Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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NOVEL STRAINS OF BACILLUS THURINGIENSIS
The present invention relates to novel bacterial
genes, and to novel strains of the bacterium Bacillus
thuringiensis; and to uses therefor.
The organism Bacillus thuringiensis produces a
protein crystal endotoxin which kills insect larvae, it
is not however toxic to mammals. It is thus very useful as
an agricultural insecticide, in particular against
Lepidoptera, Coleoptera and Diptera. Strains of Bacillus
thuringiensis have been used as agricultural insecticides
for a number of years.
The most extensively characterised strain of Bacillus
thuringiensis active against coleopteran pests is Bacillus
thuringiensis variety (var.) tenebrionis, as deposited in
the German Collection of Microorganisms (Deutsche Sammlung
von Microorganism) under the reference DSM 2803. We have
now discovered novel strains of Bacillus thuringiensis
having generally similar properties to DSM 2803, but
distinguished therefrom by specific insecticidal activity
against coleopteran larvae of the genus Diabrotica, as well
as by toxicity to lepidopteran larvae. The novel
properties of these strains appear to arise from novel
genes that they contain.
According to the present invention we provide the
novel strains JHCC 4835 and JHCC 4353 of Bacillus
thuringiensis, deposited at the National Collections of
Industrial and Marine Bacteria under the accession numbers
NCIB 40091 and 40090, respectively.
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We further provide novel 8-endotoxin genes capable of
isolation from said strains JHCC 4835 and JHCC 4353. Such
' genes may be located either on the bacterial chromosome or
on a plasmid. In a further aspect, our invention comprises
recombinant DNA homologous with the DNA sequence set out in
Figures 5-10 hereof and coding for a novel
insecticidally-active endotoxin of molecular weight about
81.2 kilodaltons (hereinafter referred to as "the 81 kD
endotoxin"). In specific embodiments of our invention,
recombinant DNA coding for insect endotoxins has been
cloned from Bacillus thuringiensis JHCC 4835 into E.coli
strains BL21/pJHll and MC1022/pJHl2, deposited at the
National Collections of Industrial and Marine Bacteria
under the accession numbers 40275 and 40278 respectively.
The endotoxin gene in the latter deposit is
lepidopteran-specific. We further provide recombinant DNA
coding for a second lepidopteran-specific endotoxin gene
derived from Bacillus thuringiensis strain JHCC 4835, which
has been deposited in the form of a bacteriophage Lambda
EMBL4 clone CL5 with the National Collections of Industrial
and Marine Bacteria under the accession number 40279.
Recombinant DNA according to our invention may
comprise genes of varying lengths encoding
insecticidally-active proteins. When cloning DNA from the
bacterial chromosome it is convenient to use bacteriophage
Lambda vectors or other cloning vectors that sequester the
recombinant DNA from host cell enzymes that might cause
homologous recombination.
We further provide novel insecticidal compositions
characterised in that they contain the b-endotoxin
produced by said strains JHCC 4835, JHCC 4353 and E.coli
BL21/pJHll, and a method of protecting plants from insect
attack which comprises exposing the larvae to a S-endotoxin
produced by the said strains JHCC 4353, JHCC 4835 and
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E.coli BL21/pJHll.
The strains JHCC 4835 and JHCC 4353 were soil
isolates from Marshall, Iowa, USA and Dallas, Iowa, USA
respectively. In colony morphology they are generally
similar to DSM 2803, and to strain HD-1 which is
insecticidal to lepidopteran larvae.
The morphology of the strains of the invention is
compared with that of known strains in Table 1.
Biochemical properties of the new and the known
strains are compared in Tables 2-4. It will be seen that
there are many similarities between the strains.
In view of these biochemical similarities it is
surprising that the gene encoding the 81 kD endotoxin in
E.coli BL21/pJHll shows very little DNA sequence homology
to the B. thuringiensis var. tenebrionis endotoxin gene of
DSM 2803. Use of the coding sequence for the _B.
thuringiensis var. tenebrionis endotoxin gene as a DNA
probe under relatively mild stringency conditions (3x
Standard Saline Citrate at 37°C.) is not sufficient to
generate a signal from the coding sequences for this
endotoxin gene in strains JHCC 4835 and JHCC 4353.
Similarly, use of the coding sequence for the
lepidopteran-specific CryIA(c) (this system of nomenclature
is described by Hofte and Whitely in Microbiol. Reviews,
53, 1989 at pages 242-255) endotoxin gene from a Bacillus
thuringiensis var, kurstaki strain is not sufficient to
generate a DNA hybridisation signal from the coding
sequence for the 81 kD endotoxin. Also, use of the novel
gene coding sequence as a DNA probe does not generate a
hybridisation signal from the tenebrionis gene or the three
CryIA genes.
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The newly-discovered B.thuringiensis strains JHCC
4835 and JHCC 4353 show a significantly different
specificity of insecticidal activity as compared with DSM
2803. In particular, 4835 and 4353 show more selective
activity against beetles than known coleopteran-active _B.
thuringiensis strains in that they are specifically
larvacidal to Diabrotica spp.. In addition, strains JHCC
4835 and JHCC 4353 are larvacidal to lepidopteran pests
whereas strain DSM 2803 is not. On the molecular level,
the newly discovered gene in Bacillus thuringiensis strains
JHCC 4835 and 4353 encode a gene product which shows a
significantly different spectrum of insecticidal activity
as compared with the coleopteran-specific endotoxin gene in
DSM 2803 or the lepidopteran-specific CryIA endotoxin genes
in HD1 and other var. kurstaki strains.
The new endotoxin gene encodes an 81.2 kilodalton
endotoxin that has a completely novel activity spectrum: it
is toxic to both lepidopteran and coleopteran larvae. This
is particularly surprising since the Bacillus thuringiensis
strain from which it is derived is not toxic to all
Coleoptera, but rather is Diabrotica-specific. Possible
explanations for this finding may include: a low
concentration of this protein in the crystal that the
microorganism produces; inaccessibility of the protein in
the crystal; presence of the toxin in the crystal as a
protoxin which is not converted to the active form in the
gut of certain insects; or other so far unrecognised
factors.
