Efficacy of Transgenic Cotton Expressing Cry1Ac and Cry1F ... - BioOne

PLANT RESISTANCE

Efficacy of Transgenic Cotton Expressing Cry1Ac and Cry1F Insecticidal Protein Against Heliothines (Lepidoptera: Noctuidae) M. WILLRICH SIEBERT,1,2 S. NOLTING,1 B. R. LEONARD,3 L. B. BRAXTON,1 J. N. ALL,4 J. W. VAN DUYN,5 J. R. BRADLEY,5 J. BACHELER,5 AND R. M. HUCKABA1

J. Econ. Entomol. 101(6): 1950Ð1959 (2008)

ABSTRACT Cotton, Gossypium hirsutum L, plants expressing Cry1Ac and Cry1F (Phytogen 440W) insecticidal crystal proteins of Bacillus thuringiensis (Bt) Berliner, were evaluated against natural populations of tobacco budworm, Heliothis virescens (F.), and bollworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), across 13 southern U.S. locations that sustained low, moderate, and high infestations. The intrinsic activity of Phytogen 440W was compared with nontreated non-Bt cotton (PSC355) and with management strategies in which supplemental insecticides targeting heliothines were applied to Phytogen 440W and to PSC355 cotton. Infestations were composed primarily of bollworm, which is the least sensitive of the heliothine complex to Cry toxins. Therefore, damage recorded in these studies was primarily due to bollworm. Greater than 75% of all test sites sustained heliothine infestations categorized as moderate to high (10.6 Ð 64.0% peak damaged bolls in nontreated PSC355). Phytogen 440W, alone or managed with supplemental insecticide applications, reduced heliothine-damaged plant terminals, squares (ßower buds), ßowers, and bolls equal to or better (1.0 Ð79.0-fold) than managing a non-Bt cotton variety with foliar insecticides across all infestation environments. Rarely (frequency of ⱕ11% averaged across structures), sprayed Phytogen 440W reduced damaged structures compared with nontreated Phytogen 440W. Protection against heliothine-induced plant damage was similar across the three levels of infestation for each viable management strategy, with exception to damaged squares for nontreated Phytogen 440W. In situations of moderate to high heliothine infestations, cotton plants expressing Cry1Ac and Cry1F may sustain higher levels of damage compared with that same variety in low infestations. No signiÞcant difference in yield was observed among heliothine management strategies within each infestation level, indicating cotton plants may compensate for those levels of plant damage. These Þndings indicate Phytogen 440W containing Cry1Ac and Cry1F provided consistent control of heliothines across a range of environments and infestation levels. KEY WORDS Bacillus thuringiensis, cotton, integrated pest management, tobacco budworm, bollworm

Tobacco budworm, Heliothis virescens (F.), and bollworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), are damaging pests of cotton, Gossypium hirsutum L., in the southern United States. Known collectively as the heliothine complex, larvae of both species cause yield losses in cotton by feeding on squares (ßower buds), ßowers, and bolls (fruit) (Leigh et al. 1996). Historically, management of this pest complex has relied almost exclusively on chemical control tactics (Martin et al. 1995, Herzog et al. 1996). However, this strategy became less effective as 1 Dow AgroSciences, LLC, 9330 Zionsville Rd., Indianapolis, IN 46268. 2 Corresponding author, e-mail: [email protected]. 3 Louisiana State University Agricultural Center, Macon Ridge Research Station, Winnsboro, LA 71295. 4 Department of Entomology, University of Georgia, Athens, GA 30602. 5 Department of Entomology, North Carolina State University, Raleigh, NC 27695.

tobacco budworm developed resistance to numerous insecticide classes, including pyrethroids (Luttrell et al. 1987, Leonard et al. 1988). Although pyrethroid insecticides continue to be an effective and economical management tool for bollworm, reduced efÞcacy and susceptibility have been reported beginning in the mid-1990s (Brown et al. 1997, Hutchinson and Weinzierl 2007, Pietrantonio et al. 2007). Insect-resistant transgenic cotton varieties have been available to producers since 1996. The Þrst commercial varieties were engineered to express the Cry1Ac protein from the bacterium, Bacillus thuringiensis Berliner (Bt). Single-protein Bt cottons revolutionized cotton insect pest management by providing complete control of tobacco budworm (MacIntosh et al. 1990, Jackson et al. 2003). Control of bollworm has been less reliable and supplemental insecticides are commonly applied to prevent economic losses (Gore et al. 2001). In addition, Cry1Ac alone does not provide satisfactory control of second-

0022-0493/08/1950Ð1959$04.00/0 䉷 2008 Entomological Society of America

December 2008 Table 1.

SIEBERT ET AL.: EVALUATION OF TRANSGENIC CRY1AC AND CRY1F COTTON

1951

Field trials evaluating the efficacy of Cry1Ac and Cry1F cotton against heliothines in the southern United States, 2003

Location

Species composition (% bollworm: % tobacco budworm)

Peak boll damagea (%) ⫾ SE

Trial categoryb

Waller, TX Tillar, AR Chula, GA Jamesville, NC Rocky Mount, NC Winnsboro, LA Portageville, MO Wayside, MS Midville, GA Elko, SC Rio Hondo, TX St. Paul, TX Shoffner, AR

Predominantly bollwormc 87:13 Predominantly bollworm 85:15 Predominantly bollworm 70:30 96:4 71:29 Predominantly bollworm 90:10 Predominantly bollworm Predominantly bollworm 88:12

48.1 ⫾ 9.3 34.4 ⫾ 2.9 25.0 ⫾ 4.4 60.0 ⫾ 7.3 64.0 ⫾ 1.5 11.3 ⫾ 4.7 20.3 ⫾ 4.9 10.6 ⫾ 2.8 20.4 ⫾ 4.9 16.8 ⫾ 3.2 6.3 ⫾ 3.3 8.1 ⫾ 1.6 5.6 ⫾ 1.2

