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Applied and Environmental Microbiology, August 2002, p. 3790-3794, Vol. 68, No. 8
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.8.3790-3794.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Control of Resistant Pink Bollworm (Pectinophora gossypiella) by Transgenic Cotton That Produces Bacillus thuringiensis Toxin Cry2Ab

Bruce E. Tabashnik,^1* Timothy J. Dennehy,¹ Maria A. Sims,¹ Karen Larkin,² Graham P. Head,³ William J. Moar,⁴ and Yves Carrière¹

Department of Entomology, University of Arizona, Tucson, Arizona 85721,¹ EnviroLogix Inc., Portland, Maine 04103,² Monsanto Co., St. Louis, Missouri 63198,³ Department of Entomology, Auburn University, Auburn, Alabama 36849⁴

Received 9 April 2002/ Accepted 29 May 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
Crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with B. thuringiensis produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (Pectinophora gossypiella), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces B. thuringiensis toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 µg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 µg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 µg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Transgenic crop plants that produce insecticidal proteins of the bacterium Bacillus thuringiensis can reduce reliance on sprayed insecticides (28). B. thuringiensis toxins kill some major crop pests, but compared with broad-spectrum insecticides, they are less harmful to nontarget organisms, including beneficial insects, wildlife, and people (4). In the B. thuringiensis crops (cotton, corn, and potato) grown commercially so far, each individual plant produces only one type of B. thuringiensis toxin. These first-generation B. thuringiensis crops, which have been used on a large scale since 1996, occupied more than 11 million hectares worldwide in 2000 (13).

The evolution of resistance by insect pests threatens the continued success of this technology. So far, no insect resistance to a B. thuringiensis crop has been reported from the field. However, many pests have evolved resistance to B. thuringiensis toxins in the laboratory, and the diamondback moth (Plutella xylostella) has evolved resistance to B. thuringiensis sprays in the field (6, 8, 31). Thus, widespread adoption of B. thuringiensis crops and persistence of B. thuringiensis toxins in transgenic plants could cause rapid evolution of resistance in pests (9).

To counter resistance, different B. thuringiensis toxins can be used simultaneously or sequentially to control a particular pest. However, these tactics are likely to be useful only if pest resistance to one toxin does not confer strong cross-resistance to the other toxins. Although multiple toxin tactics have been widely discussed and modeled (2, 9, 26, 30), data are scarce on parameters such as cross-resistance between commercially relevant B. thuringiensis crops with different toxins.

A major factor limiting the acquisition of such data is the paucity of pest strains sufficiently resistant to B. thuringiensis toxins to complete their larval development on commercially grown B. thuringiensis crops. Larvae from some resistant strains of diamondback moth can survive on B. thuringiensis canola or B. thuringiensis broccoli with Cry1A or Cry1C toxins (20, 24, 38, 39), yet these transgenic plants are not grown commercially and were developed primarily for resistance research. In contrast, laboratory selection has produced strains of pink bollworm (Pectinophora gossypiella) that can complete larval development on first-generation B. thuringiensis cotton that is grown commercially on millions of hectares (3, 16, 17, 32).

First-generation B. thuringiensis cotton produces B. thuringiensis toxin Cry1Ac, which kills larvae of some major lepidopteran pests of cotton, including pink bollworm. To broaden the range of lepidopteran pests killed (1) and to combat potential problems with resistance, second-generation B. thuringiensis cotton plants have been developed that produce B. thuringiensis toxin Cry2Ab either alone or in combination with Cry1Ac. Current plans are to commercialize plants that produce Cry1Ac plus Cry2Ab (12).

Because Cry2Ab and Cry1Ac have limited homology (4), strong cross-resistance between plants producing these toxins is not expected. Cry2Ab is toxic to some lepidopteran larvae, including the major cotton pests Heliothis virescens and Helicoverpa zea (5). The efficacy of Cry2Ab against pink bollworm has not been reported previously, but the closely related B. thuringiensis toxin Cry2Aa is highly effective against pink bollworm (14, 34). For pink bollworm, Cry1Ac and Cry2Aa bind to different sites in the midgut (14) and initial screening with artificial diet bioassays suggested that resistance to Cry1Ac does not confer strong cross-resistance to Cry2Aa (34).

