U.S. patent application number 10/394929 was filed with the patent office on 2003-10-02 for method for deploying a transgenic refuge.
Invention is credited to Pershing, Jay C., Sachs, Eric S., Sanders, Ernest F..
Application Number | 20030186813 10/394929 |
Document ID | / |
Family ID | 27399091 |
Filed Date | 2003-10-02 |
United States Patent
Application |
20030186813 |
Kind Code |
A1 |
Pershing, Jay C. ; et
al. |
October 2, 2003 |
Method for deploying a transgenic refuge
Abstract
A method to protect corn against feeding damage by one or more
pests includes the treatment of corn seed having a transgenic event
that is targeted against at least one of the pests with a pesticide
in an amount that is effective against the same or another of the
one or more pests. Seeds having such protection are also disclosed,
as well as a means for deploying a non-transgenic refuge crop into
a field of transgenic crops.
Inventors: |
Pershing, Jay C.; (Webster
Groves, MO) ; Sachs, Eric S.; (Chesterfield, MO)
; Sanders, Ernest F.; (Lake St. Louis, MO) |
Correspondence
Address: |
MONSANTO COMPANY
800 N. LINDBERGH BLVD.
ATTENTION: G.P. WUELLNER, IP PARALEGAL, (E2NA)
ST. LOUIS
MO
63167
US
|
Family ID: |
27399091 |
Appl. No.: |
10/394929 |
Filed: |
March 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10394929 |
Mar 19, 2003 |
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09972012 |
Oct 5, 2001 |
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6551962 |
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60238406 |
Oct 6, 2000 |
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60238405 |
Oct 6, 2000 |
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Current U.S.
Class: |
504/117 ;
47/1.01R; 800/279 |
Current CPC
Class: |
A01N 25/00 20130101;
A01N 51/00 20130101; Y02A 50/30 20180101; A01N 61/00 20130101; A01N
51/00 20130101; A01N 2300/00 20130101; A01N 51/00 20130101; A01N
53/00 20130101; A01N 61/00 20130101; A01N 61/00 20130101; A01N
51/00 20130101; A01N 53/00 20130101 |
Class at
Publication: |
504/117 ;
800/279; 47/1.01R |
International
Class: |
A01C 001/00; A01G
001/00; A01N 063/00; A01H 001/00 |
Claims
What is claimed is:
1. A method for deploying a non-transgenic refuge crop into a field
of transgenic pest resistant crops comprising the steps of a)
blending transgenic pest resistant crop seeds with non-transgenic
refuge crop seeds; b) ensuring a uniform mixture of transgenic and
non-transgenic crop seeds is provided; and c) planting said mix in
a field; wherein said mixture consists at least of from about 100%
to about 50% transgenic pest resistant crop seed.
2. The method of claim 1 wherein said pest resistant crop seed
comprises a first pesticidal agent and said non-transgenic refuge
crop seed is treated with a second pesticidal agent which is other
than said first pesticidal agent.
3. The method of claim 1 wherein a) said pest resistant crop seed
comprises a first pesticidal agent, and b) said pest resistant crop
seed and said non-transgenic refuge crop seed are both treated with
a second pesticidal agent which is other than said first pesticidal
agent.
4. The method of claim 2 wherein said first pesticidal agent is an
insecticidal protein selected from the group consisting of a
recombinant acyl lipid hydrolase protein, a Bacillus sphearicus
insecticidal protein, Bacillus laterosporous insecticidal protein,
a insecticidal protein derived from a Xenorhabdus bacteria species,
a insecticidal protein derived from a Photorhabdus bacteria
species, and a Bacillus thuringiensis insecticidal
.delta.-endotoxin protein or vegetative insecticidal protein (VIP),
and wherein said Bacillus thuringiensis insecticidal
.delta.-endotoxin protein or vegetative insecticidal protein is
selected from the group consisting of a Cry3Bb protein or
insecticidal variant, a tlC851 protein, a CryET70 protein, a Cry22
protein, a binary insecticidal protein CryET33 and CryET34, a
binary insecticidal protein CryET80 and CryET76, a binary
insecticidal protein tlC100 and tlC101, and a binary insecticidal
protein PS149B1.
5. The method of claim 4 wherein said first pesticidal agent is a
Bacillus thuringiensis Cry3Bb or insecticidal variant
.delta.-endotoxin protein.
6. The method of claim 2 wherein the second pesticidal agent is
selected from the group consisting of insecticides, acaricides,
nematicide, fungicides, bactericides, and herbicides.
7. The method according to claim 6 wherein the second pesticidal
agent is an insecticide.
8. The method according to claim 7 wherein the second pesticidal
agent is selected from the group consisting of pyrethrins and
synthetic pyrethroids, oxadizine derivatives, chloronicotinyls,
nitroguanidine derivatives, triazoles, organophosphates, pyrrols,
pyrazoles, phenyl pyrazoles, diacylhydrazines,
biological/fermentation products, and carbamates.
9. The method according to claim 8 wherein the second pesticidal
agent is selected from the group consisting of pyrethrins
including, 2-allyl-4-hydroxy-3-methyl-2-cyclopenten-1-one ester of
2,2-dimethyl-3-(2methyl propenyl)-cyclopropane carboxylic acid,
and/or (2-methyl-1-propenyl)-2-methoxy-4-oxo-3-(2
propenyl)-2-cyclopenten-1-yl ester and mixtures of cis and trans
isomers thereof; synthetic pyrethroids including
(s)-cyano(3-phenoxyphenyl)methyl 4-chloro alpha
(1-methylethyl)benzeneacetate (fenvalerate), (S)-cyano
(3-phenoxyphenyl) methyl (S)-4-chloro-alpha-(1-methylethyl)
benzeneacetate (esfenvalerate),
(3-phenoxyphenyl)-methyl(+)cis-trans-3-(2,2-dichoroethenyl)-2,2-dimethylc-
yclopropanecarboxylate (permethrin), (.+-.)
alpha-cyano-(3-phenoxyphenyl)
methyl(+)-cis,trans-3-(2,2-dichloroethenyl)-2,2-dimethyl-cyclopropane
carboxylate (cypermethrin), beta-cypermethrin, theta cypermethrin,
S-cyano (3-phenoxyphenyl) methyl (.+-.) cis/trans
3-(2,2-dichloroethenyl) 2,2 dimethylcyclopropane carboxylate
(zeta-cypermethrin), (s)-alpha-cyano-3-phenoxybenzyl
(IR,3R)-3-(2,2-dibromovinyl)-2,2-dimethyl cyclopropanecarboxylate
(deltamethrin), alpha-cyano-3-phenoxybenzyl 2,2,3,3, -tetramethyl
cyclopropoanecarboxylate (fenpropathrin),
(RS)-alpha-cyano-3-phenoxybenzyl(R)-2-[2-chloro-4-(trifluoromethyl)anilin-
o]-3-methylbutanoate (tau-fluvalinate),
(2,3,5,6-tetrafluoro-4-methylpheny- l)-methyl-(1 alpha, 3
alpha)-(Z)-(.+-.)-3-(2-chloro-3,3,3-trifluoro-1-prop-
enyl)-2,2-dimethylcyclopropanecarboxylate (tefluthrin),
(.+-.)-cyano (3-phenoxyphenyl) methyl
(.+-.)-4-(difluoromethoxy)-alpha-(1-methyl ethyl) benzeneacetate
(flucythrinate), cyano(4-fluoro-3-phenoxyphenyl)met- hyl
3-[2-chloro-2-(4-chlorophenyl)ethenyl]-2,2-dimethylcyclopropanecarboxy-
late (flumethrin), cyano(4-fluoro-3-phenoxyphenyl) methyl
3-(2,2-dichloroethenyl)-2,2-dimethyl-cyclopropanedarboxylate
(cyfluthrin), beta cyfluthrin, transfluthrin,
(S)-alpha-cyano-3-phenoxybe-
nzyl(Z)-(IR-cis)-2,2-dimethyl-3-[2-(2,2,2-trifluoro-trifluoromethyl-ethoxy-
carbonyl)vinyl]cyclopropane carboxylate (acrinathrin), (IR cis) S
and (IS cis) R enantiomer isomer pair of
alpha-cyano-3-phenoxybenzyl-3-(2,2dichlo-
rovinyl)-2,2-dimethylcyclopropane 2011 carboxylate
(alpha-cypermethrin),
[IR,3S)3(1'RS)(1',2',2',2'-tetrabromoethyl)]-2,2-dimethyl
cyclopropanecarboxylic acid (s)-alpha-cyano-3-phenoxybenzyl ester
(tralomethrin), cyano-(3-phenoxyphenyl) methyl
2,2-dichloro-1-(4-ethoxyph- enyl)cyclopropane carboxylate
(cycloprothrin), [1 .alpha.,
3.alpha.(Z)]-(.+-.)-cyano-(3-phenoxyphenyl)methyl
3-(2-chloro-3,3,3-trifl-
uoro-1-propenyl)-2,2-cimethylcyclopropanecarboxylate (cyhalothrin),
[1 alpha (s), 3 alpha(z)]-cyano(3-phenoxyphenyl)
methyl-3-(2-chloro-3,3,3-tr-
ifluoro-1-propenyl)-2,2-dimethylcyclopropane carboxylate (lambda
cyhalothrin), (2-methyl [1,1'-biphenyl]-3-yl) methyl
3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethyl-cycloproanexarboxyla-
te (bifenthrin),
5-1-benzyl-3-furylmethyl-d-cis(1R,3S,E)2,2-dimethyl-3-(2--
oxo,-2,2,4,5 tetrahydro thiophenylidenemethyl)cyclopropane
carboxylate (kadethrin), [5-(phenyl methyl)-3-furanyl]-3-furanyl
2,2-dimethyl-3-(2-methyl-1-propenyl) cyclopropane carboxylate
(resmethrin). (1R-trans)-[5-(phenylmethyl)-3-furanyl]methyl
2,2-dimethyl-3-(2-methyl-1-propenyl)cyclopropanecarboxylate
(bioresmethrin), 3,4,5,6-tetra
hydro-phthalimidomethyl-(IRS)-cis-trans-ch- rysanthemate
(tetramethrin), 3-phenoxybenzyl-d,I-cis,trans
2,2-dimethyl-3-(2-methylpropenyl) cyclopropane carboxylate
(phenothrin), empenthrin, cyphenothrin, prallethrin, imiprothrin,
(RS)-3-allyl-2-methyl-4-oxcyclopent-2-enyl-(1A,3R;
1R,3S)-2,2-dimethyl-3-(2-methylprop-1-enyl) cyclopropane
carboxylate (allethrin), bioallethrin, and ZXI8901; oxadiazine
derivatives including
5-(2-chloropyrid-5-ylmethyl)-3-methyl-4-nitroiminoperhydro-1,3,5-oxadiazi-
ne,
5-(2-chlorothiazol-5-ylmethyl)-3-methyl-4-nitroiminoperhydro-1,3,5-oxa-
diazine, 3-methyl-4-nitroimino-5-(1-oxido-3-pyridinomethyl)
perhydro-1,3,5-oxadiazine,
5-(2-chloro-1-oxido-5-pyridiniomethyl)-3-methy-
l-4-nitroiminoperhydro-1,3,5-oxidiazine,
3-methyl-5-(2-methylpyrid-5-ylmet-
hyl)-4-nitroiminoperhydro-1,3,5-oxadiazine, and thiamethoxam;
chloronicotinyl insecticides including acetamiprid
((E)-N-[(6-chloro-3-pyridinyl)methyl]-N'-cyano-N-methyleneimidamide),
imidacloprid
(1-[(6-chloro-3-pyridinyl)methol]-N-nitro-2-imidazolidinimim- e),
and nitenpyram
(N-[(6-chloro-3-pyridinyl)methyl]-N-ethyl-N'-methyl-2-n-
itro-1,1-ethenediamine); nitroguanidine insecticides, pyrroles;
pyrazoles chlorfenapyr
(4-bromo-2-(4-chlorophenyl)-1-ethoxymethyl-5-trifluoromethyl-
pyrrole-3-carbonitrile), fenpyroximate
((E)-1,1-dimethylethyl-4[[[[(1,3-di-
methyl-5-phenoxy-1H-pyrazole-4-yl)methylene]amino]oxy]methyl]benzoate),
and tebufenpyrad (4-chloro-N
[[4-1,1-dimethylethyl)phenyl]methyl]-3-ethyl-
-1-methyl-1H-pyrazole-5-carboxamide); phenyl pyrazoles including
fipronil (5-amino-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(1R,
S)-(trifluoromethyl)sulfinyl]-1H-pyrazole-3-carbonitrile);
diacylhydrazines including halofenozide
(4-chlorobenzoate-2-benzoyl-2-(1,- 1-dimethylethyl)-hydrazide),
methoxyfenozide (RH-2485,
N-tert-butyl-N'-(3-methoxy-o-toluoyl)-3,5-xylohydrazide), and
tebufenozide (3,5-dimethylbenzoic acid 1-(1,1-dimethylethyl)-2,
(4-ethylbenzoyl) hydrazide); triazoles including amitrole and
triazamate; biological/fermentation products including avermectin
(abamectin) and spinosad (XDE-105); organophosphate insecticides
including acephate, chlorpyrifos, chlorpyrifos-methyl , diazinon,
fenamiphos, and malathion; and carbamate insecticides including
aldicarb, carbaryl, carbofuran, oxamyl, and thiodicarb.
10. The method of claim 3 wherein said first pesticidal agent is an
insecticidal protein selected from the group consisting of a
recombinant acyl lipid hydrolase protein, a Bacillus sphearicus
insecticidal protein, Bacillus laterosporous insecticidal protein,
a insecticidal protein derived from a Xenorhabdus bacteria species,
a insecticidal protein derived from a Photorhabdus bacteria
species, and a Bacillus thuringiensis insecticidal
.delta.-endotoxin protein or vegetative insecticidal protein (VIP),
and wherein said Bacillus thuringiensis insecticidal
.delta.-endotoxin protein or vegetative insecticidal protein is
selected from the group consisting of a Cry3Bb protein or
insecticidal variant, a tlC851 protein, a CryET70 protein, a Cry22
protein, a binary insecticidal protein CryET33 and CryET34, a
binary insecticidal protein CryET80 and CryET76, a binary
insecticidal protein tlC100 and tlC101, and a binary insecticidal
protein PS149B1.
11. The method of claim 10 wherein said first pesticidal agent is a
Bacillus thuringiensis Cry3Bb or insecticidal variant
.delta.-endotoxin protein.
12. The method of claim 11 wherein the second pesticidal agent is
selected from the group consisting of insecticides, acaricides,
nematicide, fungicides, bactericides, and herbicides.
13. The method according to claim 12 wherein the second pesticidal
agent is an insecticide.
14. The method according to claim 13 wherein the second pesticidal
agent is selected from the group consisting of pyrethrins and
synthetic pyrethroids, oxadizine derivatives, chloronicotinyls,
nitroguanidine derivatives, triazoles, organophosphates, pyrrols,
pyrazoles, phenyl pyrazoles, diacylhydrazines,
biological/fermentation products, and carbamates.
