二化螟对几种杀虫剂的抗药性研究
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摘要
二化螟(Chilo suppressalis Walker)是亚洲地区最重要的水稻害虫之一。因其具有钻蛀危害特点,防治一直比较困难。近年来,随着化学农药的大量使用,导致了二化螟抗药性的发生和发展,加大了防治的难度。因此开展二化螟的抗药性监测、抗性机理研究、以及新农药制剂的研制,对开展二化螟的抗性治理工作具有重要的意义。
本研究在对四川省六个市(县)08-09两年的二化螟田间种群对近年来防治二化螟常用药剂的抗药性监测基础上,研究了二化螟田间种群产生抗药性的机制,筛选了对二化螟田间种群生物活性较高、作用机制不同单剂并进行复配,并结合稻田生态系特点进行泡腾片剂的研究。研究结果不仅有助于明确四川省二化螟的抗药性现状和不同地理种群抗药性生理生化机制,而且能为了解我国二化螟不同地理种群抗药性水平及开展抗性治理提供重要参考。本研究取得如下主要研究结果：
1.二化螟的抗药性监测
采用微量点滴法监测了四川省南部县、雨城区、彭山县、双流县、乐至县以及邛崃市6个市(县)二化螟田间种群对杀虫单、三唑磷、毒死蜱、敌百虫、氟虫腈、阿维菌素和甲维盐的抗药性。结果表明：①六个市(县)二化螟田间种群对三唑磷、杀虫单、毒死蜱已经表现出了中到高水平的抗性,通过2008～2009两年的抗性监测结果可以看出抗性呈现明显增长趋势。其中双流县二化螟种群抗性倍数增长十分显著,由2008年的43.4981倍上升到2009年的93.7325倍；六个市(县)二化螟田间种群对杀虫单的抗性倍数增长较三唑磷相对平缓,只有邛崃市的二化螟种群增长了17.5181倍；对毒死蜱抗性倍数增长最快的是彭山和双流的二化螟种群,分别增长了14.0251倍和23.0121倍。②六个市(县)二化螟田间种群对敌百虫的抗药性维持在敏感性下降或中等抗性水平,通过2008-2009两年的监测结果可以看出二化螟田间种群对其抗药性平稳中有升有降,总体变化幅度不大。其中雨城区、乐至县两地的二化螟种群抗性倍数分别增长1.3591倍和1.7558倍。南部、彭山、双流、邛崃4个市(县)二化螟种群抗性倍数分别下降2.3004倍、2.1328倍、1.1929倍和0.2349倍。③六个市(县)二化螟田间种群对氟虫腈、阿维菌素和甲维盐三种药剂的抗药性相对较低,三种药剂对二化螟具有较高的活性,其中甲维盐的活性最高。2009年的监测结果显示,LD50在0.00022～0.000535μg/larva之间。通过2008-2009两年的抗性监测结果可以看出二化螟对以上三种药剂的抗药性呈缓慢上升的趋势。其中对已经禁止在水稻上使用的氟虫腈,仍然保持较高的生物活性。相比其它药剂,二化螟对阿维菌素和甲维盐抗性增长缓慢。对阿维菌素抗性增长最快的是双流二化螟种群,2009年比2008年增长了2.479倍。对甲维盐抗性增长最为缓慢,增长最快的邛崃二化螟种群也只增长了0.5172倍。
2.二化螟田间种群抗药性生理生化机制
以二化螟田间抗性种群和室内敏感种群作为研究材料。①测定了不同品系二化螟种群羧酸酯酶(CarE)、磷酸酯酶(ACP,ALP)、乙酰胆碱酯酶(AchE)、谷胱甘肽-S-转移酶(GSTs)、多功能氧化酶(MFO)、蛋白水解酶(Proteinase)、三种保护酶(POD, SOD,CAT)活性以及粗脂(Fat)含量。结果显示田间抗性种群与室内敏感种群的各种酶系活性及粗脂含量均存在着显著性差异,说明了以上所测酶系及粗脂含量可能与二化螟产生抗性的机制有关。②二化螟种群各酶系活性与其对三唑磷、杀虫单、氟虫腈、甲维盐四种药剂的抗性水平(LD50)进行相关性分析表明：二化螟田间种群对以上四种药剂的抗性水平与GSTs, MFO, AchE, SOD, CAT活性间存在显著相关性。其中GSTs活性与二化螟对三唑磷抗性之间有极显著的相关性(r=0.86**)；MFO活性与二化螟对氟虫腈抗性之间有显著的相关性(r=0.74*); AchE活性与二化螟对杀虫单、三唑磷、氟虫腈、甲维盐抗性均呈明显得负相关性,相关系数分别为-0.86**,-0.79*,-0.60,-0.55；SOD活性与二化螟对甲维盐抗性有显著相关性(r=0.71*)；CAT活性与二化螟对杀虫单抗性有显著相关性(r=0.78*)。
3.复配剂型——泡腾片剂的研究
依据抗性监测和抗性机理研究结果,选择了两种作用机制不同,无交互抗性的杀虫单和甲维盐进行复配和剂型加工的研究。结果表明,①甲维盐和杀虫单混配具有明显的增效作用,且具有很宽的有效配比谱。室内毒力测定表明甲维盐LDso为0.5964ng/larva,杀虫单LDso为21501ng/larva,两者的毒力差异达36051.31倍。共毒系数研究表明甲维盐和杀虫单的配比在1：1000-1：36051(质量比)均有显著的增效作用。②泡腾片剂试验结果表明,片剂最佳配方为：甲维盐,0.04%；杀虫单,39.6%：分散剂NNO,3%；润湿剂Tween-80,2%;粘结剂聚乙二醇6000,3%；崩解剂：碳酸氢钠+柠檬酸(1：1),40%；填料：乳糖+硫酸钠(1：1),12%。③与市售甲维盐和杀虫单各单剂的商品药相比,其毒力效果显著。对各制剂的LD50 (ng/larva)进行比对,室内复配研制的甲维盐·杀虫单泡腾片剂的杀虫效果(LD50约为335ng/larva)显著优于市场上销售的3.6%的杀虫单大粒剂(LD50为2714ng/larva)。由于本制剂中甲维盐所占比例很小,与0.2%的甲维盐高渗可溶性粉剂(LD50为13.71ng/larva)相比,甲维盐在两种制剂中的使用量比约为0.335：13.71=1：41,因此本制剂大大减少了甲维盐的使用量。
