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Applied and Environmental Microbiology, May 1999, p. 2049-2053, Vol. 65, No. 5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Antagonism between Cry1Ac1 and Cyt1A1 Toxins of
Bacillus thuringiensis
M. Cristina
del
Rincón-Castro,
José
Barajas-Huerta, and
Jorge E.
Ibarra*
Departamento de Biotecnología y
Bioquímica, Centro de Investigación y de Estudios
Avanzados del IPN, 36500 Irapuato, Gto., Mexico
Received 13 October 1998/Accepted 12 February 1999
 |
ABSTRACT |
Most strains of the insecticidal bacterium Bacillus
thuringiensis have a combination of different protoxins in their
parasporal crystals. Some of the combinations clearly interact
synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we
describe a novel joint activity of toxins from different strains of
B. thuringiensis. In vitro bioassays in which we used pure,
trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis
subsp. israelensis, and Trichoplusia ni
BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics.
The 50% lethal concentrations (LC50s) were estimated to be
4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of
medium. When mixtures of these toxins in different proportions were
assayed, eight different LC50s were obtained. All of these LC50s were significantly higher than the expected
LC50s of the mixtures. In addition, a series of bioassays
were performed with late first-instar larvae of the cabbage looper and
pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations
of the two toxins. The estimated mean LC50 of Cry1Ac1 was
2.46 ng/cm2 of diet, while Cyt1A1 crystals exhibited no
toxicity, even at very high concentrations. The estimated mean
LC50s of Cry1Ac1 crystals were 15.69 and 19.05 ng per
cm2 of diet when these crystals were mixed with 100 and
1,000 ng of Cyt1A1 crystals per cm2 of diet, respectively.
These results indicate that there is clear antagonism between the two
toxins both in vitro and in vivo. Other joint-action analyses
corroborated these results. Although this is the second report of
antagonism between B. thuringiensis toxins, our evidence is
the first evidence of antagonism between toxins from different
subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in
vivo and in vitro. Some possible explanations for this relationship are discussed.
| |
INTRODUCTION |
Bacillus thuringiensis is
an entomopathogenic bacterium that is produced commercially and
accounts for more than 90% of the world market for biological
insecticides. During sporulation, the bacterial cells produce
proteinaceous crystalline inclusions. These crystals are composed of
protein protoxins that are modified in the insect gut to produce active
toxins, the 
-endotoxins. These proteins are toxic to lepidopteran,
dipteran, and coleopteran insects, and toxicity to nematodes, mites,
and protozoans has also been reported (17). The mode of
action of these proteins is based on solubilization and partial
proteolysis in the insect intestine, in which the activated toxins
interact with the membranes of columnar cells of the intestinal
epithelium and damage the integrity of the gut lining; this is followed
by paralysis of the host and death (10).
The amino acid or gene sequences of more than 100 different toxin
proteins (Cry proteins) have been reported to date. Because of this
sequence information, the relationships among the various Cry proteins
that are active against lepidopteran, dipteran and coleopteran species
have become apparent. In addition, a distinct group of toxins, the Cyt
proteins, which are found only in mosquitocidal strains, has been
detected. The Cyt and Cry proteins exhibit no sequence homology. Many
strains of B. thuringiensis contain different combinations
of toxins in their parasporal crystals; these toxins, such as the
toxins present in strain HD-1 of B. thuringiensis subsp.
kurstaki, may be closely related. Other strains,
particularly strains exhibiting mosquitocidal activity, contain diverse
Cry and Cyt proteins in their parasporal crystals (7).
Few workers have attempted to evaluate the combined action of toxins
that naturally occur together or experimental mixtures of different
toxins. Synergistic interactions between different -endotoxins have
been described occasionally; these interactions include the synergism
among B. thuringiensis subsp. kurstaki toxins and
the synergism among B. thuringiensis subsp.
israelensis toxins (16, 18). Other studies of
interactions between -endotoxins have revealed additive effects, and
there has been only one previous report concerning unambiguous
antagonism between closely related toxins (13).
In the present paper we describe an antagonistic relationship between
the following two quite different B. thuringiensis toxins: Cry1Ac1, a toxin with high activity toward members of the Lepidoptera, and Cyt1A1, which originated from a mosquitocidal strain but has a
broad activity range due to its cytolytic capacity.
| |
MATERIALS AND METHODS |
Strains and plasmids.
