江苏省水稻二化螟的抗药性测定及对甲胺磷的抗性机制

采用3龄幼虫点滴法测定了江苏省水稻二化螟对杀虫单和甲胺磷的抗性水平，并就二化螟对甲胺磷产生抗性的机制进行了研究，结果表明，高淳和徐州二化螟种群对杀虫单已达高抗水平，其它地区也表现为中等抗生，在所测定的6个种群中，苏州种群对甲胺磷处于低抗水平，其它种群为中抗种群，澌知体增效作用表明，氧化胡椒基丁醚（PBO）对甲胺磷有明显的增效作用，磷酸三苯酯（TPP）和顺丁烯酸二乙酸（DEM）的增效作用不明显，对离体解毒酶的研究表明，R种群幼虫多功能氧化酶（MFO）的O-脱甲基作用的活力比S幼虫高，R幼虫乙酰胆碱酯酶（AChE)的活力与S幼虫无显著差异。以甲胺磷为抑制剂的R幼虫I50是S幼虫的2.52倍，二化螟对甲胺磷的抗性机理可能与MFO的O-脱甲基作用活力增矿山和AChE的敏感性降低有关。     

水稻二化螟抗性监测的技术操作

在实践总结的基础上,提出和分析比较了水稻二化螟抗药性监测工作中的试虫准备、试剂配制,数据分析的方法,可为开展此项工作提供借鉴。     

水稻二化螟抗药性监测及防控对策*

采用点滴法测定了2007-2009年采自浙江、江苏、湖北、四川等地水稻二化螟4龄幼虫对沙蚕毒素类的杀虫单、有机磷类的三唑磷、毒死蜱、苯基吡唑类的氟虫腈及微生物源的阿维菌素等杀虫剂的抗性。结果表明：上述二化螟种群对５种杀虫剂抗性分布具有明显的地区性,其中浙江、江苏大部分地区供试种群对杀虫单、三唑磷的抗性水平达40倍以上,已普遍产生高至极高水平抗性;对毒死蜱产生的抗性水平达22倍以上,已普遍产生中等水平至高水平抗性;对氟虫腈产生的抗性水平达6倍以上,已普遍产生低水平至中等水平抗性;对阿维菌素敏感至低水平抗性;湖北孝感、四川武胜种群对５种药剂尚未产生抗性或为低水平抗性。本文还对二化螟抗性防控对策进行了讨论。     

杀虫单和甲维盐对水稻二化螟杀虫毒力室内配方筛选

通过微量点滴法,以水稻二化螟4龄幼虫为供试昆虫,测定杀虫单和甲维盐不同比例混配对水稻二化螟杀虫毒力,采用共毒因子和共度系数法进一步确定2种试验药剂的合适配比。结果表明：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LD50为0.596 4ng/larva,杀虫单LD50为21 501ng/larva,2者的毒力差异达36 051.31倍。共毒系数研究表明甲维盐和杀虫单的配比在1∶1 000～1∶36 051（质量比）均有显著的增效作用。     

水稻品种对二化螟抗性的研究

本研究对本省目前推广的47个主要稻种进行了二化螟 Chilo suppressalis(Walker)抗性测定,发现中抗品种2个,耐虫品种22个。粳稻较籼稻更抗二化螟,因而,籼、粳稻的防治指标应有所区别。     

褐飞虱对甲胺磷的抗性遗传

室内抗性筛选表明，褐飞虱对甲胺磷抗性的发展呈不规则的“S”形，第5代前抗性上升很慢，第5～15代间迅速上升，第15代后上升再次变慢。利用选育的抗甲胺磷褐飞虱品系和敏感品系，研究了褐飞虱对甲胺磷的抗性遗传形式。结果显示，正交和反交子代的显性度D值分别为0．5130和0．4673，表明抗性主效基因是不完全显性遗传的。利用剂量对数-死亡机率值曲线分析法，初步判断抗性遗传不是单基因控制的；卡方(χ^2)测验进一步证实，抗性遗传可能是两个或两个以上的基因控制。抗性筛选全过程和F5～F13的现实遗传力(h^2)分别为0．2190和0．2457，说明褐飞虱对甲胺磷抗性产生的速率较快，出现高抗性的风险较大。     

几种药剂防治二化螟田间药效试验

近年来,二化螟已成为为害钟山县水稻主要的钻蛀性螟虫.该县用于防治二化螟的主要农药有三唑磷、毒死蜱和杀虫单等,已使用多年,防效已明显下降.据何月平等人报道:在桂林地区,二化螟对三唑磷已经达到了高水平抗性,对杀虫单和毒死蜱已经达到了中等水平抗性[1].     