The Bacillus thuringiensis strains according to the
invention may be prepared in any quantity required by
fermenting a sample of NCIB 40091 or 40090 obtained from
the National Collections of Industrial and Marine Bacteria
under suitable conditions in an appropriate medium. Such
conditions and media are well known to the art. The media
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will, for example, generally contain a nitrogen source (eg
fish protein) and a carbohydrate source such as starch.
Suitable conditions include a temperature in the range
15-45°C, and an approximately neutral pH. Fermentation may
be conveniently carried out in batches, typically for
periods of 3-5 days.
E.coli strains carrying cloned endotoxin genes
according to the invention may be prepared by growing
cells to stationary phase on solid nutrient media (eg L
agar) prior to scraping cell growth from the medium
surface, lyophilising, and freezing before thawing and
weighing out the insecticidal material.
Insecticidal compositions according to the invention
may be obtained from the fermentation liquor by
concentration, for example by centrifugation or filtration
followed by addition of any desired and appropriate
formulating agents. Formulating agents which may be useful
include for example surface active agents, eg, wetting
agents: solid diluents, dispersing agents and UV
stabilisers. If desired, solid formulations may be
prepared by known methods.
The process of the invention is generally carried
out by treating (eg spraying) plants infested or liable to
infestation by insects with insecticidal compositions as
described above diluted with a diluent such as water. The
insecticidal agent is the toxic 8-endotoxin: if desired
this may be applied to the plants or insects infesting them
independently of the bacteria that produce it. Separation
of the crystalliferous protein from the bacteria Bacillus
thuringiensis, or of the cloned gene product from the
bacterium E.coli, is however generally not necessary.
Another method of carrying out the process of the
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invention is to arrange for the plant susceptible to insect
attack to produce the b-endotoxin in situ. This is done by
cloning a 8-endotoxin gene from strain NCIB 40090 or NCIB
40091, by known means; providing it with a promoter
sequence (for example the CaMV35S promoter) which will
cause expression of the gene in plants; and transforming
the plant by known methods. Suitable transformation
methods may include the use of Ti plasmid vectors for
Agrobacterium-mediated transformation of dicots, or direct
DNA uptake methods such as embryo microinjection, or use of
microprojectiles followed by protoplast regeneration. To
obtain the greatest degree of expression of the gene the
promoter sequence should be selected and engineered
appropriately and other factors (for example codon usage)
should be adapted to maximise expression in lp anta.
Coleopteran larvae which are combated by the process
of the invention may be of various species. As noted
above, the Bacillus thuringiensis strains of the invention
kill only Diabrotica, including those shown in Table 5A
below: while use of the insecticidal product from the
cloned gene of our invention will kill other coleoptera as
well.
TABLE 5A
Common Name Latin Name
Western Corn Rootworm Diabrotica virgifera virgifera
Southern Corn Rootworm Diabrotica undecim unctata
howardi
Northern Corn Rootworm Diabrotica barberi
Mexican Corn Rootworm Diabrotica virgifera zea
Banded Cucumber Beetle Diabrotica balteata
Western Spotted Cucumber Diabrotica undecimpunctata
Beetle undecimpunctata
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Lepidopteran larvae which are combated by the process
of the invention may include those listed in Table 5B.
TABLE 5B
Tobacco budworm Heliothis virescens
Corn earworm Heliothis zea
European corn borer Ostrinia nubilalis
Cabbage looper Trichoplusia ni
Diamondback moth Plutella xylostella
Fall army worm Spodoptera frugiperda
Beet army worm Spodoptera exi ua
The process of the invention may be used to protect a
wide variety of plants prone to infestation by Coleoptera
(Diabrotica, if the Bacillus thuringiensis strains are
used) or Lepidoptera. Specific examples of commercially
important plants to be protected by the invention are maize
(corn), tomatoes, potatoes, cotton, tobacco and cucurbits.
Bacillus thuringiensis JHCC 4835 and 4353 are var.
kurstaki strains according to tests with antibody to
flagellar antigens. To date, var. kurstaki strains have
been known only for their insecticidal effect on
lepidopteran larvae. Surprisingly, these strains and
indeed other ku~staki strains previously described by ICI
(e.g. strain A20 deposited at the National Collections of
Industrial and Marine Bacteria under accession number NCIB
12570
are active against
coleopteran larvae of the genus Diabrotica, in addition to
their expected activity against Lepidoptera. Moreover if
the 81 kD endotoxin gene is used as a hybridisation
probe, strongly hybridising sequences can be found in both
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chromosomal and plasmid DNA samples from other known
Bacillus thuringiensis strains. These strains include var.
kurstaki strains such as HD1, HD73 and HD241, and the var.
kenyae strain HD123. In spite of this, the 81 kD endotoxin
gene of the present invention has not been previously
described, or recognised as being present in these or other
Bacillus thuringiensis strains.
The invention may be further understood with
reference to the accompanying drawings, in which:
Figure 1 shows diagrammatically the derivation of the
cloned 81 kD endotoxin gene in the recombinant plasmid
pJHll;
Figure 2 shows diagrammatically the structure of
p,1H11, and the structures of the coleopteran-specific
tenebrionis-type gene and the CryA 6.6-type gene cloned
into the same vector system (PT712) and designated pIC 226
and pIC 228 respectively;
Figure 3 shows diagrammatically the structure of the
cloned lepidopteran-specific endotoxin gene in the
recombinant plasmid pJHl2;
Figure 4 shows diagrammatically the structure of the
cloned lepidopteran-specific endotoxin gene in the
recombinant lambda clone CLS;
Figures 5-10 show the base sequence, the amino acid
sequence, and the main restriction endonuclease recognition
sites of the 81 kD endotoxin gene carried by pJHll;
Figure 11 shows graphically the mean values of 12
separate bioassays testing the efficacy of recombinant
E.coli strain MC1022/pIC244 against first-instar larvae of
Western Corn Rootworm at 4 days after treatment;
Figure 12 shows graphically the mean values of 12
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separate bioassays testing the efficacy of recombinant
E.coli strain MC1022/pIC244 against first-instar larvae of
Western Corn Rootworm at 5 days after treatment.
With further reference to Figure 1, in this diagram,
which is not drawn to scale, N represents restriction
endonuclease NdeI, H = HindIII, E = EcoRl, D = DraI and S =
SmaI. Restriction sites above the maps are in the cloned
DNA, whereas sites below the maps are in the vector.
