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Patent 2471555 Summary

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(12) Patent: (11) CA 2471555
(54) English Title: CONTROLLING PLANT PATHOGENS WITH FUNGAL/BACTERIAL ANTAGONIST COMBINATIONS COMPRISING TRICHODERMA VIRENS AND BACILLUS AMYLOLIQUEFACIENS
(54) French Title: LUTTE CONTRE LES AGENTS PATHOGENES DE PLANTES A L'AIDE DE COMBINAISONS BACTERIENNES ET FONGIQUES ANTAGONISTES
Status: Granted
Bibliographic Data
(51) International Patent Classification (IPC):
  • A01N 63/38 (2020.01)
  • A01N 63/22 (2020.01)
  • A01C 1/06 (2006.01)
  • A01P 3/00 (2006.01)
(72) Inventors :
  • JOHNSON, THOMAS D. (United States of America)
(73) Owners :
  • NOVOZYMES BIOAG A/S (Denmark)
(71) Applicants :
  • JOHNSON, THOMAS D. (United States of America)
(74) Agent: MBM INTELLECTUAL PROPERTY AGENCY
(74) Associate agent:
(45) Issued: 2011-05-17
(22) Filed Date: 2004-06-18
(41) Open to Public Inspection: 2005-12-18
Examination requested: 2004-10-01
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): No

(30) Application Priority Data: None

Abstracts

English Abstract

Fungal/bacterial antagonist combinations, a seed coated with one of the combinations and a plant protected from plant pathogens by one of the combinations. The invention is also a fungal/bacterial antagonist combination and its use for controlling plant pathogens as a biocontrol agent, bio-pesticide or bio-fungicide. The invention also finds utility as a fungal/bacterial antagonist combination applied to the seed, stalk or leaf that results in an increase in plant yield.


French Abstract

Combinaisons de champignons et de bactéries antagonistes, semence recouverte d'une des combinaisons et plante protégée des phytopathogènes par une des combinaisons. L'invention est également une combinaison de champignons et de bactéries antagonistes; il s'agit d'un agent biologique, d'un biopesticide ou d'un biofongicide employé pour combattre les phytopathogènes. L'invention peut également être employée comme combinaison de champignons et de bactéries antagonistes appliquée sur les semences, les tiges ou les feuilles pour accroître le rendement cultural.

Claims

Note: Claims are shown in the official language in which they were submitted.





THE EMBODIMENTS OF THE INVENTION FOR WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:


1. An agricultural inoculum suitable for inoculating plant seeds, said
inoculum comprising:
Trichoderma virens isolate G1-3 (ATCC 58678);

Bacillus amyloliquefaciens strain TJ1000 (ATCC BAA-390); and

a suitable carrier that is non-phytotoxic, non-bacteriostatic, and non-
bactericidal.

2. A method of protecting a plant from disease caused by a plant pathogenic
fungus
comprising:

inoculating seeds from said plant with an effective amount of;
Trichoderma virens G1-3 (ATCC 58678) and

Bacillus amyloliquefaciens TJ1000 (ATCC BAA-390),

wherein said combination suppresses growth of plant pathogenic fungi.

3. A method of protecting a seed or a plant from disease caused by a plant
pathogenic
fungus comprising:

inoculating seeds from said plant with a composition comprising a fungal
antagonist and
a bacterial antagonist, wherein said fungal antagonist is Trichoderma virens;
and wherein said
bacterial antagonist is Bacillus amyloliquefaciens TJ1000 (ATCC BAA-390).

4. A method of protecting a seed or a plant from disease caused by a plant
pathogenic
fungus comprising:

67



inoculating seeds from said plant with a composition comprising the
agricultural
inoculum of claim 1,

wherein said inoculum suppresses growth of plant pathogenic fungi.

5. The method of claim 4 wherein the inoculum suppresses growth of the plant
pathogen
fungi Fusarium, Phythium, Phytophthora and Penicillium.

6. A method of protecting a plant from disease caused by a plant pathogenic
fungus
comprising:

inoculating said plant with a composition comprising Trichoderma virens
isolate G1-3
(ATCC 58678) and Bacillus amyloliquefaciens TJ1000 (ATCC BAA-390),

wherein said combination suppresses growth of plant pathogenic fungi.

7. A method for biologically controlling or inhibiting stalk rot or root rot
comprising:
coating seeds with an effective amount of a composition comprising Trichoderma
virens
G1-3 (ATCC 58678) and Bacillus amyloliquefaciens TJ1000 (ATCC BAA-390).

8. A process for making a composition comprising:

introducing a culture of Bacillus amyloliquefaciens TJ1000 (ATCC BAA-390) to a

growth medium about eight hours after a culture of Trichoderma virens G1-3
(ATCC 58678) is
introduced to the growth medium; and

growing the culture as a competitive culture.
68



9. A process comprising making a composition comprising:

combining a culture of Trichoderma virens G1-3 (ATCC 58678) with a culture of
Bacillus amyloliquefaciens TJ1000 (ATCC BAA-390) in a 50:50 mixture; and

applying said composition to a seed at a rate of at least 100,000 spores per
seed.

10. A method for protecting plants in a growing medium from damping off and
root rot
fungal plant disease comprising:

placing in the growing medium in the immediate vicinity of the plant to be
protected or in
contact with the plant to be protected an effective quantity of the
agricultural inoculum of claim
1.

11. A method for protecting plants from fungal plant disease comprising:

adding the agricultural inoculum of claim 1 in an effective quantity to a
substrate selected
from the group of pelletized calcium sulfate and pelletized lime; and

placing the pellet in the immediate vicinity of the plant to be protected or
in contact with
the plant to be protected.

12. The method of claim 11 further comprising:
adding another plant growth nutrient to the pellet.

13. A method for protecting a plant from fungal plant disease comprising:

adding the agricultural inoculum of claim 1 in an effective quantity to a
liquid solution;
and

69



applying the liquid solution in the immediate vicinity of the plant to be
protected or in
contact with the plant to be protected.

14. The method of claim 13 further comprising:

adding an additive to the liquid, said additive being at least one substance
selected from
the group of

a plant nutrient,

a plant micro-nutrient, and
a chemical fungicide.

15. A method for biologically controlling a plant disease caused by a plant-
colonizing
fungus, the method comprising:

inoculation of a seed of the plant with an effective amount of a microbial
inoculant
comprising a combination having all of the identifying characteristics of the
agricultural
inoculum of claim 1, said inoculation resulting in the control of said plant
disease.

16. The method of claim 15, wherein said inoculation results in the control of
more than one
plant disease.

17. A composition comprising:

Trichoderma virens isolate G1-3 (ATCC 58678); and
Bacillus amyloliquefaciens strain TJ1000 (ATCC BAA-390).



18. An antagonist for controlling plant pathogens comprising:
Trichoderma virens isolate G1-3 (ATCC 58678);

Bacillus amyloliquefaciens strain TJ1000 (ATCC BAA-390); and

a suitable carrier that is non-phytotoxic, non-bacteriostatic, and non-
bactericidal.
19. A method for culturing a plant comprising:

applying the antagonist of claim 18 to a seed or to the seedbed of the plant;
planting the seed in the seedbed;

growing the plant to yield a crop; and
harvesting the crop,

wherein said applying step increases the yield of the crop.

20. The method of claim 19 wherein the antagonist is applied to the seed or to
the seedbed of
a plant selected from the group of

a monocot, and
a dicot.

21. The method of claim 19 wherein the antagonist is applied to the seed or to
the seedbed of
a plant selected from the group of

a legume plant, and
a non-legume plant.

71



22. The method of claim 19 wherein the antagonist is applied to the seed or to
the seedbed of
a plant selected from the group of

corn,
sunflower,
soybean,
field pea, and
wheat.

23. A process comprising:

making a composition by combining a culture of Trichoderma virens G1-3 (ATCC
58678) with a culture of Bacillus amyloliquefaciens TJ1000 (ATCC BAA-390) in a
mixture; and
applying said composition to a seed, wherein said mixture ranges in
composition from 10

to 90 percent Trichoderma virens G1-3 (ATCC 58678) by culture volume and from
90 to 10
percent Bacillus amyloliquefaciens TJ1000 (ATCC BAA-390) by culture volume.

24. An antagonist for controlling plant pathogens made by combining effective
amounts of:
a strain of Trichoderma virens and

Bacillus amyloliquefaciens strain TJ1000 (ATCC BAA-390).

25. The antagonist of claim 24, further comprising a suitable carrier that is
non-phytotoxic,
non-bacteriostatic, and non-bactericidal.