草甘膦抗性杂草的田间监测

北京的大兴区北藏村镇巴园子村的苦菜、节节草对草甘膦不敏感,防效仅为40%~60%;顺义区木林镇魏家店村的马唐对草甘膦不敏感,防效仅70%;通州区潞城镇小营村的藜对草甘膦不敏感,防效仅50%;延庆县井庄镇二司村的黄花蒿、刺儿菜、狗尾草、苦荬菜对草甘膦不敏感,防效仅40%~60%;延庆县农场的黄花蒿、刺儿菜、紫花地丁、苦荬菜对草甘膦不敏感,防效仅30%~50%;山东省淄博的铁苋菜对草甘膦不敏感,防效为70%;滕州的小飞蓬、苘麻、葎草对草甘膦不敏感,防效为40%~60%;广西的杂草对草甘膦都比较敏感,防效都在88%以上;其他杂草对草甘膦较敏感。     

2017—2019年江西省二化螟种群对四种杀虫剂的抗性

为明确近年来江西省二化螟种群对常用杀虫剂的抗性水平,提出合理化用药建议,本研究采用人工饲料浸药法测定2017—2019年江西省13个县(市)二化螟田间种群对氯虫苯甲酰胺、阿维菌素、三唑磷和杀虫单的抗性。结果表明,13个二化螟种群对氯虫苯甲酰胺的抗性水平整体为中等至高水平抗性,其中有11个二化螟种群对氯虫苯甲酰胺的抗性水平整体为上升趋势,在对氯虫苯甲酰胺抗性水平增加的种群中,都昌种群的抗性倍数从2017年的44.1倍上升至2018年的65.1倍,在2019年激增至高水平抗性,抗性倍数为283.5倍,为供试种群的最高抗性倍数。13个二化螟种群对阿维菌素的抗性水平整体为中等偏下,在对阿维菌素抗性水平增加的种群中,南昌种群的抗性倍数最高,2017—2019年分别为74.8倍、108.7倍和80.6倍。13个二化螟种群对三唑磷的抗性水平整体为中等偏下,其中新干、永修、万安、大余、上高和都昌这6个种群对三唑磷的抗性水平呈波动上升的趋势,而其他7个种群对三唑磷的抗性水平为波动性变化,偶有下降。13个二化螟种群对杀虫单的抗性水平整体为中等偏下并且有明显下降趋势,其中大余、永修、泰和、会昌、新干和上高这...     

辽宁省稻瘟病菌对稻瘟灵的抗性监测及其对稻瘟病的田间防治效果

为明确辽宁省水稻稻瘟病菌Magnaporthe oryzae对稻瘟灵的抗性水平及变化动态,本研究于2017—2019年自辽宁省6个市的6个稻区采集并分离获得187株稻瘟病菌,利用菌丝生长速率法测定这187株稻瘟病菌菌株对稻瘟灵的敏感性及抗性,并采用茎叶喷雾法测定稻瘟灵对水稻稻瘟病的田间防治效果以及该药剂与其它药剂的交互抗性。结果表明：供试187株稻瘟病菌菌株对稻瘟灵的EC₅₀范围为0.94~34.96 μg/mL,左侧正态分布峰内EC₅₀平均值为5.05 μg/mL,稻瘟病菌对稻瘟灵的敏感性分布频率不符合正态分布,表明菌群中已经出现对稻瘟灵敏感性下降的亚群体。187株稻瘟病菌菌株对稻瘟灵的平均抗性倍数为3.70,抗性菌株出现频率为55.61%,平均抗性指数为0.40,抗性水平呈逐年增加趋势。2017—2019年稻瘟灵对水稻叶瘟病的防治效果分别为82.97%、78.86%和81.36%,对水稻穗颈瘟的防治效果分别为79.81%、77.68%和80.20%,说明在现有防控手段下未产生高水平抗性群体。稻瘟灵与肟菌酯、戊唑醇之间无交互抗性。与不同药剂轮换使用的前提下,稻瘟灵仍可作为主要药剂防治稻瘟病。     