The recombinant plasmids
pHT3101-Cry1Ac1 and pWF45 were used to produce the Cry1Ac1 and Cyt1A1
proteins, respectively. These plasmids were kind gifts from Brian
Federici (University of California, Riverside). Acrystaliferous strain
cry
B of B. thuringiensis was used for
transformation with the recombinant plasmids. Transformed bacteria were
grown in Tris-G supplemented with 25 µg of erythromycin per ml
(15).
Preparation of toxins.
Crystals were harvested from the
transformed strains and purified by using the procedure described by
López-Meza and Ibarra (15). Crystals were solubilized
by using 50 mM Na2CO3 at 37°C for 2 h at
pH 10.5 for Cry1Ac1 or at pH 9.5 for Cyt1A1 (1 µg of protein/ml); 25 mM dithiothreitol was also added to the preparations of Cry1Ac1. The
concentrations of proteins in supernatants were determined by the
method of Bradford (1) by using bovine serum albumin as the
standard. The solubilized protoxins were activated by using a procedure
described elsewhere (15).
Bioassays with BTI-Tn5B1-4 cells.
Cell line BTI-Tn5B1-4 of
Trichoplusia ni was generously donated by Robert R. Granados
(Boyce Thompson Institute) and was used to determine the biological
activities of the toxins. This cell line was maintained in
25-cm2 culture bottles (Falcon) at 28°C by using TC-100
medium supplemented with 10% fetal bovine serum. To determine 50%
lethal concentrations (LC50s), Cyt1A1 protein was tested at
concentrations of 3.36 to 20 ng/ml, and concentrations between 468.75 and 15,000 ng/ml were used for Cry1Ac1 protein. Four different
Cry1Ac1/Cyt1A1 ratios were also tested; these ratios were 99:1,
99.5:0.5, 99.75:0.25, and 99.875:0.125, and two replicates were tested
for each ratio. The concentrations of the toxins in the test
preparations ranged from 11.62 to 17.27 and from 1,723 to 9,905 ng/ml
for Cyt1A1 and Cry1Ac1, respectively.
The in vitro bioassays were based on the assay of Chow and Gill
(5), with minor modifications. Microtiter plates with 96 wells were inoculated with 50,000 cells/well. The cells were allowed to
adhere for 1 h at 28°C, and each well was then inoculated with a
different concentration of activated toxin(s). The cells were incubated
in the presence of the toxin(s) for 4 h at 28°C, and then the
preparations were centrifuged at 1,090 × g for 5 min. Living cells were quantified as described elsewhere (5).
Analysis.
The LC50s of toxins acting
independently were estimated by a Probit analysis (8) based
on at least three independent bioassays. When toxin mixtures were used,
eight bioassays were performed for each toxin combination. The combined
effect of the toxins was analyzed by the Tammes-Bakuniak graphical
method by using isobolograms (2). The expected toxicity of a
toxin mixture was calculated by using the following formula of
Tabashnik (18):
where LC
50(m) is the expected
LC
50, which is the harmonic mean of the LC
50s
estimated for toxins
a and
b acting
separately
and
ra and
rb are the
relative proportions of toxin
a and toxin
b in
the mixture,
respectively.
Bioassays performed with T. ni larvae.
T.
ni larvae were maintained on a semisynthetic diet under insectary
conditions by using previously described techniques (11). To
determine the LC50 of a pure crystal preparation of
Cry1Ac1, eight concentrations were tested; the maximum concentration
was 40 ng of toxin/cm2 of diet, and a dilution factor of
0.6 was used to obtain the lower concentrations. To determine the
equivalent value for pure preparations of Cyt1A1 toxin, concentrations
of 0.01, 0.1, 1.0, and 10 µg/cm2 of diet were tested. In
additional bioassays, larvae were offered a single dose consisting of 1 µg of Cyt1A1 activated toxin per larva.
The bioassays in which toxin combinations were used were performed by
using the concentrations of Cry1Ac1 toxin described
above and 1.0 or
0.1 µg of Cyt1A1 toxin per cm
2 of diet. Mortality was
recorded after incubation for 5 days under
insectary conditions. The
mortality data were subjected to a Probit
analysis.
| |
RESULTS |
Effects of toxins on BTI-Tn5B1-4 cells.
