褐飞虱对吡虫啉的抗性生化机制研究及高效复配剂的筛选毕业论文
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工 作 研 究农业开发与装备 2019年第6期摘要:近几年来国内褐飞虱爆发频率增加,造成了水稻的减产损失。
大量使用杀虫剂防治后造成了褐飞虱的抗药性大幅度上升。
综述褐飞虱的抗药性及研究进展,包括褐飞虱的发生与危害、发生原因、抗药性现状、出现抗药性原因、对几种药剂抗性的情况、抗性机理、再猖獗的现状等。
近年有研究褐飞虱的抗性及其机理、几种药剂的抗性情况等,但是仍然没有系统性的总结和比较。
鉴于此,本综述进行了上述方面的总结和整理。
关键词:褐飞虱;抗药性;治理对策1 褐飞虱的发生与危害褐飞虱Nilaparvata lugens(Stål)属半翅目同翅亚目飞虱科(Homoptera:Delphacidae),是一种典型的r-战略害虫,具有较高的内在生长速率和较强的环境适应性。
它的特点有虫体小,繁殖能力强,易大面积爆发,易变异,远距离迁飞等。
褐飞虱是亚洲和东南亚水稻种植区最具破坏性的害虫之一,自20世纪60年代以来已成为我国水稻的首要害虫(肖汉祥等,2018)。
近30年来我国褐飞虱的危害主要分为三个特征:发生面积的扩大化、爆发频率的增加、危害程度的增加(王鹏,2013)。
近年来,在中国长江流域的频繁爆发对水稻生产造成了严重威胁。
褐飞虱除了可以通过直接刺吸水稻汁液、产卵破坏植株组织外,还可以传播水稻病毒造成危害。
轻微危害可导致叶片黄化,植物高度、生长、活力、有效分蘖数量和谷物填充的减少,严重的损害可导致植物干涸和死亡,导致“燃烧幼苗”(Sangram et al,2017)。
当褐飞虱严重受损时,可导致大量水稻减产甚至无收获。
在中国,除黑龙江、内蒙古、青海和新疆外,其他省区和自治区都出现了褐飞虱,并在长江流域及其南部地区爆发。
1987、1991、1997、2005、2006年都曾出现褐飞虱特大爆发,造成巨大的经济损失。
2 褐飞虱的抗药性研究2.1 褐飞虱的抗药性现状资料显示,我国以及东南亚部分国家在化学农药使用过程中,都存在长期大面积不合理使用单一农药产品的问题。
浙江大学学报(农业与生命科学版)48(6):692~700,2022Journal of Zhejiang University (Agric.&Life Sci.)http :///agrE -mail :zdxbnsb @稻飞虱生物学、生态学及其防控技术研究进展蒯鹏,娄永根*(浙江大学农业与生物技术学院昆虫科学研究所,水稻生物学国家重点实验室/农业农村部作物病虫分子生物学重点实验室,杭州310058)摘要稻飞虱是制约我国水稻生产的一类最主要害虫,主要包括褐飞虱、白背飞虱和灰飞虱。
本文重点就稻飞虱重要遗传特性(翅型分化、繁殖力、抗药性)分子基础、水稻-稻飞虱-天敌-其他生物种间互作关系、稻飞虱灾变机制及其防控技术等方面的最新研究成果进行综述,并提出今后应进一步深入剖析稻飞虱灾变的生物学与生态学分子基础,明确集约农业与稻田生态系统抗性在微观层面的协调机制,以在集约农业背景下维持或提高稻田生态系统抗性,实现稻飞虱的可持续治理。
关键词稻飞虱;生物学特性;种间互作关系;灾变机制;可持续治理中图分类号S 435.11文献标志码A引用格式蒯鹏,娄永根.稻飞虱生物学、生态学及其防控技术研究进展[J].浙江大学学报(农业与生命科学版),2022,48(6):692-700.DOI:10.3785/j.issn.1008-9209.2022.08.221KUAI Peng,LOU Yonggen.Research advances in biology,ecology and management of rice planthoppers[J].Journal of Zhejiang University (Agriculture &Life Sciences),2022,48(6):692-700.Research advances in biology,ecology and management of rice planthoppersKUAI Peng,LOU Yonggen *(State Key Laboratory of Rice Biology/Ministry of Agriculture and Rural Affairs Key Laboratory of Molecular Biology of Crop Pathogens and Insects,Institute of Insect Sciences,College of Agriculture and Biotechnology,Zhejiang University,Hangzhou 310058,China )Abstract Rice planthoppers,mainly including Nilaparvata lugens ,Sogatella furcifera ,and Laodelphaxstriatellus ,are one of the most important insect pests of rice in China.In this review,we summarized the latest research progress on the molecular basis of important genetic characteristics (wing-morph differentiation,fecundity,insecticide resistance)of rice planthoppers,interactions among rice,rice planthoppers,natural enemies of rice planthoppers and other organisms,mechanisms underlying rice planthopper outbreak,and management of rice planthoppers.Finally,we suggest that future