Parentheses indicate sites rendered non-functional by
"filling-in" with Klenow DNA polymerase. Dashed lines
represent pUCl9 vector DNA. Dotted lines represent PT712
vector DNA in clone p,1H11 and the arrowhead represents the
bacteriophage T7 promoter. The star represents a
32P-labelled DNA fragment.
In Figure 2, the figures below the maps represent the
number of basepairs between the T7 RNA polymerase
transcriptional start site and the beginning of the open
reading frame. The large arrowhead represents the
bacteriophage Y7 promoter. The solid block in PT712
represents the cloning site; H = HindIII and S = SmaI. ApR
indicates the gene encoding resistance to ampicillin.
In Figure 3, the open box represents the cloned
fragment which is about 7 kilobasepairs in length. The
dashed lines indicate pUCl9 vector DNA and ApR is the gene
encoding ampicillin resistance. The parentheses indicate
an Ndel site which is only provisionally placed in the
region shown; other restriction sites are represented by D
- DraI, E = EcoRl, H = HindIII and N = NdeI.
With reference to Figure 4, the only EcoRl (E) sites
shown are those at which the Lambda vector and the cloned
insert fragment are joined. Open reading frames (ORFs) are
shown by arrows above the map. The numbers above the map
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are the approximate fragment lengths of selected HindIII
fragments. The Clal (c) site shown is not the only ClaI
site in the insert. The diagram is not drawn to scale; the
cloned insert fragment is approximately 16 kilobase pairs
in length.
Figures 5-10 show the base sequence, the amino-acid
sequence and the main restriction sites of the gene
encoding the 81 kD endotoxin protein and flanking DNA. The
open reading frame begins at base number 355 and ends at
- base number 2514 with the G of the termination (Ter) codon
TAG.
Figure 11 is a graphical representation of the
Western Corn Rootworm bioassay of cloned endotoxin gene
products at 4 days after treatment (DAT). Points on the
graph are mean values of percent mortality at a given rate.
Figure 12 is a graphical representation of the
Western Corn Rootworm bioassay of cloned endotoxin gene
products at 5 days after treatment (DAT). Points on the
graph are mean values of percent mortality at a given rate.
The following Examples illustrate the invention.
EXAMPLE 1
Isolation of the B. thuringiensis strain JHCC 4835
according to the invention.
Soil samples were diluted by placing 5.Og of the
sample into 45m1 of 0.5% peptone to give a 10 1 dilution
prior to emulsification. The sample was then heated to
60°C for 10 minutes in a water bath. Sequential dilutions
were then made prior to plating O.lml of the 10 3 and 10 5
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dilutions onto B. cereus selective agar plates (Bacillus
cereus agar base, Oxoid) and esculin agar plates (in
g/litre of H20: esculin 1.0; ferric citrate 0.5; peptone
10; NaCl 5; Oxoid agar 10). The plated samples were
incubated at 30°C for 5 days. Slides were made of
potential B. thuringiensis colonies, stained according to
Smirnoff's procedure and examined microscopically at 1000X
magnification for the presence of stained, parasporal
crystals.
Crystal-positive colonies were streaked onto L agar
(lOg tryptone, lOg yeast extract, 5g NaCl, lOg agar per
litre) in order to ensure a pure culture, and incubated at
30°C. Purified colonies were incubated overnight in L
broth; after incubation an equal volume of 80~ sterile
glycerol was added prior to storage at -70°C.
The strain JHCC 4353 was extracted by a similar
procedure.
EXAMPLE 2
Propagation of the B.thuringiensis strains JHCC 4835
and JHCC 4353 on solid media.
Inoculum was transferred from a glycerol storage
vial onto an L agar plate to check for purity. A
representative sweep of colonies was then used to inoculate
5m1 of broth (lOg tryptone, lOg yeast extract, 5g NaCl per
litre) prior to incubation with shaking at 30°C for 3-5
hours. One millilitre of this culture was then used to
inoculate a preparative (210 mm X 210 mm) Petri plate
containing 300 ml of CRL 1 medium agar (in g or ml/litre of
water: nutrient broth 8; glucose 6; yeast extract 5; xylose
0.5; cotton seed flour extract 30m1; corn steep liquor 3.2
ml; Mary Mendel's salt mixture lml; Oxoid agar 15). Mary
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Mendel's salt mixture is:
Mary Mendel's Salts
Distilled Water 495 ml
HC1 conc. 5 ml
FeS04 2.5 g
MnS04, H20 or MnC12.4H20 0.98 g
ZnCl2 or ZnS04.4.H20 1.76 g
Cultures were incubated for 5 days at 30°C. The
cells, spores and crystals were then harvested by scraping
confluent growth from the agar surface prior to
freeze-drying.
EXAMPLE 3
Propagation of the B. thuringiensis strain JHCC 4835
and JHCC 4353 in liquid culture according to the invention.
Inoculum was transferred from a glycerol storage vial
to a 250 ml Erylenmeyer flask containing 100 ml of CRL 1
medium (in g or ml/litre of water: nutrient broth 8;
glucose 6; yeast extract 5; xylose 0.5; cotton seed flour
extract 30m1; corn steep liquor 3.2 ml; Mary Mendel's salt
mixture 1 ml) and incubated with agitation at 30°C and 3400
rpm. After 24 hours, the entire 100 ml was used to
inoculate 1 litre of the same medium in a 2L flask; this
was incubated with agitation for 5 days at 30°C. The
cells, spores and crystals were then harvested by
centrifugation and acetone precipitated using the Dulmage
method.
EXAMPLE 4
Formulation according to the invention.
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Upon completion of the fermentation cycle, JHCC 9353
or.,lHCC 4835 bacteria can be harvested by first separating
the B: thuringiensis spores and crystals from the
fermentation broth as described in Example 2. The
recovered spores and crystals can be resuspended in 100 ml
of water and formulated into a liquid concentrate by adding
4.9g of Morwet D-425*(dispersing agent), 4.9g of Veegum HV*
(suspending agent), 4.9 ml of Tween 80*(wetting agent) and
24.4 ml of Sorbo*(anti-freezing agent). Each ingredient is
added separately in order stated above. The product is
kept at 4°C prior to use.
