Toxicity and characterization of cotton expressing Bacillus thuringiensis Cry1Ac and Cry2Ab2 proteins for control of lepidopteran pests

Cry1Ac protoxin (the active insecticidal toxin in both Bollgard and Bollgard II cotton [Gossypium hirsutum L.]), and Cry2Ab2 toxin (the second insecticidal toxin in Bollgard II cotton) were bioassayed against five of the primary lepidopteran pests of cotton by using diet incorporation. Cry1Ac was the most toxic to Heliothis virescens (F.) and Pectinophora gossypiella (Saunders), demonstrated good activity against Helicoverpa zea (Boddie), and had negligible toxicity against Spodoptera exigua (Hübner) and Spodoptera frugiperda (J. E. Smith). Cry2Ab2 was the most toxic to P. gossypiella and least toxic to S. frugiperda. Cry2Ab2 was more toxic to S. exigua and S. frugiperda than Cry1Ac. Of the three insect species most sensitive to both Bacillus thuringiensis (Bt) proteins (including H. zea), P. gossypiella was only three-fold less sensitive to Cry2Ab2 than Cry1Ac, whereas H. virescens was 40-fold less sensitive to Cry2Ab2 compared with CrylAc. Cotton plants expressing Cry1Ac only and both Cry1Ac and Cry2Ab2 proteins were characterized for toxicity against H. zea and S.frugiperda larvae in the laboratory and H. zea larvae in an environmental chamber. In no-choice assays on excised squares from plants of different ages, second instar H. zea larvae were controlled by Cry1Ac/Cry2Ab2 cotton with mortality levels of 90% and greater at 5 d compared with 30-80% mortality for Cry1Ac-only cotton, depending on plant age. Similarly, feeding on leaf discs from Cry1Ac/Cry2Ab2 cotton resulted in mortality of second instars of S.frugiperda ranging from 69 to 93%, whereas exposure to Cry1Ac-only cotton yielded 20-69% mortality, depending on plant age. When cotton blooms were infested in situ in an environmental chamber with neonate H. zea larvae previously fed on synthetic diet for 0, 24, or 48 h, 7-d flower abortion levels for Cry1Ac-only cotton were 15, 41, and 63%, respectively, whereas for Cry1Ac/Cry2Ab2 cotton, flower abortion levels were 0, 0, and 5%, respectively. Cry1Ac and Cry2Ab2 concentrations were measured within various cotton tissues of Cry1Ac-only and Cry1Ac/Cry2Ab2 plants, respectively, by using enzyme-linked immunosorbent assay. Terminal leaves significantly expressed the highest, and large leaves, calyx, and bracts expressed significantly the lowest concentrations of Cry1Ac, respectively. Ovules expressed significantly the highest, and terminal leaves, large leaves, bracts, and calyx expressed significantly (P < 0.05) the lowest concentrations of Cry2Ab2. These results help explain the observed differences between Bollgard and Bollgard II mortality against the primary lepidopteran cotton pests, and they may lead to improved scouting and resistance management practices, and to more effective control of these pests with Bt transgenic crops in the future.     

Different cross-resistance patterns in the diamondback moth (Lepidoptera: Plutellidae) resistant to Bacillus thuringiensis toxin Cry1C.

Two strains of the diamondback moth, Plutella xylostella (L.), were selected using Cry1C protoxin and transgenic broccoli plants expressing a Cry1C toxin of Bacillus thuringiensis (Bt). Both strains were resistant to Cry1C but had different cross-resistance patterns. We used 12 Bt protoxins for cross-resistance tests, including Cry1Aa, Cry1Ab, Cry1Ac, Cry1Bb, Cry1C, Cry1D, Cry1E, Cry1F, Cry1J, Cry2Ab, Cry9Aa, and Cry9C. Compared with the unselected sister strain (BCS), the resistance ratio (BR) of one strain (BCS-Cry1C-1) to the Cry1C protoxin was 1,090-fold with high level of cross-resistance to Cry1Aa, Cry1Ab, Cry1Ac, Cry1F, and Cry1J (RR > 390-fold). The cross-resistance to Cry1A, Cry1F, and Cry1J in this strain was probably related to the Cry1A resistance gene(s) that came from the initial field population and was caused by intensive sprayings of Bt products containing Cry1A protoxins. The neonates of this strain can survive on transgenic broccoli plants expressing either Cry1Ac or Cry1C toxins. The other strain (BCS-Cry1C-2) was highly resistant to Cry1C but not cross-resistant to other Bt protoxins. The neonates of this strain can survive on transgenic broccoli expressing Cry1C toxin but not Cry1Ac toxin. The gene(s) conferring resistance to Cry1C segregates independently from Cry1Ac resistance in these strains. The toxicity of Cry1E and Cry2Ab protoxins was low to all of the three strains. The overall progress of all work has resulted in a unique model system to test the stacked genes strategy for resistance management of Bt transgenic crops.     

