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Article

Effect of Blended Bt Corn Refuge on Corn Earworm (Lepidoptera: Noctuidae) Infestation and Grain Yield

by
George David Buntin
1,* and
Pamela Somerville Rowe
2
1
Department of Entomology, University of Georgia-Griffin Campus, Griffin, GA 30223, USA
2
Department of Crop and Soil Sciences, University of Georgia-Griffin Campus, Griffin, GA 30223, USA
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(10), 2246; https://doi.org/10.3390/agronomy14102246
Submission received: 2 August 2024 / Revised: 24 September 2024 / Accepted: 25 September 2024 / Published: 29 September 2024
(This article belongs to the Section Pest and Disease Management)
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Abstract

:
Blended refuges for corn-expressing toxins from Bacillus thuringiensis (Bt) Berliner controls have been approved in the United States as an alternative resistance management approach to structured refuge. This study examined the impact of blended refuges up to 30% non-Bt seed on the corn earworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), larval numbers, and kernel injury, and its effect on corn grain yield and test weights. The percentage of infested ears, larval numbers, and kernel injury of the 20% blend were not significantly different from the non-Bt and nonblended Bt for the Cry1A.105 + Cry2Ab + Cry1Fa2 treatment, but infested ears, larval numbers, and kernel injury of the nonblended Cry1A.105 + Cry2Ab treatment was lower than the comparable non-Bt hybrids, with the 20% blend being intermediate. The nonblended Cry1A.105 + Cry2Ab + Vip3Aa20 had virtually no larvae in ears and no kernel injury. Ear infestation, larval numbers, and kernel injury of the non-Bt blends with Vip3Aa20 were proportional to the percentage of non-Bt seed in the blend, and all infested ears were from the non-Bt refuge plants. Grain yield and test weight were not significantly different among nonblended or blended treatments of any Bt product tested. Results indicate losses of grain yield and test weight by corn earworm in seed blends up to 30% non-Bt seed are unlikely with infestation levels observed in this study.