Both Cyt1A1 and
Cry1Ac1 toxins caused cytolytic damage when they were tested on
monolayers of BTI-Tn5B1-4 cells (Fig. 1). The cell responses to the toxins included cell rounding, followed by
swelling and the formation of membrane vesicles and finally lysis. The
Cyt1A1 toxin was far more active than Cry1Ac1, producing complete lysis
more quickly and at lower concentrations. The typical spindle shape of
the BTI-Tn5B1-4 cells (Fig. 1a) disappeared after 1 h of exposure
to Cyt1A1 (Fig. 1b). Moreover, following the 4-h incubation period of
the assay, the cells disintegrated completely, leaving only cell debris
(Fig. 1b, arrow).
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FIG. 1.
BTI-Tn5B1-4 cell line. (a) Normal cells. (b) Cells
1 h after treatment with Cyt1A1. The arrow indicates a cell
exhibiting typical symptoms caused by this toxin.
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|
Toxicity in vitro.
When the toxins were assayed individually
with cells, the LC50s were estimated to be 11.64 ng/ml for
CytA1 and 4,957 ng/ml for Cry1Ac1 (Table
1), which indicated that Cyt1A1 was more
than 400 times more active than Cry1Ac1 with the cell line used. When mixed toxins were assayed in four different proportions, the estimated LC50s varied from 1,740 to 9,308 ng of total protein per ml
(sum of the two toxins) (Table 1). The LC50s increased as
the proportion of the Cry1Ac1 toxin increased. The narrow confidence
limits of the calculated LC50s indicate that our estimates
are very reliable. The high level of toxicity of Cyt1A1 compared to
Cry1Ac1 was also evident from the marked separation of the
corresponding regression lines and the location of the Cyt1A1 line far
to the left on the graph (Fig. 2).
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TABLE 1.
Estimated LC50s of Cry1Ac1 and Cyt1A1 toxins
acting individually and in mixtures against the BTI-Tn5B1-4 cell line
and comparison with the expected LC50s of mixtures
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FIG. 2.
Concentration-mortality relationship for independent
pure suspensions of Cry1Ac1 and Cyt1A1 toxins and for toxin mixtures,
as determined with BTI-Tn5B1-4 cells. Rep. 1 and Rep. 2, observed
regression lines obtained from bioassays 1 and 2, respectively, in
which the Cry1Ac1/Cyt1A1 ratio was 99.875:0.125.
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|
Joint-action analysis. (i) Regression lines.
When an expected
regression line was constructed by using the expected data for the
different concentrations tested and a Cry1Ac1/Cyt1A1 ratio of
99.875:0.125, this line was significantly separated (especially at the
higher concentrations) from the regression lines obtained by using data
from the bioassays performed with the same toxin ratio (Fig. 2).
(ii) Tabashnik's formula.
We compared the estimated
LC50s obtained in the bioassays in which all of the
different ratios and replicates of toxin mixtures were used with the
LC50s calculated by the formula of Tabashnik (18). The results indicated that the expected
LC50s were always significantly lower (based on their
fiducial limits; P = 0.05) than the estimated
LC50s (Table 1) and that the combinations of the two toxins
exhibited less toxicity than each toxin acting independently.
(iii) Tammes-Bakuniak graphical analysis.
All eight points for
the observed LC50s in the mixed-toxin assays fell above the
isobole generated from assays in which each toxin was used alone.
Moreover, the points were above the 95% confidence interval for the
single-toxin isobole (Fig. 3). The locations of these points on the graph indicate that there was antagonism between the two toxins.
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FIG. 3.
Isobolograms produced by the Tammes-Bakuniak graphical
method for the Cry1Ac1 and Cyt1A1 toxins acting in combination. FL,
fiducial limit.
|
|
In vivo toxicity tests.
The in vivo toxicity tests performed
with T. ni larvae indicated that Cry1Ac1 had a high level of
toxic activity (LC50, 2.41 ng/cm2 of diet),
whereas Cyt1A1 had no toxic effect even at the highest concentration
tested (10 µg/cm2 of diet) or even when the toxin was
activated (Table 2). When combinations of
these toxins were tested, however, there was a significant reduction in
the activity of Cry1Ac1. At Cyt1A1 toxin concentrations of 0.1 and 1.0 µg/cm2, the LC50s of Cry1Ac1 (15.64 and 19.05 ng/cm2) increased 6.5-fold and 8-fold, respectively.