Rice stem borer (Chilo suppressalis Walker) is one of the main rice pests in Asia. It is always difficult to control for features of rilling decayed hazard. In recent years, with the large-scale use of chemical insecticide, both the occurrence and development of resistance of Chilo suppressalis to chemical insecticide are enhanced, which increase the difficulty of prevention. So it is signality for the task of resistance management to carry out resistance monitoring, biochemical resistance mechanism and new pesticide development.
Based on the resistance monitoring of Chilo suppressalis with several kinds of common insecticides in six counties of Sichuan province between 2008 and 2009, we studied the resistant mechanism among field populations and screen for higher biological activity and different mechanisms to formulate. Meanwhile, we also involved the study on effervescent tablet, considering the ecology system. The results explicited the resistant situation, physiological and biochemical resistant mechanism among field populations. These data also provided important reference on understanding of the resistant level and the resistant management in different geographic populations in China. The main results were as follows:
1. Resistance monitoring of Chilo suppressalis
Micro-drop method was used to monitor the susceptibility of Chilo suppressalis to monosultap, triazophos, chlorpyrifos, Dipterex, fipronil, Avermectins, emamectin benzoate, collected in Nanbu, Yucheng, Pengshan, Shuangliu, Lezhi and Qionglai, respectively. The results showed as follows:
①Populations of Chilo suppressalis in Sichuan province produced medium to high level resistance to monosultap, triazophos, chlorpyrifos. Through the resistance monitoring results, we could see a clearly resistance growth. The resistance ratio in Shuangliu witnessed a significant growth, from 43.4981 in 2008 to 93.7325 in 2009. The pest resistances multiples growth to monosultap was flat in the six regions, only Qionglai increased 17.5181 times. The resistance ratio to chlorpyrifos increased fast only in Pengshan and Shuangliu,14.0251 and 23.0121 times, respectively.