26. The antagonist of claim 24 or 25, wherein the strain is Trichoderma virens
G1-21.

72



27. A method comprising:

combining a spore-forming fungal strain and a spore-forming bacterial strain
to produce a
product comprising the composition of claim 17; and

applying the product to a plant or to a part of the plant,

whereby application of the product produces yield enhancement in the plant.
28. A method comprising:

applying Trichoderma virens isolate G1-3 (ATCC 58678) and Bacillus
amyloliquefaciens
strain TJ1000 (ATCC BAA-390) to a wettable powder to produce a combination
comprising the
antagonist of claim 18; and

applying the combination to a seed,

whereby application of the combination produces a positive yield response in a
plant
growing from the seed.

29. A process comprising:

making the agricultural inoculum of claim 1; and
applying said agricultural inoculum to a seed,

wherein said agricultural inoculum ranges in composition from 1 to 99 percent
Trichoderma virens G1-3 (ATCC 58678) by culture volume and from 99 to 1
percent Bacillus
amyloliquefaciens TJ1000 (ATCC BAA-390) by culture volume.


73



30. A method for increasing the yield of a plant, the method comprising:

coating a seed of the plant with an effective amount of the agricultural
inoculum of claim
1; and

culturing the plant.

31. A composition made by combining effective amounts of:
Trichoderma virens isolate G1-3 (ATCC 58678); and
Bacillus amyloliquefaciens TJ1000 (ATCC BAA-390),

wherein the composition is effective at increasing the yield of a plant grown
from a seed to
which the composition has been applied.

32. The composition of claim 31 wherein the composition is effective at
increasing the
manganese content of the plant.

74


Description

Note: Descriptions are shown in the official language in which they were submitted.



CA 02471555 2009-06-11

CONTROLLING PLANT PATHOGENS WITH FUNGAL/BACTERIAL ANTAGONIST
COMBINATIONS COMPRISING TRICHODERMA VIRENS AND BACILLUS
AMYLOLIQUEFACIENS

Thomas D. Johnson
FIELD OF THE INVENTION

This invention relates to fungal/bacterial antagonist combinations, a seed
coated with said
combinations and a plant protected from plant pathogens by said combinations.
In particular, the
invention relates to fungal/bacterial antagonist combinations and their use
for controlling plant
pathogens.

BACKGROUND OF THE INVENTION

Early and late season stalk and root rot are major causes of crop loss. A
variety of plants
are affected, including tomatoes, peppers, turf grass, soybeans, sunflower,
wheat and corn. The
pathogens that cause these symptoms include fungi of the genera Fusarium,
Phythium,
Phytophthora and Penicillium.


One approach to solving the problem of early season damping off of plants is
treatment of
seeds with fungicides, such as captan, metalaxyl and Maxim. Although these
chemicals enhance
seed germination and seedling stand by inhibiting the pathogenic ability of
Phythium spp. (active
la


CA 02471555 2004-06-18

in cool, wet soils), they have no activity against the pathogenic fungi that
are responsible for late
season root and stalk rot.

Fusarium and Penicillium are the pathogens responsible for late season root
and stalk rot.
These pathogens prefer the warm, dry conditions that occur late in the growing
season. There is
no chemical or biological fungicide available that addresses the problem of
late season root and
stalk rot in corn. Currently, the only way to deal with this problem is to
periodically rotate to a
non-susceptible crop to reduce pathogen numbers. Corn growers can also select
hybrids that
have better "standability," but such hybrids usually have lower yields.
Unfortunately, the corn

varieties with the highest yields are usually those most susceptible to late
season root and stalk
rot.

Trichoderma is a genus of fungi that contains about 20 species. Synonyms for
the genus
name include Aleurisma and Sporoderma. Trichoderma vixens, which is also
called Gliocladium
virens, is a member of the genus. The natural habitats of these fungi include
soil and plant

material. A member of the genus, Trichoderma harzianum KRL-AG2 (ATCC 20847)
also
known as strain T-22, is used as a biocontrol agent that is applied as a seed
or soil treatment or
on cuttings and transplants. Strains of the species, Trichoderma virens, have
also been used for
control of damping off diseases in plants. For example, Gliocladium virens GL-
21 is sold under
the trade name SoilGard (formerly GlioGard).

2


CA 02471555 2004-06-18

Bacillus is a genus of rod-shaped, gram-positive, aerobic or (under some
conditions)
anaerobic bacteria. Bacillus species are widely found in soil and water and
some have been used
to control plant diseases, including root rot. Bacillus amyloliquefaciens is a
spore-forming
member of the genus. Bacillus amyloliquefaciens L.L. Campbell strain F (ATCC
23350) is the

type strain for the species. Other known and commercially available Bacillus
amyloliquefaciens
strains include those having the following ATCC accession numbers: 23842,
23843, 23844 and
23845 (Int. J. Sys. Bacteriol. 37:69-71, 1987; J. Bacteriol. 94:1124-1130,
1967).

Bacillus lentimorbus is another spore-forming member of the genus. Bacillus

lentimorbus Dutky 1940 (ATCC 14707) is the type strain for the species
(Skerman, V.B.D.,
McGowan, V., and Sneath, P.H.A., Approved lists of bacterial names. Int. J.
Syst. Bacteriol. 30:
225-420, 1980). Some researchers consider Bacillus lentimorbus to be a variety
of Bacillus
popilliae. Bacillus lentimorbus and Bacillus popilliae have recently been
reclassified as
Paenibacillus lentimorbus and Paenibacillus popilliae (Pettersson, B.,
Rippere, K.E., Yousten,

A.A. and Priest, F.G., Transfer of Bacillus lentimorbus and Bacillus popilliae
to the genus
Paenibacillus with emended descriptions of Paenibacillus lentimorbus comb.
nov. and
Paenibacilluspopilliae comb. nov., Int. J. Syst. Bacteriol. 49: 531-540,1999).
Bacillus
lentimorbus and Bacillus popilliae are the causative agents of milky disease
in Japanese beetles

and related scarab larvae and "milky spore" powders are sold under the trade
names, "Doom,"
"Milky Spore," "Japidemic" "Grub Killer" and "Grub Attack," for biocontrol of
these insects.
Background art biocontrol products have comprised the bacterium Burkholderia

cepacia, which is also known as Pseudomonas cepacia. This bacterium has been
implicated as a
3


CA 02471555 2004-06-18

human pathogen. Furthermore, it has little or no shelf life unless
refrigerated at 4 degrees
Centigrade at a minimum of 20 percent moisture.

The background art is characterized by U.S. Patent Nos. 4,476,881; 4,489,161;
4,642,131;
4,668,512; 4,678,669; 4,713,342; 4,724,147; 4,748,021; 4,818,530; 4,828,600;
4,877,738;
4,915,944; 4,952,229; 5,047,239; 5,049,379; 5,071,462; 5,068,105; 5,084,272;
5,194,258;
5,238,690; 5,260,213; 5,266,316; 5,273,749; 5,300,127; 5,344,647; 5,401,655;
5,422,107;
5,455,028; 5,409,509; 5,552,138; 5,589,381; 5,614,188; 5,628,144; 5,632,987;
5,645,831;
5,665,354; 5,667,779; 5,695,982; 5,702,701; 5,753,222; 5,852,054; 5,869,042;
5,882,641;

5,882,915; 5,906,818; 5,916,029; 5,919,447; 5,922,603; 5,972,689; 5,974,734;
5,994,117;
5,998,196; 6,015,553; 6,017,525; 6,030,610; 6,033,659; 6,060,051; and
6,103,228.

Gravely et al. in U.S. Patent No. 4,476,881 disclose a mixed complementary
culture of
bacteria and fungi that is used to degrade pectin and cellulose components of
tobacco materials.
The invention is limited in that it teaches use of different microorganisms
for a different purpose.

Papavizas in U.S. Patent No. 4,489,161 discloses a strain of the fungus
Trichoderma
viride that is an effective biocontrol agent for Fusarium wilt of
chrysanthemum. The invention is
limited in that it teaches use of a different microorganism.


Hoitinik in U.S. Patent No. 4,642,131 discloses a process for production of a
disease-
suppressive compost and a microorganism culture for use therein. The invention
is limited in
that it teaches use of different microorganisms for a different purpose.

4


CA 02471555 2004-06-18

Lewis et al. in U.S. Patent No. 4,668,512 disclose a method for preparing
pellets
containing living biocontrol fungi and nutrients. The invention is limited in
that it teaches a
process that involves use of different microorganisms.


Ricard in U.S. Patent No. 4,678,669 discloses a method of using immunizing
commensals to control soil-borne pathogens. The invention is limited in that
it teaches use of
different microorganisms.

Chet et al. in U.S. Patent No. 4,713,342 disclose a novel isolate of
Trichoderma and it
use. The invention is limited in that it teaches use of a different
microorganism.

Marois et al in U.S. Patent No. 4,724,147 disclose a method for preparing
pellets
containing living biocontrol fungi. The invention is limited in that it
teaches a process that
involves use of different microorganisms.

