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Applied and Environmental Microbiology, July 2000, p. 2804-2810, Vol. 66, No. 7
Department of Microbiology, University of
Cape Town, Rondebosch 7701,1 and South
African Sugar Association Experiment Station, Mount Edgecombe
4300,2 South Africa
Received 12 October 1999/Accepted 28 April 2000
The cry1Ac7 gene of Bacillus thuringiensis
strain 234, showing activity against the sugarcane borer Eldana
saccharina, was cloned under the control of the tac
promoter. The fusion was introduced into the broad-host-range plasmid
pKT240 and the integration vector pJFF350 and without the
tac promoter into the broad-host-range plasmids pML122 and
pKmM0. These plasmids were introduced into a Pseudomonas
fluorescens strain isolated from the phylloplane of sugarcane and
the endophytic bacterium Herbaspirillum seropedicae found
in sugarcane. The ptac-cry1Ac7 construct was introduced into the chromosome of P. fluorescens using the integration
vector pJFF350 carrying the artificial interposon Omegon-Km. Western blot analysis showed that the expression levels of the integrated cry1Ac7 gene were much higher under the control of the
tac promoter than under the control of its endogenous
promoter. It was also determined that multicopy expression in P. fluorescens and H. seropedicae of
ptac-cry1Ac7 carried on pKT240 caused plasmid instability with no detectable protein expression. In H. seropedicae,
more Cry1Ac7 toxin was produced when the gene was cloned under the control of the Nmr promoter on pML122 than in the opposite
orientation and bioassays showed that the former resulted in higher
mortality of E. saccharina larvae than the latter. P. fluorescens 14::ptac-tox resulted in higher
mortality of larvae than did P. fluorescens
14::tox. An increased toxic effect was observed
when P. fluorescens 14::ptac-tox was
combined with P. fluorescens carrying the Serratia
marcescens chitinase gene chiA, under the control of
the tac promoter, integrated into the chromosome.
The gram-positive, aerobic,
spore-forming bacterium Bacillus thuringiensis has been used
as a safe alternative and supplement to chemical pesticides for over 2 decades. It is a pathogen of insect larvae which produces highly
specific crystal inclusions during sporulation. These parasporal
crystals consist predominantly of protoxin molecules known as
As part of an integrated pest management approach to the control of
E. saccharina in South Africa, the cry1Ac7 gene
from B. thuringiensis strain 234 was previously introduced
into P. fluorescens isolate 14 (13, 33). This
organism was isolated from the surface of sugarcane leaves, stems, and
borings and shown to be a good colonizer of the phylloplane of
sugarcane. Toxicity bioassays indicated that P. fluorescens
14 clones that expressed the gene were toxic to E. saccharina larvae, and greenhouse trials showed that sugarcane
plants inoculated with the strain carrying the integrated gene were
more resistant to E. saccharina damage than were untreated controls.
Although these results were encouraging, it was felt that there was
room for further improvement in the use of recombinant bacteria for the
control of this sugarcane pest. The aim of the work presented in this
paper was to increase A further strategy to improve the biocontrol of E. saccharina involved combining P. fluorescens strains
producing the Cry1Ac7 protein and a Serratia marcescens
chitinase, ChiA. Reports have shown that coapplication of B. thuringiensis Bacterial strains, plasmids, and culture conditions.
The
bacterial strains and plasmids used in this study are listed in Table
1. Rifampin-resistant P. fluorescens 14 was grown on Luria-Bertani medium (LB) or LB medium
with agar supplemented with rifampin (100 µg/ml). The sugarcane
endophyte H. seropedicae HRC54 was provided by J. Döbereiner of the Empresa Brasiliera de Pesquisa Agnopecuaria,
Brasilia, Brazil. A spontaneous nalidixic acid-resistant mutant,
H. seropedicae Nal1, was isolated. These strains were grown
in JNFb medium, which contained, per liter, 5 g of malic acid, 0.6 ml of K2HPO4, 1.8 ml of
KH2PO4, 0.2 g of MgSO4
· 7H2O, 0.1 g of NaCl, 0.2 g of
CaCl2 · H2O, 0.066 g of FeEDTA, 2 ml of
bromothymol blue, 2 ml of micronutrients, 0.02 g of yeast extract,
and 4.5 g of KOH (pH 5.8) and was supplemented with the
appropriate antibiotic. For solid JNFb, 17 g of agar was added per
liter; for semisolid JNFb, 1.9 g of agar was added per liter with
the yeast extract omitted; and for liquid JNFb medium, 1 g of
NH4Cl was added per liter in addition to yeast extract. The
micronutrients consisted of 0.2 g of
Na2MoO4 · 2H2O, 0.235 g of
MnSO4 · H2O, 0.28 g of
H3BO3, 0.008 g of CuSO4 · 5H2O, and 0.024 g of ZnSO4 · 7H2O per 200 ml of H2O.