High High High High High Moderate Moderate Moderate Moderate Moderate Low Low Low

a

Sampled from nontreated PSC355 (non-Bt cotton). At least one sampling date per trial in which peak boll damage in nontreated PSC355 was ⬎25% or 10 Ð25% for high and moderate infestation environments, respectively. Boll damage levels ⱕ10% for all sampling dates per trial were categorized as low infestation environments. c Bollworm comprised ⬎90% of heliothine population. b

ary lepidopteran pests, including fall armyworm, Spodoptera frugiperda (J.E. Smith); beet armyworm, Spodoptera exigua (Hu¨ bner); and soybean looper, Pseudoplusia includens (Walker) (Stewart et al. 2001). Many of the current transgenic cotton varieties express a second gene encoding for an additional Bt protein. This rationale for deploying multiple insect resistance traits is to not only broaden the spectrum and increase the level of activity but also to aid in Bt resistance management (Tabashnik 1994, Gould 1998, Stewart et al. 2001). The Þrst dual-toxin Bt varieties were available during the 2003 growing season and contained Cry1Ac and Cry2Ab (Bollgard II, Monsanto Co., St. Louis, MO). In 2005, varieties containing Cry1Ac and Cry1F (WideStrike Insect Protection, Dow AgroSciences, LLC, Indianapolis, IN) were also commercially available. WideStrike varieties were developed using a standard crossing method. Cotton genotype GC510 was transformed to contain the genes that express fulllength synthetic protoxins of Cry1Ac (transformation event 3006-210-23) or Cry1F (transformation event 281-24-236). Transgenic lines were backcrossed with nontransgenic germplasm, PSC355 (Phytogen Seed Company, LLC, Dow AgroSciences, LLC). Subsequently, Cry1Ac and Cry1F-expressing cotton plants were crossed to produce a stacked product, of which Phytogen 440W (Phytogen Seed Company, Dow AgroSciences, LLC) was a selection. Cotton varieties containing Cry1Ac and Cry1F have gained in adoption since commercialization and during 2008, ⬇142,000 ha was planted in the United States (Dow AgroSciences, LLC, unpublished data). The objective of the this study was to examine the Þeld performance of Phytogen 440W, containing both Cry1Ac and Cry1F insecticidal toxins, against varying levels of heliothine infestations occurring across the mid-south and southeastern United States. The novel cotton trait was compared with a non-Bt cotton variety managed with conventional insecticides targeting lepidopteran pests. These studies have enabled the characterization of Cry1Ac and Cry1F efÞcacy against two

primary pests, occurring at varying population levels, and across a broad range of geographies. Materials and Methods Studies evaluating damage to ßowering stage Cry1Ac and Cry1F cotton plants and a non-Bt cotton variety by tobacco budworm and bollworm were conducted in 13 southern U.S. environments (locations) during 2003 (Table 1). At each test site, cultural practices including fertility, irrigation, and weed management, as recommended by state extension guidelines, were used to maintain experimental plots for optimum productivity. The entire test area was managed for nonlepidopteran insects pests, including thrips (Thysanoptera: Thripidae), aphids (Homoptera: Aphididae), stink bugs (Hemiptera: Pentatomidae), and plant bugs (Hemiptera: Miridae), by using insecticide chemistries with limited activity against lepidopteran insects. Insecticides used included aldicarb (Temik 150 g/kg, Bayer Crop Science, Research Triangle Park, NC), dicrotophos (Bidrin 480 g/liter E, Amvac Chemical Corporation), and thiamethoxam (Centric 400 g/kg WG, Syngenta Crop Protection, Greensboro, NC). Plots were planted in a split plot experimental design. The main plots consisted of one of two treatments: sprayed with conventional insecticides or nontreated. The sprayed main plots received foliar insecticide applications targeting all lepidopteran pests if at least one subplot treatment (averaged across replications) exceeded an action threshold as recommended by state extension guidelines. Nontreated main plots did not receive insecticide applications targeting lepidopteran pests. Insecticides used to maintain sprayed main plots included a coapplication of spinosad (Tracer480 g/liter SC, Dow AgroSciences, LLC) and a pyrethroid (␭-cyhalothrin, Karate 250 g/liter CS, Syngenta Crop Protection or ␤-cyßuthrin, Baythroid 240 g/liter EC, Bayer CropScience). The subplots within each main plot included Phytogen 440W and PSC355, which represent the Cry1Ac and Cry1F and non-Bt variety, respectively, and were rep-

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JOURNAL OF ECONOMIC ENTOMOLOGY

licated four times within each main plot. Subplots were 12.2 m in length by four rows (91.4 Ð101.6-cm centers). Each subplot was planted between a border of Phytogen 440W cotton (four rows) to minimize larval movement between the subplots. The resulting treatments (management strategies) evaluated were nontreated PSC355, nontreated Phytogen 440W, sprayed PSC355, and sprayed Phytogen 440W. The center two rows of each subplot were sampled (four to seven per location) weekly beginning at the onset of anthesis. On each date, four structures were sampled, including terminals, squares, ßowers, and bolls. Twenty plant terminals and 120 fruiting forms (40 squares, white ßowers, and bolls) per plot were randomly selected and examined for presence of surviving larvae and heliothine damage in each plot. A plant terminal (vegetative apex) was considered to be the uppermost mainstem node on the plant containing a leaf not fully expanded (⬇2.54 cm in diameter) and including a small square (2Ð3 mm in diameter). A terminal was recorded as damaged if leaf and/or square tissue contained small holes characteristic of heliothine damage. Squares and bolls were selected from the upper half of the plant canopy and were ⬇6 Ð 8 mm and 1.5Ð3 cm in diameter, respectively. A square or boll was considered damaged if the calyx or carpel wall, respectively, were fully penetrated. SuperÞcial external feeding on squares and bolls was not recorded as damage. White ßowers were sampled throughout the plant canopy and the presence of damage was indicated by small holes and/or consumption of ßoral reproductive components (stamen and pistil). Larvae were not distinguished between tobacco budworm and bollworm in the Þeld. However, wire cone traps (Hartstack et al. 1979) baited with artiÞcial sex pheromone lures (Hendricks et al. 1987) were used to monitor adult populations and thus for determination of the heliothine species composition present at each location. Leonard et al. (1989) determined there to be a positive correlation between the total number of tobacco budworm and bollworm male moths captured in pheromone traps and oviposition in cotton Þelds. To determine the overall effect of a management strategy, it was desirable to not only capture the intensity of infestation at each site but also the intensity over time. One method that can be used to integrate count data over time is to calculate the area under (within the) infestation pressure curve (AUIPC), which has been described by Kohler and St. Claire (2005). In the present studies, raw data were collected as counts of each damaged structure per number sampled on multiple dates. The transformed data (AUIPC) described the proportion of each plant structure damaged per day and provided a means of integrating damage incurred by plots throughout the sampling period for each location. Damage counts were converted to AUIPC before statistical analysis and each trial location resulted in a single AUIPC value (least squares mean) for each replication of each structure sampled.