Chet et al. in U.S. Patent No. 4,748,021 disclose antifungal compositions
containing
Trichoderma active against Fusarium. The invention is limited in that it
teaches use of a
different microorganism.


Marois et al. in U.S. Patent No. 4,818,530 disclose a method for preparing
pellets
containing living biocontrol fungi. The invention is limited in that it
teaches a process that
involves use of different microorganisms.

5


CA 02471555 2004-06-18

McCabe et al. in U.S. Patent No. 4,828,600 disclose a biological inoculant for
corn. The
invention is limited in that it teaches use of different microorganisms.

Handelsman et al. in U.S. Patent No. 4,877,738 disclose a new microorganism
culture
and a method for biological control of damping off and root rot. The invention
is limited in that
it teaches a process that involves use of a different microorganism, Bacillus
cereus.

Chet et al. in U.S. Patent No. 4,915,944 disclose a novel isolate of
Trichoderma and
fungicidal compositions containing it. The invention is limited in that it
teaches use of a
different microorganism.

Muir in U.S. Patent No. 4,952,229 discloses a soil and foliar supplement. The
invention
is limited in that it teaches use of different microorganisms.


Pusey in U.S. Patent No. 5,047,239 discloses a biological control agent for
fruit rot. The
invention is limited in that it teaches use of a different microorganism for a
different purpose.
Handelsman et al. in U.S. Patent No. 5,049,379 disclose a fungicidal toxin and
a method

and an inoculum for controlling root rot and damping off. The invention is
limited in that it
teaches use of a different microorganism, Bacillus cereus.

6


CA 02471555 2004-06-18

Kimura in U.S. Patent No. 5,071,462 discloses a method and apparatus for
producing an
organic fertilizer. The invention is limited in that it teaches use of
different microorganisms for a
different purpose.

Lewis et al. in U.S. Patent No. 5,068,105 disclose a fungal formulation for
biocontrol of
soil-borne plant pathogens. The invention is limited in that it teaches use of
different
microorganisms.

Speakman et al. in U.S. Patent No. 5,084,272 disclose a Trichoderma fungus and
a
fungicide that contains it. The invention is limited in that it teaches use of
a different
microorganism.

Pauu et al. in U.S. Patent No. 5,194,258 disclose a method for producing
enhanced
biocontrol agents. The invention is limited in that it teaches use of
different microorganisms.

Elad et al. in U.S. Patent No. 5,238,690 disclose a novel Trichoderma culture
and
biological compositions containing it. The invention is limited in that it
teaches use of different
microorganisms for a different purpose.

Harman et al. in U.S. Patent No. 5,260,213 disclose fused biocontrol agents.
The
invention is limited in that it teaches use of different microorganisms.

7


CA 02471555 2004-06-18

Elad et al. in U.S. Patent No. 5,266,316 disclose a novel isolate of
Trichoderma
harzianum and fungicidal compositions containing it. The invention is limited
in that it teaches
use of different microorganisms for a different purpose.

Bok et al. in U.S. Patent No. 5,273,749 disclose a process for preparing
coated microbial
pesticides and the products of the process. The invention is limited in that
it teaches use of
different microorganisms.

Williams in U.S. Patent No. 5,300,127 discloses seed coatings. The invention
is limited
in that it teaches use of different microorganisms.

Rossall in U.S. Patent No. 5,344,647 discloses a Bacillus strain that has
antimicrobial
activity. The invention is limited in that it teaches use of different
microorganisms.

Kijima et al. in U.S. Patent No. 5,401,655 disclose a process for biologically
preventing
plant diseases. The invention is limited in that it teaches use of different
microorganisms for a
different purpose.

Kubota in U.S. Patent No. 5,422,107 discloses a novel fungus and a fungicide
containing
it. The invention is limited in that it teaches use of a different
microorganism.

O'Donnell in U.S. Patent No. 5,455,028 discloses a method of inhibiting fungi.
The
invention is limited in that it teaches use of a different microorganism.

8


CA 02471555 2004-06-18

Burth et al. in U.S. Patent No. 5,409,509 disclose a seed treatment. The
invention is
limited in that it teaches use of different microorganisms.

Shanmuganathan in U.S. Patent No. 5,525,132 discloses "compositions for the

treatment/prevention of microbial diseases of fruit comprising as effective
amount of at least one
yeast strain ..." (Abstract). In Example 2, strains are tested separately and
are not combined.
The disclosed strain D9 was effective and Trichoderma viridie was not. Neither
was Bacillus
subtilis effective. The reference teaches that "it is very unpredictable
whether a particular yeast
species would be effective in the treatment/prevention of microbial disease in
fruit" (col. 2, lines
26-28).

Handelsman et al. in U.S. Patent No. 5,552,138 disclose a novel strain of
Bacillus cerus
and a method of protecting plants with the strain. The invention is limited in
that it teaches use
of a different microorganism.


Neyra et al. in U.S. Patent No. 5,589,381 disclose a novel strain of Bacillus
licheniformis
that produces an antifungal agent and a use for the strain. The invention is
limited in that it
teaches use of a different microorganism.

Urano et al. in U.S. Patent No. 5,614,188 disclose an anti-Fusarium
composition
containing strains of Bacillus sp. The invention is limited in that it teaches
use of different
microorganisms.

9


CA 02471555 2004-06-18

Eastin in U.S. Patent No. 5,628,144 discloses solid matrix priming of seeds.
The
invention is limited in that it teaches use of different microorganisms.

Payne et al. in U.S. Patent No. 5,632,987 disclose a Bacillus thuringiensis
toxin that is

active against corn rootworm larvae. The invention is limited in that it
teaches use of a different
microorganism for a different purpose.

Chilcott et al. in U.S. Patent No. 5,645,831 disclose a Bacillus thuringiensis
strain and
metabolite that are active against corn rootworm. The invention is limited in
that it teaches use
of a different microorganism for a different purpose.

Neyra et al. in U.S. Patent No. 5,665,354 disclose a novel strain of Bacillus
licheniformis
that produces an antifungal agent and a use for the strain. The invention is
limited in that it
teaches use of a different microorganism.


Kubo in U.S. Patent No. 5,667,779 discloses a fungi-inhibiting composition
comprising
Bacillus subtilis. The invention is limited in that it teaches use of a
different microorganism.
Handelsman et al. in U.S. Patent No. 5,695,982 discloses canavanine-resistant
strains of

Bacillus cereus. The invention is limited in that it teaches use of different
microorganisms.
Neyra et al. in U.S. Patent No. 5,697,186 disclose "flocculated bacterial
cells" for use "as
high-density crop inoculants" (Abstract). The reference is limited to the
flocculation of one or



CA 02471555 2004-06-18

more species of bacteria. Combination of bacteria with another type of
microorganism is not
taught by the reference. Nor, does it teach the combination of more than one
bacterium will
produce a greater response, only that the flocculation will allow the
application of more than one
bacterium for whatever reason.


O'Donnell in U.S. Patent No. 5,702,701 discloses a process for treatment of
soil and
plants with a composition containing Bacillus laterosporus. The invention is
limited in that it
teaches use of a different microorganism.

Marrone et al. in U.S. Patent No. 5,753,222 disclose an antibiotic-producing
strain of
Bacillus and methods of controlling plant diseases with it. The invention is
limited in that it
teaches use of a different microorganism.

Handelsman et al. in U.S. Patent No. 5,852,054 disclose fungicidal toxins from
a
biocontrol bacterium. The invention is limited in that it teaches use of a
different
microorganism.

Marrone et al. in U.S. Patent No. 5,869,042 disclose methods for controlling
above-
ground plant diseases. The invention is limited in that it teaches use of
different microorganisms
for a different purpose.

Shetty in U.S. Patent No. 5,882,641 discloses fruit pomice compositions and
their uses.
The invention is limited in that it teaches use of different microorganisms.

11


CA 02471555 2004-06-18

Howell in U.S. Patent No. 5,882,915 discloses viridiol-deficient strains of
Trichoderma
virens and process for making and using biocontrol agents that contain them.
The invention is
limited in that it teaches use of different microorganisms.


Heins et al. in U.S. Patent No. 5,906,818 disclose a Bacillus mycoides strain
for
controlling corn rootworm. The invention is limited in that it teaches use of
a different
microorganism for a different purpose.

Smith et al. in U.S. Patent No. 5,916,029 disclose a process for producing
seeds coated
with a microbial composition. The invention is limited in that it teaches use
of different
microorganisms.

Marrone et al. in U.S. Patent No. 5,919,447 disclose a strain of Bacillus for
controlling
plant disease. The invention is limited in that it teaches use of a different
microorganism.
Herrera-Estrella et al. in U.S. Patent No. 5,922,603 disclose a method for
obtaining

strains of Trichoderma sp. The invention is limited in that it teaches use of
a different
microorganism.


