Fangneng Huang

Success of the high-dose/refuge resistance management strategy after 15 years of Bt crop use in North America

Transgenic maize and cotton expressing Bacillus thuringiensis (Bt) toxins were first commercialized in 1996. By 2009, Bt crops were planted on ca. 47.6 Mha in 22 countries worldwide, with the USA and Canada accounting for 54% of this area. Resistance (virulence) development in target insect pests is a major threat to the sustainable use of Bt crops. Four major target pests of Bt crops in the USA and Canada – European corn borer, Ostrinia nubilalis (Hübner), southwestern corn borer, Diatraea grandiosella Dyar (both Lepidoptera: Crambidae), tobacco budworm, Heliothis virescens Fabricius (Lepidoptera: Noctuidae), and pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) – remain susceptible to Bt toxins after 15 years of intensive use of Bt maize and Bt cotton. The success in sustaining susceptibility in these major pests is associated with successful implementation of the ‘high‐dose/refuge’ insecticide resistance management (IRM) strategy: (i) Bt crop cultivars express a ‘high dose’, (ii) initial frequency of resistance alleles is very low, and (iii) a refuge is maintained nearby in the environment. Field resistance (including control failure) to a Bt crop has been clearly documented in three situations: fall armyworm [Spodoptera frugiperda JE Smith] in Puerto Rico, African stem borer [Busseola fusca Fuller (Lepidoptera: Noctuidae)] in South Africa, and P. gossypiella in India. Factors associated with these cases of field resistance include: failure to use high‐dose Bt cultivars and lack of sufficient refuge. These observations support the claim that implementation of the ‘high‐dose/refuge’ IRM strategy has been successful in substantially delaying field resistance to Bt crops.

Cry1F Resistance in Fall Armyworm Spodoptera frugiperda: Single Gene versus Pyramided Bt Maize

Evolution of insect resistance to transgenic crops containing Bacillus thuringiensis (Bt) genes is a serious threat to the sustainability of this technology. However, field resistance related to the reduced efficacy of Bt maize has not been documented in any lepidopteran pest in the mainland U.S. after 18 years of intensive Bt maize planting. Here we report compelling evidence of field resistance in the fall armyworm, Spodoptera frugiperda (J.E. Smith), to Cry1F maize (TC 3507) in the southeastern region of the U.S. An F2 screen showed a surprisingly high (0.293) Cry1F resistance allele frequency in a population collected in 2011 from non-Bt maize in south Florida. Field populations from non-Bt maize in 2012–2013 exhibited 18.8-fold to >85.4-fold resistance to purified Cry1F protein and those collected from unexpectedly damaged Bt maize plants at several locations in Florida and North Carolina had >85.4-fold resistance. In addition, reduced efficacy and control failure of Cry1F maize against natural populations of S. frugiperda were documented in field trials using Cry1F-based and pyramided Bt maize products in south Florida. The Cry1F-resistant S. frugiperda also showed a low level of cross-resistance to Cry1A.105 and related maize products, but not to Cry2Ab2 or Vip3A. The occurrence of Cry1F resistance in the U.S. mainland populations of S. frugiperda likely represents migration of insects from Puerto Rico, indicating the great challenges faced in achieving effective resistance management for long-distance migratory pests like S. frugiperda.

Comparative Susceptibility of European Corn Borer, Southwestern Corn Borer, and Sugarcane Borer (Lepidoptera: Crambidae) to Cry1Ab Protein in a Commercial Bacillus thuringiensis Corn Hybrid