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.     

Cry1Ie毒素胁迫下亚洲玉米螟的抗性发展及汰选种群对其他Bt毒素的交互抗性

亚洲玉米螟Ostrinia furnacalis (Guenée) 是危害玉米的重要害虫之一, 转Bt基因抗虫玉米为其防治提供了新的途径。然而, 靶标害虫产生抗性将严重阻碍Bt制剂及转Bt基因抗虫玉米的持续应用。明确害虫对转Bt基因玉米表达的毒素蛋白的抗性演化, 对于制定科学有效的抗性治理策略具有重要的理论和实际意义。本实验通过人工饲料汰选法研究了Bt Cry1Ie毒素胁迫下亚洲玉米螟的抗性发展及汰选14代的种群对其他Bt毒素（Cry1Ab, Cry1Ac和Cry1Fa）的交互抗性, 并观察了Cry1Ie蛋白胁迫对亚洲玉米螟生物学的影响。结果表明： 随着汰选压不断提高, 亚洲玉米螟种群对Cry1Ie毒素的敏感性逐渐下降。汰选14代后, 种群对Cry1Ie毒素的抗性水平提高了23倍。然而, Cry1Ab, Cry1Ac和Cry1Fa对所获Cry1Ie汰选种群的毒力与对敏感种群的毒力相比没有显著差异, 说明Cry1Ie汰选没有引起亚洲玉米螟对Cry1Ab, Cry1Ac和Cry1Fa毒素产生交互抗性。同时, 与敏感种群相比, Cry1Ie汰选14代的种群幼虫平均发育历期延长5.7 d, 蛹重减轻13.7%, 单雌产卵量下降40.0%。本研究结果说明, 大面积单一种植转cry1Ie基因抗虫玉米, 可能引起亚洲玉米螟产生抗性; 亚洲玉米螟Cry1Ie抗性种群对Cry1Ab, Cry1Ac和Cry1Fa没有交互抗性, 含有cry1Ie和cry1Ab, cry1Ac或cry1F双/多基因抗虫玉米, 可作为靶标害虫抗性治理的重要策略。     

Selection for Cry1F resistance in the European corn borer and cross-resistance to other Cry toxins

Evolution of resistance by insect pests is the greatest threat to the continued success of Bacillus thuringiensis (Bt) toxins used in insecticide formulations or expressed by transgenic crop plants such as Cry1F‐expressing maize [(Zea mays L.) (Poaceae)]. A strain of European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), obtained from field collections throughout the central US Corn Belt in 1996 was selected in the laboratory for resistance to Cry1F by exposure to the toxin incorporated into artificial diet. The selected strain developed more than 3000‐fold resistance to Cry1F after 35 generations of selection and readily consumed Cry1F expressing maize tissue; yet, it was as susceptible to Cry1Ab and Cry9C as the unselected control strain. Only a low level of cross‐resistance (seven‐fold) to Cry1Ac was observed. These lacks of cross‐resistance between Cry1F and Cry1Ab suggest that maize hybrids expressing these two toxins are likely to be compatible for resistance management of O. nubilalis.     

