推动我国真菌杀虫剂剂的研发及应用。
孢子悬乳剂的常温贮存期及配方优化对添加和不添加助剂(悬浮稳定剂、紫外保护剂，但不含乳化剂)的孢子悬乳剂在常温避光条件下贮存18个月，在前7个月活孢率均稳定在95％左右且不受所添加助剂的影响，至第10个月活孢率仍达75％左右。而后，含助剂悬乳剂的活孢率下降加速，至16个月时孢子基本失活；而不含助剂的活孢率在第16、17及18个月时分别还有57％、51％和42％。这表明两种孢子悬乳剂能常温保质贮存7个月，有效贮存10个月，基本适应真菌杀虫剂产业化要求。通过对多种乳化剂乳化性能及其对孢子悬乳剂常温贮存影响的比较研究，筛选出了乳化性能最佳且对孢子存活影响最小的乳化剂AE-3，按5％比例(v／v)添加能使孢子悬乳剂在1000倍稀释液中充分均匀分散，适合常规喷雾。但是，该乳化剂与球孢白僵菌分生孢子的生物学相容性仍不理想，含5％乳化剂的孢子悬乳剂仅能常温安全贮存45天(活孢率在85％以上)，随后活孢率急剧下降，到第60天使孢子基本全部失活。尽管AE-3是我们目前筛选所获最适合白僵菌的乳化剂，但仍不能长期共存，建议乳化剂AE-3与孢子悬乳剂分丌包装，田间施用前按5％比例混合均匀后再稀释喷雾。这既可充分乳化矿物油载体，又能保证孢子活力，使菌剂常温贮存的问题得到缓解。
孢子悬乳剂与亚致剂量吡虫啉混用对褐稻虱毒力的协同防效评价 按最新国际规范采用直接弥雾法，对未剂型化的孢子水悬液、剂型化的孢子悬乳液及其与低浓度吡虫啉(0.5、1.0及2.0μg／mL)混液对稻褐虱进行5次生物测定，接种剂量规范为单位面积孢子附着量(孢子数／mm~2)。每次生测设置5个剂量处理及一个相应的对照，3次重复，每重复包括30～40头3龄幼虫。所获生测数据经几率分析和时间一剂量一死亡率模型模拟分析，显示后一种分析方法更能直观且有效地揭示白僵菌与吡虫啉互作对靶标害虫控制的协同增效作用。根据五次独立生测实验数据的时间—剂量—死亡率
模拟分析，基本结果有以下几个方面。一是试虫死亡率在无菌水对照(CKI)与作为
抱子悬乳剂介质的乳油对照(CKZ)间差异不显著，但用乳油配制的抱子悬乳剂稀释
液对试虫的致病致死作用明显强于未剂型化抱子粉的水悬液，处理后5一12天基于二
者LCS。之比的相对效力提高1 .2一3 .6倍，随时间后移差距缩小。二是按有效成份在抱
子悬乳液中添加毗虫琳。.5、1.0和2.0林g/mL能大幅增强菌剂对试虫的作用，在处理
后5一12天，基于LCS。之比的相对效力分别提高1 .3一2.0倍、6.2一8.5倍和22.7一72.4倍;
抱子悬乳液中添加毗虫琳1.0和2.0协g/mL比添加0.5林留mL的相对效力分别提高
3.7一5.6倍和16.0一35.6倍，添加2.0拼g/mL比添加1.0林g/mL的相对效力提高3.0一8.4
倍，显示菌药混用的协同增效作用显著，且随用药后时间收敛。三是无论从数学意义
还是生物学意义上看，时间一剂量一死亡率模型是目前定量评价菌一药互作的最有效
工具。以上结果首次揭示了球抱白僵菌与毗虫琳控制褐稻虱的协同增效作用范围。
袍子悬乳剂田间控蚜效果评价将高纯度抱子粉兑入一种含乳化剂、悬浮稳定剂
及紫外保护剂的矿物油中，配成含抱量为100亿/mL的抱子悬乳剂(纯菌剂)，再添加
3%(W/v)的10%毗虫琳可湿剂而得菌药混配剂，于2003年5月在浙江余姚进行了防治
蔬菜蚜虫的田间药效试验和农户示范。在两块夏熟白菜地中，纯菌剂和菌药混配剂均
用水稀释兼用1000倍(10，个抱子/mL)常规喷雾2次(间隔10天)，并设清水对照和低
用量毗虫琳(与混配剂用量相同)对照。在20天的试验期间，混配剂在两块菜地的
相对防效分别达94.5%和86.2%，纯菌剂分别达86.4%和73.1%，而低用量毗虫琳处理
的最高防效仅为50%左右。两块地的纯菌剂与菌药混配剂处理中，根据喷雾期间玻片
收集到的抱子数量，菌处理的平均抱子附着量为220一255个抱子/mmZ，处理间差异不
显著(F=0.41;df=3，8;尸=0.75)。在入夏更豆、甘蓝、白菜、萝卜及梨树上用菌药混配
剂1000倍稀释液对多种蚜虫进行的农户示范试验中，一次性喷雾后5一10d内对菜蚜
防效为48%一68%，尤对梨二叉蚜的防效高达99%。以上结果表明，球抱白僵菌抱子
悬乳剂对各种蚜虫具有良好控制效果，尤以菌剂与低剂量毗虫琳混配施用效果更好。
两种杀虫真菌制剂对茶小绿叶蝉的田间防效评价于2002年盛夏在浙江遂昌一高
山茶园用球抱白僵菌和玫烟色拟青霉尸aeoilomyce:fumosoroseus的抱子悬乳剂及其与
3%(w/v)的10%毗虫琳可湿剂的混配剂对茶小绿叶蝉进行了田间药效试验，各菌剂均
稀释500倍喷雾2次，间隔12天。结果表明，两种真菌的混配剂均明显优于纯菌剂，
