【作者】 王震宇；

【导师】 吴益东；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2011， 博士

【摘要】 烟粉虱(Bemisia tabaci Gennadius)是一种重要的农业害虫,主要分布于热带以及亚热带地区,其寄主植物超过600余种,曾被冠以“超级害虫”的称号。烟粉虱不仅可以直接对寄主植物造成危害,而且可以传播多种植物病毒,对农业生产造成了严重的危害。烟粉虱是一个处于快速进化中的复合种群,存在许多生物型,各个生物型之间存在生殖隔离,在形态上难以区分,而在生活习性,适合度,传毒能力以及抗药性等方面存在一定的差别。目前报道的烟粉虱生物型超过27种,其中B型和Q型是为害最严重、呈全球性分布的生物型,而其他生物型为局部分布。B型烟粉虱先于Q型烟粉虱入侵我国农业生态系统,但近年来Q型已逐渐取代B型。吡虫啉是新烟碱类杀虫剂的代表药剂,自从1991年商品化以来,被用于防治多种作物上的害虫,由于其良好的内吸活性,尤其对刺吸式害虫效果显著。吡虫啉是一种神经毒剂,作用于昆虫的烟碱型乙酰胆碱受体(nAChR).由于其具备良好的选择毒力,对昆虫高毒而对哺乳动物低毒,所以被广泛使用。随着各类杀虫剂的不合理施用,烟粉虱逐渐对多种杀虫剂如有机磷、有机氯、氨基甲酸酯、拟除虫菊酯以及昆虫生长调节剂等产生了不同程度的抗性。在烟粉虱的防治过程中,由于吡虫啉良好的防效,使得其施用过于频繁,早在1998年西班牙南部Q型烟粉虱就对其产生了抗性。本文对我国烟粉虱生物型和抗药性水平进行了监测,对B型烟粉虱对吡虫啉抗性遗传方式、交互抗性及抗性生化机理进行了系统研究,并运用转录组测序和实时荧光定量PCR技术成功筛选出与烟粉虱吡虫啉抗性相关的2个细胞色素P450基因。本文的研究结果明确了我国田间烟粉虱的抗性和生物型的发展状况,并为烟粉虱对新烟碱类杀虫剂抗性的治理提供重要理论依据。一、烟粉虱生物型及抗药性的监测对我国东南沿海地区7个省以及新疆维吾尔自治区在内的14个点烟粉虱的生物型及抗药性进行了监测。在这14个点中共检测到两种烟粉虱生物型(B型和Q型),9个点为Q型烟粉虱,3个点为B型,2个点为B型和Q型共存。在所监测的地区,两种生物型的所有烟粉虱已经对吡虫啉(Imidacloprid)和噻虫嗪(Thiamethoxam)产生了中到高水平的抗性(与敏感种群比较,吡虫啉28至1900倍,噻虫嗪29倍至1200倍)；对αα-氯氰菊酯(alpha-cypermethrin)产生了中到高水平的抗性(22-610倍)；只有部分种群对氟虫腈(Fipronil)产生了低到中等抗性(10-25倍)；对多杀菌素(Spinosad)产生了低水平的抗性(5.7-6.4倍)；所有调查种群对阿维菌素均保持敏感。在我国东南沿海地区,Q型烟粉虱正在逐渐取代B型烟粉虱而成为优势种群；我国烟粉虱种群已经对新烟碱类杀虫剂吡虫啉和噻虫嗪产生中高水平抗性,需要限制使用；我国烟粉虱尚未对阿维菌素产生抗性,可以与其他药剂轮换使用,以延缓烟粉虱抗药性的发展。二、B型和Q型烟粉虱种群竞争与抗药性的关系在室内用棉花植株饲养情况下,对采自浙江杭州的烟粉虱B型和Q型混合种群的生物型组成以及抗药性水平进行了多代的监测。在室内饲养的F0代,两种生物型个体比例相当；在F10代,Q型已经完全被B型所取代。与F0代相比,F1o代烟粉虱种群对吡虫啉、阿维菌素和氟虫腈的抗性水平明显下降,而对αα-氯氰菊酯的抗性保持稳定。该研究表明：在没有杀虫剂选择压力的室内条件下,B型烟粉虱比Q型更具生存优势。然而在新烟碱类杀虫剂广泛使用的田问条件下,烟粉虱Q型因比B型对新烟碱类杀虫剂具有更强的抗性而获得竞争优势。三、B型烟粉虱抗吡虫啉品系的筛选、交互抗性以及抗性遗传方式利用成虫浸叶法进行了连续30代筛选,培育了1个B型烟粉虱抗吡虫啉品系NJ-Imi,其与对照品系NJ相比有490倍的抗性。研究发现,NJ-Imi品系对其他3种新烟碱类杀虫剂啶虫脒(Acetamiprid),噻虫嗪和烯啶虫胺(Nitenpyram)存在高水平的交互抗性；与杀虫单(monosultap),巴丹(Cartap)以及多杀菌素存在低水平交互抗性；与阿维菌素无交互抗性。B型烟粉虱对吡虫啉的抗性为常染色体控制的半显性遗传。四、B型烟粉虱抗吡虫啉品系的抗性生化机理以B型烟粉虱抗吡虫啉品系(NJ-Imi)和敏感品系(NJ)为试虫,通过增效剂生物测定和解毒代谢酶活力分析,研究了B型烟粉虱吡虫啉抗性的生化机理。分别在筛选过程中的地15代(G15)和第30代(G30)测定了PBO, DEM和DEF对吡虫啉的增效作用。在抗性品系中,多功能氧化酶抑制剂PBO在G15和G30分别对吡虫啉有2.5倍和2.1倍的增效作用,而在敏感品系中没有增效作用；抗性品系细胞色素P450单加氧酶活力为敏感品系的2.5倍；在抗性和敏感品系中,谷胱甘肽S-转移酶抑制剂DEM对吡虫啉都没有增效作用,谷胱甘肽S-转移酶的活力在两品系间也没有显著差异；在G15时,在抗}性和敏感品系中,酯酶抑制剂DEF对吡虫啉有2.7倍和2.5倍的增效作用,而在G30时DEF对吡虫啉没有明显的增效作用；酯酶活力在抗性品系中比敏感品系中有所降低；这表明在抗性品系中,多功能氧化酶活力的提高可能参与了吡虫啉抗性的形成。五、B型烟粉虱抗吡虫啉品系转录组的测定以及数据的分析利用Illumina (Solexa)测序平台,结合RNA-Seq技术,对B型烟粉虱抗吡虫啉品系成虫转录组进行了测序。经测序得到3700万个reads,经聚类分析后得到7万个unigenes (平均长度619bp)。基于NCBI以及其他生物信息学数据库,对组装获得的unigenes做了Contig长度分布,Scaffold-gene长度分布,Scaffold-gene功能注释,Scaffold-gene的GO分类,蛋白功能和分类预测以及Scaffold-gene代谢通路分析。同时,还分析了转录组数据中抗药性相关的基因,包括细胞色素P450基因,酯酶基因(EST),谷胱甘肽S-转移酶基因(GST)以及烟碱型乙酰胆碱受体(nAChR)。烟粉虱转录组测序数据将为烟粉虱抗药性机理研究工作奠定了良好的基础。六、与吡虫啉抗性相关的烟粉虱细胞色素P450基因筛选从B型烟粉虱转录组数据中筛选出编码细胞色素P450的序列141条(长度从100bp到2000bp以上),去掉其中较短的序列以及重复测序的序列,共得到37条编码P450基因的序列,将这些序列与NCBI的nr数据库进行比较,并根据细胞色素P450基因的命名规则进行命名,其中有2个序列已命名(CYP6CX1V1知CYP6CM1),其余35个细胞色素P450基因均为新命名的P450基因。