Susceptibility of Cry1Ab-susceptible and -resistant sugarcane borer to transgenic corn plants containing single or pyramided Bacillus thuringiensis genes

Multiple independent trials were conducted to evaluate the performance of Cry1Ab-susceptible (Cry1Ab-SS), -heterozygous (Cry1Ab-RS), and -resistant (Cry1Ab-RR) genotypes of the sugarcane borer, Diatraea saccharalis (F.), on eight commercial hybrids and six experimental corn lines. The commercial varieties included two non-Bt and six Bt corn hybrids that expressed a single Bt protein (either Cry1Ab or Cry1F) targeting above-ground lepidopteran pests. The six experimental lines consisted of two non-Bt and four Bt corn lines, two expressing just the Cry1Ab protein and two containing the pyramided-genes Cry1A.105 and Cry2Ab2 (event MON 89034). Larval mortality on non-Bt corn leaf tissue ranged from 6 to 45% after 12 d across insect genotypes. The 12 d mortality of Cry1Ab-SS on leaf tissue of commercial Cry1Ab or Cry1F corn was 96-100%, whereas it was 80-96% for Cry1Ab-RS and 68-78% for Cry1Ab-RR. On intact plants, 39-64% of larvae survived on non-Bt corn plants after 21-25 d. Larval survivorship on intact plants of commercial Cry1Ab or Cry1F corn was 0-8.1% for Cry1Ab-SS, 1.3-34% for Cry1Ab-RS, and 19-51% for Cry1Ab-RR. Larvae of Cry1Ab-RR and -RS also caused significant plant injury to most of the commercial Bt corn hybrids, especially to the Cry1Ab corn. Cry1Ab resistance in D. saccharalis was incompletely dominant on commercial Bt corn hybrids. However, both experimental lines with pyramided genes of Cry1A.105 and Cry2Ab2 provided complete control of all three insect genotypes in both leaf tissue and intact plant tests. Results of this study suggest that MON 89034 should offer a means for Bt resistance management in D. saccharalis.     

Damage and survivorship of fall armyworm (Lepidoptera: Noctuidae) on transgenic field corn expressing Bacillus thuringiensis Cry proteins

Field corn, Zea mays L., plants expressing Cry1Ab and Cry1F insecticidal crystal (Cry) proteins of Bacillus thuringiensis (Bt) Berliner are planted on considerable acreage across the Southern region of the United States. The fall armyworm, Spodoptera frugiperda (J.E. Smith), is an economically important pest during the mid-to-late season on non-Bt and some commercial Bt corn hybrids. The objective of this study was to quantify foliar injury and survivorship of fall armyworm on transgenic corn lines expressing Cry1Ab or Cry1F Bt proteins. Corn lines/hybrids expressing Cry1Ab, Cry1F, and a conventional non-Bt cultivar were evaluated against artificial infestations of fall armyworm in field trials. Larvae (second instars) of fall armyworm were placed on corn plants (V8-V10 stages). Leaf injury ratings were recorded 14 d after infestation. Hybrids expressing Cry1F had significantly lower feeding injury ratings than non-Bt corn plants. Development and survivorship of fall armyworm on Bt corn lines/hybrids were also evaluated in no-choice laboratory assays by offering freshly harvested corn leaf tissue to third instars. Transgenic corn hybrids expressing Cry1Ab or Cry1F significantly reduced growth, development, and survivorship of fall armyworm compared to those offered non-Bt corn tissue. However, 25-76% of third instars offered Bt corn leaf tissues successfully pupated and emerged as adults. These results suggest Cry1Ab has limited effects on fall armyworm; whereas Cry1F demonstrated significant reductions in foliar injury and lower survivorship compared to that on non-Bt corn tissues. Although fall armyworm is not considered a primary target for insect resistance management by the U.S. Environmental Protection Agency, these levels of survivorship could impact selection pressures across the farmscape, especially when considering that transgenic Bt cotton cultivars express similar Cry (Cry1Ac or Cry1F) proteins.     

