RESEARCH PAPER
RESEARCH PAPER
GM Crops 1:5, 337-343; November/December 2010; © 2010 Landes Bioscience
Novel Vip3A Bacillus thuringiensis (Bt) maize approaches high-dose efficacy against Helicoverpa zea (Lepidoptera: Noctuidae) under field conditions Implications for resistance management
Eric C. Burkness,1 Galen Dively,2 Terry Patton,2 Amy C. Morey1 and William D. Hutchison1,* University of Minnesota; Department of Entomology; St. Paul, MN USA; 2University of Maryland; Department of Entomology; College Park, MD USA
1
Key words: transgenic maize, GM crops, stacked events, pyramided events, Vip3A, Bt11, Spodoptera frugiperda, Ostrinia nubilalis, corn earworm
Sweet corn, Zea mays L., transformed to express a novel vegetative insecticidal protein, Vip3A (event MIR162, Syngenta Seeds, Inc.) produced by the bacterium, Bacillus thuringiensis (Bt), was evaluated over four field seasons in Maryland and two field seasons in Minnesota for efficacy against the corn earworm, Helicoverpa zea (Boddie). Hybrids expressing the Vip3A protein and pyramided in hybrids also expressing the Cry1Ab Bt protein (event Bt11, ATTRIBUTE®, Syngenta Seeds, Inc.) were compared to hybrids expressing only Cry1Ab or to genetically similar non-Bt hybrids each year. In addition to H. zea efficacy, results for Ostrinia nubilalis (Hübner) and Spodoptera frugiperda (J.E. Smith) are presented. Over all years and locations, the non-Bt hybrids, without insecticide protection, averaged between 43 and 100% ears infested with a range of 0.24 to 1.74 H. zea larvae per ear. By comparison, in the pyramided Vip3A x Cry1Ab hybrids, no larvae were found and only minimal kernel damage (likely due to other insect pests) was recorded. Hybrids expressing only Cry1Ab incurred a moderate level of H. zea feeding damage, with surviving larvae mostly limited to the first or second instar as a result of previously documented growth inhibition from Cry1Ab. These results suggest that the Vip3A protein, pyramided with Cry1Ab, appears to provide the first “high-dose” under field conditions and will be valuable for ongoing resistance management.
Introduction The corn earworm or cotton bollworm, Helicoverpa zea (Boddie), is one of the most devastating and difficult crop pests to control in United States agriculture.1 The pest colonizes over 200 host plants, many of which are economically important US crops, including maize, Zea mays L. (field and sweet corn), cotton, Gossypium hirsutum L., soybean, Glycine max L., sorghum, Sorghum bicolor L., tomato, Lycopersicum esculentum L., green bean, Phaseolus vulgaris L. and pepper, Capsicum annuum L.1-3 According to Martin et al. H. zea larvae were found on 18 of 21 vegetable and field crops included in their study, with corn and sorghum being preferred. Sweet corn is a popular fresh-market and processed vegetable crop in the US for human consumption, and is harvested relatively early during ear maturity (e.g., 70% moisture), compared to field corn grown primarily for animal and ethanol production.5,6 Consequently, sweet corn producers must rely on timely pest monitoring and effective Integrated Pest Management (IPM)
strategies to minimize kernel and ear damage by H. zea and other Lepidopteran pests.6 The fresh-market and processing industries can tolerate only minimal damage to the ears or presence of larval contaminants. Ear damage and larval contamination has also recently been exacerbated by resistance to pyrethroids in H. zea larvae.3,7 In addition, the life cycle of H. zea is conducive to causing damage. For example, in maize, most H. zea eggs are oviposited directly on corn silks; once larvae hatch, they quickly move down the silk channel and begin feeding on kernels in the ear tip. Once within the husk and kernel tissue, larvae are virtually protected from insecticidal sprays and likely avoid predation by natural enemies as well.5 One alternative to insecticidal control of H. zea in US sweet corn is the use of transgenic hybrids expressing one or more insectactive toxins from the bacterium, Bacillus thuringiensis (i.e., Bt sweet corn). Although the single event Bt11 sweet corn hybrids, expressing Cry1Ab toxin have been effective, particularly against the European corn borer, Ostrinia nubilalis (Hübner), this toxin alone does not provide 100% control of H. zea.8 Control of H. zea
*Correspondence to: William D. Hutchison; Email:
[email protected] Submitted: 12/01/10; Revised: 01/05/11; Accepted: 01/10/11 DOI: 10.4161/gmcr.1.5.14765 www.landesbioscience.com