通过传统Sanger测序法对这些序列进行了克隆和测序验证。采用实时荧光定量PCR比较了这37个P450基因mRNA在NJ-Imi抗性品系(B型)、China-YN抗性品系(Q型)和SUD-S敏感品系(非B/Q)中的表达水平。结果表明：CYP6CX4知CYP6CM1在2个抗性品系中具有10-35倍的过量表达；3个P450基因(CYP6CX2, CYP6CX3,知CYP6DZ4)在2个抗性品系具有1.9-6.7倍的过量表达；5个P450基因(CYP4C64,CYP4G69,CYP6DW2,CYP6DW3知CYP6CX5)的mRNA表达量在3个品系中相近。为了明确CYP6CX4知CYP6CM1基因过量表达与抗药性的关系,分别用区分剂量处理了China-XJ田间品系(B型)和China-NJ田间品系(Q型)烟粉虱成虫,这2个P450基因在吡虫啉处理存活成虫中mRNA表达量均明显高于未处理成虫,表明这2个P450基因过量表达与吡虫啉抗性有关。已有研究报道了CYP6CM1过量表达与吡虫啉抗性有关,CYP6CX4与吡虫啉抗性相关为本研究首次发现。更多还原

【Abstract】 The whitefly Bemisia tabaci (Gennadius) is one of the most devastating pest insects and occurs in subtropical and tropical agriculture as well as in greenhouse production systems. B. tabaci has been recorded from more than 600 plant specices. Over the last three decades, this pest has caused heavy losses of crop yield by direct feeding damage, excreting honey dew that favours sooty mould production and transmitting plant viruses. B. tabaci comprises a complex population with many biotypes, of which the B-biotype and Q-biotype are the most widespread biotypes. Both B-biotype and Q-biotype B. tabaci have invaded into China, and the Q-biotype is supplanting the B-biotype which used to be ubiquitous in China.Imidacloprid, the first commercial neonicotinoid insecticide, was introduced in 1991. As a neurotoxin, imidacloprid acts on the nicotinic acetylcholine receptor (nAChR) of insects and is selectively toxic to insects relative to mammals. Imidacloprid is used to control sucking and biting pest insects including ricehoppers, aphids, thrips, whiteflies, termites, turf insects, soil insects and some beetles. Control of B. tabaci is heavily dependent on chemical insecticides, and this pest has developed resistance to a wide range of insecticides including carbamates, organophosphates, pyrethroids, insect growth regulators and neonicotinoids. The Q-biotype B. tabaci from Spain has evolved resistance to imidacloprid since 1998.In the present study, the biotype and resistance status of B. tabaci collected from southeastern China was investigated. Inheritance mode, cross resistance spectrum, and biochemical mechanisms of imidacloprid resistance were studied in a resistant B-biotype B.tabaci strain. Combined transcriptome sequencing and real-time PCR,2 P450 genes associated with imidacloprid resistance were identified in both B-biotype and Q-biotype B. tabaci. These results will establish the technical basis of whitefly resistance management strategy in China.1. Biotype and insecticide resistance status of the whitefly B. tabaci from ChinaTwo different biotypes of B. tabaci (B-biotype and Q-biotype) were detected in south-eastern China, and the samples collected from geographical regions showed a prevalence of the Q-biotype and the coexistence of B-and Q-biotypes in some regions. Moderate to