Resistance to Toxins from Bacillus thuringiensis subsp. kurstaki ...

Vol. 59, No. 5

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1993, p. 1332-1335

0099-2240/93/051332-04$02.00/O Copyright © 1993, American Society for Microbiology

Resistance to Toxins from Bacillus thuringiensis subsp. kurstaki Causes Minimal Cross-Resistance to B. thuringiensis subsp. aizawai in the Diamondback Moth (Lepidoptera: Plutellidae)t BRUCE E. TABASHNIK,1* NAOMI FINSON,1 MARSHALL W. JOHNSON,' AND WILLIAM J. MOAR2

Department of Entomology, University of Hawaii, Honolulu, Hawaii 96822 1 and Department of Entomology, Auburn University, Auburn, Alabama 368492 Received 30 November 1992/Accepted 17 February 1993

Repeated exposure in the field followed by laboratory selection produced 1,800- to >6,800-fold resistance to formulations of Bacilus thuringiensis subsp. kurstaki in larvae of the diamondback moth, Plutella xylostella. Four toxins from B. thuringiensis subsp. kurstaki [CryIA(a), CryIA(b), CryIA(c), and CryIIA] caused significantly less mortality in resistant larvae than in susceptible larvae. Resistance to B. thuringiensis subsp. kurstaki formulations and toxins did not affect the response to CryIC toxin from B. thuringiensis subsp. aizawai. Larvae resistant to B. thuringiensis subsp. kurstaki showed threefold cross-resistance to formulations of B. thuringiensis subsp. aizawai containing CryIC and CrylA toxins. This minimal cross-resistance may be caused by resistance to CrylA toxins shared by B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai.

Insecticidal proteins from Bacillus thuringiensis are becoming increasingly important for management of pests. Technical advances, including expression of insecticidal crystal protein genes in transgenic crop plants and transgenic bacteria, are expected to increase the effectiveness of B. thuringiensis (3). At the same time, concerns about environmental hazards and widespread resistance in pests are reducing the usefulness of conventional synthetic insecticides (15, 16). Because B. thuringiensis had been used commercially for more than two decades without reports of substantial resistance in open-field populations of insect pests, some scientists predicted that resistance development was unlikely (9). However, reports of resistance to B. thuringiensis subsp. kurstaki in the diamondback moth, Plutella xylostella (L.), a global pest of cruciferous vegetables, have come from field populations in Hawaii, Florida, New York, the Philippines, and Malaysia (4, 18, 22, 24) and from greenhouse populations in Japan (29). These reports confirmed concerns that had been raised by laboratory selection for resistance to B. thuringiensis subsp. kurstaki in other members of the Lepidoptera (9, 10, 21). Although B. thuringiensis subsp. kurstaki is the most widely used subspecies of B. thuringiensis, it is only one of numerous isolates available (3). Thus, it is important to know whether resistance to B. thuingiensis subsp. kurstaki confers cross-resistance to other subspecies of B. thuin-

Dipel was associated with greater susceptibility to CryIC (31), a toxin found in some strains of B. thuringiensis subsp. aizawai and B. thuringiensis subsp. entomocidus, but not in B. thuringiensis subsp. kurstaki (1, 7). In contrast, Gould et al. (6) found that laboratory selection of the tobacco budworm, Heliothis virescens (F.), for resistance to CryIA(c), which is one of the toxins in B. thuringiensis subsp. kurstaki, increased resistance to CryIC. Studies of a strain of the diamondback moth from the Philippines suggested that repeated exposure to Dipel produced >200fold resistance to one of the toxins in Dipel [CryIA(b)], but did not affect responses to Dipel or CryIC (4). Repeated field exposure followed by laboratory selection produced resistance to Dipel in the diamondback moth in Hawaii (24-28). The objectives of this study were (i) to characterize responses of resistant diamondback moth larvae to formulations and single toxins of B. thuringiensis subsp. kurstaki and (ii) to determine whether resistance to Dipel conferred cross-resistance to B. thuringiensis subsp. aizawai and CryIC. MATERIALS AND METHODS Insects. Larvae were obtained from three laboratory colonies of the diamondback moth: LAB-P, NO-Q, and NOQA. LAB-P, a susceptible colony, was started with individuals from Pulehu, Maui, Hawaii, and had been reared in the laboratory for >80 generations without exposure to any insecticide. Resistant colonies NO-Q and NO-QA were started with individuals from a watercress farm near Pearl City, Oahu, Hawaii. Plants on this farm had been treated repeatedly with commercial formulations of B. thuringiensis subsp. kurstaki. First-generation offspring of field-collected individuals from this farm had 25-fold resistance to Dipel (24). Both NO-Q and NO-QA were selected in the laboratory for additional resistance to Dipel and were highly resistant to Dipel at the time of this study. All larvae were reared on cabbage. For additional details about the colonies and their maintenance see references 24 through 28. Formulations and toxins. We tested five formulations (Dipel, MVP, Xentari, NB200, and MYD577C) and five

giensis.