Vol. 101, no. 6

Initial observations of the data indicated that the results may be inßuenced by population levels and persistence of infestations. To maintain consistent variances when performing analyses across environments, locations, and their associated data (AUIPC values) were segregated into three categories based on the peak level (single date) of heliothine-damaged bolls sampled in nontreated PSC355 (non-Bt) plots (Table 1). Level of damaged bolls ⬎25% or 10 Ð25% on at least one sampling date per trial was considered to have a high and moderate heliothine infestation, respectively. Damaged boll levels ⱕ10% for all sampling dates per trial were categorized as low infestation environments. These ranges were arbitrarily selected to Þt the damage levels observed in the data. The AUIPC data from each location was subjected to analysis of variance (ANOVA) and analyzed using methods for split-plot designs (Littell et al. 1996). For each structure and by location, differences between the least squares means (AUIPC) for all possible pairs of treatments (management strategies) were compared. Frequencies of treatment comparison outcomes were then tabulated across locations within a level of infestation to highlight management strategies that provided signiÞcantly greater protection against heliothine damage compared with another possible management strategy. The resulting analysis described the frequency of locations in which one heliothine management strategy in a pair of strategies provided signiÞcantly better control. Within each level of heliothine infestation, AUIPC data for terminals, squares, ßowers, and bolls were combined across locations for each structure and then compared among the viable heliothine management strategies (nontreated Phytogen 440W, sprayed Phytogen 440W, and sprayed PSC335) by using ANOVA. Nontreated PSC355 was included in all trials to monitor tobacco budworm and bollworm infestations. Non-Bt cotton plots that did not receive oversprays targeting lepidopteran pests experienced high levels of insect damage and were accompanied by variances that were higher than those observed in the heliothine-managed plots. Therefore, the across location analysis of the AUIPC data within a level of infestation excluded the nontreated PSC355 treatment to avoid heterogeneous variances among the remaining treatments. The three remaining management strategies were treated as one replication of a randomized block design at each of the locations for the across location analysis (i.e., replication within a location was not included as a factor in the analysis). In addition, AUIPC data for each of the four heliothine management strategies was compared among the low, moderate, and high heliothine infestation levels for each plant structure sampled. Cotton yield (seedcotton) was harvested at seven locations (two low, three moderate, and two high infestation locations) from the entire length of the center two rows of each subplot using a mechanical picker. Seedcotton per plot (kilograms per hectare) was transformed to kilograms of lint per ha by using a standardized lint:seed ratio of 38%. Across location

December 2008


analyses of the yield data used the previously described infestation level categorizations and were conducted using methods for split plot designs (Littell et al. 1996). An ␣ value of 0.05 was used for all ANOVA procedures and mean separation tests that compared heliothine damage and cotton yield. For variables with a signiÞcant F value, means were separated using methods developed by Tukey test and for unbalanced data the TukeyÐKramer test (SAS Institute 1998). Results Heliothine infestations during 2003 were composed primarily of bollworm and ⬎75% of all test sites experienced heliothine infestations categorized as moderate to high (Table 1). Peak damaged bolls in nontreated PSC355 (non-Bt) plots for trials categorized as high, moderate, and low infestation ranged from 25.0 to 64.0, 10.6 Ð20.4, and 5.6 Ð 8.1%, respectively. Within each heliothine infestation level and for each plant structure sampled, damage was numerically greater for nontreated PSC355 compared with a similar genetic line containing Cry1Ac and Cry1F (Phytogen 440W), which was also nontreated. For locations with low heliothine infestations, levels of damaged terminals, squares, ßowers, and bolls for nontreated Phytogen 440W were 8.4-, 24.8-, 9.4-, and 53.7-fold, respectively, less than that for nontreated PSC355 (Table 2). Damaged terminals, squares, ßowers, and bolls were 7.4-, 9.2-, 8.4-, and 8.6-fold, respectively, less for nontreated Phytogen 440W compared with nontreated PSC355 for locations with moderate heliothine infestations. For locations with high infestations, damaged terminals, squares, ßowers, and bolls for nontreated Phytogen 440W was 16.5-, 8.5-, 8.7-, and 8.2fold, respectively, less than nontreated PSC355. Efficacy of Heliothine Management Strategies within an Infestation Level. In low infestation environments and when nontreated PSC355 was excluded from analysis, there were signiÞcant differences among sprayed PSC355, sprayed Phytogen 440W, or nontreated Phytogen 440W for proportion of terminals (F ⫽ 3.38; df ⫽ 2, 31; P ⫽ 0.0470) and squares (F ⫽ 9.38; df ⫽ 2, 31; P ⫽ 0.0007) damaged per day (Table 2). Terminal damage was signiÞcantly less for nontreated Phytogen 440W (3.1-fold) compared with that for sprayed PSC355 but similar to sprayed Phytogen 440W. Square damage was signiÞcantly less for nontreated and sprayed Phytogen 440W (5.6 Ð9.0-fold) compared with that for sprayed PSC355. The proportion of ßowers (F ⫽ 1.11; df ⫽ 2, 6; P ⫽ 0.3891) and bolls (F ⫽ 3.85; df ⫽ 2, 6; P ⫽ 0.0842) damaged per day was not signiÞcantly different among heliothine management strategies. In moderate infestation environments and when nontreated PSC355 was excluded from analysis, the proportion of terminals (F ⫽ 4.20; df ⫽ 2, 10.4; P ⫽ 0.0460), squares (F ⫽ 4.84; df ⫽ 2, 12; P ⫽ 0.0287), and bolls (F ⫽ 9.01; df ⫽ 2, 53; P ⫽ 0.0004) damaged was signiÞcantly different among sprayed PSC355, sprayed Phytogen 440W, and nontreated Phytogen 440W (Table 2). For terminals and squares, sprayed Phytogen

1953

Table 2. LSMean percentage of damage per day for each of four cotton plant structures by heliothines for a Bt and non-Bt cotton genotype as influenced by insecticide treatment regime and infestation level, 2003 Heliothine management strategyb,c

Heliothine infestation levela/cotton plant structure Low

Moderate

High

Nontreated PSC355 Sprayed PSC355 Nontreated Phytogen 440W Sprayed Phytogen 440W

Damaged terminals (%)d 6.36A 18.77A 28.69A 2.34aA 4.62aA 8.55aA 0.76bA 2.53abA 1.74aA 1.36abA 1.08bA 1.38aA


Damaged squares (%) 3.23B 12.06B 27.72A 1.17aA 2.93aA 5.99aA 0.13bC 1.31abB 3.28aA 0.21bA 0.40bA 0.82aA


Damaged ßowers (%) 2.63A 6.91A 19.04A 0.76aA 1.63aA 1.70aA 0.28aA 0.82aA 2.20aA 0.05aA 0.27aA 0.26aA


1.61B 0.55aA 0.03aA 0.08aA

Damaged bolls (%) 8.82B 31.50A 2.19aA 3.95aA 1.03bA 3.85aA 0.55bB 0.05aA

Means within columns, by structure, followed by the same lowercase letter are not signiÞcantly different; Uppercase letters within rows that are similar are not signiÞcantly different (P ⬍ 0.05; Tukey or TukeyÐKramer). a Averaged across three, Þve, and Þve trials classiÞed as low, moderate, and high infestation, respectively. b Nontreated PSC355 excluded from analysis comparing management strategies within a level of infestation. c Nontreated management regimes did not receive insecticide applications targeting lepidopteran pests. Sprayed management regimes received as-needed foliar insecticide applications targeting lepidopteran pests. d Values are transformed, least-squares means of proportions expressed as percentages.

