Resistance of transgenic cotton to cotton bollworm and cutworms

为了明确转BtCrylA+CP4EPSPS基因抗棉铃虫抗草甘膦棉花对棉铃虫Helicoverpa armigera(Hübner)的抗性水平及对八字地老虎Amathes c-nigrum和黄地老虎Agrotis segetum的影响,作者用转基因棉花和非转基因棉花叶片对棉铃虫、八字地老虎和黄地老虎幼虫进行了室内饲养观察.结果表明,转基因抗棉铃虫抗草甘膦棉花对新疆南部第2代棉铃虫表现为中抗,幼虫校正死亡率为70.4％;棉花叶片在整个生育期均能表达Bt毒蛋白,整个生育期Bt毒蛋白含量呈逐渐降低的趋势.转基因抗棉铃虫抗草甘膦棉花对八字地老虎幼虫有强烈的毒杀作用,可以引起幼虫大量死亡,对存活幼虫的生长发育也有明显的抑制作用,幼虫发育迟缓,不能顺利化蛹;但对黄地老虎幼虫没有明显的影响,幼虫可以正常生长.     

转基因抗虫抗除草剂棉花对棉铃虫抗性及对草甘膦耐受性研究

本论文研究了抗虫又抗除草剂棉花对草甘膦的耐受程度,比较了这一双抗性状的棉花与单抗虫棉的抗虫效果。结果表明,抗虫又抗除草剂棉花对草甘膦有较好的耐受性,四叶期喷施草甘膦后抗虫抗除草剂棉花可以安全生长,蕾期喷施草甘膦对棉花的开花率和结铃率有影响。抗虫又抗除草剂棉花和单抗虫棉对棉铃虫Helicoverpa armigera(Hübner)均具有较好的防治效果,苗期棉花叶片对棉铃虫防治效果最好,后期防治效果下降到49.2%和46.6%,吐絮期防治效果又上升到57.0%和53.1%。     

新疆棉区转Bt基因棉对棉铃虫生物学的影响

棉铃虫Helicoverpaarmigera高龄幼虫取食转Bt基因棉花组织后 ,化蛹率、羽化率、蛹重、体长均有显著下降 ,在转Bt基因棉花上棉铃虫的取食行为也受到较大的影响 ,表现为取食次数明显减少、吐丝下垂次数明显增加 ;无论是转Bt基因棉花还是常规棉花 ,棉铃虫 3龄幼虫主要分布于繁殖器官上 ,在转Bt基因棉花各繁殖器官上的分布概率为 :花 >棉铃 >棉蕾 ,常规棉花上分布概率为 :棉蕾 >棉铃 >花 ;棉铃虫高龄幼虫取食转Bt基因棉花各组织 ,成虫羽化后产卵量、卵孵化率均有明显下降 ;在较低的棉铃虫虫口密度下 ,转Bt基因棉花对棉铃虫有一定的产卵排趋性。     

Bt棉花对棉铃虫幼虫选择性行为的影响

利用“H”型嗅觉仪测定结果表明 :在棉花蕾期、花铃期 ,棉铃虫 2龄幼虫对常规棉的棉叶、嫩头的选择性明显高于转基因棉花品种 ,差异达显著或极显著水平。但 3龄幼虫对两者的选择性差异并不显著 ;而在棉花蕾期 ,果枝对棉铃虫 2龄幼虫选择性行为有一定的影响。试验初步说明Bt棉对棉铃虫低龄幼虫有一定的忌避作用 ,而对高龄幼虫忌避效应不显著。     

转(Bt Cry1Ac+CP_4EPSPS)基因抗虫抗草甘膦棉花对草甘膦的耐受性研究

本研究比较了转Bt Cry1Ac+CP_4EPSPS基因抗棉铃虫抗草甘膦棉花与常规棉花在新疆棉区对草甘膦的耐受性差异。两年的研究结果表明,转Bt Cry1Ac+CP_4EPSPS基因抗棉铃虫抗草甘膦棉花对草甘膦有较好的耐受性,而对照棉花中棉所49对草甘膦耐受性较差。苗期喷施草甘膦后转基因抗棉铃虫抗草甘膦棉花生长发育没有受到影响,而对照棉花中棉所49喷施草甘膦后生长发育受到了明显的影响,个别植株甚至死亡。转Bt Cry1Ac+CP_4EPSPS基因抗棉铃虫抗草甘膦棉花株高、真叶数、蕾数、产量等指标与对照相比差异显著。转Bt Cry1Ac+CP_4EPSPS基因棉花对草甘膦的耐受程度显著高于非转基因棉花。草甘膦对转基因抗草甘膦棉花无负面影响。     