Performance of Cry1A.105-selected fall armyworm (Lepidoptera: Noctuidae) on transgenic maize plants containing single or pyramided Bt genes

Cry1A.105 is a Cry protein expressed in some transgenic Bacillus thuringiensis (Bt) maize products. In this study, performance of five populations of fall armyworm, Spodoptera frugiperda (J.E. Smith), were evaluated on four non-Bt and eight commercial and experimental Bt maize hybrids/lines (hereafter referred as maize products). The five insect populations included one Cry1A.105-susceptible strain, two Cry1A.105-resistant strains, and two F₁ heterozygous genotypes. The eight Bt maize hybrids/lines consisted of five single-gene Bt maize products containing Cry1A.105, Cry2Ab2, Cry1F, or Cry1Ab protein, and three pyramided Bt maize products expressing Cry1A.105/Cry2Ab2, Cry1A.105/Cry2Ab2/Cry1F, or Cry1Ab/Vip3A for targeting aboveground lepidopteran maize pests. In the study, neonates of each population were tested on leaf tissues in the laboratory and whole plants in the greenhouse. Cry1A.105 and Cry1F maize killed 92.2–100% susceptible larvae in both test methods, while resistant larvae survived well on these two maize products. Performance of the two F₁ populations on Cry1A.105 and Cry1F maize varied between the two test methods. In leaf tissue bioassay, Cry1Ab maize was marginally effective against the susceptible population. In contrast, few live larvae and little leaf injury from any of the five populations were observed on Cry2Ab2 and the three pyramided Bt maize products. The results of this study showed evidence of cross resistance of the Cry1A.105-resistant S. frugiperda to Cry1F and Cry1Ab maize, but not to the Bt maize products containing Cry2Ab2 or Vip3A. Data generated from this study will be useful in developing resistance management strategies for the sustainable use of Bt maize technology.     

Susceptibility of Louisiana and Florida populations of Spodoptera frugiperda (Lepidoptera: Noctuidae) to transgenic Agrisure®Viptera™ 3111 corn

In the United States, fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), is a target species of transgenic corn (Zea mays L.) expressing pyramided Bacillus thuringiensis proteins. In 2011, a total of 150 F₂ two-parent families of S. frugiperda were established using single-pair matings of feral individuals collected from three locations in Louisiana and Florida. The objective of this study was to determine the susceptibility of these field derived families of S. frugiperda to a pyramided Bt corn hybrid containing Agrisure^®Viptera™ 3111 traits. For each F₂ family, 96 neonates were assayed on leaf tissue of Agrisure^®Viptera™ 3111 corn in the laboratory. None of the 150 families survived for 7 days on leaf tissue of the Bt corn plants. The results demonstrate that the field populations of S. frugiperda collected from Louisiana and Florida were susceptible to the pyramided Bt corn product containing Agrisure^®Viptera™ 3111 traits. The data generated in this study can be used as baseline data for resistance monitoring.     

Occurrence,distribution, and ear damage of Helicoverpa zea (Lepidoptera: Noctuidae) in mixed plantings of non-Bt and Bt corn containing Genuity® SmartStax™ traits

Protein contamination on refuge kernels due to cross-pollination from Bt corn to non-Bt corn ears is a major concern in the use of a seed mixture refuge strategy (“RIB”) for resistance management of ear-feeding pests. In this study, occurrence, distribution, and ear damage of the corn earworm, Helicoverpa zea (Boddie), were evaluated in three planting patterns of non-Bt and Bt corn plants containing Genuity^® SmartStax™ traits. The three planting patterns were 1) pure stands of 27 Bt plants; 2) pure stands of 27 non-Bt plants; and 3) one non-Bt plant in the center surrounded by 26 Bt plants. A total of six trials were conducted in open field conditions with natural infestations in 2011 and 2012. Egg populations of H. zea were distributed randomly or uniformly, and the number of eggs laid was similar between Bt and non-Bt corn ears regardless of the planting patterns, suggesting that females of H. zea have no egg-laying preference between Bt and non-Bt plants. Bt corn plants containing Genuity^® SmartStax™ traits were equally effective in the control of H. zea in pure stands of Bt corn and “RIB” plantings. Occurrence of larvae and ear damage on Bt corn were significantly lower than on non-Bt plants and there were no significant differences between pure stands of Bt and “RIB” plantings across all trials. However, the limited numbers of live larvae in the pure stands of Bt plants were distributed non-randomly, suggesting a possibility of uneven expression of Bt proteins or elevated larval movement in the pure stands of Bt plants. Larval occurrence (3rd–5th instars) and ear damage on the refuge ears in “RIB” plantings were similar to or greater than found on ears of pure stands of non-Bt plants. However, more studies are needed to understand the effect of pollen movement on the full life cycle of H. zea before a final conclusion on the refuge function of RIB planting can be made.     