440W reduced damage to levels signiÞcantly less than that of sprayed PSC355 (4.3Ð7.3-fold). Terminal and square damage was similar between nontreated Phytogen 440W and sprayed Phytogen 440W. Boll damage was signiÞcantly reduced for nontreated Phytogen 440W (2.1-fold) and sprayed Phytogen 440W (4.0fold) compared with sprayed PSC355. There were no signiÞcant differences among sprayed PSC355, sprayed Phytogen 440W, or nontreated Phytogen 440W in the percentage of damaged ßowers per day (F ⫽ 2.03; df ⫽ 2, 12; P ⫽ 0.1423). In high infestation environments and when nontreated PSC355 was excluded from analysis, there were no signiÞcant differences among sprayed PSC355, sprayed Phytogen 440W, and nontreated Phytogen 440W in the proportion of terminals (F ⫽ 1.86; df ⫽ 2, 5.94; P ⫽ 0.2362), squares (F ⫽ 1.57; df ⫽ 2, 9.06; P ⫽ 0.2594), ßowers (F ⫽ 1.90; df ⫽ 2, 9.01; P ⫽ 0.2048), and bolls (F ⫽ 1.52; df ⫽ 2, 12.1; P ⫽ 0.2581) damaged per day (Table 2). Influence of Heliothine Infestation Level on Efficacy of a Management Strategy. The proportion of damaged terminals per day among low, moderate, and

1954


high infestation environments was not signiÞcantly different for nontreated PSC355 (F ⫽ 2.34; df ⫽ 2, 8.04; P ⫽ 0.1581), sprayed PSC355 (F ⫽ 1.33; df ⫽ 2, 7.71; P ⫽ 0.3200), nontreated Phytogen 440W (F ⫽ 0.75; df ⫽ 2, 8.23; P ⫽ 0.5034), and sprayed Phytogen 440W (F ⫽ 0.07; df ⫽ 2, 8.14; P ⫽ 0.9285) (Table 2). In addition, there were no signiÞcant differences in damaged ßowers for nontreated PSC355 (F ⫽ 2.66; df ⫽ 2, 9.04; P ⫽ 0.1235), sprayed PSC355 (F ⫽ 0.34; df ⫽ 2, 8.96; P ⫽ 0.7198), nontreated Phytogen 440W (F ⫽ 3.65; df ⫽ 2, 8.68; P ⫽ 0.0709), and sprayed Phytogen 440W (F ⫽ 1.10; df ⫽ 2, 45; P ⫽ 0.3415) (Table 2) among infestation levels. The proportion of damaged squares per day was signiÞcantly different for nontreated PSC355 (F ⫽ 16.92; df ⫽ 2, 8.78; P ⫽ 0.0010) and nontreated Phytogen 440W (F ⫽ 29.48; df ⫽ 2, 45; P ⫽ 0.0001). Square damage was signiÞcantly less (3.7Ð 8.6-fold) for nontreated PSC355 in low and moderate infestation environments compared with high infestation environments. For nontreated Phytogen 440W, square damage was signiÞcantly less (10.1-fold) in low infestations compared with moderate infestation environments. In addition, square damage was signiÞcantly less (2.5-fold) for moderate infestations compared with high infestation environments. There was no signiÞcant difference in square damage for sprayed PSC355 (F ⫽ 1.14; df ⫽ 2, 9.02; P ⫽ 0.3610) and sprayed Phytogen 440W (F ⫽ 0.86; df ⫽ 2, 9.05; P ⫽ 0.4553) among infestation levels. Boll damage was signiÞcantly different for nontreated PSC355 (F ⫽ 10.44; df ⫽ 2, 9.79; P ⫽ 0.0037) among the three levels of heliothine infestations. The proportion damaged per day was signiÞcantly less for low (19.6-fold) and moderate (3.6-fold) infestations levels compared with high infestations. The proportion of bolls damaged for sprayed Phytogen 440W was signiÞcantly different (F ⫽ 4.90; df ⫽ 2, 49; P ⫽ 0.0115) among the three levels of heliothine infestations. The proportion of damaged bolls per day was signiÞcantly greater for moderate infestations environments compared with low and high infestation environments (6.9 Ð11.0-fold). There was no signiÞcant difference among low, moderate, and high infestation environments for sprayed PSC355 (F ⫽ 0.79; df ⫽ 2, 10.1; P ⫽ 0.4804) and nontreated Phytogen 440W (F ⫽ 2.50; df ⫽ 2, 9.29; P ⫽ 0.1350) among infestation levels. Comparisons between Heliothine Management Strategies. In low infestation environments when heliothine management strategies (sprayed PSC355, nontreated Phytogen 440W, and sprayed Phytogen 440W) were independently compared with nontreated PSC355, the percentage of locations in which these strategies provided signiÞcantly greater protection of damaged terminals, squares, ßowers, and bolls ranged from 33 to 100% (Fig. 1a). Sprayed Phytogen 440W provided signiÞcantly greater protection of terminals, squares, ßowers, and bolls compared with sprayed PSC355 at 33% of the locations for each structure. Terminals and ßowers were protected equally (frequencies of 0%) with nontreated Phytogen 440W and sprayed PSC355, but squares and bolls were pro-