Cook et al. in U.S. Patent No. 5,972,689 disclose methods and compositions for
control
of root diseases. The invention is limited in that it teaches use of different
microorganisms.

12


CA 02471555 2004-06-18

Eastin in U.S. Patent No. 5,974,734 discloses solid-matrix priming of seeds
with
microorganisms. The invention is limited in that it teaches use of different
microorganisms.
Handelsman et al. in U.S. Patent No. 5,998,196 disclose a method for
suppressing disease

using a novel Bacillus cereus strain. The invention is limited in that it
teaches use of a different
microorganism.

Germida et al. in U.S. Patent No. 6,015,553 disclose a Bacillus subtilis
strain for
controlling pests. The invention is limited in that it teaches use of a
different microorganism for
a different purpose.

Logan et al. in U.S. Patent No. 6,017,525 discloses a method of poultry house
litter
treatment. The invention is limited in that it teaches use of a different
microorganism for a
different purpose.


Handelsman et al. in U.S. Patent No. 6,030,610 disclose a novel strain of
Bacillus cereus.
The invention is limited in that it teaches use of a different microorganism.

Handelsman et al. in U.S. Patent No. 6,033,659 disclose a novel strain of
Bacillus cereus.
The invention is limited in that it teaches use of a different microorganism.

13


CA 02471555 2004-06-18

Heins et al. in U.S. Patent No. 6,103,228 discloses compositions and methods
for
controlling plant pests and a novel strain of Bacillus subtilis. The invention
is limited in that it
teaches use of a different microorganism.

Ocamb et al. in U.S. Patent No. 6,133,196 disclose contacting conifer seeds
with a culture
of certain bacteria, drying the seeds, planting the seeds and then treating
the plant growth
medium with a certain ectomycorrhizal fungus (Hebeloma spp.). The reference
does not teach
treating seeds with a composition comprising a fungus and a bacterium.

Branly et al. in U.S. Patent No. 6,232,270 discloses agricultural compositions
containing
bacteria. The invention is limited in that it teaches use of different
microorganisms and a
different process for their use. While the patent suggests that one bacterium
that is useful within
the disclosed invention is Bacillus amyloliquefaciens, it teaches that spores,
cultures or
suspensions of the bacterium must be combined with an agriculturally effective
ingredient of a
plant growth stunting agent or herbicide.

Moesinger in U.S. Patent No. 6,326,016 discloses plant immunization
compositions. The
invention is limited in that it teaches use of different microorganisms and a
different process for
their use. While the patent suggests that one bacterium that can be used
according to the

disclosed invention is Bacillus amyloliquefaciens, and that one fungus that
can be used according
to the invention is Trichoderma spp., it teaches production of an extract by
means of
biotechnological fermentation process performed by only one microorganism.
Furthermore, the

14


CA 02471555 2004-06-18

patent teaches that high-temperature autoclaving (e. g., one hour at +120
degrees C) and
(optionally) filtering are further steps in preparation of the extract.

Background art is also disclosed in the non-patent literature. The StratSoy
Research
Database posted on the Web (apparently in 1996) a description of a research
project entitled
Interaction of Soybean Roots with Soilborne Pathogens and Nonpathogens. The
project studied
the use of Bacillus amyloliquefaciens B94 as a biocontrol agent for plant
diseases. The
invention is limited in that it teaches use of a single different
microorganism.

An article entitled Seed's Coat of Many Microbes Wards Off Rot in Science
Update
section of the April, 1997, issue of Agricultural Research, disclosed the
mixing of helpful fungi
and bacteria to weaken or kill fungal pathogens that attack young corn
sprouts. The applicant
believes that the combination used was T. virens and Pseudomonas cepacia. This
invention is
limited for two reasons: (1) P. cepacia is implicated as causative in deaths
in cystic fibrosis

patients and, therefore, EPA registration would probably not be possible, and
(2) P. cepacia is
not a spore-forming bacterium and, therefore, the moisture content of the
combination must be
maintained at greater than about 17 percent. T. virens can be stored and
applied at low moisture
levels and does not remain in the spore state at the higher moisture levels,
making its

combination with P. cepacia impractical.

In an article authored by L.E. Datnoff et al. entitled Effect of Bacterial and
Fungal
Microorganisms to Colonize Tomato Roots, Improve Transplant Growth and Control
Fusarium
Crown and Root Rot that was posted on the Web in 2000 by the University of
Florida Southwest



CA 02471555 2004-06-18

Florida Research and Education Center, the authors discuss using a combination
of an
unidentified bacterial strain and Gliocladium virens to control Fusarium root
rot. The invention
is limited in that it teaches use of different microorganisms.

The U.S. Environmental Protection Agency Office of Pesticide Programs posted a
Biopesticide Fact Sheet on Bacillus subtilis var. arnyloliquefaciens strain
FZB24 (006480) in
May, 2000. The fact sheet noted that the strain was approved for use as a
growth enhancer and
disease suppressor. The invention is limited in that it does not teach the use
of a fungal/bacterial
combination.


No single reference and no combination of the references teach the invention
disclosed
herein. The background art does not teach combinations of microorganisms
disclosed herein,
combinations that provide a surprising consistency of performance in plant
disease control.

BRIEF SUMMARY OF THE INVENTION

A purpose of the invention is to control the plant pathogens that cause early
and late
season root and stalk rot. Another purpose is to provide for season-long
protection for plants
from the pathogens that cause early and late season root and stalk rot.
Another purpose is to

provide consistent disease control for plants. Yet another purpose is to
increase the yield of
plants and plant seed production.

16


CA 02471555 2004-06-18

One advantage of the invention is that root and stalk rot can be controlled
with a
composition that is not toxic to humans. Another advantage of the invention is
that root and
stalk rot can be controlled more economically than with chemical fungicides.
Yet another
advantage of the invention is that it provides a biocontrol agent or bio-
pesticide with extended

shelf life. Thus, a seed can be treated with the biocontrol agent and stored
for a period of months
and still host a viable biocontrol agent that will colonize the root when the
seed is placed in the
ground, germinates and grows. Furthermore, the disclosed biocontrol agent is
competitive with
natural soil microbes that occur in the rhizosphere while providing pathogen
protection for the
plant. A further advantage of the invention is that the combination of a
fungal/bacterial

antagonist is more effective in controlling fungal pathogens in the plant
rhizosphere than either a
fungal antagonist or a bacterial antagonist alone. Thus, the invention
provides an easy-to-use,
effective means of controlling plant pathogens that have been only been
controllable by rotation
management. A further advantage of the invention is that its use produces more
consistent
results than the use of either a fungal antagonist or a bacterial antagonist
alone, as shown by the

Working Examples presented herein. In fact, use of the antagonist combinations
disclosed herein
is shown to be functional when use of its individual constituent antagonists
is not.

The compositions disclosed herein may be integrated into Integrated Pest
Management
(IPM) programs, the inventive compositions may be used in combination with
other management
systems. As an alternative to synthetic agents, biocontrol agents (bio-
pesticides) offer the

advantage of containing naturally derived constituents that are safe to both
humans and the
environment. Specifically, bio- pesticides offer such advantages as being
inherently less toxic
than conventional pesticides, generally affecting only the target pest and
closely related

17


CA 02471555 2009-06-11

organisms, and are often effective in very small quantities. For these
reasons, bio-pesticides
often decompose quickly and, therefore, are ideal for use as a component of
Integrated Pest
Management (1PM) programs.

The invention is an inoculum, a seed coated with the inoculum, a plant
protected with the
inoculum, a method of producing the inoculum and a method of protecting a seed
or a plant with
the inoculum. A further embodiment of the inoculum comprises a combination of
a fungus and a
bacterium. Preferably, the fungus is a species of Trichoderma and the
bacterium is a species of
Bacillus, preferably a spore-forming strain of Bacillus. More preferably, the
fungus is

Trichoderma virens and the bacterium is Bacillus amyloliquefaciens, although
other
combinations are also envisioned. Even more preferably, the fungus is
Trichoderma virens G1-3
(ATCC 58678) and the bacterium is Bacillus amyloliquefaciens TJ1000 or 1BE
(ATCC BAA-
390).

Further embodiments of the invention comprise combining of a Trichoderma
vixens
fungus and a Bacillus amyloliquefaciens bacterium and placing this combination
on a seed or in
the vicinity of the seed or seedling. A routineer would understand that other
correct
names/designations for the specific isolates/strains involved are: T. virens
isolate G1-3 and B.
subtilis var. amyloliquefaciens strain TJ 1000. A routineer would also
understand that the names

Trichoderma virens and Gliocladium virens are synonymous. To confirm that
Trichoderma
virens G1-3 is a true Gliocladium, the ATCC listing of this organism under
ATCC Accession
No. 58678 confirms its prior classification as a Gliocladium virens. The ATCC
site lists the
claimed isolate (58678) as G1-3.

