Efficacy of Pyramided Bt Proteins Cry1F, Cry1A.105, and Cry2Ab2 Expressed in SmartStax Corn Hybrids Against Lepidopteran Insect Pests in the Northern United States Open Access

Commercial field corn (Zea mays L.) hybrids transformed to express some or all of the lepidopteran insect-resistant traits present in SmartStax corn hybrids were evaluated for insecticidal efficacy against a wide range of lepidopteran corn pests common to the northern United States, during 2008 to 2011 at locations in 15 states. SmartStax hybrids contain a pyramid of two Bacillus thuringiensis (Bt) derived events for lepidopteran control: event TC1507 expressing Cry1F protein and MON 89034 expressing Cry1A.105+Cry2Ab2. These studies focused on characterization of the relative efficacy of each event when expressed alone or in combination, and compared with non-Bt hybrid. Corn hybrids containing pyramided insecticidal proteins Cry1F+Cry1A.105+Cry2Ab2 (SmartStax) consistently showed reduced plant feeding damage by a wide range of lepidopteran larvae compared with single event and non-Bt hybrids. Corn hybrids expressing TC1507 or MON 89034 as single or pyramided events were consistently efficacious against Ostrinia nubilalis (Hübner). SmartStax hybrids had less injury from Agrotis ipsilon (Hufnagel) and Striacosta albicosta (Smith) than corn hybrids containing only event MON 89034 but were not more efficacious than single event TC1507 hybrids. Corn hybrids with event MON 89034 provided better control of Helicoverpa zea (Boddie), than event TC1507 alone. Spodoptera frugiperda (J.E. Smith) efficacy was higher for hybrids with pyramid events and single events compared with the non-Bt hybrids. The spectra of activity of events TC1507 and MON 89034 differed. The combination of TC1507 + MON 89034 provided redundant control of some pests where the spectra overlapped and thereby are expected to confer a resistance management benefit.

Planting of field corn, Zea mays L., in the United States was >97 million acres in 2012, and predicted to be slightly higher in 2013, reflecting a strong demand and commodity value (U.S. Department of Agriculture–National Agriculture Statistics Service [USDA–NASS] 2013). Total domestic supply after the 2012 harvest was slightly <11 billion bushels and predicted to be ≈14 billion bushels for 2013 harvest (USDA–NASS 2013). Corn exports are expected to account for ≈10% of total supply (USDA–NASS 2013). Collectively, these markets have supported a predicted October 2013 average farm price of US$4.40/bu, which is approximately US$3.00/bu lower than the October 2012 value (USDA–NASS 2012).

Protecting the loss of value resulting from lepidopteran pest damage to the crop is an important consideration for corn growers. In the northern U.S. Corn Belt, several important lepidopteran pests commonly cause injury to field corn. European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae); black cutworm, Agrotis ipsilon (Hufnagel); fall armyworm, Spodoptera frugiperda (J.E. Smith); corn earworm, Helicoverpa zea (Boddie); and the western bean cutworm, Striacosta albicosta (Smith) (all Lepidoptera: Noctuidae), can cause economic damage in field corn from seedling stage to maturity.

Lepidopteran corn pest management relies on multiple strategies including cultural, chemical, as well as native and transgene-mediated host plant resistance. Insecticidal proteins from Bacillus thuringiensis (Bt) heterologously expressed in plant tissues represent a relatively new management strategy, and have reduced the limitations associated with managing field corn insects with synthetic insecticides (Rice 2004). This strategy has advanced rapidly in the past 15 yr as a result of the competitive development and commercial adoption of transgenic Bt crops.

One of the first commercially available Bt proteins expressed in corn was Cry1Ab, which effectively protected the foliar tissue of the corn plant against lepidopteran feeding. Cry1Ab protein expressed in field corn is highly effective for control of larval European corn borer and southwestern corn borer (Diatraea grandiosella, Dyar), and is partially effective against corn earworm and fall armyworm (Pilcher et al. 1997; Storer et al. 2001; Buntin et al. 2004a,b; Castro et al. 2004; Buntin 2008). The subsequent deregulation by the U.S. Department of Agriculture (USDA) and registration by the U.S. Environmental Protection Agency (U.S. EPA) of event TC1507 in 2001, and its commercialization as Herculex I Insect Protection (Dow AgroSciences LLC, Indianapolis, IN) in 2003, has made it possible to control a broader spectrum of lepidopteran pests with a single event product. Event TC1507 expresses the Cry1F protein, and expanded the spectrum of lepidopteran corn pests controlled to include fall armyworm, western bean cutworm, and black cutworm in addition to the corn borers mentioned previously. Event TC1507 controlled a wide range of lepidopteran pests more effectively than non-Bt corn managed with or without supplemental foliar insecticides (Buschman et al. 2001, Royer et al. 2003, Buntin 2008, Siebert et al. 2008). This level of robust pest control was a key factor in user satisfaction and rapid product adoption, as was the reduced need for pesticide application.