Vol. 101, no. 6

tected to a greater extent with nontreated Phytogen 440W compared with sprayed PSC355 at 33% of the locations. Similarly, for all structures, there were no locations (frequencies of 0%) in which a sprayed Phytogen 440W management strategy was signiÞcantly better than a nontreated Phytogen 440W management strategy. In moderate infestation environments when heliothine management strategies (sprayed PSC355, nontreated Phytogen 440W, and sprayed Phytogen 440W) were independently compared with nontreated PSC355, the percentage of locations in which these strategies provided signiÞcantly greater protection of damaged terminals, squares, ßowers, and bolls ranged from 60 to 100% (Fig. 1b). Sprayed Phytogen 440W provided greater protection of terminals, squares, ßowers, and bolls compared with sprayed PSC355 at 40, 40, 20, and 20% of locations, respectively. Terminals, squares, ßowers, and bolls were protected equally with nontreated Phytogen 440W and sprayed PSC355 at 20% of the locations. Sprayed Phytogen 440W provided better protection against damaged terminals at 20% of the locations compared with nontreated Phytogen 440W, whereas no difference in protection (frequency of 0%) between the two management strategies including Phytogen 440W were observed for squares, ßowers, and bolls. In high infestation environments in which heliothine management strategies (sprayed PSC355, nontreated Phytogen 440W, and sprayed Phytogen 440W) were independently compared with nontreated PSC355, the percentage of locations in which these strategies provided signiÞcantly greater protection of damaged terminals, squares, ßowers, and bolls ranged from 75 to 100% (Fig. 1c). Sprayed Phytogen 440W provided greater protection of terminals, squares, ßowers, and bolls compared with sprayed PSC355 at 67, 50, 25, and 40% of the locations, respectively. Terminals, squares, and bolls were protected to a greater extent with nontreated Phytogen 440W compared with sprayed PSC355 at 67, 50, and 40% of locations, respectively. Flowers were protected equally with nontreated Phytogen 440W and sprayed PSC355 with no (0%) location differences observed. Sprayed Phytogen 440W protected against damaged ßowers and bolls at 25 and 20% of the locations, respectively, when compared with nontreated Phytogen 440W. Conversely, no differences (frequencies of 0%) in damage between the two management strategies including Phytogen 440W were observed for terminals and squares. Yield. Lint yield was not signiÞcantly different among heliothine management strategies within low infestation environments (F ⫽ 0.39; df ⫽ 1, 22.4; P ⫽ 0.5366) (Table 3). SigniÞcant differences in lint yield were detected among heliothine management strategies for the moderate (F ⫽ 13.98; df ⫽ 1, 22; P ⫽ 0.0011) and high (F ⫽ 38.34; df ⫽ 1, 14; P ⫽ 0.0001) infestation environments. In the moderate infestation environments, lint yield was signiÞcantly greater (1.3-fold) for nontreated Phytogen 440W compared with nontreated PSC355. Sprayed Phytogen 440W and sprayed

December 2008 1


1955

a

Frequency

0.8

0.6

0.4

0.2

0

0 0

0

Terminal 1

0 0 Square

0

Flower

Boll

b

Paired Comparisons

0.8

Frequency

Sprayed PHY440W > Nontreated PHY440W Nontreated PHY440W > Sprayed PSC355

0.6

Sprayed PHY440W > Sprayed PSC355 0.4

Sprayed PSC355 > Nontreated PSC355 Nontreated PHY440W > Nontreated PSC355

0.2

0

0 Terminal

0

Sprayed PHY440W > Nontreated PSC355

0

Square

Flower

Boll

1

c

Frequency

0.8

0.6

0.4

0.2

0

0

0 Terminal

0 Square

Flower

Boll

Fig. 1. Frequency of sites in which the Þrst of the pair of heliothine management strategies provided signiÞcantly greater protection (less damage) to various cotton plant structures for trials categorized as low (a), moderate (b), and high (c) infestation. Table 3. Yield for a Bt and non-Bt cotton genotype as influenced by insecticide treatment regime and heliothine infestation level, 2003 Heliothine management strategy Nontreatedb PSC355 Sprayed PSC355 Nontreated Phytogen 440W Sprayed Phytogen 440W a

kg lint/haa Low infestation

Moderate infestation

High infestation

541.9a 907.3a 691.4a 1,024.6a

865.7b 1,023.1ab 1,085.3a 1,043.4ab

1,024.3b 1,325.6a 1,268.7a 1,306.9a

Values are transformed, least-squares means of yield. Nontreated management regimes did not receive insecticide applications targeting lepidopteran pests. Sprayed management regimes received as-needed foliar insecticide applications targeting lepidopteran pests. b

PSC355 were similar to all other management strategies exposed to moderate heliothine infestations. In the high infestation environments, all heliothine management strategies provided lint yield signiÞcantly greater (1.2Ð1.3-fold) than nontreated PSC355. Discussion The activity of Cry1Ac and Cry1F insecticidal proteins, as expressed in Phytogen 440W cotton, was characterized under a range of environments and heliothine infestations levels likely to occur in the southern United States. Segmentation of the data from different locations based on peak boll damage was a

1956


reliable means of categorizing the data by insect pest abundance and for minimizing variances that were proportional to the means for damage measurements recorded for all structures. The differences observed in peak boll damage among environments compared very consistently with damage observed for terminals, squares, and ßowers in nontreated PSC355 (non-Bt) cotton. The AUIPC transformation used to integrate damage over time for the different structures was consistent with the differences observed in the peak boll damage data used for categorization. Consistency of translation, between peak boll damage observed at different locations and the AUIPC data of the categorized infestation levels, was evident when nontreated PSC355 (non-Bt) damage was compared across infestation levels. Damage was signiÞcantly greater for squares and bolls in high infestation environments compared with moderate/low infestation environments. Numeric trends of similar magnitude also were evident for damaged terminals and ßowers between high and low infestation environments for nontreated PSC355. The level of signiÞcance for terminals and ßowers was P ⫽ 0.1390 and P ⫽ 0.1362, respectively, when damage between high and low heliothine infestation levels was compared in nontreated PSC355. Frequency analysis used to compare management strategies also supported the segmentation of data into three heliothine infestation levels. The frequency, averaged across structures, in which sprayed Phytogen 440W, nontreated Phytogen 440W, and sprayed PSC355 reduced damaged structures greater than nontreated PSC355 was 58, 95, and 98% for the low, moderate, and high infestation environments, respectively. Thus, there was a corresponding increase in the frequency at which these heliothine management strategies were signiÞcantly better options than nontreated PSC355 as infestations levels increased from low to high. These results demonstrated cotton plants expressing Cry1Ac and Cry1F protein reduced heliothinedamaged terminals, squares, ßowers, and bolls under varying levels of infestation. Phytogen 440W, either nontreated or sprayed with foliar insecticides active against heliothines, sustained some level of damage to each structure. The selective, stage-speciÞc toxicity of Bt proteins in cotton plants and the difference in susceptibility between tobacco budworm and bollworm to Cry toxins resulted in varying levels of plant injury. Adult oviposition and larval eclosion is generally unaffected by Bt toxin expression in plants (Liu et al. 2002, Kumar 2004). Cotton containing Cry1Ac, Cry1Ac and Cry2Ab, and Cry1Ac and Cry1F has been documented as having no effect on heliothine adult behavior, oviposition location and frequency, and larval eclosion (Lambert et al. 1997, Jackson et al. 2003; Dow AgroSciences, LLC, unpublished data). Thus, Bt proteins in plants have no effect on initial infestation levels across geographies. Bt toxins must be consumed to be effective and therefore, some plant damage may be sustained during feeding and ingestion of the plant material until intoxication occurs. However, damage