Bt毒蛋白对棉铃虫的生长发育和相关酶活性的影响

棉铃虫Helicoverpaarmigera幼虫取食不同浓度Bt毒蛋白的人工饲料后 ,随着Bt毒蛋白浓度的升高 ,幼虫的体重增长量和蛹重下降 ,死亡率明显上升。羧酸酯酶和乙酰胆碱酯酶的活力也呈升高趋势。在棉铃虫中肠内蛋白酶中 ,类胰凝乳蛋白酶的活力随Bt蛋白浓度增大而显著升高 ;类胰蛋白酶活性在Bt蛋白浓度为 2 0 μg g时达到最高 ,随后开始下降。     

不同Bt蛋白对棉铃虫存活率和生长发育的影响

【背景】在我国Bt棉主要以Cry1Ab或Cry1Ac为主,其他新型Bt基因未被转入棉花中用来控制害虫,然而大面积种植单价Bt基因的棉花,将可能会大大增加靶标害虫对该类型Bt棉花抗性频率,因此研究其他新型Bt蛋白对靶标害虫的控制作用显得十分必要。【方法】采用蛋白混入人工饲料的生物测定方法,在室内测定了6种Bt蛋白对棉铃虫初孵幼虫的毒力,比较了浓度为1.0μg·g-1时不同Bt蛋白对棉铃虫幼虫生长发育的影响。【结果】毒力测定结果表明,不同Bt蛋白对棉铃虫初孵幼虫的毒力不同,LC50值由低到高依次为Cry1Ab 0.065μg·g-1、Cry1Ac 0.074μg·g-1、Cry2Ab 0.133μg·g-1、Cry2Aa 11.670μg·g-1、Cry1Ah 13.010μg·g-1和Cry1Ca20μg·g-1。生长发育测定结果表明,Cry1Ab和Cry1Ac对棉铃虫幼虫的生长发育影响最大,Cry2Ab次之;Cry1Ah和Cry2Aa对1龄幼虫的校正死亡率和体重抑制率差别不大,但对2龄幼虫的差异较大,Cry1Ah处理2龄幼虫后体重和生长发育参数与Cry2Ab接近,而Cry1Ca对棉铃虫幼虫生长发育几乎没影响。【结论与意义】Cry1Ah、Cry2Aa和Cry2Ab的毒力不如Cry1Ac和Cry1Ab,但仍可以作为控制棉铃虫幼虫的替代策略。     

不同Bt蛋白对棉铃虫存活率和生长发育的影响

在我国Bt棉主要以Cry1Ab或Cry1Ac为主,其他新型Bt基因未被转入棉花中用来控制害虫,然而大面积种植单价Bt基因的棉花,将可能会大大增加靶标害虫对该类型Bt棉花抗性频率,因此研究其他新型Bt蛋白对靶标害虫的控制作用显得十分必要。采用蛋白混入人工饲料的生物测定方法,在室内测定了6种Bt蛋白对棉铃虫初孵幼虫的毒力,比较了浓度为1．0μg· g-1时不同Bt蛋白对棉铃虫幼虫生长发育的影响。毒力测定结果表明,不同Bt蛋白对棉铃虫初孵幼虫的毒力不同,LC50值由低到高依次为Cry1Ab 0．065μg· g-1、Cry1Ac 0．074μg· g-1、Cry2Ab 0．133μg· g-1、Cry2Aa 11．670μg· g-1、Cry1Ah 13．010μg· g-1和Cry1Ca＞20μg· g-1。生长发育测定结果表明,Cry1Ab和Cry1Ac对棉铃虫幼虫的生长发育影响最大,Cry2Ab次之;Cry1Ah和Cry2Aa对1龄幼虫的校正死亡率和体重抑制率差别不大,但对2龄幼虫的差异较大,Cry1Ah处理2龄幼虫后体重和生长发育参数与Cry2Ab接近,而Cry1Ca对棉铃虫幼虫生长发育几乎没影响。Cry1Ah、Cry2Aa和Cry2Ab的毒力不如Cry1Ac和Cry1Ab,但仍可以作为控制棉铃虫幼虫的替代策略。     