Efficacy of SmartStax® insect-protected corn hybrids against corn rootworm: The value of pyramiding the Cry3Bb1 and Cry34/35Ab1 proteins

Monsanto Company and Dow AgroSciences have used conventional breeding techniques to develop the combined-trait corn (Zea mays L.) product MON 89034 × TC1507 × MON 88017 × DAS-59122-7 (“SmartStax^® corn”) that confers insect resistance against key lepidopteran pests and the corn rootworm complex (Diabrotica spp.), as well as herbicide tolerance. This product contains the Cry3Bb1, Cry34Ab1, and Cry35Ab1 proteins for corn rootworm (CRW) control. Replicated field trials were conducted in 2006 and 2007 to test the efficacy of Cry3Bb1, Cry34Ab1/Cry35Ab1, and SmartStax under both natural and controlled infestations. In both years, root damage and adult beetle emergence were significantly less for Bt-containing hybrids than for non-Bt controls. In trials with heavy insect pressure, adult beetle emergence (a measure of larval control) was significantly reduced for SmartStax hybrids compared with hybrids with the individual traits and with non-Bt controls. Similarly, strip-plot testing in 2012 in grower fields with high levels of root injury to non-Bt hybrids showed significantly lower feeding on SmartStax than on the non-Bt control or on either single-trait product. In grower fields where single-trait Cry3Bb1 products incurred heavy CRW damage in 2011, SmartStax provided consistent protection against CRW in 2012. The combination of these insecticidal proteins in a single plant provides better rootworm control than current single-trait Bt corn products and represents an effective approach for corn rootworm resistance management.     

Field-evolved resistance to Cry1F maize by Spodoptera frugiperda (Lepidoptera: Noctuidae) in Brazil

The Cry1F protein from Bacillus thuringiensis Berliner expressed in event TC1507 maize (Zea mays L.) was one of the most effective ways to control Spodoptera frugiperda (J. E. Smith) in Brazil. After reports of reduced effectiveness of this Bt maize event in some areas of Brazil, research was undertaken to investigate if damage to Cry1F maize was caused by resistant S. frugiperda. Additional investigations were conducted to evaluate the genetic basis of the resistance and to test if Cry1F resistant S. frugiperda selected from populations of different regions of Brazil share the same resistance locus by using complementation tests. Neonate larvae of S. frugiperda collected from TC1507 maize fields with damage in Western Bahia region in 2011 were able to survive on Cry1F maize plants under laboratory conditions and subsequently produced normal adults. Survival of Cry1F-susceptible S. frugiperda on non-Bt maize was significantly higher in leaf than plant bioassays. Resistance ratio in diet overlay bioassays was >5000-fold. A discriminating concentration of 2000 ng cm⁻² of Cry1F protein was defined for monitoring the frequency of resistance of S. frugiperda to Cry1F. Cry1F resistant S. frugiperda showed a recessive autosomal inheritance for alleles involved in resistance to Cry1F protein. In complementation tests, the resistant population from Western Bahia was crossed with the other seven resistant populations collected from different States of Brazil. F₁ larvae from each cross had the same survival at discriminating concentration of 2000 ng cm⁻² of Cry1F protein, indicating that the resistance alleles in each population were likely at the same locus. Therefore, implementation of resistance management strategies is urgent to prolong the lifetime of Cry1F for controlling S. frugiperda in Brazil.     

Movement and survival of corn rootworm in seed mixtures of SmartStax® insect-protected corn