1. Introduction

Transgenic corn, Zea mays L., expressing insecticidal toxins derived from Bacillus thuringiensis (Bt) targeting lepidopteran pests has been available in the United States for more than 25 years. Hybrids with Bt toxins initially were single-gene toxins that target the European corn borer, Ostrinia nubilalis (Hübner), and other stalk-boring lepidopteran species, including the southwestern corn borer, Diatraea grandiosella (Dyar) [1]. Original single-gene toxins and events were the Cry1Ab toxin in event MON810 (Monsanto Co., now Bayer CropScience, St. Louis, MO, USA) and event Bt11 (Syngenta Seeds, Research Triangle Park, NC, USA), which were both marketed as YieldGard® corn borer [2]. In 2002, the Cry1Fa2 toxin in event TC1507 was marketed as Herculex® 1 (Corteva, AgroSciences, Indianapolis, IN, USA) [2,3]. These events were effective against corn borers but were not highly effective in preventing kernel damage by corn earworm, Helicoverpa zea [3,4,5,6].
Since 2010, several pyramided products containing two or three Bt toxins targeting lepidopteran pests have become available in the U.S. In addition to Cry1Ab and Cry1Fa2, newer Bt toxins include Cry1A.105, Cry2Ab2, and Vip3Aa20 (Vip3A) in various combinations. Pyramided Bt products available during the period of this study include Genuity® VT Double PRO (Bayer CropScience, St. Louis, MO, USA), which contained Cry1A.105 and Cry2Ab2 toxins for lepidopterans, and SmartStax® (Corteva AgroSciences and Bayer CropScience), which contain Cry1A.105, Cry1Fa2, and Cry2Ab2 for lepidopteran pests, plus three toxins for corn rootworm control [5]. Bt products containing the Vip3Aa20 gene are Agrisure® Viptera (Syngenta Crop Sciences, RTP, NC, USA), which contains Cry1Ab and Vip3A; Optimum Leptra (Pioneer Hibred Internat., Johnston, IA, USA) with Cry1Ab + Cry1Fa2 + Vip3Aa20; and Genuity Trecepta (Bayer CropScience, St. Louis, MO, USA), which contains Cry1A.105 + Cry2Ab2 + Vip3Aa20 toxins for lepidopteran pests [2,5,7]. Bt corn products have been adopted rapidly by corn growers with transgenic corn hybrids in the United States, representing over 90% of planted corn acreage with 85% of acreage containing Bt gene varieties by 2023 [8].
Resistance management requirements for Bt products are based on the high-dose/refuge strategy where Bt toxins are expressed at high enough doses to kill >99.9% of wild-type individuals, resistance alleles are single, rare, and mostly recessive, and mating of moths from Bt and refuge plants is random [2,9]. While Bt corn was considered high dose for the initial target pest O. nubilalis, this criterion was not met for Helicoverpa zea (Boddie) [10]. The refuge requirement for pyramided Bt products is 5% in the Midwest and 20% in cotton-growing areas of the southern U.S. [11]. Originally, the refuge was a separate planting of a non-Bt hybrid in various planting arrangements [1,11]. In 2012, an integrated or blended refuge was approved for the Midwestern region, where a 5% mixture of non-Bt seed was blended with the Bt seed [11,12]. This approach is marketed as a refuge-in-a-bag (RIB) management method, which reduces the reliance on growers to plant refuges in separate areas within and near Bt corn fields [9,13].
Pyramided Bt hybrids expressing Cry1A.105 + Cry2Ab2 initially showed good levels of control of corn earworm relative to single-toxin Bt traits, with 95% control in four southern states in 2007–2009 and 97% control in the Carolinas in 2012–2013 [6]. But more recently, corn earworm has developed resistance to Cry1Ab, Cry1A.105, and Cry2Ab2 expressed in Bt corn [2,14,15,16,17]. Consequently, the only highly effective Bt corn products contain the Vip3A toxin, although resistance has been detected in a few areas [18,19]. Because of declining grower compliance in planting structured non-Bt refuges in the Southeast [20,21], there may be interest in delaying the development of resistance by increasing the amount of non-Bt refuge by deploying a blended-refuge resistance approach in cotton-growing areas [13]. If a blend approach was deployed in cotton-growing areas, a 20% blend would reflect the current requirement for a 20% structed non-Bt refuge. Towles et al. [22] demonstrated that corn earworms develop and emerge from field corn in blends of 10, 20, and 30% in proportion to the percentage of the non-Bt plants in the blend, indicating the blends can produce viable moths as part of refuge. Nevertheless, this approach in cotton-growing areas is controversial because larval movement within and among plants and variable expression of Bt toxins in kernels of refuge plants in blended plantings may expose kernel-feeding larvae to sub-lethal levels of toxins, which may enhance selection for resistance [23,24,25,26]. Corn earworms pupate in the soil and overwinter in southern areas below 40° latitude [27], so selection may continue through later generations in southern areas [26].
In addition to concerns about the blended seed approach for resistance management, there are concerns about the effect of susceptible plants in a blend on the productivity and yield potential of blended refuge corn. Wiseman [28] and reports cited therein stated that corn earworm causes losses of 2.5% annually in the U.S., with losses in the southeastern U.S. ranging from 1.5 to 16.7%. While corn earworm infestations clearly cause injury to pollen tubes, ears, and kernels, more recent studies have generally considered corn earworm injury to not cause measurable or significant yield loss when field corn is not planted late in the southern region [6,29,30,31,32]. Nevertheless, some studies have documented yield responses to Bt corn as compared with non-Bt corn in the southern U.S. [29,33,34]. Reisig et al. [35] compared the efficacy of unblended Cry1F + Cry1Ab pyramid with a 5% non-Bt blend in a study across 12 southern states and found a similar level of kernel injury by corn earworm but did not measure grain yield. A larger study examining blends of 0, 5, 10, and 20% of a Cry1F + Cry1Ab + Vip3Aa20 pyramid in nine southern states over three years found that kernel injury by corn earworm was usually greater in the 10% and 20% blends than the injury in the unblended treatment [36]. Grain yield was significantly lower in 20% blends than non-blended corn in the late-planted trials in one of three years but was not significantly different among treatments in all other experiments. Analysis by location found that only two of nine locations had greater yields in the nonblended Bt plots versus the 20% blend. Both studies [35,36] were conducted before widespread resistance to Cry1Ab and Cry2Ab had occurred [2,14,15,16,17]. The objective of current work is to document the level of ear infestation, larval numbers, and corn kernel injury by corn earworm in blends up to 30% non-Bt seed and whether blends of up to 30% non-Bt plants may reduce corn grain yield and quality as measured by test weight. The level of a 20% blend was selected for comparison in Experiment 1 because this is the largest level examined in previous studies, and 20% is the structed refuge requirements of pyramided Bt corn in the U.S. Experiment 2 examined blends of 10, 20, and 30%.