Event MON 89034 (Genuity VT Pro, Monsanto Co., St Louis, MO), deregulated and registered by the USDA and U.S. EPA, respectively, in 2008, expresses two lepidopteran-active Bt proteins, Cry1A.105 and Cry2Ab2 that confer broad-spectrum control of lepidopteran pests (Drury et al. 2008, Center for Environmental Risk Assessment [CERA] 2012). The cry1A.105 gene is a chimeric gene comprising regions encoding domains I and II of Cry1Ab and domain III of Cry1F (CERA 2012). In addition to corn borers, the spectrum of activity of Cry1A.105 includes fall armyworm, and that of Cry2Ab2 includes corn earworm (Drury et al. 2008).

The first gene expressed in corn for the control of corn rootworm produced Cry3Bb1protein (event MON 863) and was introduced in 2003. In 2005, a second Cry3Bb1 event was introduced, MON 88017, which also expressed glyphosate tolerance (YieldGard Rootworm or Genuity VT Triple Pro, Monsanto Co., St. Louis, MO; U.S. EPA 2003, 2010; Vaughn et al. 2005). In 2005, corn containing Cry34Ab1 and Cry35Ab1 as a binary Bt protein (event DAS-59122-7; Herculex RW Rootworm Protection, Dow AgroSciences LLC, in collaboration with Pioneer Hi-Bred International now known as DuPont Pioneer, a DuPont Company) was commercialized (Ellis et al. 2002, Herman et al. 2002, U.S. EPA 2005). These latter two corn rootworm events have been stacked with a lepidopteran event through conventional breeding methods to commercially offer Genuity VT Triple Pro (events MON 89034 × MON 88017) and Herculex Xtra (events TC1507 × DAS-59122-7); products that offer a broad spectrum control of lepidopteran and coleopteran rootworm pests.

Events MON 89034 and TC1507 and events MON 88017 and DAS-59122-7 have been combined through conventional breeding to create SmartStax (Dow AgroSciences LLC, Indianapolis, IN and Monsanto Company, St. Louis, MO), a trait pyramid (i.e., plants with simultaneous expression of two or more insecticidal proteins that target the same insect; Gould 1986, Sachs et al. 1996). Field corn hybrids containing these six Cry proteins (Cry1A.105, Cry2Ab2, Cry1F, Cry3Bb1, Cry34Ab1, and Cry35Ab1) broaden the spectrum of activity and aid in resistance management by providing multiple modes of action for target pest species (McGaughey and Whalon 1992; Tabashnik 1994; Gould 1998; Storer et al. 2006, 2010). Field corn hybrids containing events MON 89034, TC1507, MON 88017, and DAS-59122-7 also contain transgenes for tolerance to the herbicides glyphosate and glufosinate ammonium, thereby providing a tool for managing weeds, including an increasing number of glyphosate-resistant species (Green and Owen 2011).

The objective of the following series of trials established in the northern United States was to understand the relative efficacy (potency and spectrum) of field corn expressing Cry1F and Cry1A.105+Cry2Ab2, alone and in combination, for control of black cutworm, fall armyworm, European corn borer, corn earworm, and western bean cutworm.