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larvae on Cry1Ab maize (field or sweet corn) is typically characterized by a few surviving early instars (1st and 2nd) feeding on ear tips, with few kernels damaged.9-12 At the same time Cry1Ab corn was approved for use in the US in 1996, additional research was underway that isolated a novel vegetative insecticidal protein from B. thuringiensis—Vip3A.1315 This discovery revealed a class of insecticidal proteins with activity against a range of agriculturally important Lepidopteran larvae including black cutworm, Agrotis ipsilon (Hufnagel); fall armyworm, Spodoptera frugiperda (J.E. Smith); tobacco budworm, Heliothis virescens (Fabricius) and corn earworm, H. zea.16 Vip3A is characterized by two unique qualities. Vip3A is an exotoxin produced during the vegetative growth stage of Bt, whereas Cry proteins are produced during sporulation.16 Vip3A also shares no sequence homology with any known Cry proteins. Shared sequence homology can indicate a risk that two Bt proteins will share binding sites on the midgut of the insect and be predisposed to cross-resistance.17 Therefore, differences in production stage and lack of sequence homology with Cry proteins were indicators that Vip3A could be an important new mode of action against H. zea and other Lepidopteran pests, and thus be useful for mitigating the risk of Bt resistance.16 Subsequent research has demonstrated that there is no evidence of cross-resistance between Vip3A and Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa and Cry2A in H. virescens, or between Vip3A and Cry1Ac and Cry2Ab in H. zea.18-20 The purpose of this study was to assess the efficacy of sweet corn hybrids containing both the MIR162 event which expresses the Vip3A protein and the Bt11 event which express the Cry1Ab protein against H. zea, under a wide range of natural infestation levels in two distinct maize production regions in the US, including Minnesota (Midwest region) and Maryland (Atlantic Coast region). We also examined Vip3A x Cry1Ab efficacy against O. nubilalis and S. frugiperda, compared the efficacy of the pyramided Bt hybrids to conventional insecticide use, and discussed the implications of these results for resistance management. Results Maryland. In all years of the study, the pyramided hybrids (MIR162-Vip3A x Bt11-Cry1Ab), regardless of the parental sources of genes for event MIR162 or Bt11 being male or female, provided significant control of three major Lepidopteran pests H. zea, O. nubilalis and S. frugiperda (Table 1). In fact, there were no H. zea, O. nubilalis or S. frugiperda larvae found in a pyramided hybrid with the exception of the hybrid GSS7061 in 2010, in which 0.01 H. zea larvae per ear were found (Table 1). Bt11 by itself provided virtually 100% control of O. nubilalis for all years. While control was not complete for H. zea, Bt11 provided significant reductions and delayed developmental growth in H. zea larvae in 2008 and 2009, when compared to the non-Bt hybrid. In addition, Bt11 provided significant population suppression of S. frugiperda in 2010 but not complete control (Table 1). MIR162 by itself provided significant reductions of H. zea and S. frugiperda larvae but did not significantly reduce O. nubilalis populations. In 2007, the MIR162 treatments actually had
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significantly higher O. nubilalis infestations than the non-Bt hybrid. This phenomenon indicates that MIR162 alone does not confer efficacy against O. nubilalis, but also suggests that high populations may result from the removal of competition from the other Lepidopteran pests (Table 1). The percentage of clean ears and kernel damage results followed a similar trend to the larval results (Table 1). Pyramided hybrids had significantly higher percentages of clean ears, ranging from 97.8–100.0, and lower total area damaged by the 3 Lepidopteran pests, ranging from 0.00–0.01 cm2 (Table 1). The level of clean ears and kernel damage for hybrids with either the Bt11 or MIR162 single event depended on the composition and population pressure of pest species for a given year. Bt11 averaged 8.0–73.2 percent clean ears compared to nearly no clean ears in the non-Bt hybrids, but reduced kernel damage by 79.1–93.2 percent. Ear protection was better when O. nubilalis populations were high and H. zea and S. frugiperda were low. Similarly, MIR162 had more clean ears and less kernel damage when H. zea and S. frugiperda populations were high and O. nubilalis were low. Minnesota. In