2. Materials and Methods

2.1. Field Experiments

Two experiments were conducted from 2019 to 2022. Experiment 1 was conducted in 2019 and 2021 at the University of Georgia’s Griffin’s Bledsoe Research Farm located near Griffin, Georgia (N 33.175964 W −84.4092410), and Experiment 2 was conducted over four years at the Southwest Research and Education Center (N 32.046602 W −84.370610) located near Plains, Georgia. In both locations, tillage was conventional using chisel plowing, with 440 kg/ha of a 5-10-15 (N-P-K) granular fertilizer and an added 112 kg of N (as ammonium nitrate) incorporated via disk harrowing. Plots measured 8 rows by 12.2 m long with 76 cm row centers at Griffin and 96 cm row centers at Plains. Rows 1 and 2 and 7 and 8 in each plot were not sampled or used for yield measurements and served as borders to mitigate neighbor plot influences. Seed for various hybrid corn unblended and blended treatments was measured into single-row envelopes, and for blended treatments, seed was counted to achieve the proper percentage of blended seed for each row. Seed was planted at 79,040 seed per ha with a modified 2-row John Deere Maxi-Merge packet planter. Planting dates for Experiment 1 were 6 May 2019 and 11 May 2021, and for Experiment 2, were 30 April 2019, 14 May 2020, 23 April 2021, and 28 April 2022. Experiments each year were arranged in a randomized complete block design with four replications.
Weed control in all experiments consisted of the pre-emergence application of atrazine at 2.24 kg (AI)/ha (Aatex 4L, Syngenta, Crop Protection, Greensboro, NC, USA) plus acetochlor at 1.26 kg (AI)/ha (Warrant 3ME, Corteva AgriScience, Pittsburg, CA, USA) and glyphosate at 0.84 kg (AE)/ha, which was applied 20–25 days post-emergence. All seed was obtained from DeKalb Seeds (Bayer CropScience, St. Louis, MO, USA) and from Pioneer Hibred International Inc. (Corteva AgriScience, Johnston, IA, USA). Seed was pretreated with two to three fungicides in addition to either clothianidin at 0.5 mg per kernel (Poncho 500, Bayer CropScience, RTP, NC, USA) or thiamethoxam at 0.5 mg per kernel (Cruiser 5FS, Syngenta Crop Protection, Greensboro, NC, USA). No additional insecticides were applied. In addition to rainfall, the plots were irrigated as needed with 1.3 cm of water twice a week.
In experiment 1, four sets of three hybrid treatments of similar relative maturity and agronomic characteristics were used. In all four all, there was a non-Bt hybrid, a non-blended Bt hybrid, and a blend of 80% Bt seed and 20% non-Bt seed. The Bt products evaluated were Genuity® VT Double PRO (VT2P) containing Cry1A.105 + Cry2Ab2, Genuity® SmartStax (SStax) containing Cry1A.105 + Cry2Ab2 + Cry1F, and Genuity® Trecepta (TRE) containing Cry1A.105 + Cry2Ab2 + Vip3Aa20. The VT2P and Trecepta hybrids did not contain any Bt genes for corn rootworms, but SmartStax contains Cry3Bb1 + Cry 34Ab1 + Cry35Ab1 targeting Diabrotica corn rootworms. Diabrotica corn rootworms were not present at either location in Experiments 1 and 2. There was one set of three treatment for each of the Vt Double PRO and SmartStax hybrids, but there were two sets of hybrids with the Trecepta Bt technology for a total of 12 hybrid treatments. Treatments in Experiment 1 were the same in both years. Treatments in Experiment 2 consisted of Dekalb brand hybrids in similar agronomic backgrounds with no Bt traits, Genuity VT Double Pro, and Genuity Trecepta in non-blended and blended Trecepta with a 10, 20, or 30% non-Bt seed. The specific hybrids and treatments for both experiments are listed in Tables S1 and S2.

2.2. Data Collection

Plant stand counts were taken using rows three and four of each plot at 30 to 40 days after planting at the V5 to V7 growth stage. Infestations of fall armyworm, Spodoptera frugiperda J.E. Smith, were assessed at the same time from 2 rows per plot by recording the percentage of plants with whorl damage, and the severity of damage of each infested plant was rated using the 0 to 9 scale of Davis et al. [37], where zero is no damage and 9 is nearly total whorl and furl defoliation.
Corn earworm ear infestation was measured at stage R3 (milk stage) on 20 or 30 primary ears per plot by recording the number of small (instars 1 and 2 (<7 mm)), medium (instars 3 and 4 (7–24 mm)), and large (instars 5 and 6 (>24 mm)) larvae and ears with feeding cavities and exit holes indicating larval emergence from the ear. Corn earworm kernel/ear damage was measured at the early R6 (dent/mature) stage by measuring tip and kernel damage sustained in 20 or 30 ears per plot. Stalks were also examined at stages R3 and R5 for evidence of stalk borer feeding damage, but none was found in any experiment. Ears in the Vip3Aa20 non-blended and blended treatments in Experiment 2 that were infested with corn earworm larvae or damage were tested for Vip3Aa20 expression using QuickStix Kit gene testing strips (Envirologix Inc., Portland, ME, USA).
Grain yield and test weight were measured at maturity by harvesting 2 rows per plot, usually rows 5 and 6, where no earworm samples were taken using a Delta 2-row plot combine (Wintersteiger Inc., Salt Lake City, UT, USA) with a yield monitoring system that measures plot grain weight, test weight, and moisture content. Grain yields were calculated and adjusted to the standard 15.5% moisture content.

2.3. Statistical Analyses

Data were explored using a generalized linear mixed model analysis via SAS 9.4 (PROC MIXED) [38] SAS Institute 2013). In experiment 1, the two years were combined, and treatment was modeled as a fixed effect and year and replication as random effects. Initial analyses found that year*treatment were not significant for all variables, so the interaction was removed from the model statement. In experiment 2, results were analyzed by year with a generalized linear mixed model where treatment was a fixed effect and replication was a random effect. Results in experiment 2 were also combined over the four years in a combined analysis where year treatment and year*treatment were modeled and year was modeled as an intercept with replication as the subject. In all analyses, percentage data were transformed using an angular transformation, and count and yield data were transformed using a log10(x + 0.1) transformation. Degrees of freedom were estimated with the Kenward–Roger method. Means and standard errors calculated with PROC MEANS are reported, but statistical analyses were based on transformed LS means. Residual plots were used to verify that errors were homoscedastic responses. When significant treatment differences (α = 0.05) were found, treatments were separated using the least square means separated using Tukey’s HSD test (α = 0.05) in the PROC PLM option in SAS.

