Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR PRODUCING A POTENTIATOR OF BACILLUS.PESTICIDAL
ACTIVITY
15
1. FIELD OF THE INVENTION
The invention is related to a method of obtaining and
identifying.a factor which potentiates the pesticidal activity'
of a Bacillus related pesticide, a chemical pesticide and/or a
virus with pesticidal properties.
2. BACKGROUND OF THE INVENTION
Every year, pests detrimental to agriculture,
forestry, and public health cause losses in the millions of
dollars. various strategies have been used to control such
pests.
One strategy is the use of chemical pesticides with a
broad range or spectrum of activity. However, there are a
number of disadvantages with using chemical pesticides.
Spetifically, because of their broad spectrum.of activity, these
pesticides may destroy non-target organisms such as beneficial
insects and parasites of destructive pests. Additionally,
chemical pesticides are frequently toxic to animals and humans.
Furthermore, targeted pests frequently develop resistance when
repeatedly exposed to such substances.
Another strategy involves the use of biopesticides to
control insect, fungal and weed infestations. Biopesticides are
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naturally occurring pathogens and/or the substances produced
bythese pathogens. The advantage of using biopesticides is that
they are generally less harmful to non-target organisms and the
environment as a whole compared to chemical pesticides.
2.1. Bacillus thzrincriensis
The most widely used biopesticide is Bacillus
thuringiensis. Bacillus thuringiensis is a motile, rod-shaped,
gram-positive bacterium that is widely distributed in nature,
especially in soil and insect-rich environments. During
sporulation, Bacillus thuringiensis produces a parasporal
crystal inclusion(s) which is insecticidal upon ingestion to
susceptible insect larvae of the orders Lepidoptera, Diptera,
and Coleoptera. The inclusions may vary in shape, number, and
composition. They are comprised of one or more proteins called
delta-endotoxins, which may range in size from 27-140 kDa. The
insecticidal delta-endotoxins are generally converted by
proteases in the larval gut into smaller (truncated) toxic
polypeptides, causing midgut destruction, and ultimately, death
of the insect (HtSfte and Whiteley, 1989, Microbiological Reviews
53:242-255).
There are several Bacillus thuringiensis strains that
are widely used as biopesticides in the forestry, agricultural,
and public health areas. Bacillus thuringiensis subsp.
kurstaki and Bacil.Zus thuringiensis subsp. aizawai produce
delta-endotoxins specific for Lepidoptera. A delta-endotoxin
specific for Coleoptera is produced by Bacillus thuringiensis
subsp. tenebrionis (Krieg et al., 1988, U.S. Patent No.
4,766,203). Furthermore, Bacillus thuringiensis subsp.
israelensis produces delta-eridotoxins specific for Diptera
(Goldberg, 1979, U.S. Patent No. 4,166,112).
Other Bacillus thuringiernsis strains specific for
dipteran pests have also been described. A Bacillus
thuringiensis isolate has been disclosed which is toxic to
Diptera and Lepidoptera (Hodgman et al., 1993, FEMS Microbiology
Letters 114:17-22). SDS polyacrylamide gel electrophoresis of
the purified crystal delta-endotoxin from this isolate revealed
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three protein species which are related to CryIA(b), CryIB, and
CryIIA toxins. There has also been disclosed a Bacillus
thuringiensis isolate which produces a dipteran-active crystal
comprised of proteins with molecular weights of 140, 122, 76,
= 5 72, and 38 kDa (Payne, 1994, U.S. Patent No. 5,275,815). EPO
480,762 discloses five B.C. strains which are each active
against dipteran pests; each also have a unique crystal delta-
endotoxin pattern.
Several Bacillus thuringiensis strains have been
described which have pesticidal activity against pests other
then Lepidoptera, Coleoptera, and Diptera. Five Bacillus
thuringiensis strains have been disclosed which produce delta-
endotoxins that are toxic against nematodes (Edwards, Payne, and
Soares, 1988, Eur. Pat. Appl. No. 0 303 426 B1). There has
also been disclosed a Bacillus thuringiensis strain, PS81F,
which can be used to treat humans and animals hosting parasitic
protozoans (Thompson and Gaertner, 1991, Eur. Pat. Appl. No. 0
461 799 A2). Several Bacillus thuringiensis isolates have also
been disclosed with activity against acaride pests. These
isolates produce crystals comprised of proteins with molecular
weights in the (wide) range of 35 kDa to 155 kDa (Payne, Cannon,
and Bagley, 1992, PCT Application No. WO 92/19106). There have
also been disclosed Bacillus thuringiensis strains with activity
against pests of the order Hymenoptera (Payne, Kennedy, Randall,
Meier, and Uick, 1992, Eur. Pat. Appl. No. 0 516 306 A2); with
activity against pests of the order Hemiptera (Payne and Cannon,
1993, U.S. Patent No. 5,262,159); with activity against fluke
pests (Hickle, Sick, Schwab, Narva, and Payne, 1993., U.S. Patent
No. U.S. 5,262,399; and with activity against pests of the order
Phthiraptera (Payne and Hickle, 1993, U.S. Patent No.
5,273,746). Furthermore, another strain of Bacillus
thuringiensis subsp. kurstaki, WB3S-16, isolated from Australian
sheep wool clippings, has been disclosed that is toxic to the
biting louse Damalinia ovis, a Phthiraptera pest (Drummond,
Miller, and Pinnock, 1992, J. Invert. Path. 60:102-103).
The delta-endotoxins are encoded by cry (crystal
protein) genes which are generally located on plasmids. The cry
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genes have been divided into six classes and several subclasses
based on relative amino acid homology and pesticidal
specificity. The major classes are Lepidoptera-specific (cryi);
Lepidoptera-and Diptera-specific (cryll); Coleoj;tera-specific
( crylll) ; Diptera-specific ( czylV) (Hdite and Whiteley, 1989,
Microbiological Reviews 53:242-255); Coleoptera- and
Lepidoptera-specific (referred to as cryV genes by Tailor et
al., 1992, MoZecular Microbioiogy 6:1211-1217); and Nematode-
specific (referred to as cryV and cryVl genes by Feitelson et
al., 1992, Bio/Technology 10:271-275).
Delta-endotoxins have been produced by recombinant DNA
methods. The delta-endotoxins produced by recombinant DNA
methods may or may not be in crystal form.
Some strains of Bacillus thuringiensis have been shown
to produce a heat-stable pesticidal adenine-nucleotide analog,
known as S-exotoxin type I or thuringiensin, which is pesticidal
alone (Sebesta et al., in H.D. Burges (ed.), Microbial Control
of Pests and Plant Diseases, Academic Press, New York, 1980, pp.
249-281). i3-exotoxin type I has been found in the supernatant
of some Bacillus thuringiensis cultures. It has a molecular
weight of 701 and is comprised of adenosine, glucose, and
allaric acid (Farkas et al., 1977, Coll. Czechosslovak Chem.
Comm. 42:909-929; Luthy et al., in Kurstak (ed.), Microbial and
Viral Pesticides, Marcel Dekker, New York, 1982, pp. 35-72).
Its host range includes, but is not limited to, Musca domestica,
Mamestra configurata Walker, Tetranychus urticae, Drosophila
melanogaster, and Tetranychus cinnabarinus. The toxicity of 9-
exotoxin type I is thought to be due to inhibition of DNA-
directed RNA polymerase by competition with ATP. It has been
shown that S-exotoxin type I is encoded by a cry plasmid in five
Bacillus thuringiensis strains (Levinson et al., 1990, J.
Bacteriol. 172:3172-3179). f3-exotoxin type I was found to be
produced by Bacillus thuringiensis subsp. thuringiensis
serotype 1, Bacillus thuringiensis subsp. tolworthi serotype 9,
and Bacillus thuringiensis subsp. darmstadiensis serotype 10.
Another S-exotoxin classified as S-exotoxin type II
has been described (Levinson et al., 1990, J. Bacteriol.
