【作者】 贾变桃；

【导师】 沈晋良；

【作者基本信息】 南京农业大学 ， 农药学， 2007， 博士

【摘要】 甜菜夜蛾是一种世界性分布的重要农业害虫,其寄主范围广,抗药性强,近年在我国危害呈加重趋势。虫酰肼是目前防治甜菜夜蛾的少数几种高效药剂之一。本研究监测了不同地区甜菜夜蛾对各类杀虫剂的抗性,通过室内选育虫酰肼抗性品系,计算抗性现实遗传力,预测抗性发展速度,并进行了交互抗性,抗性遗传方式和适合度及增效剂增效作用的研究,以期为甜菜夜蛾的抗性治理提供科学依据。现将研究结果总结如下:1甜菜夜蛾的抗药性监测用浸叶法监测了江苏和河南两省几个地区甜菜夜蛾种群对各类杀虫剂的抗性,结果表明:甜菜夜蛾对几种新型杀虫剂甲维盐、甲氧虫酰肼、呋喃虫酰肼、虫酰肼、溴虫腈和茚虫威以及氨基甲酸酯类杀虫剂拉维因基本处于敏感到敏感性下降阶段,首次监测到江苏省江宁和射阳种群对阿维菌素分别具有10.4和14.2倍的中等水平抗性,江苏丰县种群对茚虫威具有6.8倍的低水平抗性。对定虫隆,只有02年采集的江苏丰县种群尚敏感,江苏射阳种群达中等水平抗性（38.3倍）,江苏江宁、河南新野和江苏阜宁种群均已达高抗（分别为195.7、365.5和407.2倍）。对有机磷杀虫剂辛硫磷、毒死蜱和敌百虫以及菊酯类杀虫剂高效氯氰菊酯和三氟氯氰菊酯产生了不同程度的抗药性,对三氟氯氰菊酯抗性最高,所测种群均达到极高抗性水平（181.1～1865.6）,辛硫磷（6.9～11.7）,为低到中等水平抗性,毒死蜱（3.6～11.4）、敌百虫（3.4～10.0）和高效氯氰菊酯（3.5～9.9）,为敏感性下降到中等或低水平抗性。2甜菜夜蛾对虫酰肼的抗性选育及抗性风险评估在室内采用饲料感染法筛选甜菜夜蛾对虫酰肼的抗性,在饲养75代期间筛选62代,与起始种群相比,抗性比为39.2倍,与室内敏感品系相比,抗性比为141.3倍。在五次连续筛选过程中,现实遗传力依次为0.0471（F₀～F₇）、0.2606(F₂₂～F₂₈)、0.3613(F₃₁～F₃₈、0.1228(F₄₅～F₅₅)和0.1849(F₅₇～F₇₅),整个筛选62代现实遗传力为0.1556。抗性发展趋势为先慢后快,再缓慢再加快。抗性发展速率随药剂杀死率和现实遗传力的提高而加快,当现实遗传力h²为0.1556,杀死率为80%～9 0%时,抗性上升10倍,需要10.2～12.9代。抗性种群筛选43次(F₅₅)后停止用药筛选,饲养21代后,与敏感品系相比,抗性由63.5倍下降到21倍,抗性下降了2倍。3甜菜夜蛾虫酰肼抗性品系对其它杀虫剂的交互抗性和增效剂增效作用在室内筛选到47和64代时分别测定了筛选种群对其它杀虫剂的敏感性,结果表明:甜菜夜蛾对虫酰肼产生高水平抗性(F₄₇为50.7倍,F₆₄为85倍)后,对定虫隆具有高水平交互抗性（抗性增长倍数RR¹＞312.5倍）;对甲氧虫酰肼具有高水平交互抗性,在F₄₇RR¹为71.4（浸叶法）和46倍（饲料感染法）;在F₆₄RR¹为123.1（浸叶法）和133倍（饲料感染法）;对呋喃虫酰肼具有低（浸叶法RR¹4.7和7.4倍）或中等水平（饲料感染法RR¹ 14.9和22.3倍）的交互抗性;对阿维菌素(F₄₇:RR¹ 13.1)和甲维盐(F₆₄:RR¹ 20.4)具有中等水平的交互抗性;对茚虫威具有低水平交互抗性（RR¹ 8.4和8.7倍）;但对溴虫腈和拉维因交互抗性不明显或没有交互抗性。分别用谷胱甘肽-S-转移酶抑制剂DEM、多功能氧化酶抑制剂PBO和酯酶抑制剂DEF对甜菜夜蛾抗性和敏感品系进行活体增效测定,结果表明:PBO对敏感和抗性品系都有一定的增效作用,增效倍数分别为1.9乖3.6倍,对抗性品系的增效作用更明显,两者增效比为1.9倍。DEM和DEF对敏感品系没有增效作用,但对抗性品系有一定的增效作用,增效比分别为1.5和1.4倍,抗性品系和敏感品系增效倍数之比分别为1.6和1.4倍。可见,多功能氧化酶在虫酰肼的抗性中起主要作用,但酯酶和谷胱甘肽转移酶也可能参与了对虫酰肼的代谢。4甜菜夜蛾对虫酰肼的抗性遗传方式甜菜夜蛾抗性和对照品系正反交后代初孵幼虫毒力分别为0.523（0.279～0.772）和0.453（0.402～0.507）mg/L,两者差异不显著（95%置信限重叠）,表明甜菜夜蛾对虫酰肼抗性为常染色体遗传。按stone（1968）报道的公式,求得正交显性度D=-0.4217,反交显性度D=-0.49 82,二者介于-1～0之间,说明甜菜夜蛾对虫酰肼的抗性为不完全隐性。同样,甜菜夜蛾抗性和敏感品系正反交后代幼虫毒力分别为0.514（0.463～0.570）和0.500（0.317～0.717）mg/L,两者差异不显著（95%置信限基本重叠）,也表明甜菜夜蛾对虫酰肼的抗性为常染色体遗传。按stone报道的公式,计算得正交和反交的显性度D值分别为-0.2651和-0.277 3,二者介于-1～0之间,也证明抗性主基因为不完全隐性。F₁与抗性亲本回交或自交后代的虫酰肼LD-P曲线与单个主基因控制假设明显不符,说明抗性为多基因控制。5甜菜夜蛾虫酰肼抗性和敏感品系的相对适合度适合度研究表明:甜菜夜蛾虫酰肼抗性品系的4～5龄幼虫的存活率下降,幼虫和蛹发育历期延长、蛹重减轻、化蛹率和产卵量降低,相对于其它品系存在一定种群繁殖劣势。计算各种群的净增殖率分别为341.3、613.0、358.4和241.6,正反交和抗性品系相对于敏感品系适合度分别为1.80、1.05和0.71,进一步表明甜菜夜蛾虫酰肼抗性品系比其它品系具有明显的生存劣势。更多还原

【Abstract】 The beet armyworm,Spodoptera exigua（H（?）bner）,is a worldwide distributed and polyphagous pest,which has developed resistance to many insecticides,including chlorinated hydrocarbons,organophosphates,carbarnates,pyrethroids,benzoylphenylureas, and Bt formulations.In recent years the damage by the beet armyworm to crops increased dramatically in China.Tebufenozide is considered as one of a few highly effective insecticides for controlling beet armyworm.In order to make good use of this insecticide and implement effective resistance management strategy,we surveyed resistance of field populations of beet armyworm from some areas of Jiangsu and Henan provinces in China to several classes of insecticides.Furthermore,the resistant strain was selected with tebufenozide,the resistance realized heritability（h²） was estimated and resistance development rate was projected.In the same time,cross-resistance,inhetitance of resitance, fitness cost and synergistic effects of three synergists to tebufenozide were studied.The results were summarized