3. Results

3.1. Results of Experiment 1

Plant stand was significantly different among treatments (F = 3.68; df = 11, 69; p = 0.0004). However, stand differences were among hybrid groups with different base genetics, whereas Bt blend treatments within each group were not significantly different. Fall armyworm whorl infestations were absent in 2019 and did not exceed 1.5% infested plants in two of the non-Bt hybrids in 2021 (Table S3).
Corn earworm exceeded 90.0% infested ears at R3 and 83.8% damaged ears at R6 in all four non-Bt hybrids (Figure 1). Blend treatments significantly affected the percentage of infested ears at R3 (F = 152.78; df = 11, 69; p < 0.0001) and the percentage of damaged ears at R6 (F = 64.00; df = 11, 69; p < 0.0001). The VT2P hybrid had a lower percentage of infested and damaged ears than the non-Bt hybrid, with the blended treatment being intermediate between the non-Bt and VT2P hybrid. The VT2P hybrid had 30.6% fewer infested ears and 16.7% fewer damaged ears than the comparable non-Bt hybrid. The SmartStax hybrid did not significantly reduce the percentage of damaged ears, and the blended treatment was not different than the non-blended SmartStax hybrid in the percentage of infested and damaged ears. Both non-blended Trecepta hybrids almost eliminated infested and damaged ears, with damaged ears averaging 0.4 ± 0.4%. Both Trecepta blended treatments had up to 20.8% infested ears and 14.2% damaged ears but were not significantly different than the non-blended Trecepta hybrid.
The total number of larvae and size distribution of larvae in blend treatments are shown in Figure 2. Treatments significantly affected total larvae per ear (F = 265.45; df = 11, 69; p < 0.0001) and numbers of small larvae (F = 40.17; df = 11, 69; p < 0.0001), medium larvae (F = 41.71; df = 11, 69; p < 0.0001), large larvae (F = 31.43; df = 11, 69; p < 0.0001), and larval exits (F = 22.55; df = 11, 69; p < 0.0001). The VT2P hybrid had significantly fewer total larvae than the comparable non-Bt hybrid, but the SmartStax hybrid did not have fewer total larvae than the comparable non-Bt hybrid. The size distribution of larvae varied, with both VT2P and SmartStax hybrids having more small larvae but fewer medium, large, and exit larvae than the comparable non-Bt hybrids. The size distribution of larvae in the blended hybrid was similar to the non-blended SmartStax hybrid but was intermediate for the VT2P and non-Bt hybrid. The Trecepta hybrids had very few larvae in the non-blended treatment, and all larvae were small- or medium-sized with only one ear with a larval exit hole. The Trecepta blends had a small but significantly more larvae than the non-blended Trecepta treatments, with larvae in all size categories.
Hybrid blend treatments also significantly affect corn ear tip (F = 93.17; df = 11, 69; p < 0.0001) and kernel (F = 107.39; df = 11, 69; p < 0.0001) damage and total ear tip plus kernel damage (F = 67.39; df = 11, 69; p < 0.0001) (Figure 3). Ear damage was not significantly different among the non-blended and blended SmartStax hybrid treatments and the comparable non-Bt hybrid. The VT2P hybrids had less total ear damage, but tip and kernel damage were not significantly less than the comparable non-Bt hybrid. The blended VT2P hybrid was intermediate and not different than either the non-blended VT2P or non-Bt hybrids. Both non-blended Trecepta hybrids had almost no tip damage and no kernel damage. The Trecepta blended treatment had both tip and kernel damage, but neither was significantly greater than the non-bended Trecepta hybrids. A selected number of ears with damage in the Trecepta blended treatment were tested for expression of Vip3Aa20 toxin, and all were negative for expression, indicating that all damaged ears in the Trecepta blends were non-Bt plants.
Hybrid blend treatments did not significantly affect grain yield (F = 0.63; df = 11, 69; p = 0.7996) (Table 1). Contrast tests of the non-blended Bt verses blended Bt treatments were not significant for the Cry protein hybrids (VT2P + SmartStax) (F = 0.45; df = 1, 69; p = 0.5129) and the Trecepta hybrids containing the Vip3Aa20 gene (F = 0.85; df = 1, 69; p = 0.3606). Hybrid blend treatments also did not significantly affect grain test weight (F = 1.32; df = 11, 69; p = 0.2300) (Table 1).

