The southwestern corn borer, Diatraea grandiosella Dyar, and sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), are two important stalk borers targeted by Bacillus thuringiensis (Bt) maize in the Americas. Practical resistance to Bt maize in D. grandiosella is emerging in the southwest region of the United States. In this study, Bt maize leaf tissue was used to screen F2 family-lines of these two stalk borers for resistance alleles (RA) to Bt maize expressing one or two of the four common Bt proteins: Cry1A.105, Cry2Ab2, Cry1F, and Cry1Ab. The family-lines were developed from single-pairing or group-mating of field-collected D. grandiosella from non-Bt maize in Tennessee and D. saccharalis from sugarcane in Louisiana during 2019–2022. The F2 screen identified one possible major RA to Cry1A.105 maize in 155 D. grandiosella and 198 D. saccharalis individuals collected from fields, while for the maize expressing one or two of the other three Bt proteins examined, no major RAs were detected in 94–208 field-collected individuals of the two species. Bayesian analysis showed that an upper-level resistance allele frequency (RAF) for major alleles to Cry1A.105 maize was 0.0063 in D. grandiosella and 0.0050 in D. saccharalis. With 95% probability, the RAF of major alleles in D. grandiosella was <0.0093 for Cry2Ab2 maize, <0.0155 for Cry1F maize, and <0.0150 for Cry1A/Cry2A maize; and for D. saccharalis, it was <0.0071 for Cry2Ab2 maize and <0.0090 for Cry1Ab maize. These findings suggest that RAFs for major alleles to the common Bt maize are still low in the two stalk borers. However, minor RAs in both species, especially to Cry2Ab2 and Cry1A.105 maize in D. saccharalis, were not rare. The results of this study, together with other data, suggest that, after 25 years of Bt maize use, the technology is still effective and field resistance to maize expressing the Cry proteins has not evolved in these two stalk borers in the mid-south region of the United States.
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