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172:3172-3179). S-exotoxin type II was found to be produced by
Bacillus thuringiensis subsp. morrisoni serotype 8ab and is
active against Leptinotarsa decemlineata. The structure of i3-
exotoxin type II is not completely known, but is significantly
different from that of S-exotoxin type I in that a pseudouridine
moiety is in the place of adenine in which attachment to the
ribose ring is at a position that would otherwise be occupied by
a proton (Levinson, in Hickle and Finch (eds.), Analytical
Chemistry of Bacillus thuringiensis, ACS Symposium Series,
Washington, D.C., 1990, pp. 114-136). Furthermore, there is
only one signal in the proton NMR spectrum corresponding to the
nucleoside base (at 7.95 ppm), and does not have a ribose-type
anomeric protein signal (5.78 ppm).
Other water soluble substances that have been isolated
from Bacillus thuringiensis include alpha-exotoxin which is
toxic against the larvae of Musca domestica (Luthy, 1980, FEMS
Microbiol. Lett. 8:1-7); gamma -exo toxins, which are various
enzymes including lecithinases, chitinases, and proteases, the
toxic effects of which are expressed only in combination with
beta-exotoxin or delta-endotoxin (Forsberg et al., 1976,
Bacillus thuringiensis: its Effects on Environmental Quality,
National Research Council of Canada, NRC Associate Committee on
Scientific Criteria for Environmental Quality, Subcomittees on
Pesticides and Related Compounds and Biological Phenomena);
sigma exotoxin which has a structure similar to beta-exotoxin,
and is also active against Leptinotarsa decemiineata (Argauer et
al., 1991, J. Entomol. Sc.i. 26:206-213); and
anhydrothuringiensin (PZystas, et al., 1975, Co11. Czechosslovak
Chem. Comm. 40:1775).
2.2. ZWITTERMICIN
A substance has been isolated from Bacillus cereus
which inhibits the growth of the plant pathogen Phytophthora
medicaginis and reduces the infection of alfalfa (see, for
example, U.S. Patent Nos. 4,877,738 and 4,878,936) . No other
activity was disclosed. The following structure has been
elucidated for zwittermicin A (He et al., Tet. Lett. 35:2499-
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2502)
OH NH2 NH2
H
N OH
H2N H (Ia).
0 OH OH OH
NH2
3. OBJECTS OF THE INVENTION
The art has strived to achieve increased mortality of
B.t. formulations. Means have included searching for new
strains with increased mortality, attempting to engineer
present strains, and attempting to design more effective
formulations by combining B.t. spores and crystals with
new pesticidal carriers chemical pesticides, or enhancers
(see, for example, U.S. Patent No. 5,250,515, a trypsin
inhibitor). It is therefore an object of the present
invention to potentiate the pesticidal activity of
pesticides.
4. SUMMARY OF THE INVENTION
The invention relates to a method for obtaining which
a factor which potentiates the pesticidal activity of a
Bacillus related pesticide comprising
(a) culturing a Bacillus strain under suitable
conditions;
(b) recovering the factor from the supernatant of the
culture of step (a).
According to one aspect of the present invention,
there is provided a method for potentiating the pesticidal
activity of a pesticide produced by a Bacillus strain,
combining said pesticide with a factor which potentiates
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the pesticidal activity, wherein said factor has the
structure Ia or salt thereof.
In a specific embodiment, the Bacillus strain is
selected from the group consisting of Bacillus subtilis,
Bacillus licheniformis, and Bacillus thuringiensis. In a
preferred embodiment, the factor is in substantially pure
form. As defined herein a"substantially pure" factor
means a factor
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which contains less than 10% of contaminants, for example,
delta-endotoxin protein. Such a substantially pure factor may
be obtained by isolating said factor, e.g., by column
chromatography.
The factor obtained is a potentiator. As defined
herein, a"potentiator is a substance which has no significant
pesticidal activity, e.g. having an LCso (LC50 is the
concentration of the substance required to kill 50% of the
pests) of more than about 3000 g/g as assayed by bioassay (see
Section 6) but acts to increase the pesticidal activity of a
Bacillus related pesticide at least about 50% and does not cause
larval stunting. As noted in Section 2, other substances
capable of enhancing pesticidal activity known in the art such
as trypsin inhibitors and exotoxins have pesticidal activity.
In a specific embodiment, the factor is water soluble.
As defined herein, a substance or compound is "water soluble" if
at least about 1 mg of a substance can be dissolved in 1 ml of
water. The factor may also potentiate the pesticidal activity
of a chemical pesticide and/or a virus with pesticidal
properties.
As defined herein, "a Bacillus related pesticide" is a
Bacillus (e.g. Bacillus thuringiensis or Bacillus subtilis)
strain, spore, or substance, e.g. protein or fragment thereof
having activity against or which kill pests or a microorganism
capable of expressing a Bacillus gene encoding a Bacillus
protein or fragment thereof having activity against or which
kill pests (e.g. Bacillus thuringiensis delta-endotoxin) and an
acceptable carrier (see Section 5.2., .fnfra, for examples of
such carriers). The pest may be, for example, an insect, a
nematode, a mite, or a snail. A microorganism capable of
expressing a Bacillus gene encoding a Bacillus protein or
fragment thereof having activity against or which kill pests
inhabits the phylloplane (the surface of the plant leaves),
and/or the rhizosphere (the soil surrounding plant roots),
and/or aquatic environments, and is capable of successfully
competing in the particular environment (crop and other insect
habitats) with the wild-type microorganisms and provide for the
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stable maintenance and expression of a Bacillus gene encoding a
Bacillus protein or fragment thereof having activity against or
which kill pests. Examples of such microorganisms include but
are not limited to bacteria, e.g. genera Bacillus, Pseudomonas,
Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces,
Rhizobium, Rhodopseudomonas, MethylophiZius, Agrobacterium,
Acetobacter, Lactobacillus, Arthrobacter, Azotobacter,
Leuconostoc, A1caligenes, and Ciostridium; algae, e.g. families
Cyanophyceae, Prochlorophyceae, Rhodophyceae, Dinophyceae,
Chrysophyceae, Prymnesiophyceae, Xanthophyceae, Raphidophyceae,
Baci.Zlariophyceae, Eustigrnatophyceae, Cryptophyceae,
Euglenophyceae, Prasinophyceae, and Chlorophyceae; and fungi,
particularly yeast, e.g. genera Saccharomyces, Czyptococcus,
Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium.
As defined herein, pesticidal activity" measures the
amount of activity against a pest through killing or stunting of
the growth of the pest or protecting the plant from pest
infestation.
The factor obtained may be formulated into a
composition comprising the factor and a pesticidal carrier as
well as the factor and a Bacillus related pesticide, chemical
pesticide and/or a virus with pesticidal properties. These
compositions may be used for controlling a pest, decreasing the
resistance of a pest to a Bacillus related pesticide comprising
exposing the pest to a composition comprising the factor and a
pesticidally acceptable carrier, or potentiating the pesticidal
activity of a Bacillus related pesticide.
The invention is also directed to a method for
identifying said factor comprising
(a) culture of a strain of Bacillus;
(b) recovering the supernatant of the culture of
(a); and
(c) assaying the supernatant of (b) for
potentiation of a Bacillus related pesticide.
5. BRTF.F DFSCRTP'-'iON OF THE FTGURES
Figure 1 schematically shows the general procedure
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used for purifying Ia.
Figure 2 shows the 13C NMR spectrum of Ia.
Figure 3 shows the proton NMR spectrum of Ia.
Figure 4 shows the results of nOe experiments on the
acetylated derivative of Ia.
6. DETAILED DESCRIPTION OF THE INVFNTION
The factor potentiates the pesticidal activity of a
Bacillus related pesticide and may have a molecular weight of
from about 350 to about 1200 or in a specific embodiment from
about 350 to about 700.