Fitness of Bt‐resistant cabbage loopers on Bt cotton plants

Development of resistance to the insecticidal toxins from Bacillus thuringiensis (Bt) in insects is the major threat to the continued success of transgenic Bt crops in agriculture. The fitness of Bt‐resistant insects on Bt and non‐Bt plants is a key parameter that determines the development of Bt resistance in insect populations. In this study, a comprehensive analysis of the fitness of Bt‐resistant Trichoplusia ni strains on Bt cotton leaves was conducted. The Bt‐resistant T. ni strains carried two genetically independent mechanisms of resistance to Bt toxins Cry1Ac and Cry2Ab. The effects of the two resistance mechanisms, individually and in combination, on the fitness of the T. ni strains on conventional non‐Bt cotton and on transgenic Bt cotton leaves expressing a single‐toxin Cry1Ac (Bollgard I) or two Bt toxins Cry1Ac and Cry2Ab (Bollgard II) were examined. The presence of Bt toxins in plants reduced the fitness of resistant insects, indicated by decreased net reproductive rate (R₀) and intrinsic rate of increase (r). The reduction in fitness in resistant T. ni on Bollgard II leaves was greater than that on Bollgard I leaves. A 12.4‐day asynchrony of adult emergence between the susceptible T. ni grown on non‐Bt cotton leaves and the dual‐toxin‐resistant T. ni on Bollgard II leaves was observed. Therefore, multitoxin Bt plants not only reduce the probability for T. ni to develop resistance but also strongly reduce the fitness of resistant insects feeding on the plants.     

A Sensitive Bioassay for the Detection of Cry1A Toxin Expression in Transgenic Cotton

In the process of development of insect resistant transgenic plants and also to evaluate the consistency in expression of the toxin under greenhouse and field conditions, immunological and bioassays are commonly used. The assay being described in this report, is based on the high levels of sensitivity of a cotton leaf feeding insect, the semilooper, Anomis flava (Fabricius) to Cry toxins (Cry1Aa, Cry1Ab and Cry1Ac). The assay is sensitive, quick and reproducible. Cry1Ac was the most toxic followed by Cry1Ab and Cry1Aa. LC 50 s of the three toxins on first instar larvae ranged from 0.79-6.08 ng cm -2 of leaf. LC 50 s of Cry1Ac for the fourth instar larvae ranged from 12.91-21.14 ng cm -2 while LC 50 s for Cry1Aa and Cry1Ab were in the range 53.0-138 ng cm -2 . The fiducial limits (at 95% probability) of the probit assay data indicated that there was no difference in response between the three different populations to each of the three toxins. The data from all assays were pooled for each of the three toxins separately and subjected to regression analysis to obtain a cumulative log dose response for first and fourth instar larvae. These can be used as standard curves to quantify toxin expression in plants based on mortality response of either first or fourth instar A. flava larvae. Apart from being used to detect expression in putative Bt cotton transgenic plants, the assay can also be used to follow the activity of Cry toxins in transgenic cotton plants in the field during the growing season.     

Field trials to evaluate effects of Bt-transgenic silage corn expressing the Cry1Ab insecticidal toxin on non-target soil arthropods in northern New England, USA

Traditionally, control of European corn borer (Ostrinia nubilalis) Hübner has been achieved through the use of chemical insecticides. With increasing emphasis on reducing pesticide inputs in agricultural production, alternative management technologies are now being used including transgenic silage corn modified to express Cry1Ab protein toxins derived from Bacillus thuringiensis (Bt) Berliner. The Cry1Ab toxin is expressed by all plant cells and throughout the growing season. Furthermore, the toxins are exuded from corn plant roots into the rhizosphere, raising concerns over possible side-effects on non-target beneficial organisms in the same habitat. In addition, detrivores are exposed to crop residues containing the toxin when incorporated into the soil. The current 2-year study (2003, 2004) evaluated effects of two silage-corn varieties: Pioneer var. 38A25 (Bt-corn expressing the Cry1Ab toxin) and Pioneer var. 38A24 (parent isoline) on species diversity and evenness of carabid beetles and Collembola. Pitfall traps were used to collect surface-dwelling species on a bimonthly schedule from April to October. Soil cores were taken once a month from April to October to sample subterranean species, which were extracted using Berlese funnels. All individuals were recorded and identified where possible to species level for analysis in the Simpson’s D and Shannon–Wiener H’ diversity indices. Evenness was measured using Simpson’s E’, after which dominant species were analyzed in a multivariate ordination analysis. Results showed Bt-corn had no negative effects on any of the organisms analyzed. There was a significant year effect on the abundance of surface-dwelling Collembola and on species diversity of soil-dwelling Collembola. Our findings suggest that crop management practices and/or environmental conditions (e.g., heavy rainfall during the 2004 growing season) had the greatest impact on species diversity and evenness, rather than the crop itself (Bt or isoline).     

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.     