high levels of resistance to two neonicotinoids were established in both biotypes (28-1900-fold to imidacloprid,29-1200-fold to thiamethoxam. Medium to high levels of resistance to alpha-cypermethrin (22-610-fold) were also detected in both biotypes. Four out of 12 populations had low to medium levels of resistance to fipronil (10-25-fold). Four out of 12 populations showed low levels of resistance to spinosad (5.7-6.4-fold). All populations tested were susceptible to abamectin. We can get the conclusion that the Q-biotype B. tabaci is supplanting the B-biotype which used to be ubiquitous in China. Field populations of both B and Q-biotypes of B. tabaci have developed high levels of resistance to imidacloprid and thiamethoxam. Abamectin is the most effective insecticide against adult B. tabaci from all populations.2. Competition between the B- and Q-biotypes of B. tabaci and its relevance to insecticide resistanceThe insecticide resistance of specimens of a mixed population of the B- and Q-biotypes of B. tabaci collected in the city of Hangzhou, Zhejiang Province was monitored for several generations. When the population was reared on cotton without insecticides for 10 generations, the proportion of B-biotype to Q-biotype individuals in the F0 population was similar; however the B-biotype completely replaced the Q-biotype in the F10 population. Resistance to imidacloprid, abamectin and fipronil in the F10 population was significantly reduced compared with the F0 population, but resistance to a-cypermethrin was similar between the F0 and F10 populations. These results indicate that the B-biotype is competitively superior to the Q-biotype in the absence of insecticide selection pressure under laboratory conditions. However, the Q-biotype is competitively superior in field conditions where neonicotinoids are widely used, presumably because it has greater potential to develop resistance to neonicotinoids than the B-biotype.3. Cross-resistance and mode of inheritance in imidacloprid-resistant B-biotype B. tabaciThe NJ-Imi strain of B-biotype B. tabaci was selected from the NJ strain with imidacloprid for 30 generations. The NJ-Imi strain exhibited 490-fold resistance to imidacloprid, high levels of cross-resistance to three other neonicotinoids, low levels of cross-resistance to monosultap, cartap and spinosad, but no cross-resistance to abamectin and cypermethrin. Imidacloprid resistance in the NJ-Imi strain was autosomal and semi-dominant.4. Biochemical mechanism of imidacloprid resistance in B-biotype B. tabaciThe biochemical mechanisms of imidacloprid resistance in B-biotype B. tabaci were studied by synergism test, detoxifing enzyme activity. The synergistic effects of PBO, DEF and DEM on imidacloprid in strain NJ were compared with strain NJ-Imi at both G15 (15th