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。     

A54外毒素与Bt毒蛋白对棉铃虫的交互作用

研究测定了A5 4胞外毒素与Bt毒蛋白对棉铃虫的交互作用。结果发现随着棉铃虫饲料中A5 4外毒素浓度的增加 ,棉铃虫幼虫的死亡率显著上升 :随着棉铃虫饲料中Bt毒蛋白浓度的增加 ,棉铃虫幼虫的死亡率也显著上升。方差分析的结果表明二者互有增效作用。     

Effect of water-deficit stress on cotton plants expressing the Bacillus thuringiensis toxin

Bacillus thuringiensis (Bt) crops require a high dosage of Bt toxin to delay development of insect resistance, in particular, when the refuge strategy is applied. This strategy is threatened by plant developmental and environmental factors that might reduce Bt toxin concentration and Bt efficacy in Bt crops. Growth of Bt (Cry1Ac) cotton under prolonged, moderate water deficit as a single stress factor was evaluated. Bt cotton plants were analysed for physiological performance, Bt toxin concentration and Bt efficacy. For performance analysis, leaf and total plant dry weight and leaf area were measured. Bt toxin concentration was determined by an immuno‐assay. Effects of Bt toxin on growth and mortality of African cotton bollworm, Helicoverpa armigera, larvae were measured in different plant organs. Leaves from young plants exposed for 30 days to moderate water deficit had both higher Bt toxin concentrations and were more effective against larvae than leaves, flowers or bolls from mature flowering plants exposed to 60 days of moderate water deficit. Although growth of Bt cotton plants under moderate water‐deficit conditions decreased Bt concentrations in leaves, flowers and bolls, this had no effect on efficacy against first‐instar cotton bollworm larvae. No significant evidence was found that moderate water deficit, as a single stress factor, decreases Bt efficacy in Bt cotton.     

Early warning of cotton bollworm resistance associated with intensive planting of Bt cotton in China

Transgenic crops producing Bacillus thuringiensis (Bt) toxins kill some key insect pests, but evolution of resistance by pests can reduce their efficacy. The predominant strategy for delaying pest resistance to Bt crops requires refuges of non-Bt host plants to promote survival of susceptible pests. To delay pest resistance to transgenic cotton producing Bt toxin Cry1Ac, farmers in the United States and Australia planted refuges of non-Bt cotton, while farmers in China have relied on "natural" refuges of non-Bt host plants other than cotton. Here we report data from a 2010 survey showing field-evolved resistance to Cry1Ac of the major target pest, cotton bollworm (Helicoverpa armigera), in northern China. Laboratory bioassay results show that susceptibility to Cry1Ac was significantly lower in 13 field populations from northern China, where Bt cotton has been planted intensively, than in two populations from sites in northwestern China where exposure to Bt cotton has been limited. Susceptibility to Bt toxin Cry2Ab did not differ between northern and northwestern China, demonstrating that resistance to Cry1Ac did not cause cross-resistance to Cry2Ab, and implying that resistance to Cry1Ac in northern China is a specific adaptation caused by exposure to this toxin in Bt cotton. Despite the resistance detected in laboratory bioassays, control failures of Bt cotton have not been reported in China. This early warning may spur proactive countermeasures, including a switch to transgenic cotton producing two or more toxins distinct from Cry1A toxins.     