3.2. Results of Experiment 2

Plant stand in the combined analysis was not significantly different among treatments (F = 1.30; df = 5, 60; p = 0.2755). Fall armyworm infestations were low in all four years and did not exceed 3.06 ± 1.23% infested plants in any hybrid treatment. Fall armyworm infestations and plant damage were not significantly (p > 0.05) different among treatments in any year, except 2020, where the non-Bt hybrid had more damaged plants than all Bt hybrid treatments (Tables S4 and S5).
Generally, over the four years, the largest corn earworm infestations and damage occurred in 2020, and the smallest occurred in 2021, with infestations in 2019 and 2022 being intermediate between these two years. In the combined analysis, the hybrid treatments significantly affected the percentage of infested ears at stage R3 (F = 261.68; df = 5, 60; p < 0.0001) and the percentage of damaged ears at stage R6 (F = 248.82; df = 5, 60; p < 0.0001) (Table 2). The VT2P hybrid significantly reduced the percentage of infested and damaged ears at R3 by a small amount. Non-blended Trecepta treatment did not have any earworms in the ears in any year. The percentage of infested and damaged ears increased in the blended treatments in relation to the increasing proportion of non-Bt seed in the blend. Ear infestations in the 10% blend were not significantly different from the non-blended Trecepta treatment, while the 30% blend had significantly greater ear infestation and damage than the non-blended and 10% blended treatments. The 20% blended was intermediate between the 10% and 30% blended treatments.
Hybrid treatments caused highly significant differences in the numbers of small larvae (F = 49.75; df = 5, 60; p < 0.0001), medium larvae (F = 45.73; df = 5, 60; p < 0.0001), large larvae (F = 36.15; df = 5, 60; p < 0.0001), exited larvae (F = 19.58; df = 5, 60; p < 0.0001), and total larvae (F = 1.33; df = 5, 60; p < 0.0001) (Figure 4). The VT2P hybrid had a similar number of total larvae as the non-Bt hybrid but had more small larvae and fewer large and exited larvae than the non-Bt hybrid. The total number of larvae was significantly greater in the blended than the non-blended Trecepta treatment, with the 20 and 30% blended having more total larvae per ear than the 10% blend. The size distribution in the three blended treatments was similar. Kernel damage was also affected by hybrid treatments (F = 74.11; df = 5, 60; p < 0.0001), but kernel damage was not significantly different between the VT2P and non-Bt hybrids (Table 2). The non-blended Trecepta treatment had no kernel damage, although a small percentage of ears had tip feeding injuries. The blended treatments had small amounts of ear damage that reflected the percentage of non-Bt seed in the blend.
Grain yields in the combined analysis were significantly different among treatments (F = 4.54; df = 5, 60; p < 0.0014), because the non-Bt hybrid yielded less than all the Bt treatments and the VT2P hybrid yielded less than the 10% Trecepta blend treatments but was not different from the other Trecepta treatments (Table 2). Grain yields within years were not significantly different (p > 0.05) in 2020, 2021, and 2022, but were lower in 2019 in the non-Bt and VT2P treatments than the non-blended and blended Trecepta treatments (Table S6). Grain yields of the non-blended and blended Trecepta treatments were not significantly different in any year and in the combined analysis. Grain test weights were not significantly different among treatments in the combined analysis (F = 1.26; df = 5, 60; p < 0.2922) (Table 2).