generaion of selection) and G30 (30th generation of selection). The oxidase inhibitor PBO showed 2.5-and 2.1-fold synergism with imidacloprid in the NJ-Imi strain at G15 and G30 respectively, but no synergism of imidacloprid efficacy in strain NJ. P450 monooxygenase activity in strain NJ-Imi (elevated 2.5-fold) was significantly higher than in strain NJ. The glutathione depleter DEM did not show appreciable synergism in combination with imidacloprid in either the NJ or the NJ-Imi strain. The glutathione S-transferase activity towards CDNB was not significantly different between strains.The esterase inhibitor DEF synergised imidacloprid both in strain NJ (2.7-fold) and in strain NJ-Imi G15 (2.5-fold), but not in strain NJ-Imi G30. Esterase activity usingα-naphthyl acetate as substrate was significantly (0.6-fold) lower in strain NJ-Imi than in strain NJ. The results indicate oxidative degradation at least to some extent in the resistant strain.5. Data analysis of the transcriptome sequencing of B-biotype B. tabaci adultsA whitefly (B-biotype) transcriptome was de novo assembled using a short read sequencing technology (Solexa). Over 37 million short reads were produced and more than 70 thousand unique sequences (mean size 619 bp) were assembled. Scaffold gene annotation, gene ontology and COG function classification were presented based on the different databases of NCBI. All these scaffold genes were further mapped to pathways. We analyzed the novel insecticide resistance-related genes including P450s, ESTs, GSTs and nAChRs from our database. Our study will provide an important basis for investigating resistance mechanisms of B. tabaci.6. Genome wide screen of imidacloprid resistance related P450 genes in B. tabaci.A total of 141 unique sequences annotated as cytochrome P450 genes (partial or full length cDNA, from～100bp to over 2000bp) were identified from the transcriptome database, and 35 new P450 genes were named based on the database of NCBI. Real-time quantitative PCR was used to analyse the relative expression of the 37 P450 genes between resistant and susceptible strains. Of these, two P450 genes (CYP6CX4 and CYP6CM1) were upregulated in two resistant strains compared with a susceptible SUD-S strain (from 10 to 35 fold). Another 3 P450 genes were also upregulated in two strains (CYP6CX2, CYP6CX3, and CYP6DZ, from 1.9 to 6.7 fold). The expression levels of five P450 genes (CYP4C64, CYP4G69, CYP6DW2, CYP6DW3, and CYP6CX5) were similar among three strains (different biotypes). An evidently elevation of expression level of CYP6CX4 and CYP-6CM1 were observed in survivors after exposure to a high dose of imidacloprid compared with untreated adults, which confirmed that overexpression of these two P450 genes are involved in imidacloprid resistance in B. tabaci.更多还原