②Populations of Chilo suppressalis in Sichuan province produced in sensitivity to decreasing or medium resistance level from 2008 to 2009 in the six regions. Compared to triazophos, the pest resistances multiples growth to Dipterex was gradual in the six regions. Yucheng and Lezhi increased 1.3591 and 1.7558 times respectively. Nanbu, Pengshan, Shuangliu and Qionglai decreased 2.3004,2.1328,1.1929 and 0.2349 times, respectively.
③The resistance of Chilo suppressalis field populations in six regions to fipronil, abamectins and emamectin benzoate was relatively low. Emamectin benzoate had higher activity:LD50=0.00022～0.000535ng/larva in 2009. Through resistance monitoring results between 2008 and 2009, it showed a slowly rising trend to the three reagents mentioned above. Fipronil, which has been banned to use for rice growth, remained showing high biological activity. The resistance of Chilo suppressali to Abamectins and Emamectin benzoate was much slower than other pesticides. The fastest growing resistance to abamectins was the Shuangliu population,2.479 times from 2008 to 2009. The slowest one was the resistance to Emamectin benzoate:the fastest growth field was Qionglai population, which only 0.5172 times.
2. Biochemical mechanism of resistance of Chilo suppressalis field populations
The research materials were from resistance population and indoor sensitive strain.①We tested the activity of CarE, ACP, ALP, AchE, GSTs, MFO, Proteinase, POD, SOD, CAT and the content of FAT in different strains. The results showed that each enzyme and crude fat content were significant different existing in the field resistance population and indoor sensitive, which might mean that it had some connect with resistant mechanism;②Comparing various enzymes activity with insect resistance level of monosultap, triazophos, fipronil, emamectin benzoate (LD50) correlation analysis, we learned that there were significant correlation between the resistance and the biology activity of AchE, GSTs, MFO, SOD, CAT. GSTs's activity and insect resistance to triazophos had significant correlation (r=0.86*); MFO's activity and insect resistance to fipronil had significant correlation (r=0.74*); AchE activity and resistanceof Chilo suppressalis to monosultap, triazophos, fipronil, emamectin benzoate were obvious negative correlation, related coefficient was -0.86**,-0.79*,-0.60,-0.55; SOD activity and resistance of Chilo suppressalis to emamectin benzoate had significant correlation (r=0.71). CAT activity and resistance of Chilo suppressalis to monosultap had significant correlation (r=0.78*).
3. Study on formulation-effervescent tablet
Based on the resistance monitoring and resistance mechanism, emamectin benzoate and monosultap were chosen for formulation and processing, which were in two different mechanism and no cross-resistance, The results were as follows:①Emamectin benzoat and monosultap mixed significant synergistic effect, and had a wide spectrum of the effective ratio. LD50 of emamectin benzoate was 0.5964ng/larva and the LD50 of monosultap was 21501ng/larva. The difference of the virulence between them was 36051.31 times. The research of co-toxicity coefficient showed that the ratio among 1:1000～1:36051 had significantl synergy effect.②The results of effervescent tablet test showed that the best recipe was:emamectin benzoate,0.04%; monosultap:39.96%; dispersant NNO,3%; Wetting agent:Tween-80,2%; Binder:polyethylene glycol 6000,3 %; Disintegrating agent:sodium bicarbonate+citric acid (1:1),40%; Packing:lactose+ sodium(1:1),12%.③Compared with single agent of emamectin benzoat and monosultap, its chemical toxic effect was more remarkable. For the preparation in the LD50 (ng/larva), the insecticidal effect of emamectin benzoate and monosultap effervescent tablet was (LD50≈335ng/larva) more significant than market 3.6% monosultap big granule (LD50=2714ng/larva). Due to the little proportion of emamectin benzoate in this tablet, when compared with the 0.2% emamectin benzoate soluble powder in LD50, the usage rate on emamectin benzoate between them was about(0.335:13.71)1:41, So it could greatly reduce the use of emamectin benzoate.