15. The method according to claim 14 wherein the second pesticidal
agent is selected from the group consisting of pyrethrins
including, 2-allyl-4-hydroxy-3-methyl-2-cyclopenten-1-one ester of
2,2-dimethyl-3-(2methyl propenyl)-cyclopropane carboxylic acid,
and/or (2-methyl-1-propenyl)-2-methoxy-4-oxo-3-(2
propenyl)-2-cyclopenten-1-yl ester and mixtures of cis and trans
isomers thereof; synthetic pyrethroids including
(s)-cyano(3-phenoxyphenyl)methyl 4-chloro alpha
(1-methylethyl)benzeneacetate (fenvalerate), (S)-cyano
(3-phenoxyphenyl) methyl (S)-4-chloro-alpha-(1-methylethyl)
benzeneacetate (esfenvalerate),
(3-phenoxyphenyl)-methyl(+)cis-trans-3-(2,2-dichoroethenyl)-2,2-dimethylc-
yclopropanecarboxylate (permethrin), (.+-.)
alpha-cyano-(3-phenoxyphenyl)
methyl(+)-cis,trans-3-(2,2-dichloroethenyl)-2,2-dimethyl-cyclopropane
carboxylate (cypermethrin), beta-cypermethrin, theta cypermethrin,
S-cyano (3-phenoxyphenyl) methyl (.+-.) cis/trans
3-(2,2-dichloroethenyl) 2,2 dimethylcyclopropane carboxylate
(zeta-cypermethrin), (s)-alpha-cyano-3-phenoxybenzyl
(IR,3R)-3-(2,2-dibromovinyl)-2,2-dimethyl cyclopropanecarboxylate
(deltamethrin), alpha-cyano-3-phenoxybenzyl 2,2,3,3, -tetramethyl
cyclopropoanecarboxylate (fenpropathrin),
(RS)-alpha-cyano-3-phenoxybenzyl(R)-2-[2-chloro-4-(trifluoromethyl)anilin-
o]-3-methylbutanoate (tau-fluvalinate),
(2,3,5,6-tetrafluoro-4-methylpheny- l)-methyl-(1 alpha, 3
alpha)-(Z)-(.+-.)-3-(2-chloro-3,3,3-trifluoro-1-prop-
enyl)-2,2-dimethylcyclopropanecarboxylate (tefluthrin),
(.+-.)-cyano (3-phenoxyphenyl) methyl
(.+-.)-4-(difluoromethoxy)-alpha-(1-methyl ethyl) benzeneacetate
(flucythrinate), cyano(4-fluoro-3-phenoxyphenyl)met- hyl
3-[2-chloro-2-(4-chlorophenyl)ethenyl-2,2-dimethylcyclopropanecarboxyl-
ate (flumethrin), cyano(4-fluoro-3-phenoxyphenyl) methyl
3-(2,2-dichloroethenyl)-2,2-dimethyl-cyclopropanedarboxylate
(cyfluthrin), beta cyfluthrin, transfluthrin,
(S)-alpha-cyano-3-phenoxybe-
nzyl(Z)-(IR-cis)-2,2-dimethyl-3-[2-(2,2,2-trifluoro-trifluoromethyl-ethoxy-
carbonyl)vinyl]cyclopropane carboxylate (acrinathrin), (IR cis) S
and (IS cis) R enantiomer isomer pair of
alpha-cyano-3-phenoxybenzyl-3-(2,2dichlo-
rovinyl)-2,2-dimethylcyclopropane carboxylate (alpha-cypermethrin),
[IR,3S)3(1'RS)(1',2',2',2'-tetrabromoethyl)]-2,2-dimethyl
cyclopropanecarboxylic acid (s)-alpha-cyano-3-phenoxybenzyl ester
(tralomethrin), cyano-(3-phenoxyphenyl) methyl
2,2-dichloro-1-(4-ethoxyph- enyl)cyclopropane carboxylate
(cycloprothrin), [1.alpha.,
3.alpha.(Z)]-(.+-.)-cyano-(3-phenoxyphenyl)methyl
3-(2-chloro-3,3,3-trifl-
uoro-1-propenyl)-2,2-cimethylcyclopropanecarboxylate (cyhalothrin),
[1 alpha (s), 3 alpha(z)]-cyano(3-phenoxyphenyl)
methyl-3-(2-chloro-3,3,3-tr-
ifluoro-1-propenyl)-2,2-dimethylcyclopropane carboxylate (lambda
cyhalothrin), (2-methyl [1,1'-biphenyl]-3-yl) methyl
3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethyl-cyclopropanecarboxyl-
ate (bifenthrin),
5-1-benzyl-3-furylmethyl-d-cis(1R,3S,E)2,2-dimethyl-3-(2-
-oxo,-2,2,4,5 tetrahydro thiophenylidenemethyl)cyclopropane
carboxylate (kadethrin), [5-(phenyl methyl)-3-furanyl]-3-furanyl
2,2-dimethyl-3-(2-methyl-1-propenyl) cyclopropane carboxylate
(resmethrin). (1R-trans)-[5-(phenylmethyl)-3-furanyl]methyl
2,2-dimethyl-3-(2-methyl-1-propenyl)cyclopropanecarboxylate
(bioresmethrin), 3,4,5,6-tetra
hydro-phthalimidomethyl-(IRS)-cis-trans-ch- rysanthemate
(tetramethrin), 3-phenoxybenzyl-d,l-cis,trans
2,2-dimethyl-3-(2-methylpropenyl) cyclopropane carboxylate
(phenothrin), empenthrin, cyphenothrin, prallethrin, imiprothrin,
(RS)-3-allyl-2-methyl-4-oxcyclopent-2-enyl-(1A,3R;
1R,3S)-2,2-dimethyl-3-(2-methylprop-1-enyl) cyclopropane
carboxylate (allethrin), bioallethrin, and ZXI8901; oxadiazine
derivatives including
5-(2-chloropyrid-5-ylmethyl)-3-methyl-4-nitroiminoperhydro-1,3,5-oxadiazi-
ne,
5-(2-chlorothiazol-5-ylmethyl)-3-methyl-4-nitroiminoperhydro-1,3,5-oxa-
diazine, 3-methyl-4-nitroimino-5-(1-oxido-3-pyridinomethyl)
perhydro-1,3,5-oxadiazine,
5-(2-chloro-1-oxido-5-pyridiniomethyl)-3-methy-
l-4-nitroiminoperhydro-1,3,5-oxidiazine,
3-methyl-5-(2-methylpyrid-5-ylmet-
hyl)-4-nitroiminoperhydro-1,3,5-oxadiazine, and thiamethoxam;
chloronicotinyl insecticides including acetamiprid
((E)-N-[(6-chloro-3-pyridinyl)methyl]-N'-cyano-N-methyleneimidamide),
imidacloprid (1-[(6-chloro-3-pyridinyl)
methol]-N-nitro-2-imidazolidinimi- me), and nitenpyram
(N-[(6-chloro-3-pyridinyl)methyl]-N-ethyl-N'-methyl-2--
nitro-1,1-ethenediamine); nitroguanidine insecticides, pyrroles;
pyrazoles chlorfenapyr
(4-bromo-2-(4-chlorophenyl)-1-ethoxymethyl-5-trifluoromethyl-
pyrrole-3-carbonitrile), fenpyroximate
((E)-1,1-dimethylethyl-4([[[(1,3-di-
methyl-5-phenoxy-1H-pyrazole-4-yl)methylene]amino]oxy]methyl]benzoate),
and tebufenpyrad
(4-chloro-N[[4-1,1-dimethylethyl)phenyl]methyl]-3-ethyl--
1-methyl-1H-pyrazole-5-carboxamide); phenyl pyrazoles including
fipronil (5-amino-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(1R,
S)-(trifluoromethyl)sulfinyl]-1H-pyrazole-3-carbonitrile);
diacylhydrazines including halofenozide
(4-chlorobenzoate-2-benzoyl-2-(1,- 1-dimethylethyl)-hydrazide),
methoxyfenozide (RH-2485,
N-tert-butyl-N'-(3-methoxy-o-toluoyl)-3,5-xylohydrazide), and
tebufenozide (3,5-dimethylbenzoic acid 1-(1,1-dimethylethyl)-2,
(4-ethylbenzoyl) hydrazide); triazoles including amitrole and
triazamate; biological/fermentation products including avermectin
(abamectin) and spinosad (XDE-105); organophosphate insecticides
including acephate, chlorpyrifos, chlorpyrifos-methyl , diazinon,
fenamiphos, and malathion; and carbamate insecticides including
aldicarb, carbaryl, carbofuran, oxamyl, and thiodicarb.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Applications Ser. Nos. 60/238,406 and 60/238,405, both
filed Oct. 6, 2000.
BACKGROUND OF THE INVENTION
[0002] (1) Field of the Invention
[0003] The present invention relates generally to the control of
pests that cause damage to crop plants, and in particular to corn
plants, by their feeding activities directed to root damage, and
more particularly to the control of such plant pests by combining a
crop plant seed comprising one or more transgenes which express one
or more proteins toxic to said plant pests in a mixture of seeds
with non-transgenic refuge crop seeds, and the treatment of such
seed with a chemical or peptide associated pesticide prior to
planting the seed.
[0004] (2) Description of the Related Art
[0005] Insects, nematodes, and related arthropods annually destroy
an estimated 15% of agricultural crops in the United States and
even more than that in developing countries. In addition,
competition with weeds and parasitic and saprophytic plants account
for even more potential yield losses.
[0006] Some of this damage occurs in the soil when plant pathogens,
insects and other such soil borne pests attack the seed after
planting. In the production of corn, for example, much of the rest
of the damage is caused by rootworms--insect pests that feed upon
or otherwise damage the plant roots; and by cutworms, European corn
borers, and other pests that feed upon or damage the above ground
parts of the plant. General descriptions of the type and mechanisms
of attack of pests on agricultural crops are provided by, for
example, Metcalf, in Destructive and Useful Insects, (1962); and
Agrios, in Plant Pathology, 3rd Ed., Academic Press (1988).
[0007] Corn is the most important grain crop in the Midwestern
United States. Among the most serious insect pests of corn in this
region are the larval forms of three species of Diabrotica beetles.
These include the Western corn rootworm, Diabrotica vergifera
vergifera LeConte, the Northern corn rootworm, Diabrotica berberi
Smith and Diabrotica berberi Lawrence, and the Southern corn
rootworm, Diabrotica undecimpunctata howardi Barber. In fact, more
chemical insecticide is used for the control of corn rootworm than
for any other pest of corn, and the total acreage treated with
chemical insecticides is greater than for any other pest in the
United States.
[0008] Corn rootworms (CRW) overwinter in the egg stage in fields
where corn was grown the previous season. The eggs hatch from late
May through June. If a corn crop is not followed by another corn
crop in the subsequent year, the larvae will die. Accordingly, the
impact of corn rootworm is felt most directly in areas where corn
is systematically followed by corn, as is typical in many areas of
the Midwestern United States.
[0009] After hatching, the larvae pass through three larval stages
or instars, during which they feed on the corn root system. About
three weeks is required for completion of the larval stage. Damage
to the corn root system caused by the feeding of larvae is the
major cause of harvest losses in corn due to corn rootworm. Corn
plants that fall over and lodge in the field after weakening or
destruction of a major part of the root system are the cause of a
major portion of this loss, since this lodged corn cannot be
harvested by conventional mechanized machinery and is left in the
field.
[0010] Following completion of larval development, the larvae
transform into immobile pupae, and thence into the adult beetles
that emerge from the soil throughout the summer, with the period of
emergence depending upon the growing location. After emergence, the
adult beetles feed for about two weeks before the females start
laying eggs. Initially, the adults feed predominantly in the same
field from which they emerged, but later will migrate to other
fields. Peak adult activity normally occurs in the U.S. Corn Belt
during late July or early August in fields planted to continuous
corn, but activity may peak later in first year or late maturing
cornfields. Rootworm beetles begin depositing eggs in cornfields
approximately two weeks after they emerge. (For more information,
see, e.g., Corn Rootworms, Field Crops Pest Management Circular
#16, Ohio Pest Management & Survey Program, The Ohio State
University, Extension Division, Columbus, Ohio; available online at
www.ag.ohio-state.edu/.abou- t.ohioline/icm-fact/fc-16.html, Sep.
13, 2000; and McGahen et al., Corn Insect Control: Com Rootworm,
PENpages number 08801502, Factsheet available from Pennsylvania
State University, State College, Pa., 1989).
[0011] There is evidence of the emergence of a new race of Corn
rootworm which ovipositions its eggs for overwinter onto adjacent
soybean plants. The most common practice in the mid-western united
states has been for fields to be rotated annually with corn,
followed the next year with soybeans, in order to manage the
development of an epidemic of corn rootworm pressure on fields of
corn. While this strategy overall has been successful in reducing
the corn rootworm feeding pressure on corn in many areas, the
evolutionary emergence of this new race of corn rootworm creates a
problem which was not anticipated and which could not have been
easily foreseen. This new race, which preferentially deposits its
eggs onto soybean fields, provides an unintended feeding pressure
on the next years' intended corn crop in the field in which
soybeans were grown the previous year, and the subsequent
requirement for insecticidal control measures which adds unintended
cost to the farmer in the form of additional labor for spraying and
additional costs of goods, further reducing the return to the
farmer on his/her investment in the crop and harvest.
[0012] One means for combating the corn rootworm pressures in the
US, in particular in view of the introduction of recombinant crops
containing genes which express proteins which are insecticidal to a
selected few intended crop pest insect species, has been the
regulatory agencies' requirement that farmers plant a
non-recombinant refuge crop which provides a means for producing a
steady and consistent population of adult insects which have never
been exposed to the recombinant pesticide pressures and so have not
had the opportunity to develop resistance as a result of the
pesticide pressure when feeding on the recombinant plants. This is
particularly true for the corn rootworm larvae as it is highly
limited in its ability to move through the soil any great distance
from the roots which are more or less adjacent to its local larval
environment within the soil. In theory, the adult insects which
emerge from the refuge environment will disperse and breed with any
insects which emerge from the recombinant fields, and if any of the
insects which emerge from the recombinant fields have developed a
level of resistance to the recombinant insecticidal proteins, the
availability of that trait in the subsequent generations will be
diluted, reducing or delaying the onset of the emergence of a race
which will be totally resistant to the recombinant insecticidal
corn plant.
[0013] The western corn rootworm, D. virigifera virigifera, is a
widely distributed pest of corn in North America, and in many
instance, chemical insecticides are indiscriminately used to keep
the numbers of rootworms below economically damaging levels. In
order to assist in the reduction of chemical insecticides used in
treatments to control the rootworm populations in crop fields,
transgenic lines of corn have been developed which produce a one of
a number of amino acid sequence variants of an insecticidal protein
produced naturally in the bacterium Bacillus thuringiensis. This
protein, generally referred to as Cry3Bb, has recently been
modified by English et al. in U.S. Pat. No. 6,023,013 and related
patents and applications, to contain one or more amino acid
sequence variations which, when tested in insect bioassay against
the corn rootworm, demonstrates a from about seven (7) to about ten
(10) increase in insecticidal activity when compared to the wild
type amino acid sequence. In particular, the enhanced expression of
a gene encoding this particular protein in root tissue in corn
provides for improved corn rootworm control without the requirement
for additional costs of goods by the farmer. In effect, a farmer
planting corn rootworm protected corn seeds would not have the
costs of labor and of chemical applications in treating fields of
corn crops to protect the fields from corn rootworm
infestation.
[0014] As indicated above, one concern is that a race of rootworm
will emerge which has developed resistance to the Cry3Bb protein
produced in the corn plants.
[0015] One strategy for combating the development of resistance is
to select a recombinant corn event which expresses high levels of
the insecticidal protein such that one or a few bites of a corn
root would cause at least total cessation of feeding and subsequent
death of the corn rootworm.
[0016] Another strategy would be to combine a second corn rootworm
specific insecticidal protein in the form of a recombinant event in
the same plant, for example a recombinant acyl lipid hydrolase or
insecticidal variant thereof (WO 01/49834), a CryEt70, a Cry22, a
CryEt33 and CryET34 binary toxin complex, a PS149B1 binary toxin
complex, or a CryET80 and CryET76 binary toxin complex, along with
a variant Cry3Bb insecticidal protein. Preferably the second toxin
or toxin complex would have a different mode of action from the
Cry3Bb variant, and preferably, if receptors were involved in the
toxicity of the insect to the recombinant protein, the receptors
for each of the two or more insecticidal proteins in the same plant
would be different so that if a change of function of a receptor or
a loss of function of a receptor developed as the cause of
resistance to the particular insecticidal protein, then it should
not and likely would not affect the insecticidal activity of the
remaining toxin which would be shown to bind to a receptor
different from the receptor causing the loss of function of one of
the two insecticidal proteins cloned into a plant.
[0017] Still another strategy would combine a chemical pesticide
with a pesticidal protein expressed in a transgenic plant. This
could conceivably take the form of a chemical seed treatment of a
recombinant seed which would allow for the dispersal into a zone
around the root of a pesticidally controlling amount of a chemical
pesticide which would protect root tissues from target pest
infestation so long as the chemical persisted or the root tissue
remained within the zone of pesticide dispersed into the soil. So
long as root tissue was within the zone of chemical pesticide
protection, a target pest such as a corn rootworm would have to
develop resistance to both forms of plant protection, i.e., to the
chemical and to the recombinant protein, in the same generation in
order to develop resistance to the combination of pesticidal
agents. Development of resistance under this particular scenario is
believed to be highly unlikely, and in fact, virtually impossible.