Helicoverpa armigera baseline susceptibility to Bacillus thuringiensis Cry toxins and resistance management for Bt cotton in India

Transgenic cotton that produces insecticidal proteins from Bacillus thuringiensis (Bt), often referred to as Bt cotton, is widely grown in many countries. Bt cotton with a single cry1A gene and stacked also with cry2A gene has provided satisfactory protection against the damage by the lepidopteran bollworms, especially the cotton bollworm, Helicoverpa armigera (Hübner) which is considered as a key pest. The baseline susceptibility of the larvae of H. armigera to Cry1Ac and other toxins carried out in many countries has provided a basis for monitoring resistance. There is no evidence of development of field-level resistance in H. armigera leading to the failure of Bt cotton crop anywhere in the world, despite the fact that Bt cotton was grown on the largest ever area of 12.1 million hectares in 2006 and its cumulative cultivation over the last 11 years has surpassed the annual cotton area in the world. Nevertheless, the Bt resistance management has become a necessity to sustain Bt cotton and other transgenic crops in view of potential of the target insects to evolve Cry toxin resistance.     

Effects of transgenic Cry1A＋CpTI cotton and Cry1Ac toxin on the parasitoid, Campoketis chlorideae （Hymenoptera：Ichneumonidae）

Abstract The effect of transgenic double genes, Cry1A + CpTI cotton and Cry1Ac toxin on the parasitoid, Campoketis chlorideae Uchida of cotton bollworm, Helicoverpa armigera (Hübner), was investigated in the laboratory. Helicoverpa armigera larvae when in the first, second and third instar could not survive if fed on transgenic cotton leaves. Consequently, C. chlorideae larvae could not complete their development if parasitizing on such hosts. After H. armigera larvae were reared on transgenic or traditional cotton leaves for 12J48 hours, they were parasitized by C. chlorideae females. Parasitized larvae continued to feed on transgenic or traditional cotton for 12–48 h. The present results showed that the body weight of larvae of the parasitoids were significantly reduced when parasitized hosts fed on transgenic cotton leaves compared to those fed on traditional cotton. Duration of egg and larvae stage were significantly prolonged, pupal and adult weight of C. chloridae was decreased when the host larvae fed on transgenic cotton leaves longer than 48 h. The development duration of C. chlorideae pupae on the hosts fed on transgenic cotton leaves in each treatment was not significantly different from those of controls. The longevity of parasitoid females and males fed with a solution containing Cry1Ac toxin was not significantly different with that of the control.     

转Bt基因抗虫棉对棉铃虫拒食作用及其机理研究

转Bt基因抗虫棉对 3 ,4,5龄棉铃虫幼虫的抗性表现形式为拒食作用 ,且随幼虫龄期的增加拒食作用明显降低 ,其中对 5龄幼虫的拒食作用很低。取食Bt棉后 ,3 ,4,5龄棉铃虫幼虫中肠消化酶比活力均较对照有所减退 ,且随幼虫龄期的增加减退率明显降低 ,其中对 5龄幼虫减退率最低。由此 ,解释了Bt棉对 3龄及 3龄以上棉铃虫幼虫抗性表现形式、抗性随不同幼虫龄期的差异性 ,及其抗性差异性的消化机理。     

Increasing tolerance to Cry1Ac cotton from cotton bollworm, Helicoverpa armigera, was confirmed in Bt cotton farming area of China

Abstract.  1. Changes in the frequency of Cry1Ac resistance genes and shifts in tolerance of cotton bollworm, Helicoverpa armigera , to the Cry1Ac toxin were assessed using bioassays of F₁ and F₂ offspring of isofemale lines from Anci County of Hebei Province (a multiple-crop system including corn, soybean, peanut, and Bt cotton) and Xiajin County of Shandong Province (an intensive Bt cotton planting area) in Northern China during 2002–2005.
2. A conservative analysis of the overall results indicated that there was a small increase in the frequency of major, non-recessive resistance genes over time.
3. The relative average development ratings [RADR – growth rate of a line on a Bt diet in proportion to the growth rate on a non-Bt (NBT) diet] of the bollworm larvae in F₁ tests increased significantly from year to year, indicating a gradual trend towards higher tolerance to Cry1Ac in the field populations.
4. There were also significant positive correlations between RADR of the lines in the F₁ generation and the RADR of their F₂ offspring, indicating that the tolerance was genetically based.
5. Quantitative genetic simulation analysis showed that resistance of H . armigera to Bt cotton in Xiajin could evolve to a high level in 11–15 years if no effective resistance management measures are carried out.     