18


CA 02471555 2009-06-11

In a further embodiment, the inoculum is produced by adding an essentially
pure culture,
a substantially pure culture, an axenic culture or a biologically pure culture
of Trichoderma
virens G1-3 to a bioreactor containing molasses-yeast extract growth medium
using a standard

inoculation technique. The medium is agitated and aerated and its temperature
is maintained at
about 28 degrees Centigrade. After the Trichoderma virens GI-3 is grown in the
medium for
about eight hours, an essentially pure culture, a substantially pure culture,
an axenic culture or a
biologically pure culture of Bacillus amyloliquefaciens TJ1000 or 1BE is added
to the medium
using a standard inoculation technique. The combined, competitive culture is
grown under the

aforementioned conditions and produces maximum cell and spore counts in
approximately seven
days. The combined culture is then used as an inoculum and is applied each
seed at a rate of no
less than about 1,000 spore counts per seed.

In a further embodiment, a solution containing an essentially pure culture, a
substantially
pure culture, an axenic culture or a biologically pure culture of the fungal
antagonist
Trichoderma virens G1-3 is combined with a solution containing an essentially
pure culture, a
substantially pure culture, an axenic culture or a biologically pure culture
of Bacillus
amyloliquefaciens TJ1000 or 1BE in a 50/50 mixture by volume and is applied to
a seed at a rate
of no less than about 10,000 spore counts per seed.


In broad terms, a further embodiment of the invention is an agricultural
inoculum suitable
for inoculating plant seeds comprising a fungal antagonist selected from the
group of
Trichoderma virens G1-3 (ATCC 58678) and mutants thereof, a bacterial
antagonist selected

19


CA 02471555 2009-06-11

from the group of Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) and
mutants
thereof, and a suitable carrier that is non-phytotoxic, non-bacteriostatic,
and non-bactericidal.
Suitable carriers include wettable clay based powders, dextrose granules or
powders, sucrose
granules or powders and maltose-dextrose granules or powders.


A further embodiment of the invention is a composition of matter comprising a
plant seed
inoculated with a combination comprising a fungal antagonist selected from the
group of
Trichoderma virens G1-3 (ATCC 58678) and mutants thereof and a bacterial
antagonist selected
from the group of Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) and
mutants

thereof, wherein said combination suppresses growth of plant pathogenic fungi.

Yet a further embodiment of the invention is a seed or plant inoculated with a
combination comprising a fungal antagonist selected from the group of
Trichoderma virens G1-3
(ATCC 58678) and mutants thereof and a bacterial antagonist selected from the
group of Bacillus
amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) and mutants thereof, wherein
the

combination suppresses growth of plant pathogenic fungi.

In broad terms, a further embodiment of the invention is a method of
protecting a plant
from disease caused by a plant pathogenic fungus comprising inoculating seeds
from said plant
with a combination comprising a fungal antagonist selected from the group of
Trichoderma

virens GI-3 (ATCC 58678) and mutants thereof and a bacterial antagonist
selected from the
group of Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) and mutants
thereof,
wherein said combination suppresses growth of plant pathogenic fungi.



CA 02471555 2009-06-11

A further embodiment of the invention is a method of protecting a seed or a
plant from
disease caused by a plant pathogenic fungus comprising inoculating seeds from
said plant with a
composition comprising a spore-forming fungal antagonist and a spore-forming
bacterial

antagonist. Preferably, the spore-forming bacterial antagonist is selected
from the group Bacillus
amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) and mutants thereof.

A further embodiment of the invention is a method of protecting a seed or a
plant from
disease caused by a plant pathogenic fungus comprising inoculating seeds from
said plant with a
composition comprising a fungal antagonist and a bacterial antagonist, wherein
said combination

suppresses growth of plant pathogenic fungi. A further embodiment is capable
of control of the
plant pathogen fungi Fusarium, Phythium, Phytophthora and Penicillium.

A further embodiment of the invention is a method of protecting a plant from
disease
caused by a plant pathogenic fungus comprising inoculating seeds from said
plant with a
composition comprising a fungal antagonist and a bacterial antagonist selected
from the group of
Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) and mutants thereof,
wherein said
combination suppresses growth of plant pathogenic fungi.

Yet A further embodiment of the invention is a method for biologically
controlling or
inhibiting stalk rot or root rot comprising coating seeds with an effective
amount of a
composition comprising Trichoderma virens G1-3 (ATCC 58678) and Bacillus
amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390).

21


CA 02471555 2009-06-11

A further embodiment of the invention is process for making a composition
comprising
introducing an essentially pure culture of Bacillus amyloliquefaciens TJ1000
or 1BE (ATCC
BAA-390) to a growth medium about eight hours after an essentially pure
culture of

Trichoderma virens G1-3 (ATCC 58678) is introduced to the growth medium and
growing the
culture as a competitive culture.

A further embodiment of the invention is a process comprising making a
composition by
combining an essentially pure culture of Trichoderma virens G1-3 (ATCC 58678)
with an

essentially pure culture of Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-
390) in a
50:50 mixture and applying said composition to a seed at a rate of at least
100,000 spores per
seed.

In one embodiment of the invention disclosed herein, the spore count applied
per seed
ranges from about 1,000 to about 1,000,000, regardless of seed size. In
another embodiment of
the invention, the spore count per seed is from about 1,000 to about 10,000.
In a further
embodiment of the invention, the spore count per seed is from about 10,000 to
about 100,000. In
a yet further embodiment of the invention, the spore count per seed is from
about 100,000 to
about 1,000,000. In a yet another embodiment of the invention, the spore count
per seed is from
about 1,000,000 to about 2,000,000.

A further embodiment of the invention is a method for protecting plants in a
growing
medium from damping off and root rot fungal plant disease comprising placing
in the growing
22


CA 02471555 2009-06-11

medium in the immediate vicinity of the plant to be protected an effective
quantity of one of the
fungal/ bacterial combinations disclosed herein.

Yet a further embodiment of the invention is a method for protecting plants
from fungal
plant disease comprising adding one of the fungal/bacterial combinations
disclosed herein in an
effective quantity to a substrate such as pelletized calcium sulfate or
pelletized lime and placing
the pellet in the immediate vicinity of the plant to be protected. The pellet
may or may not
contain other nutrients.

A further embodiment of the invention is a method for protecting plants from
fungal plant
disease comprising adding one of the fungal/bacterial combinations disclosed
herein in an
effective quantity to a liquid solution such as water and applying the liquid
solution in the
immediate vicinity of the plant to be protected. The liquid may or may not
contain additional
nutrients and may include a chemical fungicide applied to the seed such as,
for example, Maxim

or captan. The disclosed combination may also be added to a plant nutrient
(nitrogen-
phosphorus-potassium (NPK)) plus plant micro-nutrient solution that is
compatible with the
combination and applied as an in-furrow treatment.

A further embodiment of the invention is a method for biologically controlling
a plant
disease caused by a plant-colonizing fungus, the method comprising inoculating
a seed of the
plant with an effective amount of a microbial inoculant comprising a
combination of
microorganisms having all of the identifying characteristics of Trichoderma
virens G1-3 and
Bacillus amyloliquefaciens TJ1000 or 1BE, said inoculation resulting in the
control of said plant

23


CA 02471555 2009-06-11

disease. The invention is also a method according to the above further
embodiment wherein said
inoculation results in the control of more than one plant disease.

Yet a further embodiment of the invention involves combining of a spore
forming fungal
strain and a spore forming bacterial strain to enhance ease of use and
longevity of shelf life both
as a stored product and when applied to a seed. In a further embodiment, the
invention involves
applying the disclosed Trichoderma microorganism and the Bacillus
microorganism to a wettable
powder, in which form it is applied.

A further embodiment of the invention is composition of matter made by
combining: a
fungal antagonist selected from the group of Trichoderma virens G1-3 (ATCC
58678) and
mutants thereof; a bacterial antagonist selected from the group of Bacillus
amyloliquefaciens
TJ1000 or 1BE (ATCC BAA-390) and mutants thereof; and a suitable carrier that
is non-
phytotoxic, non-bacteriostatic, and non-bactericidal.


A further embodiment of the invention is an antagonist for controlling plant
pathogens
made by combining effective amounts of: a fungal antagonist selected from the
group of
Trichoderma virens G1-3 (ATCC 58678) and mutants thereof; a bacterial
antagonist selected
from the group of Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) and
mutants

thereof; and a suitable carrier that is non-phytotoxic, non-bacteriostatic,
and non-bactericidal.
Yet a further embodiment of the invention is a seed assembly made by combining
a plant
seed with effective amounts of a spore-forming bacterial antagonist and a
spore-forming fungal
24


CA 02471555 2009-06-11

antagonist. In a further embodiment, the seed is a seed of a plant selected
from the group of a
monocot, and a dicot. In a further embodiment, the seed is a seed of a plant
selected from the
group of a legume plant, and a non-legume plant. In a further embodiment, the
seed is a seed of
a plant selected from the group of corn, sunflower, soybean, field pea, and
wheat.


