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Applied and Environmental Microbiology, July 2001, p. 3216-3219, Vol. 67, No. 7
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.3216-3219.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Cross-Resistance and Stability of Resistance to Bacillus
thuringiensis Toxin Cry1C in Diamondback Moth
Yong-Biao
Liu,1,*
Bruce E.
Tabashnik,1
Susan K.
Meyer,1 and
Neil
Crickmore2
Department of Entomology, University of
Arizona, Tucson, Arizona 85721,1 and
School of Biological Sciences, University of Sussex,
Brighton, United Kingdom2
Received 22 January 2001/Accepted 2 May 2001
 |
ABSTRACT |
We tested toxins of Bacillus thuringiensis against
larvae from susceptible, Cry1C-resistant, and Cry1A-resistant strains
of diamondback moth (Plutella xylostella). The
Cry1C-resistant strain, which was derived from a field population that
had evolved resistance to B. thuringiensis subsp.
kurstaki and B. thuringiensis subsp. aizawai, was selected repeatedly with Cry1C in the
laboratory. The Cry1C-resistant strain had strong cross-resistance to
Cry1Ab, Cry1Ac, and Cry1F, low to moderate cross-resistance to Cry1Aa and Cry9Ca, and no cross-resistance to Cry1Bb, Cry1Ja, and Cry2A. Resistance to Cry1C declined when selection was relaxed. Together with
previously reported data, the new data on the cross-resistance of a
Cry1C-resistant strain reported here suggest that resistance to Cry1A
and Cry1C toxins confers little or no cross-resistance to Cry1Bb,
Cry2Aa, or Cry9Ca. Therefore, these toxins might be useful in rotations
or combinations with Cry1A and Cry1C toxins. Cry9Ca was much more
potent than Cry1Bb or Cry2Aa and thus might be especially useful
against diamondback moth.
| |
INTRODUCTION |
Because of their safety to most
nontarget organisms, spray formulations of insecticidal crystal
proteins from Bacillus thuringiensis have been used widely
to control insect pests (13). Transgenic crops producing
B. thuringiensis toxins have also been grown on millions of
hectares (4). Evolution of resistance by insects is the
greatest threat to the continued success of B. thuringiensis.
Strains of more than 10 insect species have evolved resistance to
B. thuringiensis toxins in laboratory selections (2, 14). Yet, so far, resistance has been reported in field
populations of only the diamondback moth (2, 14),
Plutella xylostella (L.) (Lepidoptera: Plutellidae), a
worldwide pest of crucifers. Many populations of the diamondback moth
have evolved resistance to spray formulations of B. thuringiensis toxins in the field (2, 14).
The first cases of resistance to B. thuringiensis in the
field were to formulations of B. thuringiensis subsp.
kurstaki containing Cry1A toxins, which had been used widely
to control the diamondback moth (14). Strains of
diamondback moth resistant to B. thuringiensis subsp.
kurstaki and Cry1A toxins do not show cross-resistance to
Cry1C (1, 18), a toxin present in spray formulations of B. thuringiensis subsp. aizawai but not of
B. thuringiensis subsp. kurstaki
(9). More recently, as the use of B. thuringiensis subsp. aizawai has increased,
field-evolved resistance of the diamondback moth to this strain and to
Cry1C has occurred (9, 11, 24, 25).
Knowledge of resistance to Cry1A and Cry1C toxins in the diamondback
moth may be useful for managing resistance of the diamondback moth and
other pests to B. thuringiensis. The most common type of
resistance to Cry1A toxins in the diamondback moth, called mode 1 resistance, entails >500-fold resistance to at least one Cry1A toxin,
recessive inheritance, little or no cross-resistance to Cry1C, and
reduced binding of at least one Cry1A toxin to midgut membrane target
sites (21). For example, the NO-QA strain of diamondback
moth from Hawaii harbors an autosomal recessive gene that confers
resistance to Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, and Cry1Ja but not to
Cry1B, Cry1C, and Cry1I (3, 19, 22). Although reduced
binding of toxin to midgut membrane target sites is the only
well-documented mechanism of resistance in diamondback moth, this mechanism does not account for all examples of diamondback moth resistance to Cry1A toxins (1, 20, 24). In laboratory studies of field-selected strains of the diamondback moth, resistance generally declined when exposure to B. thuringiensis subsp.
kurstaki stopped (15).
