Knockout of ABC transporter gene ABCA2 confers resistance to Bt toxin Cry2Ab in Helicoverpa zea.

Evolution of pest resistance reduces the benefits of widely cultivated genetically engineered crops that produce insecticidal proteins derived from Bacillus thuringiensis (Bt). Better understanding of the genetic basis of pest resistance to Bt crops is needed to monitor, manage, and counter resistance. Previous work shows that in several lepidopterans, resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2. The results here show that mutations introduced by CRISPR/Cas9 gene editing in the Helicoverpa zea (corn earworm or bollworm) gene encoding ABCA2 (HzABCA2) can cause resistance to Cry2Ab. Disruptive mutations in HzABCA2 facilitated the creation of two Cry2Ab-resistant strains. A multiple concentration bioassay with one of these strains revealed it had > 200-fold resistance to Cry2Ab relative to its parental susceptible strain. All Cry2Ab-resistant individuals tested had disruptive mutations in HzABCA2. We identified five disruptive mutations in HzABCA2 gDNA. The most common mutation was a 4-bp deletion in the expected Cas9 guide RNA target site. The results here indicate that HzABCA2 is a leading candidate for monitoring Cry2Ab resistance in field populations of H. zea.

Effects of gene flow between Bt and non-Bt plants in a seed mixture of Cry1A.105 + Cry2Ab corn on performance of corn earworm in Arizona.

BACKGROUND: Using natural populations of Helicoverpa zea from Arizona, we tested the hypotheses that gene flow between Bacillus thuringiensis (Bt) plants and non-Bt plants in a seed mixture of 10% non-Bt corn and 90% Bt corn producing Cry1A.105 and Cry2Ab reduces larval performance on ears from non-Bt plants, or increases performance on ears from Bt plants. RESULTS: Gene flow was not detected in blocks of non-Bt or Bt corn but was extensive in seed mixtures. Analyses of larval weight and abundance over a period of 3 to 4 weeks did not indicate consistent effects of gene flow on development rate and survival. However for non-Bt plants, the ear area damaged and percentage of ears with exit holes were significantly lower in the seed mixtures than blocks. By contrast, the percentage of ears with exit holes and ear damage did not differ significantly between the seed mixtures and blocks for Bt plants. Nearly 100% of the ears were damaged and the damaged area was substantial, showing that H. zea is a major ear-feeding pest in Arizona. Relative to non-Bt corn, the pyramided Bt corn did not significantly reduce the percentage of damaged ears and only reduced the ear area damaged by 21 to 39%, indicating that H. zea may have evolved resistance to Cry1A.105, Cry2Ab, or both. CONCLUSIONS: Our results indicate that gene flow between Bt and non-Bt plants in seed mixtures reduced effective refuge size, and that block refuges may be needed to manage the evolution of H. zea resistance to Bt corn in Arizona. © 2020 Society of Chemical Industry.

Gene Flow Between Bt and Non-Bt Plants in a Seed Mixture Increases Dominance of Resistance to Pyramided Bt Corn in Helicoverpa zea (Lepidoptera: Noctuidae).

For delaying evolution of pest resistance to transgenic corn producing Bacillus thuringiensis (Bt) toxins, limited data are available to compare the effectiveness of refuges of non-Bt corn planted in seed mixtures versus blocks. Here we addressed this issue in the ear-feeding pest Helicoverpa zea Boddie by measuring its survival and development in the laboratory on ears from field plots with 90% Cry1A.105 + Cry2Ab corn and 10% non-Bt corn planted in a seed mixture or blocks. We compared a strain of H. zea selected for resistance to Cry1Ac in the laboratory, its parent strain not selected in the laboratory, and their F1 progeny. The relative survival of the F1 progeny and dominance of resistance were higher on ears from Bt plants in the seed mixture than the block. Half of the kernels in ears from non-Bt plants in the seed mixture produced both Cry1A.105 and Cry2Ab. However, survival on ears from non-Bt plants did not differ between the block and seed mixture. In simulations based on the observed survival, resistance to Cry1A.105 + Cry2Ab corn evolved faster with the seed mixture than the blocks, because of the higher dominance of resistance in the seed mixture. Increasing the refuge percentage improved durability of Cry1A.105 + Cry2Ab corn more for the blocks than the seed mixture. These findings imply that, for a given percentage of non-Bt corn, resistance of H. zea and other ear-feeding pests to multi-toxin Bt corn is likely to evolve faster for seed mixtures than blocks.

