Mike W. Dunbar

Field-Evolved Resistance to Bt Maize by Western Corn Rootworm

Background Crops engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) are planted on millions of hectares annually, reducing the use of conventional insecticides and suppressing pests. However, the evolution of resistance could cut short these benefits. A primary pest targeted by Bt maize in the United States is the western corn rootworm Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). Methodology/Principal Findings We report that fields identified by farmers as having severe rootworm feeding injury to Bt maize contained populations of western corn rootworm that displayed significantly higher survival on Cry3Bb1 maize in laboratory bioassays than did western corn rootworm from fields not associated with such feeding injury. In all cases, fields experiencing severe rootworm feeding contained Cry3Bb1 maize. Interviews with farmers indicated that Cry3Bb1 maize had been grown in those fields for at least three consecutive years. There was a significant positive correlation between the number of years Cry3Bb1 maize had been grown in a field and the survival of rootworm populations on Cry3Bb1 maize in bioassays. However, there was no significant correlation among populations for survival on Cry34/35Ab1 maize and Cry3Bb1 maize, suggesting a lack of cross resistance between these Bt toxins. Conclusions/Significance This is the first report of field-evolved resistance to a Bt toxin by the western corn rootworm and by any species of Coleoptera. Insufficient planting of refuges and non-recessive inheritance of resistance may have contributed to resistance. These results suggest that improvements in resistance management and a more integrated approach to the use of Bt crops may be necessary.

Field-evolved resistance by western corn rootworm to multiple Bacillus thuringiensis toxins in transgenic maize

Significance Crops genetically engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) kill pest insects and reduce the use of conventional insecticides. However, the evolution of Bt resistance can diminishes these benefits. The western corn rootworm is a serious pest of maize and is managed with Bt maize. Beginning in 2009, western corn rootworm with resistance to maize producing the Bt toxin Cry3Bb1 imposed severe injury to Cry3Bb1 maize in Iowa. We show that cross-resistance exists between Cry3Bb1 maize and mCry3A maize and is associated with severe injury to Bt maize in farmers’ fields. These results illustrate that Bt crops producing less than a high dose of toxin against target pests may select for resistance rapidly; consequently, current approaches for managing Bt resistance should be reexamined. The widespread planting of crops genetically engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) places intense selective pressure on pest populations to evolve resistance. Western corn rootworm is a key pest of maize, and in continuous maize fields it is often managed through planting of Bt maize. During 2009 and 2010, fields were identified in Iowa in which western corn rootworm imposed severe injury to maize producing Bt toxin Cry3Bb1. Subsequent bioassays revealed Cry3Bb1 resistance in these populations. Here, we report that, during 2011, injury to Bt maize in the field expanded to include mCry3A maize in addition to Cry3Bb1 maize and that laboratory analysis of western corn rootworm from these fields found resistance to Cry3Bb1 and mCry3A and cross-resistance between these toxins. Resistance to Bt maize has persisted in Iowa, with both the number of Bt fields identified with severe root injury and the ability western corn rootworm populations to survive on Cry3Bb1 maize increasing between 2009 and 2011. Additionally, Bt maize targeting western corn rootworm does not produce a high dose of Bt toxin, and the magnitude of resistance associated with feeding injury was less than that seen in a high-dose Bt crop. These first cases of resistance by western corn rootworm highlight the vulnerability of Bt maize to further evolution of resistance from this pest and, more broadly, point to the potential of insects to develop resistance rapidly when Bt crops do not achieve a high dose of Bt toxin.

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.

Evidence of Resistance to Cry34/35Ab1 Corn by Western Corn Rootworm (Coleoptera: Chrysomelidae): Root Injury in the Field and Larval Survival in Plant-Based Bioassays

Abstract Western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a serious pest of corn in the United States, and recent management of western corn rootworm has included planting of Bt corn. Beginning in 2009, western corn rootworm populations with resistance to Cry3Bb1 corn and mCry3A corn were found in Iowa and elsewhere. To date, western corn rootworm populations have remained susceptible to corn producing Bt toxin Cry34/35Ab1. In this study, we used single-plant bioassays to test field populations of western corn rootworm for resistance to Cry34/35Ab1 corn, Cry3Bb1 corn, and mCry3A corn. Bioassays included nine rootworm populations collected from fields where severe injury to Bt corn had been observed and six control populations that had never been exposed to Bt corn. We found incomplete resistance to Cry34/35Ab1 corn among field populations collected from fields where severe injury to corn producing Cry34/35Ab1, either singly or as a pyramid, had been observed. Additionally, resistance to Cry3Bb1 corn and mCry3A corn was found among the majority of populations tested. These first cases of resistance to Cry34/35Ab1 corn, and the presence of resistance to multiple Bt toxins by western corn rootworm, highlight the potential vulnerability of Bt corn to the evolution of resistance by western corn rootworm. The use of more diversified management practices, in addition to insect resistance management, likely will be essential to sustain the viability of Bt corn for management of western corn rootworm.