二化螟dsRNA降解酶基因的克隆与表达分析

为解析二化螟Chilo suppressalis体内参与降解双链RNA(double-stranded RNA,dsRNA)的关键核酸酶的功能,克隆二化螟不同的非专一性核酸酶（non-specific nuclease,NUC）基因,并对这些基因进行生物信息学分析和组织定量表达分析,同时对dsRNA降解酶（dsRNA degrading nuclease,dsRNase）活力的组织分布进行研究。结果显示,共克隆获得5个NUC基因,其中有4个编码dsRNase亚家族基因（CsdsRNase1～CsdsRNase4）和1个编码Endonuclease G亚家族基因（CsEndoG）。5个NUC基因的开放阅读框核苷酸序列长度范围为828～1 338 bp,编码275～445个氨基酸残基,其分子量大小为31.68～49.57 kD,预测等电点为5.48～9.42。CsdsRNase1和CsdsRNase2含有信号肽序列,两者相似度极高,且与家蚕Bombyx mori和斜纹夜蛾Spodoptera litura中具有dsRNA降解酶活力的dsRNase同源聚类;CsdsRNase1和CsdsRN...     

Fipronil resistance mechanisms in the rice stem borer, Chilo suppressalis Walker

Fipronil resistance mechanisms were studied between the laboratory susceptible strain and the selective field population of rice stem borer, Chilo suppressalis Walker in the laboratory. The borer population was collected from Wenzhou county, Zhejiang province. After five generations of selection, fipronil resistance ratio was 45.3-fold compared to the susceptible strain. Synergism experiments showed that the synergistic ratios of PBO, TPP and DEF on fipronil in susceptible and resistant strains of C. suppressalis were 7.55-, 1.93- and 2.91-fold, respectively, and DEM showed no obvious synergistic action on fipronil. Activities of carboxylesterase and microsomal-O-demethylase in the resistant strain were 1.89- and 1.36-fold higher that in susceptible strain, and no significant difference of glutathione-S-transferase activity was found between the resistant and susceptible strains. The K_m and V_max experiments also demonstrated that fipronil resistance of C. suppressalis was closely relative to the enhanced activities of carboxylesterase and microsomal-O-demethylase. Moreover, cross-resistance between fipronil and other conventional insecticides and the multiple resistant properties of the original Wenzhou’s population were also discussed.     

杀虫剂对转Bt水稻靶标害虫二化螟和非靶标害虫稻飞虱种群数量的影响

为明确杀虫剂对转Bt水稻品种华恢1号(Huahui 1,HH1)靶标害虫二化螟Chilo suppressalis及其非靶标害虫白背飞虱Sogatella furcifera和褐飞虱Nilaparvata lugens种群数量的影响,于2014—2015年在江西省南昌市南昌县莲塘镇的广福试验基地进行田间试验,种植转Bt水稻和对照亲本水稻,分别对其进行施用杀虫剂和未施用杀虫剂处理,然后对经2种处理后转Bt水稻和对照亲本水稻田中靶标害虫二化螟幼虫数量及非靶标害虫白背飞虱、褐飞虱的若虫数量、成虫数量、总虫量(若虫数量和成虫数量之和)进行调查,并对这3种害虫田间种群数量的年份、水稻品种、杀虫剂影响因子进行方差分析。结果显示,转Bt水稻种植对二化螟幼虫数量影响显著,而施用杀虫剂处理未显著影响二化螟幼虫数量。转Bt水稻种植对白背飞虱和褐飞虱的若虫数量、成虫数量和总虫量无显著影响;而施用杀虫剂处理显著降低转Bt水稻及其对照亲本水稻稻田内白背飞虱和褐飞虱的若虫数量及总虫量,并显著降低白背飞虱成虫数量。2014年和2015年,施用杀虫剂和未施用杀虫剂转Bt水稻田内二化螟幼虫数量极低或未调查到;未施用杀虫剂转Bt水稻上二化螟幼虫数量仅在2014年显著低于未施用杀虫剂对照亲本水稻。2014年,未施用杀虫剂转Bt水稻和对照亲本水稻上白背飞虱总虫量最大值分别为1 037、1 376头/百丛,施用杀虫剂后分别降低至233、253头/百丛;未施用杀虫剂转Bt水稻上和对照亲本水稻上褐飞虱总虫量最大值分别为4 120、2 413头/百丛,施用杀虫剂后降低至367、410头/百丛;2015年白背飞虱和褐飞虱的发生规律与2014年相同。表明转Bt水稻可显著降低二化螟幼虫数量,但褐飞虱及白背飞虱数量没有降低,而施用杀虫剂可有效降低褐飞虱及白背飞虱数量。     

