棉铃虫对Bt生物农药早期抗性及与转Bt基因棉抗虫性的关系

用饲料感染法建立了棉铃虫Helicoverpa rmigera(Hubmer)敏感品系(SUS1)对Bt生物农药的敏感毒力基线和区分剂量,1995年测定了五省六县棉铃虫初孵幼虫对Bt生物农药的敏感性,结果表明：山东阳谷、河北邯郸、河南新乡、安徽萧县及江苏丰县棉铃虫已产生早期抗性,抗性个体百分率为5%～10%,与敏感品系相比,LC₅₀值稍有增加,但斜率b值明显变小;而江苏东台棉铃虫仍属敏感。这是国内外首次诊测到棉铃虫对Bt生物农药抗性。用棉叶喂饲法测定比较了转Bt基因棉花品系对不同种群棉铃虫的抗虫性效果,结果表明：用早期抗性的阳谷和新乡棉铃虫初孵幼虫接虫5d后平均死亡率较敏感品系下降16%～29%,说明棉铃虫对Bt农药与转Bt生物基因棉花品系间存在交互抗性。还讨论了Bt农药的抗性治理对策。     

盐城棉区棉铃虫田间种群对Bt蛋白的抗性基因频率

【背景】转Bt基因抗虫棉已经在我国进行了近20年的大规模商业化种植,产生了显著的经济和环境效益。但是,靶标害虫棉铃虫的抗性是转Bt基因抗虫棉产业健康发展所面临的最大问题,而抗性监测是解决这一问题的必要管理措施。盐城市是江苏省转基因抗虫棉的主产区,但有关该地区棉铃虫对转Bt基因抗虫棉的抗性基因频率未见报道。【方法】于2012年在盐城三龙镇和东台镇棉区采集田间棉铃虫种群,检测了初孵幼虫对花铃期转Bt基因抗虫棉中30幼嫩叶片的敏感性,用区分剂量法检测了2龄幼虫对Bt蛋白的抗性基因频率。【结果】取食转Bt基因抗虫棉叶片后,棉铃虫初孵幼虫在9 d内全部死亡;三龙镇和东台镇棉铃虫2龄幼虫对Bt蛋白的抗性基因频率分别为7.6×10-3和6.9×10-3。【结论与意义】目前,盐城棉区的棉铃虫对转Bt基因抗虫棉仍保持很高的敏感性,棉铃虫种群对Bt蛋白的抗性基因频率没有发生显著变化,但仍需持续监测。     

棉铃虫对转Bt基因棉花的行为反应

棉铃虫Helicoverpa armigera(Hübner)是转Bt基因棉花(Bt棉)最重要的靶标害虫之一。害虫对Bt棉的抗性问题是Bt棉产业持续健康发展的最主要威胁。规避行为可以减少害虫与Bt棉的接触而降低生理抗性选择压力。在本研究中,通过比较棉铃虫对Bt棉与常规棉的行为反应评估了棉铃虫对Bt棉的行为规避能力。产卵选择结果显示,棉铃虫成虫在Bt棉上的落卵量显著低于常规棉。幼虫实验结果显示,棉铃虫初孵幼虫在Bt棉植株上的居留时间显著短于在常规棉上的居留时间。无选择条件下,1龄幼虫在对Bt棉叶的取食空洞数以及总取食量均显著低于常规棉叶。综合以上的结果,认为棉铃虫对Bt棉具有一定的行为规避能力,这可能是延缓棉铃虫对Bt棉产生抗性的因素之一。本研究能够帮助有效地预测靶标害虫对Bt作物的抗性风险。     

Bt棉叶对棉铃虫抗虫性的时空变化及气象因素的影响

用叶片喂饲法,测定了转Bt基因棉不同叶位、不同生长时期的叶片对棉铃虫初孵幼虫抗虫性的时空变化,同时测定了大田和室内不同气象条件下生长的侧枝和苗期叶片对棉铃虫抗虫性的影响。结果表明：转Bt基因棉R₁₉-137株系主茎第2～10叶的抗虫性最高,初孵幼虫处理5天后的平均死亡率为97.0%～100%,为害级别在1.0～1.1级;第11～16叶的抗虫性明显下降,平均死亡率为35.6%～67.6%,存活幼虫以2龄为主。7月下旬、8月下旬测定了不同部位侧枝叶片的抗虫性,平均死亡率分别为30.9%～44.9%和10.0%～30.0%,抗虫效果进一步下降。试验结果还表明,在室内外不同气候条件下生长的Bt棉叶片的抗虫性有显著差异。讨论了Bt棉抗虫性时空变化的可能原因与Bt棉推广中气候条件的重要性。     