Our primary objective here was to determine if pink bollworm resistant to first-generation B. thuringiensis cotton with Cry1Ac could survive on second-generation B. thuringiensis cotton with Cry2Ab alone or in combination with Cry1Ac. To achieve this objective, we performed two sets of greenhouse bioassays in which a Cry1Ac-resistant strain of pink bollworm was tested on non-B. thuringiensis cotton, B. thuringiensis cotton with Cry1Ac, B. thuringiensis cotton with Cry2Ab, and two lines of B. thuringiensis cotton with Cry1Ac plus Cry2Ab. Our secondary objective was to contrast survival of Cry1Ac-resistant and -susceptible strains of pink bollworm in artificial diet bioassays with various concentrations of Cry1Ac and Cry2Aa. We performed artificial diet bioassays with Cry2Aa to enable comparisons with published data on responses to this toxin by resistant strains of several other pests (10, 11, 19, 22, 33, 37).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Pink bollworm.
Larvae were reared on wheat germ diet as described previously (29). We used six strains of pink bollworm: AZP-RO, AZP-RE, AZP99, APHIS-S, MOV97, and MOV97-R. AZP-RO is a Cry1Ac-resistant strain derived from the previously established resistant AZP-R strain as follows. Pink bollworm collected from 10 cotton fields in 1997 were used to start 10 laboratory strains (29). The AZP-R strain was created by pooling from these field-derived strains 159 individuals that survived on diet with 1, 3.2, or 10 µg of Cry1Ac per ml (32). The F5 progeny of the 159 parents of the AZP-R strain were fed a diet with 10 µg of Cry1Ac per ml. Survivors of this second round of selection were split into two subsets, AZP-RO and AZP-RE. AZP-RO was subsequently selected with 100 µg of Cry1Ac per ml of diet in every odd generation (F7, F9, F11, and so on), while AZP-RE was selected with 100 µg of Cry1Ac per ml of diet in every even generation (F6, F8, F10, and so on). AZP99 is a susceptible strain that was derived independently from the other strains studied. AZP99 was started by pooling 68 pupae and 108 adults from the parental through F3 generations of individuals collected from 15 field sites in Arizona in 1999. APHIS-S is a susceptible strain that had been reared in the laboratory for many years without exposure to toxins. MOV97 was started in 1997 from individuals collected from the Mohave Valley, Arizona. MOV97 was one of the 10 strains from which AZP-R was derived and was reared without exposure to toxins. MOV97-R was created by selecting a subset of the F10 generation of MOV97 with Cry1Ac. Subsequently, MOV97-R was selected on diet with 10 µg of Cry1Ac per ml every even generation (F12, F14, and so on).

Greenhouse bioassays.
We tested larvae from the Cry1Ac-resistant AZP-RO strain in two greenhouse bioassays (F21 in experiment 1 and F23 in experiment 2). We obtained seeds from Monsanto for five types of cotton: Deltapine 50 (Delta and Pine Land Co., Scott, Miss.), which is not transgenic; Deltapine 50B, which produces Cry1Ac; line 17180, which produces Cry2Ab; and lines 15813 and 15985, which produce Cry1Ac plus Cry2Ab. Plants were grown and tests were conducted in a greenhouse at the University of Arizona Campus Agricultural Center in Tucson. Plants were grown in 20-liter pots on drip irrigation, with halogen lighting providing a 16-h photophase. Each pot contained one to three cotton plants grown in Premiere High Performance Pro-Mix BX (Premier Horticulture Ltd., Dorval, Quebec, Canada) potting soil. Every 2 weeks, each pot was provided with ca. 16 g of Ironite (Ironite Products Co., Scottsdale, Ariz.), a fertilizer and micronutrient supplement. Plants were infested with pink bollworm eggs 106 days (experiment 1) and 84 days (experiment 2) after planting, when they had produced at least two bolls that were >14 days old (ca. 15-mm diameter) per plant. In both experiments, we used a randomized block design, with each of eight blocks containing one plant that produced Cry1Ac and one plant of each of the other three transgenic lines. In experiment 1, each block also contained a non-B. thuringiensis plant.