性诱剂与黑光灯对二化螟的诱捕效果及其影响因子

在二化螟预测预报中,性诱剂和黑光灯两种诱捕方法被广泛采用,为比较这两种方法对稻田二化螟诱捕效果的差异,于2010—2014年在浙江温岭地区进行了相关研究,并对诱芯和诱捕器种类、田间越冬幼虫量以及气象因子对二化螟诱捕效果的影响进行了研究。结果显示:对于越冬代二化螟,采用性诱剂和黑光灯诱捕的二化螟成虫数量的消长动态基本一致,但采用性诱剂诱捕的成虫峰期出现早、峰次多,而且诱捕量显著高于黑光灯诱捕;不同性诱剂诱芯与诱捕器之间的诱捕效果无显著差异;性诱剂和黑光灯两种方法诱捕的虫量与田间越冬的幼虫基数存在显著正相关关系;两种诱捕方法的诱捕虫量与气温密切相关,对于越冬代,诱捕的虫量与温度呈负相关,而其余各代的诱捕虫量则与温度成正相关。     

二化螟响应Bt杀虫蛋白Cry1Ca的小RNA转录组分析

为有效防控二化螟Chilo suppressalis,采用Illumina Solexa二代测序技术对Bt杀虫蛋白Cry1Ca处理后二化螟中肠内的小RNA序列进行测序,并对其差异表达谱进行注释与分析,并用实时荧光定量PCR技术对测序结果进行验证。结果显示,Cry1Ca对二化螟的亚致死量LC₃₀为0.061 μg/g。Cry1Ca处理和对照处理后转录组中小RNA分布峰值均为22 nt,被注释的已知小RNA比例很低,分别为7.2%和7.9%。Cry1Ca处理后,二化螟中肠内miRNA、snoRNA、snRNA、rRNA、piRNA数量均少于对照。与对照相比,Cry1Ca处理后二化螟中肠内共有358个小RNA表达上调,747个小RNA表达下调,其中有25个已知miRNA的表达量较高,其中3个已知miRNA表达量显著上调,22个已知miRNA表达量显著下调;在新预测的novel miRNA中,有23个上调表达,43个下调表达。KEGG分析结果显示,Cry1Ca诱导的miRNA影响了丝裂原活化蛋白激酶（mitogen-activated protein kinase,MAPK）通路,这个通路包含17个靶基因,其中11个靶基因可被多个miRNA调控,每个靶基因可被59~665个miRNA靶向调控,参与靶向调控miRNA的相对表达量变化倍数介于0.01倍~23 289倍之间,表明miRNA在调控二化螟防御Bt杀虫蛋白Cry1Ca方面发挥着重要作用。实时荧光定量PCR的结果与测序结果一致,表明测序结果正确。     

Interactions between the striped stem borer Chilo suppressalis (Walk.) (Lep., Pyralidae) larvae and rice plants in response to nitrogen fertilization

A screenhouse experiment was conducted to examine the damage and compensation in rice plants when injured by the striped stem borer, Chilo suppressalis (Walker), larvae at tillering stage, as well as larval survival and development of the insect at different nitrogen (N) fertilization levels. Potted plants were fertilized at late seedling stage at the rates 0, 200, 400, 600 and 800mgN/pot, respectively. More deadheads were caused as fertilization increased. Plants compensated well for injury at the fertilization concentrations of 200 and 400mgN/pot by producing new tillers, but such compensation did not take place at 600 and 800mgN/pot. Two weeks after infestation, the highest number of remaining healthy tillers was found in plants fertilized at 400mgN/pot. Larval survival varied little among the treatments 200 to 800mgN/pot. Larval weight attainment and/or developmental rate increased with increasing fertilization level from 200 to 600mgN/pot, but both declined rapidly as fertilization reached 800mgN/pot, indicating the great dependence of plant suitability on N fertilization levels. Conclusively, both the compensation response of rice plants and their suitability for C. suppressalis larvae could be significantly affected by N fertilization levels.     

