TY - JOUR
AU - Bernardi,, Oderlei
AB - Abstract The pyramided genetically modified (GM) soybean [Glycine max L. (Merr.)] MON87751 × MON87708 × MON87701 × MON89788, expressing Cry1A.105, Cry2Ab2, and Cry1Ac from Bacillus thuringiensis Berliner, was approved for commercial use in Brazil. We conducted laboratory, greenhouse, and field studies to assess the efficacy of this Bt soybean against key soybean lepidopteran pests. Neonates of Anticarsia gemmatalis (Hübner) (Lepidoptera: Erebidae), Chrysodeixis includens (Walker), and Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) were exposed to Bt proteins in diet-overlay bioassays. MON87751 × MON87708 × MON87701 × MON89788 soybean and individual components were evaluated in laboratory (leaf disc), greenhouse (high artificial infestations), and in field conditions (natural infestations). Neonates of A. gemmatalis, C. includens, and H. armigera were highly susceptible to Cry1A.105 (LC50 from 0.79 to 48.22 ng/cm2), Cry2Ab2 (LC50 from 1.24 to 8.36 ng/cm2), and Cry1Ac (LC50 from 0.15 to 5.07 ng/cm2) in diet-overlay bioassays. In laboratory leaf disc bioassays and greenhouse trials, MON87751 × MON87708 × MON87701 × MON89788 soybean as well as the individual components were highly effective in controlling A. gemmatalis, C. includens, and H. armigera. Similarly, under field conditions, the pyramided genotypes expressing Cry1A.105, Cry2Ab2, and Cry1Ac were highly effective at protecting soybean against C. includens. We concluded that the individual Bt proteins expressed by GM soybean MON87751 × MON87708 × MON87701 × MON89788 killed all or nearly all the susceptible A. gemmatalis, C. includens, and H. armigera, fulfilling one important criterion for successfully delaying resistance to pyramided Bt crops. pyramided Bt soybean, velvetbean caterpillar, soybean looper, Old World bollworm, insect resistance management Soybean [Glycine max L. (Merr.)] pest management in South America has been transformed since the 2013/2014 crop season due to the deployment of genetically modified (GM) soybean technology providing resistance to key lepidopteran pests. The stacked soybean technology MON87701 × MON89788 (Intacta RR2 PRO), expressing the Cry1Ac insecticidal Bt protein (event MON87701) and conferring tolerance to glyphosate (event MON 89788), became available to farmers and provided a robust foundation for integrated pest management (IPM) programs to protect the crop against damage caused by Anticarsia gemmatalis (Hübner, 1818) (Lepidoptera: Erebidae), Chrysodeixis includens (Walker, [1858]), and Helicoverpa armigera (Hübner, 1808) (Lepidoptera: Noctuidae) (Bernardi et al. 2012, Dourado et al. 2016). Approximately 5 yr after its initial commercialization, nearly 24 million hectares were planted with this technology during the 2017/2018 crop season, representing 41% of the total soybean plantings in Brazil, Argentina, Paraguay, and Uruguay (Brookes and Barfoot 2018, 2020). The high adoption of this technology can in part be attributed to its efficacy against major lepidopteran soybean pests such as A. gemmatalis, C. includens, and H. armigera, which fits within an insect resistance management (IRM) strategy called ‘high-dose/refuge’ (MacRae et al. 2005; Bernardi et al. 2012, 2014; Yano et al. 2016; Dourado et al. 2016). However, the high adoption of Intacta RR2 PRO on an even larger soybean-growing area in South America is imposing increasing selection pressure on target species populations. Delaying the evolution of insect resistance is the main challenge to maintaining the benefits of this technology in South America. High adoption in Brazil of Bt maize technologies to manage Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae) that did not meet the high-dose criterion, in combination with poor refuge compliance, resulted in field-evolved resistance to the Cry1F protein expressed in TC1507 maize (Farias et al. 2014), which also impacted the efficacy and durability of other Cry1-based maize technologies because of cross-resistance among these Bt proteins (Bernardi et al. 2015, Omoto et al. 2016). Therefore, developing and deploying Bt crops expressing multiple effective insecticidal proteins, with low probability of cross-resistance, is needed to delay the evolution of insect resistance (Tabashnik et al. 2009, Downes and Mahon 2012, Brévault et al. 2013). This strategy involves insertion of multiple genes encoding Bt proteins into a single plant (pyramiding) and primarily assumes that resistance to each Bt protein is independent, in other words, that there is no one mechanism in the insect pests that can confer resistance to all the insecticidal proteins in the pyramid (Roush 1998; Carrière et al. 2010, 2015). In 2018, the cultivation of the GM soybean MON87751 × MON87708 × MON87701 × MON89788 in Brazil was approved (Brazilian National Biosafety Technical Commission [CTNBio] 2018). This combination of soybean events was produced by conventional breeding of four component events: MON87751, MON87708, MON87701, and MON89788. The transgenes inserted in MON87751 (Cry1A.105, Cry2Ab2) and MON87701 (Cry1Ac) are cry genes from Bacillus thuringiensis (Berliner) that express the respective insecticidal proteins. The transgene inserted in MON89788 soybean produces CP4 EPSPS (5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium sp.), which confers tolerance to glyphosate, and MON87708 soybean produces DMO (dicamba mono-oxygenase, demethylase) derived from Stenotrophomonas maltophilia, which confers tolerance to dicamba (3,6-dichloro-2-methoxybenzoic acid). In this study, we conducted laboratory, greenhouse, and field studies to assess the efficacy of the stacked soybean MON87751 × MON87708 × MON87701 × MON89788 (and its component proteins) in Brazil against the key soybean lepidopteran pests A. gemmatalis, C. includens, and H. armigera. Material and Methods Susceptibility to Bt Proteins in Diet-Overlay Bioassays The