Resistance to Dipel, a commercial formulation of the HD-1 strain of B. thuringiensis subsp. kurstaki, conferred cross-resistance to 36 of 57 B. thuringiensis isolates tested against the Indianmeal moth, Plodia interpunctella Hubner (11). However, for 10 of 14 isolates of B. thuringiensis subsp. aizawai and one isolate of B. thuringiensis subsp. entomocidus tested, cross-resistance was minimal in the Indianmeal moth (11). Increased resistance of the Indianmeal moth to *

Corresponding author.

t This is paper

no. 3778 of the Hawaii Institute of Tropical Agriculture and Human Resources Journal Series, University of Hawaii, Honolulu, Hawaii.

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CROSS-RESISTANCE BETWEEN B. THURINGIENSIS SUBSPECIES

toxins [CryIA(a), CryIA(b), CryIA(c), CryIIA, and CryIC]. Dipel (Abbott) is a formulation of the HD-1 strain of B. thuringiensis subsp. kurstaki which contains CryIA(a), CryIA(b), CryIA(c), CryIIA, and CrylIB (1, 7). MVP (Mycogen) contains a single toxin that is very similar to the CryIA(c) toxin of B. thuringiensis subsp. kurstaki. The toxin in MVP was expressed in transgenic Pseudomonas fluorescens and encapsulated in cells of P. fluorescens that were killed after large-scale fermentation (19). Xentari (Abbott), NB200 (Entotech), and MYD577C (strain HD-276) (Mycogen) are formulations of B. thuringiensis subsp. aizawai, which typically contains CryIC, CryIA, and other toxins (7). Xentari (Abbott) contains CryIA(a), CryIA(b), CryIC, CryID, and CryIIB (1). The toxins in MYD577C and NB200 other than CryIC have not been reported.

Individual toxins were expressed in Eschenchia coli strains that each contained a single B. thuningiensis toxin gene. Toxin genes were obtained from B. thuringiensis subsp. kurstaki for CryIA(a), CryIA(b), CryIA(c), and CryIIA (14) and from B. thuringiensis subsp. aizawai for CryIC (8). The transformed E. coli strain expressing CryIC was a gift from L. Masson, National Research Council, Canada. Toxins from E. coli were expressed and purified by a modification of the procedure of Masson et al. (8) as described by Moar (14), except that all strains were grown in Terrific Broth (30) and no Renografin gradients were used. Toxin concentrations were determined by measuring total protein as described by Bradford (2), using bovine serum albumin as a standard, and then performing a visual analysis of the protein bands by using sodium dodecyl sulfate-

polyacrylamide gel electrophoresis. Bioassays. Larvae were tested for susceptibility by leaf residue bioassays at 28°C (24). In tests performed with single toxins, but not in tests performed with formulations, we added a surfactant (0.2% Triton AG98; Rohm and Haas). Each test was replicated at least four times. The mean number of larvae tested per colony for each bioassay was 267 (range, 223 to 444). Mortality was recorded at 24, 48, and 120 h. For each of the 10 materials tested, larvae from a resistant colony (NO-Q or NO-QA) and a susceptible colony (LAB-P) were tested under the same environmental conditions. Both of the resistant strains (NO-Q and NO-QA) were tested against Dipel. NO-Q larvae were tested against MYD577C. NO-QA larvae were tested against all of the other materials. Because environmental conditions before and during bioassays were the same for each colony, we concluded that any differences in responses between colonies were genetically based. Data analysis. For the five formulations and CryIC toxin, the concentrations expected to kill 50% of the larvae (LC50s), the fiducial limits, the slopes of the concentrationmortality lines, and the standard errors were estimated by probit analysis (17) as described by Tabashnik et al. (25). For these six materials, the LC50 of the resistant colony (NO-Q or NO-QA) was divided by the LC50 of the susceptible colony (LAB-P) to get an estimated resistance ratio. The difference between the values for susceptible and resistant colonies was considered statistically significant if the fiducial limits of their LC50s did not overlap. The results from the probit analysis were based on mortality at 48 h unless noted otherwise. Mortality at 48 h is strongly correlated with mortality at 24 and 120 h (25). We did not make statistical comparisons between different formulations or between formulations and toxins. Such contrasts might be confounded by differences in formulation ingredients other than toxins and by differences in the percentage of active ingredient.