The factor potentiates the pesticidal activity of a
Bacillus related pesticide at least about 1.5 fold to optionally
about 1000 fold, preferably from about 100 fold to about 400
fold. In a specific embodiment, the factor potentiates the
pesticidal activity of a Bacillus thuringiensis delta-endotoxin
including but not limited to a Cryl (including but not limited
to CryIA, CryIB, and CryIC), CryII, CryIII, CryIV, CryV, or
CxyVI protein in full-length form or a proteolytically
processed, truncated form, from about 1.5 fold to about 1000
fold. in a most specific embodiment, the factor potentiates a
B.C. delta-endotoxin from about 100 fold to about 400 fold. The
factor may also potentiate the pesticidal activity of a chemical
pesticide and/or a virus with pesticidal properties.
The factor may also be water soluble, stable in water
up to about 100 C for at least about 5 minutes, stable when
subjected to direct sunlight for at least about 10 hours, and/or
stable at a pH of about 2 for about 10 days. The factor may
have 13 carbons. Additionally, the factor may have 1H NMR
shifts at 81.5, 3.22, 3.29, 3.35, 3.43, 3.58, 3.73, 3.98, 4.07,
4.15, 4.25, 4.35.
In a most specific embodiment, said factor has the
structure Ia or salt thereof.
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OH 2 NH
2
N N OH
H2N H
O OH OH OH
NH2
The salt would be capable of potentiating a Bacillus related
pesticide.
6.1. OBTAINING THE FACTOR
The factor may be obtainable from a Bacillus strain
(e.g. Bacillus subtilis, Bacillus licheniformis, and Bacillus
thuringiensis) in shake flasks or a fermentor. In a specific
embodiment, the factor is obtainable from the supernatant of a
Bacillus thuringiensis culture including but not limited to
Bacillus thuringiensis subsp. kurstaki, Bacillus thuringiensis
subsp. aizawai, Bacillus thuringiensis subsp. galleriae,
Bacillus thuringiensis subsp. entornocidus, Bacillus
thuringiensis subsp. tenebrionis. Bacillus thuringiensis subsp.
thuringiensis, Bacillus thuringiensis subsp. alesti, Bacillus
thuringiensis subsp. canadiensis, Bacil.Zus thuringiensis subsp.
darmstadiensis, Bacillus thuringiensis subsp. dendrolimus,
Bacillus thuringiensis subsp. fini.timus, Bacillus thuringiensis
subsp. kenyae, Bacillus thuringiensis subsp. morrisoni, Bacillus
thuringiensis subsp. subtoxicus, Bacillus thuringiensis subsp.
toumanoffi and Bacillus thuringiensis subsp. israelensis. in a
preferred embodiment, the factor is obtainable from the
supernatant of Bacillus thuringiensis subsp. kurstaki, Bacillus
thuringiens.is subsp. aizawai, or Bacillus *huringiensis subsp.
gal.Zeriae or mutants thereof having substantially the same
potentiating activity. in a specific embodiment, the factor is
recovered from a cry- spo- mutant of Bacillus thuringiensis
subsp. kurstaki.
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Bacillus may be cultured using media and fermentation
techniques known in the art (see, for example, Rogoff et al.,
1969, J. invertebrate Path. 14:122-129; Dulmage et al., 1971, J.
Invertebrate Path. 18:353-358; Dulmage et al., in Microbial
Control of Pests and Plant Diseases, H.D. Burges, ed., Academic
Press, N.Y., 1980). Upon completion of the cycle, the
supernatant can be recovered by separating B.t. spores and
crystals from the culture (fermentation) broth by means well
known in the art, e.g. centrifugation and/or ultrafiltration.
The factor is contained in the supernatant which may be
recovered by means well known in the art, e.g. ultrafiltration,
evaporation, and spray-drying. This procedure is more
specifically described in the sections which follow.
Purification of the factor can be carried out by
various procedures known in the art, including but not limited
to chromatography (e.g. ion exchange, affinity, and size
exclusion column chromatography), electrophoretic procedures,
differential solubility, extraction, or any other standard
technique known in the art.
The potentiating activity of the factor of the
pesticidal activity of Bacillus related pesticide, virus having
pesticidal activity, or chemical-.pesticide against various pests
may be assayed using procedures known in the art, such as an
artificial insect diet incorporated, artificial diet overlay,
leaf painting, leaf dip, and foliar spray. Specific examples of
such assays are given in Section 7, infra.
6.2. COMPOSITIONS COMPRISING THE FACTOR
The factor obtained can be formulated alone; with a
Bacillus related pesticide, which as defined, supra, is a
Bacillus strain, spore, protein or fragment, or other
substance, thereof, with activity against or which kills pests
, or protects plants against a pest; with a chemical pesticide
and/or an entomopathogenic virus and an acceptable carrier into
a pesticidal composition(s), that is, for example, a suspension,
a solution, an emulsion, a dusting powder, a dispersible
granule, a wettable powder, an emulsifiable concentrate, an
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aerosol or impregnated granule. Examples of such Bacillus
strains include, but are not limited to, Bacillus thuringiensis
subsp. kurstaki (marketed as DIPELTM from Abbott Laboratories,
Inc., JAVELINTM from Sandoz, BIOBI'1'TM from Novo Nordisk A/S,
FORAYTM from Novo Nordisk A/S, BIOCOTTM from Novo Nordisk A/S,
MVPT,'4 from Mycogen, BACTOSPEINETM from Novo Nordisk A/S, and
THURICIDETM from Sandoz); Bacillus thuringiensis subsp. aizawai
(marketed as FLORBACTM from Novo Nordisk A/S, and XENTARITM from
Abbott Laboratories, Inc.); Bacillus thuringiensis subsp.
tenebrionis (marketed as NOVODORTM from Novo Nordisk A/S,
TRIDENTTM from Sandoz, and M-TRAKTM and M-ONETM from Mycogen) ;
Bacillus thuringiensis subsp. israelensis (marketed as either
BACTIMOSTM or SKEETALTM from Novo Nordisk A/S, TEKNARTM from
Sandoz, and VECTOBACTM from Abbott Laboratories, Inc.); Bacillus
thuringiensis kurstaki/tenebrionis (marketed as FOILTM from
Ecogen); Bacillus thuringiensis kurstaki/aizawai (marketed as
CONDORTM from Ecogen and AGREETM from Ciba-Geigy); and Bacillus
thuringiensis kurstaki/kurstaki (marketed as CUTLASSTM from
Ecogen). The Bacillus related protein may be selected from the
group including, but not limited to, Cryl, CryII, Crylil, CryIV,
CryV, and CryVI. The chemical pesticide may be, for example, an
insect growth regulator such as diflubenzuron, a carbamate such
as thiodicarb and methomyl, an organophosphate such as
chlorpyrifos, a pyrethroid such as cypermethrin and
esfenvalerate, inorganic fluorine such as cryolite, and a
pyrrole. The entomopathogenic virus may be a baculovirus, e.g.,
Autographa californica nuclear polyhedrosis virus (NPV),
Syngrapha falci fera NPV, Cydia pomonella GV (granulosis
virus), Hel.iothis zea NPV, Lymantria dispar NPV, Orgyia
pseudotsugata NPV, Spodoptera exigua NPV, Neodiprion lecontei
NPV, Neodipr.ion sertifer NPV, Harrisina brillians NPV, and
Endopiza viteana Clemens NPV.
in compositions comprising the substance and a
Bacillus related pesticide, the substance may be present in the
amount of at least about 0.1 g/BIU or 0.05 g factor per g
Bacillus delta-endotoxin and spore, optionally to about 300
g/BIU or 150 g substance per g Bacillus delta-endotoxin and
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spore, preferably 2 g/BIU or 1 g substance per g Bacillus delta-
endotoxin and spore. As defined herein "BIU is billion
international units as determined by bioassay. The bioassay
compares the sample to a standard Bacillus reference material
using Trichoplusia ni or other pest as the standard test insect.