Binding sites for Bacillus thuringiensis Cry2Ae toxin on heliothine brush border membrane vesicles are not shared with Cry1A, Cry1F, or Vip3A toxin

The use of combinations of Bacillus thuringiensis (Bt) toxins with diverse modes of action for insect pest control has been proposed as the most efficient strategy to increase target range and delay the onset of insect resistance. Considering that most cases of cross-resistance to Bt toxins in laboratory-selected insect colonies are due to alteration of common toxin binding sites, independent modes of action can be defined as toxins sharing limited or no binding sites in brush border membrane vesicles (BBMV) prepared from the target insect larvae. In this paper, we report on the specific binding of Cry2Ae toxin to binding sites on BBMV from larvae of the three most commercially relevant heliothine species, Heliothis virescens, Helicoverpa zea, and Helicoverpa armigera. Using chromatographic purification under reducing conditions before labeling, we detected specific binding of radiolabeled Cry2Ae, which allowed us to perform competition assays using Cry1Ab, Cry1Ac, Cry1Fa, Vip3A, Cry2Ae, and Cry2Ab toxins as competitors. In these assays, Cry2Ae binding sites were shared with Cry2Ab but not with the tested Cry1 or Vip3A toxins. Our data support the use of Cry2Ae toxin in combination with Cry1 or Vip3A toxins in strategies to increase target range and delay the onset of heliothine resistance.     

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.     

Variant cry1Ab entomocidal Bacillus thuringiensis toxin gene facilitates the recovery of an increased number of lepidopteran insect resistant independent rice transformants against yellow stem borer (Scirpophaga incertulus) inflicted damage

The primary technical constraint plant scientists face in generating insect resistant transgenic crops with insecticidal Bacillus thuringiensis (Bt) crystal protein (Cry) genes remains failing to generate sufficiently large numbers of effective resistant transgenic plant lines. One possible means to overcome this challenge is through deployment of a Cry toxin gene that contains high levels of insecticidal specific activity for target insect pests. In the present study, we tested this hypothesis using a natural variant of the Cry1Ab toxin under laboratory conditions that possessed increased insecticidal potency against the yellow stem borer (YSB, Scirpophaga incertulus), one of the most damaging rice insect pests. Following adoption of a stringent selection strategy for YSB resistant transgenic rice lines under field conditions, results showed recovery of a significantly higher number of YSB resistant independent transgenic plant lines with the variant cry1Ab gene relative to transgenic plant lines harbouring cry1Ab berliner gene. Structural homology modelling of the variant toxin peptide with the Cry1Aa toxin molecule, circular dichroism spectral analysis, and hydropathy plot analysis indicated that serine substitution by phenylalanine at amino acid position 223 of the Cry1Ab toxin molecule resulted in a changed role for α-helix 7 in domain I of Cry1Ab for enhanced toxicity.     

Incipient Resistance of Helicoverpa punctigera to the Cry2Ab Bt Toxin in Bollgard II? Cotton

Combinations of dissimilar insecticidal proteins (“pyramids”) within transgenic plants are predicted to delay the evolution of pest resistance for significantly longer than crops expressing a single transgene. Field-evolved resistance to Bacillus thuringiensis (Bt) transgenic crops has been reported for first generation, single-toxin varieties and the Cry1 class of proteins. Our five year data set shows a significant exponential increase in the frequency of alleles conferring Cry2Ab resistance in Australian field populations of Helicoverpa punctigera since the adoption of a second generation, two-toxin Bt cotton expressing this insecticidal protein. Furthermore, the frequency of cry2Ab resistance alleles in populations from cropping areas is 8-fold higher than that found for populations from non-cropping regions. This report of field evolved resistance to a protein in a dual-toxin Bt-crop has precisely fulfilled the intended function of monitoring for resistance; namely, to provide an early warning of increases in frequencies that may lead to potential failures of the transgenic technology. Furthermore, it demonstrates that pyramids are not ‘bullet proof’ and that rapid evolution to Bt toxins in the Cry2 class is possible.     