as follows:1 Resistance monitoring in beet armywormSusceptibility to several classes of insecticides was determined using leaf-dipping method in field populations of Spodoptera exigua from some areas of Jiangsu and Henan Provinces,in China.The results showed that the majority of populations were susceptible or decreased susceptible to a few novel insecticides,such as emamectin benzoate, methoxyfenozede,JS118,tebufenozede,chlorfenapy and indoxacarb,as well as thoidicarb, but two populations from Jiangning and Sheyang counties of Jiangsu province first developed a moderate levels of resistance to abamectin,and one population from Fengxian county of Jiangsu province had a low level of resistance to indoxacarb.Two populations from Jiangning and Funing counties of Jiangsu province and one population from Xinye county of Henan province developed very high levels resitance with 195.7-,407.2-and 365.5-fold and one population from Sheyang county of Jiangsu province had a moderate level resistance（38.3） to chlorfuazuron,and only one populaton collected from Fengxian county of Jiangsu province in 2002 showed susceptible to chlorfuazuron.All tested populations developed very high level resistance to cyhalothrin（181.1～1865.6）, low-moderate levels resistance to phoxim（6.9～11.7） and decreased susceptible to low or moderate levels resistance to chlorpyrifos（3.6～11.4）,trichlorphon（3.4～10.0） and beta-cypermethrin（3.5～9.9）.2 Resistanc selection and risk assessment to tebufenozide in beet armywormA field population of beet armyworm,Spodoptera exigua（H（?）bner）（Lepidoptera: Noctuidae）,from Shenzhen,Guangdong province was used by dietary exposure to select for resistance to tebufenozide in the laboratory.After continuous selection with tebufenozide 62 times during 75generations,a resistant strain was achieved with resistance ratio of 39.2-fold to tebufenozide when compared with the original reference strain （141.3-fold compared with a susceptible laboratory strain）.Estimation of Realized heritability（h²） of resistance in different selection stage was based on the method described by Tabashnik & McGaughey.The results suggested that h² to tebufenozide in 5 successive selection stages was 0.0471（F₀～F₈）,0.2606(F₂₂～F₂₈),0.3613(F₃₁～F₃₈),0.1228(F₄₅～F₅₅) and 0.1849(F₅₇～F₇₅),respectively,h² for the entire selection experiment was 0.1556.The time course of resistant development appeared to slow at first then speed up,afterwards become slow then speed up.The resistance development rate increases as h² and selection intensity increase.Thus,assuming h²=0.1556 and selection kills 80%～90%of the populations,then a 10-fold increase in LC₅₀ is expected in 10.2～12.9 generations.The susceptibility to tebufenozide increased 2 times when the culture was kept for 21 generation without exposure to the chemical pressure after 43 times selection.3 Cross-resistance to other insecticides of tebufenozide-selected beet armyworm and synergistic effects of synergists to tebufenozideSusceptibity to other insecticides was determined in selected population after 47 and 64 selection in the laboratory,respectively.Bioassay revealed that this resistant strain showed high levels resistance to tebufenozide(F₄₇:50.7-fold,F₆₄:85-fold),and showed high levels cross-resistance to chlorfuazuron（increasing resistance fold RR′＞312.5-fold）,high levels cross-resistance to