本研究在对四川省六个市(县)08-09两年的二化螟田间种群对近年来防治二化螟常用药剂的抗药性监测基础上,研究了二化螟田间种群产生抗药性的机制,筛选了对二化螟田间种群生物活性较高、作用机制不同单剂并进行复配,并结合稻田生态系特点进行泡腾片剂的研究。研究结果不仅有助于明确四川省二化螟的抗药性现状和不同地理种群抗药性生理生化机制,而且能为了解我国二化螟不同地理种群抗药性水平及开展抗性治理提供重要参考。本研究取得如下主要研究结果：
1.二化螟的抗药性监测
采用微量点滴法监测了四川省南部县、雨城区、彭山县、双流县、乐至县以及邛崃市6个市(县)二化螟田间种群对杀虫单、三唑磷、毒死蜱、敌百虫、氟虫腈、阿维菌素和甲维盐的抗药性。结果表明：①六个市(县)二化螟田间种群对三唑磷、杀虫单、毒死蜱已经表现出了中到高水平的抗性,通过2008～2009两年的抗性监测结果可以看出抗性呈现明显增长趋势。其中双流县二化螟种群抗性倍数增长十分显著,由2008年的43.4981倍上升到2009年的93.7325倍；六个市(县)二化螟田间种群对杀虫单的抗性倍数增长较三唑磷相对平缓,只有邛崃市的二化螟种群增长了17.5181倍；对毒死蜱抗性倍数增长最快的是彭山和双流的二化螟种群,分别增长了14.0251倍和23.0121倍。②六个市(县)二化螟田间种群对敌百虫的抗药性维持在敏感性下降或中等抗性水平,通过2008-2009两年的监测结果可以看出二化螟田间种群对其抗药性平稳中有升有降,总体变化幅度不大。其中雨城区、乐至县两地的二化螟种群抗性倍数分别增长1.3591倍和1.7558倍。南部、彭山、双流、邛崃4个市(县)二化螟种群抗性倍数分别下降2.3004倍、2.1328倍、1.1929倍和0.2349倍。③六个市(县)二化螟田间种群对氟虫腈、阿维菌素和甲维盐三种药剂的抗药性相对较低,三种药剂对二化螟具有较高的活性,其中甲维盐的活性最高。2009年的监测结果显示,LD50在0.00022～0.000535μg/larva之间。通过2008-2009两年的抗性监测结果可以看出二化螟对以上三种药剂的抗药性呈缓慢上升的趋势。其中对已经禁止在水稻上使用的氟虫腈,仍然保持较高的生物活性。相比其它药剂,二化螟对阿维菌素和甲维盐抗性增长缓慢。对阿维菌素抗性增长最快的是双流二化螟种群,2009年比2008年增长了2.479倍。对甲维盐抗性增长最为缓慢,增长最快的邛崃二化螟种群也只增长了0.5172倍。
2.二化螟田间种群抗药性生理生化机制
以二化螟田间抗性种群和室内敏感种群作为研究材料。①测定了不同品系二化螟种群羧酸酯酶(CarE)、磷酸酯酶(ACP,ALP)、乙酰胆碱酯酶(AchE)、谷胱甘肽-S-转移酶(GSTs)、多功能氧化酶(MFO)、蛋白水解酶(Proteinase)、三种保护酶(POD, SOD,CAT)活性以及粗脂(Fat)含量。结果显示田间抗性种群与室内敏感种群的各种酶系活性及粗脂含量均存在着显著性差异,说明了以上所测酶系及粗脂含量可能与二化螟产生抗性的机制有关。②二化螟种群各酶系活性与其对三唑磷、杀虫单、氟虫腈、甲维盐四种药剂的抗性水平(LD50)进行相关性分析表明：二化螟田间种群对以上四种药剂的抗性水平与GSTs, MFO, AchE, SOD, CAT活性间存在显著相关性。其中GSTs活性与二化螟对三唑磷抗性之间有极显著的相关性(r=0.86**)；MFO活性与二化螟对氟虫腈抗性之间有显著的相关性(r=0.74*); AchE活性与二化螟对杀虫单、三唑磷、氟虫腈、甲维盐抗性均呈明显得负相关性,相关系数分别为-0.86**,-0.79*,-0.60,-0.55；SOD活性与二化螟对甲维盐抗性有显著相关性(r=0.71*)；CAT活性与二化螟对杀虫单抗性有显著相关性(r=0.78*)。
3.复配剂型——泡腾片剂的研究
依据抗性监测和抗性机理研究结果,选择了两种作用机制不同,无交互抗性的杀虫单和甲维盐进行复配和剂型加工的研究。结果表明,①甲维盐和杀虫单混配具有明显的增效作用,且具有很宽的有效配比谱。室内毒力测定表明甲维盐LDso为0.5964ng/larva,杀虫单LDso为21501ng/larva,两者的毒力差异达36051.31倍。共毒系数研究表明甲维盐和杀虫单的配比在1：1000-1：36051(质量比)均有显著的增效作用。②泡腾片剂试验结果表明,片剂最佳配方为：甲维盐,0.04%；杀虫单,39.6%：分散剂NNO,3%；润湿剂Tween-80,2%;粘结剂聚乙二醇6000,3%；崩解剂：碳酸氢钠+柠檬酸(1：1),40%；填料：乳糖+硫酸钠(1：1),12%。③与市售甲维盐和杀虫单各单剂的商品药相比,其毒力效果显著。对各制剂的LD50 (ng/larva)进行比对,室内复配研制的甲维盐·杀虫单泡腾片剂的杀虫效果(LD50约为335ng/larva)显著优于市场上销售的3.6%的杀虫单大粒剂(LD50为2714ng/larva)。由于本制剂中甲维盐所占比例很小,与0.2%的甲维盐高渗可溶性粉剂(LD50为13.71ng/larva)相比,甲维盐在两种制剂中的使用量比约为0.335：13.71=1：41,因此本制剂大大减少了甲维盐的使用量。