The entomopathogenic fungus, Beauveria bassiana (Balsamo) Vuillemin, is a classic fungal biocontrol agent and has been developed into a variety of commercial formulations for insect control in the world. Technical problems involved in ambient storage, slow action, and unstable field efficacy have been for a long run obstacles to commercial development and practical use of B. bassiana-based mycoinsecticides for insect control. Based on previous studies on B. bassiana SG8702 in our laboratory, the present study was performed to determine the shelf-life of an oil-based formulation of B. bassiana conidia at ambient temperature, to search for more efficient emulsifier to ease field spray of the formulation, and to quantitatively evaluate separate or interactive effects of the emulsifiable conidial formulation and sublethal imidacloprid application rates on the control of sucking insect pests such as aphids, planthoppers and leafhoppers under laboratory and field conditions. Our objectives were to facilitate techn
ical progress in formulation and integration of B. bassiana into insect pest management systems.
Ambient storage of oil-based conidia] formulation and its optimal emulsifier for field spray. During an 18-month period of storage at ambient temperatures in dark, the oil-based B. bassiana formulation with or without additives (suspension stabilizer, UV protectant) was very stable during the first 7 months. On average, 95% of the conidia were viable at the end of 7 months, followed by >75% at the end of 10 months. Thereafter, however, the viability of the conidia in the formulation containing the additives decreased rapidly and was not detected at the end of 16 months. In contrast, the formulation not containing the additives had conidial viabilities of 57%, 51%, and 42% on months 16, 17, and 18, respectively. Apparently, the shelf-lives of the two formulations at ambient temperatures satisfied a general requirement for commercial development of mycoinsecticides. A novel emulsifier AE-3 was found to best emulsify 1000-fold aqueous dilution of the oil-based conidial formulation for field spray at a proportio
n of around 5% of the formulation. However, the biological compatibility of the emulsifier to B. bassiana conidia was less satisfactory although it was much better than other emulsifiers tested. Inclusion of the emulsifier in the conidial formulation allowed for only 45-day shelf-life of its storage at ambient temperature.