studies should further dissect the molecular basis of biology and ecology related to rice planthopper outbreak,and find the coordination mechanisms between intensified agriculture and rice ecosystem resistance at the micro level,so as to maintain or improve the rice ecosystem resistance,and achieve sustainable management of rice planthoppers in the context of intensified agriculture.Key words rice planthoppers;biological characteristics;interspecific interactions;outbreak mechanisms;sustainable management稻飞虱是危害我国和东南亚国家水稻生产的一类主要迁飞性害虫,主要包括褐飞虱(Nilaparvatalugens )、白背飞虱(Sogatella furcifera )和灰飞虱(Laodelphax striatellus ),属于半翅目(Hemiptera )DOI :10.3785/j.issn.1008-9209.2022.08.221基金项目:国家重点研发计划项目(2021YFD1401100);农业农村部现代农业产业技术体系项目(CARS -01-43)。
几种复配剂对稻飞虱的防治效果比较刘怀阿;祈建杭;陈金宏;吕敏;葛恒来【摘要】比较了6种复配剂对稻飞虱的防效,结果表明:噻嗪异丙威、吡蚜噻嗪酮、丙威毒死蜱的防效优异,吡虫乙酰甲、吡虫仲丁威的防效较好,以上5种复配剂均显著优于吡虫杀虫单,因此噻嗪异丙威、吡蚜噻嗪酮、丙威毒死蜱可作为防治稻飞虱的常规品种,吡虫乙酰甲、吡虫仲丁威作为替换品种,在防治过程中注意经常轮换药剂品种.%The comparison of the control efficacy of six pesticide mixtures against rice planthoppers was conducted in this paper. The results showed that the control efficacies of thiazide isoprocarb, pymetrozine buprofezin and isoprocarb chlorpyrifos were the best, and those of imidacloprid methacholine and imidacloprid fenobucarb were better. The control efficacies of the above five pesticide mixtures were significantly superior to that of imidacloprid monosultap. Thiazide isoprocarb, pymetrozine buprofezin and isoprocarb chlorpyrifos were recommended as the conventional varieties controlling rice planthoppers, while imidacloprid methacholine and imidacloprid fenobucarb could be used as replacement varieties. The pesticides should be frequently rotated in the pest control.【期刊名称】《江西农业学报》【年(卷),期】2013(025)001【总页数】3页(P70-72)【关键词】复配剂;稻飞虱;抗药性;防效【作者】刘怀阿;祈建杭;陈金宏;吕敏;葛恒来【作者单位】江苏里下河地区农业科学研究所,江苏扬州225007;江苏里下河地区农业科学研究所,江苏扬州225007;江苏省宝应县植保站,江苏宝应225800;江苏里下河地区农业科学研究所,江苏扬州225007;江苏里下河地区农业科学研究所,江苏扬州225007【正文语种】中文【中图分类】S435.112稻飞虱是我国水稻上重要的迁飞性害虫,主要分布于日本、朝鲜、东南亚、太平洋岛屿及澳大利亚,国内各稻区均有发生。
吡蚜酮和啶虫脒混配对褐飞虱的田间防效摘要:采用25%吡蚜酮WP和20%啶虫脒SP两种杀虫剂及其复配药剂对褐飞虱(Nilaparvata lugens)进行田间试验。
结果表明,25% 吡蚜酮WP 112.5 (a.i.)g/hm2、25% 吡蚜酮WP+20%啶虫脒SP (m∶m,4∶1)60(a.i.)g/hm2药后7 d的防治效果较好,防效分别为93.08%、93.41%;20%啶虫脒SP 22.5 (a.i.)g/ hm2药后7 d的防效为81.65%。
关键词:褐飞虱(Nilaparvata lugens);吡蚜酮;啶虫脒;防治效果褐飞虱(Nilaparvata lugens)是水稻主要害虫,虽然每年6月中下旬开始迁入湖北省稻区[1],但每年9月中下旬褐飞虱才会暴发成灾(大暴发年份除外),给农民带来巨大的损失,长期以来农民主要通过化学手段防治褐飞虱[2]。
由于农民对产量的渴望和对害虫的零容忍度,导致化学农药持续、大量或过量、不合理地使用,致使褐飞虱对常用杀虫剂产生了不同程度的抗药性[2-9]。
目前防治褐飞虱的化学药剂主要包括有机磷类、氨基甲酸酯类、新烟碱类、吡啶类、昆虫生长调节剂类等,但是褐飞虱对这几类常用杀虫剂产生了不同程度的抗药性。
为明确吡蚜酮和啶虫脒两种杀虫剂及其复配制剂对褐飞虱的防效,开展了田间试验,以期为褐飞虱的科学防治提供依据。
1 材料与方法1.1 供试药剂25%吡蚜酮WP由安徽麦兰生物化学有限公司生产;20%啶虫脒SP由山东恒利达生物科技有限公司生产。
