Field-evolved resistance to Bt maize by western corn rootworm: predictions from the laboratory and effects in the field

Crops engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) provide an effective management tool for many key insect pests. However, pest species have repeatedly demonstrated their ability to adapt to management practices. Results from laboratory selection experiments illustrate the capacity of pest species to evolve Bt resistance. Furthermore, resistance has been documented to Bt sprays in the field and greenhouse, and more recently, by some pests to Bt crops in the field. In 2009, fields were discovered in Iowa (USA) with populations of western corn rootworm, Diabrotica virgifera virgifera LeConte, that had evolved resistance to maize that produces the Bt toxin Cry3Bb1. Fields with resistant insects in 2009 had been planted to Cry3Bb1 maize for at least three consecutive years and as many as 6years. Computer simulation models predicted that the western corn rootworm might evolve resistance to Bt maize in as few as 3years. Laboratory and field data for interactions between western corn rootworm and Bt maize indicate that currently commercialized products are not high-dose events, which increases the risk of resistance evolution because non-recessive resistance traits may enhance survival on Bt maize. Furthermore, genetic analysis of laboratory strains of western corn rootworm has found non-recessive inheritance of resistance. Field studies conducted in two fields identified as harboring Cry3Bb1-resistant western corn rootworm found that survival of western corn rootworm did not differ between Cry3Bb1 maize and non-Bt maize and that root injury to Cry3Bb1 maize was higher than injury to other types of Bt maize or to maize roots protected with a soil insecticide. These first cases of field-evolved resistance to Bt maize by western corn rootworm provide an early warning and point to the need to apply better integrated pest management practices when using Bt maize to manage western corn rootworm.     

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.     

DNA screening reveals pink bollworm resistance to Bt cotton remains rare after a decade of exposure

Transgenic crops producing toxins from the bacterium Bacillus thuringiensis (Bt) kill insect pests and can reduce reliance on insecticide sprays. Although Bt cotton (Gossypium hirsutum L.) and Bt corn (Zea mays L.) covered 26 million ha worldwide in 2005, their success could be cut short by evolution of pest resistance. Monitoring the early phases of pest resistance to Bt crops is crucial, but it has been extremely difficult because bioassays usually cannot detect heterozygotes harboring one allele for resistance. We report here monitoring of resistance to Bt cotton with DNA-based screening, which detects single resistance alleles in heterozygotes. We used polymerase chain reaction primers that specifically amplify three mutant alleles of a cadherin gene linked with resistance to Bt cotton in pink bollworm, Pectinophora gossypiella (Saunders), a major pest. We screened DNA of 5,571 insects derived from 59 cotton fields in Arizona, California, and Texas during 2001-2005. No resistance alleles were detected despite a decade of exposure to Bt cotton. In conjunction with data from bioassays and field efficacy tests, the results reported here contradict predictions of rapid pest resistance to Bt crops.     

Early detection of field-evolved resistance to Bt cotton in China: cotton bollworm and pink bollworm

Transgenic crops producing Bacillus thuringiensis (Bt) toxins kill some major insect pests, but pests can evolve resistance and thereby reduce the effectiveness of such Bt crops. The main approach for slowing 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 cotton producing Bt toxin Cry1Ac, several 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. This strategy is designed for cotton bollworm (Helicoverpa armigera), which attacks many crops and is the primary target of Bt cotton in China, but it does not apply to pink bollworm (Pectinophora gossypiella), which feeds almost entirely on cotton in China. Here we review evidence of field-evolved resistance to Cry1Ac by cotton bollworm in northern China and by pink bollworm in the Yangtze River Valley of China. For both pests, results of laboratory diet bioassays reveal significantly decreased susceptibility of field populations to Cry1Ac, yet field control failures of Bt cotton have not been reported. The early detection of resistance summarized here may spur countermeasures such as planting Bt cotton that produces two or more distinct toxins, increased planting of non-Bt cotton, and integration of other management tactics together with Bt cotton.     