To take both emulsification and shelf life into account, the emulsifier should not been included in the conidial formulation until aqueous dilutions were made prior to field sprays.
Synergistic interaction between B, bassiana and imidacloprid. Interactive effects of B. bassiana SG8702 and imidacloprid were assayed on Nilaparvata lugens under laboratory conditions by spraying suspensions of unformulated conidia (assay 1) and aqueous dilutions of its emulsifiable formulation alone (assay 2) or together with imidacloprid at the sublethal application rates of a.i. 0.5, 1.0 and 2.0 g/mL (assays 3-5). Each assay included 5 conidial concentrations (no. conidia/mm2) plus a control, and replicated 3 times with 30-40 third instar brachypterous nymphs per replicate. It was found that an emulsifable, mineral oil-based liquid suited to formulate B. bassiana by enhancing fungal activity but not significantly increased background mortality of N. lugens. Synergistic effects of both agents or formulations were determined by estimating relative potencies (paired ratios of LC50 estimates on days 5-12 after spray) of assay 2 over assay 1 (1.2-3.6), assay 3 over assay 2 (1.3-2.0), assay 4 over assay 2 (6.2-8.5), assay 5 over assay 2 (22.7-72.4), assay 4 over assay 3 (3.7-5.6), assay 5 over assay 3 (16.0-35.6), and assay 5 over assay 4 (3.0-8.4). The results demonstrated in detail that a time-dose-mortality modeling method was not only mathematically but al50 biologically robust to evaluate interactive effects of B. bassiana and imidacloprid on N. lugens. Enhanced fungal formulations, compared to their counter
孢子悬乳剂的常温贮存期及配方优化对添加和不添加助剂(悬浮稳定剂、紫外保护剂，但不含乳化剂)的孢子悬乳剂在常温避光条件下贮存18个月，在前7个月活孢率均稳定在95％左右且不受所添加助剂的影响，至第10个月活孢率仍达75％左右。而后，含助剂悬乳剂的活孢率下降加速，至16个月时孢子基本失活；而不含助剂的活孢率在第16、17及18个月时分别还有57％、51％和42％。这表明两种孢子悬乳剂能常温保质贮存7个月，有效贮存10个月，基本适应真菌杀虫剂产业化要求。通过对多种乳化剂乳化性能及其对孢子悬乳剂常温贮存影响的比较研究，筛选出了乳化性能最佳且对孢子存活影响最小的乳化剂AE-3，按5％比例(v／v)添加能使孢子悬乳剂在1000倍稀释液中充分均匀分散，适合常规喷雾。但是，该乳化剂与球孢白僵菌分生孢子的生物学相容性仍不理想，含5％乳化剂的孢子悬乳剂仅能常温安全贮存45天(活孢率在85％以上)，随后活孢率急剧下降，到第60天使孢子基本全部失活。尽管AE-3是我们目前筛选所获最适合白僵菌的乳化剂，但仍不能长期共存，建议乳化剂AE-3与孢子悬乳剂分丌包装，田间施用前按5％比例混合均匀后再稀释喷雾。这既可充分乳化矿物油载体，又能保证孢子活力，使菌剂常温贮存的问题得到缓解。
孢子悬乳剂与亚致剂量吡虫啉混用对褐稻虱毒力的协同防效评价 按最新国际规范采用直接弥雾法，对未剂型化的孢子水悬液、剂型化的孢子悬乳液及其与低浓度吡虫啉(0.5、1.0及2.0μg／mL)混液对稻褐虱进行5次生物测定，接种剂量规范为单位面积孢子附着量(孢子数／mm~2)。每次生测设置5个剂量处理及一个相应的对照，3次重复，每重复包括30～40头3龄幼虫。所获生测数据经几率分析和时间一剂量一死亡率模型模拟分析，显示后一种分析方法更能直观且有效地揭示白僵菌与吡虫啉互作对靶标害虫控制的协同增效作用。根据五次独立生测实验数据的时间—剂量—死亡率