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Nonetheless, all concentrations were expressed in common units (total milligrams of material per liter; not adjusted for the percentage of active ingredient). For the four toxins from B. thuningiensis subsp. kurstaki [CryIA(a), CryIA(b), CryIA(c), and CryIIA], the probit analysis did not provide reliable estimates of LC50, primarily because of extremely low levels of mortality of resistant larvae. For these experiments, we report levels of mortality at 120 h caused by 10 and 100 mg of toxin per liter, adjusted for control mortality. We used a one-tailed Mann-Whitney U test (20) to determine whether percentage of mortality per replicate was significantly higher for susceptible larvae than for resistant larvae. At least 39 larvae (average of 10 larvae per replicate with four replicates) of each strain were tested against each concentration of toxin. The criterion for significance was P < 0.05. RESULTS Both of the selected colonies (NO-Q and NO-QA) were highly resistant to formulations of B. thuningiensis subsp. kurstaki. The Dipel LC50s for the resistant strains were more than 1,000 times greater than the Dipel LC50s for the susceptible strain (LAB-P) (Table 1). The level of resistance to MVP, which contains a single toxin similar to CryIA(c) from B. thuringiensis subsp. kurstaki, was also extremely high. At 48 hours, the LC50 for the susceptible colony was 39.5 mg of MVP per liter (Table 1), whereas 268,000 mg of MVP per liter killed none of the resistant larvae (n = 41). At 120 h, the LC50 for the susceptible colony was 5.1 mg of MVP per liter, whereas 268,000 mg of MVP per liter killed 2% of the resistant larvae. Resistant larvae exhibited significantly lower levels of mortality than susceptible larvae in response to four toxins from B. thuringiensis subsp. kurstaki (Table 2). For CryIA(a), CryIA(b), and CryIA(c), 10 and 100 mg of toxin per liter killed only 0 to 26% of the resistant larvae, compared with 94 to 100% of the susceptible larvae (Table 2). The level of resistance to CryIIA was lower than the levels of resistance to the three CryIA toxins (Table 2). The extraordinarily high level of resistance to B. thuringiensis subsp. kurstaki conferred minimal cross-resistance to formulated B. thuringiensis subsp. aizawai and no crossresistance to CryIC (Table 1). For all three formulations of B. thuringiensis subsp. aizawai, the LC50s for the colonies selected with Dipel were higher than the LC50s for the unselected, susceptible colony. In two of three of these cases (Xentari and NB200), there was a statistically significant threefold increase in the LC50 for the selected colony compared with the unselected colony (Table 1). Response to CryIC was not significantly affected by resistance to B. thuyingiensis subsp. kurstaki (Table 1). DISCUSSION Our results with the diamondback moth represent the highest levels of resistance to B. thuringiensis reported so far and the first documentation of resistance to four toxins from B. thuringiensis subsp. kurstaki. Even extremely high concentrations of B. thuringiensis subsp. kurstaki formulations and toxins had limited effects on resistant larvae, which casts doubt on the idea of overcoming resistance with ultrahigh doses (13). Diamondback moth larvae from Hawaii with strong resistance to toxins from B. thuringiensis subsp. kurstaki were not resistant to CryIC toxin, which occurs in B. thuringiensis

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APPL. ENvIRON. MICROBIOL.

TABASHNIK ET AL.

TABLE 1. Responses of diamondback moth larvae to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai Susceptible larvae

Material

Slope

Soe

B. thuringiensis subsp. kurstaki Dipel Dipel MVP B. thunngiensis subsp. aizawai Xentari NB200 MYD577C CryIC

LC50

Resistant larvaea

95%limits Fiducial

(mg/liter) Upper

38.7 71.3 39.5

27.4 49.1 22.6

50.5 101.3 96.5

1.4 1.5 1.7 1.0

28.5 15.0 5.8 14.0

18.4 11.1 3.5 4.3

42.7 20.4 9.1

0.2 0.2 0.3 0.2

LC50

95%limits Fiducial

Resistance ratiob

(mg/liter) Lower

1.9 ± 0.3c 2.2 ± 0.3 1.1 ± 0.2 ± ± ± ±

Slope

79.7

0.6 ± 0.1c 0.8 ± 0.2e

NAf 1.4 1.6 1.0 1.3

± ± ± ±

0.2 0.2 O.le 0.3

129,000d 125,000d >268,000 81.2"

38.4d 9.9 16.2

Lower

Upper

5 x 104 4 x 104 NA

9 x 106 4 x 106 NA

55.9 28.5 6.0 6.9

118.2 53.1 16.0 43.2

3,300 1,800 >6,800 3 3 2 1

a The resistant colony was NO-QA unless noted otherwise. b The resistance ratio was calculated as follows: LC50 of resistant colony/LC50 of susceptible colony. c Estimate ± standard error. d The LC50 of the resistant colony was significantly greater than the LC50 of the susceptible colony as determined by nonoverlap of fiducial limits. I The resistant colony was NO-Q. f NA, not available.