EXAMPLE 5
Cloning of plasmid-derived endotoxin genes from
B.thuringiensis strain 4835.
Endotoxin genes are cloned from covalently closed
circular (ccc) plasmid DNA prepared from B.thurigiensis
strain 4835 as follows:
A 500 ml culture of strain 4835 is grown in L broth
at 37°C, with shaking, to an absorbance value at 600 mm of
1.00 optical density (O.D) units. Cells are harvested by
centrifugation at 8000 revolutions per minute (rpm) for 10
minutes at 4°C, then re-suspended in 5m1 TE buffer [50mM
Tris HC1 pH7.6, 20mM EDTA). The resuspended cells are
added to 95m1 TE buffer containing to sodium dodecyl
sulphate (SDS) and 0.085M NaOH , pH12.4 lysin of the cell
suspension occurs during a incubation at room temperature.
lOml of 10°s SDS are then added to the lysate; the solution
is mixed gently prior to the gradual addition of lOml 2M
Tris HC1 pH7.0 with gentle mixing. 34m1 of 5M NaCl is
added and the solution is mixed well prior to overnight
* Trade-r:lark
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incubation on ice-water. The lysate is centrifuged at 9000
rpm for 15 minutes at 4°C and the supernatant carefully
transferred to a new centrifuged bottle prior to the
addition of 36m1 50% polyethylene glycol (PEG) 600 in TE
buffer. The lysate is incubated on ice-water for 3 hours
(minimum) to overnight prior to centrifugation at 10,000
rpm for 10 minutes at 4°C. The pellet is dissolved in 9 ml
TE buffer and 100,u1 5mg/ml RNA (treated at 100°C for 5
minutes, prior to use) and incubated at 45°C for 10
minutes, prior to the addition of 9.238 caesium chloride
(CsCl). After the CsCl is dissolved, 0.9m1 of 5 mg/ml
ethidium bromide is added prior to isopycnic centrifugation
of the mixture at 40,000 rpm for 48 hours at 15°C, and
isolation of the ccc DNA band. After removal of the CsCl
and ethidium bromide by conventional techniques, high
molecular weight plasmid ccc DNA (greater than 40 kilobase
pairs) is isolated by size fractionation on 10% - 40%
sucrose step gradients prior to digestion with appropriate
restriction endonucleases (ie, those which do not cleave
the DNA in the endotoxin structural gene), ligation into
appropriately digested plasmid cloning vectors (eg, pUCl8
or pUCl9), and transformation into an appropriate E.coli
host strain (the specific strain used is MC1022, which is
an ampicillin-sensitive strain of the genotype ara D139,
~(ara, leu) 7697, ~(lac Z) M15, ~al_ U, dal K, str A.
Transformants resistant to appropriate antibiotics which
select for the introduced plasmid vector were then screened
for recombinant endotoxin genes by standard DNA
hybridisation methods, using as probes the cloned
tenebrionis gene (plus flanking sequences) and a cloned
CryIA gene.
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EXAMPLE 6
Cloning of chromosomal endotoxin genes from
B.thuringiensis strain 4835.
Endotoxin genes were cloned from chromosomal DNA prepared
from strain 4835 as follows:
A 500 ml culture of strain 4835 was grown in L-broth
at 37°C, with shaking, to an Absorbance value at 600nm of
1.00 optical density units. Cells were harvested by
centrifugation at 8000 rounds per minute (rpm) for 10
minutes at 4°C, then re-suspended in 5 ml TES buffer (50mM
Tris-HC1 pH7.5, 50mM NaCl, 5mM EDTA). Cells were treated
for 30 minutes at 37°C with lysozyme (0.5 mg/ml final
concentration) and RNase (0.lmg/ml final concentration
taken from a stock solution of 5 mg/ml boiled at 100°C for
5 minutes prior to use). Lysis was completed by the
addition of Sarcosyl to give a final concentration of 0.8~
and incubation at 37°C for 60 minutes in the presence of
Pronase (0.5mg/ml final concentration taken from a stock
solution of 5mg/ml pre-incubated at 37°C for 60 minutes
prior to use). Lysate volume was adjusted to 9.0 ml in the
50mM Tris-HC1 pH 7.6, 10 mM EDTA, prior to the addition of
9.2 g caesium chloride (CsCl). After the CsCl dissolved,
1.25 ml of a 5 mg/ml solution of ethidium bromide was added
prior to isopyonic centrifugation of the mixture at 40,000
rpm for 48 hours at 15°C.
After removal of CsCl and ethidium bromide by
conventional techniques, an aliquot of purified chromosomal
DNA was partially digested with the restriction
endonuclease EcoRl prior to ligation into EcoRl-digested
bacteriophage ~ EMBL4 vector DNA. Ligation reaction
mixtures were packaged into viable phage particles using a
commercially-available kit from Amersham International PLC.
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The resultant recombinant phage particles were
selected by growth on E.coli host strain PE392, a P2
lysogen of strain LE392 which has the genotype hsd 8514
(rK ,MK+), sub E44, sub F58, lacYl or 0 (1ac12Y), coal K2,
gal T22, met B1, try R55. Recombinant phage carrying one
or more endotoxin genes were detected by hybridisation of
lysed plaques fixed to a duplicate set of nitrocellulose
filters using as probes radiolabelled fragments of a
CryIA-endotoxin gene and a 3'-terminal fragment of the gene
for the 81 kD protein.
Plaques containing endotoxin genes were purified and
characterised by restriction endonuclease mapping
techniques well known in the art.
Chromosomal endotoxin genes can also be cloned
directly into plasmid vectors (e.g. pUCl9). This may
necessitate cloning the gene in small fragments by the
technique well known in the art as "chromosome walking".
Problems with deletion events due to host-mediated
homologous recombination can be circumvented by cloning in
this manner and reconstructing the desired open reading
frame by piecing the gene together after sequencing an
appropriate number of overlapping gene fragments.
EXAMPLE 7
Solid media propagation of insecticidally-active
E.coli strains carrying cloned endotoxin genes according to
the invention.