Experiment 1 started with infestation on 27 January 2000 and ended 9 March 2000. Experiment 2 started 6 April 2000 and ended 18 May 2000. To infest B. thuringiensis cotton plants, squares (10 by 10 mm) of paper towel (Chicopee, Benson, N.C.) onto which 40 to 50 eggs had been laid by moths in the laboratory were placed under the bracts of selected bolls. Non-B. thuringiensis plants were infested with 10 eggs per boll. We used fewer eggs per boll on non-B. thuringiensis plants because in previous tests we found lower survival on B. thuringiensis plants than on non-B. thuringiensis plants. Eggs on the paper towel squares hatched, and neonate larvae entered the bolls. Seven to 12 days after infestation, we counted entrance holes on all infested bolls. Fourteen days after infestation, the bolls were enclosed in small cages constructed from 167-ml plastic cups fitted with screened lids. A small piece of paper towel was placed in each of these cages to provide a substrate on which emerging larvae pupated. Beginning 21 days after infestation, boll cages were opened twice weekly to remove and record pupae. Forty-two days after infestation, all bolls were removed, taken to the laboratory, and dissected to check for survivors. Survival was estimated as the number of survivors divided by the number of entrance holes times 100%.

From each of the five types of cotton, three to six bolls were freeze-dried and ground. We sent subsamples of the resulting powder in numbered vials to EnviroLogix (Portland, Maine) for toxin testing with immunoassays. With the exception of two bolls sampled from a single plant of line 15813 in experiment 2 in which no Cry2Ab was detected (see Results), all bolls tested had the toxin sets characteristic of their variety or line.

Artificial diet bioassays.
We tested larvae from the APHIS-S, AZP-RO, AZP-RE, MOV97, MOV97-R, and AZP99 strains in laboratory bioassays on wheat germ diet (23, 29) containing no B. thuringiensis toxin (control), Cry1Ac, or Cry2Aa. Three sets of tests were done with Cry2Aa. In the first set, started on 9 December 1999, we tested APHIS-S, AZP-RO (F19), MOV97 (F27), and MOV97-R (F24). In the second set, started on 24 August 2000, we tested APHIS-S and AZP-RE (F26). Data for APHIS-S were pooled from the two sets. In the third set, started in May 2000, we tested AZP99 (F2) and AZP-RE (F24). APHIS-S, AZP-RO (F16), MOV97 (F24), MOV97-R (F24), and AZP-RE (F26) were tested with Cry1Ac between June 1999 and March 2000.

The source of Cry1Ac was MVPII (Dow Agrosciences, San Diego, Calif.), which is a liquid formulation containing a hybrid protoxin expressed in and encapsulated by Pseudomonas fluorescens (34, 36). Cry2Aa2 (referred to as Cry2Aa for brevity) was purified from Escherichia coli that produced Cry2Aa protoxin inclusion bodies (21). The Cry2Aa used in diet bioassays and the Cry2Ab in plants (formerly called CryIIA and CryIIB, respectively) have 88% amino acid identity (5). In pairwise comparisons against five lepidopteran pests, the median lethal concentrations of Cry2Aa and Cry2Ab differed by 1.1- to 5.8-fold against any particular pest (5). Here we do not assume that toxicity to pink bollworm is the same for Cry2Aa and Cry2Ab.

In the first two sets of diet bioassays, larvae were tested individually as follows. Diet was shredded into strips and dispensed into 33-ml cups. One neonate was transferred into each cup with a fine brush (23). Cups were held in darkness at 29 ± 2°C. Twenty to 60 larvae from each strain were tested individually at each concentration, with tests performed on at least two dates for each strain and concentration. In the third set, we tested 17 groups of larvae from AZP99 (F2) and 12 groups of larvae from AZP-RE (F24) with 20 to 200 larvae per cup (533 ml) at 27 ± 2°C. Although the density per cup varied widely in the third set, percent survival was not associated with density. After 21 days in all three sets, live fourth-instar larvae and pupae were scored as survivors. To adjust for control mortality, the survival on treated diet was divided by the survival on untreated diet.