SmartStax^® insect-protected corn (Zea mays L.) contains genes for six Bacillus thuringiensis (Bt) proteins controlling both lepidopteran pests and the corn rootworm complex (Diabrotica spp.). The properties of SmartStax, particularly the multiple effective modes of action (i.e., each Bt protein provides a high level of control of the target pests with a low probability of cross-resistance among the proteins), have provided the opportunity to add to previously approved structured refuge options by combining the non-Bt refuge seed with SmartStax seed in a seed mix. Seed mixes ensure that a refuge is present in every Bt field, remove concerns about grower compliance with refuge requirements, and provide grower convenience. However, seed mixes could increase the likelihood that larval insects move between Bt and non-Bt plants and vice versa. Assessing the insect resistance management (IRM) value of a seed mix refuge requires an assessment of the amount of larval movement, and the consequences of that movement, for the key target pests. The studies here present such data for control of corn rootworm by SmartStax corn, which contains the rootworm-active protein Cry3Bb1 and the binary protein Cry34Ab1/Cry35Ab1. In a growth chamber experiment, SmartStax was most effective against first instars and significantly effective against second instars, but did not control third instars. In a field study of movement from a heavily infested non-Bt plant onto surrounding plants, a larger percentage of insects successfully dispersed from the infested plant when the surrounding plants were non-Bt plants than when they were SmartStax plants. A paired-plant study showed that few larvae migrated from infested SmartStax plants and survived on nearby non-Bt plants; larvae that migrated from infested non-Bt plants had low survival if the adjacent plants were SmartStax. Replicated field studies of plant-to-plant movement indicated that the non-Bt plants in a 5% or 10% seed mix consistently supported large populations of susceptible insects and represented a productive refuge, whereas the SmartStax plants had few or no survivors. The timing of emergence from seed mix plots containing 5% or 10% non-Bt plants was more similar to that of the non-Bt plots than that of the SmartStax plots. Thus, the available growth chamber and field data indicate that a seed mix of 5% or more will provide an effective refuge for corn rootworm in SmartStax corn.     

Low susceptibility of Spodoptera cosmioides,Spodoptera eridania and Spodoptera frugiperda (Lepidoptera: Noctuidae) to genetically-modified soybean expressing Cry1Ac protein

Spodoptera cosmioides (Walker), Spodoptera eridania (Stoll) and Spodoptera frugiperda (J. E. Smith) have caused significant damage on soybean Glycine max (L.) Merrill in Brazil. Genetically-modified MON 87701 × MON 89788 soybean that expresses the Cry1Ac protein is potentially an alternative tool for the management of these species. Purified protein bioassays were done to evaluate the susceptibility of S. cosmioides, S. eridania and S. frugiperda to Cry1Ac protein. The level of efficacy of the Bt soybean plants in controlling these species was measured through laboratory and greenhouse trials under high artificial insect infestations. The biology of these insects was evaluated over their development cycles to understand their life history when fed on Bt soybean. Purified Cry1Ac protein at the maximum concentration tested (100 μg Cry1Ac mL⁻¹ diet) resulted in low mortality of S. cosmioides and S. eridania (<13%) and intermediate mortality of S. frugiperda (50%). No significant effects of the Bt soybean plants were observed in the life table parameters of S. cosmioides and S. eridania. However, S. frugiperda fed on Bt soybean plants had a prolonged larval stage (by 5 days), reduced larvae viability, increased mean generation time (by 8 days) and reduced intrinsic rate of increase. In general, the Bt soybean plants showed poor control of Spodoptera species when evaluated by leaf-disc bioassay and greenhouse trials. Consequently, other control tactics must be used in combination with MON 87701 × MON 89788 soybean in the field for the efficient management of S. cosmioides, S. eridania and S. frugiperda.     

Genetic basis of Cry1F resistance in two Brazilian populations of fall armyworm,Spodoptera frugiperda

Large-scale adoption of transgenic crops expressing genes from Bacillus thuringiensis (Bt) imposes high selection pressure for evolution of field-relevant resistance that can reduce pest control efficacy, such as reported for Cry1F maize (Zea mays L.) in populations of fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), of Puerto Rico, Brazil, and the United States. As part of our effort to improve fall armyworm resistance management to Bt crops, here we determined the genetic basis of Cry1F resistance in two S. frugiperda strains originated from field collections in different regions of Brazil and further selected in the laboratory for high levels of resistance to Cry1F maize. Continuous exposure to the TC1507 event for 11 generations resulted in more than 183-fold resistance to Cry1F in the two strains studied, and such a high resistance level enabled the insects to complete larval development on the Bt maize plants. Genetic analyses using concentration-response bioassays with progenies from reciprocal crosses between resistant and susceptible insects indicated that the inheritance of the resistance is autosomal, recessive and without maternal effects. Backcross of the F₁ progeny with the parental resistant strains revealed that the resistance in the two selected strains is conferred by a single locus or set of tightly linked loci. These results support some of the assumptions of the strategy in use for fall armyworm resistance management to Bt Cry1F maize, but survival rates of heterozygotes on the Bt plants were higher than 5%, showing that the Cry1F maize does not produce a high dose of the insecticidal protein for S. frugiperda. Additionally, we detected a delay in larval development time that may favor assortative mating of individuals carrying resistance alleles. These findings are consistent with the rapid evolution of Cry1F resistance in certain field populations of fall armyworm. Implications for resistance management of S. frugiperda to Bt maize are discussed.     