The potency is determined by dividing the reference standard
LCSO then multiplying by the reference standard potency.
In another embodiment, the composition may comprise
the factor in substantially pure form or a supernatant from
Bacillus in dry, concentrated, or liquid form and a
pesticidally acceptable carrier, examples of which are
disclosed, infra. This composition may be applied separately to
a plant, e.g., transgenic plants. Specifically, the composition
may be applied to a plant previously containing and expressing a
Bacillus thuringiensis gene. in another embodiment, the
composition may be applied to a plant previously exposed to a
Bacillus thuringiensis composition. in another embodiment, the
composition may be applied to other environments of a dipteran
pest(s), e.g., water or soil. The substance is present in the
composition at a concentration of about 0.001% to about 60%
(w/w).
The composition comprising the substance and a
pesticidally acceptable carrier in addition to controlling a
pest may also be used to decrease the resistance of a pest to a
pesticide. Alternatively, the composition may be used to
potentiate a Bacillus related pesticide. The composition in one
embodiment may be applied at the same time as the pesticide in
an amount of at least about 2 g substance/BIU up to optionally
about 300 g substance/BIU. In another embodiment, the
composition may be applied up to about 24 hours after the
pesticide as an adjuvant to extend the efficacy of residual
pesticide.
Such compositions disclosed above may be obtained by
the addition of a surface active agent, an inert carrier, a
preservative, a humectant, a feeding stimulant, an attractant,
an encapsulating agent, a binder, an emulsifier, a dye, a U.V.
protectant, a buffer, a flow agent, or other component to
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facilitate product handling and application for particular
target pests.
Suitable surface-active agents include anionic
compounds such as a carboxylate, for example, a metal
carboxylate of a long chain fatty acid; a N-acylsarcosinate;
mono or di-esters of phosphoric acid with fatty alcohol
ethoxylates or salts of such esters; fatty alcohol sulphates
such as sodium dodecyl sulphate, sodium octadecyl sulphate or
sodium cetyl sulphate; ethoxylated fatty alcohol sulphates;
ethoxylated alkylphenol sulphates; lignin sulphonates;
petroleum sulphonates; alkyl aryl sulphonates such as alkyl-
benzene sulphonates or lower alkylnaphthalene sulphonates, e.g.,
butyl-naphthalene sulphonate; salts or sulphonated naphthalene-
formaldehyde condensates; salts of sulphonated phenol-
formaldehyde condensates; or more complex sulphonates such as
the amide sulphonates, e.g., the sulphonated condensation
product of oleic acid and N-methyl taurine or the dialkyl
sulphosuccinates, e.g., the sodium sulphonate or dioctyl
succinate. Non-ionic agents include condensation products of
fatty acid esters, fatty alcohols, fatty acid amides or fatty-
alkyl- or alkenyl-substituted phenols with ethylene oxide, fatty
esters of polyhydric alcohol ethers, e.g., sorbitan fatty acid
esters, condensation products of such esters with ethylene
oxide, e.g., polyoxyethylene sorbitar fatty acid esters, block
copolymers of ethylene oxide and propylene oxide, acetylenic
glycols such as 2,4,7,9-tetraethyl-5-decyn-4,7-diol, or
ethoxylated acetylenic glycols. Examples of a cationic surface-
active agent include, for instance, an aliphatic mono-, di-, or
polyamine as an acetate, naphthenate or oleate; an oxygen-
containing amine such as an amine oxide of polyoxyethylene
alkylamine; an amide-linked amine prepared by the condensation
of a carboxylic acid with a di- or polyamine; or a quaternary
ammonium salt.
Examples of inert materials include inorganic minerals
such as kaolin, mica, gypsum, fertilizer, phyllosilicates,
carbonates, sulfates, or phosphates; organic materials such as
sugar, starches, or cyclodextrins; or botanical materials such
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as wood products, cork, powdered corncobs, rice hulls, peanut
hulls, and walnut shells.
The compositions of the present invention can be in a
suitable form for direct application or as a concentrate or
primary composition which requires dilution with a suitable
quantity of water or other diluent before application. The
pesticidal concentration will vary depending upon the nature of
the particular formulation, specifically, whether it is a
concentrate or to be used directly. The composition contains 1
to 98% of a solid or liquid inert carrier, and 0 to 50%,
preferably 0.1 to 50% of a surfactant. These compositions will
be administered at the labeled rate for the commercial product,
preferably about 0.01 pound to 5.0 pounds per acre when in dry
form and at about 0.01 pint to 25 pints per acre when in liquid
form.
In a further embodiment, the Bacillus thuringiensis
crystal delta-endotoxin and/or factor can be treated prior to
formulation to prolong the pesticidal activity when applied to
the environment of a target pest as long as the pretreatment is
not deleterious to the crystal delta-endotoxin or substance.
Such treatment can be by chemical and/or physical means as long
as the treatment does not deleteriously affect the properties of
the composition(s). Examples of chemical reagents include, but
are not limited to, halogenating agents; aldehydes such as
formaldehyde and glutaraldehyde; anti-infectives, such as
zephiran chloride; alcohols, such as isopropranol and ethanol;
and histological fixatives, such as Bouin's fixative and Helly's
fixative (see, for example, Humason, Animal Tissue Techniques,
W.H. Freeman and Co., 1967).
The compositions of the invention can be applied
directly to the plant by, for example, spraying or dusting at
the time when the pest has begun to appear on the plant or
= before the appearance of pests as a protective measure. Plants
to be protected within the scope of the present invention
include, but are not limited to, cereals (wheat, barley, rye,
oats, rice, sorghum and related crops), beets (sugar beet and
fodder beet), drupes, pomes and soft fruit (apples, pears,
CA 02223034 1997-12-01
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plums, peaches, almonds, cherries, strawberries, raspberries,
and blackberries), leguminous plants (alfalfa, beans, lentils,
peas, soybeans), oil plants (rape, mustard, poppy, olives,
sunflowers, coconuts, castor oil plants, cocoa beans,
groundnuts), cucumber plants (cucumber, marrows, melons), fibre
plants (cotton, flax, hemp, jute), citrus fruit (oranges,
lemons, grapefruit, mandarins), vegetables (spinach, lettuce,
asparagus, cabbages and other brassicae, carrots, onions,
tomatoes, potatoes), lauraceae (avocados, cinnamon, camphor),
deciduous trees and conifers (linden-trees, yew-trees, oak-
trees, alders, poplars, birch-trees, firs, larches, pines), or
plants such as maize, turf plants, tobacco, nuts, coffee, sugar
cane, tea, vines, hops, bananas and natural rubber plants, as
well as ornamentals. The composition can be applied by foliar,
furrow, broadcast granule, "lay-by", or soil drench application.
It is generally important to obtain good control of pests in the
early stages of plant growth as this is the time when the plant
can be most severely damaged. The spray or dust can
conveniently contain another pesticide if this is thought
necessary. In a preferred embodiment, the composition of the
invention is applied directly to the plant.
The compositions of the present invention can also be
applied directly to ponds, lakes, streams, rivers, still water,
and other areas subject to infestation by dipteran pests,
especially pests o.f concern to public health. The composition
can be applied by spraying, dusting, springling, or the like.