模拟分析，基本结果有以下几个方面。一是试虫死亡率在无菌水对照(CKI)与作为
抱子悬乳剂介质的乳油对照(CKZ)间差异不显著，但用乳油配制的抱子悬乳剂稀释
液对试虫的致病致死作用明显强于未剂型化抱子粉的水悬液，处理后5一12天基于二
者LCS。之比的相对效力提高1 .2一3 .6倍，随时间后移差距缩小。二是按有效成份在抱
子悬乳液中添加毗虫琳。.5、1.0和2.0林g/mL能大幅增强菌剂对试虫的作用，在处理
后5一12天，基于LCS。之比的相对效力分别提高1 .3一2.0倍、6.2一8.5倍和22.7一72.4倍;
抱子悬乳液中添加毗虫琳1.0和2.0协g/mL比添加0.5林留mL的相对效力分别提高
3.7一5.6倍和16.0一35.6倍，添加2.0拼g/mL比添加1.0林g/mL的相对效力提高3.0一8.4
倍，显示菌药混用的协同增效作用显著，且随用药后时间收敛。三是无论从数学意义
还是生物学意义上看，时间一剂量一死亡率模型是目前定量评价菌一药互作的最有效
工具。以上结果首次揭示了球抱白僵菌与毗虫琳控制褐稻虱的协同增效作用范围。
袍子悬乳剂田间控蚜效果评价将高纯度抱子粉兑入一种含乳化剂、悬浮稳定剂
及紫外保护剂的矿物油中，配成含抱量为100亿/mL的抱子悬乳剂(纯菌剂)，再添加
3%(W/v)的10%毗虫琳可湿剂而得菌药混配剂，于2003年5月在浙江余姚进行了防治
蔬菜蚜虫的田间药效试验和农户示范。在两块夏熟白菜地中，纯菌剂和菌药混配剂均
用水稀释兼用1000倍(10，个抱子/mL)常规喷雾2次(间隔10天)，并设清水对照和低
用量毗虫琳(与混配剂用量相同)对照。在20天的试验期间，混配剂在两块菜地的
相对防效分别达94.5%和86.2%，纯菌剂分别达86.4%和73.1%，而低用量毗虫琳处理
的最高防效仅为50%左右。两块地的纯菌剂与菌药混配剂处理中，根据喷雾期间玻片
收集到的抱子数量，菌处理的平均抱子附着量为220一255个抱子/mmZ，处理间差异不
显著(F=0.41;df=3，8;尸=0.75)。在入夏更豆、甘蓝、白菜、萝卜及梨树上用菌药混配
剂1000倍稀释液对多种蚜虫进行的农户示范试验中，一次性喷雾后5一10d内对菜蚜
防效为48%一68%，尤对梨二叉蚜的防效高达99%。以上结果表明，球抱白僵菌抱子
悬乳剂对各种蚜虫具有良好控制效果，尤以菌剂与低剂量毗虫琳混配施用效果更好。
两种杀虫真菌制剂对茶小绿叶蝉的田间防效评价于2002年盛夏在浙江遂昌一高
山茶园用球抱白僵菌和玫烟色拟青霉尸aeoilomyce:fumosoroseus的抱子悬乳剂及其与
3%(w/v)的10%毗虫琳可湿剂的混配剂对茶小绿叶蝉进行了田间药效试验，各菌剂均
稀释500倍喷雾2次，间隔12天。结果表明，两种真菌的混配剂均明显优于纯菌剂，
The entomopathogenic fungus, Beauveria bassiana (Balsamo) Vuillemin, is a classic fungal biocontrol agent and has been developed into a variety of commercial formulations for insect control in the world. Technical problems involved in ambient storage, slow action, and unstable field efficacy have been for a long run obstacles to commercial development and practical use of B. bassiana-based mycoinsecticides for insect control. Based on previous studies on B. bassiana SG8702 in our laboratory, the present study was performed to determine the shelf-life of an oil-based formulation of B. bassiana conidia at ambient temperature, to search for more efficient emulsifier to ease field spray of the formulation, and to quantitatively evaluate separate or interactive effects of the emulsifiable conidial formulation and sublethal imidacloprid application rates on the control of sucking insect pests such as aphids, planthoppers and leafhoppers under laboratory and field conditions. Our objectives were to facilitate techn
ical progress in formulation and integration of B. bassiana into insect pest management systems.