Effects of seasonal changes in cotton plants on the evolution of resistance to pyramided cotton producing the Bt toxins Cry1Ac and Cry1F in Helicoverpa zea.

BACKGROUND: In pests with inherently low susceptibility to Bacillus thuringiensis (Bt) toxins, seasonal declines in the concentration of Bt toxins in transgenic crops could accelerate evolution of resistance by increasing the dominance of resistance. Here, we evaluated Helicoverpa zea survival on young and old cotton plants that produced the Bt toxins Cry1Ac and Cry1F or did not produce Bt toxins. RESULTS: Using a strain selected for resistance to Cry1Ac in the laboratory, its parent strain that was not selected in the laboratory, and their F1 progeny, we showed that resistance to Cry1Ac + Cry1F cotton was partially dominant on young and old plants. On Cry1Ac + Cry1F cotton, redundant killing was incomplete on young plants but nearly complete on old plants. No significant fitness costs on non-Bt cotton occurred on young plants, but large recessive costs affected survival on old plants. Simulation models incorporating the empirical data showed that the seasonal changes in fitness could delay resistance to Cry1Ac + Cry1F cotton by inducing low equilibrium frequencies of resistance alleles when refuges are sufficiently large. CONCLUSION: Our results suggest that including effects of seasonal changes in fitness of pests on Bt crops and refuge plants can enhance resistance risk assessment and resistance management. © 2017 Society of Chemical Industry.

Cross-resistance to toxins used in pyramided Bt crops and resistance to Bt sprays in Helicoverpa zea.

To delay evolution of resistance by insect pests, farmers are rapidly increasing their use of transgenic crops producing two or more Bacillus thuringiensis (Bt) toxins that kill the same pest. A key condition favoring durability of these "pyramided" crops is the absence of cross-resistance between toxins. Here we evaluated cross-resistance in the major lepidopteran pest Helicoverpa zea (Boddie) to Bt toxins used in pyramids. In the laboratory, we selected a strain of this pest with Bt toxin Cry1Ac followed by selection with MVP II, a formulation containing a hybrid protoxin that is identical to Cry1Ac in the active portion of the toxin and 98.5% identical overall. We calculated the resistance ratio as the EC50 (concentration causing mortality or failure to develop beyond the first instar of 50% of larvae) for the laboratory-selected strain divided by the EC50 for its field-derived parent strain that was not selected in the laboratory. The resistance ratio was 20.0-33.9 (mean=27.0) for MVP II, 57.0 for Cry1Ac, 51.3 for Cry1A.105, 22.4 for Cry1Ab, 3.3 for Cry2Ab, 1.8 for Cry1Fa, and 1.6 for Vip3Aa. Resistance ratios were 2.9 for DiPel ES and 2.0 for Agree VG, which are commercial Bt spray formulations containing Cry1Ac, other Bt toxins, and Bt spores. By the conservative criterion of non-overlap of 95% fiducial limits, the EC50 was significantly higher for the selected strain than its parent strain for MVP II, Cry1Ac, Cry1A.105, Cry1Ab, Cry2Ab and DiPel ES. For Cry1Fa, Vip3Aa, and Agree VG, significantly lower susceptibility to a high concentration indicated low cross-resistance. The resistance ratio for toxins other than Cry1Ac was associated with their amino acid sequence similarity to Cry1Ac in domain II. Resistance to Cry1Ac and the observed cross-resistance to other Bt toxins could accelerate evolution of H. zea resistance to currently registered Bt sprays and pyramided Bt crops.