The compositions of the present invention may be
effective against insect pests of the order Lepidoptera, e.g.,
Achroia grisella, Acleris gloverana, Acleris variana, Adoxophyes
orana, Agrotis ipsilon, Alabama argillacea, Alsophila pometaria,
Amyelois transitella, Anagasta kuehniella, Anarsia lineatella,
Anisota senatoria, Antheraea pernyi, Anticarsia gemmatalis,
Archips sp., Argyrotaenia sp., Athetis mindara, Bombyx mori,
Bucculatrix thurberiella, Cadra cautella, Choristoneura sp.,
Cochylis hospes, Colias eurytheme, Corcyra cephalonica, Cydia
lati ferreanus, Cydia pomonella, Datana integerrima, Dendrolimus
sibericus, Desmia funeralis, Diaphania hyalinata, Diaphania
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nitidalis, Diatraea grandiosella, Diatraea saccharalis, Ennomos
subsignaria, Eoreuma loftini, Ephestia elutella, Erannis
tiliaria, Estigmene acrea, Eulia salubricola, Eupoecilia
ambiguella, Euproctis chrysorrhoea, Euxoa messoria, Galleria
mellonella, Grapholita znolesta, Harrisina americana, Helicoverpa
subflexa, Helicoverpa zea, Heliothis virescens, Hemileuca
oliviae, Homoeosoma electellurn, Hyphantria cunea, Keiferia
Iycopersicella, Lambdina fiscellaria fiscellaria, Lambdina
fiscellaria lugubrosa, Leucoma salicis, Lobesia botrana,
Loxostege sticticalis, Lymantria dispar, Macalla thyrsisalis,
Malacosoma sp., Mamestra brassicae, Mamestra configurata,
Manduca quinquernaculata, Manduca sexta, Maruca testulalis,
Melanchra picta, Operophtera brumata, Orgyia sp., Ostrinia
nubilalis, Paleacrita vernata, Papilio cresphontes, Pectinophora
gossypiella, Phryganidia californica, Phyllonorycter
blancaz-della, Pieris napi, Pieris rapae, Plathypena scabz-a,
Platynota flouendana, Platynota sultana, Platyptilia
carduidactyla, Plodia interpunctella, Plutella xylostella,
Pontia protodice, Pseudaletia unipuncta, Pseudoplusia includens,
Sabulodes aegrotata, Schizura concinna, Sitotroga cerea.Zella,
Spilonota ocellana, Spodoptera sp., Thaurnstopoea pityocampa,
Tineola bisselliella, Trichoplusia ni, Udea rubigalis, xylomyges
curialis, Yponomeuta padella; order Diptera, e.g., Aedes sp.,
Andes vittatus, pnastrepha ludens, Anastrepha suspensa,
Anopheles barberi, Anopheles quadrimaculatus, Armigeres
subalbatus, Calliphora stygian, Calliphora vicina, Ceratitis
capitata, Chironomus tentans, Chrysomya rufifacies, Coch3iomyia
macellaria, Culex sp., Culiseta inornata, Dacus oleae, Delia
antiqua, Delia platura, Delia radicum, Drosophila melanogaster,
Eupeodes corollae, Glossina austeni, Glossina brevipalpis,
Glossina fuscipes, Glossina morsitans centralis, Glossina
morsitans morsitans, Glossina moristans submorsitans, Glossina
pallidipes, Glossina palpalis gambiensis, Glossina palpalis
palpalis, Glossina tachinoides, Haelnagogus equinus, Haematobia
irri tans, Hypoder:na bovis, Hypoderma lineatum, Leucopis ninae,
Lucilia cuprina, Lucilia sericata, Lutzomyia longlpaipis,
Lutzomyia shannoni, Lycoriella mali, Mayetiola destructor, Musca
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autumnalis, Musca domestica, Neobellieria sp., Nephrotoma
suturalis, Ophyra aenescens, Phaenicia sericata, Phlebotomus
sp., Phormia regina, Sabethes cyaneus, Sarcophaga bullata,
Scatophaga stercoraria, Stomoxys calcitrans, Toxorhynchites
amboinensis, Tripteroides bambusa. However, the compositions of
the invention may also be effective against insect pests of the
order Coleoptera, e.g., Leptinotarsa sp., Acanthoscelides
obtectus, Callosobruchus chinensis, Epilachna varivestis,
Pyrrhalta luteola, Cylas formicarius elegantulus, Listronotus
oregonensis, Sitophilus sp., Cyclocephala borealis, Cyclocephala
immaculata, Macrodactylus subspinosus, Popillia japonica,
Rhizotrogus majalis, AZphitobius diaperinus, Palorus ratzeburg.i,
Tenebrio molitor, Tenebrio obscurus, Tribolium castaneum,
Tribolium confusum, Tribolius destructor; Acari, e.g.,
Oligonychus pratensis, Panonychus u1mi, Tetranychus urticae;
Hymenoptera, e.g., Iridomyrmex humilis, Solenopsis invicta;
Isoptera, e.g., Reticulitermes hesperus, Reticulitermes
flavipes, Coptotermes formosanus, Zootermopsis angusticollis,
Neotermes connexus, Incisitermes minor, Incisitermes immigrans;
Siphonaptera, e.g., Ceratophyllus gallinae, Ceratophyllus niger,
Nosopsyllus fasciatus, Leptopsylla segnis, Ctenocephalides
canis, Ctenocephalides felis, Echicnophaga gallinacea, Pu1ex
irritans, Xenopsylla cheopis, Xenopsylla vexabilis, Tunga
penetrans; and Tylenchida, e.g., Melodidogyne incognita,
Pratylenchus penetrans.
The following examples are presented by way of
illustration, not by way of limitation.
7. EXAMPLE= CHARACTERIZATION OF Ia
As detailed herein, 2a is recovered and purified. The
characterization of Ia is detailed infra.
7.1. RRC = RY AND PURIFICATION OF Ia
B. thuringiensis subsp. kurstaki strain EMCC0086
(deposited with the NRRL as B-21147) is fermented for 72 hours
at 30 C in a medium comprised of a carbon source such as starch,
hydrolyzed starch, or glucose and a nitrogen source such as
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protein, hydrolyzed protein, or corn steep liquor. The
production of Ia is detected at 13 hours into the fermentation.
Peak activity is found to be at approximately 30 hours.
Supernatant from a B. thuringiensis subsp. kurstaki
fermentation is recovered by centrifugation and then is
clarified by u:ltrafiltration through a 30 kDa MW-CO membrane
using a Rhone Poulenc UF system. The 30 kDa filtration removed
any remaining cell debris, crystal delta-endotoxin, spores, and
soluble protein greater than 30 kDa molecular mass. The
permeate is concentrated 10 fold by evaporation. The permeate
is centrifuged and then 0.2}.t,filtered to further remove
insolubles from the broth, leaving a clear broth containing Ia.
The purification of Ia to homogeneity is achieved
using a multi-step purification procedure shown schematically in
Figure 1. in conjunction with the recovery protocol outlined
above, the purification proceeded with a 5 kDa ultrafiltration
step. The permeate from the 5 kDa ultrafiltration is adsorbed
to a Sulfopropyl (SP) cation exchange resin and eluted with an
ammonium acetate solution. The compound is then concentrated
approximately 30X by lyophilization, and the salt and other
contaminants are removed with a BioRad P2 size exclusion column.
The pool from the P2 column is run over a high resolution SP
HPLC cation exchange column which yielded a homogeneous
compound. The contaminating salt is removed by repeated
lyophilization.
Activity is monitored by a Spodoptera exigua micro-
bioassay, and purity is determined by capillary electrophoresis.
Sample consisting of 50 l of Ia and 50 l of CryIA(c) protein
(15 g/ml) purified from BIOBITTM FC (100 l), is applied to
individual wells of a jelly tray containing 500 l of solidified
artificial insect diet. The trays containing the various
samples are air dried. Two to four 2nd or early 3rd instar
Spodoptera exigua are added to the wells containing the-dried
sample. The wells*are sealed with mylar poked with holes and
are incubated for 2-3 days at 30 C. Degree of stunting and
percent mortality are then recorded. Typically, 5 replicate
wells are run for each sample.