Effects of refuges on the evolution of resistance to transgenic corn by the western corn rootworm, Diabrotica virgifera virgifera LeConte

BACKGROUND Diabrotica virgifera virgifera LeConte is a major pest of corn and causes over a billion dollars of economic loss annually through yield reductions and management costs. Corn producing toxins derived from Bacillus thuringiensis (Bt) has been developed to help manage D. v. virgifera. However, previous studies have demonstrated the ability of this species to evolve resistance to Bt toxins in both laboratory and field settings. RESULTS We used an experimental evolution approach to test the refuge strategies for delaying resistance of D. v. virgifera to corn producing Bt toxin Cry34/35Ab1. In the absence of refuges, D. v. virgifera developed resistance to Bt corn after three generations of selection. In some cases, non-Bt refuges reduced the level of resistance compared with the strain selected in the absence of refuges, but refuge strains did show reduced susceptibility to Bt corn compared with the unselected strain. CONCLUSIONS In this study, non-Bt refuges delayed resistance to Bt corn by D. v. virgifera in some cases but not others. Combining the refuge strategy with pyramids of multiple Bt toxins and applying other pest management strategies will likely be necessary to delay resistance of D. v. virgifera to Bt corn.

Interactions Among Bt Maize, Entomopathogens, and Rootworm Species (Coleoptera: Chrysomelidae) in the Field: Effects on Survival, Yield, and Root Injury

ABSTRACT A 2 yr field study was conducted to determine how a blend of entomopathogens interacted with Bt maize to affect mortality of Diabrotica spp. (Coleoptera: Chrysomelidae), root injury to maize (Zea maize L.) and yield. The blend of entomopathogens included two entomopathogenic nematodes, Steinernema carpocapsae Weiser and Heterorhabditis bacteriophora Poinar, and one entomopathogenic fungus, Metarhizium brunneum (Metschnikoff) Sorokin. Bt maize (event DAS59122–7, which produces Bt toxin Cry34/35Ab1) decreased root injury and survival of western corn rootworm (Diabrotica virgifera virgifera LeConte) and northern corn rootworm (Diabrotica barberi Smith & Lawrence) but did not affect yield. During year 1 of the study, when rootworm abundance was high, entomopathogens in combination with Bt maize led to a significant reduction in root injury. In year 2 of the study, when rootworm abundance was lower, entomopathogens significantly decreased injury to non-Bt maize roots, but had no effect on Bt maize roots. Yield was significantly increased by the addition of entomopathogens to the soil. Entomopathogens did not decrease survival of corn rootworm species. The results suggest that soil-borne entomopathogens can complement Bt maize by protecting roots from feeding injury from corn rootworm when pest abundance is high, and can decrease root injury to non-Bt maize when rootworm abundance is low. In addition, this study also showed that the addition of entomopathogens to soil contributed to an overall increase in yield.

Abundance and Distribution of Western and Northern Corn Rootworm (Diabrotica spp.) and Prevalence of Rotation Resistance in Eastern Iowa

ABSTRACT The western corn rootworm Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) and the northern corn rootworm Diabrotica barberi Smith & Lawrence (Coleoptera: Chrysomelidae) are major pests of corn (Zea mays L.). Historically, crop rotation has been an effective management strategy, but both species have adapted to crop rotation in the Midwest. For both species in eastern Iowa, we measured abundance and prevalence of rotation resistance using sticky traps and emergence cages in fields of corn and soybean (Glycine max L.). Based on currently available data, we calculated the economic thresholds for these pests at two Diabrotica spp. per trap per day in cornfields and 1.5 D. v. virgifera per trap per day in soybean fields. The economic injury level of rotation-resistant D. barberi was determined to be 3.5 adult insects per emergence cage per year. Peak abundance of rootworm adults in cornfields was below economic thresholds in the majority of fields sampled, suggesting that management of rootworm larvae in continuous cornfields may not always be necessary. Rotation-resistant D. barberi was found throughout eastern Iowa using emergence cages in first-year cornfields, however, the abundance was below levels expected to impose economic injury in 14 of 17 fields evaluated. The presence of rotation-resistant D. v. virgifera, as measured by the occurrence of this insect in soybean fields, occurred only in northeastern Iowa and was also below the economic threshold. These data suggests that crop rotation remains a viable pest management strategy in eastern Iowa.