Only root tissue which would grow beyond the zone of dispersal of
the chemical pesticide treatment would be subject to only one form
of protection.
[0018] In present conventional agricultural practice, in cases
where corn follows corn, it is normal for an insecticide to be
applied to protect the corn root system from severe feeding by
rootworm larvae. Conventional practice is to treat for the adult
beetles or to treat for the larvae. Examples of conventional
treatment formulations for adult beetles include the application of
carbaryl insecticides (e.g., SEVIN.RTM. 80S at 1.0-2.0 lbs
active/acre); fenvalerate or esfenvalerate (e.g., PYDRIN.RTM. 2.4EC
at 0.1 to 0.2 lbs active/acre, or ASANA.RTM. 0.66EC at 0.03 to 0.05
lbs active/acre); malathion (57% E at 0.9 lbs active/acre);
permethrin (e.g., AMBUSH.RTM. 2.0EC at 0.1 to 0.2 lbs active/acre,
or POUNCE.RTM. 3.2EC at 0.1 to 0.2 lbs active ingredient/acre); or
PENNCAP-M.RTM. at 0.25-0.5 lbs active/acre.
[0019] To treat for CRW larvae, conventional practice is to apply a
soil insecticide either at or after planting, but preferably as
close to egg hatching as possible. Conventional treatments include
carbofuran insecticides (e.g., FURADAN.RTM. 15G at 8 oz/1000 ft of
row); chloropyrifos (e.g., LORSBAN.RTM. 15G at 8 oz/1000 ft of
row); fonophos (e.g., DYFONATE.RTM. 20G at 4.5 to 6.0 oz/1000 ft of
row); phorate (e.g., THIMET.RTM. 20G at 6 oz/1000 ft of row);
terbufos (e.g., COUNTER.RTM. 15G at 8 oz/1000 ft of row); or
tefluthrin (e.g., FORCE.RTM. 3G at 4 to 5 oz/1000 ft of row).
[0020] Many of the chemical pesticides listed above are known to be
harmful to humans and to animals in general. The environmental harm
that these pesticides cause is often exacerbated due to the
practice of applying the pesticides by foliar spraying or direct
application to the surface of the soil. Wind-drift, leaching, and
runoff can cause the migration of a large fraction of the pesticide
out of the desired zone of activity and into surface waters and
direct contact with birds, animals and humans.
[0021] Because of concern about the impact of chemical pesticides
on public health and the health of the environment, significant
efforts have been made to find ways to reduce the amount of
chemical pesticides that are used. Recently, much of this effort
has focused on the development of transgenic crops that are
engineered to express insect toxicants derived from microorganisms.
For example, U.S. Pat. No. 5,877,012 to Estruch et aL discloses the
cloning and expression of proteins from such organisms as Bacillus,
Pseudomonas, Clavibacter and Rhizobium into plants to obtain
transgenic plants with resistance to such pests as black cutworms,
armyworms, several borers and other insect pests. Publication
WO/EP97/07089 by Privalle et aL teaches the transformation of
monocotyledons, such as corn, with a recombinant DNA sequence
encoding peroxidase for the protection of the plant from feeding by
corn borers, earworms and cutworms. Jansens et aL., in Crop Sci.,
37(5):1616-1624 (1997), reported the production of transgenic corn
containing a gene encoding a crystalline protein from Bacillus
thuringiensis (Bt) that controlled both generations of the European
corn borer. U. S. Patent Nos. 5,625,136 and 5,859,336 to Koziel et
aL reported that the transformation of corn with a gene from B.
thuringiensis that encoded for delta-endotoxins provided the
transgenic corn with improved resistance to European corn borer. A
comprehensive report of field trials of transgenic corn that
expresses an insecticidal protein from B. thuringiensis has been
provided by Armstrong et al., in Crop Science, 35(2):550-557
(1995).
[0022] It was known that wild-type Bt .delta.-endotoxins had low
activity against coleopteran insects, and Kreig et al., in 1983,
reported the first isolation of a coleopteran-toxic B.
thuringiensis strain. (See U.S. Pat. No. 4,766,203). U.S. Pat. Nos.
4,797,279 and 4,910,016, also disclosed wild-type and hybrid B.
thuringiensis strains that produced proteins having some
coleopteran activity. More recently, however, amino acid sequence
variant forms of Cry3Bb have been developed that have significantly
higher levels of corn rootworm activity than the activity of the
wild type Cry3Bb protein (See, e.g., U.S. Pat. Ser. Nos. 6,023,013,
6,060,594, and 6,063,597).
[0023] However, it is not known at present whether any transgenic
plant expressing a single insecticide directed to controlling corn
rootworms will be sufficiently effective to protect corn from
damage by corn rootworm in heavily infested fields in which crop
rotation on an annual basis is not practiced. In fact, the total
control of corn rootworm damage by any one transgenic event may not
be desirable in the long term, because of the potential for the
development of resistant strains of the target pest.
[0024] Another alternative to the conventional forms of pesticide
application is the treatment of plant seeds with pesticides. The
use of fungicides or nematicides to protect seeds, and young roots
and shoots from attack after planting and sprouting, and the use of
low levels of insecticides for the protection of, for example, corn
seed from wireworm, has been used for some time. Seed treatment
with pesticides has the advantages of providing for the protection
of the seeds, while minimizing the amount of pesticide required and
limiting the amount of contact with the pesticide and the number of
different field applications necessary to attain control of the
pests in the field.
[0025] Other examples of the control of pests by applying
insecticides directly to plant seed are provided in, for example,
U.S. Pat. No. 5,696,144, which discloses that the European corn
borer caused less feeding damage to corn plants grown from seed
treated with a 1-arylpyrazole compound at a rate of 500 g per
quintal of seed than control plants grown from untreated seed. In
addition, U.S. Pat. No. 5,876,739 to Turnblad et al. (and its
parent, U.S. Pat. No. 5,849,320) disclose a method for controlling
soil-borne insects which involves treating seeds with a coating
containing one or more polymeric binders and an insecticide. This
reference provides a list of insecticides that it identifies as
candidates for use in this coating and also names a number of
potential target insects. However, while the 5,876,739 patent
states that treating corn seed with a coating containing a
particular insecticide protects corn roots from damage by the corn
rootworm, it does not indicate or otherwise suggest that such
treatment could be used with recombinant seed.
[0026] The treatment of recombinant seed with nitroimino- or
nitroguanidino-compound pesticides has previously been suggested
(See, e.g., WO 99/35913), and insecticides such as thiamethoxam,
imidacloprid, thiacloprid, and TI-435 (clothianidin) were
identified as being preferred. In the PCT publication, the use of
these insecticides was suggested for the seeds of a number of
different plant species, and for such seeds having any one of a
long list of potential recombinant insecticidal traits. However,
that reference provided no guidance as to the details of how such
treatments might be effected--such as the amounts of active
ingredient that would be necessary per unit amount of seed--and no
examples that would give reason to believe that the proposed
treatments would actually provide suitable protection.
[0027] Therefore, although recent developments in genetic
engineering of plants have improved the ability to protect plants
from pests without using chemical pesticides, and while such
techniques as the treatment of seeds with pesticides have reducing
the harmful effects of pesticides on the environment, numerous
problems remain that limit the successful application of these
methods under actual field conditions. Accordingly, it would be
useful to provide an improved method for the protection of plants,
especially corn plants, from feeding damage by pests. It would be
particularly useful if such method would reduce the required
application rate of conventional chemical pesticides, and also if
it would limit the number of separate field operations that were
required for crop planting and cultivation.
[0028] In addition, it would be useful to have a method of
deploying a transgenic refuge required by the regulatory agencies
in a field of transgenic crops instead of peripheral to a field of
transgenic crops.
BRIEF SUMMARY OF THE INVENTION
[0029] Briefly, therefore, the present invention is directed to a
novel method for protecting a transgenic corn plant against feeding
damage by one or more pests, the method comprising providing a seed
for the transgenic corn plant which seed comprises a transgenic
event having activity against at least one of the one or more
pests; and treating the seed with an effective amount of a
pesticide.
[0030] The present invention is also directed to a novel seed of a
transgenic corn plant that provides increased resistance to the
resulting corn plant against feeding damage by one or more pests,
comprising a transgenic event having activity against at least one
of the one or more pests, which seed has been treated with an
effective amount of a pesticide.
[0031] The present invention is also directed to a novel transgenic
corn seed that has been treated by the method of the present
invention.
[0032] The present invention is also directed to a method for
deploying a non-transgenic refuge crop into a field of recombinant
pest resistant crops, effectively reducing the labor, costs, and
management required to deploy a refuge into a field adjacent to,
along side of, or within a close proximity to a field of
recombinant crops. Such a refuge could be either a mixture of
recombinant pest resistant seeds and non-recombinant seeds each
treated with a seed coating comprising a chemical insecticide,
nematicide, herbicide, or fungicide alone or in combination, each
present in an amount effective for reducing or eliminating pest
infestation within a diffusible zone around the site into which the
roots of the germinated or sprouted seed would introgress, and
through which the root system of the germinated or sprouted seed
would grow without inhibition or delay in growth and development to
said root system as a result of the chemical insecticide,
nematicide, herbicide, or fungicide composition or coating.
[0033] Among the several advantages found to be achieved by the
present invention, therefore, may be noted the provision of an
improved method for the protection of plants, especially corn
plants, from feeding damage by pests; the provision of such a
method that reduces the required application rate of conventional
chemical pesticides; also the provision of such a method that
limits the number of separate field operations that were required
for crop planting and cultivation; and also the provision of a
method for deploying a non-transgenic refuge crop into a field of
transgenic crops.
DETAILED DESCRIPTION OF THE INVENTION
[0034] As used herein, the term "corn" means Zea mays or maize and
includes all plant varieties that can be bred with corn, including
wild maize species.
[0035] As used herein, the term "comprising" means "including but
not limited to".
[0036] As used herein, the terms pest, pesticide, and pesticidal
are meant to be interchangeable and inclusive of the following
terms: for example, insect, insecticide, and insecticidal when
referring to an insect pest; or with the terms, for example,
nematode, nematicide, and nematicidal when referring to a nematode
pest; or with acaric, acaricide, and acaricidal when referring to
an acaric pest; or with fungus or fungal, fungicide, and fungicidal
or equivalent terms such as mycotic, and mycocidal when referring
to fungal or related pests; or with plant or herb, planticide or
herbicide, or planticidal or herbicidal when referring to a plant
or a herb pest.
[0037] As used herein, the term "transgenic refuge" refers to the
requirement of a resistance management plan for reducing or
eliminating the likelihood of development of resistance to one or
more insecticides that are either present within a recombinant
plant or present adjacent to one or more parts or tissues of a
plant.
[0038] In accordance with the present invention, it has been
discovered that corn plants, and by analogy any other plant, can be
protected against feeding damage by one or more pests by a method
that includes providing a transgenic corn seed encoding an
insecticidal protein that has activity against at least one of the
pests and then treating the transgenic corn seed with an effective
amount of a pesticide. For example, it has been found that the
combination of a transgenic corn seed exhibiting bioactivity
against corn rootworm as a result of the production of an
insecticidal amount of an insecticidal protein within the cells of
the corn seed and treatment of the seed with certain chemical or
protein pesticides provides unexpectedly synergistic advantages to
seeds having such treatment, including unexpectedly superior
efficacy for protection against damage to the resulting corn plant
by corn rootworm. In particular, it is shown herein that transgenic
corn seeds exhibiting bioactivity against corn rootworms as a
result of the production of an amino acid variant of a Cry3Bb
.delta.-endotoxin exhibiting improved insecticidal activity
compared to the native Cry3Bb protein, in combination with the
treatment of such seeds with imidacloprid, was unexpectedly
superior to either the transgenic event alone, or to treatment with
imidacloprid alone, in protecting resulting corn plants against
more severe levels of damage by corn rootworm--levels of damage
that are known to reduce corn yield.
[0039] Corn plants and seeds that have been engineered to include
exogenous genes derived from Bacillus thuringiensis that encode for
the expression of Cry3 .delta.-endotoxins having activity against
Coleopteran pests are known, as are methods for the treatment of
seeds (even some transgenic seeds) with pesticides. Such useful
Cry3 proteins include but are not limited to Cry3A proteins, Cry3B
proteins, and Cry3C proteins. In addition, other insecticidal
proteins are specifically contemplated to be effective in the
compositions and methods of the present invention. For example,
recombinant forms of acyl lipid hydrolases known as patatins are
effective as insecticidal agents (WO 01/49834); and tlC851,
CryET70, and Cry22 are effective in controlling corn rootworms
(U.S. application Ser. No. 09/853,533 filed May 11, 2001). Also,
the binary toxins CryET33 and CryET34 (WO 98/13498), tlC100 and
tlC101 (U.S. Provisional Application Ser. No. 60/232,099 filed Sep.
12, 2000), CryET80 and CryET76 (WO 00/66742), and PS149B1
(Moellenbeck et al., 2001, Nat. Biotechnol. 19:668-672) have all
demonstrated corn rootworm controlling activity. However, it had
not been realized until the present invention that certain
effective amounts of certain chemical or protein pesticides could
be used to treat recombinant corn seeds expressing an insecticidal
protein, with the result that the combination would be unexpectedly
superior in increasing the efficacy of both the pesticide and the
transgene, and would provide the additional advantages of
increasing the ability to match pesticidal activity against pest
pressure, decreasing cost of treatment and/or application,
increasing safety of seed handling, and decreasing environmental
impact of either or both the event and the pesticide.
[0040] In particular, it has been found that the treatment of a
transgenic corn seeds that are capable of expressing certain
modified Cry3Bb proteins with from about 100 gm to about 400 gm of
certain pesticides per 100 kg of seed provided unexpectedly
superior protection against corn rootworm. In addition, it is
believed that such combinations are also effective to protect the
emergent corn plants against damage by black cutworm. The seeds of
the present invention are also believed to have the property of
decreasing the cost of pesticide use, because less of the pesticide
can be used to obtain a required amount of protection than if the
innovative method is not used. Moreover, because less pesticide is
used and because it is applied prior to planting and without a
separate field application, it is believed that the subject method
is therefore safer to the operator and to the environment, and is
potentially less expensive than conventional methods.
[0041] When it is said that some effects are "synergistic", it is
meant to include the synergistic effects of the combination on the
pesticidal activity (or efficacy) of the combination of the
transgenic event and the pesticide. However, it is not intended
that such synergistic effects be limited to the pesticidal
activity, but that they should also include such unexpected
advantages as increased scope of activity, advantageous activity
profile as related to type and amount of damage reduction,
decreased cost of pesticide and application, decreased pesticide
distribution in the environment, decreased pesticide exposure of
personnel who produce, handle and plant corn seeds, and other
advantages known to those skilled in the art.
[0042] The present invention also provides an advantage of
increasing the ability to match pesticidal activity against pest
pressure. This refers to the ability to design the combination of
the transgenic event and the pesticide treatment so that the seed
or the resulting plant is provided with effective pesticidal
activity during the period when feeding pressure from the target
pest on the seed or plant reaches its maximum. By way of example,
when a pesticide such as imidacloprid or terfluthrin is applied to
a corn seed having a corn rootworm transgenic event, the pesticide
can be applied in a coating designed to provide controlled release
of the pesticide. The release rate can be selected so that the
pesticide provides protection against such other pests as, for
example, black cutworm, at the post emergence stage of corn, while
the transgenic event provides corn rootworm protection at a later
stage of plant development--when such protection is needed.
[0043] As used herein, the terms "pesticidal effect" and
"pesticidal activity", or "activity" refer to a toxic effect
against a pest. The terms "activity against (one or more) pests",
also have the same meaning. When it is said that a seed or plant is
"protected against feeding damage by one or more pests", it is
meant that such seed or plant possesses a feature having direct or
indirect action on one or more pests that results in reduced
feeding damage by such pest or pests on the seeds, roots, shoots
and foliage of plants having such feature as compared to the
feeding damage caused under the same conditions to plants not
having such feature. Such direct or indirect actions include
inducing death of the pest, repelling the pest from the plant
seeds, roots, shoots and/or foliage, inhibiting feeding of the pest
on, or the laying of its eggs on, the plant seeds, roots, shoots
and/or foliage, and inhibiting or preventing reproduction of the
pest.