Comparisons between diamondback moth resistance to Cry1A and Cry1C are
beginning to reveal some key differences and similarities. Unlike
resistance to Cry1A, reduced binding was not a major mechanism of
resistance to Cry1C in field-selected strains from Malaysia, Hawaii, or
Florida (10, 24, 25). In Hawaii and Florida strains, the
dominance of resistance to Cry1C increased as concentration decreased
and dominance was intermediate at the concentration killing 50% of the
larvae tested (LC50) (7, 25). Like most cases
of resistance to B. thuringiensis subsp.
kurstaki, resistance to B. thuringiensis subsp.
aizawai (24) and to Cry1C (25) declined when selection stopped.
Two methods for delaying resistance to B. thuringiensis are
to rotate or combine B. thuringiensis toxins that are
unlikely to produce cross-resistance to each other. However, in
contrast to the many published studies about cross-resistance
associated with Cry1A resistance, little has been reported about
cross-resistance associated with diamondback moth resistance to Cry1C.
Thus, the primary objective of the present study was to determine the
cross-resistance pattern of the Cry1C-resistant NO-95C strain of
diamondback moth from Hawaii. We also examined the stability of Cry1C resistance.
| |
MATERIALS AND METHODS |
Insects.
We used three strains of diamondback moth:
resistant strain NO-QA, resistant strain NO-95C, and susceptible strain
LAB-PS (8). NO-QA was derived in 1989 and NO-95C was
derived in 1995 from the NO field population in Hawaii, which had
evolved resistance first to B. thuringiensis subsp.
kurstaki and Cry1A toxins (16, 17) and later to
B. thuringiensis subsp. aizawai and Cry1C
(9). NO-QA was selected repeatedly with Dipel, a
spore-crystal formulation of B. thuringiensis subsp.
kurstaki, and was resistant to B. thuringiensis subsp. kurstaki, Cry1A toxins, Cry1F, and Cry1J
(20). NO-95C was selected repeatedly with MYX833-4C1
(referred to hereafter as Cry1C), a liquid formulation containing Cry1C
protoxin produced and encapsulated by transgenic Pseudomonas
fluorescens (Dow Agrosciences, San Diego, Calif.). Larvae were
reared on cabbage plants.
B. thuringiensis toxins.
We obtained from
Ecogen powders containing spores and crystals of the following B. thuringiensis proteins: Cry1Aa, Cry1Ab, Cry1Ac, Cry1Bb, Cry1Ca,
Cry1Fa, Cry1Ja, and Cry2Aa. Plant Genetic Systems provided powder
containing purified Cry9Ca toxin. The powders were diluted with
distilled water containing 0.2% Triton AG-98 (a surfactant; Rohm & Haas, Philadelphia, Pa.) for bioassays.
Bioassays.
We used leaf-residue bioassays (9).
One week after eggs were placed on cabbage plants, larvae were used for
bioassays. Ten third-instar larvae were placed on each treated leaf
disk. For each bioassay, four replicates (40 larvae) were tested at each concentration for each toxin and strain. After 2 days, fresh untreated leaves were added to each petri dish. Mortality was recorded
at 5 days. Bioassays and rearing were conducted at 28°C and a
photoperiod of 14:10 (L:D) h.
Two sets of bioassays were conducted for most toxins. In the first set,
we screened all toxins except Cry9Ca at two or three concentrations
against susceptible strain LAB-PS and resistant strain NO-95C. For
Cry1Bb, Cry1Ja, and Cry2Aa, resistant strain NO-QA was also tested in
the first set of bioassays. In the second set of bioassays, we
estimated LC50s for all toxins except Cry2Aa and
Cry1Bb by testing at five concentrations, including a control against
the NO-95C and LAB-PS strains. LC50s for Cry2Aa
and Cry1Bb were not estimated because of their low toxicity and our
limited quantities of these toxins.