Data analysis.
In the comparison from experiment 1 between nontransgenic cotton (Deltapine 50) and B. thuringiensis cotton with Cry1Ac (Deltapine 50B), we used multiple regression analysis (27) to determine whether the survival of AZP-RO (arcsine-square root transformed) was affected by the cotton variety, block, or number of entrance holes per boll. To determine whether Cry2Ab affected the survival of AZP-RO in experiments 1 and 2, we contrasted the survival on B. thuringiensis cotton with Cry1Ac (Deltapine 50B) and that on B. thuringiensis cotton in the three lines with Cry2Ab alone or with Cry1Ac plus Cry2Ab (lines 17180, 15813, and 15985). Because survival did not differ between experiments 1 and 2 (see Results), we pooled the data from the two experiments and used a randomization test (25) to check the statistical significance of the effects of Cry2Ab on survival. The survival values (n = 8) were sampled at random with replacement 1,000 times, with the difference between the mean of the randomized survival values on B. thuringiensis cotton with Cry1Ac (n = 2) and the mean of the randomized survival values on the three lines with Cry2Ab alone or with Cry2Ab plus Cry1Ac (n = 6) being calculated each time. Using the distribution of the 1,000 differences calculated, we estimated the probability of obtaining a difference equal to or greater than the actual mean difference observed in the two experiments (i.e., 22.9%). We also used a randomization test with 1,000 repetitions to determine whether the survival of individually tested larvae differed significantly between the two susceptible strains (APHIS-S and MOV97) and the three Cry1Ac-resistant strains (MOV97-R, AZP-RO, and AZP-RE) on artificial diet with 0.32 or 1.0 µg of Cry2Aa per ml. In the final analysis, we used a randomization test to determine whether the survival of larvae in replicated groups on artificial diet with 1.0 µg of Cry2Aa per ml differed significantly between the susceptible AZP99 strain and the resistant AZP-RE strain.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Greenhouse bioassays.
Two independent sets of greenhouse bioassays showed that larvae from a pink bollworm strain that survived on B. thuringiensis cotton with Cry1Ac had little or no survival on B. thuringiensis cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. In experiment 1, the mean survival of Cry1Ac-resistant pink bollworm larvae from strain AZP-RO was 20.4% on non-B. thuringiensis cotton compared with 22.4% on B. thuringiensis cotton that produced Cry1Ac (Table 1). In the comparison between non-B. thuringiensis cotton and B. thuringiensis cotton with Cry1Ac, survival was not affected by cotton variety (F = 0.017; df = 1, 30; P = 0.90), block (F = 0.98; df = 7, 30; P = 0.46), or number of entrance holes per boll (F = 0.30; df = 1, 30; P = 0.58). These results show that Cry1Ac in the bolls of B. thuringiensis cotton did not kill resistant larvae. In contrast, B. thuringiensis cotton producing Cry2Ab alone or Cry1Ac plus Cry2Ab killed all of the resistant larvae tested in experiment 1 (Table 1).

View this table: [in this window] [in a new window]

TABLE 1. Survival of Cry1Ac-resistant pink bollworm on nontransgenic cotton and B. thuringiensis cotton with Cry1Ac, Cry2Ab, or Cry1Ac plus Cry2Ab

In experiment 2, the mean survival of Cry1Ac-resistant larvae was 24.0% on B. thuringiensis cotton with Cry1Ac. No larvae survived on B. thuringiensis cotton with Cry2Ab alone or on B. thuringiensis cotton line 15985 with Cry1Ac plus Cry2Ab (Table 1). The mean survival on B. thuringiensis cotton line 15813 with Cry1Ac plus Cry2Ab was 1.8%.

The survival of Cry1Ac-resistant larvae on the four types of B. thuringiensis cotton (Deltapine 50B and lines 17180, 15813, and 15985) did not differ between experiments 1 and 2 (t = 0.035; df = 6; P = 0.97). Pooling results from experiments 1 and 2 showed that the average survival of Cry1Ac-resistant larvae was higher on B. thuringiensis cotton with Cry1Ac (23.2%) than on the three lines of B. thuringiensis cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab (0.3%) (randomization test, 1,000 repetitions, P = 0.008; see Materials and Methods).

The 11 survivors on B. thuringiensis cotton line 15813 with Cry1Ac plus Cry2Ab in experiment 2 were found on two bolls of a single plant. We were not able to rear progeny from the 11 survivors to determine if they had genetically based resistance to Cry2Ab. However, we did not detect Cry2Ab in two bolls of the single plant from which survivors emerged. Thus, we suspect that an absence or reduced concentration of Cry2Ab enabled survival on the aforementioned plant.