4. Discussion

The corn hybrids containing the Vip3Aa20 toxin had virtually no infested ears and no kernel damage. The Vip3Aa20 blends had infested ears up to the proportion of non-Bt seed in the blend. Gene checking of damaged ears in Experiment 2 indicated that infested and damaged ears within the blends with Bt seed containing the Vip3Aa20 toxin were the non-Bt plants within the blend. The SmartStax Bt hybrid did not reduce corn earworm infestations or damage. The VT2P corn had less infested ears, larval numbers, and damage than comparable non-Bt corn, but the levels of reductions were limited. Nevertheless, the size distribution of larvae in the non-blended Bt corn suggests that the Bt toxins in VT2P and SmartStax delayed larvae development but did not substantially reduce the number of larvae completing development [39]. The lack of effective control by VT2P and SmartStax is indicative of the resistance previously reported to the Cry1Ab and Cry2Ab toxins by corn earworm [2,14,15,16,19]. While the Vip3Aa20 toxin is highly effective in corn for corn earworm control, resistance is a concern because resistance alleles have been detected in corn earworm populations in the southern U.S [18,19].
Registration of Bt-expressing corn requires a resistance management plan that includes a refuge of non-Bt corn [12,40]. The goal of the resistance management plan is to delay the development of resistance in target pests thereby extending the effectiveness of Bt products [12]. A non-Bt refuge can be arranged as a structured refuge by planting non-Bt refuge in a separate block but near the Bt crops or planting non-Bt strips within the Bt field, or by mixing refuge plants in a blend of both refuge and Bt seed [9,22,41]. The later approach of mixing non-Bt with Bt seed is referred to as an integrated refuge or blended refuge and is marketed as refuge-in-a-bag or RIB. Structured refuges require growers to actively implement the refuge during the planting process and effective resistance management is based on good grower compliance [13]. However, grower compliance in the southern region is low with one study reporting only 38.3 to 44.3% of corn growers planted corn refuges [42]. A key advantage of the blended refuge approach is that the refuge is automatically deployed when growers plant blended seed thereby removing the need for the grower to actively deploy the refuge [9,13]. A blended refuge also maximizes refuge adult mixing in the field which reduces the change of Bt-resistant moths mating when compared with a separate block refuge or refuge that is not immediately adjacent to the Bt field [12].
However, one negative aspect of blended refuge is possible larval movement between Bt and non-Bt plants which may allow larvae to avoid a lethal dose of toxin [23,25,43]. In addition, cross-pollination of refuge plants with nearby Bt plants can create a mosaic of Bt expression in kernels of refuge ears in a blend [24,26]. In this case, refuge moths emerging from a blended Bt refuge may be selected for resistance as compared with those from a larger separate block refuge. One question about blended refuges is whether the blends produce refuge moths in adequate numbers to serve as an effective refuge. Towles et al. [22] found that H. zea moth emergence from refuge blends was proportional to the percentage of the non-Bt blend and that moth emergence timing was not significantly delayed in seed blends as compared with a structured refuge. This suggests in areas with low structured refuge compliance that a seed blended refuge may provide an alternative resistance management approach for H. zea in Bt corn [22].
The effect of seed blends on grain yield of Bt corn was examined in a large multi-state project before widespread resistance was reported in corn earworm to Cry Bt traits [36]. In that study, corn earworm injury usually was greater in the 10 and 20% blends, but grain yield was only significantly reduced in the late-planted trials in one of three years between the 0% and 20% blend, with no significant effect on yield by 5 and 10% blends. We found no significant yield differences between non-blended Bt corn and blends up to 30% non-Bt seed even though corn earworms caused ear kernel damage in the refuge blends at levels up to the proportion of non-Bt seed in the refuge blend treatments. All the Bt products evaluated in this study are effective against most other lepidopteran pests such as fall armyworm and stalk borers [2,7], but blends of 30% or higher may be problematic because other lepidopteran pests may attack the non-Bt plants within a blend and cause economic damage.
Several studies have found variable effects of Bt corn on yield, but yield was not associated with corn earworm kernel injury [6,29,30,44]. Olivi et al. [31] concluded that 60-plus kernels need to be injured before measurable yield loss occurs. Another study with simulated earworm kernel injury also found that a loss of 30 kernels per ear at the blister or milk stage caused no significant yield loss and a loss of about 50 kernels per ear caused no yield loss at the dough stage [44]. The lack of yield loss was due to compensation of increasing kernel weight in some cases. A generally accepted conversion is 1 cm2 of area of kernel damaged equals 4 injured kernels [45] so about 12.5 to 15 cm2 of kernel area damaged would be needed to cause measurable yield loss. In the current study, the area of kernel damage in the non-Bt hybrids did not exceed 5 cm2 in Experiment 1 and 5.60 ± 0.98 cm2 in Experiment 2. The larger number in Experiment 2 indicated about 22.4 ± 3.9 kernels per ear were injured in the non-Bt hybrid which is substantially less than the 50 to 60 kernels needed to cause measurable yield loss. The older Cry products, VT2P and SmartStax, did not control corn earworm infestations, reduce corn kernel damage, or prevent yield loss. Indeed, the SmartStax blend did not yield significantly less than the yields of either the pure SmartStax or the non-Bt counterpart. The number of injured kernels in the Trecepta blend treatments would be about 0.84 ± 0.28, 2.21 ± 0.56, and 3.12 ± 0.80 kernels per ear for the 10%, 20% and 30% blend, respectively indicating that Trecepta blends had much less kernel damage than would be needed to cause measurable yield loss from corn earworm. The lack of measurable yield loss of blends examined in this study is consistent with the lack of yield loss from corn earworm injury in other studies using non-blended seed [6,30,32,34]. Nevertheless, if Bt blends are being considered for use as a resistance management tool in other regions, they should be evaluated under local environmental conditions to assess the impact on target and non-target pests and agricultural production practices.

5. Conclusions

The current study demonstrates that blends of Bt and non-Bt seed up to 30% of non-Bt seed of corn with Cry1A.105+ Cry2Ab, Cry1A.105 + Cry1Fa2 + Cry2Ab and Cry1A.105 + Cry2Ab + Vip3Aa20 toxins will not adversely affect corn grain yield and test weight in areas where corn earworm is the predominate ear-feeding insect and corn stalk borers are not present. Economic losses from ear injury by corn earworm in seed blends up to 30% non-Bt seed is unlikely.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14102246/s1. Table S1. Treatments and hybrids used in Experiment 1 in both years. Table S2. Treatments and hybrids used in Experiment 2 in four years. Table S3. Mean (±SEM) fall armyworm percent infested plants and damage rating at stage V5-V7 of non-blended and blended Bt corn in Experiment 1 in 2021. Table S4. Mean (±SEM) fall armyworm percent infested plants of non-blended and blended Bt corn in Experiment 2 over four years. Table S5. Mean (±SEM) fall armyworm damage rating per plant of non-blended and blended Bt corn in Experiment 2 over four years. Table S6. Mean (±SEM) grain yield of non-blended and blended Bt corn in Experiment 2 over four years.