Materials and Methods

Field trials were designed to evaluate insect damage from leaf-, stalk-, and ear-infesting insects to Bt and non-Bt corn hybrids. These trials were conducted between 2008 and 2011 at locations in 15 states in the northern United States (Table 1). Depending on the year and location, trials compared Bt events, both alone and in pyramid, and a non-Bt corn hybrid. To minimize the effects of corn genotypes, near-isogenic corn hybrids were evaluated, which expressed one of the following treatments: 1) Cry1F (event TC1507 or TC1507 × DAS-59122-7); 2) Cry1A.105 + Cry2Ab2 (MON 89034 × MON 88017); 3) SmartStax, a breeding combination containing Cry1F and Cry1A.105 + Cry2Ab2 (MON 89034 × TC1507 × MON 88017 × DAS-59122-7, also including Cry34Ab1 or Cry35Ab1 and Cry3Bb1); and 4) a non-Bt corn hybrid (containing event NK603 and conferring only glyphosate herbicide resistance). Corn hybrid treatments were evaluated against the following target pests: European corn borer, fall armyworm, corn earworm, black cutworm, and western bean cutworm. Seed was treated with commercial fungicides and 250 mg a.i. per kernel of either thiamethoxam (Syngenta, Greensboro, NC) or clothianidin (Bayer CropSciences, Research Triangle Park, NC). Trial areas received no soil or foliar fungicide or insecticide applications; commercial herbicides were applied consistent with local weed management practices. Plot size was four rows spaced 76.2 cm apart and 6.1 m in length, and were organized using randomized complete block design. Treatments were replicated four times for each trial, with the exception that some black cutworm trials had six replications. Black cutworm plots were single row and 3 m in length with individual plants or groups of plants surrounded by a barrier to contain the larvae. Trials were managed using locally accepted agronomic production practices.

All of the insects used in these studies were obtained from commercial insectaries (Benzon Research, Calisle, PA, or French Agricultural Research Inc., Lamberton, MN) and had no exposure to insecticides or Bt crops within the past 12 yr, except for western bean cutworm eggs that were field collected annually from Nebraska corn fields.

Black Cutworm.

Plants were infested at V1 corn growth stage (Ritchie et al. 1993) by placing two, third-instar larvae at the base of each plant. Larvae were confined to the area of the seedlings by placing barriers (steel barriers, 30 cm in height and enclosing 3-m × 76-cm area) around each plot or around individual plants (PVC pipe, 15 cm in diameter by 15 cm in height above soil line). At 7 and 14 d after infestation, plant injury was assessed by counting the number of plants that were severed at the soil surface (i.e., number of plants cut). Plant injury counts were transformed into percent stand reduction values for each plot based on the number of plants injured versus the total number infested plants.

Fall Armyworm.

Fall armyworm injury to corn whorls was assessed by estimating injury to whorl tissue. A minimum of 10 plants at the V6–V8 growth stage plants in the center of the plot were infested using one of three following methods: 1) a single egg mass containing approximately 40 eggs was laid on wax paper and placed deep within the whorl, 2) a mixture of corn grit containing 20–30 neonate larvae were deposited in whorls using the “bazooka” method (Davis and Oswalt 1979), or 3) 15 first-instar or five second-instar were placed into each whorl using a paint brush. Larger larvae infested within whorls were fed an artificial diet before infestation in the field. Leaf-feeding injury was evaluated at 14 d after infestation using the 0–9 rating scale (Davis et al. 1992).

European Corn Borer.

European corn borer first generation injury to corn whorls was assessed 14 d after infestation on 10–20 plants per plot. All plants were artificially infested with 50 neonate larvae using the corn grit method, where larvae were applied within the corn whorl at the V6–V8 corn growth stage. Infested plants were evaluated for leaf feeding using the 1–9 scale (Guthrie et al. 1960).

European corn borer second generation injury to corn stalks was assessed on a different set of 10–20 plants in each plot that were artificially infested with 100 neonate larvae using the corn grit method. Larvae were applied to the corn leaf axil located above and below the ear node at the VT–R1corn growth stage, where by larvae mixed with corn grits were volumetrically applied to corn plants at a rate of 50 larvae per leaf axil (axil above and below ear leaf axil). Between 14 to 21 d after infestation, individual stalks of infested plants were dissected and assessed for injury by recording the number of tunnels and the length (centimeter) of each tunnel.

Corn Earworm and Western Bean Cutworm.

Protection against ear-feeding pests was evaluated after natural, artificial, or both infestations, of corn earworm or western bean cutworm, whereby species were in separate field trials. At late R2 corn growth stage, five, first- or second-instar were applied to the silks of 10 corn ears per plot for each species. At trial locations with natural mixed species, pollination shoot bags were placed over the ear to prevent artificially infested ears from being contaminated with natural infestations. Between 14 to 21 d after infestation, 10–20 ears per plot were evaluated by determining the number of larvae per ear and area of kernels consumed per ear (square centimeter).