Diet bioassays.
Tests in which larvae from six strains of pink bollworm were fed artificial diet showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. In diet bioassays where larvae were tested individually (Table 2), the MOV97-R, AZP-RO, and AZP-RE strains of pink bollworm had >90% survival on diet with 10 µg of Cry1Ac per ml, but no larvae survived on diet with 3.2 or 10 µg of Cry2Aa per ml. In addition, when individually tested larvae ate diet with 0.32 µg of Cry2Aa per ml, the average survival did not differ between the three resistant strains (MOV97-R, AZP-RO, and AZP-RE) and the two susceptible strains (APHIS-S and MOV-97) (Table 2; randomization test, 1,000 repetitions, P = 0.137). However, survival at 1 µg of Cry2Aa per ml in individual diet bioassays ranged from 5.1 to 10.0% for the three Cry1Ac-resistant strains, whereas the two susceptible strains had 0% survival (Table 2). At this concentration, the average survival was significantly higher for the three resistant strains than for the two susceptible strains (Table 2; randomization test, 1,000 repetitions, P = 0.027). In diet bioassays where larvae were tested in replicated groups, survival at 1 µg of Cry2Aa per ml was greater for the resistant strain AZP-RE (mean = 2.2%; standard error = 0.5%; n = 12 groups) than for the susceptible strain AZP99 (mean = 0%; standard error = 0%; n = 17 groups) (randomization test, 1,000 repetitions, P < 0.001).

View this table: [in this window] [in a new window]

TABLE 2. Survival of pink bollworm strains on diet treated with Cry1Ac or Cry2Aa

Top Abstract Introduction Materials and Methods Results Discussion References

 
The finding here that survival of the resistant AZP-RO strain was not lower on B. thuringiensis cotton with Cry1Ac (22.4%) than on non-B. thuringiensis cotton (20.4%) differs from earlier reports that our laboratory-selected resistant strains of pink bollworm had considerably lower survival on B. thuringiensis cotton with Cry1Ac than on non-B. thuringiensis cotton (16, 17, 32). In one previous set of greenhouse tests with the AZP-R strain in September and October 1998, survival on B. thuringiensis cotton relative to that on non-B. thuringiensis cotton was 40% (3.1% on B. thuringiensis cotton with Cry1Ac versus 7.8% on non-B. thuringiensis cotton) (32), and in another previous set from September 1999, AZP-R survival on B. thuringiensis cotton with Cry1Ac relative to that on non-B. thuringiensis cotton was 46% (5.3% on B. thuringiensis cotton with Cry1Ac versus 11.5% on non-B. thuringiensis cotton) (17). Independent results from artificial diet bioassays show that as laboratory selection with Cry1Ac continued, survival at an extremely high concentration of Cry1Ac (320 µg per ml of diet) increased from 6% in June and July 1998 to 76% in September 1999 and to 86% in March 2000 (36). Thus, increased resistance to Cry1Ac might have contributed to higher survival on plants with Cry1Ac in the greenhouse tests reported here, which were started in January and April 2000 (see Materials and Methods). Note, however, that the survival of resistant larvae on non-B. thuringiensis cotton was higher here (20.4%) than in previous tests (7.8 and 11.5%). Thus, conditions in the present set of tests apparently were more favorable than those in previous tests for pink bollworm survival on non-B. thuringiensis cotton. Such favorable conditions, which might involve the quality of cotton plants or other environmental factors, may tend to reduce the difference in survival between non-B. thuringiensis cotton and B. thuringiensis cotton with Cry1Ac for resistant larvae.

The results reported here show that pink bollworm resistance to Cry1Ac did not confer strong cross-resistance to Cry2Ab. Cry1Ac-resistant larvae had little or no survival in bolls of second-generation B. thuringiensis cotton plants producing Cry2Ab alone or Cry1Ac plus Cry2Ab (mean survival = 0.3%). The only survivors on second-generation B. thuringiensis cotton were found on a single plant (line 15813 with Cry1Ac plus Cry2Ab) that had a reduced concentration of Cry2Ab in some bolls. If such variation occurred in a commercial variety, a small percentage of plants might be damaged in the field. B. thuringiensis cotton line 15985, which produces Cry1Ac plus Cry2Ab and is slated for commercialization, killed all larvae tested (n = 1,182 entrance holes). B. thuringiensis cotton line 17180, which produces only Cry2Ab, also killed all larvae tested (n = 1,108 entrance holes).

Cry1Ac-resistant pink bollworm also were not strongly cross- resistant to Cry2Aa in diet. These results confirm previous findings that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa in the APHIS-98R and AZP-R strains of pink bollworm (34). We can exclude strong cross-resistance to Cry2Aa, because no larvae survived at 3.2 or 10 µg of Cry2Aa per ml despite >90% survival of Cry1Ac-resistant strains at 10 µg of Cry1Ac per ml. With 1.0 µg of Cry2Aa per ml, the survival of the three Cry1Ac-resistant strains tested here (AZP-RE, AZP-RO, and MOV97-R) was 2 to 10%, whereas the survival of the three susceptible strains was 0%. Thus, the results reported here suggest weak cross-resistance to Cry2Aa at this relatively low concentration. In a previous test, however, the survival of a fourth Cry1Ac-resistant strain (APHIS-98R) was 0% at 1.0 µg of Cry2Aa per ml (34).