[0044] The term "insecticidal activity" has the same meaning as
pesticidal activity, except it is limited to those instances where
the pest is an insect. Except where specifically noted, when the
term "pesticide" is used herein, that term refers to a chemical
pesticide that is supplied externally to the seed, and it is not
meant to include active agents that are produced by the particular
seed or the plant that grows from the particular seed. However, the
terms "pesticidal activity" and "insecticidal activity" can be used
with reference to the activity of either, or both, an externally
supplied pesticide and/or an agent that is produced by the seed or
the plant.
[0045] One feature of the present invention is a seed of a
transgenic corn plant. As used herein, the terms "transgenic corn
plant" mean a corn plant or progeny thereof derived from a
transformed corn plant cell or protoplast, wherein the plant DNA
contains an introduced exogenous DNA molecule not originally
present in a native, non-transgenic plant of the same strain.
[0046] It is preferred that the seed contains an exogenous gene
derived from a strain of Bacillus thuringiensis, and in particular,
it is preferred that the exogenous gene is one that encodes an
insecticidal .delta.-endotoxin derived from B. thuringiensis. Such
.delta.-endotoxins are described in WO 99/31248, and include the
Cry3 toxins. It is preferred that the .delta.-endotoxins of the
present invention include the Cry3B proteins, and even more
preferred that the .delta.-endotoxins include the
coleopteran-active Cry3Bb proteins. However, as indicated herein,
other insecticidal proteins have been shown to be effective,
including but not limited to tlC851, CryET70, Cry22, binary
insecticidal proteins CryET33 and CryET34, CryET80 and CryET76,
tlC100 and tlC101, and PS149B1, as well as insecticidal proteins
derived from Xenorhabdus and Photorhabdus bacteria species,
Bacillus laterosporous species, and Bacillus sphearicus species.
The nomenclature of the B. thuringiensis insecticidal crystal
proteins was set forth by Hofte and Whitely, Microbiol. Rev.,
53.242-255, 1989. This nomenclature has been revised, and the
revised nomenclature can be found at http://epunix.biols.susx.ac.-
uk/Home/Neil-Crickmore/Bt/index.html. The revised nomenclature will
be used herein to describe transgenic event features and the
.delta.-endotoxin proteins encoded by the transgenic event.
[0047] When the terms "transgenic event" are used herein, such
terms are meant to refer to the genetically engineered DNA that is
described above, but also to include the protein(s) that are
encoded by the modified gene. A transgenic event in a corn seed, or
corn plant, therefore, includes the ability to express a protein.
When it is said that a "transgenic event has activity against a
pest", it is to be understood that it is the protein that is
encoded by the gene that actually has such activity when the
protein is expressed and brought into contact with the pest.
[0048] The term "transgenic event" is also meant herein to include
recombinant plants produced by transformation of plant cells with
heterologous DNA, i.e., a nucleic acid construct that includes a
transgene of interest, regeneration of a population of plants
resulting from the insertion of the transgene into the genome of
the plant, and selection of a particular plant characterized by
insertion into a particular genome location. The term "event"
refers to the original transformant and progeny of the transformant
that include the heterologous DNA. The term "event" also refers to
progeny produced by a sexual outcross between the transformant and
another variety that include the heterologous DNA. Even after
repeated back-crossing to a recurrent parent, the inserted DNA and
flanking DNA from the transformed parent is present in the progeny
of the cross at the same chromosomal location. The term "event"
also refers to DNA from the original transformant comprising the
inserted DNA and flanking genomic sequence immediately adjacent to
the inserted DNA that would be expected to be transferred to a
progeny that receives inserted DNA including the transgene of
interest as the result of a sexual cross of one parental line that
includes the inserted DNA (e.g., the original transformant and
progeny resulting from selfing) and a parental line that does not
contain the inserted DNA.
[0049] It is also to be understood that two different transgenic
plants can also be mated to produce offspring that contain two
independently segregating added, exogenous genes. Selfing of
appropriate progeny can produce plants that are homozygous for both
added, exogenous genes. Back-crossing to a parental plant and
out-crossing with a non-transgenic plant are also contemplated, as
is vegetative propagation. Descriptions of other breeding methods
that are commonly used for different traits and crops can be found
in one of several references, e.g., Fehr, in Breeding Methods for
Cultivar Development, Wilcox J. ed., American Society of Agronomy,
Madison, Wis. (1987).
[0050] WO 99/31248 describes methods for genetically engineering B.
thuringiensis .delta.-endotoxin genes so that modified
.delta.-endotoxins can be expressed. The modified
.delta.-endotoxins differ from the wild-type proteins by having
specific amino acid substitutions, additions or deletions as
compared with the proteins produced by the wild-type organism. Such
modified .delta.-endotoxins are identified herein by the use of an
asterisk (*), or by reference to a specific protein by its
identifying number. Thus, a genetically modified Cry3
.delta.-endotoxin would be expressed as Cry3*, one of which is, for
example, Cry3Bb.11231.
[0051] Some of the modified .delta.-endotoxins that are described
in WO 99/31248 were found to have enhanced activity against
coleopteran insects, and in particular against Diabrotica spp.,
including corn rootworm. As used herein, the terms "enhanced
activity" refer to the increased insecticidal activity of a
modified toxin as compared with the activity of the same toxin
without the amino acid modifications when both are tested under the
same conditions. In particular, it was found that Cry3*
.delta.-endotoxins exhibited enhanced activity against corn
rootworm, and are therefore preferred for use in the present
invention. More preferred are Cry3B* .delta.-endotoxins, and even
more preferred are Cry3Bb* .delta.-endotoxins. Even more preferred
transgenic events are those that comprise the ability to express
modified .delta.-endotoxins Cry3Bb.11231 (which was deposited on
May 27, 1997 as NRRL Accession Number B-21769) and Cry3Bb.11098
(which was deposited on Nov. 28, 1997 as NRRL Accession Number
B-21093). Amino acid sequences for these two preferred proteins are
given in WO 99/31248, as are the nucleotide sequences that encode
them. Transgenic plants known as transgenic events herein derived
from the insertion of a DNA sequence designed to express the Cry3Bb
variant protein 11231 were designated as transgenic event No.
MON853. Transgenic plants known as transgenic events herein derived
from the insertion of a DNA sequence designed to express the Cry3Bb
variant protein 11098 were designated as transgenic event No.
MON863.
[0052] It has also been found that a preferred use of the present
invention is for reducing pest feeding damage when used in
combination with seeds having transgenic events that have certain
levels of effectiveness against such pest. To illustrate which
levels of effectiveness are preferred, the following example will
use the Iowa Root Rating Method (Hills and Peters, J. Econ.
EntomoL., 64:764-765, 1971), which measures corn rootworm feeding
damage to corn roots on a 1-6 scale. In the rating, 1=no damage or
only a few minor feeding scars; 2=feeding scars evident but no
roots eaten off to within 11/2 inch of the plant; 3=several roots
eaten off to within 11/2 inch of the plant, but never the
equivalent of an entire node of roots is destroyed; 4=one root node
completely destroyed; 5=two root nodes completely destroyed; and
6=three or more root nodes destroyed. A destroyed root is defined
as a root that has been pruned to within 11/2 inch of the base.
Pruned roots do not have to originate from a single node, but all
pruned roots must equal the equivalent of a full node to count as a
destroyed node.
[0053] As used herein, a transgenic event is within the preferred
range of effectiveness level against a target pest if that event
reduces feeding damage by that pest by a certain amount as compared
with the same crop without the transgenic event, but does not
prevent substantially all damage by the target pest. For example,
if 10% of transgenic corn suffered corn rootworm damage of 4 or
higher on the Iowa 1-6 Scale, while 80% of non-transgenic corn
suffered damage of 4 or higher, then it could be said that the
damage to the transgenic corn was (10/80).times.100=12.5% of that
of the non-transgenic corn. For the purposes of the present
invention, it will be understood that a transgenic event in corn is
within the preferred range of effectiveness level if corn having
such event suffers from about 5% to about 50% of the damage
suffered by non-transgenic corn due to the same pest under the same
conditions. It is more preferred that corn having such transgenic
event suffers from about 10% to about 40% of the damage suffered by
non-transgenic corn by the same pest under the same conditions,
even more preferred is damage of from about 15% to about 30%, and
yet more preferred is damage of from about 20% to about 30% of the
damage suffered by non-transgenic corn by the same pest under the
same conditions. As used herein, when the term "about" is used to
describe the degree of damage to corn, it is to be understood that
the degree of damage can be above or below the limits described by
as much as 1% or 2% and still be considered to be within the ranges
described. By way of example, a level of 4.5% damage would be
regarded as being "about 5%".
[0054] Without wishing to be bound to this or any other theory, it
is believed that the pesticidal seed treatment can provide
significant advantages when combined with a transgenic event that
provides protection that is within the preferred effectiveness
range against a target pest. In addition, it is believed that there
are situations that are well known to those having skill in the
art, where it is advantageous to have such transgenic events within
the preferred range of effectiveness.
[0055] The present invention also includes seeds and plants having
more that one transgenic event. Such combinations are referred to
as "stacked" transgenic events. These stacked transgenic events can
be events that are directed at the same target pest, or they can be
directed at different target pests. In one preferred method, a seed
having the ability to express a Cry 3 protein also has the ability
to express at least one other insecticidal protein that is
different from a Cry 3 protein.
[0056] In another preferred method, the seed having the ability to
express a Cry 3 protein also has a transgenic event that provides
herbicide tolerance. It is more preferred that the transgenic event
that provides herbicide tolerance is an event that provides
resistance to glyphosate, N-(phosphonomethyl) glycine, including
the isopropylamine salt form of such herbicide, even more preferred
is the transgenic event that is effective to provide the herbicide
resistance of ROUNDUP READY.RTM. plants and seeds available from
Monsanto Co., St. Louis, Mo.
[0057] In the present method, a corn seed having a transgenic event
is treated with a pesticide.
[0058] Pesticides suitable for use in the invention include
pyrethrins and synthetic pyrethroids; oxadizine derivatives;
chloronicotinyls; nitroguanidine derivatives; triazoles;
organophosphates; pyrrols; pyrazoles; phenyl pyrazoles;
diacylhydrazines; biological/fermentation products; and carbamates.
Known pesticides within these categories are listed in The
Pesticide Manual, 11th Ed., C. D. S. Tomlin, Ed., British Crop
Protection Council, Farnham, Surry, UK (1997).
[0059] Pyrethroids that are useful in the present composition
include pyrethrins and synthetic pyrethroids. The pyrethrins that
are preferred for use in the present method include, without
limitation, 2-allyl-4-hydroxy-3-methyl-2-cyclopenten-1-one ester of
2,2-dimethyl-3-(2methyl propenyl)-cyclopropane carboxylic acid,
and/or (2-methyl-1-propenyl)-2-methoxy-4-oxo-3-(2
propenyl)-2-cyclopenten-1-yl ester and mixtures of cis and trans
isomers thereof (Chemical Abstracts Service Registry Number ("CAS
RN") 8003-34-7).
[0060] Synthetic pyrethroids that are preferred for use in the
present invention include
[0061] (s)-cyano(3-phenoxyphenyl)methyl 4-chloro alpha
(I-methylethyl)benzeneacetate (fenvalerate, CAS RN 51630-58-1),
(S)-cyano (3-phenoxyphenyl) methyl
(S)-4-chloro-alpha-(1-methylethyl) benzeneacetate (esfenvalerate,
CAS RN 66230-04-4),
(3-phenoxyphenyl)-methyl(+)cis-trans-3-(2,2-dichoroethenyl)-2,2-dimethylc-
yclopropanecarboxylate (permethrin, CAS RN 52645-53-1), (.+-.)
alpha-cyano-(3-phenoxyphenyl)
methyl(+)-cis,trans-3-(2,2-dichloroethenyl)-
-2,2-dimethyl-cyclopropane carboxylate (cypermethrin, CAS RN
52315-07-8), (beta-cypermethrin, CAS RN 65731-84-2), (theta
cypermethrin, CAS RN 71697-59-1), S-cyano (3-phenoxyphenyl) methyl
(.+-.) cis/trans 3-(2,2-dichloroethenyl) 2,2 dimethylcyclopropane
carboxylate (zeta-cypermethrin, CAS RN 52315-07-8),
(s)-alpha-cyano-3-phenoxybenzyl
(IR,3R)-3-(2,2-dibromovinyl)-2,2-dimethyl cyclopropanecarboxylate
(deltgamethrin, CAS RN 52918-63-5), alpha-cyano-3-phenoxybenzyl
2,2,3,3, -tetramethyl cyclopropoanecarboxylate (fenpropathrin, CAS
RN 64257-84-7),
(RS)-alpha-cyano-3-phenoxybenzyl(R)-2-[2-chloro-4-(trifluoromethyl)anilin-
o]-3-methylbutanoate (tau-fluvalinate, CAS RN 102851-06-9),
(2,3,5,6-tetrafluoro-4-methylphenyl)-methyl-(1 alpha, 3
alpha)-(Z)-(35
)-3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethylcyclopropanecarboxy-
late (tefluthrin, CAS RN 79538-32-2), (.+-.)-cyano
(3-phenoxyphenyl) methyl (.+-.)-4-(difluoromethoxy)-alpha-(1-methyl
ethyl) benzeneacetate (flucythrinate, CAS RN 70124-77-5),
cyano(4-fluoro-3-phenoxyphenyl)methyl
3-[2-chloro-2-(4-chlorophenyl)ethenyl]-2,2-dimethylcyclopropanecarboxylat-
e (flumethrin, CAS RN 69770-45-2), cyano(4-fluoro-3-phenoxyphenyl)
methyl 3-(2,2-dichloroethenyl)-2,2-dimethyl-cyclopropanedarboxylate
(cyfluthrin, CAS RN 68359-37-5), (beta cyfluthrin, CAS RN
68359-37-5), (transfluthrin, CAS RN 118712-89-3),
(S)-alpha-cyano-3-phenoxybenzyl
(Z)-(lR-cis)-2,2-dimethyl-3-[2-(2,2,2-trifluoro-trifluoromethyl-ethoxycar-
bonyl)vinyl]cyclopropane carboxylate (acrinathrin, CAS RN
101007-06-1), (IR cis) S and (IS cis) R enantiomer isomer pair of
alpha-cyano-3-phenoxybenzyl-3-(2,2dichlorovinyl)-2,2-dimethylcyclopropane
carboxylate (alpha-cypermethrin, CAS RN 67375-30-8),
[IR,3S)3(1'RS)(1',2',2',2'-tetrabromoethyl)]-2,2-dimethyl
cyclopropanecarboxylic acid (s)-alpha-cyano-3-phenoxybenzyl ester
(tralomethrin, CAS RN 66841-25-6), cyano-(3-phenoxyphenyl) methyl
2,2-dichloro-1-(4-ethoxyphenyl)cyclopropane
carboxylate(cycloprothrin, CAS RN 63935-38-6), [1.alpha.,
3.alpha.(Z)]-(.+-.)-cyano-(3-phenoxyphenyl- )methyl
3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-cimethylcyclopropaneca-
rboxylate (cyhalothrin, CAS RN 68085-85-8), [1 alpha (s), 3
alpha(z)]-cyano(3-phenoxyphenyl)
methyl-3-(2-chloro-3,3,3-trifluoro-1-pro-
penyl)-2,2-dimethylcyclopropane carboxylate (lambda cyhalothrin,
CAS RN 91465-08-6), (2-methyl [1,1'-biphenyl]-3-yl) methyl
3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethyl-cyclopropanecarboxyl-
ate (bifenthrin, CAS RN 82657-04-3),
5-1-benzyl-3-furylmethyl-d-cis(1
R,3S,E)2,2-dimethyl-3-(2-oxo,-2,2,4,5 tetrahydro
thiophenylidenemethyl)cy- clopropane carboxylate (kadethrin,
RU15525, CAS RN 58769-20-3), [5-(phenyl
methyl)-3-furanyl]-3-furanyl 2,2-dimethyl-3-(2-methyl-1-propenyl)
cyclopropane carboxylate (resmethrin, GAS RN 10453-86-8),
(1R-trans)-[5-(phenylmethyl)-3-furanyl]methyl
2,2-dimethyl-3-(2-methyl-1-- propenyl)cyclopropanecarboxylate
(bioresmethrin, GAS RN 28434-01-7), 3,4,5,6-tetra
hydro-phthalimidomethyl-(lRS)-cis-trans-chrysanthemate
(tetramethrin, CAS RN 7696-12-0), 3-phenoxybenzyl-d,l-cis,trans
2,2-dimethyl-3-(2-methylpropenyl) cyclopropane carboxylate
(phenothrin, GAS RN 26002-80-2); (empenthrin, GAS RN 54406-48-3);
(cyphenothrin; GAS RN 39515-40-7), (prallethrin, GAS RN
23031-36-9), (imiprothrin, GAS RN 72963-72-5),
(RS)-3-allyl-2-methyl-4-oxcyclopent-2-enyl-(1A,3R;
1R,3S)-2,2-dimethyl-3-(2-methylprop-1-enyl) cyclopropane
carboxylate (allethrin, GAS RN 584-79-2), (bioallethrin, GAS RN
584-79-2), and (ZXI8901, GAS RN 160791-64-0). It is believed that
mixtures of one or more of the aforementioned synthetic pyrethroids
can also be used in the present invention. Particularly preferred
synthetic pyrethroids are tefluthrin, lambda cyhalothrin,
bifenthrin, permethrin and cyfluthrin. Even more preferred
synthetic pyrethroids are tefluthrin and lambda cyhalothrin, and
yet more preferred is tefluthrin.