Cry1Ac, Cry1A.105, and Cry2Ab2 purified insecticidal proteins were produced by Bayer U.S. Crop Science (Chesterfield, MO) at 91%, 80%, and 87% purity, respectively. The activity of these Bt proteins was evaluated against susceptible reference strains of A. gemmatalis, C. includens, and H. armigera. The A. gemmatalis and C. includens susceptible strains have been maintained in the laboratory for >8 yr and H. armigera for approximately 2 yr free of selection pressure by Bt proteins and insecticides. Bioassays were performed with an artificial diet (adapted from Greene et al. 1976), commonly used for rearing Lepidoptera larvae. After preparation, the diet was poured into 128-well bioassay trays (BIO-BA-128; CD International Inc., Pitman, NJ; 1 ml/well) and allowed to gel for 30 min inside a laminar air flow cabinet under ultraviolet light. Afterward, 5–7 concentrations of Cry1Ac (0.020–83.30 ng/cm2), Cry1A.105 (0.080–666.00 ng/cm2), and Cry2Ab2 (0.080–83.30 ng/cm2) were prepared by dilution in TX buffer (0.005%): Triton X-100, 10 mM Tris-HCL, pH 7.4 and 0.01 mM tetraacetic diamine ethylene acid (EDTA), pH 8.0. The control treatment was composed only of TX buffer. After the diet had solidified, Bt protein and control treatments were applied on the diet surface with a repeater pipette (100 µl/well). The diet surface area in each well was 1.5 cm2. After a drying period (~60 min), one neonate (<24 h old) was added to each well using a fine paintbrush. The trays were sealed with self-adhesive plastic sheets (BIO-CV-16; CD International Inc.) that allowed for gas exchange and then placed in a climatic chamber (temperature 27 ± 1°C, 60 ± 10% relative humidity, and 14 h:10 h light:dark photoperiod). A total of three to four replicates of 16 neonates/species/concentration of each Bt protein were tested. Mortality was assessed at 6 d. Larvae without movement were considered dead. Efficacy in Leaf Disc Bioassays Leaf discs of MON87751 × MON87701 (expressing Cry2Ab2, Cry1A.105, and Cry1Ac), MON87751 (expressing Cry2Ab2 and Cry1A.105), MON87701 (expressing Cry1Ac), A845232 (expressing Cry1A.105), A844620 (expressing Cry2Ab2), and a near-isogenic negative check were grown in a greenhouse (all varieties were near-isolines). When plants reached the V4, R1–R2, and R5 growth stages, the last completely expanded leaves were removed from the upper third of the plant. Leaf discs 1.2 cm in diameter were cut using a metallic cutter and placed on a gel mixture of agar–water at 2.5% agar (1 ml/well) in 24-well acrylic plates (Corning, Tewksbury, MA). Leaf discs were separated from the water–agar layer by filter paper. One neonate (<24 h old) of a given species was placed on each leaf disc using a fine brush. Plates were sealed with plastic film (Magipack) and placed in a climatic chamber (temperature 25 ± 1°C, 60 ± 10% RH, and 14 h:10 h light:dark photoperiod). The experimental design was completely randomized with four replicates per treatment. Each replicate consisted of 24 neonates, for a total of 120 neonates tested for each species (A. gemmatalis, C. includens, and H. armigera). Mortality was recorded at 5 d. Efficacy Trials in Greenhouse The Bt soybean genotypes and near-isogenic negative check used in the leaf disc bioassays were also tested with high infestations of C. includens and H. armigera under greenhouse conditions. For each insect species, the experiments comprised six treatments consisting of MON87751 × MON87701 (expressing Cry2Ab2, Cry1A.105, and Cry1Ac), MON87751 (expressing Cry2Ab2 and Cry1A.105), MON87701 (expressing Cry1Ac), A845232 (expressing Cry1A.105), A844620 (expressing Cry2Ab2), and a near-isogenic negative check. Experiments were seeded in 14 December 2014 at a density of 13 seeds per meter, in a randomized block design. Four blocks were planted for each experiment, each with two soybean rows of each treatment per block. Within each block, the soybean lines (2.0 m row length × 0.4 m between rows) represented the experimental replicates (plots). From the V6 growth stage until the end of the study, the plants were kept in screened nylon cages 13.0 m long × 3.5 m wide × 2.9 m tall. When the plants reached the R1–R2 growth stage, 3,000 C. includens or H. armigera pupae were subdivided into four groups of 750 pupae, packed in open acrylic boxes (11 × 11 cm) and then placed in the respective cage at four points (one point of infestation per block) on 1-m-tall wooden stands. The larval and pupal incidence and defoliation assessments were made at 30 d after adult emergence. Larval and pupal incidence were estimated by counting the number of larvae or pupae in a 1-m row of soybeans and expressed as the number per meter. The percentage of pods damaged by H. armigera was evaluated for the 13 central plants of each row by counting the total pods and damaged pods on each plant. To assess defoliation, 26 plants per plot were evaluated at random, with the percentage of defoliation estimated by comparing the leaves with a soybean defoliation scale (Hammond et al. 2014). Efficacy in Field Trial In field conditions, the efficacy of the GM soybean MON87751 × MON87708 × MON87701 × MON89788 was evaluated under natural lepidopteran infestations. The trial was carried out at a Bayer Crop Science Brazil experimental station located at Não-Me-Toque, RS during the 2018/2019 soybean season. Three treatments planted were the following: MON87751 × MON87708 × MON87701 × MON89788 (expressing Cry2Ab2, Cry1A.105, and Cry1Ac), Intacta RR2 PRO soybean (expressing Cry1Ac), and an isogenic negative check. The experimental design had randomized blocks with four replicates (plots) per treatment. The area of each plot was 32 m2 (8.0 m length × 4.0 m width) with eight soybean rows (8.0 m length × 0.5 m between rows). Larval incidence and defoliation (%) were monitored every 7 d. These evaluations started when the target pests were first seen in the experimental area and continued until the soybeans