Author Contributions

Conceptualization, P.S.R. and G.D.B.; P.S.R. and G.D.B. prepared treatments, conducted experiments, and analyzed and interpreted data; resources and data curation, G.D.B.; writing—original draft preparation, P.S.R.; writing—review and editing, G.D.B.; supervision, project administration, and funding acquisition, G.D.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded in part each year by a grant from Bayer CropScience. We also thank Bayer CropScience and Pioneer Hibred International for providing corn seed. This research also was funded in part by Hatch project funds (accession no. 1009397) from the U.S. Department of Agriculture National Institute of Food and Agriculture.

Data Availability Statement

Data is available upon request from the corresponding author.

Acknowledgments

We thank Conor Fair for statistical advice and Brett Byous, William Yancy Barton, Suzanne Deeb, Anna Agi, and Joshua Joyner for technical assistance.

Conflicts of Interest

The authors declare no financial or other conflicts of interest.

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Figure 1. Experiment 1: Mean (±SEM) percentage of H. zea infested ears at stage R3 and damaged ears at stage R6 in sets of non-blended Bt and non-Bt corn and a 20% blend (RIB) of Bt corn. VT2P = VT Double PRO; SST = SmartStax; and TRE = Trecepta. Horizontal lines indicate groups of treatments with similar base genetics. Means with the same lower-case letters are not significantly different at R3 and the same upper-case letter are not significantly different at R6 (Tukey’s HSD test, α = 0.05).
Figure 1. Experiment 1: Mean (±SEM) percentage of H. zea infested ears at stage R3 and damaged ears at stage R6 in sets of non-blended Bt and non-Bt corn and a 20% blend (RIB) of Bt corn. VT2P = VT Double PRO; SST = SmartStax; and TRE = Trecepta. Horizontal lines indicate groups of treatments with similar base genetics. Means with the same lower-case letters are not significantly different at R3 and the same upper-case letter are not significantly different at R6 (Tukey’s HSD test, α = 0.05).
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Figure 2. Experiment 1: Mean (±SEM) number of H. zea larvae per ear by larval size category in sets of non-blended Bt and non-Bt corn and a 20% blend (RIB) of Bt corn. VT2P = VT Double PRO; SST = SmartStax; and TRE = Trecepta. Horizontal lines indicate groups of treatments with similar base genetics. Total means with the same upper-case letter are not significantly different, and means within the larval size category with the same lower-case letter are not significantly different (Tukey’s HSD test, α = 0.05).
Figure 2. Experiment 1: Mean (±SEM) number of H. zea larvae per ear by larval size category in sets of non-blended Bt and non-Bt corn and a 20% blend (RIB) of Bt corn. VT2P = VT Double PRO; SST = SmartStax; and TRE = Trecepta. Horizontal lines indicate groups of treatments with similar base genetics. Total means with the same upper-case letter are not significantly different, and means within the larval size category with the same lower-case letter are not significantly different (Tukey’s HSD test, α = 0.05).
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Figure 3. Experiment 1: Mean (±SEM) area of ear tip (top) and kernel damage (bottom) by H. zea larvae in sets of non-blended Bt and non-Bt corn and a 20% blend (RIB) of Bt corn in Experiment 1. VT2P = VT Double PRO; SST = SmartStax; and TRE = Trecepta. Horizontal lines indicate groups of treatments with similar base genetics. Total means with the same upper-case letter are not significantly different, and means within the larval size category with the same lower-case letter are not significantly different (Tukey’s HSD test, α = 0.05).
Figure 3. Experiment 1: Mean (±SEM) area of ear tip (top) and kernel damage (bottom) by H. zea larvae in sets of non-blended Bt and non-Bt corn and a 20% blend (RIB) of Bt corn in Experiment 1. VT2P = VT Double PRO; SST = SmartStax; and TRE = Trecepta. Horizontal lines indicate groups of treatments with similar base genetics. Total means with the same upper-case letter are not significantly different, and means within the larval size category with the same lower-case letter are not significantly different (Tukey’s HSD test, α = 0.05).