Inoculum was transferred from a glycerol storage vial
to L agar Petri plates containing antibiotics suitable for
selection of the cloning vector. Inoculated plates were
incubated 24 - 72 hours to allow for the appearance of
characteristic colonial morphology. A selection of single
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colonies of the correct appearance (e.g. rough colonies in
the case of E.coli strain BL21/pJHll carrying the cloned
the 81 kD endotoxin gene) was used to inoculate a small
volume of L broth [15g Tryptone, 7.5g yeast extract, 7.5g
NaCl per 1500 ml total volume] containing an antibiotic
(e. g. ampicillin) suitable for selection for the plasmid
vector carrying the cloned endotoxin gene. Cultures were
grown to an an Absorbance value at 600nm of 0.5 - 0.7 O.D.
units. One millilitre (ml) of culture was used to
inoculate, by spreading with a glass "spreader", a
preparative (i.e. 245mm X 245mm X 20mm) Petri plate
containing L agar [L broth as above supplemented with 16g
Oxoid agar, an appropriate antibiotic and IPTG to a final
concentration of 120 microgram/ml.]. Preparative plates
were incubated overnight at 37°C. Bacterial growth was
scraped from the preparative plates using a glass spreader.
The scraped product, pooled from several plates if
necessary, was transferred to a sterile plastic container
and frozen for 2 hours at -20°C prior to lyophilisation
for 16 - 18 hours. The material was stored at -20°C. The
dried product is crushed into an even powder prior to use
as an insecticidal material in insect bioassays.
EXAMPLE 8
Purification of the novel 81.2 kilodalton endotoxin
protein from the recombinant E.coli strain MC1022/pJHll.
E.coli strain MC1022/p,1H11 was prepared on solid media as
described in Example 7, but the scraped cell mass was
stored at -20°C without lyophilisation. Frozen cells were
thawed on ice prior to disruption by sonication at an
amplitude of 14 microns for 9 X 20 seconds using a 1 cm
diameter probe. The sonicated cells were then centrifuged
at 9300 x g at 4°C to remove unbroken cells, prior to
high-speed centrifugation (100,000 x g for 60 minutes at
4°C) to remove membranes. The high-speed extract was then
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subjected to ion-exchange chromatography over DEAE-
Sepharose at pH 8Ø The column was then eluted with a
0-500-mM NaCl gradient, and fractions monitored by
SDS-PAGE. Fractions containing the 81.2 kD protein were
pooled, dialysed against lOmM Tris pH8.0, and subjected to
a second FPLC ion-exchange chromatography step, again
eluting the bound proteins with a 0- 500 mM NaCl gradient.
Fractions containing the partially-purified 81.2 kD protein
were identified and pooled prior to further purification by
gel filtration chromatography. This process results in an
endotoxin protein which is 90% pure and which may be used
(with or without a concentration step) in insect bioassays.
Examples 9 and 10 illustrate the activity of the
novel B. thuringiensis strains of the invention against
different Diabrotica spp.
EXAMPLE 9
Efficacy of larvacidal activity of B.thuringiensis
strain JHCC 4835 against Western Corn Rootworm (Diabrotica
virgifera virgifera).
For each B.thuringiensis strain, a mixture of spores
and crystals was prepared by incubating the organism at
30°C for 5 days on 210 mm X 210 mm Petri plates as in
Example 2, scraping confluent growth from the agar surface
and freeze drying. For tests on first instar larvae of
Western Corn Rootworm (Diabrotica virgifera virgifera),
freeze dried spores and crystals were mixed sterile water
and a sterile sucrose solution to give the treatment rates
indicated in Table 6 in parts per million (ppm) and a final
sucrose concentration of 2.5%. The solubilised spore
crystal (treatment) mixture was homogeneously dispersed by
sonication in a water bath sonicator for 5 minutes. The
* Trade-mark
CA 02054843 1999-09-17
PS35271 -
- 19 -
treatment was then vortexed and applied as 0.075 ml of
solution to a disk l.5cm in diameter cut from "Teri towels"*
(Kimberly Clark product #34770). One test consisted of 5
Teri towel disks with applied treatment, each placed in a
separate plastic Falcon test dish prior to infestation with
5 first instar larvae per dish. Tests were placed in a
closed styrofoam box with a moistened Teri towel as a
humidity source; the box was incubated in a room held at
78oF - 80°F for 3 or more days after treatment (DAT) prior
to evaluation of the bioassay. The conditions inside the
Styrofoam box were 74°F - 76°F and 80% relative humidity.
Tests were evaluated using a dissecting microscope. The
efficacy of these treatments at various concentrations
(rates) is shown in Table 6.
EXAMPLE 10
Efficacy of larvacidal activity of B.thuringiensis
strain JHCC 4835 against Southern Corn Rootworm (Diabrotica
undecimpunctata howardi).
For each B.thuringiensis strain, a mixture of spores and
crystals was prepared by incubating the organism at 30°C
for 5 days on 210 mm X 210 mm Petri plates as in Example 2,
scraping confluent growth from the agar surface and freeze
drying. Tests on first instar Southern Corn Rootworm
(Diabrotica undecimpunctata howardi) were set up, incubated
and evaluated as described in Example 9. The efficacy of
these treatments at various concentrations (rates) is shown
in Table 7.
EXAMPLE 11
Specificity of insecticidal activity of
B.thuringiensis strains ,1HCC 4835 and JHCC 4353.
* Trade-mark
PS35271
- 2~ - 205484 3
A mixture of spores and crystals was prepared by
incubating the organism at 30°C for 5 days on 210 mm X 210
mm Petrie plates as in Example 2, scraping confluent growth
from the agar surface and freeze-drying. Freeze-dried
spores and crystals were mixed with a sterile 2.5% sucrose
solution for tests on first-instar Southern Corn Rootworm
(Diabrotica undecimpunctata howardi) larvae. Freeze-dried
spores and crystals were mixed with sterile H20 and
presented on potato leaves dipped in this suspension for
tests on first-instar Colorado potato beetle (Leptinotarsa
decemlineata) larvae. Freeze-dried spores and crystals
were mixed with sterile H20 and presented on cotton
cotyledons dipped in this suspension for tests on Boll
Weevil (Anthonomus grandis) adults. The efficacy of these
preparations at various concentrations in parts per million
(ppm) is shown in Table 8. Comparison of the activity
spectrum B.thuringiensis variety tenebrionis (DSM 2803)
with those of strains JHCC 4835 and JHCC 4353 shows the
more selective effect of the latter two strains (Table 8).