Selection and crosses with susceptible strain.
Our goals
were to increase the resistance to Cry1C and to reduce the frequency of
the multitoxin resistance gene (3, 19) that confers
resistance to Cry1A toxins, Cry1F, and Cry1J. To increase resistance to
Cry1C, NO-95C was selected with Cry1C (MYX833-4C1) 22 times in 75 generations of laboratory rearing (Table
1). In each selection, 300 to 600 third-instar larvae were fed leaf disks that had been treated with
Cry1C (2.5 to 10 ml/liter) by using the method described above for the
bioassays. Mortality of Cry1C-selected larvae ranged from 41 to 99%
(Table 1). Adult survivors from treated leaf disks were pooled to
produce progeny for the next generation.
To reduce the frequency of the multitoxin resistance gene, we crossed
NO-95C with susceptible strain LAB-PS at generations
19 and 31 of
NO-95C. Mature larvae from each strain were sexed
(
6). We
pooled about 50 LAB-PS males with about 50 NO-95C females
for one cross
and pooled about 50 LAB-PS females with about 50
NO-95C males for the
reciprocal cross. F
1 offspring from the two
crosses were mixed and allowed to mate to produce
F
2 offspring.
Selection with Cry1C resumed with
the F
2 from the
crosses.
Stability of Cry1C resistance.
To evaluate the stability of
Cry1C resistance, we compared mortality at single concentrations (Table
1) and resistance ratios (see below) during the course of selection.
LC50s of Cry1C for NO-95C and a paired
susceptible strain were estimated at generations 1 and 2 (9), generations 8 and 10 (7), generations 40 and 45 pooled (10), and generation 60. Between the first
and second measurements of LC50 from generation 2 to 8, NO-95C was selected four times at 5 ml/liter. Between the last
two measurements of LC50 from generation 45 to
60, NO-95C was selected with Cry1C three times at 5 ml/liter.
Data analysis.
Mortality was adjusted for mortality in
controls using Abbott's method. We used probit analysis
(12) to estimate LC50s and their
95% fiducial limits (FLs) and slopes of concentration-mortality lines
and their standard errors (SE). LC50s were
considered significantly different if their 95% FLs did not overlap.
Resistance ratios were calculated as the LC50 for
the resistant strain divided by the LC50 for
LAB-PS.
| |
RESULTS |
Cross-resistance.
Compared with the susceptible LAB-PS strain,
the Cry1C-selected NO-95C strain had a 11-fold resistance to Cry1Ca in
powder and a 17-fold resistance to Cry1C in a liquid formulation (Table 2). NO-95C showed strong
cross-resistance to Cry1Ab, Cry1Ac, and Cry1Fa, low to moderate
cross-resistance to Cry1Aa and Cry9Ca, and no cross-resistance to
Cry1Bb, Cry1Ja, and Cry2Aa (Tables 2 and
3). In contrast to NO-95C, the NO-QA
strain selected with B. thuringiensis subsp.
kurstaki showed strong cross-resistance to Cry1Ja (Table 3).
NO-QA was not cross-resistant to Cry1Ba or Cry2Aa, which were least
potent to susceptible larvae of any of the toxins tested (Table 3).
Stability of Cry1C resistance.
Resistance to Cry1C in NO-95C
declined when selection with Cry1C stopped for many generations (Tables
1 and 4). During the course of selection,
variation in concentration and crosses with LAB-PS in generations 19 and 31 contributed to fluctuations in mortality (Table 1). However,
after 13 generations without selection and without crossing to LAB-PS,
mortality at 5 ml of Cry1C per liter increased from 52.2% at
generation 62 to 99.5% at generation 75 (Table 1). NO-95C was selected
four times at 5 ml of Cry1C per liter in six generations between the
first and second measurements of LC50. Compared
with a susceptible strain, the resistance of NO-95C to Cry1C increased
from 22-fold (generations 1 and 2 pooled) to 76-fold (generation 8). In
contrast, between generations 45 to 60, only three selections occurred
and the resistance ratio declined from 48 to 17 (Table 4).