1.2 试验设计1.2.1 试验处理试验共设置4个处理:25% 吡蚜酮WP 112.5 (a.i.)g/hm2;20%啶虫脒SP 22.5 (a.i.)g/hm2;25% 吡蚜酮WP+20%啶虫脒SP(m∶m,4∶1,下同)60 (a.i.)g/hm2;清水对照。
每个处理重复4次,共16个小区,每个小区面积50 m2,随机排列。
1.2.2 试验地点及施药方法试验地点在湖北省武穴市梅川镇李四村进行,试验田为中稻田,水稻品种为广两优籼66,水稻采用育苗移栽方式,所有试验小区的栽培条件(土壤类型、低洼地、肥料、播栽期、密度、生育期、水层管理)均匀一致。
浙江省水稻三种飞虱对杀虫剂的敏感性测定何月平;张珏锋;肖鹏飞;陈列忠;陈建明【摘要】为了调查浙江省水稻飞虱抗药性现状,采用稻茎浸渍法测定了2010-2011年采自浙江省嘉兴市褐飞虱田间种群、杭州市白背飞虱种群以及长兴市和嘉兴市灰飞虱种群对几种常用杀虫剂的敏感性.测定结果表明,嘉兴种群褐飞虱对吡虫啉分别产生了615.9~814.2倍抗性,对噻虫嗪产生了66.2倍抗性,对噻嗪酮具有13.0倍抗性,对烯啶虫胺尚敏感;相对于2008年种群,2010年杭州白背飞虱种群对毒死蜱的敏感性降低了9.5倍,说明白背飞虱对毒死蜱的抗性上升很快;相对于2007年种群,2010-2011年长兴灰飞虱对噻虫嗪、毒死蜱和吡虫啉的敏感性分别降低了1.6,2.5和2.3倍;噻虫嗪和毒死蜱对长兴种群灰飞虱的毒力略高于嘉兴种群.根据敏感性测定结果,提出了针对防治水稻飞虱的田间用药和抗药性治理的参考意见.%To investigate the current status of the resistance to insecticides of rice planthoppers in Zhejiang province, rice stem-dipping method was used for detecting the susceptibilities to insecticides of brown planthopper, Nilaparvata lugens(St(a)l) , collected from Jiaxing city, and white-backed planthopper, Sogatella furcifera (Horváth) , collected from Hangzhou city, and small brown planthopper, Laodelphax striatellus(Fallén) , collected from Jiaxing city and Changxing city. The results showed that the Jiaxing population of N. lugens had developed a high level of resistance to imidacloprid with 615. 9 -814. 2 fold, and to thiamethoxam with 66. 2 fold, and to buprofezin with 13. 0 fold, and still susceptible to nitenpyram. The susceptibility of the Hangzhou population of S. furcifera to chlorpyrifos was dropped 9. 5 times from 2008 to 2010, which suggested a rapidevolution of resistance of S. furcifera to chlorpyrifos. The susceptibilities of the Changxing population of L. striatellus to thiamethoxam, chlorpyrifos and imidacloprid were reduced 1. 6, 2. 5 and 2. 3 fold, respectively, from 2007 to 2010 or 2011, and toxicities of thiamethoxam and chlorpyrifos against the Changxing population of L. striatellus were slightly higher than those against the Jiaxing population. Based on the bioassay data, strategies of the application of insecticide in field and insecticide resistance management for the control of rice planthoppers were also proposed.【期刊名称】《浙江农业学报》【年(卷),期】2012(024)004【总页数】5页(P642-646)【关键词】褐飞虱;白背飞虱;灰飞虱;敏感性;抗药性【作者】何月平;张珏锋;肖鹏飞;陈列忠;陈建明【作者单位】浙江省植物有害生物防控重点实验室省部共建国家重点实验室培育基地,浙江省农业科学院植物保护与微生物研究所,浙江杭州310021;浙江省植物有害生物防控重点实验室省部共建国家重点实验室培育基地,浙江省农业科学院植物保护与微生物研究所,浙江杭州310021;浙江省植物有害生物防控重点实验室省部共建国家重点实验室培育基地,浙江省农业科学院植物保护与微生物研究所,浙江杭州310021;浙江省植物有害生物防控重点实验室省部共建国家重点实验室培育基地,浙江省农业科学院植物保护与微生物研究所,浙江杭州310021;浙江省植物有害生物防控重点实验室省部共建国家重点实验室培育基地,浙江省农业科学院植物保护与微生物研究所,浙江杭州310021【正文语种】中文【中图分类】Q965.9水稻飞虱(褐飞虱、白背飞虱和灰飞虱)是威胁我国粮食安全最大的生物灾害。