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7.2. STRUCTURE ELUCIDATION
The active compound is found to be water soluble but
is not soluble in organic solvents. It is positively charged
and reacted with ninhydrin as evidenced by silica thin layer
chromatography. 13C and proton NMR of the-compound are shown in
Figures 2 and 3, respectively. 13C NMR experiments revealed the
presence of 13 carbons (referenced to 3-[trimethylsilyl
propionic acid). A DEPT experiment determined that there are
three quaternary carbons (C), seven methines (CH), three
methylenes (CH2) and no methyl groups (CH3). Using proton
coupling experiments such as 1-D decoupling and COSY, one large
spin system containing eight carbons is identified. in
addition, a smaller spin system consisting of two carbons is
present. A carbon proton correlation experiment (HMBC) enabled
assignment of each proton resonance in the molecule to its
attached carbon.
Treatment of the active compound (13 mg) with acetic
anhydride in pyridine resulted in the formation of an acetylated
derivative which is much less polar. This derivative is
purified by HPLC to give 3 mg of pure acetylated derivative.
Mass spectroscopy analysis revealed that the derivative has 7
acetates and a molecular weight of 690, which gives a molecular
weight of 396 for the active compound and indicates that an even
number of nitrogens are present. Also, fragments containing 6
acetates and 5 acetates are detected. High resolution data for
5 and 6 acetate daughter ions are 645.2594 (6 acetates) and
607.2519 (5 acetates) which indicate the following molecular
formula for Ia, C13H28N608.
Treatment of the active compound (13 mg) with 6 N HC1
gave a derivative which is ninhydrin positive. These results
indicate the presence of amide bonds. The derivative had the
same Rf value as determined by thin layer chromatography as 2,3-
diaminopropionic acid. These results along with NMR data,
suggest the presence of 2,3 diaminopropionic acid.
Another technique used to analyzed Ia is nOe (Nuclear
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Overhauser Effect) which can detect proximity of protons to one
another through space. nOe is carried out on an acetylated
derivative of Ia. In a two dimensional nOe experiment (NOESY),
NOEs are observed between an N-H proton at 8.06 ppm and the 5.17
proton (Figure 4).
The following structure has been elucidated for Ia
OH 2 NH2
H
N~ OH
H2N H i
O OH OH OH
NH2
It can be classified as a ureido amide. Constituents include 2
amides, a urea, two aminos, and five hydroxyls. It contains
seven chiral centers.
7.3. PROPERTIES OF Ia
The isolated Ia is found to potentiate the activity of
Bacillus thuringiensis subsp. kurstaki and Bacillus
thuringiensis subsp. aizawai crystal delta-endotoxin pesticidal
proteins toward Spodoptera exigua regardless of the form of the
pesticidal proteins. The pesticidal activity of formulated
B.t.k., isolated crystals, full-length (130 kDa molecular mass)
or truncated CryIA proteins (-65 kDa molecular mass) are all
potentiated. The activity of Cryll and CryIC inclusions are
also potentiated. it is also found to potentiate the activity
of the individual truncated CzyIA(a), (b), and (c) proteins.
Incubation time of Ia with the Cry protein is not found to be
critical for bioactivity. However, Ia is inactive alone. The
level of potentiation is found to be 100-200 fold for the
truncated CrylA proteins, Cryll and CryIC inclusions and
approximately 320 fold with full-length CryIA(c) (see Tables I
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and II respectively). Specifically,-for full-length protein,
0.75 g/ml CryIA(c) produced the same insect mortality/stunt
score when Ia is included as 240 g/ml of CryIA(c) alone. in
the case of the truncated CryIA(c), an OD280 of 0.0006 gave the
same stunt score in combination with Ia as the same sample of
CryIA(c) tested alone with an OD280 of 0.075. CryII inclusions,
at a concentration of 0.6 g/ml gave the same stunt score and
similar mortality in combination with Ia as Cryll protein alone
at 75 g/ml, a 125 fold potentiation. CzyIC inclusions, at 0.3
g/ml with the addition of Ia gave similar mortality and stunt
score as 75 g/ml of the CryIC protein alone, which reflects a
250 fold level of potentiation. The concentration of CryIA
protein that produced stunting yielded mortality on addition of
Ia.
Ia is found to be stable by bioassay as described in
Section 7.1. upon boiling for 5 minutes, but loses all activity
upon autoclaving (>190C). Further, it is stable when subjected
to direct sunlight for at least 10 hours. Ia is stable at pH 2
for 3 days, but unstable at pH 12. It is found to lose all
activity when exposed to periodic acid or concentrated HC1.
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TABLE I
POTENTIATION EFFECTS OF Ia WITH PURIFIED TRUNCATED Bt PROTEIN
Bt Protein Soodontera EXicrua
Tvipe OD280 3& Mortality* Stunt Scoret
CxyIa(a) 0.055 - 0/5 2.2
0.040 - 0/5 2.2
0.020 - 0/5 2.0
0.020 + 2/5 0.0
0.010 + 0/5 0.2
0.005 + 0/5 0.0
0.0025 + 0/5 0.4
0.0012 + 0/5 1.8
0.0006 + 0/5 1.6
CryIA(c) 0.075 - 0/5 3.4
0.040 - 0/5 2.6
0.020 - 0/5 2.8
0.020 + 1/5 0.0
0.010 + 0/5 0.2
0.005 + 1/5 0.0
0.0025 + 2/5 2.0
0.0012 + 0/5 1.0
0.0006 + 1/5 1.0
None NA + 0/5 4.0
None NA - 0/5 4.0
* Mortality =# insects dead/# total insects after 2 days
t Stunt score is defined by the average size of the live insect larvae at
the end of the bioassay: 4.0 = untreated control, 3.0 = 75% size of
untreated control, 2.0 = 50% size of untreated control, 1.0 = 25% size of
untreated control, 0.0 = no growth or size unchanged from start of
experiment.
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TABLE II
POTENTIATION EFFECTS OF Ia WITH Bt PROTEIN
Bt Protein 9podoAtera E:ciaua
Type LLcr/ml. JA Mortality* - Stunt Scoret
CryIA(c) 240 - 1/5 0.5
120 - - - 0/5 2.2
60 - 0/5 2.2
30 - 0/5 4.0
60 + 5/5 -
30 + 5/5
+ 4/5 0.0
3 + 4/5 1.0
0.8 + 2/5 1.6
15 Cryli 300 - 1/5 0.8
150 - 2/5 0.7
75 - 1/5 0.2
38 - 0/5 0.8
19 - 0/5 1.6
9 - 0/5 1.8
5 - 1/5 4.0
38 + 3/5 1.0
19 + 2/5 0.5
9 + 3/5 0.0
5 + 1/5 0.5
2.4 + 1/5 0.0
1.2 + 3/5 0.5
0.6 + 2/5 0.3
CryiI 300 - 2/5 0.3
150 - 2/5 0.0
75 - 1/5 0.8
38 - 0/5 3.2
38 + 5/5 ---
3 5 19 + 5/5 ---
9 + 5/5 5 + 4/5 0.0
2.4 + 1/5 0.0
1.2 + 5/5 ---
4 0 0.6 + 3/5 1.5
0.3 + 2/5 1.3
None NA - 0/5 4.0
None NA + 0/5 4.0
* Mortality =# insects dead/# total insects after 2 days
t Stunt score is defined by the average size of the live insect larvae at
the end of the bioassay: 4.0 = untreated control, 3.0 = 75% size of
untreated control, 2.0 = 50% size of untreated control, 1.0 = 25% size of
untreated control, 0.0 = no growth or size unchanged from start of
experiment.
7.4. EVALUATION OF OTHER SUBSPECIES OF Bacillus
thur7ncr7ensis AND OTHER SPECIES OF BacilZi
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Several Bacillus species are evaluated for production
of Ia. The strains are fermented for 72 hours at 30 C in a
medium comprised of a carbon source such as starch, hydrolyzed
starch, or glucose and a nitrogen source such protein,
hydrolyzed protein, or corn steep liquor. The supernatants are
tested for Ia production using the Spodoptera exigua micro-
bioassay described supra. B. thuringiensis subsp. aizawai
strain EMCC0087 (deposited with the NRRL as NRRL B-21148) and B.
thuringiensis subsp. galleriae (deposited with the NRRL) are
found to produce Ia in about the same concentration as B.
thuringiensis subsp. kurstaki.