Effect of Soybean Varieties on Survival and Fecundity of Western Corn Rootworm

ABSTRACT The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a major pest of corn (Zea mays L.) in North America and has evolved resistance to crop rotation by ovipositing in alternate crops such as soybeans [Glycine max (L.) Merr.]. Through experiments with plants grown in the greenhouse and the field, we tested whether soybeans with resistance to the soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), affected survival, fecundity, and consumption of soybean for D. v. virgifera. Soybean varieties tested included those types resistant to A. glycines (Rag1 and rag1/rag3) and a susceptible near isoline of the Rag1 variety. Females were provided with a diet of corn tissue for 4 d after which they were fed a diet of tissue from one of three soybean varieties for 4 d, starved for 4 d, or fed corn tissue. When fed greenhouse grown plants, strains differed significantly in survival and consumption, but consumption did not differ by variety of soybean. Diet treatment only affected fecundity; individuals fed corn continuously had greater fecundity than those individuals fed soybeans. In the experiment with plants grown in the field, leaf consumption differed among strains and individuals fed corn continuously had greater fecundity than the other treatments. Soybean varieties with Rag1 and rag1/rag3 resistance to A. glycines did not appear to affect the fitness of D. v. virgifera. Thus, planting of these A. glycines-resistant soybean varieties should not directly affect the spread of rotation-resistant D. v. virgifera.

Limited Impact of a Fall-Seeded, Spring-Terminated Rye Cover Crop on Beneficial Arthropods

Abstract Cover crops are beneficial to agroecosystems because they decrease soil erosion and nutrient loss while increasing within-field plant diversity. Greater plant diversity within cropping systems can positively affect beneficial arthropod communities. We hypothesized that increasing plant diversity within annually rotated corn and soybean with the addition of a rye cover crop would positively affect the beneficial ground and canopy-dwelling communities compared with rotated corn and soybean grown without a cover crop. From 2011 through 2013, arthropod communities were measured at two locations in Iowa four times throughout each growing season. Pitfall traps were used to sample ground-dwelling arthropods within the corn and soybean plots and sweep nets were used to measure the beneficial arthropods in soybean canopies. Beneficial arthropods captured were identified to either class, order, or family. In both corn and soybean, community composition and total community activity density and abundance did not differ between plots that included the rye cover crop and plots without the rye cover crop. Most taxa did not significantly respond to the presence of the rye cover crop when analyzed individually, with the exceptions of Carabidae and Gryllidae sampled from soybean pitfall traps. Activity density of Carabidae was significantly greater in soybean plots that included a rye cover crop, while activity density of Gryllidae was significantly reduced in plots with the rye cover crop. Although a rye cover crop may be agronomically beneficial, there may be only limited effects on beneficial arthropods when added within an annual rotation of corn and soybean.

Increased Risk of Insect Injury to Corn Following Rye Cover Crop

Abstract Decreased pest pressure is sometimes associated with more diverse agroecosystems, including the addition of a rye cover crop (Secale cereale L.). However, not all pests respond similarly to greater vegetational diversity. Polyphagous pests, such as true armyworm (Mythimna unipuncta Haworth), black cutworm (Agrotis ipsilon Hufnagel), and common stalk borer (Papaipema nebris Guenee), whose host range includes rye have the potential to cause injury to crops following a rye cover crop. The objectives of this study were to compare the abundance of early-season insect pests and injury to corn (Zea mays L.) from fields with and without a rye cover crop on commercial farms. Fields were sampled weekly to quantify adult and larval pests and feeding injury to corn plants from mid-April until corn reached V8 stage, during 2014 and 2015. Measurements within fields were collected along transects that extended perpendicularly from field edges into the interior of cornfields. Adult true armyworm and adult black cutworm were captured around all cornfields, but most lepidopteran larvae captured within cornfields were true armyworm and common stalk borer. Cornfields with a rye cover crop had significantly greater abundance of true armyworm and greater proportion of injured corn. Both true armyworm abundance and feeding injury were significantly greater in the interior of cornfields with rye. Common stalk borer abundance did not differ between cornfields with or without rye cover. Farmers planting corn following a rye cover crop should be aware of the potential for increased presence of true armyworm and for greater injury to corn.