Insect resistance to transgenic Bt crops: lessons from the laboratory and field

Transgenic crops that produce insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) grew on >62 million ha worldwide from 1996 to 2002. Despite expectations that pests would rapidly evolve resistance to such Bt crops, increases in the frequency of resistance caused by exposure to Bt crops in the field have not yet been documented. In laboratory and greenhouse tests, however, at least seven resistant laboratory strains of three pests (Plutella xylostella [L.], Pectinophora gossypiella [Saunders], and Helicoverpa armigera [Hübner]) have completed development on Bt crops. In contrast, several other laboratory strains with 70- to 10,100-fold resistance to Bt toxins in diet did not survive on Bt crops. Monitoring of field populations in regions with high adoption of Bt crops has not yet detected increases in resistance frequency. Resistance monitoring examples include Ostrinia nubilalis (Hübner) in the United States (6 yr), P. gossypiella in Arizona (5 yr), H. armigera in northern China (3 yr), and Helicoverpa zea (Boddie) in North Carolina (2 yr). Key factors delaying resistance to Bt crops are probably refuges of non-Bt host plants that enable survival of susceptible pests, low initial resistance allele frequencies, recessive inheritance of resistance to Bt crops, costs associated with resistance that reduce fitness of resistant individuals relative to susceptible individuals on non-Bt hosts ("fitness costs"), and disadvantages suffered by resistant strains on Bt hosts relative to their performance on non-Bt hosts ("incomplete resistance"). The relative importance of these factors varies among pest-Bt crop systems, and violations of key assumptions of the refuge strategy (low resistance allele frequency and recessive inheritance) may occur in some cases. The success of Bt crops exceeds expectations of many, but does not preclude resistance problems in the future.     

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.     

Insect resistance to Bt crops: evidence versus theory

Evolution of insect resistance threatens the continued success of transgenic crops producing Bacillus thuringiensis (Bt) toxins that kill pests. The approach used most widely to delay insect resistance to Bt crops is the refuge strategy, which requires refuges of host plants without Bt toxins near Bt crops to promote survival of susceptible pests. However, large-scale tests of the refuge strategy have been problematic. Analysis of more than a decade of global monitoring data reveals that the frequency of resistance alleles has increased substantially in some field populations of Helicoverpa zea, but not in five other major pests in Australia, China, Spain and the United States. The resistance of H. zea to Bt toxin Cry1Ac in transgenic cotton has not caused widespread crop failures, in part because other tactics augment control of this pest. The field outcomes documented with monitoring data are consistent with the theory underlying the refuge strategy, suggesting that refuges have helped to delay resistance.     

Refuges in reverse: the spread of Bacillus thuringiensis resistance to unselected greenhouse populations of cabbage loopers Trichoplusia ni

1 The dispersal of susceptible insects between refuges and Bacillus thuringiensis (Bt) treated fields is the key to resistance management of Bt crops. Here we describe the opposite situation; the movement of Bt resistant Trichoplusia ni moths from over‐wintered, greenhouse populations in British Columbia (BC) exposed to high Bt use to neighbouring greenhouses where Bt sprays have not been used. 2 The spread of Bt resistance to non‐selected populations of T. ni, and the resulting increase in resistance, indicates a surprising level of dispersal of resistant moths among greenhouses even in the face of fitness costs. 3 Field populations of T. ni in BC are seasonal migrants from regions of California where Bt cotton is grown. In 2006, field populations surveyed along the migration path from California through Oregon were highly susceptible to Bt insecticides and, thus, showed no indication of selection for resistance among these source populations. 4 The arrival of the immigrant moths provides a potential source of susceptible individuals to dilute the levels of resistance in greenhouse populations in BC later in the summer, but this has not occurred. Thus, field populations in BC do not appear to serve as refuges to combat Bt resistance in greenhouse populations.     