subsp. aizawai and B. thuringiensis subsp. entomocidus (1, 7). These findings are consistent with a previous report that a strain of diamondback moth from the Philippines that was resistant to CryIA(b) toxin from B. thuringiensis subsp. kurstaki was not resistant to CryIC (4). Because reduced binding of toxin to brush border membrane epithelium is a mechanism of resistance in the Phillipines strain of diamondback moth (4), we suspect that a similar mechanism is also important in the diamondback moth from Hawaii. The Philippines strain was not resistant to Dipel (4), which contains several toxins in addition to CryIA(b) (1, 7). Thus, it was not certain whether resistance to Dipel would cause cross-resistance to CryIC. Our data suggest that even exceptionally high levels of resistance to Dipel and another formulation of B. thuringiensis subsp. kurstaki had no effect on the response to CryIC in the diamondback moth. This conclusion differs from previous results obtained with the Indianmeal moth and the tobacco budworm. Selection with Dipel increased the level of Indianmeal moth susceptibility to CryIC by about fourfold (31), whereas selection with CryIA(c) caused cross-resistance to CryIC in the tobacco budworm (6). Together, these data suggest that one cannot generalize about patterns of cross-resistance to B. thunngiensis toxins across insect species, even within the Lepidoptera. Although cross-resistance to CryIC was not seen in the TABLE 2. Responses of diamondback moth larvae to toxins from B. thuringiensis subsp. kurstaki Mortality (%) at a concn of': Toxin

CryIA(a)

CryIA(b) CryIA(c)

CryllA

10 mg/liter Susceptible Resistant larvae larvaeb

100 100 94 22

0 10 0 6

100 mg/liter Susceptible Resistant larvae larvaeb

100 100 100 82

6 26 9 41

a Percentage of mortality at 120 h, adjusted for the control percentage of mortality. b The resistant colony was NO-QA in all cases.

diamondback moth, we did find minor cross-resistance to formulations of B. thuringiensis subsp. aizawai. In addition, a field-collected strain of diamondback moth from Malaysia exhibited 100-fold resistance to B. thuringiensis subsp. kurstaki and 3-fold resistance to B. thuringiensis subsp. aizawai (22). For the Indianmeal moth, resistance to B. thuringiensis subsp. kurstaki also caused at least minor increases in resistance to all 14 isolates of B. thuringiensis subsp. aizawai tested by McGaughey and Johnson (11). The CrylA toxins in B. thuringiensis subsp. aizawai that are also found in B. thuringiensis subsp. kurstaki (1, 7) probably account for much of the observed cross-resistance. Our data suggest that CryIA toxins had little or no effect on Dipel-resistant larvae, yet they were highly toxic to susceptible larvae. Therefore, formulations of B. thunrngiensis subsp. aizawai, which have CrylA toxins as well as CryIC toxin (1, 7), were more toxic to susceptible strains than resistant strains of diamondback moth. CryID, which occurs in some strains of B. thunngiensis subsp. aizawai, but not in B. thuringiensis subsp. kurstaki (1, 7), exhibited low levels of toxicity to the diamondback moth (4). A complete understanding of this system will require knowledge of the proportions of each toxin in B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai, the toxicity of these compounds for susceptible and resistant larvae, and their interactions (23). Available evidence suggests that lepidopteran pests can evolve resistance to subspecies of B. thuringiensis that produce CryIC toxin. In laboratory tests, the Indianmeal moth readily developed resistance to two isolates of B. thuringiensis subsp. aizawai, one isolate of B. thuringiensis subsp. entomocidus, and a mixture of B. thunrngiensis subsp. aizawai and B. thuringiensis subsp. kurstaki (12). The results of laboratory bioassays of diamondback moth larvae suggest that two field populations in Thailand have become resistant to B. thuingiensis subsp. aizawai (5). Because B. thuringiensis subsp. aizawai contains CryIC and CryIA toxins, we hypothesize that selection with B. thuringiensis subsp. aizawai should cause stronger cross-resistance to B. thuringiensis subsp. kurstaki than the minimal cross-resistance to B. thuringiensis subsp. aizawai caused by selection with B. thuringiensis subsp. kurstaki.

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ACKNOWLEDGMENTS We thank W. McGaughey for his thoughtful comments and I. Mok and C. Y. Yap for technical assistance. We also thank L. Masson, Abbott Laboratories, Entotech, and Mycogen for providing insecticidal materials for testing. This research was supported by U.S. Department of Agriculture grant HAW00947H, by U.S. Department of Agriculture CSRS Special Grant in Tropical/Subtropical Agriculture 92-34135-7314, by the U.S. Department of Agriculture Western Region Pesticide Impact Assessment Program, by a grant from the B. t. Management Working Group, and by gifts from Abbott Laboratories and Entotech.

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