【作者】 温玉丛；

【导师】 韩召军；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2009， 博士

【摘要】 褐飞虱Nilaparvata lugens Stal (Brown planthopper, BPH)是一种重要的迁飞性水稻害虫,生产上主要以化学防治为主。虽然褐飞虱对有机氯、有机磷、氨基甲酸酯和拟除虫菊酯类杀虫剂的抗性时有报道,但通常是局部的中等水平抗性,只有对吡虫啉的抗性发生面广,抗性水平高,而且在大范围内导致田间用药失败。为了寻找吡虫啉的替代药剂,实现对褐飞虱的有效治理,本文在前期研究褐飞虱对吡虫啉室内筛选抗性的基础上,就褐飞虱对吡虫啉的田间抗性进行了系统研究。一、褐飞虱对常规杀虫剂的敏感基线和田间抗性监测通过杀虫剂毒力测定方法建立了褐飞虱对四类九种常规杀虫剂的敏感基线,并以此为基准分别对2005年和2008年采自安庆,桂林,江浦和吴江的四个地理种群的褐飞虱进行了田间抗药性监测。结果发现,褐飞虱敏感品系对九种药剂中敏感性较高的依次是呋虫胺,吡虫啉,丁硫克百威,吡蚜酮,啶虫醚和氟虫腈。对田间褐飞虱的抗药性监测发现,不同地理种群间褐飞虱对同种药剂的抗性水平没有显著差异(除2005年对吡虫啉抗性外),但是褐飞虱对不同药剂的抗性发展水平是不同的。2005年监测到褐飞虱对吡虫啉的抗性已达到了中高抗和极高抗水平(抗性倍数26.6-147.8倍),对乙酰甲胺磷处于低抗阶段(<10倍),而对氟虫腈,丁硫克百威和噻嗪酮的抗性状态仍处于敏感水平。2008年监测结果显示田间褐飞虱已表现出多重抗性,对吡虫啉抗性仍为高抗水平(61.1-87.3),同时对吡蚜酮,啶虫醚和噻嗪酮(12.2-23.8)和丁硫克百威(24.9-64.5)也表现出中等水平抗性,对有机磷类两种药剂的抗性处于低抗阶段(4.9-14.3),而对呋虫胺和氟虫腈两种药剂仍为敏感性状态。二、褐飞虱对吡虫啉田间抗性的稳定性和发生趋势为进一步研究褐飞虱对吡虫啉田间抗性的稳定性和抗性发展趋势,对吴江种群(RR-147.8倍)分别进行了敏感性恢复和抗性再筛选实验。结果表明,田间褐飞虱抗性种群在停止药剂筛选的情况下,抗性水平显著下降,尤其是前几代更为明显,19代后从147.8倍降到21.9倍。室内用吡虫啉再筛选可以使抗性进一步上升,19代时后抗性水平从147.8倍上升到380.9倍。但当种群达到极高水平后,继续连代筛选,短期内抗性没有明显变化,进行隔代筛选则抗性有所下降,而停止用药则抗性下降迅速,6代后从380.9倍降到157.7倍。综合以上现象说明,褐飞虱对吡虫啉的抗性是不稳定的而且抗性越高越不稳定,但是在持续的药剂压力下抗性仍会上升,并维持在极高抗水平。三、田间吡虫啉抗性褐飞虱的交互抗性分析通过测定四大类八种常规杀虫剂对褐飞虱田间抗性品系、室内筛选的敏感和抗性品系的毒力水平,分析了褐飞虱抗吡虫啉品系对几种杀虫剂的交互抗性。结果表明,对吡虫啉高抗的褐飞虱品系并非对所有的新烟碱类杀虫剂均表现出明显的交互抗性,在测试的新烟碱类杀虫剂中,仅对吡蚜酮和啶虫醚有显著的交互抗性,而对呋虫胺没有交互抗性。测定结果还发现,吡虫啉抗性品系对其它类型的药剂如乙酰甲胺磷、氟虫腈、噻嗪酮和丁硫克百威等均没有显著的交互抗性。由此认为吡虫啉的交互抗性谱相对较窄,仅限于同类中的某些药剂,因而生产上可以通过没有交互抗性的药剂,实施交替使用或混用来延缓抗性的产生。四、褐飞虱对吡虫啉抗感品系间相对适合度研究本研究分别构建了褐飞虱对吡虫啉的敏感品系、敏感恢复品系和抗性品系的实验种群生命表,利用种群趋势指数对比分析了抗性品系的相对适合度。结果发现,褐飞虱对吡虫啉产生抗性后,抗性品系(380.96倍)的种群数量趋势指数仅有25.6,除了性比之外,各项观测指标均显著低于敏感品系,尤其是产卵量和孵化率,均不到敏感品系的一半,其相对适合度只有0.191。然而敏感恢复品系(21.92倍)的种群数量趋势指数则达到119.5,仅在高龄若虫存活率和成虫羽化率两个指标上显著低于敏感品系,其相对适合度为0.889。这表明在没有药剂的选择压力的情况下,敏感褐飞虱具有极显著的种群增长优势,杂合种群对吡虫啉的抗性具有不稳定性。因此生产上可以通过避免褐飞虱接触吡虫啉或与吡虫啉有交互抗性的杀虫剂,恢复其敏感性。五、田间褐飞虱对吡虫啉抗性的生化机理研究本研究首先分析了酯酶、谷光甘肽转移酶和P450细胞色素氧化酶的抑制剂磷酸三苯酯、顺丁烯二酸二乙酯和胡椒基丁醚在褐飞虱敏感品系、抗性品系和敏感性恢复品系中对吡虫啉的增效作用,结果分析表明,细胞色素P450升高是褐飞虱对吡虫啉田间抗性升高的重要生化机制,EST可能与吡虫啉的极高水平的抗性有关,而GST则与抗性没有明显的关系。为了更进一步了解其生化机理,本研究分别测定了P450和EST的活力随着抗性水平的上升和衰退所发生的变化,结果发现不同褐飞虱抗性水平与P450活力的相关系数高达0.95(P<0.01,DPS6.50),而与EST活力的变化相关性并不大。因此认为细胞色素P450的活力升高是褐飞虱对吡虫啉田间抗性产生的关键因素。另外通过对2005年田间四个地理种群P450活力的测定和2008年田间种群对代谢酶(P450,EST和GST)的活力测定,验证了P450活力的升高是田间褐飞虱对吡虫啉抗性升高的主要生化机制,同时表明这也是2008年田间褐飞虱多重抗性发展的重要因素。六、褐飞虱乙酰胆碱受体a亚基功能区片段克隆及氨基酸多态性分析利用简并引物通过半巢式PCR克隆了褐飞虱乙酰胆碱受体a亚基功能区的cDNA片段,测序分析并与其它已报道的乙酰胆碱受体a亚基对比,发现这些片段均具有nAChR基因家族的典型特征,并与昆虫烟碱型乙酰胆碱受体的相应亚基具有很高的相似性,高达75-92%。因此证实所克隆的为褐飞虱nAchR的四个a亚基功能区的cDNA片段,并分别命名为：N1α1-like (FJ628418), N1α2-like (FJ557108), N1α3-like (FJ557109)和N1α4-like (FJ557110)。为验证之前报道的抗性突变位点(室内筛选品系获得)和进一步研究田间褐飞虱对吡虫啉高抗品系的靶标抗性机制,根据已获得的序列设计特异引物分别对褐飞虱抗感品系的四个a亚基功能区的cDNA片段进行序列比较,发现与敏感品系相比高抗品系的序列具有频率较高的氨基酸多态性,但目前没有检测到抗性密切相关的氨基酸变异。七、褐飞虱细胞色素P450基因的表达量及其与抗药性的关系分析根据GenBank所公布的褐飞虱基因序列Actin、CYP6AX1和CYP6AY1以及本实验室所获得的相关基因序列(CYP3A4、CYP4C1、CYP6A2、CYP4G1和CYP6A20)设计引物进行实时定量PCR,然后以Actin为持家基因,并以敏感品系CYP3A4基因的表达量为对照,通过比较CT值法(即2-ΔΔCT)获得了相关CYP450基因mRNA在褐飞虱抗感品系中的相对表达量。结果发现除CYP4G1和CYP6AX1的表达量在敏感品系略高外,其余基因的表达量都以抗性品系较高,其中CYP6AY1最为显著,是敏感品系的21.70倍。结合生化机理分析认为,褐飞虱对吡虫啉的田间抗性与CYP6AY1的过量表达有关。更多还原