Delaying corn rootworm resistance to Bt corn

Transgenic crops producing Bacillus thuringiensis (Bt) toxins for insect control have been successful, but their efficacy is reduced when pests evolve resistance. To delay pest resistance to Bt crops, the U.S. Environmental Protection Agency (EPA) has required refuges of host plants that do not produce Bt toxins to promote survival of susceptible pests. Such refuges are expected to be most effective if the Bt plants deliver a dose of toxin high enough to kill nearly all hybrid progeny produced by matings between resistant and susceptible pests. In 2003, the EPA first registered corn, Zea mays L., producing a Bt toxin (Cry3Bb1) that kills western corn rootworm, Diabrotica virgifera virgifera LeConte, one of the most economically important crop pests in the United States. The EPA requires minimum refuges of 20% for Cry3Bb1 corn and 5% for corn producing two Bt toxins active against corn rootworms. We conclude that the current refuge requirements are not adequate, because Bt corn hybrids active against corn rootworms do not meet the high-dose standard, and western corn rootworm has rapidly evolved resistance to Cry3Bb1 corn in the laboratory, greenhouse, and field. Accordingly, we recommend increasing the minimum refuge for Bt corn targeting corn rootworms to 50% for plants producing one toxin active against these pests and to 20% for plants producing two toxins active against these pests. Increasing the minimum refuge percentage can help to delay pest resistance, encourage integrated pest management, and promote more sustainable crop protection.     

How to cope with insect resistance to Bt toxins?

Transgenic Bt crops producing insecticidal crystalline proteins from Bacillus thuringiensis, so-called Cry toxins, have proved useful in controlling insect pests. However, the future of Bt crops is threatened by the evolution of insect resistance. Understanding how Bt toxins work and how insects become resistant will provide the basis for taking measures to counter resistance. Here we review possible mechanisms of resistance and different strategies to cope with resistance, such as expression of several toxins with different modes of action in the same plant, modified Cry toxins active against resistant insects, and the potential use of Cyt toxins or a fragment of cadherin receptor. These approaches should provide the means to assure the successful use of Bt crops for an extended period of time.     

The halo effect: suppression of pink bollworm on non-bt cotton by bt cotton in china

In some previously reported cases, transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) have suppressed insect pests not only in fields planted with such crops, but also regionally on host plants that do not produce Bt toxins. Here we used 16 years of field data to determine if Bt cotton caused this "halo effect" against pink bollworm (Pectinophora gossypiella) in six provinces of the Yangtze River Valley of China. In this region, the percentage of cotton hectares planted with Bt cotton increased from 9% in 2000 to 94% in 2009 and 2010. We found that Bt cotton significantly decreased the population density of pink bollworm on non-Bt cotton, with net decreases of 91% for eggs and 95% for larvae on non-Bt cotton after 11 years of Bt cotton use. Insecticide sprays targeting pink bollworm and cotton bollworm (Helicoverpa armigera) decreased by 69%. Previously reported evidence of the early stages of evolution of pink bollworm resistance to Bt cotton in China has raised concerns that if unchecked, such resistance could eventually diminish or eliminate the benefits of Bt cotton. The results reported here suggest that it might be possible to find a percentage of Bt cotton lower than the current level that causes sufficient regional pest suppression and reduces the risk of resistance.     

Suppressing resistance to Bt cotton with sterile insect releases

Genetically engineered crops that produce insecticidal toxins from Bacillus thuringiensis (Bt) are grown widely for pest control. However, insect adaptation can reduce the toxins' efficacy. The predominant strategy for delaying pest resistance to Bt crops requires refuges of non-Bt host plants to provide susceptible insects to mate with resistant insects. Variable farmer compliance is one of the limitations of this approach. Here we report the benefits of an alternative strategy where sterile insects are released to mate with resistant insects and refuges are scarce or absent. Computer simulations show that this approach works in principle against pests with recessive or dominant inheritance of resistance. During a large-scale, four-year field deployment of this strategy in Arizona, resistance of pink bollworm (Pectinophora gossypiella) to Bt cotton did not increase. A multitactic eradication program that included the release of sterile moths reduced pink bollworm abundance by >99%, while eliminating insecticide sprays against this key invasive pest.     

Parallel evolution of Bacillus thuringiensis toxin resistance in lepidoptera

Despite the prominent and worldwide use of Bacillus thuringiensis (Bt) insecticidal toxins in agriculture, knowledge of the mechanism by which they kill pests remains incomplete. Here we report genetic mapping of a membrane transporter (ABCC2) to a locus controlling Bt Cry1Ac toxin resistance in two lepidopterans, implying that this protein plays a critical role in Bt function.     