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TABLE 2.
Comparison of LC50s estimated from bioassays
performed with first-instar larvae of T. ni, in which
Cry1Ac1 was used alone and in combination with two concentrations
of Cyt1A1
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|
The antagonistic effect of Cyt1A1 was also evident when the regression
lines from the Probit analysis were compared (Fig.
4). The line for Cry1Ac1 acting alone
shifted markedly to the
right when this toxin was mixed with 0.1 and
1.0 µg of Cyt1A1
per cm
2.
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FIG. 4.
Concentration-mortality relationship for the Cry1Ac1
toxin acting alone or mixed with Cyt1A1, as determined with
first-instar T. ni larvae. The mixtures contained different
concentrations of Cry1Ac1 and 1.0 µg (combination I) or 0.1 µg
(combination II) of Cyt1A1 toxin per cm2.
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| |
DISCUSSION |
This paper is the first report which describes the combined action
of the Cry1Ac1 and Cyt1A1 toxins of B. thuringiensis both in
vitro and in vivo. All three techniques employed in this study revealed
that there is some antagonism between these two toxins. The Cry1Ac1
toxin was very toxic to T. ni larvae, while Cyt1A1 exhibited
no obvious toxicity even at the highest concentration tested. In spite
of the known lack of specificity of Cyt1A1, receptors determine the
susceptibility of mosquito larvae to Cyt1A1 (9); hence, the
lack of activity of Cyt1A1 against T. ni larvae may be due
to the absence of specific cellular receptors for this toxin on the
surfaces of columnar cells of the T. ni larval midgut. The
difference in toxicity may also be related to differences in the
proteases of T. ni and mosquito larvae, which may differ in
their abilities to cleave the Cyt1A1 protoxin. However, no toxicity was
observed even when Cyt1A1 was activated with trypsin. For the moment,
these explanations are merely speculative; however, they are consistent
with previous results obtained when Cyt1A1 was tested against
Manduca sexta larvae (12). As a result of this
work, interesting data might emerge from similar assays conducted with
mosquito larvae and mosquito cell lines.
Both toxins caused cell lysis in vitro, which was expected. Unlike the
in vivo test results, however, Cyt1A1 toxin exhibited much higher lytic
activity than Cry1Ac1 exhibited with T. ni cells in vitro.
This may have been due to the origin of the cell line, which was cloned
from embryonic tissue rather than differentiated epithelial tissue,
which is the principal target for this toxin (10). These
results clearly show the serious limitation of using undifferentiated
cell lines to assess the toxicity or the mode of action of Cry toxins.
In contrast, the Cyt1A1 toxin appears to act as a nonspecific detergent
with very different types of insect cells (9), as it is
known that Cyt1A1 interacts with phospholipids in organized bilayers
(19) and that this interaction catalyzes insertion of Cyt1A1
into the membrane without the mediation of specific membrane proteins
(14).
Such effects have been studied with diverse insect cell lines,
including cell lines derived from Aedes aegypti, Aedes
albopictus, Anopheles stephensi, Culex
quinquefasciatus, Choristoneura fumiferana, Helicoverpa zea, Spodoptera frugiperda,
Mamestra brassicae, and Lymantria dispar (4,
20). Toxic activity of Cyt1A1 has not been observed previously
with BTI-Tn5B1-4 cells, but the effects observed were similar to the
effects observed with other insect cell lines challenged with similar
concentrations of toxin (4, 20).
Additive and synergistic effects have been observed previously with the
toxins of B. thuringiensis. Some studies on the interactions of toxins found in B. thuringiensis subsp.
israelensis have revealed evident synergism when the toxins
act in different combinations against Aedes aegypti,
Culex quinquefasciatus, and Anopheles stephensi mosquito larvae; an exception is the additive effect observed with
Cry4B1 and Cry11A1 (3, 16, 18). Among the lepidopteran toxins, the Cry1Ac1 toxin of B. thuringiensis subsp.
kurstaki exhibited synergism in combination with the Cry1Aa1
toxin from the same subspecies when the toxins were tested against
L. dispar larvae (13). Other reports of synergism
between B. thuringiensis toxins are unclear (21).