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Biocontrol of the Sugarcane Borer Eldana saccharina by
Expression of the Bacillus thuringiensis cry1Ac7 and
Serratia marcescens chiA Genes in
Sugarcane-Associated Bacteria
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-endotoxins, Cry toxins, or Cry proteins. The crystal inclusions
dissolve in the larval midgut, where one or more protoxins are released
and proteolytically converted into smaller toxic polypeptides. The
activated toxins are highly specific to the insect and very specific in
their activity (14). Despite the success of conventional
B. thuringiensis-based products, they have several
disadvantages as bioinsecticides. In the case of the sugarcane borer
Eldana saccharina Walker (Lepidoptera: Pyralidae), a
widespread sugarcane pest which causes considerable crop loss in the
cane-growing areas of South Africa and Swaziland, these include
instability in the environment and on the surface of sugarcane, as well
as difficulty in reaching the internal regions where the larvae feed.
The use of recombinant DNA technology has provided solutions to the
problems through the development of two approaches, namely, genetically
modified microorganisms and transgenic plants (18, 21, 22, 25,
26).
-endotoxin expression by cloning the
cry1Ac7 gene under the control of the tac
promoter with subsequent integration of the cassette into the
chromosome of P. fluorescens 14. In addition, since
recombinant P. fluorescens 14 populations are not stably
maintained on sugarcane over long periods (33), the
potential of endophytic bacteria present in the interior regions of
healthy sugarcane plants that express the gene as a biocontrol agent
was investigated. Of particular interest is the gram-negative,
obligately endophytic, nitrogen-fixing bacterium Herbaspirillum
seropedicae, which has been isolated only from monocotyledonous
plants such as sugarcane, rice, sorghum, maize, 13 different
graminaceous weeds, and the roots of a pigeonpea plant (3, 6,
7). The use of an endophytic bacterium was also seen as a
possible solution to the problem of inaccessibility of conventional
B. thuringiensis-based products to the interior regions of
the plant. The advantages of using these recombinant endophytes is
their high stability in sugarcane and the ability to be transferred to
subsequent generations via sugarcane setts (4, 6, 7).
-endotoxins and bacterial chitinases
significantly increased the insecticidal effect of the former against
insect larvae (28, 31). It is believed that the chitinase
causes perforations in the chitin-containing peritrophic membrane of
the larvae, thereby increasing the accessibility of the midgut
membranes to the -endotoxin (28). The introduction of
both Cry and ChiA into bacteria or plants offers great potential for
increasing the insecticidal activity in transgenic systems where the
Cry toxins are expressed at low levels and/or in a crystalline form
(28).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Bacterial strains and plasmids used in this study
70°C.
Molecular techniques. Molecular techniques were performed as described by Sambrook et al. (29).
Western blot analysis.
Determination of the expression of
the cry1Ac7 gene in P. fluorescens 14 and
H. seropedicae Nal1 was carried out by Western blot
analysis. Cell extracts were prepared from 1 ml of stationary-phase cultures by resuspending cell pellets in 100 µl of denaturing loading
buffer (20). Samples (20 µl) were loaded onto a denaturing gradient (10 to 5%) acrylamide gel, and the proteins were separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by
the method of Laemmli (20). For the quantitative analysis of
Cry1Ac7 production, Escherichia coli cultures were grown
overnight at 30°C in LB medium supplemented with the appropriate
antibiotic, diluted 100-fold, grown to mid-exponential phase at 37°C,
and induced with 0.3 mM
isopropyl-
-D-thiogalactopyranoside (IPTG) as described
by Ausubel et al. (1). Uninduced controls were prepared by
dividing the mid-exponential-phase (optical density at 600 nm, 0.4)
culture in two before adding IPTG. Samples from both uninduced and
induced cultures were removed at various time intervals after
induction. Samples (1 ml) of cultures induced for 24 h were
sonicated, and the protein concentration was determined by the method
of Bradford (5). Volumes of denatured cell extracts representing 50 µg of protein were separated by SDS-PAGE.