both years, pyramided hybrids significantly reduced H. zea and O. nubilalis compared to the non-Bt hybrids and provided 100 percent clean ears with no kernel damage detected (Table 2). The single event of Bt11 provided complete control of O. nubilalis and suppressed the development of H. zea larvae where 100 and 90 percent of the larvae in 2008 and 2010, respectively, were less then 3rd instar. Conversely, the nonBt hybrids in 2008 and 2010 averaged 79.6 and 84.3 percent H. zea larvae that were 3rd instar or greater, respectively (Table 2). Non-Bt hybrids averaged 55.0 and 70.8 percent O. nubilalis larvae that were 3rd instar or greater in 2008 and 2010, respectively. Pyramided hybrids and Bt11 by itself both provided significant reductions in kernel damage with no damage detected in the pyramided hybrids. A non-Bt hybrid was treated with a foliar insecticide in 2010 and did not provide a significant reduction in H. zea or O. nubilalis populations compared to the non-Bt hybrid GH2395 but did significantly reduce O. nubilalis compared to the non-Bt hybrid Garrison. However, populations of H. zea were higher in the non-Bt hybrid that received multiple foliar insecticide applications, compared to the non-Bt hybrid Garrison; this outcome was likely due to recent problems with pyrethroid control of H. zea in the Midwest region.7 Both the pyramided hybrids and Bt11 by itself provided significant levels of H. zea and O. nubilalis population reduction compared to the non-Bt hybrid with foliar insecticide applications (Table 2). Maryland and Minnesota. When efficacy data are combined for both Maryland and Minnesota from 2007 to 2010 (Fig. 1) we see that for H. zea, both the pyramided hybrids and MIR162 by itself provide significant reductions in larval populations compared to the non-Bt hybrid and Bt11 (Fig. 1A). However, for O. nubilalis, MIR162 by itself did not provide any significant reduction in larval population compared to the non-Bt hybrid (Fig. 1B), suggesting a lack of Vip3A activity against O. nubilalis. However, both the pyramided hybrid and Bt11 by itself provided high levels of control for O. nubilalis larvae. Despite having too few replicates to allow analysis of S. frugiperda data,
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Table 1. Efficacy of transgenic sweet corn hybrids on H. zea, O. nubilalis and S. frugiperda larvae Beltsville, MD, 2007–2010, US Mean total number of larvae per ear
O. nubilalis
S. frugiperda
% Clean earsa
Total damaged kernel area (cm2)/ear
0.00 ± 0.00c
0.00 ± 0.00c
-
97.8 ± 2.2a
0.01 ± 0.01d
MIR162 (♂) x Bt11 (♀)
0.00 ± 0.00
0.00 ± 0.00
c
-
a
98.9 ± 1.1
0.00 ± 0.00d
Bt11
1.11 ± 0.11a
0.00 ± 0.00c
-
24.7 ± 4.2b
0.90 ± 0.14c
1.02 ± 0.18
b,c
2.79 ± 0.40b
b,c
3.10 ± 0.36b
Hybrid (Event)
H. zea
MIR162 (♀) x Bt11 (♂)
2007
MIR162 (♂)
0.04 ± 0.03
MIR162 (♀)
c
-
20.6 ± 8.4
0.07 ± 0.03
0.89 ± 0.12
a
-
13.6 ± 5.9
Non-Bt
1.74 ± 0.15a
0.49 ± 0.11b
-
1.7 ± 1.7c
GSS 7061 (MIR162 x Bt11)
0.00 ± 0.00d
0.00 ± 0.00b
-
100.0 ± 0.0a
0.00 ± 0.00c
GH 2390 (MIR162 x Bt11)
0.00 ± 0.00
d
0.00 ± 0.00
b
-
100.0 ± 0.0
0.00 ± 0.00c
GSS0966 (Bt11)
0.31 ± 0.04
c
0.00 ± 0.00
b
-
73.2 ± 4.4
GH 2395 (Non-Bt)
1.54 ± 0.10a
0.03 ± 0.02ab
-
2.0 ± 1.2c
Garrison (Non-Bt)
1.15 ± 0.22
0.06 ± 0.02
GSS7061 (MIR162 x Bt11)
0.00 ± 0.00d
GSS0966 (Bt11)
bc b
a
8.81 ± 0.54a
2008 a
b
0.09 ± 0.02b 3.83 ± 0.28a
-
c
16.0 ± 12.1
3.47 ± 0.36a
0.00 ± 0.00b
0.00 ± 0.00b
100.0 ± 0.0a
0.00 ± 0.00d
0.74 ± 0.04b
0.00 ± 0.00b
0.06 ± 0.02a
28.0 ± 2.7c
0.59 ± 0.04b
GSS7041 (MIR162)
c
0.04 ± 0.02
0.31 ± 0.03
a
0.00 ± 0.00
b
74.9 ± 2.9
0.23 ± 0.06c
Garrison (Non-Bt)
1.23 ± 0.14
a
0.12 ± 0.10
a,b
0.05 ± 0.02
a
0.0 ± 0.0
GSS7061 (MIR162 x Bt11)
0.01 ± 0.01b
0.00 ± 0.00b
0.00 ± 0.00c
99.0 ± 1.0a
0.00 ± 0.00c
GH 2390 (MIR162 x Bt11)
0.00 ± 0.00b
0.00 ± 0.00b
0.00 ± 0.00c
100.0 ± 0.0a
0.00 ± 0.00c
WH0809 (Bt11)
0.76 ± 0.06
a
0.01 ± 0.01
0.50 ± 0.15
8.0 ± 1.6
b
3.81 ± 1.13b
Avalon (Non-Bt)
a
0.70 ± 0.11
6.90 ± 0.30
0.0 ± 0.0
b
18.23 ± 2.11a
b
a
2009
b
d
8.71 ± 0.59a
2010
b
b
1.53 ± 0.30
a
a
Means ± SEM within columns within each year followed by the same letter are not significantly different (p > 0.05), Protected Least significant difference Test (LSD). Mean percentage of marketable ears for fresh market and processing were transformed using the arcsine transformation and insect count and kernel damage data were transformed using the rank transformation to obtain mean separations using LSD (p = 0.05); untransformed means are presented. aPercentage of ears with no kernel damage present.