A further embodiment of the invention is method for culturing a plant
comprising:
applying an antagonist disclosed herein to a seed or to the seedbed of the
plant; planting the seed
in the seedbed; growing the plant to yield a crop; and harvesting the crop;
wherein said applying
step increases the yield of the crop. In a further embodiment, the antagonist
is applied to the seed
or to the seedbed of a plant selected from the group of a monocot, and a
dicot. In a further

embodiment, the antagonist is applied to the seed or to the seedbed of a plant
selected from the
group of a legume plant, and a non-legume plant. In a further embodiment, the
antagonist is
applied to the seed or to the seedbed of a plant selected from the group of
corn, sunflower,
soybean, field pea, and wheat.


Plant species that may be treated with the disclosed invention include
commercial crops
species, e.g., barley, oat, millet, alfalfa. The disclosed invention may also
be used to treat
legumous plants (e.g., soybeans, alfalfa, and peas and non-legumous plants
(e.g., corn, wheat,
and cotton). The disclosed invention may also be used to treat angiosperms and
cereals.


Yet a further embodiment is a process comprising: making a composition by
combining
an essentially pure culture of Trichoderma virens G1-3 (ATCC 58678) with an
essentially pure
culture of Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) in a
mixture; and



CA 02471555 2009-06-11

applying said composition to a seed; wherein said mixture ranges in
composition from 10 to 90
percent Trichoderma virens G1-3 (ATCC 58678) by volume and from 90 to 10
percent Bacillus
amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) by volume.

Yet a further embodiment of the invention is a process comprising: making a
composition by combining an essentially pure culture of Trichoderma virens G1-
3 (ATCC
58678) with a plurality of essentially pure cultures of bacteria in a mixture;
and applying said
composition to a seed; wherein said mixture ranges in composition from 10 to
90 percent
Trichoderma virens G1-3 (ATCC 58678) by volume.


In one embodiment of the invention the mixture ranges in composition from 10
to 90
percent Trichoderma virens G1-3 (ATCC 58678) by volume and from 90 to 10
percent Bacillus
amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) by volume. In another
embodiment of the
invention, the mixture comprises about 20 percent Trichoderma virens G1-3
(ATCC 58678) by

volume 80 percent Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) by
volume. In
a further embodiment of the invention, the mixture comprises about 30 percent
Trichoderma
virens G1-3 (ATCC 58678) by volume 70 percent Bacillus amyloliquefaciens
TJ1000 or 1BE
(ATCC BAA-390) by volume. In a yet further embodiment of the invention, the
mixture
comprises about 40 percent Trichoderma virens G1-3 (ATCC 58678) by volume 60
percent

Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) by volume.

A further embodiment of the invention is an antagonist for controlling plant
pathogens
made by combining effective amounts of. a fungal antagonist selected from the
group of a strain
26


CA 02471555 2009-06-11

of Trichoderma virens and mutants thereof; a bacterial antagonist selected
from the group of
Bacillus subtilis var. amyloliquefaciens strain TJI000 (ATCC BAA-390) and
mutants thereof;
and a suitable carrier that is non-phytotoxic, non-bacteriostatic, and non-
bactericidal. Preferably,
the strain is Trichoderma vixens G121, which is presently EPA registered.


In a further embodiment, the invention is an antagonist for controlling plant
pathogens
made by combining effective amounts of: a fungal antagonist selected from the
group of
Trichoderma virens G1-3 (ATCC 58678) and mutants thereof; a plurality of
bacterial
antagonists; and a suitable carrier that is non-phytotoxic, non-
bacteriostatic, and non-bactericidal.

Preferably, the plurality of bacterial antagonists comprises a strain of
Erwinia carotovora and/or
a strain of Bacillus lentimorbus.

In a preferred embodiment, the invention is a method comprising: combining a
spore-
forming fungal strain and a spore-forming bacterial strain to produce a
product comprising a
composition of matter disclosed herein; and applying the product to a plant or
to a part of the
plant; whereby application of the product produces yield enhancement in the
plant.

In another preferred embodiment, the invention is a method comprising:
applying a
Trichoderma spp. microorganism and a Bacillus spp. microorganism to a wettable
powder to

produce a combination comprising an antagonist disclosed herein; and applying
the combination
to a seed; whereby application of the combination produces a positive yield
response in a plant
growing from the seed.

27


CA 02471555 2009-06-11

In yet another preferred embodiment, the invention is a process comprising:
making an
agricultural inoculum disclosed herein; and applying said agricultural
inoculum to a seed;
wherein said agricultural inoculum ranges in composition from 1 to 99 percent
Trichoderma
virens G1-3 (ATCC 58678) by culture volume and from 99 to 1 percent Bacillus

amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) by culture volume.

In another preferred embodiment, the invention is a composition of matter
comprising:

a plant seed inoculated with an agricultural inoculum disclosed herein;
wherein said combination
increases the yield of the plant. In another preferred embodiment, the
invention is a method for
increasing the yield of a plant, the method comprising: coating a seed of the
plant with an

effective amount of an agricultural inoculum disclosed herein; and culturing
the plant.

In another preferred embodiment, the invention is a composition made by
combining
effective amounts of: a spore-forming fungal antagonist; and a spore-forming
bacterial

antagonist; wherein the spore-forming fungal antagonist does not produce a
substance that
substantially inhibits the growth of the spore-forming bacterial antagonist
and the spore-forming
bacterial antagonist does not produce a substance that substantially inhibits
the growth of the
spore-forming fungal antagonist; and wherein the composition is effective at
increasing the yield
of a plant grown from a seed to which the composition has been applied.
Preferably, the

composition is effective at increasing the manganese content of the plant

The compositions of the present invention can be used for controlling fungal
infestations
by applying an effective amount of the composition or a formulation thereof,
either at one point
28


CA 02471555 2004-06-18

in time or throughout the plant/crop cycle via multiple applications. The
formulation may be
applied to the locus to be protected for example by spraying, atomizing,
vaporizing, scattering,
dusting, coating, watering, squirting, sprinkling, pouring, fumigating, and
the like. The dosage of
the bioagent(s) applied may be dependant upon factors such as the type of
fungal pest, the carrier

used, the method of application (e.g., seed, plant application or soil
delivery) and climate
conditions for application (e.g., indoors, arid, humid, windy, cold, hot,
controlled), or the type of
formulation (e.g., aerosol, liquid, or solid).

Biocontrol agents comprising the disclosed compositions may be applied in
agricultural,
horticultural and seedling nursery environments. This generally includes
application of agents to
soil, seeds, whole plants, or plant parts (including, but not limited to,
roots, tubers, stems, flowers
and leaves). Bio-pesticide or microbial combinations may be used alone,
however, they may
additionally be formulated into conventional products such as dust, granule,
microgranule, pellet,
wettable powder, flowable powder, emulsion, microcapsule, oil, or aerosol. To
improve or

stabilize the effects of the bio-pesticide, the agent may be blended with
suitable adjuvants and
then used as such or after dilution if necessary.

A worker skilled in the art would recognize that the bioagent(s) may be
formulated for
seed treatment either as a pre-treatment for storage or sowing. The seed may
form part of a

pelleted composition or, alternatively, may be soaked, sprayed, dusted or
fumigated with the
inventive compositions. Additionally, the inventive compositions may be
applied to the soil or
turf, a plant, crop, or a plantation. Some areas may additionally require that
the invention provide
for slow-release materials such that the agent is designed to have an extended
release period.

29


CA 02471555 2004-06-18

In use, the invention disclosed herein may comprise the application of an
aqueous or a
non-aqueous spray composition to the crop. For example, the inventive
composition may be
applied to the soil, or to a plant part (e.g., stalk, root or leaf), or both,
as an aqueous spray

containing spray adjuvants such as surfactants and emulsified agricultural
crop oils which insure
that the agent is deposited as a droplet which wets the stalk or leaf and is
retained on the plant so
that agent can be absorbed.

The skilled artisan would realize that the inventive compositions may be
applied in

combination with nutrients (fertilizers) or herbicides or both, or may form
part of a formulation
comprising the inventive composition in combination with a fertilizer or
herbicide or both. Such
a formulation may be manufactured in the form of a liquid, a coating, a pellet
or in any format
known in the art.

The skilled artisan would realize that the inventive compositions may be
applied to seeds
as part of stratification, desiccation, hormonal treatment, or a mechanical
process to encourage
germination or to terminate dormancy. Treatments including the inventive
agents in combination
with hormones, PEG, or varying temperature, or in combination with mechanical
manipulation of
the seed (i.e. piercing), are contemplated.