Influences of Cry1Ac Broccoli on Larval Survival and Oviposition of Diamondback Moth

Larval survival and oviposition behavior of three genotypes of diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), (homozygous Cry1Ac-susceptibile, Cry1Ac-resistant, and their F₁ hybrids), on transgenic Bacillus thuringiensis (Bt) broccoli expressing different levels of Cry1Ac protein were evaluated in laboratory. These Bt broccoli lines were designated as relative low, medium, and high, respectively, according to the Cry1Ac content. Untransformed brocccoli plants were used as control. Larval survival of diamondback moth on non-Bt leaves was not significantly different among the three genotypes. The Cry1Ac-resistant larvae could survive on the low level of Bt broccoli plants, while Cry1Ac-susceptible and F₁ larvae could not survive on them. The three genotypes of P. xylostella larvae could not survive on medium and high levels of Bt broccoli. In oviposition choice tests, there was no significant difference in the number of eggs laid by the three P. xylostella genotypes among different Bt broccoli plants. The development of Cry1Ac-susceptible and Cry1Ac-resistant P. xylostella on intact Bt plants was also tested in greenhouse. All susceptible P. xylostella larvae died on all Bt plants, while resistant larvae could survive on broccoli, which expresses low Cry1Ac protein under greenhouse conditions. The results of the greenhouse trials were similar to that of laboratory tests. This study indicated that high dose of Bt toxins in broccoli cultivars or germplasm lines is required for effective resistance management.     

Comparative susceptibility of Ostrinia furnacalis, Ostrinia nubilalis and Diatraea saccharalis (Lepidoptera: Crambidae) to Bacillus thuringiensis Cry1 toxins

Transgenic corn hybrids that express toxins from Bacillus thuringiensis (Bt) are highly effective against the European corn borer, Ostrinia nubilalis (Hübner), and the closely related Asian corn borer, Ostrinia furnacalis (Guenée). Since the registration of Bt corn hybrids in the U.S. in 1996, there has been a great deal of information generated on O. nubilalis. However, relatively little information exists for O. furnacalis. To help determine whether the information generated for O. nubilalis can be leveraged for decisions regarding the use of transgenic Bt corn against O. furnacalis, experiments were designed to determine whether the pattern of sensitivity to various Bt Cry1 toxins is similar between the two species. Test insects included laboratory-reared O. furnacalis originating from Malaysia, a Bt-susceptible laboratory colony of O. nubilalis maintained at the University of Nebraska-Lincoln (UNL) and an out-group consisting of the sugarcane borer, Diatraea saccharalis (F.), from Louisiana which represents a different genus from the same family. O. furnacalis and O. nubilalis exhibited a similar pattern of susceptibility to all the Cry1 toxins and were highly susceptible to the range of Bt toxins tested including Cry1Aa, Cry1Ab, Cry1Ac and Cry1F. Both of the Ostrinia species were more tolerant to Cry1Ba compared with D. saccharalis, although sensitivity of O. furnacalis was intermediate and did not differ significantly from that of O. nubilalis and D. saccharalis. D. saccharalis was also susceptible to the range of toxins tested but unlike the two Ostrinia species, was more tolerant to Cry1F and more susceptible to Cry1Ba. These results indicate that both of the Ostrinia corn borer species are similar in sensitivity to the Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1F toxins, thus suggesting shared toxin receptors and mechanisms of toxicity for the two species.     

Frequency of Bacillus thuringiensis Cry1A.105 resistance alleles in field populations of the fall armyworm,Spodoptera frugiperda,in Louisiana and Florida