when combining locations and years, it is still clear that the pyramided hybrids and MIR162 by itself provide a high level of efficacy against S. frugiperda larvae (Fig. 1C). The percentage of clean ears also indicates the high level of efficacy provided by the pyramided hybrid (Fig. 1D). The pyramided hybrid provides nearly 100 percent clean ears, which is significantly higher than both of the single event hybrids and the non-Bt hybrid. Discussion As the management of H. zea in US sweet corn continues to be challenged by various obstacles such as pyrethroid resistance (see Table 2), the pyramided hybrids (MIR162 x Bt11) can clearly provide a highly effective and more targeted sustainable approach to Lepidopteran insect control in sweet corn.3,21 Compared to the single gene Bt11 event, this combination of Bt proteins significantly increases control efficacy against a broader spectrum of Lepidopteran pests for several reasons. First, the MIR162 event
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has been shown to express a high dose of Vip3A protein against S. frugiperda and a “near high dose” against H. zea.22 Second, the average expression per ear in the endosperm of the kernels is higher due to the segregation pattern of the two independent genes encoding the Cry1Ab and Vip3A proteins compared to the segregation pattern of a single gene. For example, ATTRIBUTE Cry1Ab sweet corn hybrids are hemizygous for the Bt11 trait. Due to open pollination and gene segregation in the ear, approximately 75% of the kernels per ear will express the Bt11 trait (50% hemizygous and 25% homozygous) while 25% of kernels will not inherit the gene.23 This is true for any single insect resistance trait sold as a hemizygous hybrid. Hybrids containing two unlinked insect resistance traits, such as the pyramided Bt11 x MIR162 hybrids will have only 6% of the kernels that will not inherit at least one trait, with 94% expressing either the Cry1Ab, the Vip3A or both insecticidal proteins. This is an important point because H. zea larvae hatching later in the crop cycle can invade the ear without feeding on silk tissue, depending on the
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Table 2. Efficacy of transgenic sweet corn hybrids on H. zea and O. nubilalis larvae Rosemount, MN, 2008 and 2010, US. Mean number of larvae per ear
Marketable ears (%)
Late instar H. zeaa
Total H. zeab
Late instar O. nubilalisc
Total O. nubilalisd
Cleane
Processingf
Total number of damaged kernels/ earg
GSS 7061 (MIR162 x Bt11)
0.00 ± 0.00b
0.00 ± 0.00c
0.00 ± 0.00c
0.00 ± 0.00c
100.0 ± 0.0a
100.0 ± 0.0a
0.00 ± 0.00c
GH 2390 (MIR162 x Bt11)
0.00 ± 0.00b
0.00 ± 0.00c
0.00 ± 0.00c
0.00 ± 0.00c
100.0 ± 0.0a
100.0 ± 0.0a
0.00 ± 0.00c
GSS 0966 (Bt11)
0.00 ± 0.00
b
0.05 ± 0.03
0.00 ± 0.00
c
0.00 ± 0.00
c
96.0 ± 1.6
100.0 ± 0.0
0.05 ± 0.03b
GH 2395 (Non-Bt)
0.32 ± 0.10
a
0.40 ± 0.09
b
b
0.08 ± 0.02
55.0 ± 6.6
Garrison (Non-Bt)
0.19 ± 0.05a
0.24 ± 0.04a
0.12 ± 0.03a
0.20 ± 0.06a
57.0 ± 7.2c
GSS 7061 (MIR162 x Bt11)
0.00 ± 0.00d
0.00 ± 0.00e
0.00 ± 0.00c
0.00 ± 0.00c
GH 2390 (MIR162 x Bt11)
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
c
GSS 0966 (Bt11)
0.05 ± 0.02c
0.54 ± 0.10d
0.00 ± 0.00c
0.00 ± 0.00c
51.3 ± 5.2b
95.0 ± 2.0b
1.45 ± 0.21d
GH 2395 (Non-Bt)
1.20 ± 0.10
1.44 ± 0.06
a
0.15 ± 0.12
0.20 ± 0.10
b
5.0 ± 3.5
c
10.0 ± 4.6
d
14.55 ± 0.95a
Garrison (Non-Bt)
0.81 ± 0.09
c
0.34 ± 0.15
0.51 ± 0.14
a
5.0 ± 2.0
c
12.5 ± 4.3
d
11.85 ± 1.47b
Garrison (Non-Bt) + Foliar Insecticide1
0.79 ± 0.05b
Hybrid (Event) 2008
b a
0.04 ± 0.00
b
b
3.16 ± 1.24a
72.0 ± 5.9b
2.22 ± 0.42a
100.0 ± 0.0a
100.0 ± 0.0a
0.00 ± 0.00e
100.0 ± 0.0
100.0 ± 0.0
0.00 ± 0.00e
c
65.0 ± 7.6
a
2010 d
a b
0.95 ± 0.10
e
1.18 ± 0.05b
c
b a
0.06 ± 0.02b
0.15 ± 0.04b
7.5 ± 1.4c
a
a
27.5 ± 4.3c
7.73 ± 0.67c
Means ± SEM within columns followed by the same letter are not significantly different (p > 0.05), Protected Least significant difference Test (LSD). Mean percentage of marketable ears for fresh market and processing were transformed using the arcsine transformation and count data were transformed using the rank transformation to obtain mean separations using LSD (p = 0.05); untransformed means are presented. 1Foliar insecticide applications were made on 8/12; 8/16; 8/23 with lambda-cyhalothrin (Warrior II at a rate of 1.92 oz/ac). aIncludes H. zea that are 3–6th instar in the tip, side, or butt of ear. bIncludes all H. zea instars in the husk, silk, tip, side, butt, or shank of the ear. cIncludes O. nubilalis that are 3–5th instar in the tip, side, or butt of the ear. dIncludes all O. nubilalis instars in the husk, silk, tip, side, butt or shank of the ear. ePercentage of ears with neither kernel damage nor larvae present. fPercentage of ears with only small larvae (1–2 instar O. nubilalis and/or 1–2 instar H. zea) and/or damage limited to the tip; no damage or larvae on the side or butt of the ear (US). gTotal number of kernels damaged/ear in the tip, side or butt by O. nubilalis and/or H. zea.