Bacterial transformation by electroporation and conjugation.
Broad-host-range plasmids and the integration vector carrying the
ptac-cry1Ac7 cassette were electroporated into P. fluorescens 14 and H. seropedicae Nal1 using a
modification of the method of Waalwijk et al. (36). Cells
harvested at mid-exponential phase and washed three times in 300 mM
sucrose were electroporated in 40-µl volumes with 1 to 3 µg of DNA
using a Bio-Rad Gene Pulser and controller set at 25 µF, 2.5 kV, and
200 
. Phenotypic expression was carried out at 30°C for 2 h
in 1 ml of LB or JNFb medium (for P. fluorescens and
H. seropedicae, respectively). The cells were plated
undiluted onto LB medium with agar or JNFb solid medium supplemented
with kanamycin (100 µg/ml) and grown at 30°C. To increase the
electroporation efficiency of H. seropedicae, the method
described by Wirth et al. (37) was attempted.
Plasmid stability.
The stability of the pML122-derived
plasmids pMT7 and pMT11 carrying the cry1Ac7 gene in
H. seropedicae Nal1 was assessed. The strains were grown in
liquid JNFb medium supplemented with kanamycin (100 µg/ml) to
stationary phase at 30°C, diluted to 10
6 bacteria per
20 ml of JNFb medium without antibiotics, and grown to stationary
phase. This cycle of growth and dilution was performed five times. The
cells from a 106 dilution were plated onto JNFb plates
after each cycle of growth. One hundred of the resulting CFU were
patched onto JNFb plates supplemented with kanamycin (100 µg/ml). The
percentage of patched colonies which grew on these plates was recorded.
This experiment was performed three times.
Southern blot analysis. Southern blot analysis was used to demonstrate integration of the Omegon-Km-ptac-cry1Ac7 cassette into the chromosome of P. fluorescens 14 clones. Total bacterial DNA was isolated as described by Ausubel et al. (1). Probes were labeled with digoxigenin-11-dUTP (Boehringer Mannheim) by the random primed DNA labeling method in accordance with the manufacturer's instructions. Southern blot analysis was carried out by the method described in the digoxigenin-11-dUTP system user's guide.
Toxicity bioassays. Weighed quantities of the various freeze-dried bacterial preparations expressing the cry1Ac7 and chiA genes were mixed with weighed quantities of an artificial insect diet (11) such that a known amount of bacterial preparation per gram of diet was obtained. Aliquots (0.2 g) were added to Eppendorf tubes, and five 2-week-old E. saccharina neonate larvae were placed in each tube. Each treatment comprised five tubes. Mortality of the larvae was recorded every 24 h for 5 days. An analysis of variance was done with one between-subjects variable (treatment) and one within-subjects variable (time). As the response variable, mortality, was a proportion, it had to be transformed using the usual variance-stabilizing transformation for proportions, namely, arcsine square root (22). Multiple comparisons were made using Fisher's least-significant-difference procedure (15).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Introduction of the cry1Ac7 gene of B. thuringiensis isolate 234 into expression, broad-host-range, and integration vectors and Cry1Ac7 expression. In order to improve the expression of the cry1Ac7 gene, referred to as tox in this report, from pGH37D-1 (13) for subsequent biocontrol use, it was cloned into the plasmid pKK223-3 for expression under the control of the tac promoter. The 3.7-kb NdeI fragment of pGH37D-1, carrying the cry1Ac7 gene under the control of its own promoter, was cloned into the SmaI site of pKK223-3. The resulting construct was called ptac-tox. The 4-kb BamHI fragment of ptac-tox was cloned into the BamHI site of the broad-host-range plasmid pKT240, yielding pKTT (pKT240tactox). Overexpression of a chitinase gene on pKT240 was shown to be highly unstable in H. seropedicae and P. fluorescens (data not shown), and the highly expressed cry1Ac7 gene on pKTT had a detrimental effect on the latter strain (see below). It was therefore decided to clone the cry1Ac7 gene under the control of its own promoter into the broad-host-range plasmids pML122 and pKmM0 (32), a Kmr derivative of the broad-host-range plasmid pDER405 (27) shown to be stably maintained in Herbaspirillum spp. (data not shown). The 3.7-kb blunted NdeI fragment of pGH37D-1 was cloned into the blunted EcoRI site of pML122 in both orientations with respect to the Nmr promoter present on the vector, resulting in the plasmids pMT7 and pMT11 (pML122tox). In pMT7, the cry1Ac7 gene was cloned under the control of the Nmr promoter in addition to its endogenous promoter while in pMT11, the cry1Ac7 gene was under the control of its own promoter. The fragment was cloned into the EcoRV site of pKmM0 to yield pKmM0tox. For cloning into the integration vector, the BamHI ptac-cry1Ac7 fragment from ptac-tox was made blunt and cloned into the blunted NdeI site of the integration vector pJFF350, which carries the artificial interposon Omegon-Km, generating the Omegon-Km-ptac-cry1Ac7 cassette on pJTT (pJFF350tactox).