Vol. 101, no. 6

levels observed for the management strategies in the current study are more likely related to incomplete control of bollworm. To date, Cry1Ac alone provides near complete control of tobacco budworm (Jackson et al. 2003), but it is less active against bollworm (Luttrell et al. 1999, Perlak et al. 2001). Samples of surviving larvae on cotton plants containing Cry1Ac and Cry1F in Þeld trials have consistently been identiÞed as bollworm. Tobacco budworm larvae have not been observed to complete development on cotton plants containing Cry1Ac, including Cry1Ac and Cry1F cotton (Dow AgroSciences, LLC, unpublished data). Therefore, the plant damage reported in these studies is likely to have been induced by bollworm. Although fall armyworm and beet armyworm may injure cotton fruiting structures similar to that for bollworm (Leigh et al. 1996), larvae of these species were not observed across all locations during the sampling period in which heliothines were present. For each vegetative and fruiting structure within a category of infestation, the level of protection provided by the two proteins was statistically equal to or better than that observed on non-Bt cotton managed with conventional insecticides targeting heliothine pests. Although few statistical differences were observed between sprayed PSC355 and Phytogen 440W (nontreated or sprayed), damage was generally numerically lower with the variety containing Cry1Ac and Cry1F. Furthermore, damage levels for nontreated Phytogen 440W were always statistically equal to sprayed Phytogen 440W, regardless of plant structure. Differences among management strategies for each damaged structure were not detected under high infestations. In a few instances, statistical differences were observed in low and moderate infestation environments. This is likely related to the frequency of applications. In high infestation environments, a greater number of applications (Þve to eight among locations) were required to reduce the levels of infestations compared with that in moderate and low infestation locations (three to Þve among locations). Furthermore, these multiple applications were applied at shorter spray intervals in high infestation environments, which are typically more effective against bollworm compared with a single application applied on an action threshold. Thus, protection of terminals, squares, ßowers, and bolls likely was more equal among heliothine management strategies in the high infestations environments compared with low and moderate infestation environments. These results, in which nontreated Phytogen 440W, sprayed Phytogen 440W, and sprayed PSC355 provided similar levels of control, are in contrast to another series of studies. Jackson et al. (2003) determined another two-gene cotton cultivar, Cry1Ac and Cry2Ab (Bollgard II), provided similar levels of control in a nontreated or pyrethroid-treated regime but provided greater control compared with that for a pyrethroid-treated conventional cotton variety. Differences between the two studies may be related to heliothine infestation levels, differences in the response of target lepidopteran spe-

December 2008


cies to various Bt proteins (Luttrell et al. 1999) and combinations of proteins, or the insecticides used to manage heliothines. In the current study, a lepidopteran-speciÞc insecticide and a pyrethroid were coapplied. This may have improved control of bollworm, as well as other lepidopteran pests that attack fruit and foliage, including soybean looper and Spodoptera spp. Across the three levels of infestation, heliothineinduced damage to each of the three management strategies was statistically similar, with exception of square damage for nontreated Phytogen 440W and boll damage for sprayed Phytogen 440W. Greater boll damage for sprayed Phytogen 440W exposed to moderate infestations is likely attributed to application frequency and interval as discussed previously. Square damage was different across levels of infestation for nontreated Phytogen 440W. This provided evidence that in situations of moderate to high heliothine infestations, cotton containing Cry1Ac and Cry1F may sustain more damage as compared with that same variety exposed to low heliothine infestations. Overall, these results indicated management strategies provided equal levels of protection from heliothineinduced damage across geographies and infestation levels. These studies conÞrmed that each of the three heliothine management strategies (exclusive use of Bt cotton, managing non-Bt cotton with supplemental insecticides, or managing Bt cotton with supplemental insecticides) evaluated are viable pest control options compared with nontreated non-Bt cotton across environments. Cry1Ac and Cry1F cotton (nontreated or sprayed) and PSC355 cotton managed with conventional insecticides protected against damage more (95Ð98% averaged across structures) frequently in moderate and high infestations environments and to a lesser (58% averaged across structures) extent in low infestation environments compared with nontreated PSC355. Managing heliothines with Phytogen 440W alone, or in combination with insecticides targeting heliothines, provided better control more (25Ð 42% averaged across structures) frequently than with non-Bt cotton (PSC355) managed with conventional insecticides across all infestation environments. The frequency of occurrence in which sprayed Phytogen 440W provides a greater beneÞt than nontreated Phytogen 440W was low (ⱕ11% averaged across structures) across all infestation environments. Lint yield data support the results observed inseason for terminal, square, ßower, and boll damage. Yields among management strategies were not significantly different compared within each heliothine infestation level. These results provided additional conÞrmation for the near parity among strategies for managing heliothines in cotton. Heliothine infestation level did inßuence the extent to which a heliothine management strategy provided a greater beneÞt than not managing heliothines on non-Bt cotton. There was no difference among treatments at the low infestation level and only nontreated Phytogen 440W provided yields signiÞcantly greater than nontreated PSC355 at the moderate infestation level. In contrast, yields for