【Abstract】 The brown planthopper (BPH), Nilaparvata lugens Stal, is one of the most important migratory rice pests, and its control is emphasized and largely relied on insecticids. Though resistance to organochlorines, organophosphates, carbamates and pyrethroids had been reported from time to time in this pest, no high resistance widely occurred and resulted in failture in field control. The only exception is the resistance to imidacloprid recently occured. In order to seek efficient substitute of imidacloprid and achieve a good control of BPH, the development and the mechanism of imidacloprid resistance in field population were purchased based on the previous studies with laboratory selected imidacloprid resistance.1. Establishment of the relative susceptible baseline data to common insecticides and resistance monitoring in field populations of BPHThe susceptibility of BPH to 9 insecticides of five different categories was determined with laboratory susceptible strain and the base line data were obtained. Based on these data, four field populations (Anqing, Jiangpu, Wujiang and Guilin) collected from paddy field in 2005 and 2008 were monitored for resistance to various insecticides. The results revealed that dinotefuran, imidacloprid, carbosulfan, pymetrozine, acetamiprid and fipronil have high relative toxicity index to susceptible strain among the nine insecticides. Various field populations usually showed similar resistance to the same insecticide, except for resistance to imidacloprid in 2005. But it is sure that resistance of field populations varied with different insecticides and monitoring time. BPH of field populations in 2005 had developed moderate to high level of resistance to imidacloprid (RR 26.6-147.8) and low level to acephate(RR<10), but no resistance to fipronil, carbosulfan, and buprofezin. Whereas in 2008, multiple resistances had been developed. Beside the high level of resistance to imidacloprid was maintained (61.1-87.3), moderate level of resistance to pymetrozine, acetamiprid and buprofezin (12.2-23.8), moderate to high level of resistance to carbosulfan (24.9-64.5), and low level of resistance to acephate and Chlorpyrifos(4.9-14.3) were developed, leaving only dinotefuran and fipronil as sensitive ones among the insecticides tested.2. Susceptibility recovery of field resistant population and further selectionTo declare the stability and dynamics of imidacloprid resistance in BPH, the hoppers from WJ field resistant population (RR 147.8) were separated into two groups and subjected to parallel treatments, susceptibility recovery and further resistance selection with imidacloprid. The results showed that susceptibility recovery by breeding without contacting any insecticides had a dramatic decline in its imidacloprid resistance (RR from 147.8 to 21.9 in 19 generations), especially in the first several generations. On the other hand, the further selection made the resistance increased (from 147.8-fold to 381.0-fold in 19 generations) and a high resistant strain (R19) was