转Bt基因抗虫棉田棉铃虫消长规律及危害特点

通过对转 Bt基因抗虫棉田棉铃虫的系统调查研究表明 :(1)转 Bt基因抗虫棉对棉铃虫蛾、卵无抗性表现 ,田间落卵量与常规棉田无明显差异 ,相反 ,二代棉铃虫百株累计卵量倒比常规棉处理区增加了 2 16～ 2 74粒 ,增长幅度为 30 %～ 38% ;(2 )转 Bt基因抗虫棉对二代棉铃虫幼虫的抗性及控制作用较强 ,基本不用进行药剂防治 ,但对三代棉虫幼虫的抗性控制效果明显下降 ,田间 3龄以上幼虫平均达2 0～ 4 0头 /百株 ,多者达 6 0头以上 ,仍需进行必要的药剂防治 ;(3)棉铃虫在转 Bt基因抗虫棉株上的危害特点与常规棉株有明显的差异 ,表现在幼虫和危害症状均较隐蔽 ,不易被发现。     

棉铃虫对转Bt基因棉的抗性筛选及遗传方式的研究

通过室内筛选获得了棉铃虫Helicoverpa armigera (Hübner)对转Bt基因棉的抗性种群,并在此基础上进行了抗性遗传方式的研究。试验结果表明：经16代筛选,棉铃虫对转Bt基因棉的抗性倍数上升到43.3倍。筛选过程中,棉铃虫7日龄幼虫的体重和成虫羽化率变化明显,被选为确定筛选剂量的标准。敏感与抗性棉铃虫杂交,正交、反交的显性度都小于0、杂交过程中雌雄性比基本接近1∶1、假设抗性基因为单基因时的χ²值较低,因此,初步认为棉铃虫对转Bt基因棉的抗性是常染色体单基因控制的不完全隐性遗传。     

棉铃虫对Bt杀虫剂的抗性遗传方式

随着转基因棉花种植面积的日益增加,棉铃虫Helicoverpa armigera（Hübner)对Bt的抗性已经成为一个不容忽视的问题。发展转多价基因作物是当前缓解害虫对Bt抗性的最有效措施。本研究以经室内多年筛选的、抗性倍数达2 000多倍的Bt杀虫剂（含多种蛋白）抗性品系为材料,通过生物测定和不同的杂交试验,测定棉铃虫对Bt杀虫剂的抗性遗传方式,以期为Bt生物农药的抗性治理提供一定的依据,同时为制定棉铃虫对转多基因作物的抗性治理策略提供一定的参考。对敏感亲本和抗性亲本杂交产生的F1代的研究结果表明,杂交品系的抗性倍数分别为22.2倍和24.6倍;抗性显性度D值均小于0,分别为-0.20和-0.17,抗性为常染色体不完全隐性遗传。对4种回交后代和2种自交后代F2的研究结果表明,实际死亡率与期望死亡率差异较大,说明抗性是由单基因多个位点或多基因控制。     

抗Bt棉棉铃虫幼虫Bt受体氨肽酶N(APN2)基因克隆

通过对Bt棉抗性和敏感棉铃虫幼虫中肠氨肽酶N的克隆和测序 ,鉴定了氨肽酶N基因家族的 1个成员Haapn2 ,其cDNA序列具有 3209个核苷酸 ,含有 3096bp的开放阅读框 ,编码产生1032个氨基酸的蛋白质。其推定氨基酸序列具有锌结合模体HEXXHX₁₈E ,N-末端具有 1 7个氨基酸的疏水性信号序列 ,C-末端还具有 2 2个氨基酸的糖基磷酯酰肌醇 (GPI)添加信号肽。比对抗性和敏感棉铃虫cDNA的开放阅读框 ,抗性品系的开放阅读框中 ,有 5 7个点突变 ,共导致了 1 5个氨基酸的改变 ,其中 2个突变 (谷氨酰胺₁₃₇→谷氨酸、缬氨酸₁₃₇→苏氨酸 )位于Cry1A毒素结合区域 ,可能与棉铃虫对转Bt基因棉产生抗性有关。报道的氨肽酶cDNA序列已提交GenBank ,AY346383和AY2 795 35分别是Bt抗性和敏感品系的Haapn2。     