Larval mortality and development for rotation‐resistant and rotation‐susceptible populations of western corn rootworm on Bt corn

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is one of the most economically important insect pests threatening the production of corn, Zea mays (L.), in the United States. Throughout its history, this insect has displayed considerable adaptability by overcoming a variety of pest management tactics, including the cultural practice of annual crop rotation. Since first reported in Illinois in the late 1980s, populations of the rotation‐resistant western corn rootworm have spread over a wide area of the eastern Corn Belt. Currently, little information is available concerning the interaction of rotation resistance with the use of genetically modified corn expressing insecticidal toxins from Bacillus thuringiensis Berliner (Bt), a popular tactic for preventing larval injury and its associated yield loss. The goal of this greenhouse experiment was to determine whether rotation‐resistant and rotation‐susceptible western corn rootworm larvae differ with respect to survival or development when exposed to single‐ or dual‐toxin (pyramided) Bt corn. Individual corn plants were infested with 225 near‐hatch eggs at the V5 (five leaf collar) growth stage. Larvae developed undisturbed on the root systems for 17 days, after which they were recovered using Berlese–Tullgren funnels. Surviving larvae were counted to estimate mortality, and head capsule widths were measured to assess development. Rotation‐resistant and rotation‐susceptible larvae had statistically similar mean levels of mortality and head capsule widths when exposed to both single‐toxin (Cry3Bb1 or Cry34/35Ab1) and pyramided (Cry3Bb1+ Cry34/35Ab1) Bt corn, suggesting that these two populations do not differ with respect to survival or development when exposed to Bt corn. Additionally, the statistically similar mean levels of mortality for larvae exposed to single‐toxin and pyramided Bt corn suggest that pyramided Bt hybrids containing the Cry3Bb1 and Cry34/35Ab1 toxins do not result in additive mortality for western corn rootworm larvae. Implications for management of this economically important pest are discussed.     

DiPel-selected Ostrinia nubilalis larvae are not resistant to transgenic corn expressing Bacillus thuringiensis Cry1Ab

The survival of KS-SC DiPel-resistant and -susceptible European corn borer, Ostrinia nubilalis (Hübner), was evaluated on different tissues from corn, Zea mays L., hybrids, including a nontransgenic and two transgenic corn plants (events MON810 and Bt11) expressing high doses of Bacillus thuringiensis (Bt) Cry1Ab. The survival of Bt-resistant and -susceptible third instars was similar after a 5-d exposure to transgenic plant tissues. Survivors eventually died when returned to Bt corn tissues, but many were able to continue development when transferred to non-Bt corn tissues. Survival of resistant and susceptible larvae also was evaluated in bioassays with dilutions of leaf extracts from the three corn hybrids incorporated in an artificial diet. In these assays, survival was significantly higher for resistant O. nubilalis neonates at three of the five dilutions compared with the susceptible strain, but the resistance ratio was only 2.2- and 2.4-fold for MON810 and Bt11, respectively. The data demonstrate that Bt-resistant and unselected control O. nubilalis larvae were similar in susceptibility to MON810 and Bt11 event corn hybrids. Although we were unable to evaluate the Cry1Ab protein that larvae were exposed to in the transgenic tissue because of company restrictions, Cry1Ab protoxin produced in Escherichia coli was incubated with extracts from non-Bt corn leaves to simulate the in planta effect on the transgenic protein. Cry1Ab protoxin was hydrolyzed rapidly by enzymes in the corn extract into peptide fragments with molecular masses ranging from 132 to 74 kDa, and eventually 58 kDa. Overall, these data suggest that plant enzymes hydrolyze transgenic toxin to one that is functionally activated. Therefore, resistant insect populations with reduced proteinase activity do not seem to pose a threat to the efficacy of commercial MON810 and Bt11 corn hybrids.     

Increased frequency of pink bollworm resistance to Bt toxin Cry1Ac in China

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) kill some key insect pests, but evolution of resistance by pests can reduce their efficacy. The main approach for delaying pest adaptation to Bt crops uses non-Bt host plants as "refuges" to increase survival of susceptible pests. To delay evolution of pest resistance to transgenic cotton producing Bt toxin Cry1Ac, the United States and some other countries have required refuges of non-Bt cotton, while farmers in China have relied on "natural" refuges of non-Bt host plants other than cotton. The "natural" refuge strategy focuses on cotton bollworm (Helicoverpa armigera), the primary target of Bt cotton in China that attacks many crops, but it does not apply to another major pest, pink bollworm (Pectinophora gossypiella), which feeds almost entirely on cotton in China. Here we report data showing field-evolved resistance to Cry1Ac by pink bollworm in the Yangtze River Valley of China. Laboratory bioassay data from 51 field-derived strains show that the susceptibility to Cry1Ac was significantly lower during 2008 to 2010 than 2005 to 2007. The percentage of field populations yielding one or more survivors at a diagnostic concentration of Cry1Ac increased from 0% in 2005-2007 to 56% in 2008-2010. However, the median survival at the diagnostic concentration was only 1.6% from 2008 to 2010 and failure of Bt cotton to control pink bollworm has not been reported in China. The early detection of resistance reported here may promote proactive countermeasures, such as a switch to transgenic cotton producing toxins distinct from Cry1A toxins, increased planting of non-Bt cotton, and integration of other management tactics together with Bt cotton.     