developed. However, after nineteen generations selected, it seemed to be difficult to select a higher imidacloprid resistance. Selections in every two generations made a slow decline in resistance and stopping selection made a quick decline (RR from 380.9-fold to157.7-fold in 6 generations). So, all of these results indicated that imidacloprid resistance was not stable, especial the resistance at high level.3. Cross-resistance in field population of BPH resistant to imidaclopridThe cross-resistance to various insecticides was analysized by testing of the toxicity of eight insecticides of four different categories on the susceptible and resistant strains and the field resistant population of BPH. The results indicated that the BPH with high resistance to imidacloprid did not show obvious cross-resistance to all the neonicotinoid insecticides. Only pymetrozine and acetamiprid amongthe insecticides tested were confirmed to be cross-resistant. To Dinotefuran and other non-neonicotinoid insecticides, such as acephate, fipronil, buprofezin and carbosulfan, no significant cross-resistance was found. In conclusion, imidacloprid might have a narrow of cross-resistance spectrum. Thus, it means that the alternate and mixed use of substitutes with little cross-resistance could be selected to delay the resistance development inthis pest.4. The relative fitness of BPH resistant and susceptible to imidacloprid The relative fitness of resistant BPH has been studied by comparing the population trend index, which was obtained through constructing life tables of susceptible, susceptibility-recovery and resistant strains. The results showed that the resistant strain (RR 380.96) had very lower population trend index (25.6) with dramaticly poor fecundity and hatchability, which were less than half that of susceptible strain. The relative fitness index calculated was only 0.191. However, the population trend and relative fitness index was 119.5 and 0.889, respectively in susceptibility recovery strain (RR 21.92). Among the tested indexes, only two (survival rate and emergence rate) were found decreased. These indicated that susceptible BPH had advantages over resistant ones under the circumstance without imidacloprid selection pressure, and further revealed that imidacloprid resistance in heterozagous populations was not stable. So, the susceptibility of BPH could recovery if they were kept from contacting with imidacloprid or other insecticides exhibiting cross-resistance.5. Biochemical mechanisms for imidacloprid resistance in field population of BPHThe synergisms of TPP, PBO and DEM, the inhibitors of esterase (EST), P450 monooxydase and glutathione S-transferase (GST), on imidacloprid in the susceptible, susceptibility recovery and resistant strains were first tested. The results revealed that the enhanced P450-detoxification could be an important biochemical mechanism for imidacloprid resistance in field populations, EST could also play some roles for super high level resistance, but GST seemed not. For further demonstration of