转Bt基因棉花对棉铃虫不同龄期幼虫的杀虫活性和抑制生长作用

以我国培育的转Bt基因棉花(简称Bt棉)为材料,系统测定了Bt棉叶片对棉铃虫 Helicoverpa armigera(Hubner)不同龄期幼虫的杀虫活性和抑制生长的作用。结果表明,Bt棉的杀虫活性随着龄期的增大而趋于降低,其中处理3d只对1龄幼虫有较高效果;从1～4龄开始连续取食Bt棉后均不能化蛹,5龄幼虫则能正常化蛹。3龄幼虫取食Bt棉叶3d后的体重与初始体重之比为0.94,而取食常规棉叶的相应比值为5.48,对幼虫生长的抑制作用明显。在28℃条件下用Bt棉饲养棉铃虫1～3龄幼虫3d,对幼虫的致死率显著高于25℃处理,对3龄幼虫抑制生长的作用也显著提高。研究结果可为确定棉铃虫对Bt棉的抗性监测与治理技术以及Bt棉的田间应用技术提供依据。     

Bt杀虫蛋白对不同品系棉铃虫和中红侧沟茧蜂生长发育的影响

以棉铃虫Helicoverpa armigera (Hübner)室内敏感品系和田间品系为寄主,研究了亚致死浓度的Bt杀虫蛋白对中红侧沟茧蜂Microplitis mediator (Haliday)生长发育的影响。结果表明： 当寄主一直取食,或者在被寄生前12小时开始取食含Bt杀虫蛋白浓度为0,0.5,1.0, 2.0,4.0,8.0 μg/g的饲料时,与对照相比,中红侧沟茧蜂的卵-幼虫历期延长,茧重和成虫体重降低,成虫寿命缩短,但对茧期没有明显影响。Bt杀虫蛋白能有效抑制两个棉铃虫品系幼虫的生长,显著降低棉铃虫蛹重;当Bt蛋白浓度为4.0 μg/g时,显著降低棉铃虫化蛹率。用转双基因抗虫棉SGK321（表达Cry1A+CpTI蛋白）饲喂两个棉铃虫品系初孵幼虫,室内品系的第2、3、4和5天校正死亡率分别为48.5%、87.8%、96.6%和 95.8%,显著高于田间品系（30.9%、59.6%、80.9%及86.1%）。本研究表明,不论是田间品系还是室内品系,棉铃虫取食含Bt杀虫蛋白的饲料后,对中红侧沟茧蜂的生长发育都具有显著的负面作用。     

Effects of Bt cotton and crylac toxin on survival and development of pink bollworm (Lepidoptera: Gelechiidae).

We evaluated the effects of Bacillus thuringiensis (Bt) toxin CrylAc on survival and development of a susceptible strain and laboratory-selected resistant strains of pink bollworm, Pectinophora gossypiella (Saunders). For susceptible and resistant strains tested on artificial diet, increases in CrylAc concentration reduced developmental rate and pupal weight. In greenhouse tests, survival of resistant larvae on transgenic cotton that produces CrylAc (Bt cotton) was 46% relative to their survival on non-Bt cotton. In contrast, Bt cotton killed all susceptible larvae tested. F1 hybrid progeny of resistant and susceptible adults did not survive on Bt cotton, which indicates recessive inheritance of resistance. Compared with resistant or susceptible larvae reared on non-Bt cotton, resistant larvae reared on Bt cotton had lower survival and slower development, and achieved lower pupal weight and fecundity. Recessive resistance to Bt cotton is consistent with one of the basic assumptions of the refuge strategy for delaying resistance to Bt cotton. Whereas slower development of resistant insects on Bt cotton could increase the probability of mating between resistant adults and accelerate resistance, negative effects of Bt cotton on the survival and development of resistant larvae could delay evolution of resistance.     

不同抗性水平棉铃虫地理种群的基因交流对抗性演化的影响

采自我国长江流域棉区的棉铃虫Helicoverpa armigera(Hubner)四川简阳种群、湖北武穴种群、湖南衡阳种群、江苏南京种群和浙江杭州种群对辛硫磷、三氟氯氰菊酯和硫丹的抗性水平分别为8.05～38.80、2.86～290.00和5.87～19.83倍。抗性较高的简阳种群和较低的杭州种群正、反交F₁代对上述3种农药的抗性为13.16和16.69倍、10.43和19.00倍及12.42和10.21倍,抗性遗传的显性系数为-0.2220、-0.2147和0.0700,表明对辛硫磷、三氟氯氰菊酯抗性呈不完全隐性方式遗传,而硫丹抗性则为半隐性遗传。P1自交F2代对辛硫磷、三氟氯氰菊酯和硫丹的抗性水平分别为17.66、7.57和9.03倍,F₁代对杭州种群回交F₂代的抗性倍数分别为8.79、3.Oo和6.61。其结果,高抗区和低抗区棉铃虫种群的基因流动将导致对三种农药抗性水平明显下降。     