The expression of the cry1Ac7 gene in E. coli JM105 was determined by Western blot analysis, and it was evident that it is expressed from the tac promoter in ptac-tox at levels considerably higher than from its own promoter (results not shown).Construction of the ptac-chiA cassette and introduction into P. fluorescens Rif1. For high-level expression of the S. marcescens chiA gene in gram-negative bacteria for the biocontrol of phytopathogenic fungi, the gene was cloned under the control of the tac promoter and introduced into the chromosome of isolate P. fluorescens Rif1 as described by Downing and Thomson (8).
Construction of P. fluorescens 14 and H. seropedicae Nal1 strains expressing the cry1Ac7
gene.
The plasmids pKTT and pJTT, carrying the
ptac-cry1Ac7 cassette on pKT240 and the integration vector
pJFF350, respectively, were introduced into P. fluorescens
14 by electroporation. P. fluorescens 14(pKTT)
electrotransformants grew poorly in liquid medium, indicating that
constitutive expression of the cry1Ac7 gene at high levels
had a lethal effect on this organism. To circumvent this problem and to
prevent horizontal transfer of the gene to other bacterial species, the
Omegon-Km-ptac-cry1Ac7 cassette was inserted into the
chromosome and integration was confirmed by Southern blot analysis.
Total DNA of P. fluorescens
14::ptac-cry1Ac7 was cut with EcoRI and
probed with the 4-kb BamHI fragment of ptac-tox
carrying the ptac-cry1Ac7 cassette (Fig.
1). Four fragments of 3.5, 3.3, 0.7, and
0.2 kb hybridized to EcoRI-restricted pJTT. In all of the
clones analyzed, two EcoRI fragments of 0.7 and 0.2 kb,
corresponding to the fragments internal to the
Omegon-Km-ptac-cry1Ac7 cassette, and two of different sizes
greater than 3.5 and 3.3 kb, hybridized to the probe. Random, single
integration of the cassette was indicated by the fact that the two
larger EcoRI fragments were of different sizes and only two
of the larger EcoRI fragments were detected in these clones.
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Expression of the -endotoxin gene.
Expression of the
cry1Ac7 gene in P. fluorescens 14 and H. seropedicae Nal1 was determined by quantitative Western blot
analysis. The 134-kDa Cry1Ac7 protein was not detected in P. fluorescens 14 (pKTT) clones carrying the ptac-cry1Ac7
cassette on pKT240 (Fig. 2A, lane 4).
However, this gene, under the control of its endogenous promoter on
pKT240 and pDER405, was expressed in P. fluorescens 14 at
toxin protein levels of 3.5 and 2.2%, respectively, of the total
proteins (12). This implied that constitutive expression of
cry1Ac7 at high levels in P. fluorescens 14(pKTT)
must have resulted in the accumulation of mutants defective in
cry1Ac7 expression after overnight growth.
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mutants resulting from high levels of the
constitutively expressed cry1Ac7 gene in H. seropedicae(pKTT). In contrast, H. seropedicae(pMT7) clones with the cry1Ac7 gene downstream of the
Nmr promoter on pML122 produced higher levels of the
Cry1Ac7 protein than did H. seropedicae(pMT11) clones with
the gene in the opposite orientation with respect to this promoter
(Fig. 2B). This indicated that expression of the gene in the former
clones was under the control of the Nmr promoter, which
could explain the high levels of instability of this plasmid. Labes et
al. (19) reported that the Nmr promoter was an
efficient and more effective promoter than the tac promoter
for overexpression of foreign genes in soil bacteria, including
Pseudomonas spp. The Cry1Ac7 protein was also detected in
strains carrying pKmM0-tox (results not shown).