Statistical Analysis.

The mean and ±SE were determined for all evaluations, and are reported in the results tables. Efficacy data for black cutworm, corn earworm, western bean cutworm, and European corn borer (stalk injury) were analyzed using a generalized linear mixed model procedure (SAS Institute 2008). The least-squares means were used for Tukey-Kramer mean separation tests to distinguish treatment differences. Fall armyworm and European corn borer leaf-injury rating data were analyzed by determining the frequency of occurrence of the leaf-injury rating data in the species rating scale categories. These frequency data were analyzed using a generalized linear mixed model procedure for a multinomial response distribution with a cumulative logit link function (SAS GLIMMIX; SAS Institute 2008). Odds ratios were compared among treatments to distinguish treatment differences, and determine which treatments provided better plant protection by having a higher frequency of lower leaf-injury ratings. Location, replicate within location and treatment by location factors were considered random in all models. All of the analyses were performed using an alpha level of 0.05.

Results and Discussion

Black Cutworm.

Percent plant stand reduction in seedling stage corn was significantly less for hybrids containing Cry1F and the pyramid of Cry1F+Cry1A.105+Cry2Ab2 proteins compared with the non-Bt and Cry1A.105+Cry2Ab2 hybrids for both evaluation intervals. The Cry1F single and pyramid hybrids protected plants and maintained mean plant stands of >85%, even under a high infestation level and confinement on the plant (Table 2). There were no significant differences in percent stand reduction between the hybrids containing Cry1F and Cry1F+Cry1A.105+Cry2Ab2. These results are consistent with other field studies in which Cry1F-expressing corn hybrids consistently had the highest plant populations after a naturally occurring black cutworm infestation (Kullik et al. 2011). Stand reduction by black cutworm was similar in the two non-Cry1F hybrids (non-Bt and Cry1A.105+Cry2Ab2) by 14 d after infestation. The Cry1A.105+Cry2Ab2 proteins did not add to the efficacy of Cry1F. The combination of Cry1F in corn and a seed-applied insecticide provides consistent protection against black cutworm.

Fall Armyworm.

Whorl feeding was significantly reduced for all three Bt-expressing hybrids compared with the non-Bt hybrids (Tables 3 and 4). Cry1F or Cry1A.105+Cry2Ab2 each provided control of fall armyworm that was not statistically different from the pyramided protein hybrid (Cry1F+Cry1A.105+Cry2Ab2). Each of the single event components of SmartStax hybrids provides high levels of control; therefore, it is unlikely that a statistically significant reduction in fall armyworm feeding would be observed when the events are combined (although in this study SmartStax provided the greatest protection numerically). These results are consistent with the performance of these Bt corn products exposed to fall armyworm infestations in the southern United States (Buntin 2008; Drury et al. 2008; Siebert et al. 2008, 2012; Hardke 2011). The results for these studies suggest that pyramided proteins provide redundant protection to fall armyworm larvae meaning that insects are exposed to lethal doses of more than one Bt protein. With differences in the modes of action of the component proteins (Storer et al. 2012a,b), SmartStax is expected to provide improved resistance management compared with single event products.

European Corn Borer.

All corn hybrids containing single or pyramided Bt proteins were highly efficacious against European corn borer and showed no evidence of larval feeding. In contrast to the non-Bt corn hybrid, no leaf feeding or corn stalk tunneling was observed in the Bt hybrids (Table 5). These results were expected as single event products and are believed to satisfy the “high dose” definition (>99.99% mortality of susceptible larvae; U.S. EPA 2001). The three proteins pyramided in SmartStax trait each provide high levels of European corn borer larval mortality, and likely provide redundant protection with associated resistance management benefits because of the expected high mortality of insects that are resistant to one of the proteins (Storer et al. 2012b).

Corn Earworm.