[0062] Insecticides that are oxadiazine derivatives are useful in
the subject method. The oxadizine derivatives that are preferred
for use in the present invention are those that are identified in
U.S. Pat. No. 5,852,012. More preferred oxadiazine derivatives are
5-(2-chloropyrid-5-ylmethyl)-3-methyl-4-nitroiminoperhydro-1,3,5-oxadiazi-
ne,
5-(2-chlorothiazol-5-ylmethyl)-3-methyl-4-nitroiminoperhydro-1,3,5-oxa-
diazine, 3-methyl-4-nitroimino-5-(1-oxido-3-pyridinomethyl)
perhydro-1,3,5-oxadiazine,
5-(2-chloro-1-oxido-5-pyridiniomethyl)-3-methy-
l-4-nitroiminoperhydro-1,3,5-oxidiazine; and
3-methyl-5-(2-methylpyrid-5-y-
lmethyl)-4-nitroiminoperhydro-1,3,5-oxadiazine. Even more preferred
is thiamethoxam (CAS RN 153719-23-4).
[0063] Chloronicotinyl insecticides are also useful in the subject
method. Chloronicotinyls that are preferred for use in the subject
composition are described in U.S. Pat. No. 5,952,358, and include
acetamiprid
((E)-N-[(6-chloro-3-pyridinyl)methyl]-N'-cyano-N-methyleneimidamide,
CAS RN 135410-20-7), imidacloprid
(1-[(6-chloro-3-pyridinyl)methol]-N-nitro-2- -imidazolidinimime,
CAS RN 138261-41-3), and nitenpyram
(N-[(6-chloro-3-pyridinyl)methyl]-N-ethyl-N'-methyl-2-nitro-1,1-ethenedia-
mine, CAS RN 120738-89-8).
[0064] Nitroguanidine insecticides are useful in the present
method. Such nitroguanidines can include those described in U.S.
Pat. Nos. 5,633,375, 5,034,404 and 5,245,040.
[0065] Pyrrols, pyrazoles and phenyl pyrazoles that are useful in
the present method include those that are described in U.S. Pat.
5,952,358. Preferred pyrazoles include chlorfenapyr
(4-bromo-2-(4-chlorophenyl)-1-et-
hoxymethyl-5-trifluoromethylpyrrole-3-carbonitrile, CAS RN
122453-73-0), fenpyroximate
((E)-1,1-dimethylethyl-4[[[[(1,3-dimethyl-5-phenoxy-1H-pyra-
zole-4-yl)methylene]amino]oxy]methyl]benzoate, CAS RN 111812-58-9),
and tebufenpyrad
(4-chloro-N[[4-1,1-dimethylethyl)phenyl]methyl]-3-ethyl-1-me-
thyl-1H-pyrazole-5-carboxamide, CAS RN 119168-77-3). A preferred
phenyl pyrazole is fipronil
(5-amino-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-- [(1R
,S)-(trifluoromethyl)sulfinyl]-1H-pyrazole-3-carbonitrile, CAS RN
120068-37-3).
[0066] Diacylhydrazines that are useful in the present invention
include halofenozide
(4-chlorobenzoate-2-benzoyl-2-(1,1-dimethylethyl)-hydrazide, CAS RN
112226-61-6), methoxyfenozide (RH-2485; N-tert-butyl-N'-(3-methoxy-
-o-toluoyl)-3,5-xylohydrazide, CAS RN 161050-58-4), and
tebufenozide (3,5-dimethylbenzoic acid 1-(1,1-dimethylethyl)-2,
(4-ethylbenzoyl)hydrazide, CAS RN 112410-23-8).
[0067] Triazoles, such as amitrole (CAS RN 61-82-5) and triazamate
are useful in the nethod of the present invention. A preferred
triazole is triazamate (ethyl
[[1-[(dimethylamino)carbonyl]-3-(1,1-dimethylethyl)-1H--
1,2,4-triazol-5-yl]thio]acetate, CAS RN 112143-82-5).
[0068] Biological/fermentation products, such as avermectin
(abamectin, CAS RN 71751-41-2) and spinosad (XDE-105, CAS RN
131929-60-7) are useful in the present method.
[0069] Organophosphate insecticides are also useful as one of the
components of the present method. Preferred organophophate
insecticides include acephate (CAS RN 30560-19-1), chlorpyrifos
(CAS RN 2921-88-2), chlorpyrifos-methyl (CAS RN 5598-13-0),
diazinon (CAS RN 333-41-5), fenamiphos (CAS RN 22224-92-6), and
malathion (CAS RN 121-75-5).
[0070] In addition, carbamate insecticides are useful in the
subject method. Preferred carbamate insecticides are aldicarb (CAS
RN 116-06-3), carbaryl (CAS RN 63-25-2), carbofuran (CAS RN
1563-66-2), oxamyl (CAS RN 23135-22-0) and thiodicarb (CAS RN
59669-26-0).
[0071] When an insecticide is described herein, it is to be
understood that the description is intended to include salt forms
of the insecticide as well as any isomeric and/or tautomeric form
of the insecticide that exhibits the same insecticidal activity as
the form of the insecticide that is described.
[0072] The insecticides that are useful in the present method can
be of any grade or purity that pass in the trade as such
insecticide. Other materials that accompany the insecticides in
commercial preparations as impurities can be tolerated in the
subject methods and compositions, as long as such other materials
do not destabilize the composition or significantly reduce or
destroy the activity of any of the insecticide components or the
transgenic event against the target pest(s). One of ordinary skill
in the art of the production of insecticides can readily identify
those impurities that can be tolerated and those that cannot.
[0073] It has been found that the present method is useful to
protect seeds and plants against a wide array of agricultural
pests, including insects, mites, fungi, yeasts, molds, bacteria,
nematodes, weeds, and parasitic and saprophytic plants.
[0074] When an insect is the target pest for the present invention,
such pests include but are not limited to:
[0075] from the order Lepidoptera, for example,
[0076] Acleris spp., Adoxophyes spp., Aegeria spp., Agrotis spp.,
Alabama argillaceae, Amylois spp., Anticarsia gemmatalis, Archips
spp, Argyrotaenia spp., Autographa spp., Busseola fusca, Cadra
cautella, Carposina nipponensis, Chilo spp., Choristoneura spp.,
Clysia ambiguella, Cnaphalocrocis spp., Cnephasia spp., Cochylis
spp., Coleophora spp., Crocidolomia binotalis, Cryptophlebia
leucotreta, Cydia spp., Diatraea spp., Diparopsis castanea, Earias
spp., Ephestia spp., Eucosma spp., Eupoecilia ambiguella, Euproctis
spp., Euxoa spp., Grapholita spp., Hedya nubiferana, Heliothis
spp., Hellula undalis, Hyphantria cunea, Keiferia lycopersicella,
Leucoptera scitella, Lithocollethis spp., Lobesia botrana,
Lymantria spp., Lyonetia spp., Malacosoma spp., Mamestra brassicae,
Manduca sexta, Operophtera spp., Ostrinia Nubilalis, Pammene spp.,
Pandemis spp., Panolis flammea, Pectinophora gossypiella,
Phthorimaea operculella, Pieris rapae, Pieris spp., Plutella
xylostella, Prays spp., Scirpophaga spp., Sesamia spp.,
Sparganothis spp., Spodoptera spp., Synanthedon spp., Thaumetopoea
spp., Tortrix spp., Trichoplusia ni and Yponomeuta spp.;
[0077] from the order Coleoptera, for example,
[0078] Agriotes spp., Anthonomus spp., Atomaria linearis,
Chaetocnema tibialis, Cosmopolites spp., Curculio spp., Dermestes
spp., Diabrotica spp., Epilachna spp., Eremnus spp., Leptinotarsa
decemlineata, Lissorhoptrus spp., Melolontha spp., Orycaephilus
spp., Otiorhynchus spp., Phlyctinus spp., Popillia spp., Psylliodes
spp., Rhizopertha spp., Scarabeidae, Sitophilus spp., Sitotroga
spp., Tenebrio spp., Tribolium spp. and Trogoderma spp.;
[0079] from the order Orthoptera, for example,
[0080] Blatta spp., Blattella spp., Gryllotalpa spp., Leucophaea
maderae, Locusta spp., Periplaneta ssp., and Schistocerca spp.;
[0081] from the order Isoptera, for example,
[0082] Reticulitemes ssp;
[0083] from the order Psocoptera, for example,
[0084] Liposcelis spp.;
[0085] from the order Anoplura, for example,
[0086] Haematopinus spp., Linognathus spp., Pediculus spp.,
Pemphigus spp. and Phylloxera spp.;
[0087] from the order Mallophaga, for example,
[0088] Damalinea spp. and Trichodectes spp.;
[0089] from the order Thysanoptera, for example,
[0090] Franklinella spp., Hercinothrips spp., Taeniothrips spp.,
Thrips palmi, Thrips tabaci and Scirtothrips aurantii;
[0091] from the order Heteroptera, for example,
[0092] Cimex spp., Distantiella theobroma, Dysdercus spp.,
Euchistus spp., Eurygaster spp., Leptocorisa spp., Nezara spp.,
Piesma spp., Rhodnius spp., Sahlbergella singularis, Scotinophara
spp. and Triatoma spp.;
[0093] from the order Homoptera, for example,
[0094] Aleurothrixus floccosus, Aleyrodes brassicae, Aonidiella
spp., Aphididae, Aphis spp., Aspidiotus spp., Bemisia tabaci,
Ceroplaster spp., Chrysomphalus aonidium, Chrysomphalus
dictyospermi, Coccus hesperidum, Empoasca spp., Eriosoma larigerum,
Erythroneura spp., Gascardia spp., Laodelphax spp., Lacanium corni,
Lepidosaphes spp., Macrosiphus spp., Myzus spp., Nehotettix spp.,
Nilaparvata spp., Paratoria spp., Pemphigus spp., Planococcus spp.,
Pseudaulacaspis spp., Pseudococcus spp., Psylla ssp., Pulvinaria
aethiopica, Quadraspidiotus spp., Rhopalosiphum spp., Saissetia
spp., Scaphoideus spp., Schizaphis spp., Sitobion spp.,
Trialeurodes vaporariorum, Trioza erytreae and Unaspis citri;
[0095] from the order Hymenoptera, for example,
[0096] Acromyrmex, Atta spp., Cephus spp., Diprion spp.,
Diprionidae, Gilpinia polytoma, Hoplocampa spp., Lasius sppp.,
Monomorium pharaonis, Neodiprion spp, Solenopsis spp. and Vespa
ssp.;
[0097] from the order Diptera, for example,
[0098] Aedes spp., Antherigona soccata, Bibio hortulanus,
Calliphora erythrocephala, Ceratitis spp., Chrysomyia spp., Culex
spp., Cuterebra spp., Dacus spp., Drosophila melanogaster, Fannia
spp., Gastrophilus spp., Glossina spp., Hypoderma spp., Hyppobosca
spp., Liriomysa spp., Lucilia spp., Melanagromyza spp., Musca ssp.,
Oestrus spp., Orseolia spp., Oscinella frit, Pegomyia hyoscyami,
Phorbia spp., Rhagoletis pomonella, Sciara spp., Stomoxys spp.,
Tabanus spp., Tannia spp. and Tipula spp.,
[0099] from the order Siphonaptera, for example,
[0100] Ceratophyllus spp. und Xenopsylla cheopis and
[0101] from the order Thysanura, for example,
[0102] Lepisma saccharina.
[0103] It has been found that the present invention is particularly
effective when the insect pest is a Diabrotica spp., and especially
when the pest is Diabrotica virgifera, Diabrotica barberi, or
Diabrotica undecimpunctata.
[0104] Another application wherein the present invention is
believed to be particularly effective is when the pesticide has
activity against a weed or a parasitic or saprophytic plant and the
transgenic event has activity against a member selected from the
group consisting of Diabrotica virgifera, Diabrotica barberiand
Diabrotica undecimpunctata. This is believed to be more preferred
useful when the weed or a parasitic or saprophytic plant is the
African plant known as "Striga", and even more preferred when the
pesticide is ROUNDUP.RTM. (available from Monsanto Company).
[0105] In the method of the present invention, the pesticide is
applied to a transgenic corn seed. Although it is believed that the
present method can be applied to a transgenic corn seed in any
physiological state, it is preferred that the seed be in a
sufficiently durable state that it incurs no damage during the
treatment process. Typically, the seed would be a seed that had
been harvested from the field; removed from the plant; and
separated from any cob, stalk, outer husk, and surrounding pulp or
other non-seed plant material. The seed would preferably also be
biologically stable to the extent that the treatment would cause no
biological damage to the seed. In one embodiment, for example, the
treatment can be applied to seed corn that has been harvested,
cleaned and dried to a moisture content below about 15% by weight.
In an alternative embodiment, the seed can be one that has been
dried and then primed with water and/or another material and then
re-dried before or during the treatment with the pesticide. Within
the limitations just described, it is believed that the treatment
can be applied to the seed at any time between harvest of the seed
and sowing of the seed. As used herein, the term "unsown seed" is
meant to include seed at any period between the harvest of the seed
and the sowing of the seed in the ground for the purpose of
germination and growth of the plant.
[0106] When it is said that unsown seed is "treated" with the
pesticide, such treatment is not meant to include those practices
in which the pesticide is applied to the soil, rather than to the
seed. For example, such treatments as the application of the
pesticide in bands, "T"-bands, or in-furrow, at the same time as
the seed is sowed are not considered to be included in the present
invention.
[0107] The pesticide, or combination of pesticides, can be applied
"neat", that is, without any diluting or additional components
present. However, the pesticide is typically applied to the seeds
in the form of a pesticide formulation. This formulation may
contain one or more other desirable components including but not
limited to liquid diluents, binders to serve as a matrix for the
pesticide, fillers for protecting the seeds during stress
conditions, and plasticizers to improve flexibility, adhesion
and/or spreadability of the coating. In addition, for oily
pesticide formulations containing little or no filler, it may be
desirable to add to the formulation drying agents such as calcium
carbonate, kaolin or bentonite clay, perlite, diatomaceous earth or
any other adsorbent material. Use of such components in seed
treatments is known in the art. See, e.g., U.S. Pat. No. 5,876,739.
The skilled artisan can readily select desirable components to use
in the pesticide formulation depending on the seed type to be
treated and the particular pesticide that is selected. In addition,
readily available commercial formulations of known pesticides may
be used, as demonstrated in the examples below.
[0108] The seeds may also be treated with one or more of the
following ingredients: other pesticides, including compounds which
act only below the ground; fungicides, such as captan, thiram,
metalaxyl, (methoxam=resolved isomer of metalaxyl), fludioxonil,
oxadixyl, and isomers of each of those materials, and the like;
herbicides, including compounds selected from carbamates,
thiocarbamates, acetamides, triazines, dinitroanilines, glycerol
ethers, pyridazinones, uracils, phenoxys, ureas, and benzoic acids;
herbicidal safeners such as benzoxazine, benzhydryl derivatives,
N,N-diallyl dichloroacetamide, various dihaloacyl, oxazolidinyl and
thiazolidinyl compounds, ethanone, naphthalic anhydride compounds,
and oxime derivatives; fertilizers; and biocontrol agents such as
naturally-occurring or recombinant bacteria and fungi from the
genera Rhizobium, Bacillus, Pseudomonas, Serratia, Trichoderma,
Glomus, Gliocladium and mycorrhizal fungi. These ingredients may be
added as a separate layer on the seed or alternatively may be added
as part of the pesticide composition.