ear tip coverage and tightness of the silk channel. Reducing the number of non-protein expressing kernels increases the average expression per ear as well as the likelihood of larval mortality via consumption of protein expressing kernels. The use of transgenic hybrids ideally fits an IPM approach by combining host plant resistance and different modes of action of a reduced risk bioinsecticide to facilitate resistance management.24 Reductions in foliar insecticide use may also help slow or even allow the reversal of pyrethroid resistance in H. zea through the reduction in selection pressure and the removal of potentially resistant individuals. A similar case could be made for H. zea and the potential development of resistance to the Bt11 event where the use of MIR162 will lessen the selection pressure placed on H. zea and slow the development of resistance to Bt11.16,18,19 Not only does the use of MIR162 allow greater efficacy against Lepidopteran pests in sweet corn in the short term but also allows for the continued use of efficacious control options like pyrethroid insecticides in crops where transgenic hybrids are not available. The significant reductions in foliar insecticide applications afforded by the pyramided hybrids will lead to less worker exposure and environmental risk. Studies have shown that the Cry1Ab-expressing sweet corn and Vip3A-expressing field corn have no direct adverse effects on the beneficial arthropod community and that effects caused by insecticide applications are far more disruptive.25-28 Pyramided hybrids of sweet corn will not be insect free depending on the pest composition in the particular production
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region, so regular monitoring of insects not affected by the expressed Cry1Ab or Vip3A proteins continues to be essential for successful IPM. For example, kernel feeding by sap beetle and silk fly larvae and silk feeding by corn rootworm adults can cause ear quality problems and may need to be controlled with supplemental insecticide applications. Conversely, some secondary pests may be less likely to infest ears that lack damage by Lepidopteran pests but further investigation would be required to verify this assertion. In summary, pyramided Bt11 x MIR162 sweet corn provides a dual mode of action for managing several key Lepidopteran pests of maize. In addition to supplementing current control options, Vip3A also provides the ability to maximize the usefulness of the current control technologies available today and in the future. However, the use of Vip3A and other insecticidal proteins will continue to require a keen focus on stewardship and research to maintain the effectiveness of these technologies in the future by mitigating resistance development.13,24,29 Materials and Methods The stacked sweet corn hybrids used in this study are a product of traditional breeding involving separate crosses of Bt11 and MIR162 maize inbreds with an inbred of sweet corn and then successively back-crossing and selfing to fix the genetic elements in a homozygous elite inbred line. Bt11 maize was created through gene gun transformation of protoplasts from an inbred
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Figure 1. Mean number of larvae per ear for H. zea (A) and O. nubilalis (B) using transgenic sweet corn hybrids. Data summarized for combined years and locations for Rosemount, MN (2008 and 2010) and Beltsville, MD (2007-2010), U.S. Numbers above each bar in parentheses indicate the number of observations for a given variable and treatment combination. Mean number of larvae per ear for S. frugiperda (C), and percentage of clean ears (D) using transgenic sweet corn hybrids. Data summarized for combined years and locations for Rosemount, MN (2008 and 2010) and Beltsville, MD (2007-2010), U.S. Numbers above each bar in parentheses indicate the number of observations for a given variable and treatment combination.