Fall armyworm, Spodoptera frugiperda (J.E. Smith), is a major pest of many crops and a cross-crop target of transgenic maize, cotton, and soybean containing Bacillus thuringiensis (Bt) genes. Some of the current Bt maize products for controlling lepidopteran species contain the Bt event MON 89034. The objective of this study was to determine the frequency of resistance alleles in field populations of S. frugiperda collected from Louisiana and Florida, U.S. to Cry1A.105, one of the two Bt genes in MON 89034. A total of 150 F₂ two-parent families of S. frugiperda were established using single-pair mating of field-collected individuals in 2011, which included 79 families from two locations in Louisiana and 71 families from one location in Florida. F₂ screen was conducted to detect resistance alleles in these families to Cry1A.105 protein in maize plants. Four out of the 79 Louisiana and 14 out of the 71 Florida families were identified to possess resistance alleles to the Cry1A.105 maize plants. Thus, the corresponding frequency of resistance alleles to Cry1A.105 maize was estimated to be 0.0158 with a 95% credibility interval (CI) of 0.0052–0.0323 for the Louisiana populations and 0.0559 with a 95% CI of 0.0319–0.0868 for the Florida populations. The resistant families survived on whole Cry1A.105 maize plants and demonstrated a significant level (>116-fold) of resistance to the Cry1A.105 protein in a diet-incorporated bioassay. These findings suggest that resistance allele frequency in S. frugiperda to single-gene Cry1A.105 maize in the U.S. southeast region apparently is not rare, most likely due to the selection of Cry1F resistance and its cross-resistance to Cry1A.105.     

A preliminary study on the effects of a transgenic corn event on the non-target pest Dalbulus maidis (Hemiptera: Cicadellidae)

The inclusion of the cry gene in corn may produce direct effects on non-target pests. Our research was focused on the relationship between Bt corn germplasm, expressing the cry1F protein to control the fall armyworm [Spodoptera frugiperda (Noctuidae)], and a non-target pest, the corn leafhopper [Dalbulus maidis (Cicadellidae)]. The aim of this contribution was to elucidate if Bt corn plants have influence on the oviposition preference of the leafhopper and to evaluate the effect of the transgenic plant on the hatching rate of egg. Female corn leafhoppers were released in cages each containing two potted plants in the V2 stage: a Bt germplasm and the corresponding isogenic hybrid. Laid eggs were counted and the number of hatched nymphs recorded. D. maidis females oviposited and laid more eggs in Bt plants. The egg hatching rate was negatively affected by the Bt germplasm. In addition, a field study was conducted in order to determine the abundance of D. maidis adults in Bt corn and the corresponding non-Bt isoline. Two corn plots sown with the same germplasms as used in the laboratory bioassays were sampled weekly. In the field, the population of the corn leafhopper was higher in the Bt corn plot than in the non-Bt isoline. Possible hypotheses for the differences in abundance of the vector in the field are: a) that pleiotropic effects of Bt corn could attract adults; b) the existence of a possible direct competition between the corn leafhopper and the target pest in order to utilize the whorls of corn plants as refuge and feeding sites, so the high populations of the vector could be due to the large supply of healthy whorls in the transgenic plot; and/or c) a differential attack of natural enemies occurring in non-Bt plots.     

Pest resistance to Cry1Ab Bt maize: Field resistance,contributing factors and lessons from South Africa

This paper documents the historical development of resistance of the African maize stem borer, Busseola fusca (Fuller) (Lepidoptera: Noctuidae) to Bt maize (Zea mays L.). This pest was one of the first to evolve resistance to Bt maize expressing Cry1Ab protein. A time-line of events and contributing factors are presented, from the commencement of efficacy testing through to the present situation, where the Cry1Ab toxin has lost its efficacy against B. fusca at many localities throughout the maize producing region, and single-gene Bt maize events often require insecticide treatments for which farmers are compensated. Significant levels of pest survival on Bt maize was observed in the first season after commercial release in 1998 and confirmed seven years later. Reduced selection pressure on the target pest is the objective of insect resistance management (IRM), and strategies to accomplish this should receive highest priority. Where resistance is prevalent, the only viable options to reduce selection pressure are withdrawal of the product and/or enforcement of high-dose/refuge requirements. The latter action may however be of no value under conditions where resistance is prevalent, since the value of refugia to an IRM strategy may be compromised. Remedial actions taken in South Africa included the propagation and enforcement of refuge compliance followed by the release of pyramided maize hybrids in 2011. These pyramids combine Cry1A.105 and Cry2Ab2 toxin-producing transgenes, replacing the ineffective single-transgene. However, it remains uncertain if cross-resistance occurs between Cry1A.105/Cry2Ab2 and the closely related Cry1Ab toxin, and for how long this pyramided event will endure. Cultivation of Cry1Ab-expressing hybrids continues in areas where resistance levels have been confirmed to be high. In retrospect, this case provides lessons regarding IRM, not only in South Africa, but wherever Bt crops are being introduced.     