The efficacy of B.thuringiensis strain JHCC 4835 in
the control of various lepidopteran larvae is illustrated
in Examples 12 - 15.
EXAMPLE 12
Efficacy of B.thuringiensis strain JHCC 4835 in the
control of various lepidopteran larvae.
A mixture of spores and crystals was prepared as in
Example 2, and mixed with an appropriate conventional
artificial insect diet. Results are shown in Table 9
below. Comparison of the efficacy of B.thuringiensis
variety tenebrionis (DSM 2803) with that of strain JHCC
4835 shows that only strain 4835, and the known var.
205484 3
- 21 -
kurstaki strain JHCC 4360, are insecticidal to lepidopteran
larvae (Table 9).
EXAMPLE 13
Efficacy of B.thuringiensis strain JHCC 4835 in the
control of Fall Army Worm (Spodoptera frugiperda).
A mixture of spores and crystals was prepared as in
Example 2, and mixed with an appropriate conventional
artificial insect diet. Results are shown in Table 10
below. Comparison of the efficacy of B.thuringiensis strain
JHCC 4580 (an isolate very similar to var. tenebrionis)
with that of strain JHCC 4835 shows that only strain 4835,
and the known kurstaki strain JHCC 4360, are insecticidal
to S.frugiperda (Table 10).
EXAMPLE 14
Efficacy of B.thuringiensis strain JHCC 4835 in the
control of Beet Army Worm (Spodoptera exigua).
A mixture of spores and crystals was prepared as in
Example 2, and mixed with an appropriate conventional
artificial insect diet. Comparison of the efficacy of
B.thuringiensis strain JHCC 4580 (an isolate very similar
to var. tenebrionis) with that of strain JHCC 4835 showed
that only strain 4835, and the known kurstaki strain JHCC
4360, are insecticidal to S.exigua.
EXAMPLE 15
Efficacy of Bacillus thuringiensis strains JHCC 4835
and 4353 in the control of Heliothis zea.
PS35271
- 22 - 205484 3
A mixture of spores and crystals was prepared as in
Example 2, and mixed with an appropriate conventional
artificial insect diet. Control of larvae obtained is shown
in Table 12 below.
The efficacy and novel larvacidal activity spectrum
of recombinant E.coli cells carrying the cloned endotoxin
gene encoding the 81.2kD protein are illustrated in
Examples 16 - 18
EXAMPLE 16
Efficacy of the larvacidal activity of the 81 kD
endotoxin expressed by recombinant E.coli strain
MC1022/pJHll in controlling European Corn Borer (Ostrinia
nubilalis).
E.coli strain MC1022/pJHll was prepared on solid
media as described in Example 7. Freeze-dried cells were
thawed and mixed with an appropriate conventional
artificial insect diet to give the final treatment
concentration in parts per million (ppm) shown in Table 13.
Tests were infested with first instar European corn borer
larvae and evaluated at 6 days after treatment (DAT).
E.coli strains carrying the recombinant plasmid with the 81
kD endotoxin gene (pJHll) and those carrying the CryIA 6.6
type lepidopteran- specific gene (pIC228) were
insecticidal, whereas those carrying the vector only
(PT712) or the tenebrionis-type gene (pIC226) were not.
EXAMPLE 17
Efficacy of the larvacidal activity of the 81 kD
endotoxin expressed by recombinant E.coli strain
MC1022/pJHll in controlling Colorado Potato Beetle
(Leptinotarsa decemlineata).
PS35271
-23-205483
E.coli strain MC1022/pJHll was prepared on solid
media as described in Example 7. Freeze-dried cells were
thawed, mixed with sterile H20 and presented on potato
leaves dipped in this suspension for tests on first-instar
larvae of Colorado Potato Beetles (Leptinotarsa
decemlineata) to give the final treatment concentration in
parts per million (ppm) shown in Table 14. E.coli strains
carrying the recombinant plasmid with the 81 kD endotoxin
gene (pJHll) and those carrying the tenebrionis-type gene
(pIC226) were insecticidal whereas those carrying the
vector only (PT712) or the CryIA 6.6 type
lepidopteran-specific gene (pIC228) were not.
EXAMPLE 18
Efficacy of the larvacidal activity of the 81 kD
endotoxin expressed by recombinant E.coli strain
MC1022/pJHll in controlling Western Corn Rootworms
(Diabrotica virgifera virgifera).
E.coli strain MC1022/pJHll was prepared on solid media as
described in Example 7. For tests on first instar larvae
of Western Corn Rootworm (Diabrotica virgifera virgifera),
freeze dried cells were thawed, mixed with sterile water
and a sterile sucrose solution to give the treatment rates
indicated and a final sucrose concentration of 2.5~. The
solubilised cell (treatment) mixture was homogeneously
dispersed by sonication in a water bath sonicator for 5
minutes. The treatment was then vortexed and applied as
0.075 ml of solution to a disk l.5cm in diameter cut from
"Teri towels" (Kimberly Clark product #34770) as described
in Example 9 to give the final treatment concentration in
parts per million (ppm) shown in Tables 15 & 16. These
tests were read at 4 and 5 DAT and the results were
subjected to statistical analysis. Results are presented
. _ 205484 3
- 24 -
graphically in Figures 11 & 12 and indicate that E.coli
strains carrying the recombinant plasmid with the 81 kD
endotoxin gene (pJHll) and those carrying the
tenebrionis-type gene (pIC226) were insecticidal whereas
those carrying the vector only (PT712) or the CryIA 6.6
type lepidopteran-specific gene (pIC228) were not; the
differences in activity between these two groups of strains
(pJHll and pIC226 versus the vector PT712 and pIC228) are
statistically significant.
The efficacy and novel larvacidal activity spectrum
of the partially-purified and purified novel 81.2 kD
endotoxin protein are illustrated in Examples 19 - 21.
EXAMPLE 19
Efficacy of the larvacidal activity of the
partially-purified and purified 81 kD endotoxin in
controlling European Corn Borer (Ostrinia nubilalis).