文献综述吡虫啉农药废水处理方法一、前言我国是农药生产和使用大国,农药行业在我国国民经济中占有重要地位。
近年来,传统农药由于残留毒性大、效能低,正在被对环境更加友好的新一代农药逐步替代。
吡虫啉以其高效、低毒、低残留的特点,成为新一代农药的代表,具有良好的市场前景。
但是,对其生产废水的处理却并无成熟的工艺可循,该废水的不达标排放,对环境造成了严重的污染。
因此,对吡虫啉生产废水治理工艺的研究不仅可以为企业排忧解难,还可以为高浓度难降解有机废水的治理寻求一种有效的处理手段。
吡虫啉,又名咪蚜胺(1-(6-氯-3-吡啶基甲基)-N-硝基亚咪唑烷-2-基胺),英文名Imidacloprid,是一种高效、低毒、低残留的仿生物杀虫剂,他可应用于松茸,大米,鸡肉,猪肉,牛肉,大蒜,洋葱,苹果,板栗,桃,大葱,甘蓝,胡萝卜,番茄,草莓,芦笋,其他,大豆,蘑菇,玉米,花生,茶叶等农产品。
但是其生产废水中含有大量丙烯腈、甲苯、DMF及少量的2-氯-5-氯甲基吡啶等,具有毒性大、成分复杂、难降解有机物浓度高、治理难度大等特点,属于典型的高浓度难降解毒性有机废水,直接排放会严重污染环境。
国内农药废水的治理始于上世纪六七十年代80 年代后逐步展开。
目前农药废水的处理技术概括可分为物化法、化学法和生化法等。
物化法常作为预处理手段,用来回收废水中的有用成分,或对难生物降解物进行处理,达到去除有机物、提高可生化性、降低生化处理负荷、提高处理效率的目的。
化学法常作为生化处理的预处理方法使用,主要有药剂氧化法、光催化氧化法、湿式氧化法、微电解法和超临界水氧化技术。
1.药剂氧化法包括氯氧化法、Fenton 试剂法、臭氧氧化法等。
2.光催化氧化技术是利用锐钛型二氧化钛在紫外光的照射下产生氧化性极强的 OH将有机物质转化为CO2 、H2O 以及无机物,降解速度快,无二次污染。
3.湿式氧化法是在一定温度和压力下向废水中通入氧气或空气,将水中有机物分解为小分子无机物及残存有机物的方法。
褐飞虱对吡虫啉的抗性选育及毒力测定摘要:为研究褐飞虱对吡虫啉的抗性规律,进行了褐飞虱对吡虫啉的逐代抗性选育及室内毒力测定研究。
从敏感品系开始选育,共饲养了23代,其中前18代为连续施药汰选,后5代为不施药的继代培养。
室内生物活性测定结果表明,随抗性选育代数的增加,lc50增大,抗性水平不断提高,其中以18代最大,lc50为101.49 mg/l,抗性为敏感品系的181.2倍;无药剂筛选的18-22代褐飞虱对吡虫啉的抗药性缓慢下降,其抗性由181.2倍下降到151.5倍。
研究结果表明,合理控制吡虫啉的施用浓度及施用频率可以适当延长药剂的使用寿命。
关键词:褐飞虱;吡虫啉;抗性选育;毒力测定中图分类号:s435.112.3 文献标识码:a 文章编号:0439-8114(2013)04-0817-03resistance selection and toxicity test of brown planthopper to imidaclopriddong de-zhen,yuan jing,xing jia-hua,yu ji-ping,chen jie (zhejiang research institute of chemical industry company limited,hangzhou 310023,china)abstract: in order to investigate the rules of the resistance of brown planthopper to imidacloprid, the resistance selection and toxicity test were studied in laboratory generation by generation. with sensitive strains,23 generations were selected continuously, in which the former 18 generations were selected with imidacloprid,whereas the latter 5 generations were without pesticides. the result showed that the values of lc50 and the resistance levels increased gradually generation by generation, of which the f18 generation was the highest, lc50 was 101.49 mg/l, and the resistance was 180.3 times as the sensitive strains. the resistance slowly fell to 151.5 times in the 18-22 th generations without pesticides. our results showed that it was necessary to control the concentration and frequency of imidacloprid, which would be useful for lengthening their service life.key words: brown planthopper; imidacloprid; resistance selection; toxicity test褐飞虱[nilaparvata lugens(stal)]属同翅目飞虱科,是亚洲水稻生产的重要害虫,随季风迁移,对环境具有较强的适应性,在外界条件适宜时容易暴发成灾[1-4]。