Ia is also produced in B. subtilis, B. cereus, B.C.
subsp. alesti, B.C. subsp. canadiensis, B.C. subsp.
darmstadiensis, B.C. subsp. dendrolimus, B.L. subsp.
entomocidus, B.C. subsp. finitimus, B.C. subsp. israelensis,
B.C. subsp. kenyae, B.C. subsp. morrisoni, B.C. subsp.
subtoxicus, B.C. subsp. tenebrionis, B.C. subsp. thuringiensis,
and B.L. subsp. tournanoffi, B. cereus, B. subtilis, and B.
thuringiensis subsp. kurstaki cry- spo- mutant as determined by
capillary electrophoresis.
Specifically, a Beckman P/ACE Capillary
Electrophoresis System equipped with a 50 m x 57 cm uncoated
capillary, 0.2 M phosphate pH 6.8 buffer, voltage at 15KV, and
detection at 200 nm is used for quantifying the level of Ia.
Sample volumes are 20 nl with a run time of 25 minutes.
A standard curve is generated using purified Ia as the
standard at levels of 1.25 mg/ml, 0.625 mg/ml, 0.3125 mg/ml,
0.156 mg/ml, and 0.078 mg/ml. A linear calibration curve is
generated. The resultant y = mx + b equation is used to
generate the concentration of Ia in each sample.
Before each run, the capillary is flushed with running
buffer (0.2 M phosphate, pH 6.8) for three minutes. After each
25 minute run, the capillary is flushed with 1 N NaOH for 1
minute, filtered HPLC water for 1 minute, 0.5 M phosphoric acid
for 3 minutes, and filter HPLC water for 1 minute. The area
under each peak is integrated and the peak area is determined
and a final concentration is calculated from the standard curve.
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7.5. FyAI.,UATION OF B t PRODUCTS
The amount of Ia present in various commercially
available B.C. products is determined by capillary
electrophoresis described in Section 6.4, supra. BACTOSPEINET-q,
JAVELINTM, NOVODORTi''M, SPHERIMOST"M, BACTIMOSTM, FORAYTM, FLORBACTM
and BIOBITTM are obtained from Novo Nordisk A/S. XENTARITM and
DIPELTM are obtained from Abbott Laboratories. AGREETM is
obtained from Ciba-Geigy; MVPTM is obtained from Mycogen and
CUTLASSTM is obtained from Ecogen.
The results are shown in Table III, infra and indicate
that Ia is present in varying quantities ranging from less than
0.001 g Ia/BIU to 0.071 g Ia/BIU.
TABLE III
Ia IN Bacillus thurirngiensis PRODUCTS
PRODUCT type Lot Number Potency Ia g/BIU
AVELINTM WG Btk 9942281 32000 IU/mg .071
ENTARITM Bta 58715PG 15000 IU/mg .06
GREETM Bta%Btk RA208004 25000 IU/mg .033
BIOBITTM HPWP Btk 5012 48950 U/mgPIA .018
BIOBITTM FC Btk AG46669071 8 BIU/L .013
ORAYTM 48B Btk BBN7018 12.6 BIU/L .012
IPELTM Btk 58739PG 32,000 IU/mg .011
ORAYTM 76B Btk 20.0 BIU/L .007
3ACTOSPEINETM Btk BOB001 123653 IU/mg .003
3ACTOSPEINET''' Btk KX02A 100,000 IU/mg .003
3ACTOSPEINETM Btk WP 16,000 IU/mg <.001
OVODORTM Btt 9024 16.3 Million 9.5 x10-9 g/LTU
TU/qt
LORBACTx Bta 082-31-1 30,000 U/mg E <.001
PHERIMOSTM B. sphr BSN006 none
V PTM Btk 21193542 none
UTLASSTM Btk/Btk none
BACTIMOSTM Bti BIB0024 11,700 IU/mg none
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7. 6. DT.aTF IDTCORPORATIODT BIOASSAYS
B.t.k. activity is determined by an artificial diet
incorporation bioassay using third instar Spodoptera exigua
larvae, second instar Helicoverpa zea larvae, third instar
Spodoptera frugiperda larvae, second instar Heliothis virescens
larvae, third instar Trichoplusia ni larvae, third instar
Pseudoplusia includens larvae, third instar Plutella xylostella
larvae, third instar Spodoptera littoralis, and third instar
Mamestra brassicae larvae.
To determine the level of potentiation by adding Ia to
B.C. products, and establish the range of insects that are
affected, diet incorporation bioassays are performed. In the
experiments with high concentrations of Ia against Spodoptera
exigua (7.4-23.7 g Ia/BIU), purified Ia (70% active ingredient,
30% acetate counter ion) is used to potentiate BIOBITTM FC (FC
represents flowable concentrate) . The remaining data presented
in Table IV shows the potentiation of BIOBITTM HPWP (high
potency wettable powder) with Ia (0.658% active ingredient). S.
littoralis and M. brassicae are tested using FLORBACTM HPWP and
Ia. -
The various B.L. products are weighed and Ia is added
to give 0.1 to 237 g Ia/BIU. The volume is adjusted with 0.1%
TweenTM. The samples are sonicated for 1 minute and then
diluted to final volume. Neat samples (without Ia) and
reference substances are prepared as well. Reference substances
include B.t.k. HD-1-S-1980 (obtained from the NRRL) which is
assigned a potency of 16,000 international units (IU) per
milligram and JAVELINTm WG which has been assigned a potency of
53,000 Spodoptera Units/mg (SU).
Standard artificial diet composed of water, agar,
sugar, casein, wheat germ, methyl paraben, sorbic acid, linseed
oil, cellulose, salts, and vitamins are prepared in a 20 L
heated kettle. This provides enough diet to test 10 to 12
samples with seven different concentrations of each test
substance. The B.C. solutions are serially diluted to give 16
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WO 96/39037
ml aliquots. Each aliquot is added to 184 g of molten diet.
The mixture is subsequently homogenized and then poured into a
plastic tray bearing 40 individual cells. Three control trays
are prepared for each batch of diet. Once the diet has cooled
and solidified, one insect of a known age (2-3 instar) is added
to each cell, and the trays are covered with a perforated sheet
of clear mylar. The trays are placed on racks and incubated for
four days at 28'C and 65% relative humidity.
After four days, insect mortality is rated. Each tray
is given a sharp blow against a table top, and larvae that did
not move are counted as dead. Percent mortality is calculated
and the data is analyzed via parallel probit analysis. LC50s,
LC90s, the slope of the regression lines, coefficient of
variation, and potencies are estimated. Samples are run a
minimum of 3 times or until three potencies are within 20% of a
calculated mean for each sample. To calculate the increase in
activity associated with each concentration of Ia, the LCSO of
the B.t./Ia sample is corrected to reflect the amount of B.C. in
the sample. The LC50s of the paired neat samples are divided by
the corrected LC50 values to give the fold reduction in LCso
associated with ia.
The following procedure is used to assay for Lobesia
bothrana. vine grapes attacked by Lobesia bothrarna are
collected in an unsprayed field and larva is removed. A
dilution series of Ia (250 g/ml, 500 g/ml, and 1000 g/ml) is
made in water. One larva is put in the middle of the petri
dish. If the larva is observed to drink, it is moved into a
petri dish with freshly cut grape berries. The larvae are
stored at 22'C for 3-4 days.
As shown in Table IV, significant reductions in LC50s
are observed for all species.