In contrast, previous reports of antagonism between B. thuringiensis toxins have been scarce. A clear antagonistic effect was observed with the Cry1Aa1 and Cry1Ab1 toxins of B. thuringiensis subsp. kurstaki when they were tested
against L. dispar larvae (13). However, ambiguous
antagonism between the B. thuringiensis subsp.
israelensis Cry4 and Cry11 toxins (the nomenclature of the
toxins has changed since this study was performed) (6) was
reported when they were tested against Aedes aegypti larvae (18), and the same combination was later reported to have an additive effect (16). The present study differs from the
previous studies in that it assessed in vitro and in vivo interactions between toxins from different subspecies (B. thuringiensis
subsp. kurstaki and B. thuringiensis subsp.
israelensis). The mechanism behind the antagonism observed
in the previous studies and the present study is not known. It is
possible that toxins may interact physically to form a complex, thus
blocking one or more of the active sites on one or more molecules.
Alternatively, antagonism may be a result of competition for space (not
receptors) on the cell surface. Such hypotheses require experimental
confirmation of the underlying cause(s) of the antagonism, which was
observed in this study both in vitro and in vivo.
In the search for a new combination of toxins, workers generally look
for potentiation of each toxin when the toxins act jointly. It is clear
that a natural combination of Cyt and mosquitocidal Cry proteins
significantly improves the insecticidal activity of B. thuringiensis subsp. israelensis; however, a
combination consisting of Cyt proteins and lepidopteran-active Cry
proteins had never been tested. Such combinations may improve the
ability of B. thuringiensis to control crop pests even if
the toxins are present in very different strains, as recombinant
strains with combinations of toxins can be developed. However, as
important as finding synergistic effects among toxins is, the discovery of antagonism is relevant, not only because new joint-action studies may help improve our understanding of the mode of action of different B. thuringiensis toxins but also because using a combination
of these toxins in a bioinsecticide is now senseless.
| |
ACKNOWLEDGMENTS |
We thank Brian Federici and R. R. Granados for
providing crucial material, Trevor Williams for revising the
manuscript, and Javier Luévano-Borroel and Joel E. López-Meza for technical assistance.
| |
FOOTNOTES |
*
Corresponding author. CINVESTAV-IPN, Apartado Postal
629, 36500 Irapuato, Gto., Mexico. Phone: 52-462-39643. Fax:
52-462-45996. E-mail:
jibarra{at}irapuato.ira.cinvestav.mx.
| |
REFERENCES |
| 1.
|
Bradford, M. M.
1976.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:248-254[Medline].
|
| 2.
|
Busvine, J. R.
1971.
A critical review of the techniques for testing insecticides.
Commonwealth Agricultural Bureaux, London, England.
|
| 3.
|
Chang, C.,
S. M. Dai,
R. Frutos,
B. A. Federici, and S. Gill.
1992.
Properties of a 72-kilodalton mosquitocidal protein from Bacillus thuringiensis subsp. morrisoni PG-14 expressed in B. thuringiensis subsp. kurstaki by using the shuttle vector pHT3101.
Appl. Environ. Microbiol.
58:507-512[Abstract/Free Full Text].
|
| 4.
|
Chilcott, C. N., and D. J. Ellar.
1988.
Comparative toxicity of Bacillus thuringiensis var. israelensis crystal proteins in vivo and in vitro.
J. Gen. Microbiol.
134:2551-2558[Abstract/Free Full Text].
|
| 5.
|
Chow, E., and S. S. Gill.
1989.
A rapid colorimetric assay to evaluate the effects of Bacillus thuringiensis toxins on cultured insect cells.
J. Tissue Cult. Methods
12:39-42.
|
| 6.
|
Crickmore, N.,
D. R. Zeigler,
J. Feitelson,
E. Schnepf,
J. Van Rie,
D. Lereclus,
J. Baum, and D. H. Dean.
1998.
Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins.
Microbiol. Mol. Biol. Rev.
62:807-813[Abstract/Free Full Text].
|
| 7.
|
Federici, B. A.,
P. Luthy, and J. E. Ibarra.
1990.
Parasporal body of Bacillus thuringiensis israelensis, p. 17-65.