the biochemical mechanisms, the changes in the activity of P450 and EST among different strains were analyzed. Of which, the correlation index of the P450 activity and imidacloprid resistance among different strains is as high as 0.95 (P<0.01, DPS6.50), but the EST activity did not correlate with imidacloprid resistance level. It implied that the enhancement of P450 activity was the key factor for imidacloprid resistance in BPH field populations. Furthermore, the P450 activity in four field strains collected in 2005 and the activity of three detoxification enzymes (P450, EST and GST) in the strains collected in 2008 were also tested, respectively. These results also proved that higher activity of P450 was the main biochemical mechanism for imidacloprid resistance in BPH and also for multiple resistances in the populations tested in 2008.6. Cloning of the functional extracellular regions of nAChR a-subunit genes and amino acid polymorphisms analysis With degenerate primers designed for nAChR a-subunit genes, half nest PCR was carried out and four cDNA fragments of the nicotinic acetylcholine receptor (nAChR) genes were cloned from BPH. Further sequence analyzed and comparison with previously reported sequences of nAChR genes confirmed that these fragments had a typical characteristic of nAChR a-subunit and high similarity with other insect a-subunits (75-92%). So, the four cloned putative nAChR subunits were designated as Nlal-like (FJ628418), Nlα2-like (FJ557108), Nlα3-like (FJ557109) and Nlα4-like (FJ557110). For checking the resistance mutation previously reported in laboratory selected strains and studying the mechanism for target resistance in BPH field population, the functional regions of the four nAChR a-subunits were cloned and the cDNA sequences from resistant and susceptible individuals were compared. The results revealed that each gene contained some amino acid polymorphisms. However, most of them occurred in very low frequence and no mutation was found to be associated with imidacloprid resistance.7. Quantitative expression of cytochrome P450 genes and the relationship with imidacloprid resistance in Nilaparvata lugensAccording to the published sequences of Actin, CYP6AX1 and CYP6AY1 in GenBank, and other related gene sequences (CYP3A4, CYP4C1, CYP6A2, CYP4G1 and CYP6A20) obtained in our laboratory, primers were designed for real-time quantitative PCR. Then considered Actin as the housekeeping gene and sensitive CYP3A4 gene expression as control, the relative mRNA expression of various CYP450 genes in resistant and susceptible strains of BPH were estimated by CT comparison method. It was found that most CYP450 genes had higher level of relative mRNA expression in resistant strain. Especially, the gene of CYP6AY1 was 21.7 folds higher in resistant strain than that in susceptible strain. Combination of the previous results in biochemical mechanism analysis revealed that the over-expression of CYP6AY1 might associate with the high resistance to imidacloprid in field populations of BPH.更多还原