methoxyfenozide,RR′71.4（leaf-dipping method） and RR′46（dietary exposure） in F₄₇ and RR′123.1（leaf-dipping method） and RR′133（dietary exposure） in F₆₄, low（RR′4.7 and 7.4-fold with leaf-dipping method） or moderate（RR′14.9 and 22.3-fold with dietary exposure） levels cross-resistance to JS118,moderate levels cross-resistance to abamectin(F₄₇:RR′13.1)and emamectin benzoate(F₆₄:RR′20.4),low levels crossresistance to indoxacarb（RR′8.4 and 8.7）,a little cross-resistance to chlorfenapy（RR′1.9 and 1.3）,and no cross-resistance to thiodicarb（RR′0.3）. Synergism experiment in vitro with DEM,PBO and DEF was performed to see the roles of detoxication enzymes in tebufenozide resistance.The results revealed that PBO had significant synergistic effects on tebufenozide in resistant（SR 3.6） and susceptible strain （SR1.9）.Synergistic ratio between resistant and susceptible strains was 1.9.DEM and DEF had apparent synergistic effects in resistant strain（SR 1.6 and 1.4）,but no effect in susceptible strain.This implied that mixed-function oxidase played an important role in resistance to tebufenozide,but esterase and glutathione-S-tranferase might be involved in the tebufenozide metabolism.4 Resistance inheritance to tebufenozide in beet armywormLogit regression analysis of F-1 reciprocal crosses between resistant and un-selected strain of beet armyworm indicated that LC₅₀ of the progenies from F₁ and F₁′were 0.523（0.279～0.772） and 0.453（0.402～0.507） mg/L,respectively,and there were no significant difference between the two LC₅₀s.This results showed the gene（or genes） that confers resistance to tebufenozide was on autosome.According to the formula of Stone（1968）,the dominance degree（D） of F₁ and F₁′were -0.4217 and -0.4982,respectively.This indicated that resistance was inherited as an incompeletely recessive trait（-1＞D＞0）.Similarly,Logit regression analysis of F₁ reciprocal crosses between resistant and laboratory susceptible strain of beet armyworm indicated that LC₅₀ of the progenies from F₁ and F₁′were 0.514 （0.463～0.570） and 0.500（0.317～0.717） mg/L,respectively,and there were no significant difference between the two LC₅₀s.This results also showed the gene（or genes） that confers resistance to tebufenozide was on autosome.According to the formula of Stone（1968）,the dominace degree（D） of F₁ and F₁′were -0.2651 and -0.2773,respectively.This indicated that resistance was inhetited as an incompeletely recessive trait（-1＞D＞0）.LD-P curves for progenies of two backcrosses and F₂,F₂′were apparently different from correspongding expected curves assuming monogenic inheritance.Theχ² analysis confirmed that observed mortalities failed to fit the monogenetic model,which supports the conclusion that resistance to tebufenozide conferred by more than one factor.5 Fitness cost of resistance to tebufenozide in beet armywormThe results of the relative fitness indicated that the resistant strain possessed significantly biological disadvantages,including lower survivor rate of fourth to fifth instar,prolonged larval and pupal duration,lighter pupa weight,lower pupation and egg number laid per female when compared with other strains.Intrinsic rate of natural increase of resistant, susceptible strains and RS,SR were 341.3,241.6,613.0 and 358.4,respectively.The resistant strain and RS,SR were calculated to have 0.71,1.80 and 1.05 of fitness value relative to susceptible strain.更多还原