The compatibility of a nucleopolyhedrosis virus control with resistance management for Bacillus thuringiensis: co-infection and cross-resistance studies with the diamondback moth, Plutella xylostella

The use of genetically modified crops expressing Bacillus thuringiensis (Bt) toxins can lead to the reduction in application of broad-spectrum pesticides and an increased opportunity for supplementary biological control. Bt microbial sprays are also used by organic growers or as part of integrated pest management programs that rely on the use of natural enemies. In both applications the evolution of resistance to Bt toxins is a potential problem. Natural enemies (pathogens or insects) acting in combination with toxins can accelerate or decelerate the evolution of resistance to Bt. In the present study we investigated whether the use of a nucleopolyhedrovirus (AcMNPV) could potentially affect the evolution of resistance to the Bt toxin Cry1Ac in Plutella xylostella. At low toxin doses there was evidence for antagonistic interactions between AcMNPV and Cry1Ac resistant and susceptible insects. However, this antagonism was much stronger and more widespread for susceptible larvae; interactions were generally not distinguishable from additive for resistant larvae. Selection for resistance to Cry1Ac in two populations of P. xylostella with differing resistance mechanisms did not produce any correlated changes in resistance to AcMNPV. Stronger antagonistic interactions between Bt and AcMNPV on susceptible rather than resistant larvae can decrease the relative fitness between Bt-resistant and susceptible larvae. These interactions and the lack of cross-resistance between virus and toxin suggest that the use of NPV is compatible with resistance management to Bt products.     

Fitness costs and maternal effects associated with resistance to transgenic cotton in the pink bollworm (Lepidoptera: Gelechiidae).

Transgenic cotton producing a Bacillus thuringiensis (Bt) toxin is widely used for controlling the pink bollworm, Perctinophora gossypiella (Saunders). We compared performance of pink bollworm strains resistant to Bt cotton with performance of their susceptible counterparts on non-Bt cotton. We found fitness costs that reduced survival on non-Bt cotton by an average of 51.5% in two resistant strains relative to the susceptible strains. The survival cost was recessive in one set of crosses between a resistant strain and the susceptible strain from which it was derived. However, crosses involving an unrelated resistant and susceptible strain indicated that the survival cost could be dominant. Development time on non-Bt cotton did not differ between the two related resistant and susceptible strains. A slight recessive cost affecting development time was suggested by comparison of the unrelated resistant and susceptible strains. Maternal effects transmitted by parents that had eaten Bt-treated artificial diet as larvae had negative effects on embryogenesis, adult fertility, or both, and reduced the ability of neonates to enter cotton bolls. These results provide further evidence that fitness costs associated with the evolution of resistance to Bt cotton are substantial in the pink bollworm.     

Resistance of Trichoplusia ni Populations Selected by Bacillus thuringiensis Sprays to Cotton Plants Expressing Pyramided Bacillus thuringiensis Toxins Cry1Ac and Cry2Ab

Two populations of Trichoplusia ni that had developed resistance to Bacillus thuringiensis sprays (Bt sprays) in commercial greenhouse vegetable production were tested for resistance to Bt cotton (BollGard II) plants expressing pyramided Cry1Ac and Cry2Ab. The T. ni colonies resistant to Bacillus thuringiensis serovar kurstaki formulations were not only resistant to the Bt toxin Cry1Ac, as previously reported, but also had a high frequency of Cry2Ab-resistant alleles, exhibiting ca. 20% survival on BollGard II foliage. BollGard II-resistant T. ni strains were established by selection with BollGard II foliage to further remove Cry2Ab-sensitive alleles in the T. ni populations. The BollGard II-resistant strains showed incomplete resistance to BollGard II, with adjusted survival values of 0.50 to 0.78 after 7 days. The resistance to the dual-toxin cotton plants was conferred by two genetically independent resistance mechanisms: one to Cry1Ac and one to Cry2Ab. The 50% lethal concentration of Cry2Ab for the resistant strain was at least 1,467-fold that for the susceptible T. ni strain. The resistance to Cry2Ab in resistant T. ni was an autosomally inherited, incompletely recessive monogenic trait. Results from this study indicate that insect populations under selection by Bt sprays in agriculture can be resistant to multiple Bt toxins and may potentially confer resistance to multitoxin Bt crops.     