Monitoring Cry1Ab susceptibility in Asian corn borer (Lepidoptera: Crambidae) on Bt corn in the Philippines

Between 2002 and 2004, collections of egg masses of Asian corn borer (ACB), Ostrinia furnacalis (Guenée) were made from corn-planting sites on the major Philippine islands of Luzon (Laguna, Pangasinan, Camarines Sur and Isabela provinces) and Mindanao (Bukidnon and South Cotabato provinces). The resulting neonates were bioassayed for susceptibility to Bacillus thuringiensis (Bt) Cry1Ab protein. The median lethal concentrations (LC₅₀s) for the different collections ranged from 0.42 to 2.37 ng/cm². The bioassay results suggest that Philippine corn borer populations were highly susceptible to Cry1Ab protein prior to the widespread deployment of Bt corn. The upper limit of the estimated LC₉₉ (104 ng/cm²) from the pooled bioassay data was selected as a candidate diagnostic concentration and subsequently tested on eleven ACB populations. Results of the validation assays showed that the mortality response of all the tested ACB populations was higher than the expected mortality (99%). Therefore, the concentration of 104 ng/cm² was used to monitor susceptibility in ACB populations in the Philippines. Monitoring of field populations during 2009 in areas where Bt corn had been grown for 3 years found some enhanced survival of neonates at the diagnostic concentration but progeny of the diagnostic-concentration survivors did not survive on Bt corn, indicating that ACB populations in the Philippines remain susceptible to Cry1Ab-containing Bt corn hybrids.     

Agronomic assessment of Bt trait and seed or soil-applied insecticides on the control of corn rootworm and yield

Corn rootworm (Diabrotica spp.) has become the most concern and widespread insect pest of corn (Zea mays L.) production in North America. Two field experiments were conducted to assess the agronomic and yield performance of transgenic rootworm trait, Bacillus thuringiensis (Bt) Cry3Bb, seed-coating treatment, and a soil-applied insecticide under natural corn rootworm infestation. Experiment 1 compared a conventional corn hybrid with and without insecticide (Force 3G) with its near isoline Bt hybrid from 2003 to 2005, on a clay loam soil. Experiment 2 investigated the same treatments as in Experiment 1 plus an additional seed-coated Poncho treatment on a sandy loam in 2004 and 2005. Rootworm population before the crop anthesis, root node injury and root:shoot dry weight ratio during the early grain filling stage, and stalk lodging and grain yield were determined. Our data showed that rootworm population diminished over the 3 years owing to rootworm displacement and adverse weather conditions. At the clay loam site, both Force 3G and the Bt hybrid significantly reduced the larval populations, root injury and lodging score, and increased root:shoot ratio. Over the 3 years, grain yields of the Bt hybrid were 11–66% greater than the untreated non-Bt isoline hybrid; yield of the non-Bt hybrid treated with Force 3G was also significantly greater than the same untreated non-Bt hybrid in 2 of 3 years. Despite less root node injury in the first rows of non-Bt plants adjacent to the Bt plots was observed, yield benefit of this effect remained to be proven. On sandy loam soil, the larval population was very low and there were no differences in root node injury and plant lodging among all the four treatments in either 2004 or 2005. The yield of the Bt hybrid was up to 10% greater than its non-Bt isoline hybrid treated or not with an insecticide in 1 year. Our data showed that Bt rootworm seed technology was effective to control the rootworm larvae and protected grain yield under severe infestation. Furthermore, our data suggest that some of the gain in Bt hybrid yield may be attributed to the genetic transformation as observed in sandy loam soil experiment. In all cases, corn producers should be aware of the pest history, rootworm pressure in relation to economic threshold, soil type and the expected cost-to-benefit ratio before deciding to adopt any protective measures.     