[0109] Preferably, the amount of the novel composition or other
ingredients used in the seed treatment should not inhibit
generation of the seed, or cause phytotoxic damage to the seed.
[0110] The pesticide formulation that is used to treat the
transgenic corn seed in the present invention can be in the form of
a suspension; emulsion; slurry of particles in an aqueous medium
(e.g., water); wettable powder; wettable granules (dry flowable);
and dry granules. If formulated as a suspension or slurry, the
concentration of the active ingredient in the formulation is
preferably about 0.5% to about 99% by weight (w/w), preferably
5-40%.
[0111] As mentioned above, other conventional inactive or inert
ingredients can be incorporated into the formulation. Such inert
ingredients include but are not limited to: conventional sticking
agents, dispersing agents such as methylcellulose (Methocel A15LV
or Methocel A15C, for example, serve as combined
dispersant/sticking agents for use in seed treatments), polyvinyl
alcohol (e.g., Elvanol 51-05), lecithin (e.g., Yelkinol P),
polymeric dispersants (e.g., polyvinylpyrrolidone/vin- yl acetate
PVPNA S-630), thickeners (e.g., clay thickeners such as Van Gel B
to improve viscosity and reduce settling of particle suspensions),
emulsion stabilizers, surfactants, antifreeze compounds (e.g.,
urea), dyes, colorants, and the like. Further inert ingredients
useful in the present invention can be found in McCutcheon's,
vol.1, "Emulsifiers and Detergents," MC Publishing Company, Glen
Rock, N.J., U.S.A., 1996. Additional inert ingredients useful in
the present invention can be found in McCutcheon's, vol. 2,
"Functional Materials," MC Publishing Company, Glen Rock, N.J.,
U.S.A., 1996.
[0112] The pesticides and pesticide formulations of the present
invention can be applied to seeds by any standard seed treatment
methodology, including but not limited to mixing in a container
(e.g., a bottle or bag), mechanical application, tumbling,
spraying, and immersion. Any conventional active or inert material
can be used for contacting seeds with pesticides according to the
present invention, such as conventional film-coating materials
including but not limited to water-based film coating materials
such as Sepiret (Seppic, Inc., Fairfield, N.J.) and Opacoat
(Berwind Pharm. Services, Westpoint, Pa.).
[0113] The subject pesticides can be applied to a seed as a
component of a seed coating. Seed coating methods and compositions
that are known in the art are useful when they are modified by the
addition of one of the embodiments of the combination of pesticides
of the present invention. Such coating methods and apparatus for
their application are disclosed in, for example, U.S. Pat. Nos.
5,918,413, 5,891,246, 5,554,445, 5,389,399, 5,107,787, 5,080,925,
4,759,945 and 4,465,017. Seed coating compositions are disclosed,
for example, in U.S. Pat. Nos. 5,939,356, 5,882,713, 5,876,739,
5,849,320, 5,834,447, 5,791,084, 5,661,103, 5,622,003, 5,580,544,
5,328,942, 5,300,127, 4,735,015, 4,634,587, 4,383,391, 4,372,080,
4,339,456, 4,272,417 and 4,245,432, among others.
[0114] Useful seed coatings contain one or more binders and at
least one of the subject combinations of pesticides.
[0115] Binders that are useful in the present invention preferably
comprise an adhesive polymer that may be natural or synthetic and
is without phytotoxic effect on the seed to be coated. The binder
may be selected from polyvinyl acetates; polyvinyl acetate
copolymers; ethylene vinyl acetate (EVA) copolymers; polyvinyl
alcohols; polyvinyl alcohol copolymers; celluloses, including
ethylcelluloses, methylcelluloses, hydroxymethylcelluloses,
hydroxypropylcelluloses and carboxymethylcellulose;
polyvinylpyrolidones; polysaccharides, including starch, modified
starch, dextrins, maltodextrins, alginate and chitosans; fats;
oils; proteins, including gelatin and zeins; gum arabics; shellacs;
vinylidene chloride and vinylidene chloride copolymers; calcium
lignosulfonates; acrylic copolymers; polyvinylacrylates;
polyethylene oxide; acrylamide polymers and copolymers;
polyhydroxyethyl acrylate, methylacrylamide monomers; and
polychloroprene.
[0116] It is preferred that the binder be selected so that it can
serve as a matrix for the subject pesticides. While the binders
disclosed above may all be useful as a matrix, the specific binder
will depend upon the properties of the combination of pesticides.
The term "matrix", as used herein, means a continuous solid phase
of one or more binder compounds throughout which is distributed as
a discontinuous phase one or more of the subject pesticides.
Optionally, a filler and/or other components can also be present in
the matrix. The term matrix is to be understood to include what may
be viewed as a matrix system, a reservoir system or a
microencapsulated system. In general, a matrix system consists of
pesticides of the present invention and filler uniformly dispersed
within a polymer, while a reservoir system consists of a separate
phase comprising the subject pesticides, that is physically
dispersed within a surrounding, rate-limiting, polymeric phase.
Microencapsulation includes the coating of small particles or
droplets of liquid, but also to dispersions in a solid matrix.
[0117] The amount of binder in the coating can vary, but will be in
the range of about 0.01 to about 25% of the weight of the seed,
more preferably from about 0.05 to about 15%, and even more
preferably from about 0.1% to about 10%.
[0118] As mentioned above, the matrix can optionally include a
filler. The filler can be an absorbent or an inert filler, such as
are known in the art, and may include woodflours, clays, activated
carbon, sugars, diatomaceous earth, cereal flours, fine-grain
inorganic solids, calcium carbonate, and the like. Clays and
inorganic solids, which may be used, include calcium bentonite,
kaolin, china clay, talc, perlite, mica, vermiculite, silicas,
quartz powder, montmorillonite and mixtures thereof. Sugars, which
may be useful, include dextrin and maltodextrin. Cereal flours
include wheat flour, oat flour and barley flour.
[0119] The filler is selected so that it will provide a proper
microclimate for the seed, for example the filler is used to
increase the loading rate of the active ingredients and to adjust
the control-release of the active ingredients. The filler can aid
in the production or process of coating the seed. The amount of
filler can vary, but generally the weight of the filler components
will be in the range of about 0.05 to about 75% of the seed weight,
more preferably about 0.1 to about 50%, and even more preferably
about 0.5% to 15%.
[0120] The pesticides that are useful in the coating are those
pesticides that are described herein. The amount of pesticide that
is used for the treatment of the seed will vary depending upon the
type of seed and the type of active ingredients, but the treatment
will comprise contacting the seeds with an amount of the
combination of pesticides that is pesticidally effective. When
insects are the target pest, that amount will be an amount of the
insecticide that is insecticidally effective. As used herein, an
insecticidally effective amount means that amount of insecticide
that will kill insect pests in the larvae or pupal state of growth,
or will consistently reduce or retard the amount of damage produced
by insect pests.
[0121] In general, the amount of pesticide that is applied to the
seed in the treatment will range from about 10 gm to about 2000 gm
of the active ingredient of the pesticide per 100 kg of the weight
of the seed. Preferably, the amount of pesticide will be within the
range of about 50 gm to about 1000 gm active per 100 kg of seed,
more preferably within the range of about 100 gm to about 600 gm
active per 100 kg of seed, and even more preferably within the
range of about 200 gm to about 500 gm of active per 100 kg of seed
weight. Alternatively, it has been found to be preferred that the
amount of the pesticide be over about 60 gm of the active
ingredient of the pesticide per 100 kg of the seed, and more
preferably over about 80 gm per 100 kg of seed.
[0122] In preferred embodiments of the present invention the
transgenic event comprises the ability to express a Cry3Bb.11231
protein or a Cry3Bb.11098 protein, and the pesticide is selected
from either imidacloprid or tefluthrin.
[0123] The pesticides that are used in the treatment must not
inhibit germination of the seed and should be efficacious in
protecting the seed and/or the plant during that time in the target
insect's life cycle in which it causes injury to the seed or plant.
In general, the coating will be efficacious for approximately 0 to
120 days after sowing.
[0124] The pesticides of the subject invention can be applied to
the seed in the form of a coating. The use of a coating is
particularly effective in accommodating high pesticidal loads, as
can be required to treat typically refractory pests, such as corn
rootworm, while at the same time preventing unacceptable
phytotoxicity due to the increased pesticidal load.
[0125] Optionally, a plasticizer can be used in the coating
formulation. Plasticizers are typically used to make the film that
is formed by the coating layer more flexible, to improve adhesion
and spreadability, and to improve the speed of processing. Improved
film flexibility is important to minimize chipping, breakage or
flaking during storage, handling or sowing processes. Many
plasticizers may be used, however, useful plasticizers include
polyethylene glycol, glycerol, butylbenzylphthalate, glycol
benzoates and related compounds. The range of plasticizer in the
coating layer will be in the range of from bout 0.1 to about 20% by
weight.
[0126] When the pesticide used in the coating is an oily type
formulation and little or no filler is present, it may be useful to
hasten the drying process by drying the formulation. This optional
step may be accomplished by means will known in the art and can
include the addition of calcium carbonate, kaolin or bentonite
clay, perlite, diatomaceous earth, or any absorbent material that
is added preferably concurrently with the pesticidal coating layer
to absorb the oil or excess moisture. The amount of calcium
carbonate or related compounds necessary to effectively provide a
dry coating will be in the range of about 0.5 to about 10% of the
weight of the seed.
[0127] The coatings formed with the pesticide are preferably of the
type that are capable of effecting a slow rate of release of the
pesticide by diffusion or movement through the matrix to the
surrounding medium.
[0128] In addition to the coating layer, the seed may be treated
with one or more of the following ingredients: other pesticides
including fungicides and herbicides; herbicidal safeners;
fertilizers and/or biocontrol agents. These ingredients may be
added as a separate layer or alternatively may be added in the
pesticidal coating layer.
[0129] The pesticide formulation may be applied to the seeds using
conventional coating techniques and machines, such as fluidized bed
techniques, the roller mill method, rotostatic seed treaters, and
drum coaters. Other methods, such as spouted beds may also be
useful. The seeds may be presized before coating. After coating,
the seeds are typically dried and then transferred to a sizing
machine for sizing. Such procedures are known in the art.
[0130] The pesticide-treated seeds may also be enveloped with a
film overcoating to protect the pesticide coating. Such
overcoatings are known in the art and may be applied using
conventional fluidized bed and drum film coating techniques.
[0131] In another embodiment of the present invention, a pesticide
can be introduced onto or into a seed by use of solid matrix
priming. For example, a quantity of the pesticide can be mixed with
a solid matrix material and then the seed can be placed into
contact with the solid matrix material for a period to allow the
pesticide to be introduced to the seed. The seed can then
optionally be separated from the solid matrix material and stored
or used, or the mixture of solid matrix material plus seed can be
stored or planted directly. Solid matrix materials which are useful
in the present invention include polyacrylamide, starch, clay,
silica, alumina, soil, sand, polyurea, polyacrylate, or any other
material capable of absorbing or adsorbing the pesticide for a time
and releasing that pesticide into or onto the seed. It is useful to
make sure that the pesticide and the solid matrix material are
compatible with each other. For example, the solid matrix material
should be chosen so that it can release the pesticide at a
reasonable rate, for example over a period of minutes, hours, or
days.
[0132] The present invention further embodies imbibition as another
method of treating seed with the pesticide. For example, plant seed
can be combined for a period of time with a solution comprising
from about 1% by weight to about 75% by weight of the pesticide in
a solvent such as water. Preferably the concentration of the
solution is from about 5% by weight to about 50% by weight, more
preferably from about 10% by weight to about 25% by weight. During
the period that the seed is combined with the solution, the seed
takes up (imbibes) a portion of the pesticide. Optionally, the
mixture of plant seed and solution can be agitated, for example by
shaking, rolling, tumbling, or other means. After imbibition, the
seed can be separated from the solution and optionally dried, for
example by patting or air drying.
[0133] In yet another embodiment, a powdered pesticide can be mixed
directly with seed. Optionally, a sticking agent can be used to
adhere the powder to the seed surface. For example, a quantity of
seed can be mixed with a sticking agent and optionally agitated to
encourage uniform coating of the seed with the sticking agent. The
seed coated with the sticking agent can then be mixed with the
powdered pesticide. The mixture can be agitated, for example by
tumbling, to encourage contact of the sticking agent with the
powdered pesticide, thereby causing the powdered pesticide to stick
to the seed.
[0134] The present invention also provides a transgenic corn seed
that has been treated with a pesticide by the method described
above.
[0135] The treated seeds of the present invention can be used for
the propagation of corn plants in the same manner as conventional
treated corn seed. The treated seeds can be stored, handled, sowed
and tilled in the same manner as any other pesticide treated seed.
Appropriate safety measures should be taken to limit contact of the
treated seed with humans, food or feed materials, water and birds
and wild or domestic animals.
[0136] In a preferred embodiment, the invention is an insect
transgenic seed mix refuge strategy, i.e., 10% non-transgenic seed,
combined with an insecticidal seed treatment. The combination of
seed mix refuge strategy in combination with a seed treatment
allows for protection of the non-transgenic plants in the mixture
and provides a second mode of action for the transgenic seeds. The
combination refuge strategy and second mode of action are optimal
in delaying the onset of resistance development. This assumes that
larvae would survive to adults on the non-transgenic plants and at
the same time that these plants are sufficiently protected by the
seed treatment. The seed treatment may be on all seed or only on
the non-transgenic seed within the mix.
[0137] The words "seed mix refuge strategy" is intended to refer to
a means for deploying into a field of crops some percentage of the
seeds which sprout and develop into mature refuge plants but do not
contain the transgene, thus allowing susceptible adults to survive.
Although this strategy may be acceptable on low to moderate levels
of insect pressure, under very high levels of insect pressure the
non-protected plants, i.e. refuge plants, may be damaged such that
this insect resistance management strategy is not commercially
viable. By combining the mix seed refuge strategy with a seed
treatment, the non-transgenic plants are sufficiently protected but
still allow for larval survivorship to adults, and the seed mix
refuge strategy becomes commercially viable under all levels of
insect pressure. At the same time, two modes of action are
achieved, assuring the longest possible term for commercial
viability and utility of the transgenic crop seeds with a minimal
risk to the development of resistance races of insects.
[0138] In the regulatory environment that currently exists today,
obtaining the approval of an appropriate regulatory agency for
commercialization of a recombinant plant generally requires that a
percentage of all of the crop that is planted by a particular
farmer intending to plant a crop containing a recombinant trait
which effects the survival of particular insect pests be planted as
a refuge of non-recombinant or non-transgenic crops, or crops which
do not contain the ability to inhibit the development and growth of
the particular insect pest by the same mode of action. In fact, it
is preferred by the regulatory agencies that the refuge crop be
planted with a non-transgenic crop, and it is further required that
the refuge be planted as a block separate and apart from the
recombinant crops. In addition, the percentage of the total crop
planted is required to be at least 1% refuge, more preferably
between from about 2 to about 5% refuge, even more preferably
between from about 5% to about 10% refuge, and more preferably
still between from about 10% to about 20% refuge or more depending
on the amount of insect pressure expected for a particular
geographic location and depending also on the type of crop plant
subject to regulatory requirements. Such practices cause added
expense for farmers in terms of their input into labor and
financial expenses, and are difficult to police. Even though
farmers are required to purchase enough non-recombinant seed to
plant the required refuge along with any recombinant seed purchase,
the added labor for planting and segregating the refuge and the
likely lower yields within the refuge as a result of greater insect
infestation is a disincentive for the farmer to comply with the
regulatory requirements. Thus, a seed mix containing the requisite
refuge amount of non-transgenic seed, and which is treated with an
insecticide to protect the refuge plants from infestation, would be
a commercially acceptable means for ensuring compliance with
regulatory agency refuge strategies.
[0139] Advantages of a seed mix deployable refuge strategy over a
block refuge strategy includes elimination of the issues around
enforcement and compliance, simplicity, and complementarity with
block refuge strategies required for other insect resistance
traits. Furthermore by adding a seed treatment to the seed mix
deployable refuge strategy, no plants are left unprotected in the
field and a second mode of action is uniformly introduced to
function along with the transgenic insect control means.