maize line and regeneration on selective medium. The plasmid vector contained a truncated synthetic cry1Ab gene encoding Cry1Ab endotoxin and the phosphinothricin acetyltransferase (pat) gene as the selectable marker. MIR162 maize was produced by Agrobacterium tumefaciens-mediated transformation of immature embryos. The transformation vector included the vip3Aa19 gene encoding Vip3A protein and the phosphomannose-isomerase (pmi) gene as the selectable marker. Furthermore, constitutive expression of the Cry1Ab gene was controlled by the 35S promoter derived from cauliflower mosaic virus (CaMV) and the Vip3A gene expression was controlled by the Zea mays polyubiquitin gene (ZmUbiInt). Detailed descriptions of the transformation process are given by CERA.30
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Maryland. From 2007–2010 sweet corn trials were conducted to evaluate the efficacy of hybrids containing single and pyramided genes of MIR162 and Bt11 events. Trials were conducted at the Central Maryland Research and Education Center in Beltsville, MD. Each year plots were arranged in a randomized complete block design with four replications and managed according to standard sweet corn growing practices, including overhead or drip irrigation and no insecticides were applied. A genetically similar non-Bt hybrid was included as a negative control in all experiments and also planted as a buffer between plots and around the perimeter of the study sites. In 2007, plots consisting of 2 rows spaced 0.76 m (30 in) apart and 7.62 m (25 ft) long were planted by hand on 12 June.
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Treatments were hybrids expressing Cry1Ab endotoxin (Bt11 event), Vip3A exotoxin (MIR162 event), or both toxins pyramided in hybrids created by crossings of different gene donors. In 2008, plots consisting of 4 rows spaced 0.76 m apart and 9.14 m (30 ft) long were planted by hand on 20 June. Three treatments included the pyramided hybrid (SPS1020) expressing Cry1Ab (Bt11 event) and Vip3A (MIR162 event) insecticidal proteins, single gene hybrid (SPS1007L) expressing Cry1Ab (Bt11 event) alone, and the genetically similar non-Bt hybrid (SPS1008L). In 2009 and 2010, plots consisted of 4 rows spaced 0.76 m apart and 9.14 m long and were planted by hand on 25 June and 2 June, respectively. Four treatments included hybrids expressing Cry1Ab endotoxin (Bt11), Vip3A exotoxin (MIR 162) (in 2009 only), both events pyramided (Bt11 x MIR162), and the non-Bt hybrid. All primary ears in each plot were examined at peak maturity for fresh market sweet corn on 21 August 2007, 3 September 2008, 9 September 2009 and 6 September 2010. The number of ears harvested ranged from 14–38, 25–69, 25–50 and 10–25 in 2007, 2008, 2009 and 2010, respectively. At harvest the percentage of clean ears, extent of kernel damage and/or consumption and the number of H. zea, O. nubilalis and S. frugiperda larvae found in ears were recorded. Minnesota. In 2008 and 2010, sweet corn trials were conducted to evaluate the efficacy of hybrids containing single and pyramided genes of MIR162 and Bt11 events. Trials were conducted at the University of Minnesota Outreach, Research and Education Park in Rosemount, MN. In both years plots were arranged in a randomized complete block design with four replicates. A skip row between plots and 3.04 m (10 ft) alleys between replicates were maintained throughout the study. Plots were managed following standard growing practices for fresh market sweet corn in Minnesota.31 In 2008 and 2010, plots consisting of 4 rows spaced 0.76 m and 7.62 m long were planted on 19 June and 21 June, respectively. References 1.