1957

all heliothine management strategies were signiÞcantly greater than nontreated non-Bt cotton in high infestation environments. These yield results are similar to the paired comparison analysis that indicated a greater (1.7-fold) frequency in high versus low infestation environments at which viable management strategies would provide better control compared with nontreated PSC355. Thus, as infestations levels increase, beneÞts (protection against structure damage and yield loss) to heliothine control provided by each management strategy will also increase compared with nontreated PSC355. Yield results from our study are also different from Jackson et al. (2003) in which insecticide-treated conventional cotton yielded signiÞcantly less than treated or nontreated Cry1Ac and Cry2Ab transgenic cotton. Similar to the damage results described previously, differential efÞcacy between Bt protein combinations or synthetic chemical classes used to manage sprayed management strategies could have inßuenced the outcome of the two studies. Although statistical and even numerical differences in plant damage did occur among heliothine management strategies, fruit compensation by the plant may have allowed for the yield difference among treatments to be minimized across infestation levels in these studies. The ability of cotton to compensate for fruit loss from insects has been widely documented (Brook et al. 1992, Jones et al. 1996, Heitholt 1999). Or, cotton plants may have been insensitive to the ranges of damage sustained to the various structures among the management options. Additionally, these results suggest current action thresholds used for initiating treatments on both non-Bt and Bt cotton are conservative and that bollworm rarely caused signiÞcant yield loss on a cotton variety containing Cry1Ac and Cry1F. Results from studies conducted by Gore et al. (2008) generated similar conclusions. The present study and that by Gore et al. (2008) did not evaluate effects of heliothine management on crop maturity. The presence and timing of signiÞcant insect infestations can inßuence management decisions during crop development and delay maturity (Gore et al. 2000). These results further support the numerous beneÞts of transgenic technologies compared with conventional insecticides that have been cited. Bt cotton plants are protected from caterpillar damage by expressing transgenic protein throughout plant tissue types and the duration of plant development. This protection avoids most problems associated with the use of conventional insecticides such as application timing, spray deposition and coverage, environmental degradation of the insecticide, and off-target concerns (Navon 2000, Castro 2002). An advantage though to managing non-Bt cotton with conventional insecticides is that selection of insecticides and application timing may coincide with the presence of single or multiple pests, such as plant bugs or stink bugs, at an action threshold rather than selection of a transgenic technology at-planting before pest presence (Funderburk and Higley1994, Edge et al. 2001). A risk considered from Bt cotton use has been the cost of the

1958


technology fee, which can be greater than the costs of conventional broad spectrum insecticide in years when target pest infestations are relatively low (Edge et al. 2001). However, there is no way to predict at the time of planting the location and intensity of heliothine infestations that may occur later in the season. In conclusion, the results from these studies conducted across the southern United States indicate transgenic cotton expressing Cry1Ac and Cry1F are effective against heliothines. The expression of these proteins provided control equal to or better than a management option in which a non-Bt cotton variety was managed with conventional insecticides. In addition, cotton expressing Cry1Ac and Cry1F only rarely beneÞted with signiÞcantly greater protection of terminals, squares, ßowers, and bolls when used in combination with conventional insecticides active against lepidopteran pests. Furthermore, different management options tested generally performed consistently across varying levels of heliothine pressure. However, insect control provided by heliothine management strategies had a greater value as infestations levels increase. State extension recommendations suggest that varieties containing Bt toxins be monitored and supplemental insecticides applied on an as-needed basis when local action thresholds for heliothines are exceeded (Bacheler 2006, Parker et al. 2007, Bagwell and Leonard 2008). These recommendations are largely based on the varying levels of damage observed across environments and levels of infestations. Our results have demonstrated that cotton varieties containing a combination of Cry1Ac and Cry1F proteins were an effective tool for managing economically important tobacco budworm and bollworm across a range of geographies. Acknowledgments We acknowledge the efforts of our many colleagues at Dow AgroSciences, LLC; Louisiana State University AgCenter; University of Georgia; North Carolina State University; and private researchers who contributed to this project. We thank N. Storer and G. Thompson of Dow AgroSciences, LLC for reviewing the manuscript.

References Cited Bacheler, J. 2006. Managing insects on cotton. North Carolina Cooperative Extension Service. Publ. No. AG-417. Bagwell, R. D., and B. R. Leonard. 2008. Louisiana recommendations for control of cotton insects, pp. 112Ð117. In 2008 Insect pest management guide. Louisiana Cooperative Extension Service. Publ. No. 1838. Brook, K. D., A. B. Hearn, and C. F. Kelly. 1992. Response of cotton, Gossypium hirsutum L., to damage by insect pests in Australia: manual simulation of damage. J. Econ. Entomol. 85: 1368Ð1377. Brown, T. M., P. K. Bryson, D. S. Brickle, J. T. Walker, and M. J. Sullivan. 1997. Pyrethroid-resistant Helicoverpa zea in cotton in South Carolina. Resistant Pest Manag. Newsl. 9: 26Ð27. Castro, B. A. 2002. Evaluation of Bacillus thuringiensis Þeld corn for management of Louisiana corn pests. Ph.D. dissertation, Louisiana State University, Baton Rouge.