醚菊酯、吡虫啉、噻虫嗪及其混配对褐飞虱的毒力测定郁艳;李明;李荣玉;樊龙飞;卢春【期刊名称】《广东农业科学》【年(卷),期】2015(042)001【摘要】采用点滴法测定了吡虫啉、噻虫嗪、醚菊酯及其混配对贵州3个县(黄平、开阳、桐梓)褐飞虱三龄若虫的毒力,并通过交互测定法筛选出对褐飞虱具有明显增效作用的优选配方.结果表明:噻虫嗪、吡虫啉和醚菊酯对3县稻飞虱的LD50范围分别为0.00125~0.00175、0.00135~0.00169、0.01739~0.01850 μg/头;与敏感品系相比,褐飞虱对吡虫啉表现出中等水平抗性(19.9~24.9倍),而对醚菊酯仍表现出敏感性.吡虫啉与醚菊酯和噻虫嗪与醚菊酯有效成分分别以1∶5和1∶7混配时对褐飞虱具有较高的增效作用,共毒系数(CTC)分别为166.93~198.23和174.29~188.74,其结果可为防治褐飞虱药剂汰选、混配用药及制剂研发提供科学依据.【总页数】5页(P73-77)【作者】郁艳;李明;李荣玉;樊龙飞;卢春【作者单位】贵州大学生命科学学院,贵州贵阳550025;贵州大学作物保护研究所,贵州贵阳550025;贵州大学精细化工研究开发中心,贵州贵阳550025;贵州大学生命科学学院,贵州贵阳550025;贵州大学精细化工研究开发中心,贵州贵阳550025【正文语种】中文【中图分类】S482.3【相关文献】1.10%噻虫嗪·杀虫单缓释粒剂防控水稻害虫褐飞虱·二化螟·稻纵卷叶螟效果 [J], 郭志刚;郭振营2.醚菊酯、噻虫嗪及其混配对贵州三地褐飞虱的毒力及田间防治效果 [J], 徐雪凌;李明;李荣玉;韩晶波;尹显慧3.烯啶虫胺、醚菊酯及其混剂对褐飞虱的毒力测定 [J], 凌炎;钟勇;韦淑丹;尹文兵;龙丽萍4.褐飞虱对噻嗪酮和噻虫嗪呈中等至高水平抗药性 [J], 张帅;张绍明;5.噻虫嗪对褐飞虱的毒力及解毒代谢酶活性的影响 [J], 张钰明; 向兴; 王学贵因版权原因,仅展示原文概要,查看原文内容请购买。
目录目录摘要 (I)ABSTRACT (III)第一章文献综述 (1)1 吡虫啉抗性研究进展 (1)1.1 吡虫啉简介 (1)1.2 吡虫啉抗性产生现状 (2)1.3 吡虫啉抗性机理研究概况 (3)2 复配杀虫剂 (5)2.1 复配杀虫剂简介 (6)2.2 复配杀虫剂的评价方法(研究方法) (6)2.3 国内吡虫啉复配杀虫剂的研究情况 (8)3 本文复配所用杀虫剂简介 (10)3.1 吡蚜酮简介 (10)3.2 呋虫胺简介 (12)4 研究目的与意义 (13)4.1 褐飞虱对吡虫啉的抗性生化机制 (13)4.2 褐飞虱对吡蚜酮的生测体系的建立 (14)4.3 吡虫啉增效复配剂的筛选 (14)4.4 吡虫啉替代药剂呋虫胺的增效复配 (15)第二章褐飞虱Nilaparvata lugens (Stål)对吡虫啉的抗性生化机制研究 (17)1 材料和方法 (17)1.1 供试虫源及主要试剂 (17)1.2 毒力的生物测定 (18)1.3 mRNA水平的检测 (18)1.4 CYP6AY1基因在大肠杆菌(E. coli)上的表达和膜蛋白的提取 (20)1.5 酶活的测定 (20)1.6 RNA干扰 (21)2 结果 (21)2.1 生物测定和增效作用的评价 (21)2.2 细胞色素P450单加氧酶系mRNA水平的检测 (22)2.3 CYP6AY1基因的功能性表达以及吡虫啉的代谢 (23)2.4 CYP6AY1的RNA干扰 (24)2.5 田间种群褐飞虱的抗性水平以及CYP6AY1基因的mRNA表达水平的测定 (25)3 讨论 (26)第三章飞虱对吡蚜酮的生测体系的建立 (29)1 材料和装置 (29)1.1 试验用虫 (29)1.2 试验药剂 (29)1.3 试验装置 (30)2. 生物测定方法及流程 (31)2.1 生测若虫的准备 (31)2.2 吡蚜酮母液的配制 (31)2.3 吡蚜酮浸苗处理药剂的配制 (31)2.4 浸药处理 (32)2.5 生测小苗的接虫前处理 (32)2.6 接虫 (33)2.7 结果检查与数据处理 (33)3. 试验条件参数分析 (35)3.1 有机溶剂的选择及对生物测定的影响 (35)3.2 药剂处理时间的选择 (36)3.3 表面活性剂Triton X-100的选择及对生物测定的影响 (38)3.4 浸药时间对生物测定的影响 (39)3.5 对照死亡率及试验准确度分析 (39)4 讨论 (40)第四章吡虫啉-吡蚜酮高效复配剂的筛选及田间药效试验 (43)1 材料与方法 (43)1.1 试验材料 (43)1.2 药剂的配制方法 (44)1.3 药剂毒力的生物测定 (44)1.4 增效复配配比的筛选方法 (45)1.5 大田药效试验方法 (45)1.6 数据分析 (46)2 结果与分析 (46)2.1 吡虫啉-吡蚜酮增效复配比例的筛选 (47)目录2.2 吡虫啉-吡蚜酮增效复配大田药效试验 (50)3 讨论 (54)第五章呋虫胺-吡蚜酮复配剂的筛选 (57)1 材料与方法 (57)1.1 试验材料 (57)1.2 药剂的配制方法 (58)1.3 药剂毒力的生物测定 (58)1.4 增效复配配比的筛选方法 (58)1.5 数据分析 (58)2 结果与分析 (58)2.1 毒力回归曲线的测定 (58)2.2 Mansour法初步评价联合毒力 (59)2.3 孙云沛法评价呋虫胺-吡蚜酮不同配比的联合毒力 (60)3 讨论 (63)全文总结 (65)参考文献 (67)附录:发表的学术论文 (75)致谢 (77)摘要褐飞虱对吡虫啉的抗性生化机制研究及高效复配剂的筛选摘要吡虫啉(Imidacloprid)是近20年来发展最快、使用最多的新烟碱类杀虫剂,其作用机制为有选择性地作用于昆虫神经系统中的乙酰胆碱受体,破坏昆虫中枢神经的正常传导,扰乱昆虫的正常神经活动使其处于极度兴奋状态,逐渐麻痹直至死亡。
自从吡虫啉作为新烟碱类第一种杀虫剂在1991年被投入市场,由于其很好的效果和持效性,很快就成为了防治褐飞虱Nilaparvata lugens (Stål)最主要的杀虫剂之一。
但是,亚洲多个国家的田间褐飞虱种群调查显示,自2006年以来,褐飞虱对吡虫啉的田间抗性,不论在抗性强度还是在地理分布上都产生了相当大的增加。
2009年以来,我国农业部门建议暂停使用吡虫啉单剂防治褐飞虱。
但是鉴于吡虫啉较低的防治成本和对与褐飞虱混合发生的白背飞虱和灰飞虱具有较好的防效,吡虫啉还不可避免地与褐飞虱的防治联系在一起,部分地区仍然是重要的药剂品种。
国内外很多学者在褐飞虱对吡虫啉抗性机制方面进行了深入研究。