Partially-purified and purified 81 kD endotoxin
protein was prepared from freeze-dried recombinant E.coli
cells MC1022/pJHll as described in Example 8. Fractions
from the second FPLC ion-exchange column were designated
MonoQ A, B, and C and contained about 50%, 50%, and 25%
81.2 kD endotoxin protein respectively. These fractions
were added to conventional artificial insect diet to give
the treatment rates in ppm shown in Table 17 in bioassays
to test insecticidal activity on first-instar larvae of
European corn borer (Ostrinia nubilalis). The results in
Table 19 show that all fractions were active in producing
either mortality or stunting of larval growth. Purified
81.2 kD protein was also tested and found to be
insecticidal to European corn borer larvae and to stunt
larval growth (Table 18).
__.
,~ _ _ _._. ..__. ~ _.~ ~.--_--..__.
2054~84~ 3
- - 25 -
EXAMPLE 20
Efficacy of the larvacidal activity of the
partially-purified and purified 81 kD endotoxin in
controlling Colorado Potato Beetle (Leptinotarsa
decemlineata).
Partially-purified and purified 81.2 kD endotoxin
protein was prepared from freeze-dried recombinant E.coli
cells MC1022//pJHll as described in Example 8. Fractions
from the second, FPLC ion-exchange column were designated
MonoQ A, B, and C and contained about 50%, 50%, and 25%
81.2 kD endotoxin protein respectively. These fractions
and the purified 81.2 kD protein were mixed with sterile
H20 and presented on potato leaves dipped in this
suspension for tests on first-instar larvae of Colorado
Potato Beetles (Leptinotarsa decemlineata) to give the
final treatment concentration in parts per million (ppm)
shown in Table 19. The results in Table 19 show that all
fractions were insecticidal to Colorado Potato Beetle
larvae.
EXAMPLE 21
Efficacy of the larvacidal activity of the
partially-purified and purified 81 kD endotoxin in
controlling Western Corn Rootworms (Diabrotica virgifera
virgifera).
Partially-purified and purified 81 kD novel endotoxin
protein was prepared from freeze-dried recombinant E.coli
cells MC1022/pJHll as described in Example 8. Fractions
from the second, FPLC ion-exchange column were designated
MonoQ A, B, and C and contained about 50%, 50%, and 25%
81.2 kD endotoxin protein respectively. These fractions
205484 3
- - 26 -
and the purified 81.2 kD protein were mixed with sterile
water and a sterile sucrose solution to give the treatment
rates indicated in Table 20, and a final sucrose
concentration of 2.5$. Tests on first-instar larvae of
Western Corn Rootworm were carried out as described in
Example 18. The results in Table 20 indicate that the 81.2
kD endotoxin is insecticidal to Western Corn Rootworm
larvae.
The following microorganisms and clones referred to
in this specification have been deposited at the National
Collections of Industrial and Marine Bacteria, 23 St.
Machar Drive, Aberdeen AB2 1RY, Scotland:
Name Degosit Number Date
Bacillus thuringiensis
A20 12570 20 October 1987
JHCC 4835 40091 7 December 1988
JHCC 4353 90090 7 December 1988
E.coli
BL21/pJHll 40275 6 April 1990
MC1022/p,1H12 40278 .. 24 April 1990
Bacteriophage Lambda
EMBL4 clone
CL5 40279 26 April 1990
PS35271
-27- 205484 3
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PS35271
-28- 205484 3
TABLE 2
Biochemical Markers on Microtitre Plate
Reagent HD-1 DSM JHCC JHCC
2803 4353 4835
Glycerol - - - -
Erythritol - - - -
D-arabinose - - - -
L-arabinose - - - -
Ribose + +/- + +
D-xylose - - - -
L-xylose - - - -
Adonitol - - - -
~--methyl-xyloside - - - -
Galactose - - - -
D-glucose + + + +
D-fructose + + + +
D-mannose - + - -
L-sorbose - - - -
Rhamnose - - - -
Dulcitol - - - -
Inositol - - - -
Mannitol - - - -
Sorbitol - - - -
a-methyl-D-mannoside- - - -
a-methyl-D-glucoside- - - -
N acetyl glucosamine+ + + +
Amygdaline - - - -
Arbutine + + + +
Esculine + +/- + +
Salicine + - + +
Cellobiose + - + +
Maltose + + + +
Lactose - - - -
Melibiose - - - -
Saccharose - + - -
Trehalose + + + +
Inuline - - - -
Melezitose - - - -
D-raffinose - - - -
PS35271
-29- 205484 3
TABLE 2 CONTINUED
Reagent HD-1 DSM JHCC JHCC
2803 4353 4835
Amidon + + + +
Glycogene + + + +
Xylitol - - -
~-gentiobiose - - -
D-turanose - - -
D-lyxose - - -
D-tagatose - - -
D-fucose - - -
L-fucose - - -
D-arabitol - - -
L-arabitol - - _
Gluconate - - -
2-ceto-gluconate - - -
5-ceto-gluconate - - -
Ortho-nitro-phenyl - - -
galactoside (ONPG)
Arginine (ADC- + + + +
arginine dihydrolase)
Lysine (LDH-lysine+ - - -
Decarboxylase)
Sodium Citrate + + +
(citrate utilisation)
Sodium Thiosulphate - - -
(H2S production)
Urea (urease) + - + +
Tryptophane - - -
(deaminase detection)
Tryptophane (indole - - -
production)
Sodium Pyruvate + + + +
(VP)
Gelatine (Gelatinase)+ + + +
N03-N02 Reduction + - + +
Ornithine - - -
decarboxylase (ODC)
+ - Positive Reaction
- - Negative Reaction
+/- - Weak Reaction
PS35271
-3~- 205484 3
TABLE 3
Biochemical Markers on ID-IDENT Plates
Reagent HD-1 DSM JHCC JHCC
2803 4353 4835
2-naphthyl-phosphate- - - -
2-naphthyl-butyrate + + + +
2-naphthyl-caprylate+ + + +
2-naphthyl-myristate+ + + +
L-leucyl-2- + + + +
naphthylamide .