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Figure 4. Experiment 2: Mean (±SEM) number of H. zea larvae per ear by larval size category in non-blended Bt and non-Bt corn and three blends (RIB) of Bt corn. VT2P = VT Double PRO; TRE = Trecepta; and RIB = Trecepta Blend. Total means with the same upper-case letter are not significantly different, and means within the larval size category with the same lower-case letter are not significantly different (Tukey’s HSD test, α = 0.05).
Figure 4. Experiment 2: Mean (±SEM) number of H. zea larvae per ear by larval size category in non-blended Bt and non-Bt corn and three blends (RIB) of Bt corn. VT2P = VT Double PRO; TRE = Trecepta; and RIB = Trecepta Blend. Total means with the same upper-case letter are not significantly different, and means within the larval size category with the same lower-case letter are not significantly different (Tukey’s HSD test, α = 0.05).
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Table 1. Mean (±SEM) grain yield and test weight of non-blended and blended Bt corn in Experiment 1 combined over two years.
Table 1. Mean (±SEM) grain yield and test weight of non-blended and blended Bt corn in Experiment 1 combined over two years.
Hybrid NameBt Treatment aBt ToxinsGrain Yield
(kg ha−1)		Grain Test Weight
(kg hL−1)
DKC 6694NoneNone13,666 ± 363 a74.0 ± 2.0 a
DKC 6697VT Double PROCry1A.105 + Cry2Ab213,978 ± 495 a74.0 ± 1.6 a
DKC 6697/6694VT Double PRO—20%RIBCry1A.105 + Cry2Ab214,595 ± 711 a73.0 ± 1.9 a
DKC 6205NoneNone14,167 ± 548 a72.9 ± 1.5 a
DKC 6208SmartStaxCry1A.105 + Cry2Ab2 + Cry1Fa213,584 ± 763 a73.9 ± 0.9 a
DKC 6205/6208SmartStax—20%RIBCry1A.105 + Cry2Ab2 + Cry1Fa213,662 ± 458 a73.7 ± 1.1 a
DKC 6694NoneNone14,174 ± 925 a73.9 ± 1.9 a
DKC 6629Trecepta1Cry1A.105 + Cry2Ab2 + Vip3Aa2014,934 ± 729 a75.4 ± 1.7 a
DKC 6629/6694Trecepta1—20%RIBCry1A.105 + Cry2Ab2 + Vip3Aa2014,380 ± 596 a74.2 ± 1.2 a
DKC 6824NoneNone14,521 ± 672 a73.1 ± 1.4 a
DKC 6799Trecepta2Cry1A.105 + Cry2Ab2 + Vip3Aa2014,279 ± 639 a71.8 ± 1.2 a
DKC6799/6824Trecepta2—20%RIBCry1A.105 + Cry2Ab2 + Vip3Aa2013,860 ± 907 a73.4 ± 1.3 a
Notes: Means within columns with the same letters are not significantly different (Tukey’s HSD test, α = 0.05). a RIB = refuge in a bag or blended refuge.
Table 2. Mean (±SEM) of infested and damaged ears, damaged kernels, grain yield, and test weight of non-blended and blended Bt corn in Experiment 2.
Table 2. Mean (±SEM) of infested and damaged ears, damaged kernels, grain yield, and test weight of non-blended and blended Bt corn in Experiment 2.
Bt Treatment aBt ToxinsInfested Ears
at R3 (%)		Damaged Ears at R6 (%)Damaged Kernels per
Ear (cm2)		Grain Yield (kg ha−1)Grain Test Weight
(kg hL−1)
Non-BtNone87.50 ± 4.77 a75.63 ± 7.89 a5.60 ± 0.98 a11,554 ± 268 c71.9 ± 0.5 a
Genuity
VTDouble PRO		Cry1A.105 + Cry2Ab269.38 ± 7.37 b60.63 ± 8.32 b4.09 ± 0.79 a11,822 ± 289 bc73.4 ± 0.8 a
Genuity Trecepta
0% Blend		Cry1A.105 + Cry2Ab2 + Vip3Aa200.31 ± 0.31 e0.31 ± 0.31 d0 d12,352 ± 370 ab72.4 ± 0.7 a
Genuity Trecepta—10% BlendCry1A.105 + Cry2Ab2 + Vip3Aa209.06 ± 1.53 de5.94 ± 1.23 d0.21 ± 0.07 c12,522 ± 257 a72.8 ± 0.8 a
Genuity Trecepta—20% BlendCry1A.105 + Cry2Ab2 + Vip3Aa2016.88 ± 2.77 cd 11.56 ± 2.64 cd0.53 ± 0.14 bc12,245 ± 332 abc72.8 ± 0.8 a
Genuity Trecepta—30% BlendCry1A.105 + Cry2Ab2 + Vip3Aa2024.69 ± 2.87 c17.81 ± 2.85 c0.78 ± 0.20 b12,237 ± 267 abc71.8 ± 0.8 a
Notes: Means within columns with the same letters are not significantly different (Tukey’s HSD test, α = 0.05).
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Buntin, G.D.; Rowe, P.S. Effect of Blended Bt Corn Refuge on Corn Earworm (Lepidoptera: Noctuidae) Infestation and Grain Yield. Agronomy 2024, 14, 2246. https://doi.org/10.3390/agronomy14102246

AMA Style

Buntin GD, Rowe PS. Effect of Blended Bt Corn Refuge on Corn Earworm (Lepidoptera: Noctuidae) Infestation and Grain Yield. Agronomy. 2024; 14(10):2246. https://doi.org/10.3390/agronomy14102246

Chicago/Turabian Style

Buntin, George David, and Pamela Somerville Rowe. 2024. "Effect of Blended Bt Corn Refuge on Corn Earworm (Lepidoptera: Noctuidae) Infestation and Grain Yield" Agronomy 14, no. 10: 2246. https://doi.org/10.3390/agronomy14102246

APA Style

Buntin, G. D., & Rowe, P. S. (2024). Effect of Blended Bt Corn Refuge on Corn Earworm (Lepidoptera: Noctuidae) Infestation and Grain Yield. Agronomy, 14(10), 2246. https://doi.org/10.3390/agronomy14102246

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