Next‐generation transgenic cotton: pyramiding RNAi and Bt counters insect resistance

Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are extensively cultivated worldwide. To counter rapidly increasing pest resistance to crops that produce single Bt toxins, transgenic plant ‘pyramids’ producing two or more Bt toxins that kill the same pest have been widely adopted. However, cross‐resistance and antagonism between Bt toxins limit the sustainability of this approach. Here we describe development and testing of the first pyramids of cotton combining protection from a Bt toxin and RNA interference (RNAi). We developed two types of transgenic cotton plants producing double‐stranded RNA (dsRNA) from the global lepidopteran pest Helicoverpa armigera designed to interfere with its metabolism of juvenile hormone (JH). We focused on suppression of JH acid methyltransferase (JHAMT), which is crucial for JH synthesis, and JH‐binding protein (JHBP), which transports JH to organs. In 2015 and 2016, we tested larvae from a Bt‐resistant strain and a related susceptible strain of H. armigera on seven types of cotton: two controls, Bt cotton, two types of RNAi cotton (targeting JHAMT or JHBP) and two pyramids (Bt cotton plus each type of RNAi). Both types of RNAi cotton were effective against Bt‐resistant insects. Bt cotton and RNAi acted independently against the susceptible strain. In computer simulations of conditions in northern China, where millions of farmers grow Bt cotton as well as abundant non‐transgenic host plants of H. armigera, pyramided cotton combining a Bt toxin and RNAi substantially delayed resistance relative to using Bt cotton alone.     

Relative fitness of Cry1A-resistant and -susceptible Helicoverpa armigera (Lepidoptera: Noctuidae) on conventional and transgenic cotton

Glasshouse and laboratory experiments were conducted to evaluate the relative fitness of Cry1A-susceptible and laboratory-selected resistant strains of Helicoverpa armigera (Hübner). Life history parameters of H. armigera larvae feeding on young cotton plants showed a significant developmental delay of up to 7 d for the resistant strain compared with the susceptible strain on non-Bacillus thuringiensis (Bt) cotton. This fitness cost was not evident on artificial diet. There was no developmental delay in the F1 hybrid progeny from the reciprocal backcross of the resistant and susceptible strains, indicating that the fitness cost is recessive. In two cohorts tested, survival to pupation of resistant larvae on Bt cotton expressing Cry1Ac was 54 and 51% lower than on non-Bt cotton, whereas all susceptible and F1 larvae tested on Cry1Ac cotton were killed. Mortality of susceptible larvae occurred in the first or second instar, whereas the F1 larvae were able to develop to later instars before dying, demonstrating that resistance is incompletely recessive. The intrinsic rate of increase was reduced by >50% in the resistant strain on Cry1Ac cotton compared with the susceptible strain on non-Bt cotton. There was a significant reduction in the survival of postdiapausal adults from the resistant strain and the F1 strains, indicating that there is a nonrecessive overwintering cost associated with Cry1A resistance in H. armigera.     

Fitness of Cry1A-resistant and -susceptible Helicoverpa armigera (Lepidoptera: Noctuidae) on transgenic cotton with reduced levels of Cry1Ac

The performance of Helicoverpa armigera (Hübner) on 15-wk-old cotton plants was compared for a susceptible strain, a near-isogenic laboratory-selected strain, and F1 progeny of the two strains. Glasshouse experiments were conducted to test the three insect types on conventional plants and transgenic plants that produced the Bacillus thuringiensis (Bt) toxin Cry1Ac. At the time of testing (15 wk), the Cry1Ac concentration in cotton leaves was 75% lower than at 4 wk. On these plants, < 10% of susceptible larvae reached the fifth instar, and none survived to pupation. In contrast, survival to adulthood on Cry1Ac cotton was 62% for resistant larvae and 39% for F1 larvae. These results show that inheritance of resistance to 15-wk-old Cry1Ac cotton is partially dominant, in contrast to results previously obtained on 4-wk-old Cry1Ac cotton. Growth and survival of resistant insects were similar on Cry1Ac cotton and on non-Bt cotton, but F1 insects developed more slowly on Cry1Ac cotton than on non-Bt cotton. Survival was lower and development was slower for resistant larvae than for susceptible and F1 larvae on non-Bt cotton. These results show recessive fitness costs are associated with resistance to Cry1Ac.