Kennedy GG, Storer NP. Life systems of polyphagous arthropod pests in temporally unstable cropping systems. Annu Rev Entomol 2000; 45:467-93. 2. Foster R, Flood B, Eds. Vegetable insect management. Willoughby, OH: Meister Media Worldwide 2005; 264. 3. Hutchison WD, Burkness EC, Jensen B, Leonard BR, Temple J, Cook DR, et al. Evidence for decreasing Helicoverpa zea susceptibility to pyrethroid insecticides in the midwestern United States. Plant Health Prog 2007; DOI: 10.1094/PHP-2007-0719-02-RV. 4. Martin PB, Lingren PD, Greene GL. Relative abundance and host preferences of cabbage looper, soybean looper, tobacco budworm and corn earworm on crops grown in northern Florida. Environ Entomol 1976; 5:878-82. 5. Hutchison WD, Flood B, Wyman JA. Advances in United States sweet corn and snap bean insect pest management. In: Horowitz AR, Ishaaya I, Eds. Insect Pest Management. Heidelberg, Germany: SpringerVerlag 2004; 247-78. 6. Flood B, Foster R, Hutchison B, Sweet corn PS. In: Foster R, Flood B, Eds. Vegetable insect management. Willoughby, OH: Meister Media Worldwide 2005; 38-63.
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Treatments in both years consisted of 2 non-Bt hybrids, a Bt11 hybrid, 2 hybrids with MIR162 and Bt11 pyramided and in 2010 a non-Bt hybrid sprayed with the foliar insecticide lambda-cyhalothrin (Warrior II with zeon technology ® 2CS; Syngenta Crop Protection, Inc., Greensboro, NC) at 0.034 kg AI/ha (1.92 oz/ac) was included. Plots that received foliar insecticides had treatments applied 8/12/10 (~70% plants with silk); 8/16/10; 8/23/10 with a CO2 backpack sprayer and 10 ft boom with 6-Teejet 8002 flat fan nozzles with no screens. The sprayer was calibrated to deliver 233.8 l/ha (25 gpa) water at 242 kPa (35 psi). Primary ears in each plot were examined at peak maturity for fresh market sweet corn on 3 September 2008 and 30 August 2010. The number of ears harvested in each plot was 25 and 20 in 2008 and 2010, respectively. At harvest the percentage of clean ears, extent of kernel damage and/or consumption and the number of H. zea and O. nubilalis larvae found in ears were recorded. Data analysis. Data for each year were analyzed using a one-way analysis of variance and a protected Least Significant Difference Test (LSD) (p=0.05) for mean separation.32 Both insect counts and kernel damage data were transformed using a RANK procedure (non-parametric) that accounts for the non-normal distribution of data for Bt and non-Bt hybrid comparisons.32 For non-normal populations, multiple comparisons procedures are more robust and have more power when rank-transformed data are used.33 Proportion data were transformed using the arcsine transformation.32 Untransformed data are presented. Acknowledgements
We thank A. Hanson and Suzanne Wold-Burkness, University of Minnesota, and A. Miller, University of Maryland for technical assistance with conducting this study. We also thank Michele Gardiner, Ryan Walker and Ryan Kurtz at Syngenta for seed and technical support. This research was supported by Syngenta, Maryland Agric. Expt, Station and the Rapid Agricultural Response Fund, University of Minnesota Agric. Expt. Station.
7. Jacobson A, Foster R, Krupke C, Hutchison W, Pittendrigh B, Weinzierl R. Resistance to pyrethroid insecticides in Helicoverpa zea (Lepidoptera: Noctuidae) in Indiana and Illinois. J Econ Entomol 2009; 102:2289-95. 8. Burkness EC, Hutchison WD, Weinzierl RA, Wedberg JL, Wold S, Shaw JT. Efficacy and risk efficiency of sweet corn hybrids expressing a Bacillus thuringiensis toxin for Lepidopteran pest management in the Midwestern US. Crop Protection 2002; 21:157-69. 9. Horner TA, Dively GP, Herbert DA. Development, survival and fitness performance of Helicoverpa zea (Lepidoptera: Noctuidae) in MON-810 Bt field corn. J Econ Entomol 2003; 96:914-24. 10. O’Rourke PK, Hutchison WD. Developmental delay and evidence for reduced cannibalism in corn earworm (Lepidoptera: Noctuidae) larvae feeding on transgenic Bt sweet corn. J Entomol Sci 2004; 39:294-7. 11. Hutchison WD, Storer NP. Expanded use of pyramided transgenic maize hybrids expressing novel Bacillus thuringiensis toxins in the Southern US: Potential for areawide suppression of Helicoverpa zea (Lepidoptera: Noctuidae) in the Mississippi delta. Southwest Entomol 2010; 35:403-8. 12. Dively G. Does it pay to grow Bt sweet corn? In: Your role in growing Attribute® sweet corn. Syngenta Seeds grower guide 2010; http://www.rogersadvantage.com/ pdf/AttributeGrowerGuide1.pdf.