Abstract One field strain each of the European corn borer, Ostrinia nubilalis (Hübner); southwestern corn borer, Diatraea grandiosella Dyar; and sugarcane borer, Diatraea saccharalis (F.); were collected from cornfields in northeastern Louisiana. Susceptibilities of the field strain and a corresponding laboratory strain of the three borer species to Cry1Ab protein in DK69-70 Bacillus thuringiensis (Bt) corn hybrid were determined by exposing neonates to intact leaf tissues from whorl stage plants or by feeding neonates or third instars on a meridic diet treated with different concentrations of Cry1Ab protein extracted from Bt corn leaves. Mortality and growth of larvae were evaluated after 2 and 4 d posttreatment in the bioassays by using intact leaf tissues or after 7 d in the bioassays by using diet incorporating Cry1Ab protein. D. saccharalis was the least susceptible species to Cry1Ab protein among the three species, followed by D. grandiosella, whereas O. nubilalis was most susceptible. The 2-d mortality of D. saccharalis neonates on intact Bt leaf tissues was lower than that of O. nubilalis and D. grandiosella. All neonates of O. nubilalis were killed on the diet treated with Cry1Ab protein at 0.5 and 1 mg/kg. The mortality of D. grandiosella was >75% at 1 mg/kg, but it was <6% for D. saccharalis at 1 mg/kg. The LC50 values of D. saccharalis were 3- and 11-fold higher than those of D. grandiosella and O. nubilalis, respectively. The LC90 values of D. saccharalis were 8- and 32-fold higher than those of D. grandiosella and O. nubilalis, respectively. Larval growth of the three species on Cry1Ab-treated diet was inhibited, but the inhibition was greater for O. nubilalis and D. grandiosella than for D. saccharalis. The lower susceptibility of D. saccharalis to Cry1Ab protein suggests that it is necessary to verify if a high-dose Bt corn for O. nubilalis and D. grandiosella is also a high dose for D. saccharalis.

Sugarcane borer (Lepidoptera: Crambidae) resistance to transgenic Bacillus thuringiensis maize.

Abstract Transgenic maize, Zea mays L., expressing the Bacillus thuringiensis (Bt) Cry1Ab toxin has been planted to extensive areas across the United States and several other countries, but no resistance has been documented in field populations of lepidopteran target pests. This article describes the first report of resistance alleles to commercially available Cry1Ab Bt maize in a Louisiana population of sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae). Two hundred thirteen two-parent isolines of D. saccharalis were screened for Cry1Ab resistance on Bt maize leaf tissue using an F2 screening technique. Larvae representing three isolines survived >15 d on Bt tissue in the F2 generation. The second generation backcross progeny (B1F2) derived from isoline 52 completed larval development on Bt maize in the greenhouse. Segregation and resistance frequency analysis associated with isoline 52 suggested that Bt resistance is probably determined by a nearly completely recessive allele at a single locus. With this assumption, the estimated resistance allele frequency in this population is 0.0023, within a 95% confidence interval of 0.0003–0.0064.

Survival of Kansas Dipel-Resistant European Corn Borer (Lepidoptera: Crambidae) on Bt and Non-Bt Corn Hybrids

Abstract The Kansas Dipel-resistant and susceptible European corn borer, Ostrinia nubilalis (Hübner), were evaluated in the greenhouse on different Bt transgenic events expressed in corn hybrids. There were important differences in the resistance offered by the different Bt event corn hybrids. Hybrid comparison tests indicate that these Dipel-resistant first-instar European corn borer were not able to survive to adulthood on whorl-stage MON810, Bt11, or 176 Bt event corn plants. Third instars did not survive to adulthood on whorl-stage MON810 or Bt11 event corn plants but a small number of fifth instars were found on whorl-stage DBT418 plants infested with Dipel-resistant larvae. First and third instars of these Dipel-resistant European corn borers caused more leaf-feeding damage and more tunneling on whorl-stage Bt-corn plants than did the Dipel-susceptible European corn borers. However, in the single Bt corn hybrid test, there was no survival of the Dipel-resistant European corn borers on DK580BtX or MAX454 Bt plants 35 to 42 d after they had been infested with first instars. These results demonstrate that the current Kansas selection of Dipel-resistant European corn borer strain cannot establish reproducing populations in the tested Bt corn lines and hybrids.