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TABLE IV
Diet Incorporation Bioassays
Increase in activity
Insect ct Ia oer BIU Fold reduction in LC.C
LQ
Spodoptera exigua 0.1 1.5
(BIOBITTM HPWP) 0.2 1.7
2.0 4.3
4.0 7.5
Spodoptera exigua 7.4 13
(BIOBITTM FC) 15 26
30 34
118 59
237 79
Spodoptera frugiperda 0.2 2.2
(BIOBITTM HPWP) 0.8 3.9
2.0 7.2
4.0 11.6
Trichoplusia ni 0.1 1.1
(BIOBIT- HPWP) 0.2 1.2
2.0 2.0
4.0 3.1
Pseudoplusia includens 0.1 0
(BIOBITTM HPWP) 0.2 1.2
0.8 2.1
2.0 2.4
4.0 3.4
Plutella xylostella 0.2 1.6
(BIOBITTM HPWP) 0.8 1.3
2.0 1.4
4.0 1.9
Helicoverpa zea 3.2 12.6
(BIOBITTM HPWP)
Heliothis virescens 3.2 4.2
(BIOBITTM HPWP)
Lobesia bothrana 2.0 3.0
(BIOBITTM HPWP)
Spodoptera Iittoral.is 2.0 8.6
(FLORBACTM HPWP)
Mamestra brassicae 2.0 4.9
( FLORBACTM HPWP)
The potentiation of various products on Spodoptera
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exigua by Ia is determined using diet incorporation bioassays
described supra. Amounts of Ia added/BIU product are shown in
Table v, infra. Ia/B.t. product mixture is incorporated into an
agar-based wheat germ casein diet. The insects are placed on
the diet for four days and held at 28'C. Mortality is recorded and analyzed
using probit analysis. LC50, LC90 and potency are
calculated from matched product lacking Ia. The results shown
in Table V indicate that Ia potentiate various B.t.k. and B.t.a.
products obtained from various sources. The B.t. strains
contained in these products are described in Section 5.2.,
supra.
TABLE V
Potentiation of B.t. Products on Spodoptera exigua
1 5 a Ia per BIU Increase in activity
Product Fold reduction in LC.;O
BACTOSPEINETM WP 0.4 1.04
1.7 2.3
CONDORTM 0.4 2.4
1.7 5.1
AGREETm 0.4 1.1
1.7 1.6
CUTLAS STM 0.4 1.1
1.7 2.5
MVPTM 0.4 6.0
1.7 7.7
2.0 12.1
FLORBACTM HPWP 0.2 1.1
0.8 2.0
DIPELTM 2X 0.2 1.2
0.8 2.3
2.0 3.9
JAVELINTM WG 0.2 0
0.8 1.08
2.0 2.9
XENTARITM 0.2 1.2
0.8 1.6
2.0 2.4
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7.7. FOLIAR BIOASSAYS
Foliar bioassays are performed with second.instar
Spodoptera exigua larvae on broccoli plants using BIOBITTM FC
and Ia. The ratio of Ia to BIOBITTM FC is the same 2 g ia/BIU
BIOBITTM FC. The treatments are applied to broccoli plants via
a track sprayer in a carrier volume of 20 gallons per acre.
Leaves are excised from the plants after the spray deposit had
dried, and infested with second instar Spodoptera exigua larvae.
The results are shown in Table Vi, infra. 100% mortality is
observed at a rate of 8.7 BIU/hectare BIOBITTM FC + Ia, while
BIOBITTM FC alone killed 92% of the larvae at 58.8 BIU/hectare
and 8% at 17.6 BIU/hectare. Treated plants are also placed in
direct sunlight for eight hours, after which leaves are excised
and infested. After eight hours in sunlight, BIOBITTM FC alone
at 58.8 BIU/hectare gave 27% mortality, while BIOBITTM FC + Ia
gave 100% mortality at 8.7 BIU/hectare.
A foliar assay done with early fourth instar larvae
had BIOBITTM FC alone with 75% mortality at 52 BIU/hectare, and
BIOBITTM FC (FC is flowable concentrate) + Ia gave 100%
mortality at 13 BIU/hectare.
TABLE VI
Foliar Bioassavs
Treatment BTU/hectare % mortali.tv larval instar
BIOBITTM FC 58.8 92% 2
BIOBITTM FC 17.6 8% 2
BIOBITTM FC + Ia 8.7 100% 2
BIOBITTM FC +
8hr sunlight 58.8 27% 2
BIOBITTM FC + Ia
+ 8hr sunlight 8.7 100% 2
BIOBITTM FC 52 75% 4
BIOBITTM FC + Ia 13 100% 4
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7.8. FTELD TRIALS
Field trials on garbonzo beans (Spodoptera exigua)
demonstrated that BIOBITTM FC alone at 70 BIU/hectare gave 51%
control while 2 g Ia/B1U BIOBITTM FC at 40 BIU/hectare provided
89% control (relative to no treatment). JAVELINTM WG at 45
BIU/hectare gave 51% control.
Field trials on sweet corn (Spodoptera frugiperda)
demonstrated that at 39.5 BIU/hectare, 2 g Ia/BIU BIOBITTM FC
provided 84% control.
7.9. RESISTANCE RATIOS
Colonies of susceptible and resistant Plutella
xylostella are bioassayed. Resistant moths are field collected
samples from Florida that have developed B.C. resistance
following intensive exposure to JAVELINTM WG. BIOBITTM HPWP with
Ia is analyzed using a leaf-dip bioassay. Resistance to
JAVELINTM and XENTARITM is assayed without Ia. Six cm diameter
cabbage leaf disks are dipped for 10 seconds into one of eight
different concentrations of B.C. products or B.t./Ia
formulations. Concentrations range from 1 to 1000 ppm. The
leaf disks are allowed to air dry for two hours and placed in
plastic petri dishes with second instar (0.2 to 0.4 mg) larvae.
Twenty five insects/dose/day are replicated twice to give 50
insects/dose. After 72 hours at 27'C, mortality is recorded.
Dose mortality regression is analyzed with probit analysis.
Resistance ratios are calculated by dividing the LCSO and LCyo
values of the susceptible moths. The results are shown in Table
VII and indicate that the BIOBITTM HPWP potentiates with 2 g
Ia/BIU and 4 g Ia/BIU. Specifically, with 4 g Ia/BIU there is a
2 fold decrease in the LCso resistance ratio and a 10 fold
decrease in the LC9o resistance ratio.
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TABLE VII
Plutella xylostella (B.t.k. Resistant) Resistance Ratios
LC50 RR LC90 RR
PRODUCT TESTED
JAVELINTM WG 302.6 3829.7
BIOBITTM HPWP 20.5 98.5
23.2 88.0
2.0 g Ia/BIU
BIOBITTM HPWP
4.0 g Ia/BIU 10.4 11.5
BIOBITTM HPWP
XENTARITM 9.7 8.2
The invention described and claimed herein is not to
be limited in scope by the specific embodiments herein
disclosed, since these embodiments are intended as illustrations
of several aspects of the invention. Any equivalent embodiments
are intended to be within the scope of this invention. Indeed,
various modifications of the invention in addition to those
shown and described herein will become apparent to those skilled
in the art from the foregoing description. Such modifications
are also intended to fall within the scope of the appended
claims.
8. DEPOSIT OF MICROORGANISMS
The following strains of Bacillus thuringiensis have
been deposited according to the Budapest Treaty in the
Agricultural Research Service Patent Culture Collection (NRRL),
Northern Regional Research Center, 1815 University Street,
Peoria, Illinois, 61604, USA.
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Strain Accession Number Deposit Date
EMCC0086 NRRL B-21147 October 6, 1993
The strains have been deposited under conditions that
assure that access to the culture will be available during the
pendency of this patent application to one determined by the
Commissioner of Patents and Trademarks to be entitled thereto
under 37 C.F.R. 1.14 and 35 U.S.C. 122. The deposit
represents a substantially pure culture of each deposited
strain. The deposit is available as required by foreign patent
laws in countries wherein counterparts of the subject
application, or its progeny are filed. However, it should be
understood that the availability of a deposit does not
constitute a license to practice the subject invention in
derogation of patent rights granted by governmental action.
34