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13. Hutchison WD, Burkness EC, Mitchell PD, Moon RD, Leslie TW, Fleischer SJ, et al. Areawide suppression of European corn borer with Bt maize reaps savings to non-Bt maize growers. Science 2010; 330:222-5. 14. Estruch JJ, Warren GW, Mullins MA, Nye GJ, Craig JA, Koziel MG. Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against Lepidopteran insects. Proc Natl Acad Sci USA 1996; 93:5389-94. 15. Yu C, Mullins MA, Warren GW, Koziel MG, Estruch JJ. The Bacillus thuringiensis vegetative insecticidal protein Vip3A lyses midgut epithelium cells of susceptible insects. App Environ Micro 1997; 63:532-53. 16. Kurtz RW. A review of Vip3A mode of action and effects on Bt cry protein-resistant colonies of Lepidopteran larvae. Southwest Entomol 2010; 35:391-4. 17. Tabashnik BE. Evolution of resistance to Bacillus thuringiensis. Annu Rev Entomol 1994; 39:47-79. 18. Jackson RE, Marcus MA, Gould F, Bradley JR, Van Duyn JW. Cross-resistance responses of Cry1Acselected Heliothis virescens (Lepidoptera: Noctuidae) to the Bacillus thuringiensis protein Vip3A. J Econ Entomol 2007; 100:180-6. 19. Anilkumar KJ, Rodrigo-Simón A, Ferré J, PusztaiCarey M, Sivasupramaniam S, Moar WJ. Production and characterization of Bacillus thuringiensis Cry1Acresistant cotton bollworm Helicoverpa zea (Boddie). Appl Environ Microbiol 2008; 74:462-9.
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20. Lee MK, Miles P, Chen JS. Brush border membrane binding properties of Bacillus thuringiensis Vip3A to Heliothis virescens and Helicoverpa zea midguts. Biochem Biophys Res Commun 2006; 339:1043-7. 21. Burkness EC, Galvan TL, Hutchison WD. Optimizing Helicoverpa zea (Lepidoptera: Noctuidae) insecticidal efficacy in Minnesota sweet corn: a logistic regression to assess timing parameters. J Econ Entomol 2009; 102:677-84. 22. US Environmental Protection Agency (EPA). Bacillus thuringiensis Vip3Aa20 insecticidal protein and the genetic material necessary for its production (via elements of vector pNOV1300) in event MIR 162 maize (OECD Unique Identifier: SYN-IR162-4). Biopesticides Registration Action Document (BRAD) 2009; 175; http://www.epa.gov/oppbppd1/biopesticides/ingredients/tech_docs/brad_006599.pdf 23. Chilcutt CF, Tabashnik BE. Contamination of refuges by Bacillus thuringiensis toxin genes from transgenic maize. Proc Nat Acad Sci 2004; 101:7526-9.
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24. Romeis J, Shelton AM, Kennedy GG, Eds. Integration of insect-resistant genetically modified crops within IPM programs. Springer 2008; 441. 25. Wold SJ, Burkness EC, Hutchison WD, Venette RC. In-field monitoring of beneficial insect populations in transgenic corn expressing a Bacillus thuringiensis toxin. J Entomol Sci 2001; 36:177-87. 26. Rose R, Dively G. Effects of insecticide-treated and Lepidopteran-active Bt transgenic sweet corn on the abundance and diversity of arthropods. Environ Entomol 2008; 36:1254-68. 27. Dively G. Impact of transgenic VIP3a x Cry1ab Lepidopteran-resistant field corn on the nontarget arthropod community. Environ Entomol 2005; 34:1267-91. 28. Raybould A, Vlachos D. Non-target organism effects tests on Vip3A and their application to the ecological risk assessment for cultivation of MIR162 maize. Trans Res 2010; DOI: 10.1007/s11248-010-9442-1.
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29. Onstad DW, (Ed.). Insect Resistance Management: Biology, Economics and Prediction. Elsevier, Amsterdam, Netherlands 2008; 305. 30. CERA. GM Crop Database. Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington DC 2010; http://cera-gmc.org/index. php?action=gm_crop_database. 31. Fritz VA, Tong CB, Rosen CJ, Wright JA. Sweet corn (vegetable crop management) 2010; http://www. extension.umn.edu/distribution/cropsystems/DC7061. html. 32. SAS Institute Inc., SAS/STAT® 9.2 User’s Guide. Cary, NC: SAS Institute Inc 2008. 33. Conover WJ, Iman RL. Rank transformations as a bridge between parametric and nonparametric statistics. The Am Statist 1981; 35:124-9.
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