Evidence of field-evolved resistance to Cry1Ac-expressing Bt cotton in Helicoverpa armigera (Lepidoptera: Noctuidae) in northern China

BACKGROUND Evolution of resistance threatens the continued success of transgenic crops expressing insecticidal proteins. One of the key factors for successful resistance management is the timely implementation of monitoring programmes to detect early changes of resistance allele frequency in field populations. F(1)/F(2) screen, dose-response bioassays and field survey were used to monitor resistance to the Cry1Ac-expressing cotton in a field population of Helicoverpa armigera (Hübner), the primary target of transgenic Bt cotton in China. RESULTS Field survey showed an increased trend of egg populations of H. armigera on Bt cotton in the Qiuxian area from 2003 to 2007. By using the F(2) screening procedure, the resistance allele frequency in the Qiuxian (Hebei, China) population of H. armigera collected during 2007 was estimated to be 0.075 (95% CI: 0.053-0.100), which was 12 times greater than that estimated 9 years ago. Dose-response bioassay with the field population collected from the same area showed a significant resistance level (11-fold) to Cry1Ac toxin compared to a laboratory susceptible strain. CONCLUSION This study documented a case of field-evolved resistance in H. armigera after several years of intensive planting of Bt cotton. Proactive tactics must be adopted to prevent further increase of resistance gene frequency in the Qiuxian region.

Susceptibility of Cry1Ab-resistant and -susceptible sugarcane borer (Lepidoptera: Crambidae) to four Bacillus thuringiensis toxins.

Sugarcane borer, Diatraea saccharalis (F.), is a primary corn stalk borer pest targeted by transgenic corn expressing Bacillus thuringiensis (Bt) proteins in many areas of the mid-southern region of the United States. Recently, genes encoding for Cry1A.105 and Cry2Ab2 Bt proteins were transferred into corn plants (event MON 89034) for controlling lepidopteran pests. This new generation of Bt corn with stacked-genes of Cry1A.105 and Cry2Ab2 will become commercially available in 2009. Susceptibility of Cry1Ab-susceptible and -resistant strains of D. saccharalis were evaluated on four selected Bt proteins including Cry1Aa, Cry1Ac, Cry1A.105, and Cry2Ab2. The Cry1Ab-resistant strain is capable of completing its larval development on commercial Cry1Ab-expressing corn plants. Neonates of D. saccharalis were assayed on a meridic diet containing one of the four Cry proteins. Larval mortality, body weight, and number of surviving larvae that did not gain significant weight (<0.1mg per larva) were recorded after 7 days. Cry1Aa was the most toxic protein against both insect strains, followed in decreasing potency by Cry1A.105, Cry1Ac, and Cry2Ab2. Using practical mortality (larvae either died or no significant weight gain after 7 days), the median lethal concentration (LC(50)) of the Cry1Ab-resistant strain was estimated to be >80-, 45-, 4.1-, and -0.5-fold greater than that of the susceptible strain to Cry1Aa, Cry1Ac, Cry1A.105 and Cry2Ab2 proteins, respectively. This information should be useful to support the commercialization of the new Bt corn event MON 89034 for managing D. saccharalis in the mid-southern region of the United States.

Effects of delayed mating on reproductive performance of Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae)

Virgin male and female Indianmeal moths, Plodia interpunctella (H . ubner), were mated soon after emergence or delayed from mating for 1–5 d, to determine the effect of age at mating on the number of spermatophores transferred by males to females, number of eggs laid (fecundity), egg viability, and adult longevity. When male and female moths were mated without delay, all females mated successfully. On average, each mated female had 2.2 spermatophores and laid 161 eggs. About 99% of the eggs laid were viable. Fecundity and egg viability were significantly and positively correlated with the number of spermatophores/female. For each day that mating of virgin male or female P. interpunctella was delayed, fecundity decreased by about 25 eggs. Egg viability decreased by 22%/d only when females were delayed from mating. Males delayed from mating for 5 d were unable to inseminate females. However, about 23% of females had one spermatophore when females or both males and females were delayed from mating for 5 d. On average, 22–39 non-viable eggs were laid by a female in treatments where male or female moths were delayed from mating for 5 d. Similarly, unmated (virgin) females laid 33 non-viable eggs. These results suggest that methods that can delay and disrupt mating may be effective behavioral strategies for managing this important pest of stored commodities and processed foods. r 2002 Elsevier Science Ltd. All rights reserved.