L-valyl-2 + + + +
naphthylamide
L-cystyl-2- + + + +
naphthylamide
N-benzoyl-DL-arginine0 + + +
-2-naphthylamide
N-glutaryl- 0 + + +
phenylalanine-2-
naphthylamine
2-naphthyl-phosphate+ + + +
naphthol-AS-B1- + + + +
phosphate
PS35271
-31- 205484 3
TABLE 3 CONTINUED
Reagent HD-1 DSM JHCC JHCC
2830 4353 4835
6-bromo-2-naphthyl-aD- - - - -
galactopyranoside
2-naphthyl-SD- - - -
galactopyranoside
Naphtol-AS-B1-SD - - - -
glucuronide
2-naphthyl-aD- + + + +
glucopyranoside
6-bromo-2-naphthyl-~D-
+ - + +
glucopyranoside
1-naphthyl-N-acetyl-SD- - - - -
glucosaminide
6-Bromo-2-naphthyl-aD- - - - -
mannopyranoside
2-naphthyl-aL- - - - -
fucopyranoside
ID-IDENT is a Trade Mark of API Analytab Products
PS35271
-32_ 2054843
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PS35271
- 33 -
TABLE 6
205484 3
Strain % Mortality
~m SCRW perm CPB ~m BW
3 6 3 DAT 3 DAT
DAT DAT
DSM 2803 4800 8 92 200 100 1200 87
JHCC 4835 4800 38 92 200 7 1200 13
JHCC 4353 4800 12 68 200 0 1200 13
UNTREATED - 0 4 - 0 - 20
CONTROL
ppm = Parts per million
SCRW = Southern Corn Rootworm
CPB = Colorado Potato Beetle
BW = Boll Weevil
(RF) _ % Reduction Feeding
PS35271
-34_205484 3
TABLE 7
Diabrotica virgifera
virgifera
Expt B.thuringiensis% Mortality
No at 3 days after
treatment
Test Larvae* Untreated Controls*
1 4835 88 4
4353 72 16
2 4835 50 4
4353 60 8
* 25 first-instar larvae per test group
PS35271
-35- 205484 3
TABLE 8
Bt Strain Southern CornBoll Weevil Colorado Potato
Rootworm Beetle
3 DAT 6 DAT 3 DAT 3 DAT
4800 ppm 1200 ppm 200 ppm
DSM 2803 8 92 87 100
tenebrionis
4835 38 92 13 7
4353 12 68 13 0
Control 0 4 ZO 0
RESULTS = % MORTALITY
DAT = DAYS AFTER TREATMENT
PS35271 2 0 5 4 8 4 3
36 -
TABLE 9
Bt Strain Rate H.zea T.ni P.xylostella
~PPm)
4360 5 85 95 100
kurstaki
4835 25 100 100 100
250 100 - -
4580 25 0 0 0
tenebrionis
type 250 5 - -
Control - 0 0 10
RESULTS = % MORTALITY AT 4 DAYS AFTER TREATMENT
PS35271
37 205484 3
TABLE 10
Bt STRAINS VERSUS Spodoptera Frugiperda
AT 6 DAYS AFTER TREATMENT
4580 4835 4360 Control
tenebrionis kurstaki
PREP 0 92 84 3
1
PREP 0 60 80 3
2
PREP 0 92 88 3
3
PREP 8 100 100 3
4
RESULTS EXPRESSED AS % MORTALITY AT 80 PARTS PER MILLION
PS35271
_.. -38- 205484 3
TABLE 12
B.t. STRAINS VERSUS Heliothis Zea
AT 6 DAYS AFTER TREATMENT
. 4580 4835 4360
tenebrionis kurstaki
1 2 1 2 1 2
PREP 1 4 8 100 96 100 100
PREP 2 4 0 60 34 96 100
PREP 3 9 0 100 100 100 100
PREP 4 0 4 100 100 100 100
CONTROL 1 = 3.5% CONTROL 2 = 2%
RESULTS EXPRESSED AS % MORTALITY AT 80 PARTS PER MILLION
PS35271
._ -39- 205484 3
TABLE 13
EUROPEAN CORN BORER BIOASSAYS
1ST Experiments Prep
Number
Rate/%R. S.
1 2 5 6 7 8
p1C228 500ppm 30 30 63 5 10 75
%R.S. 100 100 100 100 100 100
pJHll 500ppm 15 75 85 72 85 80
%R.S. 100 100 100 100 100 100
p1C226 500ppm 0 0 10 5 0 10
%R.S. 0 0 11 6 0 0
PT712 500ppm 0 0 10 0 0 0
%R.S. 0 0 17 5 0 0
Control 0 0 8 3 0 8
%R.S. 0 3 11 0 0 3
4835F2 lOppm - - 100 90 80 100
%R.S. - - xxx 100 100 xxx
RESULTS = % MORTALITY AT 6 DAT
%R. S. _ % SURVIVORS OF REDUCED SIZE
PS35271
-4~- 205484 3
TABLE 14
COLORADO POTATO BEETLE BIOASSAYS
PREP NUMBER
SAMPLE RATE 1 2 5 6 7 $
p1C226 5000ppm 84 84 60 53 27 93
pJHll 5000ppm 84 100 60 93 79 87
PT712 5000ppm 0 17 7 14 7 14
p1C228 5000ppm 0 4 13 7 0 23
Control ------- 0 0 7 7 0 13
4580F2 40ppm - - 100 93 100 73
RESULTS = % MORTALITY AT 3 DAYS AFTER TREATMENT
PS35271
_41- 2054843
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TABLE 19
8lkD PROTEIN VERSUS COLORADO POTATO BEETLE
PREP 1
Mono Q Fractions B.t. Strain
Control A B C 8lkD 4580
Rate (ppm): -- 330 213 270 -- 40
0 47 21 47 -- 80
PREP 2
Mono Q Fractions B.t. Strain
Control A B C 8lkD 4580
Rate (ppm): -- 466 366 342 148 40
0 87 67 87 33 100
PREP 3
Mono Q Fractions B.t. Strain
Control A B C 8lkD 4580
Rate (ppm): -- -- -- 588 257 40
0 -- -- 60 73 80
Results= % Mortality at 3 Days After Treatment
PS35271
47 205484 3
TABLE 20
81 kD PROTEIN VERSUS WESTERN CORN ROOTWORM
Mortality at:
Sample Rate 3 DAT 4 DAT
8lkD Protein 900ppm 98 100
Tris Control - 0 0
Control (2) - 0 0
DAT = DAYS AFTER TREATMENT