In
H. deBarjac, and D. J. Sutherland (ed.), Bacterial control of mosquitoes & black flies. Rutgers University Press, New Brunswick, N.J.
|
| 8.
|
Finney, D. J.
1977.
Probit analysis, 3rd ed.
Cambridge University Press. Cambridge, Cambridge, United Kingdom.
|
| 9.
|
Gill, S. S.,
G. J. P. Singh, and J. M. Hornung.
1987.
Cell membrane interaction of Bacillus thuringiensis subsp. israelensis cytolytic toxins.
Infect. Immun.
55:1300-1308[Abstract/Free Full Text].
|
| 10.
|
Gill, S. S.,
E. A. Cowles, and P. V. Pietrantonio.
1992.
The mode of action of Bacillus thuringiensis endotoxins.
Annu. Rev. Entomol.
37:615-636[Medline].
|
| 11.
|
Guy, R. H.,
N. C. Lepla,
J. R. Lye,
C. W. Green,
S. L. Barrette, and K. H. Hollien.
1985.
Trichoplusia ni, p. 487-494.
In
P. Singh, and R. F. Moore (ed.), Handbook of insect rearing, vol. II. Elsevier, Amsterdam, The Netherlands.
|
| 12.
|
Koni, P. A., and D. J. Ellar.
1994.
Biochemical characterization of Bacillus thuringiensis cytolytic -endotoxins.
Microbiology
140:1869-1880[Abstract/Free Full Text].
|
| 13.
|
Lee, M. K.,
A. Curtiss,
E. Alcantara, and D. H. Dean.
1996.
Synergistic effect of the Bacillus thuringiensis toxins CryIAa and CryIAc on the gypsy moth, Lymantria dispar.
Appl. Environ. Microbiol.
62:583-586[Abstract].
|
| 14.
|
Li, J.,
P. A. Koni, and D. J. Ellar.
1996.
Structure of the mosquitocidal -endotoxin CytB from Bacillus thuringiensis subsp. kyushuensis and implications for membrane pore formation.
J. Mol. Biol.
257:129-152[Medline].
|
| 15.
|
López-Meza, J. E., and J. E. Ibarra.
1996.
Characterization of a novel strain of Bacillus thuringiensis.
Appl. Environ. Microbiol.
62:1306-1310[Abstract].
|
| 16.
|
Poncet, S.,
A. Delécluse,
A. Klier, and G. Rapoport.
1995.
Evaluation of synergistic interactions among the CryIVA, CryIVB, and CryIVD toxic components of B. thuringiensis subsp. israelensis crystals.
J. Invertebr. Pathol.
66:131-135.
|
| 17.
|
Schnepf, E.,
N. Crickmore,
J. Van Rie,
D. Lereclus,
J. Baum,
J. Feitelson,
D. R. Zeigler, and D. H. Dean.
1998.
Bacillus thuringiensis and its pesticidal crystal proteins.
Microbiol. Mol. Biol. Rev.
62:775-806[Abstract/Free Full Text].
|
| 18.
|
Tabashnik, B. E.
1992.
Evaluation of synergism among Bacillus thuringiensis toxins.
Appl. Environ. Microbiol.
58:3343-3346[Abstract/Free Full Text].
|
| 19.
|
Thomas, W. E., and D. J. Ellar.
1983.
Mechanism of action of Bacillus thuringiensis var. israelensis insecticidal -endotoxin.
FEBS Lett.
154:362-368[Medline].
|
| 20.
|
Thomas, W. E., and D. J. Ellar.
1983.
Bacillus thuringiensis var. israelensis crystal -endotoxin: effects on insect and mammalian cells in vitro and in vivo.
J. Cell Sci.
60:181-197[Abstract].
|
| 21.
|
Van Frankenhuyzen, K.,
J. L. Gringorten,
R. E. Miine,
D. Gauthier,
M. Pusztai,
R. Brousseau, and L. Masson.
1991.
Specificity of activated CryIA proteins from Bacillus thuringiensis subsp. kurstaki HD-1 for defoliating forest Lepidoptera.
Appl. Environ. Microbiol.
57:1650-1655[Abstract/Free Full Text].
|
Applied and Environmental Microbiology, May 1999, p. 2049-2053, Vol. 65, No. 5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