靶标不敏感性方面,本课题组在室内筛选的抗吡虫啉褐飞虱品系中,在吡虫啉作用靶标烟碱型乙酰胆碱受体(Nicotinic Acetylcholine Receptors)两个α亚基中发现了Y151S 点突变,与褐飞虱对吡虫啉的抗性密切相关。
代谢抗性方面,无论是室内抗性品系还是田间种群,都发现细胞色素P450酶系活性的上升是褐飞虱对吡虫啉抗性的主要生化机制,虽然具体的P450基因还未能鉴定出来。
本文首次从mRNA 表达水平研究了不同P450基因在褐飞虱对吡虫啉抗性产生中的作用,发现了吡虫啉代谢的关键基因。
另外为了充分发挥吡虫啉的防治效果、减少田间用药量,将吡虫啉与吡蚜酮复配,筛选出了两个吡虫啉的高效复配配比,并通过田间药效试验验证了增效效果。
呋虫胺是吡虫啉未来的替代药剂,同样属于新烟碱类杀虫剂,在褐飞虱防治中,是吡虫啉的最佳替换品种,为此本文还筛选了呋虫胺与吡蚜酮的高效复配,增效效果显著。
一、P450酶系在褐飞虱对吡虫啉抗性中的作用本文使用qRT-PCR技术比较了14条P450基因/片段在吡虫啉敏感(Sus)和抗性品系(Res)褐飞虱中的mRNA水平,发现了抗性品系中有6个P450基因在表达量上显著高于敏感品系。
其中,CYP6AY1基因的表达量差异最显著,抗性品系是敏感品系的17.9倍。
通过大肠杆菌原核表达CYP6AY1蛋白,发现CYP6AY1可以高效代谢吡虫啉(峰值活性为37.6 µg/mg蛋白/min)。
采用RNAi 对抗性品系褐飞虱的CYP6AY1基因进行干扰,降低其mRNA水平时,该品系对吡虫啉的抗性下降。
进一步的研究表明,褐飞虱不同田间种群中CYP6AY1的表达水平均有不同程度的上升,而且抗性倍数越高,CYP6AY1表达水平上升的倍数越大。
CYP6AY1基因是第一个从褐飞虱中发现对吡虫啉抗性起重要作用的P450家族成员。
二、吡虫啉-吡蚜酮增效复配剂的筛选由于吡蚜酮独特的杀虫机理,击倒活性弱,持效期长,对其生物测定需要较长的处理时间,传统的稻苗浸渍法不能满足吡蚜酮生物测定的需求。
本文为了满足后续复配筛选的需要,对已有的稻苗浸渍法进行改进以以满足吡蚜酮生物测定的需求,设计了一套吡蚜酮生物测定的流程,能够非常稳定、准确地测定吡蚜酮的毒力。
在建立了标准生物测定方法的基础上,筛选了吡虫啉和吡蚜酮复配的最佳增效配比,得到了两个增效作用显著的复配比例,吡虫啉与吡蚜酮含量之比分别为3:2和1:3,共毒系数分别达到了410.38和616.17。
通过田间药效试验验证了这两个增效复配在田间使用中的增效效果,两个复配比例在田间对稻飞虱成虫和若虫的防治效果均好于等剂量的两种单剂,并且实现了速效性和持效性的统一。
三、呋虫胺增效复配剂的筛选呋虫胺是一种新的新烟碱类杀虫剂,目前已经在世界多个国家推向市场并获得了很好的效果,对稻飞虱等半翅目害虫表现出良好的防效。
虽然目前还没有引入国内市场,但不久的将来肯定会在国内登记使用,将在褐飞虱的防治中起十分重要的作用,成为吡虫啉的高效代替品种。
本文通过生物测定,将呋虫胺与吡蚜酮复配筛选,得到了2个增效复配比例,呋虫胺:吡蚜酮分别是1:7和4:175,共毒系数分别达到了3712.62 和3206.61,体现出极高水平的增效作用。
本增效复配剂的发明不仅可以充分发挥呋虫胺的药效,减少药量、节约成本,还能够延缓害虫对呋虫胺抗性的产生和发展,符合农药科学使用的要求。
关键词:褐飞虱;吡虫啉;抗性;P450单加氧酶;杀虫剂增效复配ABSTRACTBIOCHEMICAL MECHANISMS OF IMIDACLOPRID RESISTANCE IN NILAP ARVATA LUGENS AND SELECTION OF HIGH EFFICIENT INSECTICIDE MIXTURES WITHIMIDACLOPRIDABSTRACTDuring recent years, imidacloprid is the fastest growing pesticide of neonicotinoid insecticides. Imidacloprid selectively acts on insect nicotinic acetylcholine receptors and destructs the normal conduction of the insect central nervous system. Then the insect will be at a state of ecstasy because of disturbed nervous activities and gradual paralysis until death. As soon as imidacloprid was introduced to the market as the first neonicotinoid insecticide in 1991, it becomes one of the most common insecticides against Nilaparvata lugens, principally owing to its efficacy and long-lasting effect. However, recent surveys of N. lugens field populations from several Asian countries revealed that the intensity and geographical distribution of imidacloprid resistance in the field has increased substantially since 2006. Chinese agricultural sectors suggested the suspending use of imidacloprid singly against N. lugens. However, because of the lower control costs and high control efficiency against other rice planthoppers, imidacloprid will