[0140] The seed mix deployable transgenic refuge strategy is
particularly significant for corn rootworm resistant transgenic
corn, for which a seed mix refuge strategy may be the only feasible
means of deploying a refuge for the production of susceptible corn
rootworms that will mate with any resistant individuals which may
survive upon feeding on a corn rootworm resistant transgenic plant.
By combining a seed treatment with the corn rootworm transgenic and
non-transgenic seed in a mix, the seed mix refuge strategy would
then be commercially viable, because the non-transgenic seed would
be sufficiently protected by the seed treatment and still allow for
sufficient numbers of larvae to survive to adults while continuing
to provide for susceptible adult insects emerging from the field of
crops.
[0141] This invention eliminates the necessity for grower
application of chemical or other insecticides to the refuge to
protect the plants as would be the case in a block refuge strategy.
In the absence of seed treatment on the transgenic seeds in such a
mix, the transgenic seeds sprout and send their roots outward and
downward. Target insects which feed on these roots necessarily
succumb to the levels of the insecticidal protein preferentially
produced in the root tissue of the plant. In this scenario, the
seeds comprising the non-transgenic refuge mixed uniformly into the
seed mix deployable refuge mixture can either be treated with a
chemical insecticide or left untreated. Of course the untreated
refuge seed in the mixture would be entirely susceptible to insect
infestation, generally resulting in a yield loss with respect to
the percentage of refuge seed contained within the mixture.
Ideally, however, the refuge seed would be treated with a
composition which contains at least one and perhaps two or more
insecticidal agents selected from the group consisting of chemical
insecticide and biologically derived insecticidal agents such as
Bacillus thuringiensis insecticidal .delta.-endotoxin protein or
vegetative insecticidal protein (VIP), Bacillus sphearicus
insecticidal protein, Bacillus laterosporous insecticidal protein,
insecticidal proteins derived from Xenorhabdus and Photorhabdus
bacteria species, and insecticidal proteins which have been
specifically demonstrated to be effective, including but not
limited to tlC851, CryET70, Cry22, and binary insecticidal proteins
CryET33 and CryET34, CryET80 and CryET76, tlC100 and tlC101, and
PS149B1. Treated refuge seeds within the mix would sprout when
planted and the roots would grow outward and downward away from the
soil surface. Commensurate with planting and exposure to the
moisture in the soil, the treatment composition on the seed would
disperse into the microenvironment of the seed in the soil,
providing a decreasing concentration of insecticidal agent as a
second mode of action through which the young root tissue would
have to extend in order to be susceptible to insect feeding.
Ordinarily, the microenvironment into which the insecticidal agents
would disperse would be from about 1 to about 5 centimeters from
the point of dispersion, or from about 1 to about 10 centimeters
from the point of dispersion, said point of dispersion being
defined as the centerpoint of the seed mass within the soil at the
time of germination. Ordinarily, an insecticidally effective dose
of the chemical or protein agent contained within the seed
treatment would be required to extend outward for some distance
from the centerpoint of seed mass within the soil at the time of
germination. That effective dose would be required to be within the
dispersal zone around the seed mass, generally being from about 1
to about 5 centimeters from the point of dispersion, and more
preferably from about 1 to about 10 centimeters from the point of
dispersion, and even more preferably from about 2 to about 10
centimeters from the point of dispersion.
[0142] The more preferable means of deploying a transgenic refuge
into a field of recombinant crops would comprise a seed mixture
comprising from about 1% to about 10% refuge seed or more
preferably from about 1 to about 20% refuge seed. This embodiment
encompasses the treatment of all seeds contained within the
mixture, such that the afore mentioned dispersal zone around the
center of mass of any of the seeds planted into the soil would
suffice. It is also envisioned that regulatory requirements would
mandate a refuge requirement greater than the aforementioned 20%,
and it is intended . that those greater requirements for refuge be
included within the scope of this invention.
[0143] Preferred embodiments of the invention are described in the
following examples. Other embodiments within the scope of the
claims herein will be apparent to one skilled in the art from
consideration of the specification or practice of the invention as
disclosed herein. It is intended that the specification, together
with the examples, be considered exemplary only, with the scope and
spirit of the invention being indicated by the claims which follow
the examples.
[0144] The following examples describe preferred embodiments of the
invention. Other embodiments within the scope of the claims herein
will be apparent to one skilled in the art from consideration of
the specification or practice of the invention as disclosed herein.
It is intended that the specification, together with the examples,
be considered exemplary only, with the scope and spirit of the
invention being indicated by the claims which follow the examples.
In the examples all percentages are given on a weight basis unless
otherwise indicated.
EXAMPLE 1
[0145] Production of transgenic corn seed effective against corn
rootworm and treatment of such seed with imidacloprid (Gaucho.RTM.)
and tefluthrin (Raze.RTM.).
[0146] Corn seeds were prepared to express amino acid sequence
variant proteins of a Coleopteran inhibitory Bacillus thuringiensis
Cry3Bb .delta.-endotoxin (Cry3Bb.11231 (MON853) or Cry3Bb.11098
(MON863)) by the methods described for these respective events in
WO 99/31248. Such variant proteins have been shown to exhibit
improved levels of bioactivity in controlling pests such as
Diabrotica species. (U.S. Pat. Serial No. 6,063,597).
[0147] Corn transformation event MON853 contains a nucleotide
sequence which has not been optimized for plant expression. The
insecticidal Cry3Bb protein variant produced by the MON853 event
has been shown to exhibit improved insecticidal activity, in
particular directed against corn rootworms. While it is not
preferred that a nucleotide sequence encoding an insecticidal
protein from Bacillus thuringiensis be introduced into a plant
without first being modified to remove sequences which cause the
resulting protein to be produced inefficiently, it is believed that
the coding sequence within event MON853 functions to produce
effective insecticidal activity in part because the length of the
amino acid sequence which comprises a Cry3Bb variant protein is
about half of what a lepidopteran effective insecticidal Bacillus
thuringiensis Cry protein, and because the MON853 variant protein,
Cry3Bb.11231 has from about 3 to about 10 fold greater bioactivity
against corn rootworms than the native Cry3Bb protein derived from
Bacillus thuringiensis. Native Bacillus thuringiensis nucleotide
sequences encoding truncated Cry proteins exhibiting lepidopteran
inhibitory bioactivity are about the same size as the sequence
encoding Cry3Bb variants exemplified in these examples herein, and
have been shown to be expressed at very low but ineffective levels
in some plants.
[0148] Corn transformation event MON863 contains a modified
nucleotide sequence which has been optimized for plant expression.
The insecticidal Cry3Bb protein variant produced by the MON863
event, designated Cry3Bb.11098, has been shown to exhibit improved
insecticidal activity, in particular directed against corn
rootworms. MON863 exhibits better corn rootworm control than MON853
with or without seed treatment, more likely than not because the
MON863 event contains a modified sequence encoding a variant Cry3Bb
protein, 11098, similar in insecticidal activity to the variant
Cry3Bb protein 11231 in event MON853, but which is expressed more
efficiently from the modified coding sequence.
[0149] Corn seeds of the same hybrid species, with and without the
respective transgenic events, were treated with either imidacloprid
(available as GAUCHO.RTM. from Bayer Corp.) or tefluthrin
(available as RAZE.RTM. from Wilbur-Ellis Co., Great Falls, Mont.;
Walla Walla, Wash.) as follows. A seed treatment formulation of the
desired pesticide was prepared by mixing a measured amount in water
as a carrier and applying the formulation for one minute at room
temperature to a measured weight of corn seed in a rotostatic seed
treater. The respective weights of the pesticide preparation and
the corn seed were calculated to provide the desired rate of
treatment of pesticide on the seed. The pesticide was mixed into
sufficient water to permit efficient distribution of the
formulation to all of the seeds in the batch while minimizing loss
of treatment formulation due to lack of uptake of the formulation
by the seeds. Treated seeds were allowed to sit uncapped for at
least four hours before planting.
[0150] When the seeds were treated with imidacloprid, a sufficient
amount of Gaucho.RTM. 600 FS (containing 48.7% by weight
imidacloprid; available from the Gustafson LLC) was thoroughly
mixed into water to form a seed treatment formulation, and the
formulation was applied to a weight of corn seed to provide
treatment levels of 300 grams imidacloprid per 100 kg of seed (0.75
mg imidacloprid/kernel), or 400 grams imidacloprid per 100 kg of
seed (1.0 mg imidacloprid/kernel).
[0151] When the seeds were treated with tefluthrin, a sufficient
amount of Raze.RTM. 2.5 FS (containing 26.8% by weight tefluthrin;
available from Wilbur-Ellis Co.,) was thoroughly mixed into water
to form a seed treatment formulation, and the formulation was
applied to a weight of corn seed to provide treatment levels of 300
grams active tefluthrin per 100 kg of seed (0.75 mg
tefluthrin/kernel).
EXAMPLE 2
[0152] Field trials for the determination of efficacy of transgenic
event Cry3Bb.11231 in corn seed in combination with corn root worm
pesticide seed treatments against western and northern corn
rootworm.
[0153] Field trials were run in accordance with pertinent protocols
and in conformance with USDA notification requirements. The purpose
of the trials was to determine the efficacy of transgenic event
Cry3Bb.11231 in corn seed in combination with corn root worm seed
treatments against western and northern corn root worm.
[0154] For each growing site that was selected, the plot design
included the following:
1 Row spacing: 30 inches Plot size: 4 rows .times. 20 Plant
density: 2.0 seed/foot Hybrid used: LH198 .times. LH185 or RX670
Replicates: 4 Design: Randomized complete block Locations: 4 Larvae
source: natural infestations supplemented by artificial infestation
of corn rootworm eggs at 400 eggs/ft (growth stage V2)
[0155] The following seed treatment combinations were used for each
growing area:
2 Pesticide and amount (grams AI/100 No. Corn Seed Type kg seed or
mg ai/kernel) 1 Isohybrid None, other than low levels for wire worm
protection 2 Cry3Bb.11231 None, other than low levels for wire worm
protection 3 Cry3Bb.11231 Gaucho .RTM. 600 FS @ 300 gm AI/100 kg or
.75 mg AI/kernel 4 Cry3Bb.11231 Gaucho .RTM. 600 FS @ 400 gm AI/100
kg or 1.0 mg AI/kernel 5 Cry3Bb.11231 Raze .RTM. 2.5 FS @ 300 gm
AI/100 kg or .75 mg AI/kernel 6 Isohybrid Force .RTM. 3G @ 0.014 gm
AI/m, or 0.15 oz AI/1000 ft row, applied as a 5" band on the soil
surface at the time of planting. 7 Isohybrid Lorsban .RTM. 15G
(chlorpyrifos; available from DowElanco) @ 0.11 gm AI/m, or 1.2 oz
AI/1000 ft row, applied as a 5" band on the soil surface at the
time of planting.
[0156] All seed treatments with pesticides were carried out as
described in Example 1. In seed treatment number 1 and 2,
Gaucho.RTM. was used for wire worm protection, but at levels
sufficiently low that it would be expected to have no effect on
corn rootworms (i.e., at a treatment level of about 60 gm of
active/100 kg seed or 0.16 mg active/kernel), otherwise, seed
receiving treatment number 2 had only transgenic event Cry3Bb.11231
and no pesticide treatment that would be expected to be effective
against corn rootworm.
[0157] For seeds having treatments numbered 3 through 5, the
pesticides were applied by the methods described in Example 1. For
seeds having treatment numbers 6 and 7, commercially available
Force.RTM. 3G and Lorsban.RTM. 15G were applied to the soil in a 5"
band at the time of sowing. The levels of application are as shown
and are within the ranges recommended for standard commercial
practice.
[0158] Corn seeds to be tested were planted and grown at four
different locations across four Midwestern states in the United
States corn belt according to the protocol described above.
[0159] The determination of damage by corn rootworm was made
according to the following protocol. At stage V4-V6, an evaluation
of early stand was made by counting the number of plants per acre.
At stage VT-R1, an evaluation of corn rootworm damage was carried
out by methods that are well known in the industry, and damage by
corn rootworm was reported according to the Iowa 1-6 rating system.
In that system, the root systems of 10 corn plants per plot are
recovered and scored using the 1-6 rating scale, where: 1=no injury
or only a few minor feeding scars, 2=feeding injury evident, but no
roots eaten back to 11/2 inches of the plant, 3=at least one root
eaten off to within 11/2 inches of the plant, but never an entire
node of roots destroyed, 4=one node of roots eaten back to within
11/2 inches of the plant, 5=two nodes (circles) of roots eaten back
to within 11/2 inches of the plant, 6=three nodes (circles) of
roots eaten back to within 11/2 inches of the plant.
3TABLE 1 Corn rootworm damage to isohybrid corn plants having
conventional surface banding treatments and corn plants having
transgenic event Cry3Bb.11231 alone and in combination with seed
treatment with selected pesticides at four growing locations. MEANS
SEED SITE SITE SITE SITE ACROSS NO. A B C D LOCATIONS 1 4.3 4.0 4.0
4.2 4.1 2 2.5 2.4 2.2 2.0 2.3 3 2.1 2.3 2.5 1.9 2.2 4 1.8 2.3 2.2
1.8 2.0 5 2.3 2.3 2.6 1.8 2.2 6 2.7 2.1 2.6 1.9 2.3 7 3.3 2.4 2.5
1.8 2.5
[0160] From the data of Table 1, it can be seen that transgenic
seeds that were treated with either imidacloprid or tefluthrin at
any level were more resestant to corn rootworm damage than the
transgenic seeds without such pesticide treatment. Moreover, all
combination treatments (of transgenic event plus pesticide
treatment) were more efficacious that conventional surface banding
with either FORCE.RTM. or LORSBAN.RTM..
[0161] Therefore, it can be concluded that the treatment of a corn
seed having a transgenic event with either imidacloprid or
tefluthrin provides improved resistance over that provided by
either the transgenic event alone, or hybrid seed that has also
received a standard pesticide surface banding treatment at
planting.
EXAMPLE 3
[0162] Field trials for the determination of efficacy of transgenic
event Cry3Bb.11231 in corn seed in combination with imidacloprid
seed treatments against western and northern corn rootworm.
[0163] A field trial was established and completed in accordance
with pertinent protocols and in conformance with USDA notification
requirements. The purpose of the trial was to determine the
efficacy of transgenic event Cry3Bb.11231 in corn seed in
combination with corn rootworm seed treatments using
imidacloprid.
[0164] For each growing site that was selected, the plot design
included the following:
4 Row spacing: 30 inches Plot size: 4 rows .times. 20 Plant
density: 2.0 seed/foot Hybrid used: LH198 .times. LH185 or RX670
Replicates: 4 Design: Randomized complete block Locations: 4 Larvae
source: natural infestations supplemented by artificial infestation
of corn rootworm eggs at 400 eggs/ft (growth stage V2)
[0165] The following seed treatment combinations were used for each
growing area:
5 Pesticide and amount (grams AI/100 No. Corn Seed Type kg seed or
mg ai/kernel) 1 Isohybrid None, other than low levels for wire worm
protection 2 Cry3Bb.11231 None, other than low levels for wire worm
protection 3 Cry3Bb.11231 Gaucho .RTM. 600 FS @ 300 gm AI/100 kg or
.75 mg AI/kernel 4 Cry3Bb.11231 Gaucho .RTM. 600 FS @ 400 gm AI/100
kg or 1.0 mg AI/kernel 5 Cry3Bb.11231 Raze .RTM. 2.5 FS @ 300 gm
AI/100 kg or .75 mg AI/kernel 6 Isohybrid Force .RTM. 3G @ 0.014 gm
AI/m, or 0.15 oz AI/1000 ft row, applied as a 5" band on the soil
surface at the time of planting. 7 Isohybrid Lorsban .RTM. 15G
(chlorpyrifos; available from DowElanco) @ 0.11 gm AI/m, or 1.2 oz
AI/1000 ft row, applied as a 5" band on the soil surface at the
time of planting.
[0166] All seed treatments with pesticides were carried out as
described in Example 1. In seed treatment number 1 and 2,
Gaucho.RTM. was used for wire worm protection, but at levels
sufficiently low that it would be expected to have no effect on
corn rootworms (i.e., at a treatment level of about 60 gm of
active/100 kg seed or 0.16 mg active/kernel), otherwise, seed
receiving treatment number 2 had only transgenic event Cry3Bb.11231
and no pesticide treatment that would be expected to be effective
against corn rootworm.