Interactions between the striped stem borer Chilo suppressalis (Walk.) (Lep., Pyralidae) larvae and rice plants in response to nitrogen fertilization

A screenhouse experiment was conducted to examine the damage and compensation in rice plants when injured by the striped stem borer, Chilo suppressalis (Walker), larvae at tillering stage, as well as larval survival and development of the insect at different nitrogen (N) fertilization levels. Potted plants were fertilized at late seedling stage at the rates 0, 200, 400, 600 and 800mgN/pot, respectively. More deadheads were caused as fertilization increased. Plants compensated well for injury at the fertilization concentrations of 200 and 400mgN/pot by producing new tillers, but such compensation did not take place at 600 and 800mgN/pot. Two weeks after infestation, the highest number of remaining healthy tillers was found in plants fertilized at 400mgN/pot. Larval survival varied little among the treatments 200 to 800mgN/pot. Larval weight attainment and/or developmental rate increased with increasing fertilization level from 200 to 600mgN/pot, but both declined rapidly as fertilization reached 800mgN/pot, indicating the great dependence of plant suitability on N fertilization levels. Conclusively, both the compensation response of rice plants and their suitability for C. suppressalis larvae could be significantly affected by N fertilization levels.     

Triazophos resistance mechanisms in the rice stem borer (Chilo suppressalis Walker)

A field population of the rice stem borer (Chilo suppressalis Walker) with 203.3-fold resistance to triazophos was collected. After 8-generation of continuous selection with triazophos in laboratory, resistance increased to 787.2-fold, and at the same time, the resistance to isocarbophos and methamidophos was also enhanced by 1.9- and 1.4-fold, respectively, implying some cross-resistance between triazophos and these two organophosphate insecticides. Resistance to abamectin was slightly enhanced by triazophos selection, and fipronil and methomyl decreased. Synergism experiments in vivo with TPP, PBO, and DEM were performed to gain a potential indication of roles of detoxicating enzymes in triazophos resistance. The synergism results revealed that TPP (SR, 1.92) and PBO (SR 1.63) had significant synergistic effects on triazophos in resistant rice borers. While DEM (SR 0.83) showed no effects. Assays of enzyme activity in vitro demonstrated that the resistant strain had higher activity of esterase and microsomal O-demethylase than the susceptible strain (1.20- and 1.30-fold, respectively). For glutathione S-transferase activity, no difference was found between the resistant and the susceptible strain when DCNB was used as substrate. However, 1.28-fold higher activity was observed in the resistant strain when CDNB was used. These results showed that esterase and microsomal-O-demethylase play some roles in the resistance. Some iso-enzyme of glutathione S-transferase may involve in the resistance to other insecticides, for this resistant strain was selected from a field population with multiple resistance background. Acetylcholinesterase as the triazophos target was also compared. The results revealed significant differences between the resistant and susceptible strain. The V_max and K_m of the enzyme in resistant strain was only 32 and 65% that in the susceptible strain, respectively. Inhibition tests in vitro showed that I₅₀ of triazophos on AChE of the resistant strain was 2.52-fold higher. Therefore, insensitive AChE may also involved in triazophos resistance mechanism of rice stem borer.     

二化螟对双酰胺类杀虫剂抗性的分子机制研究进展

新型双酰胺类杀虫剂已广泛用于保障水稻生产,而二化螟作为危害水稻生产的钻蛀性害虫,已经对该类杀虫剂产生了抗性,明确该类杀虫剂抗性分子机制,可为二化螟抗性快速检测和绿色防控提供技术支撑。本文在总结二化螟对双酰胺类杀虫剂抗性现状的基础上,重点综述了近年来有关其抗性分子机制研究的进展,主要包括解毒酶和转运蛋白基因过表达介导的代谢抗性,以及鱼尼丁受体基因突变介导的靶标抗性;指出了该研究领域在抗性分子检测、抗性新基因鉴定、抗性基因调控网络和多重抗性机制等方面存在的问题,并展望了其发展方向,认为：利用高通量测序技术检测害虫种群抗药性;利用多组学技术鉴定新抗性基因及调控网络,以探明多重抗性机制;将反向遗传学工具放射性配基结合及电生理技术深入验证抗性基因功能;需开发靶向抗性基因的dsRNA转基因作物、纳米农药及选择性新型化学杀虫剂,以达到杀虫剂减施增效的目的。