senesced. The incidence of larvae of Lepidoptera soybean pests was determined by beating the plants of the four center rows of each plot on a beating cloth (1.0 m length × 0.50 m width). The larval incidence of target species was converted into larvae per meter. For the evaluation of defoliation, 10 plants per row were randomly evaluated in the four center rows. The percentage of defoliation per plant was estimated by comparing the leaves to a defoliation scale for soybean (Hammond et al. 2014). Statistical Analysis To assess the relative toxicity of Cry1Ac, Cry1A.105, and Cry2Ab2 Bt proteins against larvae of key lepidopteran species tested, the LC50 and LC90 lethal concentrations and their 95% confidence intervals (CIs) were estimated using Probit analysis in SAS 9.1 (PROC PROBIT, SAS Institute 2000). A likelihood ratio test was used to test the hypothesis that the LC values were equal (Robertson et al. 2007). If the hypothesis was rejected, pairwise comparisons were performed, and significant differences were declared if the 95% CIs did not overlap (Savin et al. 1977). The data from leaf disc bioassays (percentage of mortality), greenhouse trials (larval incidence, percentage of defoliation, and pods damaged), and field trial (larval incidence and percentage of defoliation) were subjected to analysis of variance (REML-ANOVA) using the PROC MIXED procedure in SAS 9.1 (SAS Institute 2000). Treatment differences were determined with a Least-Square Means Statement (LSMEANS option of PROC MIXED) followed by Tukey’s honestly significant difference test (P < 0.05) in SAS 9.1 (SAS Institute 2000). Results Susceptibility to Bt Proteins in Diet-Overlay Bioassays The Cry1A.105, Cry2Ab2, and Cry1Ac proteins had high toxicity against neonates of A. gemmatalis, C. includens, and H. armigera. The estimated LC50 and LC90 values of Cry1A.105 (0.79 and 2.23 ng/cm2, respectively) and Cry1Ac (0.15 and 3.05 ng/cm2, respectively) for A. gemmatalis indicated that these Bt proteins were more active against this species than against the other species evaluated, while these proteins had similar biological activity (based on LC50 values) to C. includens and H. armigera (Table 1). When exposed to Cry2Ab2, neonates of C. includens and A. gemmatalis had similar susceptibility (based on overlapping CIs for the LC50 values) and were more susceptible to Cry2Ab2 than H. armigera. Table 1. Concentration-mortality response (LC; ng/cm2) of A. gemmatalis, C. includes, and H. armigera neonates exposed to Bt proteins overlaid on artificial diet Species . n . Slope ± SE . LC50 (95% CI)a,b . LC90 (95% CI)a,b . χ 2c . dfd . Cry1A.105 Anticarsia gemmatalis 192 2.85 ± 0.42 0.79 (0.60–1.02) a 2.23 (1.64–3.58) a 5.07 4 Chrysodeixis includens 336 0.88 ± 0.11 28.68 (15.63–54.61) b 590.26 (338.21–817.64) c 4.45 3 Helicoverpa armigera 208 4.05 ± 1.60 48.22 (19.23–68.14) b 99.90 (71.06–140.73) b 5.62 4 Cry2Ab2 Anticarsia gemmatalis 186 1.51 ± 0.47 1.79 (0.80–4.89) a 12.57 (3.73–18.89) a 9.43 4 Chrysodeixis includes 176 2.14 ± 0.68 1.24 (0.19–2.16) a 4.95 (3.10–12.27) a 2.17 3 Helicoverpa armigera 208 1.84 ± 0.33 8.36 (4.90–13.38) b 41.67 (24.32–94.23) b 0.14 3 Cry1Ac Anticarsia gemmatalis 175 0.99 ± 0.20 0.15 (0.06–0.28) a 3.05 (1.29–18.38) a 2.86 4 Chrysodeixis includens 240 1.94 ± 0.39 4.98 (2.63–7.78) b 22.86 (14.04–53.91) b 1.26 4 Helicoverpa armigera 271 0.96 ± 0.14 5.07 (2.87–8.33) b 91.02 (52.97–174.16) b 4.74 3 Species . n . Slope ± SE . LC50 (95% CI)a,b . LC90 (95% CI)a,b . χ 2c . dfd . Cry1A.105 Anticarsia gemmatalis 192 2.85 ± 0.42 0.79 (0.60–1.02) a 2.23 (1.64–3.58) a 5.07 4 Chrysodeixis includens 336 0.88 ± 0.11 28.68 (15.63–54.61) b 590.26 (338.21–817.64) c 4.45 3 Helicoverpa armigera 208 4.05 ± 1.60 48.22 (19.23–68.14) b 99.90 (71.06–140.73) b 5.62 4 Cry2Ab2 Anticarsia gemmatalis 186 1.51 ± 0.47 1.79 (0.80–4.89) a 12.57 (3.73–18.89) a 9.43 4 Chrysodeixis includes 176 2.14 ± 0.68 1.24 (0.19–2.16) a 4.95 (3.10–12.27) a 2.17 3 Helicoverpa armigera 208 1.84 ± 0.33 8.36 (4.90–13.38) b 41.67 (24.32–94.23) b 0.14 3 Cry1Ac Anticarsia gemmatalis 175 0.99 ± 0.20 0.15 (0.06–0.28) a 3.05 (1.29–18.38) a 2.86 4 Chrysodeixis includens 240 1.94 ± 0.39 4.98 (2.63–7.78) b 22.86 (14.04–53.91) b 1.26 4 Helicoverpa armigera 271 0.96 ± 0.14 5.07 (2.87–8.33) b 91.02 (52.97–174.16) b 4.74 3 aLC50: concentration of Bt protein (ng/cm2) required to kill 50% of larvae in the observation period of 6 d. Similarly, LC90 is the concentration of Bt protein required to kill 90% of larvae tested. bLC50 and LC90 values designated by different letters in each Bt protein are significantly different due to non-overlap of 95% CIs. cP > 0.05 in goodness of fit. dDegrees of freedom. Open in new tab Table 1. Concentration-mortality response (LC; ng/cm2) of A. gemmatalis, C. includes, and H. armigera neonates exposed to Bt proteins overlaid on artificial diet Species . n . Slope ± SE . LC50 (95% CI)a,b . LC90 (95% CI)a,b . χ 2c . dfd . Cry1A.105 Anticarsia gemmatalis 192 2.85 ± 0.42 0.79 (0.60–1.02) a 2.23 (1.64–3.58) a 5.07 4 Chrysodeixis includens 336 0.88 ± 0.11 28.68 (15.63–54.61) b 590.26 (338.21–817.64) c 4.45 3 Helicoverpa armigera 208 4.05 ± 1.60 48.22 (19.23–68.14) b 99.90 (71.06–140.73) b 5.62 4 Cry2Ab2 Anticarsia gemmatalis 186 1.51 ± 0.47 1.79 (0.80–4.89) a 12.57 (3.73–18.89) a 9.43 4 Chrysodeixis includes 176 2.14 ± 0.68 1.24 (0.19–2.16) a 4.95 (3.10–12.27) a 2.17 3 Helicoverpa armigera 208 1.84 ± 0.33 8.36 (4.90–13.38) b 41.67 (24.32–94.23) b 0.14 3 Cry1Ac Anticarsia gemmatalis 175 0.99 ± 0.20 0.15 (0.06–0.28) a 3.05 (1.29–18.38) a 2.86 4 Chrysodeixis includens 240 1.94 ± 0.39 4.98 (2.63–7.78) b 22.86 (14.04–53.91) b 1.26 4 Helicoverpa armigera 271 0.96 ± 0.14 5.07 (2.87–8.33) b 91.02 (52.97–174.16) b 4.74 3 Species . n . Slope ± SE . LC50 (95% CI)a,b . LC90 (95% CI)a,b . χ 2c . dfd . Cry1A.105 Anticarsia gemmatalis 192 2.85 ± 0.42 0.79 (0.60–1.02) a 2.23 (1.64–3.58) a 5.07 4 Chrysodeixis includens 336 0.88 ± 0.11 