be still an important insecticide against N. lugens.By now, many scholars have studied both biochemical and target insensitivity mechanisms of imidacloprid resistance in N. lugens. The previous studies in our laboratory on lab-selected imidacloprid resistance in N. lugens have identified a single point mutation (Y151S) within the extracellular agonist-binding domain of two nAChR α subunits. As to biochemical mechanisms, both in laboratory resistant strain and field populations, the increased cytochrome P450 monooxygenase activity is reported as the important or major mechanism for the imidacloprid resistance in N. lugens. However, the P450 gene(s) responsible for imidacloprid resistance has not been identified in N. lugens until now. In order to find out the biochemical mechanisms of imidacloprid resistance in N. lugens, we detected the expression of different P450 genes at mRNA levels and compared between Res and Sus strains. In addition, in order to utilize imidacloprid with the highest effection and reduce the field dosage of imidacloprid, we screened out two optimum mixture ratios withimidacloprid and pymetrozine, and field trials confirmed the synergistic effects of these two mixtures. Belong to neonicotinoid insecticides, dinotefuran is one of alternative compounds of imidacloprid in the future. We also have screened out two high efficient insecticide mixtures of dinotefuran and pymetrozine, and the field trials will be soon performed to confirm the control effects in fields..1.Biochemical mechanisms of imidacloprid resistance in Nilaparvata lugensWe analyzed mRNA levels of 14 P450 monooxygenases by qRT-PCR with gene specific primers and compared between Sus and Res strains. Significant higher mRNA levels of six P450 genes were found in Res strain when compared to Sus strain. For the biggest difference (17.94-fold) in mRNA levels between Res and Sus strain, CYP6AY1was selected to perform functional study in vitro and in vivo. By expressing in E. coli cells, CYP6AY1 was found to metabolize imidacloprid with high activity, with the peak activity of 37.6 µg/mg protein/min. When CYP6AY1 mRNA levels in Res strain was reduced by RNA interference, imidacloprid susceptivity was recovered. In four field populations with different resistance levels, high levels of CYP6AY1transcript were also found. It is the first time that the biochemical mechanisms of imidacloprid resistance in N. lugens were detected at genetic level.2. Screening of high efficient insecticide mixtures of imidacloprid and pymetrozinePymetrozine is an important insecticide used to control N. lugens recently, when imidacloprid