Interaction of Bacillus thuringiensis δ-endotoxins with Midgut Brush Border Membrane Vesicles of Helicoverpa armigera

Pesticidal activity of different Bacillus thuringiensis (Bt) δ-endotoxins, Cry1Aa, Cry1Ab, Cry1Ac and Cry2A, were investigated against Helicoverpa armigera infesting cotton crop worldwide. Cry1Ac toxin was found to be the most potent toxin towards H. armigera. All selected Bt toxins were found stable in vitro processing by midgut juice of H. armigera. Saturation and competition binding experiments were performed with iodine-125 labeled proteins and brush border membrane vesicles prepared from the midgut of H. armigera. The results show saturable, specific and high affinity of all toxins except for Cry2A. Both the toxins were bound with low binding affinity but with high binding site concentration. Heterologous competition experiments showed that Cry1Aa, Cry1Ab and Cry1Ac recognized or share the same binding site which is different from that of Cry2A. The data suggest that development of multiple toxin system in transgenic plants with toxin pyramiding, which recognize different binding sites, may be useful in the deployment strategies to decrease the rate of pest adaptation to Bt toxins in transgenic plants.     

Expression of Cry1Ab and Cry2Ab by a Polycistronic Transgene with a Self-Cleavage Peptide in Rice

Insect resistance to Bacillus thuringiensis (Bt) crystal protein is a major threat to the long-term use of transgenic Bt crops. Gene stacking is a readily deployable strategy to delay the development of insect resistance while it may also broaden insecticidal spectrum. Here, we report the creation of transgenic rice expressing discrete Cry1Ab and Cry2Ab simultaneously from a single expression cassette using 2A self-cleaving peptides, which are autonomous elements from virus guiding the polycistronic viral gene expression in eukaryotes. The synthetic coding sequences of Cry1Ab and Cry2Ab, linked by the coding sequence of a 2A peptide from either foot and mouth disease virus or porcine teschovirus-1, regardless of order, were all expressed as discrete Cry1Ab and Cry2Ab at high levels in the transgenic rice. Insect bioassays demonstrated that the transgenic plants were highly resistant to lepidopteran pests. This study suggested that 2A peptide can be utilized to express multiple Bt genes at high levels in transgenic crops.     

Resistance to Bacillus thuringiensis toxin Cry2Ab in a strain of Helicoverpa armigera (Lepidoptera: Noctuidae) in Australia

Transgenic cotton, Gossypium hirsutum L., expressing the crylAc and cry2Ab genes from Bacillus thuringiensis (Bt) Berliner variety kurstaki in a pyramid (Bollgard II) was widely planted for the first time in Australia during the 2004-2005 growing season. Before the first commercial Bollgard II crops, limited amounts of cotton expressing only the crylAc gene (Ingard) was grown for seven seasons. No field failures due to resistance to CrylAc toxin were observed during that period and a monitoring program indicated that the frequency of genes conferring high level resistance to the CrylAc toxin were rare in the major pest of cotton, Helicoverpa armigera (Htibner) (Lepidoptera: Noctuidae). Before the deployment of Bollgard II, an allele conferring resistance to Cry2Ab toxin was detected in field-collected H. armigera. We established a colony (designated SP15) consisting of homozygous resistant individuals and examined their characteristics through comparison with individuals from a Bt-susceptible laboratory colony (GR). Through specific crosses and bioassays, we established that the resistance present in SP15 was due to a single autosomal gene. The resistance was recessive. Homozygotes were highly resistant to Cry2Ab toxin, so much so, that we were unable to induce significant mortality at the maximum concentration of toxin available. Homozygotes also were unaffected when fed leaves of a cotton variety expressing the cry2Ab gene. Although cross-resistant to Cry2Aa toxin, SP15 was susceptible to CrylAc and to the Bt product DiPel.