Evaluation of Transgenic Bacillus thuringiensis Corn Hybrids Against Cry1Ab-Susceptible and -Resistant Sugarcane Borer (Lepidoptera: Crambidae)

Abstract A Louisiana strain of the sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), was selected for resistance to the Cry1Ab protein of Bacillus thuringiensis (Bt) by using an F2 screening procedure. Survival of Bt-resistant, -susceptible, and -heterozygous genotypes of sugarcane borer was evaluated on vegetative and reproductive stages of five non-Bt and seven Bt field corn, Zea mays L., hybrids in a greenhouse study. Larval survival was recorded 21 d after infestation of neonates on potted plants. Larval survival across the three sugarcane borer genotypes and five non-Bt corn hybrids after 21 d ranged from 23.6 ± 5.2% (mean ± SEM) to 57.5 ± 5.2%. Mean survival of Cry1Ab-resistant larvae on vegetative and reproductive plant stages was 12 and 21%, respectively. During the vegetative stages, all seven Bt corn hybrids were highly efficacious against Cry1Ab-susceptible and -heterozygous genotypes of sugarcane borer, with a larval survival rate of <2% for the Bt-susceptible genotype and ≤5% for the heterozygotes. However, 8–18% of the heterozygous genotype survived on reproductive stage plants for four of the seven Bt corn hybrids tested. The variation in performance of Bt corn cultivars at vegetative and reproductive growth stages against Cry 1Ab resistant sugarcane borer suggests differential seasonal expression that may hasten resistance in the field. Bt corn hybrids expressing a “high dose” for European corn borer, Ostrinia nubilalis (Hübner), may not produce a sufficient high dose for the sugarcane borer.

Susceptibility of Field Populations of the Fall Armyworm (Lepidoptera: Noctuidae) from Florida and Puerto Rico to Purified Cry1f Protein and Corn Leaf Tissue Containing Single and Pyramided Bt Genes

ABSTRACT Larval survival of Cry1F-susceptible (FL), -resistant (PR and Cry1F-RR), and -heterozygous (FL x PR and Cry1F-RS) populations of the fall armyworm, Spodoptera frugiperda (J. E. Smith) to purified Cry1F protein and corn leaf tissue of 7 Bacillus thuringiensis (Bt) corn hybrids and 5 non-Bt corn hybrids was evaluated in the laboratory. The 7 Bt corn hybrids represent 5 Bt corn traits: Herculex®I, which expresses a single Bt protein (Cry1F), and Genuity® VT Double Pro™, VT Triple Pro™, SmartStax™, and Agrisure® Viptera® 3111, which contain ≥ 2 pyramided Bt genes. The original FL and PR populations were collected from corn fields in 2011 in Florida and Puerto Rico, respectively. Diet-incorporation bioassays showed that FL was susceptible to Cry1F protein with a LC50 value of 0.13–0.23 µg/g, while PR was highly resistant to Cry1F protein (> 137-fold). FL was also susceptible to all 7 Bt corn hybrids with a 7-day mortality of > 95%, while PR and a backcrossed and reselected population, Cry1F-RR, were highly resistant to Cry1F corn leaf tissue. The resistance was recessive or incompletely recessive. None of the 5 populations of S. frugiperda could survive on Viptera™ 3111, suggesting this Bt corn trait can completely overcome the resistance and thus should provide a means of managing Cry1F resistance in S. frugiperda. However, Cry1F-RR exhibited a significant cross-resistance to the leaf tissue of the other 3 pyramided Bt corn traits. The possible cross-resistance between single-gene and pyramided Bt corn products suggest that careful selection of Bt genes is essential in use of gene pyramiding strategy for resistance management.