resistance becomes more serious. Pymetrozine is with the unique action mode, with good sustained effects, but without knockdown activity, which shows that the bioassay on pymetrozine needs long treating time. In order to find out a suitable bioassay method for pymetrozine against N. lugens, the traditional rice seedlings impregnation method was improved to meet the requirements of pymetrozine. The improved method can determine the toxicity of pymetrozine stably and accurately.Joint–toxicity of imidacloprid and pymetrozine against N. lugens has been tested by the above methods. The result indicated that the mixture of imidacloprid and pymetrozine showed the significant synergistic effects. Two ratios have co-toxicity coefficient of 410.38 and 616.17 with the mixture ratios of3:2 and 1:3 for imidacloprid and pymetrozine. Subsequently, field trials confirmed the synergistic effects of these two ratios. The control efficiency of these two ratios against adultsABSTRACTand nymphs in field were better than the single insecticide at the same dose. The ratios were also good at the quick effects and sustained effects.3. Screening of high efficient insecticide mixtures of dinotefuran and pymetrozineDinotefuran has been marketed in many countries against the hemipteran insect pests as a new neonicotinoid insecticide, including N. lugens. Although dinotefuran has not been registered in China, it is believed that dinotefuran will be released soon in Chinese market as the alternative of imidacloprid and play important roles in N. lugens control. In order to use dinotefuran rationally and avoid the quick resistance development in N. lugens and other rice planthoppers, we try to screen the possible insecticide mixtures with dinotefuran. Because of the quantity of the tested insects, we tested the joint–toxicity of dinotefuran and pymetrozine on Sogatella fureifera, a rice planthoppers normally being together with N. lugens in rice filed. The results showed that the mixtures of dinotefuran and pymetrozine showed significant synergistic effect s. Two best ratios have co-toxicity coefficient of 3712.62 and 3206.61, with the ratios of 1:7 and 4:175 for dinotefuran and pymetrozine. These efficient mixtures of dinotefuran and pymetrozine could reduce the usage of dinotefuran in field, lower the production const and delay the resistance development in rice planthoppers.KEY WORDS: Nilaparvata lugens; Imidacloprid; Insecticide resistance; P450 monooxygenase; High efficient insecticide mixture第一章文献综述第一章文献综述褐飞虱Nilaparvata lugens(Stål)(Brown planthopper,BPH)属半翅目飞虱科,常与白背飞虱、灰飞虱、稻叶蝉等混合发生,是危害水稻最严重的害虫之一(巫国瑞等,1987)。