Ambient storage of oil-based conidia] formulation and its optimal emulsifier for field spray. During an 18-month period of storage at ambient temperatures in dark, the oil-based B. bassiana formulation with or without additives (suspension stabilizer, UV protectant) was very stable during the first 7 months. On average, 95% of the conidia were viable at the end of 7 months, followed by >75% at the end of 10 months. Thereafter, however, the viability of the conidia in the formulation containing the additives decreased rapidly and was not detected at the end of 16 months. In contrast, the formulation not containing the additives had conidial viabilities of 57%, 51%, and 42% on months 16, 17, and 18, respectively. Apparently, the shelf-lives of the two formulations at ambient temperatures satisfied a general requirement for commercial development of mycoinsecticides. A novel emulsifier AE-3 was found to best emulsify 1000-fold aqueous dilution of the oil-based conidial formulation for field spray at a proportio
n of around 5% of the formulation. However, the biological compatibility of the emulsifier to B. bassiana conidia was less satisfactory although it was much better than other emulsifiers tested. Inclusion of the emulsifier in the conidial formulation allowed for only 45-day shelf-life of its storage at ambient temperature.
To take both emulsification and shelf life into account, the emulsifier should not been included in the conidial formulation until aqueous dilutions were made prior to field sprays.
Synergistic interaction between B, bassiana and imidacloprid. Interactive effects of B. bassiana SG8702 and imidacloprid were assayed on Nilaparvata lugens under laboratory conditions by spraying suspensions of unformulated conidia (assay 1) and aqueous dilutions of its emulsifiable formulation alone (assay 2) or together with imidacloprid at the sublethal application rates of a.i. 0.5, 1.0 and 2.0 g/mL (assays 3-5). Each assay included 5 conidial concentrations (no. conidia/mm2) plus a control, and replicated 3 times with 30-40 third instar brachypterous nymphs per replicate. It was found that an emulsifable, mineral oil-based liquid suited to formulate B. bassiana by enhancing fungal activity but not significantly increased background mortality of N. lugens. Synergistic effects of both agents or formulations were determined by estimating relative potencies (paired ratios of LC50 estimates on days 5-12 after spray) of assay 2 over assay 1 (1.2-3.6), assay 3 over assay 2 (1.3-2.0), assay 4 over assay 2 (6.2-8.5), assay 5 over assay 2 (22.7-72.4), assay 4 over assay 3 (3.7-5.6), assay 5 over assay 3 (16.0-35.6), and assay 5 over assay 4 (3.0-8.4). The results demonstrated in detail that a time-dose-mortality modeling method was not only mathematically but al50 biologically robust to evaluate interactive effects of B. bassiana and imidacloprid on N. lugens. Enhanced fungal formulations, compared to their counter
引文
丁珊，王成树，樊美珍等．1997．白僵菌混合粉剂的工业性试验设计．中国虫生真菌研究与应用，北京：中国农业科技出版社．4：150-153．
丁珊，汤坚，王成树等．1996．灭幼脲与白僵菌的相容性及增效作用的研究．安徽农业大学学报，23(3)：366-370．
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