Quantification of Bacillus thuringiensis Cry1Ab protein in tissues of YieldGard (MON810) corn hybrids tested at multiple field locations in India

Transgenically expressed Bacillus thuringiensis insecticidal-protein Cry1Ab was quantified in target tissues of insect feeding of several YieldGard^® corn hybrids. The Cry1Ab protein is intended to protect corn plants from two economically important stem borers, Chilo partellus and Sesamia inferens. A total of seven YieldGard hybrids, all with MON810 event, were field-tested in a total of fourteen locations during the dry season (October–March) of 2005/2006 and wet season (May–October) of 2006. S. inferens and C. partellus oviposit on leaves of young corn plants, 15–60 days after emergence (DAE). The neonates initially feed by scraping the leaf lamina before migrating to bore into the stem. Thus high concentrations of Cry1Ab in whorl leaf and stem tissues would ensure effective control of the borers. The mean tissue Cry1Ab concentrations during the oviposition window of the borers (15–60 DAE), ranged from 50.05 to 21.01 ppm in whorl leaf, and between 9.26 and 3.47 ppm in stem tissue during the same period in the dry season of 2005/06. Similarly, Cry1Ab concentrations in whorl leaf and stem between 15 and 60 DAE during the wet season of 2006 ranged between 19.30 to 11.08 and 14.28 to 4.69 ppm, respectively. The baseline-sensitivity data of these insects to Cry1Ab in laboratory assays was determined. The concentrations of Cry1Ab in the target tissues as studied in seven YieldGard hybrids tested suggest effective management of the two borers. This paper also provides a summary of the expression of the Cry1Ab gene in various genetic backgrounds.     

Prevalence of Helicoverpa zea (Lepidoptera: Noctuidae) on late season volunteer corn in Mississippi: Implications on Bt resistance management

The southern United States has a long growing period between corn, Zea mays L., harvest and first winter frost, so volunteer corn which germinates after harvest has a growing period sufficient for corn earworm, Helicoverpa zea (Boddie) and fall armyworm, Spodoptera frugiperda (J. E. Smith) to feed on these plants. However, lower air temperatures can limit larval development on late season volunteer corn and thereby successful pupation. Here we explore the suitability of late season volunteer corn for larval development and the potential contribution of H. zea larvae to the overwintering population. Our survey revealed the occurrence of volunteer corn in high densities, with monthly mean densities ranging from 56,000 to 143,000 plants ha⁻¹. H. zea larvae were found feeding on both vegetative and reproductive stage plants while S. frugiperda were only found on vegetative stage plants. An analysis of H. zea growing degree day (GDD) accumulations based on Mississippi weather data from 1980 to 2010 revealed that sufficient GDD to reach prepupation would always be accumulated before first frost if oviposition occurred by 9 September, with the probability of successful pupation decreasing rapidly thereafter. However, most of the H. zea larvae were oviposited after this, and could not reach pupation. Because S. frugiperda cannot overwinter in Mississippi, their ability to pupate was not examined. Low suitability of whorl stage corn for H. zea development coupled with low larval densities during this stage effectively diminish the number of larvae that complete development on late season volunteer transgenic corn expressing genes from the soil bacterium, Bacillus thuringiensis (Bt). This limits the Bt resistance risk posed by larvae developing on late season volunteer corn in all but the most southern locations in the US.     

Functional dominance of different aged larvae of Bt-resistant Spodoptera frugiperda (Lepidoptera: Noctuidae) on transgenic maize expressing Vip3Aa20 protein

Fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith), is the main target pest of transgenic maize expressing insecticidal proteins from Bacillus thuringiensis Berliner (Bt) in Brazil. To optimize resistance management strategies, we evaluated the functional dominance of different aged larvae of Bt-resistant FAW on Vip3Aa20 maize. We measured the survival and development of Vip3Aa20-resistant, -heterozygote, and -susceptible strains on MIR162 (expressing Vip3Aa20) and Bt11 × MIR162 × GA21 (expressing Vip3Aa20 and Cry1Ab) maize. The resistant strain, from neonate to sixth instar, showed more than 72% survival on Vip3Aa20 maize. From surviving larvae, more than 64 and 54% developed to pupae and adults, respectively. In contrast, heterozygote and susceptible strains showed no larval survival up to fourth instar, and less than 25% larval survival in the fifth and sixth instar on Vip3Aa20 maize. These larvae produced less than 21% of pupae and adults. The development time of FAW strains from neonate-to-adult exposed to Vip3Aa20 maize was similar; however, the resistant strain showed an increase of ∼ 2 d when compared to those fed only non-Bt maize. In summary, the resistance of S. frugiperda to Vip3Aa20 maize is functionally recessive from neonate up to fourth instar larvae. However, high larval survival of resistant strain and some survival of heterozygote larvae in advanced instars on Vip3Aa20 maize were observed. These results will be important for designing insect resistance management to Bt maize plants expressing Vip3Aa20 protein in Brazil.