Genetics of resistance to transgenic Bacillus thuringiensis poplars in Chrysomela tremulae (Coleoptera: Chrysomelidae)

The area under genetically engineered plants producing Bacillus thuringiensis (Bt) toxins is steadily increasing. This increase has magnified the risk of alleles conferring resistance to these toxins being selected in natural populations of target insect pests. The speed at which this selection is likely to occur depends on the genetic characteristics of Bt resistance. We selected a strain of the beetle Chrysomela tremulae Fabricius on a transgenic Bt poplar clone Populus tremula L. x Populus tremuloides Michx producing high levels of B. thuringiensis Cry3Aa toxin. This strain was derived from an isofemale line that generated some F2 offspring that actively fed on this Bt poplar clone. The resistance ratio of the strain was >6400. Susceptibility had decreased to such an extent that the mortality of beetles of the strain fed Bt poplar leaves was similar to that of beetles fed nontransgenic poplar leaves. Genetic crosses between susceptible, resistant, and F1 hybrids showed that resistance to the Cry3Aa toxin was almost completely recessive (D(LC) = 0.07) and conferred by a single autosomal gene. The concentration of Cry3Aa produced in the transgenic Bt poplar used in this study was 6.34 times higher than the LC99 of the F1 hybrids, accounting for the complete recessivity (D(ML) = 0) of survival on Bt poplar leaves. Overall, the genetic characteristics of the resistance of C. tremulae to the Cry3Aa toxin are consistent with the assumptions underlying the high-dose refuge strategy, which aims to decrease the selection of Bt resistance alleles in natural target pest populations.     

Western corn rootworm and Bt maize: challenges of pest resistance in the field

Crops genetically engineered to produce insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) manage many key insect pests while reducing the use of conventional insecticides. One of the primary pests targeted by Bt maize in the United States is the western corn rootworm, Diabrotica virgifera virgifera LeConte. Beginning in 2009, populations of western corn rootworm were identified in Iowa, USA that imposed severe root injury to Cry3Bb1 maize. Subsequent laboratory bioassays revealed that these populations were resistant to Cry3Bb1 maize, with survival on Cry3Bb1 maize that was three times higher than populations not associated with such injury. Here we report the results of research that began in 2010 when western corn rootworm were sampled from 14 fields in Iowa, half of which had root injury to Cry3Bb1 maize of greater than 1 node. Of these samples, sufficient eggs were collected to conduct bioassays on seven populations. Laboratory bioassays revealed that these 2010 populations had survival on Cry3Bb1 maize that was 11 times higher and significantly greater than that of control populations, which were brought into the laboratory prior to the commercialization of Bt maize for control of corn rootworm. Additionally, the developmental delays observed for control populations on Cry3Bb1 maize were greatly diminished for 2010 populations. All 2010 populations evaluated in bioassays came from fields with a history of continuous maize production and between 3 and 7 y of Cry3Bb1 maize cultivation. Resistance to Cry34/35Ab1 maize was not detected and there was no correlation between survival on Cry3Bb1 maize and Cry34/35Ab1 maize, suggesting a lack of cross resistance between these Bt toxins. Effectively dealing with the challenge of field-evolved resistance to Bt maize by western corn rootworm will require better adherence to the principles of integrated pest management.     