28.68 (15.63–54.61) b 590.26 (338.21–817.64) c 4.45 3 Helicoverpa armigera 208 4.05 ± 1.60 48.22 (19.23–68.14) b 99.90 (71.06–140.73) b 5.62 4 Cry2Ab2 Anticarsia gemmatalis 186 1.51 ± 0.47 1.79 (0.80–4.89) a 12.57 (3.73–18.89) a 9.43 4 Chrysodeixis includes 176 2.14 ± 0.68 1.24 (0.19–2.16) a 4.95 (3.10–12.27) a 2.17 3 Helicoverpa armigera 208 1.84 ± 0.33 8.36 (4.90–13.38) b 41.67 (24.32–94.23) b 0.14 3 Cry1Ac Anticarsia gemmatalis 175 0.99 ± 0.20 0.15 (0.06–0.28) a 3.05 (1.29–18.38) a 2.86 4 Chrysodeixis includens 240 1.94 ± 0.39 4.98 (2.63–7.78) b 22.86 (14.04–53.91) b 1.26 4 Helicoverpa armigera 271 0.96 ± 0.14 5.07 (2.87–8.33) b 91.02 (52.97–174.16) b 4.74 3 aLC50: concentration of Bt protein (ng/cm2) required to kill 50% of larvae in the observation period of 6 d. Similarly, LC90 is the concentration of Bt protein required to kill 90% of larvae tested. bLC50 and LC90 values designated by different letters in each Bt protein are significantly different due to non-overlap of 95% CIs. cP > 0.05 in goodness of fit. dDegrees of freedom. Open in new tab Efficacy in Leaf Disc Bioassays Neonates of A. gemmatalis, C. includens, and H. armigera presented high mortality when exposed to the Bt proteins Cry1A.105, Cry2Ab2, and Cry1Ac expressed individually or combined in GM soybean genotypes (Table 2). At 5 d, except for H. armigera on GMA844620 expressing only Cry2Ab2 (97.9% mortality at R1–R2 growth stage), there was complete mortality of all species on all the Bt-expressing materials, while mortality ranged from 3.1% to 18.8% on the negative check. Table 2. Neonate mortality (%) of A. gemmatalis, C. includes, and H. armigera on leaf discs of Bt soybean genotypes and an isogenic negative check in laboratory bioassays . . Soybean growth stagea . Soybean genotype . Bt protein expressed . V4 . R1–R2 . R5 . Anticarsia gemmatalis MON87751 × MON87701 Cry1A.105, Cry2Ab2, Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87751 Cry1A.105, Cry2Ab2 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87701 Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A845232 Cry1A.105 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A844620 Cry2Ab2 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b Isogenic negative check None 3.1 ± 1.2 a 13.5 ± 6.7 a 4.2 ± 0.0 a F value 961.0 168.5 1840.8 df 5, 18 5, 18 5, 18 (P > F) ANOVA <0.0001 <0.0001 <0.0001 Chrysodeixis includens MON87751 × MON87701 Cry1A.105, Cry2Ab2, Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87751 Cry1A.105, Cry2Ab2 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87701 Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A845232 Cry1A.105 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A844620 Cry2Ab2 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b Isogenic negative check None 10.4 ± 3.6 a 10.4 ± 4.3 a 9.4 ± 3.1 a F value 619.8 73.8 842.1 df 5, 18 5, 18 5, 18 (P > F) ANOVA <0.0001 <0.0001 <0.0001 Helicoverpa armigera MON87751 × MON87701 Cry1A.105, Cry2Ab2, Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87751 Cry1A.105, Cry2Ab2 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87701 Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A845232 Cry1A.105 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A844620 Cry2Ab2 100.0 ± 0.0 b 97.9 ± 1.2 b 100.0 ± 0.0 b Isogenic negative check None 6.3 ± 1.2 a 18.8 ± 2.1 a 14.6 ± 5.6 a F value 6274.2 1131.3 555.7 df 5, 18 5, 18 5, 18 (P > F) ANOVA <0.0001 <0.0001 <0.0001 . . Soybean growth stagea . Soybean genotype . Bt protein expressed . V4 . R1–R2 . R5 . Anticarsia gemmatalis MON87751 × MON87701 Cry1A.105, Cry2Ab2, Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87751 Cry1A.105, Cry2Ab2 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87701 Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A845232 Cry1A.105 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A844620 Cry2Ab2 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b Isogenic negative check None 3.1 ± 1.2 a 13.5 ± 6.7 a 4.2 ± 0.0 a F value 961.0 168.5 1840.8 df 5, 18 5, 18 5, 18 (P > F) ANOVA <0.0001 <0.0001 <0.0001 Chrysodeixis includens MON87751 × MON87701 Cry1A.105, Cry2Ab2, Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87751 Cry1A.105, Cry2Ab2 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87701 Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A845232 Cry1A.105 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A844620 Cry2Ab2 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b Isogenic negative check None 10.4 ± 3.6 a 10.4 ± 4.3 a 9.4 ± 3.1 a F value 619.8 73.8 842.1 df 5, 18 5, 18 5, 18 (P > F) ANOVA <0.0001 <0.0001 <0.0001 Helicoverpa armigera MON87751 × MON87701 Cry1A.105, Cry2Ab2, Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87751 Cry1A.105, Cry2Ab2 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87701 Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A845232 Cry1A.105 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A844620 Cry2Ab2 100.0 ± 0.0 b 97.9 ± 1.2 b 100.0 ± 0.0 b Isogenic negative check None 6.3 ± 1.2 a 18.8 ± 2.1 a 14.6 ± 5.6 a F value 6274.2 1131.3 555.7 df 5, 18 5, 18 5, 18 (P > F) ANOVA <0.0001 <0.0001 <0.0001 aValues represent means ± SE after correction based on isogenic negative check. There were statistically significant differences (Tukey’s honestly significant difference test at P < 0.05) between Bt soybean plants and the isogenic negative check at all soybean growth stages and for all lepidopteran species evaluated. Open in new tab Table 2. Neonate mortality (%) of A. gemmatalis, C. includes, and H. armigera on leaf discs of Bt soybean genotypes and an isogenic negative check in laboratory bioassays . . Soybean growth stagea . Soybean genotype . Bt protein expressed . V4 . R1–R2 . R5 . Anticarsia gemmatalis MON87751 × MON87701 Cry1A.105, Cry2Ab2, Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87751 Cry1A.105, Cry2Ab2 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87701 Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A845232 Cry1A.105 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A844620 Cry2Ab2 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b Isogenic negative check None 3.1 ± 1.2 a 13.5 ± 6.7 a 4.2 ± 0.0 a F value 961.0 168.5 1840.8 df 5, 18 5, 18 5, 18 (P > F) ANOVA <0.0001 <0.0001 <0.0001 Chrysodeixis includens MON87751 × MON87701 Cry1A.105, Cry2Ab2, Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87751 Cry1A.105, Cry2Ab2 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87701 Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A845232 Cry1A.105 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A844620 Cry2Ab2 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b Isogenic negative check None 10.4 ± 3.6 a 10.4 ± 4.3 a 9.4 ± 3.1 a F value 619.8 73.8 842.1 df 5, 18 5, 18 5, 18 (P > F) ANOVA <0.0001 <0.0001 <0.0001 Helicoverpa armigera MON87751 × MON87701 Cry1A.105, Cry2Ab2, Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87751 Cry1A.105, Cry2Ab2 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87701 Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A845232 Cry1A.105 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A844620 Cry2Ab2 100.0 ± 0.0 b 97.9 ± 1.2 b 100.0 ± 0.0 b Isogenic negative check None 6.3 ± 1.2 a 18.8 ± 2.1 a 14.6 ± 5.6 a F value 6274.2 1131.3 555.7 df 5, 18 5, 18 5, 18 (P > F) ANOVA <0.0001 <0.0001 <0.0001 . . Soybean growth stagea . Soybean genotype . Bt protein expressed . V4 . R1–R2 . R5 . Anticarsia gemmatalis MON87751 × MON87701 Cry1A.105, Cry2Ab2, Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87751 Cry1A.105, Cry2Ab2 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87701 Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A845232 Cry1A.105 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A844620 Cry2Ab2 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b Isogenic negative check None 3.1 ± 1.2 a 13.5 ± 6.7 a 4.2 ± 0.0 a F value 961.0 168.5 1840.8 df 5, 18 5, 18 5, 18 (P > F) ANOVA <0.0001 <0.0001 <0.0001 Chrysodeixis includens MON87751 × MON87701 Cry1A.105, Cry2Ab2, Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87751 Cry1A.105, Cry2Ab2 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87701 Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A845232 Cry1A.105 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A844620 Cry2Ab2 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b Isogenic negative check None 10.4 ± 3.6 a 10.4 ± 4.3 a 9.4 ± 3.1 a F value 619.8 73.8 842.1 df 5, 18 5, 18 5, 18 (P > F) ANOVA <0.0001 <0.0001 <0.0001 Helicoverpa armigera MON87751 × MON87701 Cry1A.105, Cry2Ab2, Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87751 Cry1A.105, Cry2Ab2 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b MON87701 Cry1Ac 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A845232 Cry1A.105 100.0 ± 0.0 b 100.0 ± 0.0 b 100.0 ± 0.0 b A844620 Cry2Ab2 100.0 ± 0.0 b 97.9 ± 1.2 b 100.0 ± 0.0 b Isogenic negative check None 6.3 ± 1.2 a 18.8 ± 2.1 a 14.6 ± 5.6 a F value 6274.2 1131.3 555.7 df 5, 18 5, 18 5, 18 (P > F) ANOVA <0.0001 <0.0001 <0.0001 aValues represent means ± SE after correction based on isogenic negative check. There were statistically significant differences (Tukey’s honestly significant difference test at P < 0.05) between Bt soybean plants and the isogenic negative check at all soybean growth stages and for all lepidopteran species evaluated. Open in new tab Efficacy Trials in Greenhouse Larval and pupal incidence of C. includens was insignificant on both single-trait and pyramided soybean genotypes, resulting in limited defoliation (Table 3). In contrast, high larval and pupae incidence were found on the isogenic negative check (17.9 and 27.9 larvae and pupae per meter, respectively), causing 50% defoliation, which was the maximum value on the scale used to assess this endpoint. Larval incidence of H. armigera was 0.2 or fewer larvae per meter on Bt soybeans, while the isogenic negative check had more than 33 larvae per meter (Table 4). The low larval incidence on the Bt soybean genotypes caused minimal defoliation (near zero) and pod damage (2% or lower). These small damages may be attributed to larval movement from non-Bt soybean onto Bt soybean plants. In contrast, the high larval incidence on non-Bt soybean resulted in 50% defoliation and almost 100% of pods damaged by H. armigera. Table 3. Larval and pupal incidence (larvae and pupae per meter of row) and damage (% defoliation) on Bt soybean plants compared with isogenic negative checks after infestation with C. includens in a greenhouse trial during the 2014/2015 season (Santa Cruz das Palmeiras, São Paulo, Brazil) Soybean genotype . Bt protein expressed . Larval incidencea . Pupal incidencea . Defoliation (%)a . MON87751 × MON87701 Cry1A.105, Cry2Ab2, Cry1Ac 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a MON87751 Cry1A.105, Cry2Ab2 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a MON87701 Cry1Ac 0.0 ± 0.0 a 0.0 ± 0.0 a 1.0 ± 1.0 a A845232 Cry1A.105 0.0 ± 0.0 a 0.1 ± 0.1 a 0.0 ± 0.0 a A844620 Cry2Ab2 0.0 ± 0.0 a 0.0 ± 0.0 a 0.1 ± 0.1 a Isogenic negative check None 17.9 ± 5.8 b 27.9 ± 2.2 b 50.0 ± 0.0 b F value 9.3 131.7 269985.0 df 5, 15 5, 15 5, 15 (P > F) ANOVA 0.0004 <0.0001 <0.0001 Soybean genotype . Bt protein expressed . Larval incidencea . Pupal incidencea . Defoliation (%)a . MON87751 × MON87701 Cry1A.105, Cry2Ab2, Cry1Ac 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a MON87751 Cry1A.105, Cry2Ab2 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a MON87701 Cry1Ac 0.0 ± 0.0 a 0.0 ± 0.0 a 1.0 ± 1.0 a A845232 Cry1A.105 0.0 ± 0.0 a 0.1 ± 0.1 a 0.0 ± 0.0 a A844620 Cry2Ab2 0.0 ± 0.0 a 0.0 ± 0.0 a 0.1 ± 0.1 a Isogenic negative check None 17.9 ± 5.8 b 27.9 ± 2.2 b 50.0 ± 0.0 b F value 9.3 131.7 269985.0 df 5, 15 5, 15 5, 15 (P > F) ANOVA 0.0004 <0.0001 <0.0001 aValues represent means ± SE. There were statistically significant differences (Tukey’s honestly significant difference test at P < 0.05) between Bt soybean plants and the isogenic negative check for all the performance measurements. Open in new tab Table 3. Larval and pupal incidence (larvae and pupae per meter of row) and damage (% defoliation) on Bt soybean plants compared with isogenic negative checks after infestation with C. includens in a greenhouse trial during the 2014/2015 season (Santa Cruz das Palmeiras, São Paulo, Brazil) Soybean genotype . Bt protein expressed . Larval incidencea . Pupal incidencea . Defoliation (%)a . MON87751 × MON87701 Cry1A.105, Cry2Ab2, Cry1Ac 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a MON87751 Cry1A.105, Cry2Ab2 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a MON87701 Cry1Ac 0.0 ± 0.0 a 0.0 ± 0.0 a 1.0 ± 1.0 a A845232 Cry1A.105 0.0 ± 0.0 a 0.1 ± 0.1 a 0.0 ± 0.0 a A844620 Cry2Ab2 0.0 ± 0.0 a 0.0 ± 0.0 a 0.1 ± 0.1 a Isogenic negative check None 17.9 ± 5.8 b 27.9 ± 2.2 b 50.0 ± 0.0 b F value 9.3 131.7 269985.0 df 5, 15 5, 15 5, 15 (P > F) ANOVA 0.0004 <0.0001 <0.0001 Soybean genotype . Bt protein expressed . Larval incidencea . Pupal incidencea . Defoliation (%)a . MON87751 × MON87701 Cry1A.105, Cry2Ab2, Cry1Ac 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a MON87751 Cry1A.105, Cry2Ab2 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a MON87701 Cry1Ac 0.0 ± 0.0 a 0.0 ± 0.0 a 1.0 ± 1.0 a A845232 Cry1A.105 0.0 ± 0.0 a 0.1 ± 0.1 a 0.0 ± 0.0 a A844620 Cry2Ab2 0.0 ± 0.0 a 0.0 ± 0.0 a 0.1 ± 0.1 a Isogenic negative check None 17.9 ± 5.8 b 27.9 ± 2.2 b 50.0 ± 0.0 b F value 9.3 131.7 269985.0 df 5, 15 5, 15 5, 15 (P > F) ANOVA 0.0004 <0.0001 <0.0001 aValues represent means ± SE. There were statistically significant differences (Tukey’s honestly significant difference test at P < 0.05) between Bt soybean plants and the isogenic negative check for all the performance measurements. Open in new tab Table 4. Larval incidence (larvae per meter of row) and damage (% defoliation and pods damaged) on Bt soybean plants compared with isogenic negative checks after infestation with H. armigera in a greenhouse trial during the 2014/2015 season (Santa Cruz das Palmeiras, São Paulo, Brazil) Soybean genotype . Bt protein expressed . Larval incidencea . Defoliation (%)a . Pods damaged (%)a . MON87751 × MON87701 Cry1A.105, Cry2Ab2, Cry1Ac 0.0 ± 0.0 a 0.5 ± 0.5 a 2.0 ± 1.6 a MON87751 Cry1A.105, Cry2Ab2 0.0 ± 0.0 a 0.1 ± 0.1 a 1.1 ± 1.1 a MON87701 Cry1Ac 0.0 ± 0.0 a 0.0 ± 0.0 a 1.6 ± 0.9 a A845232 Cry1A.105 0.0 ± 0.0 a 0.2 ± 0.1 a 1.1 ± 1.1 a A844620 Cry2Ab2 0.2 ± 0.2 a 0.6 ± 0.6 a 1.6 ± 1.0 a Isogenic negative checks None 33.6 ± 8.9 b 50.0 ± 0.0 b 99.9 ± 0.1 b F value 14.4 43148.5 1634.9 df 5, 15 5, 15 5, 16 (P > F) ANOVA <0.0001 <0.0001 <0.0001 Soybean genotype . Bt protein expressed . Larval incidencea . Defoliation (%)a . Pods damaged (%)a . MON87751 × MON87701 Cry1A.105, Cry2Ab2, Cry1Ac 0.0 ± 0.0 a 0.5 ± 0.5 a 2.0 ± 1.6 a MON87751 Cry1A.105, Cry2Ab2 0.0 ± 0.0 a 0.1 ± 0.1 a 1.1 ± 1.1 a MON87701 Cry1Ac 0.0 ± 0.0 a 0.0 ± 0.0 a 1.6 ± 0.9 a A845232 Cry1A.105 0.0 ± 0.0 a 0.2 ± 0.1 a 1.1 ± 1.1 a A844620 Cry2Ab2 0.2 ± 0.2 a 0.6 ± 0.6 a 1.6 ± 1.0 a Isogenic negative checks None 33.6 ± 8.9 b 50.0 ± 0.0 b 99.9 ± 0.1 b F value 14.4 43148.5 1634.9 df 5, 15 5, 15 5, 16 (P > F) ANOVA <0.0001 <0.0001 <0.0001 aValues represent means ± SE. There were statistically significant differences (Tukey’s honestly significant difference test at P < 0.05) between Bt soybean plants and the isogenic negative check for all the performance measurements. Open in new tab Table 4. Larval incidence (larvae per meter of row) and damage (% defoliation and pods damaged) on Bt soybean plants compared with isogenic negative checks after infestation with H. armigera in a greenhouse trial during the 2014/2015 season (Santa Cruz das Palmeiras, São Paulo, Brazil) Soybean genotype . Bt protein expressed . Larval incidencea . Defoliation (%)a . Pods damaged (%)a . MON87751 × MON87701 Cry1A.105, Cry2Ab2, Cry1Ac 0.0 ± 0.0 a 0.5 ± 0.5 a 2.0 ± 1.6 a MON87751 Cry1A.105, Cry2Ab2 0.0 ± 0.0 a 0.1 ± 0.1 a 1.1 ± 1.1 a MON87701 Cry1Ac 0.0 ± 0.0 a 0.0 ± 0.0 a 1.6 ± 0.9 a A845232 Cry1A.105 0.0 ± 0.0 a 0.2 ± 0.1 a 1.1 ± 1.1 a A844620 Cry2Ab2 0.2 ± 0.2 a 0.6 ± 0.6 a 1.6 ± 1.0 a Isogenic negative checks None 33.6 ± 8.9 b 50.0 ± 0.0 b 99.9 ± 0.1 b F value 14.4 43148.5 1634.9 df 5, 15 5, 15 5, 16 (P > F) ANOVA <0.0001 <0.0001 <0.0001 Soybean genotype . Bt protein expressed . Larval incidencea . Defoliation (%)a . Pods damaged (%)a . MON87751 × MON87701 Cry1A.105, Cry2Ab2, Cry1Ac 0.0 ± 0.0 a 0.5 ± 0.5 a 2.0 ± 1.6 a MON87751 Cry1A.105, Cry2Ab2 0.0 ± 0.0 a 0.1 ± 0.1 a 1.1 ± 1.1 a MON87701 Cry1Ac 0.0 ± 0.0 a 0.0 ± 0.0 a 1.6 ± 0.9 a A845232 Cry1A.105 0.0 ± 0.0 a 0.2 ± 0.1 a 1.1 ± 1.1 a A844620 Cry2Ab2 0.2 ± 0.2 a 0.6 ± 0.6 a 1.6 ± 1.0 a Isogenic negative checks None 33.6 ± 8.9 b 50.0 ± 0.0 b 99.9 ± 0.1 b F value 14.4 43148.5 1634.9 df 5, 15 5, 15 5, 16 (P > F) ANOVA <0.0001 <0.0001 <0.0001 aValues represent means ± SE. There were statistically significant differences (Tukey’s honestly significant difference test at P < 0.05) between Bt soybean plants and the isogenic negative check for all the performance measurements. Open in new tab Efficacy in Field Trial The only larvae of Lepidoptera soybean pests were determined to be C. includens in the non-Bt control plots (Table 5). Significant differences in larval incidence of C. includens were detected from the R1 to R6 growth stages between MON87751 × MON87708 × MON87701 × MON89788 and MON87701 × MON89788 (Intacta RR2 PRO soybeans) compared to the isogenic negative check (zero incidence compared to 0.5–0.6 larvae per meter). Defoliation also was significantly different from the R1 to R6 growth stages for MON87751 × MON87708 × MON87701 × MON89788 and MON87701 × MON89788 compared to the isogenic negative check, reaching 0.2%, 0.9%, and 4.8% of defoliation, respectively. The defoliation observed in Bt soybeans