Effect of Cry1Ac7+ P. fluorescens and
H. seropedicae strains on E. saccharina
larvae.
The biological activity of Cry1Ac7+ P. fluorescens and H. seropedicae strains was determined
in toxicity bioassays using 3 mg of freeze-dried bacteria per g of diet
(Table 2). The results, at the 5%
significance level, showed that P. fluorescens
14::ptac-cry1Ac7 was significantly different from
P. fluorescens 14::cry1Ac7. Both P. fluorescens 14::cry1Ac7 and
14::ptac-cry1Ac7 were significantly different from
the parental strain, which was not significantly different from the
untreated control. H. seropedicae Nal1(pMT7) was
significantly different from H. seropedicae Nal1, which
was not significantly different from the control. H. seropedicae Nal1(pMT11) was different from H. seropedicae Nal1, but the sample size was not large enough to
declare significance at the 5% level.
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Effect of P. fluorescens strains expressing the
cry1Ac7 and chiA genes on E. saccharina larvae.
P. fluorescens
14::ptac-cry1Ac7 and P. fluorescens
Rif1::ptac-chiA were combined at different
concentrations and used in toxicity bioassays (Table
3). Mortality was determined after 2 and
5 days. The results, at the 5% level of significance, show that when
the chitinase-expressing strain was added at either 0.3 or 30 mg/g of
diet along with the Cry1Ac7-expressing strain at 0.3 mg/g of diet,
there was a significantly increased toxic effect. The reason for the
lack of increased toxicity when the chitinase-expressing strain was
added at 3.0 mg/g of diet along with the Cry1Ac7-expressing strain at
0.3 mg/g of diet is most likely the smaller sample size of this
experiment (n = 4). Although the chitinase-expressing strain showed toxicity at 30 mg/g of diet, it did not do so at 0.3 or 3 mg/g of diet. However, there was a significant increase in toxicity
when it was mixed with the Cry1Ac7-expressing strain at 0.3 mg/g of
diet.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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B. thuringiensis cry genes have been introduced into bacteria other than B. thuringiensis, such as the root colonizers P. fluorescens and Agrobacterium radiobacter and Ancylobacter aquaticus, a bacterium isolated from aquatic habitats. These strains were toxic against the larvae of the tobacco hornworm (Manduca sexta), the malaria mosquito Anopheles stephensi, and the leatherjacket (Tipula oleracea) (16, 23, 24, 35). The introduction of the cryIA(c) gene from B. thuringiensis subsp. kurstaki into the chromosome of Clavibacter xyli subsp. cynodontis, which naturally colonizes the xylem of Bermuda grass, is the only report of the use of a genetically modified endophyte as a biocontrol agent. This recombinant endophyte was shown to colonize corn and was tested for its effectiveness against the European corn borer (Ostrinia nubilalis). Moderate control of this pest was achieved by expression of the toxin gene chromosomally integrated into the endophyte (21). However, integration of endotoxin gene sequences into the chromosome of C. xyli subsp. cynodontis was unstable and segregant colonies made up 15% of the colonies isolated from corn at the end of the growing season. The authors suggested that the loss of the integrated gene could serve as an environmental safety feature (35). As little research had been done on the suitability of endophytic bacteria as biocontrol agents to date, it was of interest to investigate the potential of the sugarcane endophytes A. diazotrophicus and H. seropedicae, in addition to the phylloplane bacterium P. fluorescens 14, engineered to express a B. thuringiensis cry gene against the sugarcane borer E. saccharina.
To improve expression of the cry1Ac7 gene, we cloned it
under the control of the strong tac promoter and used the
vector pJFF350, which carries the artificial interposon Omegon-Km
(9), to integrate it into the chromosome of P. fluorescens 14. On this plasmid, the interposon is flanked by
two synthetic inverted 28-bp repeats of IS1, which can
transpose if IS1 gene products are supplied. The vector has
an origin of transfer which allows mobilization into gram-negative
bacteria. It also carries a disabled IS1 element which
enables transposition of the Omegon-Km cassette although it cannot
transpose itself, resulting in stably integrated genes. Although the
stability of the ptac-cry1Ac7 cassette in P. fluorescens 14 was not investigated, Herrera (12)
showed that the Omegon-Km-cry cassette in P. fluorescens 14 was stably integrated for at least 100 generations
and stable integration of cry genes into root-colonizing P. fluorescens strains using a transposon
Tn5-mediated system or suicide vectors for integration by
homologous recombination have been described in the literature
(23, 24, 35).