In parallel with the results for pink bollworm, resistance to Cry1A toxins did not confer strong cross-resistance to Cry2Aa in the NO-QA (33) and Loxahatchee (37) strains of diamondback moth. In the YHD2 strain of H. virescens, 10,000-fold resistance to Cry1Ac caused only 5- to 25-fold cross-resistance to Cry2Aa (10). However, in the CP73 strain of H. virescens, selection with Cry1Ac produced 50-fold resistance to Cry1Ac and 53-fold cross-resistance to Cry2Aa (11). Whereas most cases of resistance to Cry1A toxins in Lepidoptera are associated with reduced binding of toxin to midgut membrane target sites (35), resistance in the CP73 strain is apparently caused by a different mechanism that confers broader and lower levels of resistance (7, 11, 18). In addition, selection of Indianmeal moth (Plodia interpunctella) with various naturally occurring combinations of Cry1 toxins produced 5- to 24-fold resistance to Cry2Aa (19), and selection of beet armyworm (Spodoptera exigua) with Cry1C produced 73-fold cross-resistance to Cry2Aa (22). Cross-resistance to Cry2Ab occurred in a diamondback moth strain (BCS-Cry1C-1) resistant to Cry1C and Cry1Ac but not in a related strain (BCS-Cry1C-2) resistant to Cry1C but not Cry1Ac (39).

The lack of strong cross-resistance to Cry2A toxins exhibited by Cry1Ac-resistant pink bollworm implies that genes conferring high levels of resistance to Cry2A toxins will evolve independently of those conferring resistance to Cry1Ac. Nonetheless, we expect that field populations of targeted pests contain genes for high levels of resistance to Cry2A toxins. For example, selection with Cry2Aa produced 393-fold resistance to Cry2Aa in H. virescens (15).

Models predict that the greatest benefits of combining toxins in single plants (sometimes called "pyramiding" or "stacking") are achieved when no cross-resistance occurs, resistance to each toxin is rare and recessive, and refuges of plants without toxins are present (9, 26, 30). Results reported here show no strong cross-resistance to Cry2Ab or Cry2Aa in pink bollworm resistant to Cry1Ac but suggest weak cross-resistance to Cry2Aa. Preliminary tests also suggest weak cross-resistance of the AZP-RE strain to Cry2Ab in artificial diet. Simulation results suggest that weak cross-resistance would have a relatively minor effect on the evolution of resistance (2).

Unlike the relatively minor effect of weak cross-resistance, deviations from recessive inheritance and increases in the frequency of resistance greatly diminish the expected advantage of combining toxins relative to deploying them sequentially (9, 26, 30). The inheritance of pink bollworm resistance to B. thuringiensis cotton with Cry1Ac is recessive (16, 17, 32), but the inheritance of resistance to Cry2A toxins in pink bollworm has not been examined. To evaluate the risk of resistance to Cry2Ab in pink bollworm and the potential benefits of combining versus rotating Cry1Ac and Cry2Ab toxins, estimates of the frequency and analysis of the characteristics of genes conferring resistance to Cry2Ab are needed. Furthermore, increases in the frequency of pink bollworm resistance to Cry1Ac between now and the deployment of second-generation B. thuringiensis cotton would reduce the benefits of combining Cry1Ac with Cry2Ab.

 
We thank Juan Ferré, Ruud de Maagd, and Mike Caprio for comments; Stacy Anderson, Danny Holley, and the staff of the Extension Arthropod Resistance Management Laboratory for technical assistance; Jim Baum for amino acid sequence alignments; and Dow Agrosciences for providing MVPII.

Support was provided by USDA-NRI grant 99-35302-8300, Monsanto, and the University of Arizona.

 
* Corresponding author. Mailing address: Department of Entomology, University of Arizona, Tucson, AZ 85721. Phone: (520) 621-1141. Fax: (520) 621-1150. E-mail: brucet{at}ag.arizona.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, August 2002, p. 3790-3794, Vol. 68, No. 8
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.8.3790-3794.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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