| |
DISCUSSION |
Cross-resistance to B. thuringiensis toxins differs
between the NO-95C and NO-QA strains of the diamondback moth. Both
strains originated from the same watercress farm in Hawaii
(9), but NO-95C was selected in the laboratory with Cry1C,
while NO-QA was selected with B. thuringiensis subsp.
kurstaki. NO-95C evolved resistance to Cry1C, but NO-QA did
not (9). NO-95C had little cross-resistance to Cry1Aa,
whereas NO-QA was extremely resistant to Cry1Aa (20). In
contrast to the strong cross-resistance of NO-QA to Cry1Ja seen
here (Table 3) and previously (18, 20, 22), NO-95C was not
cross-resistant to Cry1Ja. Cross-resistance of NO-95C was 19-fold for
Cry1Ab and 20-fold for Cry1Ac (Table 2), which is higher than the
cross-resistance of NO-95C to Cry1Aa but much lower than the
>1,000-fold resistance of NO-QA to these toxins (17, 20).
Previous work showed that NO-95C resistance to Cry1Ab was inherited
independently from its resistance to Cry1C (7). Thus, resistance to Cry1Ab in NO-95C suggests that, despite two crosses with
the susceptible LAB-PS strain, we did not completely eliminate a
gene or genes conferring resistance to Cry1Ab (9) from
NO-95C. Nonetheless, the lack of cross-resistance to Cry1Ja and
relatively low cross-resistance to Cry1A toxins imply that NO-95C did
not have a high frequency of the multitoxin resistance gene that
confers resistance to Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, and Cry1Ja in
NO-QA (3, 19, 22).
Resistance to Cry1C in NO-95C was at most 76-fold greater, which is
much lower than the over 60,000-fold-greater resistance to Cry1C in the
Cry1C-Sel strain of diamondback moth from Florida (25).
Nonetheless, resistance to Cry1C was unstable in both NO-95C (Tables 1
and 4) and Cry1C-Sel (25). Unstable resistance is a
desirable characteristic for resistance management that can be exploited by rotating different B. thuringiensis toxins or by rotating B. thuringiensis toxins with other pest management agents.
Studies of cross-resistance in the NO-95C strain and in various other
resistant strains of the diamondback moth (1, 5, 18, 23)
suggest that resistance to Cry1A and Cry1C toxins confers little or no
cross-resistance to Cry1Bb, Cry2Aa, or Cry9Ca. Therefore, these toxins
might be useful in rotations or combinations with Cry1A and Cry1C
toxins. Cry9Ca was much more potent than Cry1Bb or Cry2Aa and thus
might be especially useful against the diamondback moth.
| |
ACKNOWLEDGMENTS |
We thank L. Anstine, J. Barnard, R. Biggs, S. Borgquist, W. Chang, M. Choo, N. Finson, B. Helvig, J. Riley, M. Silva, A. Taguchi, and J. Tuitele for technical assistance. We thank Juan Ferré for
thoughtful comments on the manuscript. We also thank Dow Agrosciences, Ecogen, and Plant Genetics Systems for providing insecticidal materials
for testing.
This study was supported by a USDA Western Regional PIAP grant,
USDA/CSRS Special Grant 95-34135-1771, USDA NRI grant 96-35302-3470, and the University of Arizona.
| |
FOOTNOTES |
*
Corresponding author. Present address: USDA, ARS, US
Agricultural Research Station, 1636 East Alisal St., Salinas, CA 93905. Phone: (831) 755-2825. Fax: (831) 755-2814. E-mail:
yb_liu{at}yahoo.com.
| |
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Applied and Environmental Microbiology, July 2001, p. 3216-3219, Vol. 67, No. 7
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.3216-3219.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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(2001). Variation in Susceptibility to Bacillus thuringiensis Toxins among Unselected Strains of Plutella xylostella. Appl. Environ. Microbiol.
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Abstract
Introduction