Our results proved that the tac promoter is capable of
operating efficiently in Pseudomonas and is responsible for
the increased levels of expression of the gene. We are unaware of any
reports of a cry1A(c) gene under the control of the
tac promoter having been integrated into the chromosome of a
Pseudomonas sp. Quantitative analysis of the -endotoxin
by enzyme-linked immunosorbent assay in P. fluorescens 14:: ptac-cry1Ac7 clones was not
determined, but Herrera et al. (13) showed that P. fluorescens 14::cry1Ac7 clones produced high
levels of Cry1Ac7 protein similar to those produced by
pKT240-cry1Ac7 clones, representing 3.7 and 3.5% of the
total protein, respectively. These levels were comparable to those of
0.5 to 1% reported by Obuckowicz (23) for a similar cry gene in root-colonizing pseudomonads. Ge et al.
(10) reported that expression of the cry1A(c)73
gene from its own promoter in E. coli was 0.24% of the
total cellular protein and that from the tac promoter after
induction was about 50% of the total cellular protein. In another
system, expression of a B. thuringiensis subsp. aizawai 130-kDa protein gene from the tac
promoter in E. coli was 38% of the total cellular protein
compared to 3% of the total cellular protein when expressed from its
own promoter (25). Therefore, it is believed that the levels
of the Cry1Ac7 protein expressed from the tac promoter in
P. fluorescens would be considerably greater than 3.7%.
This would only result if the ptac-cry1Ac7 cassette were
present in the cell as a single integrated copy, as it is clear from
the nonexpressing mutants of P. fluorescens carrying the
cassette on the multicopy plasmid pKTT that only a certain level of
expression is tolerated by these cells.
Toxicity bioassays showed that increased expression of the cry1Ac7 gene in both P. fluorescens 14 and H. seropedicae improves the control of E. saccharina larvae. However, it is important to consider that although increased expression leads to increased toxicity, it can also be a burden on bacterial cells, resulting in the accumulation of nonexpressing mutants or in lethality. All of these factors need to be taken into account when planning strategies for biological control of E. saccharina in sugarcane.
Synergistic insecticidal effects with combined B. thuringiensis suspensions and chitinase or chitinase-producing bacteria, as well as the combined effects of a Cry1C protein and S. marcescens ChiA, have been demonstrated previously (28, 31). The addition of both B. thuringiensis and chitinase increased the insecticidal effect on Chonstoneura fumiferana larvae significantly. Perforation of the peritrophic membrane by ChiA caused an increase in the toxicity of Cry1C, possibly due to an increase in the numbers of Cry1C toxin molecules binding to the membrane receptors present in the epithelium of the insect larvae. A Cry1C concentration of 20 µg/ml was required for a maximum toxic effect on larvae in the absence of chitinase, whereas only 3 µg of Cry1C per ml was needed for the same toxic effect in the presence of ChiA.
Our results demonstrate that by cointroduction of cry1Ac7 and chitinase genes into strains of P. fluorescens, increased biocontrol of insect pests could be achieved, requiring lower levels of Cry1Ac7 protein expression. This is advantageous, since lower expression may enable the bacteria to compete better in the environment with a diminished risk of generation of resistant larval populations resulting from exposure to high levels of Cry protein. The optimum, effective concentrations of the recombinant strains, as well as the synergistic toxic effect of H. seropedicae strains producing the Cry1Ac7 protein and chitinase, need to be investigated.
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ACKNOWLEDGMENTS |
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We thank Imke Hansen-Wester for construction of pKmM0-tox and its expression in H. seropedicae, Gillian Mimmack for statistical analysis of the data, Barbara Huckett for assistance with bioassays, and Helena Boshoff for helpful discussions.
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FOOTNOTES |
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* Corresponding author. Mailing address: Microbiology Department, University of Cape Town, Private Bag Rondebosch 7701, South Africa. Phone: 27 (21) 650 3269/70. Fax: 27 (21) 689 7573. E-mail: jat{at}molbiol.uct.ac.za.
Present address: Molecular Biology Unit, The South African
Institute for Medical Research, Johannesburg, South Africa.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, July 2000, p. 2804-2810, Vol. 66, No. 7
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