may be attributed to nontarget species such as some Spodoptera species and defoliating beetles. Table 5. Larval incidence of C. includens and defoliation on Bt soybean genotypes compared with isogenic negative check after natural infestation in field trial during the 2018/2019 soybean season (Não-Me-Toque, Rio Grande do Sul, Brazil) Soybean genotype . Bt protein expressed . Soybean growth stagea . . . R1–R2 R3–R4 R5–R6 Larval incidence (larvae per meter of row)b MON87751 × MON87708 × MON87701 × MON89788 Cry1A.105, Cry2Ab2, Cry1Ac 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a MON87701 × MON89788 Cry1Ac 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a Isogenic negative check None 0.5 ± 0.1 b 0.6 ± 0.1 b 0.6 ± 0.1 b F value 307.5 37.4 11.5 df 2, 6 2, 6 2, 6 (P > F) ANOVA <0.0001 0.0004 0.0089 Defoliation (%) MON87751 × MON87708 × MON87701 × MON89788 Cry1A.105, Cry2Ab2, Cry1Ac 0.0 ± 0.0 a 0.2 ± 0.1 a 0.2 ± 0.1 a MON87701 × MON89788 Cry1Ac 0.4 ± 0.1 b 0.9 ± 0.2 b 0.5 ± 0.1 b Isogenic negative check None 1.5 ± 0.1 c 4.4 ± 0.1 c 4.8 ± 0.2 c F value 2061.0 486.6 750.1 df 2, 6 2, 6 2, 6 (P > F) ANOVA <0.0001 <0.0001 <0.0001 Soybean genotype . Bt protein expressed . Soybean growth stagea . . . R1–R2 R3–R4 R5–R6 Larval incidence (larvae per meter of row)b MON87751 × MON87708 × MON87701 × MON89788 Cry1A.105, Cry2Ab2, Cry1Ac 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a MON87701 × MON89788 Cry1Ac 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a Isogenic negative check None 0.5 ± 0.1 b 0.6 ± 0.1 b 0.6 ± 0.1 b F value 307.5 37.4 11.5 df 2, 6 2, 6 2, 6 (P > F) ANOVA <0.0001 0.0004 0.0089 Defoliation (%) MON87751 × MON87708 × MON87701 × MON89788 Cry1A.105, Cry2Ab2, Cry1Ac 0.0 ± 0.0 a 0.2 ± 0.1 a 0.2 ± 0.1 a MON87701 × MON89788 Cry1Ac 0.4 ± 0.1 b 0.9 ± 0.2 b 0.5 ± 0.1 b Isogenic negative check None 1.5 ± 0.1 c 4.4 ± 0.1 c 4.8 ± 0.2 c F value 2061.0 486.6 750.1 df 2, 6 2, 6 2, 6 (P > F) ANOVA <0.0001 <0.0001 <0.0001 aValues represent means ± SE. There were statistically significant differences (Tukey’s honestly significant difference test at P < 0.05) between Bt soybean plants and the isogenic negative check for all the performance measurements. bThe only key lepidopteran soybean pest was determined to be C. includens in the non-Bt control plots. Open in new tab Table 5. Larval incidence of C. includens and defoliation on Bt soybean genotypes compared with isogenic negative check after natural infestation in field trial during the 2018/2019 soybean season (Não-Me-Toque, Rio Grande do Sul, Brazil) Soybean genotype . Bt protein expressed . Soybean growth stagea . . . R1–R2 R3–R4 R5–R6 Larval incidence (larvae per meter of row)b MON87751 × MON87708 × MON87701 × MON89788 Cry1A.105, Cry2Ab2, Cry1Ac 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a MON87701 × MON89788 Cry1Ac 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a Isogenic negative check None 0.5 ± 0.1 b 0.6 ± 0.1 b 0.6 ± 0.1 b F value 307.5 37.4 11.5 df 2, 6 2, 6 2, 6 (P > F) ANOVA <0.0001 0.0004 0.0089 Defoliation (%) MON87751 × MON87708 × MON87701 × MON89788 Cry1A.105, Cry2Ab2, Cry1Ac 0.0 ± 0.0 a 0.2 ± 0.1 a 0.2 ± 0.1 a MON87701 × MON89788 Cry1Ac 0.4 ± 0.1 b 0.9 ± 0.2 b 0.5 ± 0.1 b Isogenic negative check None 1.5 ± 0.1 c 4.4 ± 0.1 c 4.8 ± 0.2 c F value 2061.0 486.6 750.1 df 2, 6 2, 6 2, 6 (P > F) ANOVA <0.0001 <0.0001 <0.0001 Soybean genotype . Bt protein expressed . Soybean growth stagea . . . R1–R2 R3–R4 R5–R6 Larval incidence (larvae per meter of row)b MON87751 × MON87708 × MON87701 × MON89788 Cry1A.105, Cry2Ab2, Cry1Ac 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a MON87701 × MON89788 Cry1Ac 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a Isogenic negative check None 0.5 ± 0.1 b 0.6 ± 0.1 b 0.6 ± 0.1 b F value 307.5 37.4 11.5 df 2, 6 2, 6 2, 6 (P > F) ANOVA <0.0001 0.0004 0.0089 Defoliation (%) MON87751 × MON87708 × MON87701 × MON89788 Cry1A.105, Cry2Ab2, Cry1Ac 0.0 ± 0.0 a 0.2 ± 0.1 a 0.2 ± 0.1 a MON87701 × MON89788 Cry1Ac 0.4 ± 0.1 b 0.9 ± 0.2 b 0.5 ± 0.1 b Isogenic negative check None 1.5 ± 0.1 c 4.4 ± 0.1 c 4.8 ± 0.2 c F value 2061.0 486.6 750.1 df 2, 6 2, 6 2, 6 (P > F) ANOVA <0.0001 <0.0001 <0.0001 aValues represent means ± SE. There were statistically significant differences (Tukey’s honestly significant difference test at P < 0.05) between Bt soybean plants and the isogenic negative check for all the performance measurements. bThe only key lepidopteran soybean pest was determined to be C. includens in the non-Bt control plots. Open in new tab Discussion A pyramiding strategy to manage insect resistance to Bt crops, which combines two or more insecticidal proteins active against the same insect, represents a robust strategy to manage lepidopteran pests by conferring higher protection and delaying resistance relative to single-mechanism-of-action technologies (Roush 1998, Head and Greenplate 2012, Carrière et al. 2016). Pyramided Bt crops rely on the assumption that each insecticidal protein acts individually in a way that would kill all insects susceptible to that insecticidal protein, including those insects that are resistant to the other protein(s) expressed in the plant (Roush 1997, 1998). This concept was named ‘redundant killing’ (Comins 1986, Gould 1986, Roush 1998). Therefore, a central question about the ability of pyramids to delay the onset of insect resistance is the extent to which individuals resistant to one Bt protein are killed by the other(s) in the Bt plant (Roush 1997). Another important requirement of the pyramiding strategy to manage insect resistance is to achieve high levels of mortality of susceptible insects with the individual proteins in the pyramid when expressed in plants (Roush 1998, Head and Greenplate 2012, Brévault et al. 2013, Carrière et al. 2016). Thus, to evaluate the killing power of pyramiding Cry1A.105, Cry2Ab2, and Cry1Ac proteins in a soybean plant, we measured biological activity and efficacy against