Sustained susceptibility of pink bollworm to Bt cotton in the United States

Evolution of resistance by pests can reduce the benefits of transgenic crops that produce toxins from Bacillus thuringiensis (Bt) for insect control. One of the world's most important cotton pests, pink bollworm (Pectinophora gossypiella), has been targeted for control by transgenic cotton producing Bt toxin Cry1Ac in several countries for more than a decade. In China, the frequency of resistance to Cry1Ac has increased, but control failures have not been reported. In western India, pink bollworm resistance to Cry1Ac has caused widespread control failures of Bt cotton. By contrast, in the state of Arizona in the southwestern United States, monitoring data from bioassays and DNA screening demonstrate sustained susceptibility to Cry1Ac for 16 y. From 1996-2005, the main factors that delayed resistance in Arizona appear to be abundant refuges of non-Bt cotton, recessive inheritance of resistance, fitness costs associated with resistance and incomplete resistance. From 2006-2011, refuge abundance was greatly reduced in Arizona, while mass releases of sterile pink bollworm moths were made to delay resistance as part of a multi-tactic eradication program. Sustained susceptibility of pink bollworm to Bt cotton in Arizona has provided a cornerstone for the pink bollworm eradication program and for integrated pest management in cotton. Reduced insecticide use against pink bollworm and other cotton pests has yielded economic benefits for growers, as well as broad environmental and health benefits. We encourage increased efforts to combine Bt crops with other tactics in integrated pest management programs.     

盐碱土壤转Bt基因棉花外源蛋白表达量时空变化及对抗虫性的影响

盐碱地是潜在的可利用耕地资源,但土壤盐碱化严重制约了农业生产的可持续发展。基于棉花机械化程度低、劳动力成本和生产资料投入剧增、比较效益下降和实施粮食生产安全战略等因素影响,我国长江流域和黄河流域棉花面积锐减,种植区域向内陆盐碱旱地或滨海盐碱地转移,但目前针对盐碱地转Bt基因棉种植可能带来的生态安全性问题研究甚少,正成为国内外研究的焦点和热点。伴随着棉花向盐碱地大面积转移种植趋势,检测盐胁迫是否影响转基因抗虫棉抗虫性,明确其影响程度,直接关系到转基因抗虫棉种植的安全性,也是目前抗虫棉扩大生产中迫切需要解决的问题。以非转基因棉花为对照,分别在低盐、中盐和高盐土壤种植的棉花的苗期、蕾期和花铃期采样,室内测定了转Bt基因棉花叶片对棉铃虫幼虫校正死亡率和外源蛋白表达量。研究结果发现盐分胁迫下转Bt基因棉花苗期叶片对棉铃虫幼虫校正死亡率下降了9.22%—47.46%,蕾期下降了31.61%—45.42%,花铃期下降了3.59%—18.52%;土壤盐分显著降低了转Bt基因棉花叶片中外源蛋白的表达量,苗期功能叶外源蛋白表达量下降了7.66%—29.86%;蕾期下降了3.77%—36.85%;花铃期下降了18.13%—41.02%;相关性分析表明,盐分胁迫条件下转Bt基因棉花叶片中外源蛋白表达量与其对棉铃虫抗性程度存在正相关关系。结果表明,盐碱土壤显著降低了转Bt基因棉花叶片外源杀虫蛋白表达量,从而导致转Bt基因棉花叶片对棉铃虫的抗虫性下降。研究土壤盐分对转Bt基因棉花对棉铃虫的影响及其作用机制,可为建立盐碱地转Bt基因棉花田害虫综合防控技术体系、转Bt基因棉花环境安全评价及转Bt基因棉安全管理提供依据。     

Control of resistant pink bollworm (Pectinophora gossypiella) by transgenic cotton that produces Bacillus thuringiensis toxin Cry2Ab

Crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with B. thuringiensis produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (Pectinophora gossypiella), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces B. thuringiensis toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 microg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 microg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 microg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended.