Extended investigation of field-evolved resistance of the corn earworm Helicoverpa zea(Lepidoptera: Noctuidae) to Bacillus thuringiensis Cry1A.105 and Cry2Ab2 proteins in thesoutheastern United States.

Previous studies have reported that the corn earworm/bollworm, Helicoverpa zea (Boddie), has developed field resistance to pyramided Bacillus thuringiensis (Bt) Cry1A/Cry2A maize and cotton in certain areas of the southeastern United States. The objective of the current study was to determine the current status and distribution of the resistance to Cry1A.105 and Cry2Ab2 in H. zea. In the study, 31 H. zea populations were collected from major maize planting areas across seven southeastern states of the United States during 2018 and 2019 and assayed against the two Bt proteins. Diet over-lay bioassays showed that most of the populations collected during the two years were significantly resistant to the Cry1A.105 protein. Most of the populations collected during 2019 were also resistant to Cry2Ab2, while significant variances were observed in the susceptibility of the populations collected during 2018 to Cry2Ab2. The results showed that Cry1A.105 and Cry2Ab2 resistance in H. zea is widely distributed in the regions sampled. The resistance to Cry1A.105 appeared to have plateaued, while selection for Cry2Ab2 resistance is likely still occurring. Thus, effective measures for mitigating the Cry1A/Cry2A resistance need to be developed and implemented to ensure the sustainable use of Bt crop biotechnology.

Field-evolved resistance of Helicoverpa zea (Boddie) to transgenic maize expressing pyramided Cry1A.105/Cry2Ab2 proteins in northeast Louisiana, the United States.

The corn earworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), is a major target pest of pyramided Bt maize and cotton in the U.S. In 2017 and 2018, notable ear damage and larval survival of H. zea were observed on pyramided Cry1A.105/Cry2Ab2 maize in some fields in northeast Louisiana, U.S. The objective of this study was to determine if the field control problem was due to resistance development to the Bt proteins in plants. A total of 15 H. zea populations were collected from Bt and non-Bt maize plants in 2017 and 2018 in multiple locations in Louisiana, Florida, and Georgia. Diet-overlay bioassays showed that LC50s of Cry1A.105 and Cry2Ab2 for populations collected from the areas with control problems in northeast Louisiana were as much as >1623- and 88-fold greater than that of a susceptible strain, respectively. In addition, two field trials in 2018 validated that Cry1A.105/Cry2Ab2 maize failed in managing natural H. zea populations, while Bt maize containing Vip3A was effective in northeast Louisiana. Results of the study documented that the observed field control problems of Cry1A.105/Cry2Ab2 maize against H. zea in northeast Louisiana were due to resistance development of the insect to the Bt proteins in plants. This is the first documentation of field-evolved resistance to pyramided Bt maize in a target insect species in southern U.S. However, susceptibility levels to Cry1A.105 and Cry2Ab2 varied greatly among populations collected from the three states, suggesting uneven distributions of the resistance in the region.

Performance of Bt-susceptible and -heterozygous genotypes of Spodoptera frugiperda (J.E. Smith) possessing single- or dual-gene resistance alleles in sequential feedings of non-Bt and Cry1A.105/Cry2Ab2 maize leaf tissues.

We simulated larval feeding behavior in seed blends of non-Bt and Bt maize to determine if seed blends create more favorable conditions for heterozygous-resistant insects over their Bt-susceptible counterparts. Survival, growth, development, and progeny production of four genotypes of the fall armyworm, Spodoptera frugiperda, Bt-susceptible (aabb), Cry1A.105 heterozygous resistant (Aabb), Cry2Ab2 heterozygous resistant (aaBb), and Cry1A.105/Cry2Ab2 heterozygous resistant (AaBb), were evaluated in eight feeding sequences (Seq 1-8) of non-Bt and MON89034 Bt maize leaf tissue expressing the Cry1A.105 and Cry2Ab2 proteins. We report variation in the performance of the four genotypes across the feeding sequences and biological parameters measured. Three heterozygous genotypes generally outperformed the susceptible genotype in larval survival, pupation rate, pupal weight, and progeny production. The performance was greater for Aabb over aaBb, AaBb over Aabb or aaBb, in two of the feeding sequences. The findings of this study could have important implications in assessing the risk of seed blends as refuge plantings for Bt crop resistance management where resistance in the target pest is not functionally recessive.

Fitness costs and inheritance of Cry2Ab2 resistance in Spodoptera frugiperda (J.E. Smith).

Cry2Ab2 is a Bacillus thuringiensis (Bt) protein expressed in transgenic corn and cotton targeting above-ground lepidopteran pests including the fall armyworm, Spodoptera frugiperda (J.E. Smith). The objective of this study was to characterize fitness costs and inheritance of Cry2Ab2 resistance in S. frugiperda. To determine if fitness costs were associated with the resistance, life history parameters (larval survival, growth, development and egg production) of Cry2Ab2-resistant, -susceptible, and two reciprocal F1 colonies of S. frugiperda were assayed on non-toxic diet and non-Bt corn leaf tissue. The results showed that there were no significant differences among the four insect colonies for all the biological parameters measured with few exceptions, suggesting that the resistance in the colony was not associated with significant fitness costs in the test conditions. To examine the inheritance of resistance, susceptibilities of the resistant and susceptible parents, as well as eight additional colonies generated from various genetic crosses, were assayed using Cry2Ab2-treated diet and Cry2Ab2 corn leaf tissue. The Cry2Ab2 resistance in S. frugiperda in the colony was inherited as a single autosomal recessive or incompletely recessive gene. The results of the study suggest a potential risk of resistance development in S. frugiperda to the Cry2Ab2 protein and thus effective management strategies should be implemented for the sustainable use of the Bt corn technology for pest management.

F2 screen for resistance to Bacillus thuringiensis Cry2Ab2-maize in field populations of Spodoptera frugiperda (Lepidoptera: Noctuidae) from the southern United States.

The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), is a target pest of transgenic maize and cotton expressing Bacillus thuringiensis (Bt) proteins in both North and South America. In 2013 and 2014, a total of 215 F2 two-parent families of S. frugiperda were established using single-pair mating of field individuals collected from seven locations in four states of the southern U.S.: Texas, Louisiana, Georgia, and Florida. The objective of the investigation was to detect resistance alleles in field populations to Cry2Ab2, a common Bt protein produced in transgenic maize and cotton. For each F2 family, 128 F2 neonates were screened on leaf tissue of Cry2Ab2 maize plants in the laboratory. A conservative estimate of the frequency of major Cry2Ab2 resistance alleles in S. frugiperda from the four states was 0.0023 with a 95% credibility interval of 0.0003-0.0064. In addition, six families were considered to likely possess minor resistance alleles at a frequency of 0.0082 with a 95% credibility interval of 0.0033-0.0152. One F2 family from Georgia (GA-15) was confirmed to possess a major resistance allele to the Cry2Ab2 protein. Larvae from this family survived well on whole maize plants expressing Cry2Ab2 protein and demonstrated a significant level (>15-fold) of resistance when fed with the same protein incorporated in a meridic diet. The detection of the major resistance allele along with the relatively abundant minor resistance alleles revealed in this study may have important implications for resistance management.

Characterizing the sublethal effects of SmartStax PRO dietary exposure on life history traits of the western corn rootworm, Diabrotica virgifera virgifera LeConte.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is an economically important pest of field corn (Zea mays L.) across the United States (U.S.) Corn Belt. Repeated use of transgenic hybrids expressing Bacillus thuringiensis (Bt) proteins has selected for field-evolved resistance to all current rootworm-active Bt proteins. The newest product available for WCR management is SmartStax® PRO, a rootworm-active pyramid containing Cry3Bb1, Cry34/35Ab1 [now reclassified as Gpp34Ab1/Tpp35Ab1] and a new mode of action, DvSnf7 dsRNA. Understanding the fitness of adult WCR after dietary exposure to SmartStax® PRO will identify potential impacts on WCR population dynamics and inform efforts to optimize resistance management strategies. Therefore, the objective of the present study was to characterize the effect of SmartStax® PRO dietary exposure on WCR life history traits. Adult WCR were collected during 2018 and 2019 from emergence tents placed over replicated field plots of SmartStax® PRO or non-rootworm Bt corn at a site with a history of rootworm-Bt trait use and suspected resistance to Cry3Bb1 and Cry34/35Ab1. Adult survival was reduced by 97.1-99.7% in SmartStax® PRO plots relative to the non-rootworm Bt corn plots during the study. Individual male/female pairs were fed different diets of ear tissue to simulate lifetime or adult exposure. Life history parameters measured included adult longevity, adult head capsule width, lifetime female egg production, and egg viability. Results indicate that lifetime or adult exposure to SmartStax® PRO significantly reduced adult longevity and lifetime egg production. Larval exposure to SmartStax® PRO significantly reduced WCR adult size. Results from this study collectively suggest that SmartStax® PRO may negatively impact WCR life history traits, which may lead to reduced population growth when deployed in an area with WCR resistance to Bt traits.

Performance of Bt-susceptible and -heterozygous dual-gene resistant genotypes of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) in seed blends of non-Bt and pyramided Bt maize.

A seed blend refuge has been implemented in the U.S. Corn Belt for Bt maize resistance management. The fall armyworm, Spodoptera frugiperda (J.E. Smith), is a target pest of Bt maize in the Americas. The larvae of this pest are mobile, which may affect the efficacy of seed blend refuges. In this study, field and greenhouse trials were conducted to determine the performance of Bt-susceptible (aabb) and -heterozygous dual-gene-resistant (AaBb) genotypes of S. frugiperda in seed blends of non-Bt and pyramided Bt maize. Three field trials evaluated larval survival, larval growth, and plant injury with aabb in seed blends of Bt maize expressing Cry1A.105/Cry2Ab2/Vip3A with 0-30% non-Bt seeds. Greenhouse tests investigated the performance of aabb and AaBb in seed blends of Cry1A.105/Cry2Ab2 with 0-30% non-Bt seeds. In pure non-Bt maize plots, after 9-13 d of neonates being released on the plants, 0.39 and 0.65 larvae/plant survived with leaf injury ratings of 4.7 and 5.9 (Davis's 1-9 scale) in the field and greenhouse, respectively. In contrast, live larvae and plant injury were virtually not observed on Bt plants across all planting patterns. Larval occurrence and plant injury by aabb on non-Bt plants were similar between seed blends and pure non-Bt plantings, suggesting that the blended refuges could provide an equivalent susceptible population as structured refuge under the test conditions. In the greenhouse, the two insect genotypes in seed blends performed similarly, indicating that the seed blends did not provide more favorable conditions for AaBb over aabb. The information generated from this study should be useful in managing S. frugiperda and evaluating if send blends could be suitable refuge options for Bt resistance management in the regions where the insect is a primary target pest.

Inheritance of Bacillus thuringiensis Cry2Ab2 protein resistance in Helicoverpa zea (Lepidoptera: Noctuidae).

BACKGROUND: The corn earworm, Helicoverpa zea (Boddie), is a major target pest of pyramided Bt corn and cotton in the United States. Field-evolved practical resistance to Cry1 and Cry2 proteins in H. zea has been documented in multiple locations in the United States. Understanding the genetic basis of Bt resistance is essential in developing insect resistance management (IRM) strategies for the sustainable use of the Bt crop technology. In this study, we characterized the genetic bases of Cry2Ab2 resistance in H. zea using diet-overlay bioassays with two different forms of Cry2Ab2 protein. RESULTS: Laboratory bioassays using a Cry2Ab2-resistant (RR) strain, a susceptible (SS) strain, as well as cross and backcross strains, revealed that resistance to Cry2Ab2 was autosomally inherited and controlled by more than one locus. In diet bioassays, the dominance of Cry2Ab2 resistance in H. zea varied from incompletely recessive to incompletely dominant across all tested Cry2Ab2 concentrations of either Bt corn leaf powder or solubilized protein. On leaf tissue of TwinLink cotton (expressing Cry1Ab and Cry2Ae), Cry2Ab2 resistance in H. zea was completely dominant. CONCLUSION: These results have significant implications for understanding the widespread field-evolved resistance of H. zea against Cry1 and Cry2 proteins in Bt corn and cotton and should be useful in developing effective IRM strategies for H. zea. © 2020 Society of Chemical Industry.

Extended evaluation of Bt protein cross-pollination in seed blend plantings on survival, growth, and development of Helicoverpa zea feeding on refuge ears.

BACKGROUND: A seed blend, also called refuge in a bag (RIB), is used as a strategy to provide refuge for planting pyramided Bt corn in the U.S. Corn Belt. A major concern for the RIB strategy is cross-pollination between Bt and refuge plants, which may reduce susceptible populations of ear-feeding insects like Helicoverpa zea and affect the evolution of resistance. Previous studies showed a 5:95% (Non-Bt: Bt) RIB would be unlikely to provide sufficient refuge populations of H. zea for resistance management. In this study, we extended our research and conducted multiple trials to determine if three RIB plantings with greater refuge sizes (10, 20, and 30% refuge) could provide sufficient H. zea susceptible populations to delay resistance development. RESULTS: Experimental results showed that cross-pollination in 10:90%, 20:80%, and 30:70% RIB plantings still significantly reduced larval survival, delayed larval development and decreased the pupal size of H. zea on refuge plants. Regression analysis showed that the percent reduction of neonate-to-adult survivorship, relative to the survivorship on pure non-Bt ears, was significantly negatively correlated with the refuge percentage. CONCLUSION: These findings suggest that, approximately a 15% non-Bt refuge plants in RIB plantings could produce a similar number of susceptible adult moths as a 5% structured refuge planting, while an approximately 30% non-Bt refuge plant in RIB plantings could provide a similar number of susceptible moths as a 20% structured refuge planting. Information generated from this study should be useful in refining resistance management strategies for Bt crop technologies. © 2019 Society of Chemical Industry.

Phenotypic performance of nine genotypes of Cry1A.105/Cry2Ab2 dual-gene resistant fall armyworm on non-Bt and MON 89034 maize.

BACKGROUND: The fall armyworm, Spodoptera frugiperda (J.E. Smith), is the first target pest that has developed resistance to Bt crops across several countries. Leaf tissue and whole plant assays were employed to determine the survival, development, progeny production, and net reproductive rate of all nine possible genotypes of Cry1A.105/Cry2Ab2-dual gene resistant S. frugiperda on non-Bt and MON 89034 maize expressing the Cry1A.105/Cry2Ab2 proteins. RESULTS: The homozygous resistant genotype was highly resistant to Bt plants. Genotypes possessing only Cry2Ab2 resistance alleles (RAs) or two Cry1A.105 RAs only were susceptible to MON 89034 with a functional dominance level (DML ) of 0.0-0.07 on MON 89034 plants. In contrast, genotypes containing two Cry1A.105 plus one Cry2Ab2 RA performed well on Bt plants, with a DML of 0.11-0.78. Significant numbers of survivors on Bt plants were also observed for genotypes containing a single Cry1A.105 RA, or a Cry1A.105 plus one or two Cry2Ab2 RAs, with a DML of 0.0-0.47. CONCLUSIONS: The fitness of individual resistant genes on pyramided Bt plants varied in the dual-gene resistance system. Genotypes containing more Cry1A.105 RAs performed better than those possessing more Cry2Ab2 RAs. The functional dominance level of an individual resistant gene in this system is related to the DML level in its corresponding single-gene system. Data generated from this study should fill gaps in understanding dual-/multiple-gene Bt resistance, as well as providing useful information for refining resistance modeling, improving resistance risk assessment, and developing management strategies for the sustainable use of pyramided Bt maize technology. © 2019 Society of Chemical Industry.