Kernel consumption was significantly reduced in corn hybrids containing Cry1A.105+Cry2Ab2 and the pyramid of Cry1F+Cry1A.105+Cry2Ab2 proteins compared with the corn hybrid containing Cry1F and the non-Bt hybrid (Table 6). Single and pyramid hybrids expressing Cry1A.105+Cry2Ab2 reduced the kernel area consumed in corn ears by >98%. The Cry1F hybrids also had significantly lesser corn kernel consumption than the non-Bt hybrids; although the level of protection was significantly lower than that for the Cry1A.105+Cry2Ab2 hybrids. These results are consistent with previous reports in which corn containing Cry1F reduced ear infestations of corn earworm relative to a non-Bt corn hybrid (Buntin 2008), and with commercial labels for Cry1F that claim suppression of corn earworm rather than control (Dow AgroSciences 2007). These data are consistent with the findings of Siebert et al. (2012), which demonstrate that corn hybrids containing Cry1F combined with Cry1A.105+Cry2Ab2 have improved corn earworm protection compared with Cry1F alone.

Western Bean Cutworm.

Corn kernel consumption was significantly lesser for hybrids containing Cry1F and the pyramid of Cry1F+Cry1A.105+Cry2Ab2 proteins than the non-Bt and Cry1A.105+Cry2Ab2 hybrids (Table 6). There was no significant difference in kernel consumption between corn hybrids with TC1507 event containing Cry1F protein. The two non-Cry1F hybrids (non-Bt and Cry1A.105+Cry2Ab2) did not differ significantly in kernel consumption by western bean cutworm. The ear feeding efficacy data is consistent with the findings of previous reports in demonstrating that Cry1F protein provides effective management of western bean cutworm (Catangui and Berg 2006, Eichenseer et al. 2008, Hutchinson et al. 2011). Cry1F protein represents only a single mode of action in the pyramid corn hybrids (SmartStax). If western bean cutworm continues to expand its range and persistence in corn growing areas (Michel et al. 2010), then additional pest management tools may be warranted to extend the durability of Cry1F against this pest.

Evaluations conducted from 2008 to 2011 to characterize the insect spectrum of control and efficacy of Bt traits present in SmartStax against several important lepidopteran corn pests common to the northern United States. SmartStax hybrids contain a pyramid of two Bt events for lepidoptera control: event TC1507 expressing Cry1F protein and MON 89034 expressing Cry1A.105+Cry2Ab2. Field corn hybrids containing pyramided insecticidal proteins Cry1F+Cry1A.105+Cry2Ab2 (SmartStax) were consistently more efficacious in reducing a wide range of lepidopteran-mediated plant injury or damage than either single event and compared with non-Bt hybrids. In general, TC1507 and MON 89034 individually provide complementary spectra of pest control. When pyramided as SmartStax, the resulting hybrids provided broader corn pest control than either single event. For several key pests, the data suggest there is redundant protection, that is, susceptible larvae encounter lethal doses of more than one Bt protein. This is expected to confer a resistance management benefit as, in the absence of significant cross-resistance, insects that are resistant to one of the Bt proteins will continue to be controlled by the other Bt proteins when feeding on SmartStax (Storer et al. 2012b). However, the durability and utility of Bt proteins in SmartStax will continue to require active resistance management measures, including resistance monitoring and the use of refuges. Grower compliance with refuge requirements is enhanced through the blended refuge concept. Seed blends with SmartStax are available in the market (Refuge Advanced powered by SmartStax, Dow AgroSciences LLC, and Genuity SmartStax RIB Complete, Monsanto Company, St. Louis, MO) providing a convenient and simple solution that ensures refuge compliance.

Acknowledgments

We acknowledge the efforts of our many colleagues at Dow AgroSciences, Monsanto Company, Colorado State University, Iowa State University, Michigan State University, Ohio State University, Pennsylvania State University, Purdue University, South Dakota State University, University of Delaware, University of Illinois, University of Kentucky, University of Minnesota, University of Nebraska, University of Wisconsin, and private researchers who contributed to this project. We thank Jon Babcock, Gary Thompson, Scott Hutchins, Simran Trana, Andrea Borucki, Joe Miller, and Kari Lukasik of Dow AgroSciences for reviewing the manuscript. Herculex and Refuge Advanced are trademarks of The Dow Chemical Company (“Dow”) or an affiliated company of Dow. Herculex developed in collaboration with Pioneer Hi-Bred International, known as DuPont Pioneer of the DuPont Company. Roundup Ready, Genuity, SmartStax, YieldGard, VT Triple Pro, and RIB Complete are registered trademarks of Monsanto Technology LLC. SmartStax multi-event technology developed by Dow AgroSciences and Monsanto.

References Cited