Rice stem borer (Chilo suppressalis Walker) is one of the main rice pests in Asia. It is always difficult to control for features of rilling decayed hazard. In recent years, with the large-scale use of chemical insecticide, both the occurrence and development of resistance of Chilo suppressalis to chemical insecticide are enhanced, which increase the difficulty of prevention. So it is signality for the task of resistance management to carry out resistance monitoring, biochemical resistance mechanism and new pesticide development.
Based on the resistance monitoring of Chilo suppressalis with several kinds of common insecticides in six counties of Sichuan province between 2008 and 2009, we studied the resistant mechanism among field populations and screen for higher biological activity and different mechanisms to formulate. Meanwhile, we also involved the study on effervescent tablet, considering the ecology system. The results explicited the resistant situation, physiological and biochemical resistant mechanism among field populations. These data also provided important reference on understanding of the resistant level and the resistant management in different geographic populations in China. The main results were as follows:
1. Resistance monitoring of Chilo suppressalis
Micro-drop method was used to monitor the susceptibility of Chilo suppressalis to monosultap, triazophos, chlorpyrifos, Dipterex, fipronil, Avermectins, emamectin benzoate, collected in Nanbu, Yucheng, Pengshan, Shuangliu, Lezhi and Qionglai, respectively. The results showed as follows:
①Populations of Chilo suppressalis in Sichuan province produced medium to high level resistance to monosultap, triazophos, chlorpyrifos. Through the resistance monitoring results, we could see a clearly resistance growth. The resistance ratio in Shuangliu witnessed a significant growth, from 43.4981 in 2008 to 93.7325 in 2009. The pest resistances multiples growth to monosultap was flat in the six regions, only Qionglai increased 17.5181 times. The resistance ratio to chlorpyrifos increased fast only in Pengshan and Shuangliu,14.0251 and 23.0121 times, respectively.