Bacillus thuringiensis: a biotechnology model

This paper is on the different biotechnological approaches that have been used to improve Bacillus thuringiensis (Bt) for the control of agricultural insect pests and have contributed to the successful use of this biological control agent; it describes how a better knowledge of the high diversity of Bt strains and toxins genes together with the development of efficient host-vector systems has made it possible to overcome a number of the problems associated with Bt based insect control measures. First we present an overview of the biology of Bt and of the mode of action of its insecticidal toxins. We then describe some of the progress that has been made in furthering our knowledge of the genetics of Bt and of its insecticidal toxin genes and in the understanding of their regulation. The paper then deals with the use of recombinant DNA technology to develop new Bt strains for more effective pest control or to introduce the genes encoding partial-endotoxins directly into plants to produce insect-resistant trangenic plants. Several examples describing how biotechnology has been used to increase the production of insecticidal proteins in Bt or their persistence in the field by protecting them against UV degradation are presented and discussed. Finally, based on our knowledge of the mechanism of transposition of the Bt transposon Tn4430, we describe the construction of a new generation of recombinant strains of Bt, from which antibiotic resistance genes and other non-Bt DNA sequences were selectively eliminated, using a new generation of site-specific recombination vectors. In the future, continuing improvement of first generation products and research into new sources of resistance is essential to ensure the long-term control of insect pests. Chimeric toxins could also be produced so as to increase toxin activity or direct resistance towards a particular type of insect. The search for new insecticidal toxins, in Bt or other microorganisms, may also provide new weapons for the fight against insect damage.     

Efficacy of genetically modified Bt toxins against insects with different genetic mechanisms of resistance

Transgenic crops that produce Bacillus thuringiensis (Bt) toxins are grown widely for pest control, but insect adaptation can reduce their efficacy. The genetically modified Bt toxins Cry1AbMod and Cry1AcMod were designed to counter insect resistance to native Bt toxins Cry1Ab and Cry1Ac. Previous results suggested that the modified toxins would be effective only if resistance was linked with mutations in genes encoding toxin-binding cadherin proteins. Here we report evidence from five major crop pests refuting this hypothesis. Relative to native toxins, the potency of modified toxins was >350-fold higher against resistant strains of Plutella xylostella and Ostrinia nubilalis in which resistance was not linked with cadherin mutations. Conversely, the modified toxins provided little or no advantage against some resistant strains of three other pests with altered cadherin. Independent of the presence of cadherin mutations, the relative potency of the modified toxins was generally higher against the most resistant strains.     

Initial frequency of alleles conferring resistance to Bacillus thuringiensis poplar in a field population of Chrysomela tremulae

Globally, the estimated total area planted with transgenic plants producing Bacillus thuringiensis (Bt) toxins was 12 million hectares in 2001. The risk of target pests becoming resistant to these toxins has led to the implementation of resistance-management strategies. The efficiency and sustainability of these strategies, including the high-dose plus refuge strategy currently recommended for North American maize, depend on the initial frequency of resistance alleles. In this study, we estimated the initial frequencies of alleles conferring resistance to transgenic Bt poplars producing Cry3A in a natural population of the poplar pest Chrysomela tremulae (Coleoptera: Chrysomelidae). We used the F(2) screen method developed for detecting resistance alleles in natural pest populations. At least three parents of the 270 lines tested were heterozygous for a major Bt resistance allele. We estimated mean resistance-allele frequency for the period 1999-2001 at 0.0037 (95% confidence interval = 0.00045-0.0080) with a detection probability of 90%. These results demonstrate that (i) the F(2) screen method can be used to detect major alleles conferring resistance to Bt-producing plants in insects and (ii) the initial frequency of alleles conferring resistance to Bt toxin can be close to the highest theoretical values that are expected prior to the use of Bt plants if considering fitness costs and typical mutation rates.     

Understanding successful resistance management: the European corn borer and Bt corn in the United States

The European corn borer, Ostrinia nubilalis Hübner (Lepidoptera: Crambidae) has been a major pest of corn and other crops in North America since its accidental introduction nearly a hundred years ago. Wide adoption of transgenic corn hybrids that express toxins from Bacillus thuringiensis, referred to as Bt corn, has suppressed corn borer populations and reduced the pest status of this insect in parts of the Corn Belt. Continued suppression of this pest, however, will depend on managing potential resistance to Bt corn, currently through the high-dose refuge (HDR) strategy. In this review, we describe what has been learned with regard to O. nubilalis resistance to Bt toxins either through laboratory selection experiments or isolation of resistance from field populations. We also describe the essential components of the HDR strategy as they relate to O. nubilalis biology and ecology. Additionally, recent developments in insect resistance management (IRM) specific to O. nubilalis that may affect the continued sustainability of this technology are considered.