Further aspects of the invention will become apparent from consideration of
the drawings
and the ensuing description of further embodiments of the invention. A person
skilled in the art
will realize that other embodiments of the invention are possible and that the
details of the



CA 02471555 2009-06-11

invention can be modified in a number of respects, all without departing from
the inventive
concept. Thus, the following drawings and description are to be regarded as
illustrative in nature
and not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The features of the invention will be better understood by reference to the
accompanying
drawings which illustrate presently further embodiments of the invention. In
the drawings:

Fig. 1 is a plot that compares the incidence of stalk rot in TJ1300-treated
plots versus the
incidence of stalk rot in control plots.

Fig. 2 is a plot that compares final plant populations in TJ1300-treated plots
versus final
plant populations in control plots.

DETAILED DESCRIPTION OF THE INVENTION

A further embodiment of the invention comprises the fungus Trichoderma virens
G1-3
(ATCC 58678). This microorganism may be obtained from the American Type
Culture
Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852-1776.

A further embodiment of the invention also comprises the bacterium Bacillus
lentimorbus
TJ 1000, which is renamed herein Bacillus amyloliquefaciens TJ1000 or 1BE,
based on a more
accurate determination of the name of Bacillus species that occurred before
this patent

31


CA 02471555 2009-06-11

application was filed. This microorganism was deposited with the ATTC on
October 31, 2001,
and was assigned accession number ATCC BAA-390.

A further embodiment of the invention involves combining an essentially pure
culture of
Trichoderma virens G1-3 (ATCC 58678) and an essentially pure culture of
Bacillus
amyloliquefaciens TJ1000 or IBE (ATCC BAA-390) in a competitive culture
process. The
competitive culture process involves adding the Bacillus amyloliquefaciens
TJ1000 or 1BE
(ATCC BAA-390) to a growth medium about eight hours after the Trichoderma
virens G1-3
(ATCC 58678) was added to the medium. The combined culture is then applied to
a seed, for

example, a corn seed. The combination grown in a competitive culture provides
protection for
seeds and plants and is especially effective in a high-stress, high-fungal
pathogen environment
during the early stages of plant development.

A further embodiment of the invention involves growing an essentially pure
culture of
Trichoderma virens G1-3 (ATCC 58678) and an essentially pure culture of
Bacillus
amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) separately for five days. After
the cultures
are grown separately, the compositions that contain them are combined in a
50/50 combination
by volume and then the combination is applied to a seed, for example, a corn
seed. The
combined cultures are applied to a seed provides protection for seeds and
plants from fungal

pathogens. This combination is especially effective under conditions that are
less stressful to the
plant.

32


CA 02471555 2009-06-11

A further step in the process involves applying either of the above
combinations to a seed
involves adding an aqueous solution comprising 30 grams/liter of molasses to
the solution
containing the combination to produce an appropriate spore count in the
resulting composition.
The resulting composition is then applied to the seed as a liquid mist to
achieve optimum

application rates per seed using the molasses as an adhesive to adhere the
spores to the seed.
In a further embodiment, the bioreactor used to culture the microorganism
cultures is a
New Brunswick Bioflow III bioreactor. For optimal results, the agitation
setting of the bioreactor
is set at about 350 rpm, the aeration setting of the bioreactor is set at
about 3.0 with an aeration

air pressure of about 15 pounds per square inch and the temperature setting is
set at about 28
degrees Centigrade. The further growth medium for each of the individual
cultures and the
combined competitive culture comprises about 30 grams per liter of molasses
and about 5 grams
per liter of yeast extract and is referred to as a MYE medium. In A further
embodiment, the
medium contains about 5 milliliters of antifoam. In a further embodiment,
spore production is

measured by counting spores using a hemacytometer manufactured by Hausser
Scientific.

A variety of seed treatments or no seed treatment may be practiced before the
seed is
inoculated with the disclosed inoculum. In some further embodiments, seed
treatments include
osmotic priming and pre-germination of the seed. Because Trichoderma virens G1-
3 and

Bacillus amyloliquefaciens TJ1000 or 1BE are spore formers, the disclosed
inoculum does not
require high moisture levels for survival and, therefore, can be applied to
seed and other
materials without a sticker, such as those sold under the trade names Pelgel
(LipaTech), Keltrol'm
(Xanthan) Cellprill or Bond.

33


CA 02471555 2009-06-11

In a further embodiment, the invention involves combining of a spore forming
fungal
strain and a spore forming bacterial strain to enhance ease of use and
longevity of shelf life both
as a stored product and when applied to a seed. In A further embodiment, the
invention involves
applying the disclosed Trichoderma microorganism and the disclosed Bacillus
microorganism to
a wettable powder, and marketing the wettable powder.

First Greenhouse Working Example

Greenhouse testing was conducted to determine the effectiveness of the
disclosed

biocontrol agents. Treated and untreated corn seeds were grown in soil
infested with seven
percent Fusarium infested wheat seed. In this testing, the following treatment
codes were used:
CONTROL - Nothing on the seed

TJ 1000-- Bacillus amyloliquefaciens TJ1000 or 1BE
TJ 0300--Trichoderma virens G1-3

TJ 1300-50/50 combination of Trichoderma virens G1-3 and Bacillus
amyloliquefaciens
TJ 1000 or I BE

TJ 1310--competitive culture of Trichoderma virens G1-3 and Bacillus
amyloliquefaciens
TJ1000 or 1BE, resulting in a 70/30 ratio of Trichoderma to Bacillus


34


CA 02471555 2009-06-11

The results of greenhouse testing are presented in Table 0. The rating scale
used was 9 =
worst plant protection and 1 = best plant protection. Seed treated with
biocontrol organisms
grown in competitive culture showed an increase in plant protection over seed
treatments with
the same biological control organisms grown in non-competitive culture. The
biocontrol agents
were applied to the seed without a sticker.

Table 0. Greenhouse Testing Results

Treatment Replication 1 Replication 2 Replication 3 Average
Control 9 7 6 7.3
TJ 0300 6 5 5 5.3

TJ 1000 7 6 5 6
TJ 1300 6 5 6 5.6
TJ 1310 1 3 3 2.3
Field Trials Working Example

In a subsequent experiment, field trials were conducted at seven locations
throughout the
U.S. Site locations included Arizona, Colorado, Kansas, Montana, North Dakota
and two South
Dakota locations. At each location, the trial contained a CONTROL that was
treated with the
industry-standard chemical treatment, MAXIM. All cultures used in the trial
were grown in
MYE broth for five days. Bacillus amyloliquefaciens TJ1000 or 1BE was cultured
individually

(non-competitive) and with Trichoderma virens GI-3 (competitive culture).
Trichoderma virens
G1-3 and Bacillus amyloliquefaciens TJ1000 or 1BE were also grown in non-
competitive culture
were also applied to the same seed to test the effectiveness of non-
competitive culture versus



CA 02471555 2004-06-18

competitive culture. Corn seeds were treated to give a final concentration of
1,000,000,000
bacterial/fungal spores per acre. Seed treatment was done with a Gustafson
benchtop seed
treater, Model BLT.

The plot location in Kansas was severely damaged by early dry conditions and
the plot
was terminated prior to harvest. The Colorado location was damaged due to
machine damage
prior to harvest. Colorado yield data were collected but were extremely
variable and were not
included in the analyzed data set. The Colorado stalk rot data were included
in the data set.

The value of the Stalk Rot variable was determined by counting ten plants in a
row,
determining the number of root rot/stalk rot infected plants and expressing
that number as a
percentage. As illustrated in Fig. 1, in six trials, the average infection
rate in the control was
55.13 percent versus 38.62 percent in the entries treated with the
fungal/bacterial combination,
TJ1300. The data revealed an average reduction of disease incidence of 30
percent with the

Colorado location showing a reduction of over 60 percent.

The value of the Final Population variable was determined by a conducting a
physical
count of the plants in a measured area and converting to a per acre count. As
illustrated in Fig. 2,
the average increase in final plant population was 3,742 plants per acre or an
increase of 12.2

percent. This increased population was the result of controlling the disease
early and having less
plant death throughout the season.

36


CA 02471555 2009-06-11

Use of TJ1300 resulted in an average yield benefit of 5.35 bushels per acre.
Average yield
was determined from eight trials: 4 in South Dakota, 1 in North Dakota, 2 in
Arizona, and 1 in
Montana.