key target insect pests using purified proteins and plant tissue assays. The LC50 values of Cry1Ac revealed that this Bt protein had higher toxicity against A. gemmatalis than C. includens and H. armigera. The same trend was observed for Cry1A.105; based on LC50 values, A. gemmatalis was ~60-fold more susceptible to Cry1A.105 than C. includens and ~35 fold more susceptible to Cry1A.105 than H. armigera. High susceptibility to Cry1 proteins has been previously reported for A. gemmatalis, which has been shown in previous studies to be more susceptible than C. includens (Bobrowski et al. 2002, Bernardi et al. 2012) and H. armigera (Dourado et al. 2016). Neonates of H. armigera were less susceptible to Cry2Ab2, with higher LC50 and LC90 values, than A. gemmatalis and C. includens. Overall, our results indicate that the primary soybean pests in Brazil—A. gemmatalis, C. includens, and H. armigera—all exhibited relative high susceptibility to Cry1Ac, Cry1A.105, and Cry2Ab2. Mathematical modeling indicated that the concentration of each insecticidal protein in a Bt pyramid must be sufficient to kill a high percentage (~95%) of susceptible individuals to substantially delay resistance (Roush 1998). The high mortality levels obtained for A. gemmatalis, C. includens, and H. armigera larvae from susceptible colonies feeding on soybeans plants expressing only Cry1A.105, Cry2Ab2, or Cry1Ac indicate that each of these proteins are capable of providing high levels of susceptible target pest control in plants. However, these efficacy results do not capture other important elements for assessing resistance risk, for example, the susceptibility of field populations of target pests and potential for cross-resistance. Due to the current absence of resistant colonies for testing, our assessment strategy used susceptible insects. Resistance, and cross-resistance between Bt proteins, could reduce the redundant killing power of a pyramid because individuals resistant to one insecticidal protein could also survive exposure to other(s) in the pyramid (Carrière et al. 2016). The potential for cross-resistance among Cry1 proteins has been documented in several lepidopteran species (Hernández-Rodríguez et al. 2013, Bernardi et al. 2015, Zhang et al. 2014, Grimi et al. 2018, Wang et al. 2019). Therefore, it will be important to establish resistant strains of the key lepidopteran soybean pests to understand the potential for cross-resistance among the individual components of MON87751 × MON87708 × MON87701 × MON89788 soybean. Moreover, the susceptibility of field populations of target pests to Cry1A.105 and Cry2Ab2 needs to be characterized; susceptibility to Cry1Ac has already been reported for A. gemmatalis, C. includens, and H. armigera in Brazil (Yano 2012, Yano et al. 2016, Dourado et al. 2016). Despite its potential IPM and IRM benefits, the effectiveness and durability of MON87751 × MON87708 × MON87701 × MON89788 soybean may be compromised if resistance to one of the Bt proteins is established at the field level (Onstad and Meinke 2010, Head and Greenplate 2012). In fact, relevant field exposure to Cry1Ac, an important component of MON87751 × MON87708 × MON87701 × MON89788, is ongoing because of the widespread plantings of MON87701 × MON89788 (Intacta RR2 PRO, a single-mode-of-action Bt plant expressing only Cry1Ac) in Brazil, Argentina, Paraguay, and Uruguay (Brookes and Barfoot 2018, 2020). Despite its high level of adoption in recent years, MON87701 × MON89788 continues to provide effective control of the major lepidopteran soybean pests, particularly C. includens and A. gemmatalis. Based on sampling of lepidopteran pests in soybean fields in Brazil, we have not been able to detect H. armigera in significant densities in recent years. The Cry1Ac resistance allele frequency baseline established during the precommercial period of Intacta RR2 PRO, crop season 2014/2015, indicated low initial resistance allele (R) frequencies in C. includens populations for Cry1Ac in Brazil (MON87701 × MON89788) (estimated R frequency = 0.0004; Yano et al. 2016). Using the same methodology as Yano et al. (2016), the Cry1Ac resistance allele frequency in C. includens field populations has been systematically monitored across crop seasons and regions in Brazil. The increasing adoption of MON87701 × MON89788 soybean in Brazilian fields has not resulted in a shift in resistance allele frequency, which was estimated during the crop season 2019/2020 at similarly low levels to those established during the precommercial phase (Bayer Crop Science, Patrick M. Dourado (P.M.D.)). The maintenance of low Cry1Ac resistance allele frequencies in C. includens in Brazil after 5 yr of commercial use of MON87701 × MON89788 soybean can be attributed to the fit of the technology to the high-dose concept (MacRae et al. 2005; Bernardi et al. 2012, 2014; Yano et al. 2016) and the maintenance of reasonable levels of refuge compliance at the field level. Brazilian growers planting MON87701 × MON89788 soybean are recommended to plant at least 20% of their soybean area with non-Bt soybean varieties (structured refuge) within 800 m of their Bt soybean (Brazilian National Biosafety Technical Commission [CTNBio] 2010). Nevertheless, the development of new, effective pyramided Bt soybean technologies, such as MON87751 × MON87708 × MON87701 × MON89788, may play an important role in delaying resistance and sustaining the benefits of Bt soybean in South American countries. 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TI - Performance of Genetically Modified Soybean Expressing the Cry1A.105, Cry2Ab2, and Cry1Ac Proteins Against Key Lepidopteran Pests in Brazil
JF - Journal of Economic Entomology
DO - 10.1093/jee/toaa236
DA - 2020-12-09
UR - https://www.deepdyve.com/lp/oxford-university-press/performance-of-genetically-modified-soybean-expressing-the-cry1a-105-IcHiRkfoxc
SP - 2883
EP - 2889
VL - 113
IS - 6
DP - DeepDyve
ER -