[0167] For seeds having treatments numbered 3 through 5, the
pesticides were applied by the methods described in Example 1. For
seeds having treatment numbers 6 and 7, commercially available
Force.RTM. 3G and Lorsban.RTM. 15G were applied to the soil in a 5"
band at the time of sowing. The levels of application are as shown
and are within the ranges recommended for standard commercial
practice.
[0168] Corn seeds to be tested were planted and grown at four
different locations across several Midwestern states in the United
States corn belt according to the protocol described above.
[0169] The determination of damage by corn root worm was made
according to the protocol described in Example 2.
6TABLE 2 Corn rootworm damage to isohybrid corn plants and corn
plants having transgenic event Cry3Bb.11231 alone and in
combination with seed treatment with imidacloprid pesticide at
different growing locations. CORN ROOTWORM DAMAGE IN EACH IOWA
CLASS (IOWA 1-6 PERCENT SCALE) GRAND OF TREATMENT 1 2 3 4 5 6 TOTAL
CONTROL Isohybrid 0 3 16 36 21 4 80 100 Cry3Bb.11231 5 51 23 1 0 0
80 31.2 Imidacloprid @ 3 15 36 21 5 0 80 80.5 400 g/100 kg of seed
Cry3Bb.11231 13 53 14 0 0 0 80 18.2 with Imidacloprid @ 400 gm/100
kg of seed FORCE .RTM. 3G 3 58 34 3 0 0 98 39.2 surface band at
planting LORSBAN .RTM. 6 39 38 16 1 0 100 57.1 15G surface band at
planting Notes: a. Data for the isohybrid control was taken as the
same as determined for a related protocol that was carried out in
an adjoining plot.
[0170] The data showed that both the transgenic event alone and
seed treatment with imidacloprid alone provided some level of
protection against corn rootworm damage above the untreated
isohybrid control. At higher levels of damage (i.e., damage levels
4-6), corn having the transgenic event suffered 4.7% of the damage
of the non-transgenic control. Since 4.7% would be considered to be
about 5%, the Cry3Bb.11231 event was considered to be within a
preferred effectiveness range of about 5% to about 50% of the
damage of the non-transgenic control.
[0171] Imidacloprid seed treatment alone at 400 gm/100 kg was
effective against corn rootworm damage, but the effectiveness of
imidacloprid was lower than the effectiveness of the transgenic
event alone. The combination of treatment with imidacloprid of the
transgenic seed was more effective against rootworm damage than the
pesticide treatment alone or the transgenic event alone. Moreover,
the combination of Cry3Bb.11231 with imidacloprid at 400 gm/100 kg
of seed provided better protection than the commercial standard
treatment of either FORCE.RTM. or LORSBAN.RTM. applied as a surface
band at planting.
[0172] The advantages of the present treatment of transgenic seed
with imidacloprid include the simplification of planting, by
removing the requirement for separate application of the pesticide.
Furthermore, planting is easier and safer, since the planter does
not have to handle a concentrated pesticide.
[0173] The combination of imidacloprid seed treatment with corn
seed having a Cry3Bb.11231 transgenic event was tested for possible
synergy at a level of rootworm damage of 3-6. In the first test,
shown in Table 2, the percentage of test plants having damage
levels of from 3 to 6, on the Iowa 1-6 Scale, was determined for
the control and for seeds treated with the pesticide at two levels,
and for seeds having the transgenic event, alone and in
combination. The following formula was then used to calculate a
"synergy threshold":
(% of control Cry3Bb.11231)*(% of control imidacloprid
treatment)/100.
[0174] This threshold was compared against the percent of control
for the treatment combination (i.e., Cry3Bb.11231 with imidacloprid
@ 400 gm/100 kg). If the treatment combination percent of control
was below the threshold, then it was concluded that there was
synergy. If the treatment combination percent of control was above
the threshold, then it was concluded that synergy was not
demonstrated for that combination.
[0175] It was believed that the measurement of rootworm damage at
higher damage levels (i.e., levels 3-6) is a useful indicator that
correlates with subsequent yield loss due to such damage. The
reason for this is that rootworm damage at levels 1 and 2 seldom
causes corn plants to fall over and lodge, and such minimal root
loss is not believed to reduce the number or weight of kernels per
ear. However, root damage at levels of 3 and above increasingly
causes lodging and loss of yield. Therefore, it is believed that
the summed damage levels of 3-6 (and in some cases, 4-6 and 5 and
6), provides a useful indication of the effect of corn rootworm
damage on subsequent corn yield.
7TABLE 3 Efficacy of seed treatment with imidacloprid alone and in
combination with corn transgenic event Cry3Bb.11231 against corn
rootworm damage at levels 3-6 on the Iowa 1-6 Scale. NUMBER OF
PLANTS PERCENT HAVING OF THRESHOLD TREATMENT 3-6 DAMAGE LEVEL
CONTROL SYNERGY Untreated 96.1 100 -- Control Cry3Bb.11231 40 31.2
-- Imidacloprid @ 71.7 80.5 -- 400 gm/100 kg Cry3Bb.11231 24 18.2
25.1 with imidacloprid @ 400 gm/100 kg FORCE 3G as 40.7 39.2 --
surface band LORSBAN 60.8 57.1 -- 15G as surface band
[0176] This analysis indicated that the combination of the corn
Cry3Bb.11231 transgenic event with seed treatment with imidacloprid
at 400 gm/100 kg was synergistic and unexpectedly efficacious
against corn rootworm damage at the 3-6 level. Accordingly, it was
concluded that the combination of the transgenic event with the
imidacloprid seed treatment provided significant advantages over
the use of either method alone, and that such protection was
unexpectedly superior in efficacy against severe damage by corn
rootworm.
[0177] It was also believed to be noteworthy that the combination
of imidacloprid and transgenic event provided protection against
severe corn rootworm damage at levels that were far better than
that provided by either FORCE.RTM. or LORSBAN.RTM. applied as
surface bands.
EXAMPLE 4
[0178] Field trials for the determination of efficacy of transgenic
event Cry3Bb.11231 in corn seed in combination with tefluthrin
pesticide seed treatments against western and northern corn
rootworm.
[0179] A field trial for the determination of efficacy of the
combination of transgenic event Cry3Bb.11231 in corn seed with
tefluthrin (available as RAZE.RTM. from Wilbur-Ellis Company) could
be carried out according to the same protocol as described in
Example 3, except that tefluthrin would be substituted for
imidacloprid in each case where imidacloprid was used at levels
expected to be effective against corn rootworm (e.g., at levels of
higher than 60 gm/100 kg seed). If desirable, it would be
permissible to continue to use imidacloprid at levels of 60 gm/100
kg, or less, for wireworm protection.
[0180] It would be expected that the combination of tefluthrin seed
treatment with a transgenic event in corn seed having effectiveness
against corn rootworm would provide synergistic protection similar
to that shown in Example 3 for the combination of imidacloprid and
Cry3Bb.11231.
EXAMPLE 5
[0181] This example illustrates the use of a seed mixture
containing various ratios of transgenic and non-transgenic seeds to
deploy a transgenic refuge, with or without seed treatments,
provides an effective means for allowing adequate survival of
susceptible corn rootworms in fields of recombinant crops to
prevent or slow the rate of resistance evolution and still reduce
economic loss due to corn rootworm infestation.
[0182] The western corn rootworm (WCR), D. virgifera virgifera, is
a widely distributed pest of maize in North America. In many
instances, insecticides are indiscriminately used to reduce their
numbers below an economically damaging level. To assist in the
reduction of insecticides used against the WCR, the inventors
herein have utilized a transgenic line of maize expressing the
Cry3Bb insecticidal protein. Upon ingestion by the rootworm, this
protein forms pores in the midgut cells causing swelling and lysis
of these cells and eventually death to the feeding worm. One
concern is that the WCR will evolve resistance to the protein which
will potentially spread throughout the rootworm's distribution and
population. Deploying a transgenic refuge by planting seed mixtures
of transgenic and non-transgenic maize may be a reliable resistance
management strategy for controlling corn rootworms. The inventors
herein have investigated the technical feasibility of a resistance
management program that uses in-field seed mixes containing various
proportions of transgenic and non-transgenic seed, i.e., T:NT, in
combination with a new seed treatment technology to prevent
substantial damage to non-transgenic maize provided in the mix. If
effective, this methodology could provide growers with greater
yields at lower cost and labor requirements, and could
simultaneously provide a means for preventing or managing the
development of resistant strains of CRW. The underlying assumption
is that planting of a mix comprising transgenic vs. non-transgenic
(T:NT) seed at the appropriate ratios allows adequate survival of
susceptible CRW in the fields to prevent or slow the rate of
resistance evolution and still reduce economic losses due to CRW
infestation.
[0183] This method utilized a factorial design having five ration
levels of transgenic vs non-transgenic seed in a mix, consisting of
100:0 T:NT, 90:10 T:NT, 80:20 T:NT, 60:40 T:NT, and 0:100 T:NT. Two
levels of WCR egg infestation were utilized at the V2-V3 plant
growth stage, consisting of 500 eggs per thirty centimeter row and
1000 eggs per thirty centimeter row, which were designated as low
and high infestation rates, respectively. Two levels of seed
treatment were utilized, similar to what was used in the examples
above. One treatment level consisted of Gaucho (imidocloprid) at 60
grams per 100 kilogram of seed and was designated as WWST. The
other treatment level consisted of clothianidine at 200 milligrams
on non-transgenic (NT) seed, Gaucho on the transgenic seed (T),
designated CRWST1, and 100% non-transgenic (NT) seed mix. Four
additional treatments were used for comparison purposes only, and
were not included at all in the ANOVA's. One of these additional
treatments consisted of T80NT20 at a low and high level of egg
infestation, with imidocloprid applied at 30 grams per 100
kilograms of seed on transgenic (T) seed and Gaucho on the
non-transgenic seed (NT), designated as the CRWST2 treatments.
Another of these additional treatments consisted of two 100%
non-transgenic (NT) trials at low and high levels of egg
infestation, treated only with Force3G insecticide, which is the
conventional means presently in commercial use for treating corn
rootworm infestation. All treatments were replicated four times
over 96 plots, and the seeds were hand planted to verify the proper
transgenic vs non-transgenic (T:NT) rations. Emergence cages
covered five plants, exemplifying the total plot of T:NT at various
ratios. A Hills & peters 1-6 damage rating scale, as indicated
herein, was used to score the damage to roots near the end of the
adult emergence cycle, using ten plants per rep out of a total of
800 plants.
[0184] Over all of the treatments, significantly more female WCR
emerged than male WCR (4972 female vs 2823 male), using a paired
t-test, in which t=-7.82, df=79, and P<0.0001. It was determined
that there was no significant interactions among seed treatments,
egg rates, and ratios of transgenic to non-transgenic maize. Seed
treatment had no significant effect on the mean number of WCR
emerging, however, it was determined that significantly more
(F=18.65, df=1.57, P<0.0001) WCR emerged from caged infested at
the high level (4447 total, 111.2.+-.19.4) than at the low level of
infestation (3348 total, 83.7.+-.18.3). The mean number of WCR
emerging from the different seed ratios differed significantly
(F=105.34, df=4.57, P<0.01). All pairwise comparisons were
significantly different (df=57, P<0.0001) based on t-tests on
Lease Squares Means using Bonferroni adjustments to control Type 1
errors (alpha=0.05), except for WCR emerging from the T90:NT10 and
T80:NT20 ratio studies. The fewest number of WCR emerged from the
T100NT0 ratio study and the highest number from the T0NT100 ratio
study. The mean number of WCR emerging from the T80NT20 maize
treated blend with CRWST2 (29.3.+-.6.2) was comparable to the mean
number emerging from the T80NT20 maize (35.9.+-.4.8) treated blend
with CRWST1 and WWST. The mean number of WCR emerging from the
maize treated with Force3G (98.3.+-.16.6) was comparable to the
mean number emerging from the T60NT40 ratio study
(93.6.+-.12.2).
[0185] Seed treatment had no significant effect on mean root damage
rating, however, it was determined that a significant interaction
between egg infestation rates and ratios of transgenic to
non-transgenic maize (F=5.35, df=1.776, P<0.001). Based on
t-tests on Least Squares Means using Bonferroni adjustments to
control Type 1 errors (alpha=0.05), most pairwise comparisons were
significantly different (df=776, P<0.0001). Exceptions include
root damage rating from the low egg infestation rate at T0:NT100
and high egg infestation rate at T0:NT100, low egg infestation rate
at T100:NT0, and high egg infestation rate at T100:NT0. The lowest
root damage ratings were obtained from the T100NT0 ratio studies
and the highest root damage rating was observed in the T0NT100
ratio study. The mean root damage rating from the maize treated
with CRWST2 (1.61.+-.0.10) was comparable to the mean root damage
rating from the T80NT20 ratio study (1.81.+-.0.07). Similarly, the
mean root damage rating from the maize treated with Force3G
(2.81.+-.0.09) was comparable to the mean root damage rating from
the T60NT40 ratio study (2.74.+-.0.09).
[0186] More females emerged than males. Whether this is due to
differential mortality on the sexes caused by the transgenic maize
or some other phenomenon is not clear. Further investigations into
the sex ratio of WCR is necessary to elucidate any sexually biased
effects caused by the transgenic maize.
[0187] The number of emerging WCR differed among the ratios of
transgenic maize to non-transgenic maize. The ratios T100NT0,
T90NT10, and T80NT20 were the most effective at reducing rootworm
populations. These three ratios had the least number of emerged
beetles. As expected, the non-transgenic maize had little or no
controlling effect on beetle numbers. The CRWST1 had no significant
impact on reducing the number of emerging WCR or on root damage
rating. Similar numbers of WCR emerged from both the T60NT40 ratio
studies and the maize treated with Force3G, which may explain the
similar amount of damage to maize roots for these two
treatments.
[0188] Root damage greatly exceeded economically acceptable levels
(RDR 3.0) for the T0NT100 maize plots, and only slightly for the
T60NT40 ratio studies at high egg infestation rates. The least
amounts of root damage occurred to plants in the T100NT0, T90NT10,
and T80NT20 ratio studies. Maize planted at these ratios never
exceeded the economically damaging root damage rating level of 3.0
on the Iowa Hills&Peters scale.
[0189] One concern about the commercial release of transgenic maize
for control of CRW is the evolution of resistance by the rootworms.
One means for managing the development of resistance is to require
that producers and growers plant a refuge to maintain resistant
alleles at a low frequency. This disclosure illustrates a seed mix
refuge option. The data in this example illustrates that a T90NT10
and a T80NT20 ratio seed mix maintained root damage levels below
the economically damaging levels and produced similar numbers of
adult beetles. A T60NT40 ratio only exceeded economically damaging
levels under high levels of insect infestation and was comparable
to the conventionally used insecticide Force3G. The combination of
a seed treatment along with the deployment of refuge seed in a mix
of transgenic seeds is therefore a useful strategy for prolonging
the onset of resistance to either the seed treatment or to the
recombinant insect inhibitory trait contained within the plant
tissue.
[0190] These results demonstrate that all ratios including
transgenic maize were as effective as the traditional method of
applying insecticides to maintain WCR root damage levels below
economically damaging levels. Most of the transgenic:non-transgenic
ratios performed much better than the traditional method. Only the
100% non-transgenic maize had consistent root damage ratings
exceeding the economic threshold. Using a seed mix of transgenic
and non-transgenic seed in various proportions, in particular in
combination with seed treatments providing a second mode of action,
for planting in a crop in a field, can reduce the onset of
resistance in the target insect pests.
[0191] All references cited in this specification, including
without limitation all papers, publications, presentations, texts,
reports, manuscripts, brochures, internet postings, journal
articles, periodicals, and the like, are hereby incorporated by
reference. The discussion of the references herein is intended
merely to summarize the assertions made by their authors and no
admission is made that any reference constitutes prior art.
Applicants reserve the right to challenge the accuracy and
pertinence of the cited references.
[0192] In view of the above, it will be seen that the several
advantages of the invention are achieved and other advantageous
results attained.
[0193] As various changes could be made in the above methods and
compositions without departing from the scope of the invention, it
is intended that all matter contained in the above description
shall be interpreted as illustrative and not in a limiting
sense.
* * * * *
References