②Populations of Chilo suppressalis in Sichuan province produced in sensitivity to decreasing or medium resistance level from 2008 to 2009 in the six regions. Compared to triazophos, the pest resistances multiples growth to Dipterex was gradual in the six regions. Yucheng and Lezhi increased 1.3591 and 1.7558 times respectively. Nanbu, Pengshan, Shuangliu and Qionglai decreased 2.3004,2.1328,1.1929 and 0.2349 times, respectively.
③The resistance of Chilo suppressalis field populations in six regions to fipronil, abamectins and emamectin benzoate was relatively low. Emamectin benzoate had higher activity:LD50=0.00022～0.000535ng/larva in 2009. Through resistance monitoring results between 2008 and 2009, it showed a slowly rising trend to the three reagents mentioned above. Fipronil, which has been banned to use for rice growth, remained showing high biological activity. The resistance of Chilo suppressali to Abamectins and Emamectin benzoate was much slower than other pesticides. The fastest growing resistance to abamectins was the Shuangliu population,2.479 times from 2008 to 2009. The slowest one was the resistance to Emamectin benzoate:the fastest growth field was Qionglai population, which only 0.5172 times.
2. Biochemical mechanism of resistance of Chilo suppressalis field populations
The research materials were from resistance population and indoor sensitive strain.①We tested the activity of CarE, ACP, ALP, AchE, GSTs, MFO, Proteinase, POD, SOD, CAT and the content of FAT in different strains. The results showed that each enzyme and crude fat content were significant different existing in the field resistance population and indoor sensitive, which might mean that it had some connect with resistant mechanism;②Comparing various enzymes activity with insect resistance level of monosultap, triazophos, fipronil, emamectin benzoate (LD50) correlation analysis, we learned that there were significant correlation between the resistance and the biology activity of AchE, GSTs, MFO, SOD, CAT. GSTs's activity and insect resistance to triazophos had significant correlation (r=0.86*); MFO's activity and insect resistance to fipronil had significant correlation (r=0.74*); AchE activity and resistanceof Chilo suppressalis to monosultap, triazophos, fipronil, emamectin benzoate were obvious negative correlation, related coefficient was -0.86**,-0.79*,-0.60,-0.55; SOD activity and resistance of Chilo suppressalis to emamectin benzoate had significant correlation (r=0.71). CAT activity and resistance of Chilo suppressalis to monosultap had significant correlation (r=0.78*).
3. Study on formulation-effervescent tablet
Based on the resistance monitoring and resistance mechanism, emamectin benzoate and monosultap were chosen for formulation and processing, which were in two different mechanism and no cross-resistance, The results were as follows:①Emamectin benzoat and monosultap mixed significant synergistic effect, and had a wide spectrum of the effective ratio. LD50 of emamectin benzoate was 0.5964ng/larva and the LD50 of monosultap was 21501ng/larva. The difference of the virulence between them was 36051.31 times. The research of co-toxicity coefficient showed that the ratio among 1:1000～1:36051 had significantl synergy effect.②The results of effervescent tablet test showed that the best recipe was:emamectin benzoate,0.04%; monosultap:39.96%; dispersant NNO,3%; Wetting agent:Tween-80,2%; Binder:polyethylene glycol 6000,3 %; Disintegrating agent:sodium bicarbonate+citric acid (1:1),40%; Packing:lactose+ sodium(1:1),12%.③Compared with single agent of emamectin benzoat and monosultap, its chemical toxic effect was more remarkable. For the preparation in the LD50 (ng/larva), the insecticidal effect of emamectin benzoate and monosultap effervescent tablet was (LD50≈335ng/larva) more significant than market 3.6% monosultap big granule (LD50=2714ng/larva). Due to the little proportion of emamectin benzoate in this tablet, when compared with the 0.2% emamectin benzoate soluble powder in LD50, the usage rate on emamectin benzoate between them was about(0.335:13.71)1:41, So it could greatly reduce the use of emamectin benzoate.
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