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Edge, J. M., J. H. Benedict, J. P. Carroll, and H. K. Reding. 2001. Bollgard cotton: an assessment of global economic, environmental, and social beneÞts. J. Cotton Sci. 5: 121Ð 136. Funderburk, J. E., and L. G. Higley. 1994. Management of arthropod pests, pp. 199 Ð228. In J. L. HatÞeld and D. L. Karlen [eds.], Sustainable agriculture systems. Lewis Publishers, Boca Raton, FL. Gore, J., B. R. Leonard, E. Burris, D. R. Cook, and J. H. Fife. 2000. Maturity and yield response of non-transgenic and transgenic Bt cotton to simulated bollworm injury. J. Cotton Sci. 4: 152Ð160. Gore, J., B. R. Leonard, and J. J. Adamczyk. 2001. Bollworm (Lepidoptera: Noctuidae) survival on ÔBollgardÕ and ÔBollgard IIÕ cotton ßower bud and ßower components. J. Econ. Entomol. 94: 1445Ð1451. Gore, J., J. J. Adamczyk, Jr., A. Catchot, and R. Jackson. 2008. Yield response of dual-toxin Bt cottons to bollworm, Helicoverpa zea, infestations. J. Econ. Entomol. 101: 1594 Ð 1599. Gould, F. 1998. Sustainability of transgenic insecticidal cultivars: integrating pest genetics and ecology. Annu. Rev. Entomol. 43: 701Ð726. Hartstack, A. W., J. A. Witz, and D. R. Buck. 1979. Moth traps for the tobacco budworm. J. Econ. Entomol. 75: 519 Ð522. Heitholt, J. J. 1999. Cotton: factors associated with assimilation capacity, ßower production, boll set, and yield, pp. 235Ð269. In D. L. Smith and C. Hamel [eds.], Crop yield: physiology and processes. Springer, Berlin, Germany. Hendricks, D. E., T. N. Shaver, and J. L. Goodenough. 1987. Development of bioassay of molded polyvinyl chloride substrates for dispensing tobacco budworm (Lepidoptera: Noctuidae) sex pheromone bait formulations. Environ. Entomol. 16: 605Ð 613. Herzog, G. A., J. B. Graves, J. T. Reed, W. P. Scott, T. F. Watson. 1996. Chemical control, pp. 447Ð 469. In E. G. King, J. R. Phillips, and R. J. Coleman [eds.], Cotton insects and mites: characterization and management. The Cotton Foundation Publisher, Memphis, TN. Hutchinson, W. D., and R. A. Weinzierl. 2007. Increasing concerns about corn earworm susceptibility to pyrethroids in the midwestern USA. Plant Health Progress. (http://www.plantmanagementnetwork.org/sub/php/ symposium/hzea/intro/). Jackson, R. E., J. R. Bradley, and J. W. VanDuyn. 2003. Field performance of transgenic cottons expressing one or two Bacillus thuringiensis endotoxins against bollworm, Helicoverpa zea (Boddie). J. Cotton Sci. 7: 57Ð 64. Jones, M. A., R. Wells, and D. S. Guthrie. 1996. Cotton response to seasonal patterns of ßower removal: I. Yield and Þber quality. Crop Sci. 36: 633Ð 638. Kohler, G. R. and D. A. St. Claire. 2005. Variation for resistance to aphids (Homoptera: Aphididae) among tomato inbred backcross lines derived from wild Lycopersicon species. J. Econ. Entomol. 98: 988 Ð995. Kumar, H. 2004. Orientation, feeding, and ovipositional behavior of diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), on transgenic cabbage expressing Cry1Ab toxin of Bacillus thuringiensis (Berliner). Environ. Entomol. 33: 1025Ð1031. Lambert, A. L., J. R. Bradley, Jr., and J. W. Van Duyn. 1997. Interactions of Helicoverpa zea and Bt cotton in North Carolina, pp. 870 Ð 873. In Proceedings, 1997 Beltwide Cotton Conferences, 7Ð10 January 1997, New Orleans, LA. National Cotton Council, Memphis, TN. Leigh, T. F., S. H. Roach, and T. F. Watson. 1996. Biology and ecology of important insect and mite pests of cotton,

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pp. 17Ð162. In E. G. King, J. R. Phillips, and R. J. Coleman [eds.], Cotton insects and mites: characterization and management. The Cotton Foundation Publisher, Memphis, TN. Leonard, B. R., J. B. Graves, T. C. Sparks, and A. M. Pavloff. 1988. Variation in resistance of Þeld populations of tobacco budworm and bollworm (Lepidoptera: Noctuidae) to selected insecticides. J. Econ. Entomol. 81: 1521Ð1528. Leonard, B. R., J. B. Graves, E. Burris, A. M. Pavloff, and G. Church. 1989. Heliothis spp. (Lepidoptera: Noctuidae) captures in pheromone traps: species composition and relationship to oviposition in cotton. J. Econ. Entomol. 82: 574 Ð579. Littell, R. C., G. A. Milliken, W. S. Stroup, and R. D. Wolfinger. 1996. SAS system for mixed models. SAS Institute, Cary, NC. Liu, Y., B. E. Tabashnik, T. J. Dennehy, Y. Carrie`re, M. A. Sims, and S. K. Meyer. 2002. Oviposition on and mining in bolls of Bt and non-Bt cotton by resistant and susceptible pink bollworm (Lepidoptera: Gelechiidae). J. Econ. Entomol. 95: 143Ð149. Luttrell, R. T., R. T. Roush, A. Ali, J. S. Mink, M. R. Reid, and G. L. Snodgrass. 1987. Pyrethroid resistance in Þeld populations of Heliothis virescens (Lepidoptera: Noctuidae) in Mississippi in 1986. J. Econ. Entomol. 80: 985Ð989. Luttrell, R. G., L. Wan, and K. Knighten. 1999. Variation in susceptibility of noctuid (Lepidoptera) larvae attacking cotton and soybean to puriÞed endotoxin proteins and commercial formulations of Bacillus thuringiensis. J. Econ. Entomol. 92:21Ð32. MacIntosh, S. C., T. B. Stone, S. R. Sims, P. L. Hunst, J. T. Greenplate, P. G. Marrone, F. J. Perlak, D. A. Fischoff, and R. L. Fuchs. 1990. SpeciÞcity and efÞcacy of puriÞed Bacillus thuringiensis proteins against agronomically important insects. J. Invertebr. Pathol. 56: 258 Ð266.

1959

Martin, S. H., G. W. Elzen, J. B. Graves, S. Micinski, B. R. Leonard, and E. Burris. 1995. Toxicological response of tobacco budworm (Lepidoptera: Noctuidae) from Louisiana, Mississippi, and Texas to selected insecticides. J. Econ. Entomol. 88: 505Ð511. Navon, A. 2000. Bacillus thuringiensis insecticides in crop protectionÑreality and prospects. Crop Prot. 19: 669 Ð 676. Parker, R. D., D. D. Fromme, A. E. Knutson, M. Jungman, and C. G. Sansone. 2007. Managing cotton insects in the southern, eastern, and blacklands areas of Texas, 2007. Texas Cooperative Extension. Publ. E-5 4-07. Perlak, F. J., M. Oppenhuizen, K. Gustafson, R. Voth, S. Sivasupramaniam, D. Heering, B. Carey, R. A. Ihrig, and J. K. Roberts. 2001. Development and commercial use of Bollgard䉸 cotton on the USAÐ early promises versus todayÕs reality. Plant J. 27: 489 Ð501. Pietrantonio, P. V., T. A. Junek, R. Parker, D. Mott, K. Siders, N. Troxclair, J. Vargas-Camplis, J. K. Westbrook, and V. A. Vassiliou. 2007. Detection and evolution of resistance to the pyrethroid cypermethrin in Helicoverpa zea (Lepidoptera: Noctuidae) populations in Texas. J. Econ. Entomol. 36: 1174 Ð1188. SAS Institute. 1998. SAS userÕs manual, version 8. SAS Institute, Cary, NC. Stewart, S. D., J. J. Adamczyk, Jr., K. S. Knighten, and F. M. Davis. 2001. Impact of Bt cottons expressing one or two insecticidal proteins of Bacillus thuringiensis Berliner on growth and survival on noctuid (Lepidoptera) larvae. J. Econ. Entomol. 94: 752Ð760. Tabashnik, B. E. 1994. Evolution of resistance to Bacillus thuringiensis. Annu. Rev. Entomol. 39: 47Ð 49. Received 28 April 2008; accepted 21 August 2008.