Second Greenhouse Working Example

Greenhouse Methods: All test cultures were grown in MYE (three percent
Molasses, 0.5
percent Yeast Extract) broth for five days. Bacteria were grown up
individually (non-
competitive) and with T virens G1-3 (competitive culture). T. virens GI-3 was
also grown in a

non-competitive culture for testing. T. virens G1-3 and test bacteria grown in
non-competitive
culture were also applied to the same seed to test the effectiveness of non-
competitive culture
versus competitive culture. Corn seeds were treated to give a final
concentration of 1 x 109
bacteria/fungal spores (may also be referred to a Colony Forming Units or CFU)
per acre. Seed
treatment was done with a Gustafson Benchtop Seed Treater, Model BLT. Seeds
were grown in

soil infested with seven percent Fusarium-infested wheat seed. After four
weeks, plant heights
were taken as well as plant biomass. Plant heights were taken by measuring
from the soil line to
the tallest leaf, biomass of the plants was taken by cutting the plants at the
soil line and then
weighing plants on analytical scale. The treatment matrix was as follows:

Control - No pathogen added to soil.
Control - With pathogen added to soil.

TJ 1000 - Bacillus amyloliquefaciens TJ 1000 or 1 BE
TJ0300 - Trichoderma vixens G1-3

TJ2000 - Erwinia carotovora

37


CA 02471555 2009-06-11

TJ1300-B. amyloliquefaciens TJ1000 or 1BE and T. vixens G1-3 (non-competitive)
TJ2300 - E. carotovora and T. virens G1-3 (non-competitive)

TJ1310 - B. amyloliquefaciens TJ1000 or 1BE and T. virens G1-3 (competitive)
TJ1-2310 - B. amyloliquefaciens TJ1000 or 1BE , E. carotovora and T virens G1-
3
(competitive)

TJ23 10 - E. carotovora and T virens G1-3 (competitive)

Determination of CFU (Colony Forming Units) concentrations in competitive
cultures:
Competitive cultures grown for five days. CFU counts of each organism were
performed using a
hemacytometer (Hausser Scientific) under light microscopy 5000x magnification.
This method
was used to determine the CFU counts in the greenhouse and field trials.

Enumeration through plate counts: Competitive cultures were grown for five
days in
submerged culture then 200 milliliters (ml) of the culture was harvested and
aliquoted into four
50 ml centrifuge tubes. After centrifugation at 10,000 revolutions per minute
(rpm) for 10

minutes resulting pellets were washed twice in equal volumes of D2H20. Pellets
were then re-
suspended in 25 ml of saline. One ml samples were diluted 10-1 to 10-8 and
plated onto potato
dextrose agar (PDA) plates. Colonies are then counted and correlated with the
dilution rates to
determine CFU per ml of culture broth.


Results: All of the biocontrol agents in this experiment produced significant
plant
biomass increases over the pathogen-treated control and all of the treatments
were numerically
greater than the control plants in soil that contained no pathogen. The
effects of bacterial/fungal
38


CA 02471555 2004-06-18

combination TJ 1310 and the bacterial treatment TJ 1000 were significantly
greater than both
controls in the experiment.

Table 1. Demonstration of the Effectiveness of Biological Combinations and
Individual
Bacteria and Individual Fungal Treatments on Increasing the Biomass of

Greenhouse-Grown Corn Seedlings in Pathogen-Treated Soil vs. the Untreated
Control

Treatment Ratio Rank Biomass (grams)
Control Path 0/0 10 3.62 a
Control No Path 0/0 9 7.25 ab
TJ 1300 50/50 8 8.67 b
TJ 2310 30/70 7 9.04 b
TJ 2000 100/0 6 10.73 b
TJ 1-2310 20/20/60 5 11.37 b
TJ 2300 50/50 4 11.41 b
TJ 0300 0/100 3 11.53 b
TJ 1310 30/70 2 12.24 be
TJ 1000 100/0 1 12.89 be
CV % 33.9
LSD (0.05) 4.55
Combinations Field Trial Working Example

Materials and Methods: A field trial was conducted using the corn variety NK
3030Bt
using the following biological treatments of the seed at a rate of
approximately 106 CFU per
seed. The seed was planted at a seeding rate of 25,000 seeds per acre in 30-
inch rows in a
randomized, replicated block. Each entry was replicated four times. The
pathogen levels were
natural populations at a location near Groton, SD. The entries were as
follows:

Control: Maxim Seed treatment (Maxim is a trademark of Syngenta Crop
Protection)
TJ 1000 - Bacillus amyloliquefaciens TJ1000 or 1BE

39


CA 02471555 2009-06-11
TJ 0300 - Trichoderma virens GI-3

TJ 1300 - 50/50 combination of B. amyloliquefaciens TJ1000 or 1BE and T.
virens GI-3
TJ 1310 - Coculture 30/70 combination of B. amyloliquefaciens TJ1000 or 1BE
and T.
virens G1-3

TJ 66/300 - 50/50 combination of Bacillus amyloliquefaciens TJ1000 or 1BE and
T.
virens G1-3

Results: The trial produced significant yield response over the control with
the entries TJ
0300, TJ 1300, and TJ 1310. The combinations TJ 1300 and TJ 1310 produced a
yield response
numerically greater than that of TJ 0300. The effects of bacterial/fungal
combination TJ 66/300
and the bacterial treatment TJ 1000 were numerically greater than the control
but not

significantly greater. The results are presented in Table 2.

Conclusion: The bacterial/fungal combinations of entries TJ 1300 and TJ 1310
are the
most effective biocontrol treatments in the trial for increasing the yield of
corn.

Table 2. Effect of Biological Seed Treatment on Yield of Corn Variety N3030 Bt
under
Field Conditions.

Treatment Ratio Rank Location Trial Yield
Control Maxim 0/0 6 Groton,SD Seed Treat 164.8 a
TJ 1000 100/0 4 Groton,SD Seed Treat 175.1 ab
TJ 0300 0/100 3 Groton,SD Seed Treat 179.5 be
TJ 1300 50/50 2 Groton,SD Seed Treat 183.3 be
TJ 1310 30/70 1 Groton,SD Seed Treat 189.8 c
TJ 66/300 50/50 5 Groton,SD Seed Treat 173.2 ab
CV% 13.54
LSD 0.05 12.5



CA 02471555 2009-06-11

50/50 Combination Field Trial Working Example

Materials and Methods: A field trial was conducted using the corn variety NK
3030Bt
using the following biological treatments of the seed at a rate of
approximately 106 CFU per

seed. The seed was planted at a seeding rate of 25,000 seeds per acre in 30-
inch rows in a
randomized replicated block. Each entry was replicated four times. The
pathogen levels were
natural populations at a location near Groton, SD. The entries were as
follows:

Control: Maxim Seed treatment (Maxim is a trademark of Syngenta Crop
Protection)

TJ 1300 - 50/50 combination of B. amyloliquefaciens TJ1000 or 1BE and T.
virens G1-3

Results: As indicated in Table 3, the trial produced a significant response in
the yield of
the seed treated with the biocontrol agent TJ 1300 (described above) as
compared with the
untreated control.

Table 3. Effect of Biological Seed Treatment on Yield of Corn Variety NK
3030Bt under
Field Conditions.

Treatment Ratio Rep Location Yield
Control 0/0 1 Groton, SD 156.8
Control 0/0 2 Groton, SD 163.3
Control 0/0 3 Groton, SD 151.0
Average 0/0 Groton, SD 157.03 a
1300 50/50 1 Groton, SD 184.3
1300 50/50 2 Groton, SD 179.1
1300 50/50 3 Groton, SD 177.3
Average 50/50 Groton, SD 180.21 b
CV% 5.65
LSD (0.05%) 9.04

41


CA 02471555 2009-06-11

Application Rate Field Trial Working Example

Materials and Methods: A field trial was conducted using the corn variety
NK2555 using
the TJ 1300 (50/50 combination of B. amyloliquefaciens TJ1000 or 1BE and T
virens G1-3)

biological treatments of the seed at variable rates. The purpose of the trial
was to identify the
most effective application rate for the bacterial/fungal combination of TJ
1300. The lx rate was
approximately 1X106 CFU per seed. The seed was planted at a seeding rate of
25,000 seeds per
acre in 30-inch rows in a randomized, replicated block. Each entry was
replicated four times.
The pathogen levels were natural populations at a location near Groton, SD.
The entries were as
follows:

Control - Maxim (Maxim is a trademark of Syngenta Crop Protection)
0.5x rate

1 x rate
1.5x rate
2x rate

Results: All of the biocontrol treatments in this experiment resulted in
significant yield
response over the control with the 1.5x rate producing significantly better
results than the 2x rate.
The results of this trial, presented in Table 4, indicated that the most
efficacious application rate
of the biocontrol agent TJ 1300 was approximately 1.5X106 per seed.

42


CA 02471555 2004-06-18

Table 4. Effect of TJ1300 Biological Seed Treatment on Yield of Corn Variety
N2555 at
Variable Rates

Treatment Ratio Rank Location Trial Yield
Control 0/0 5 Groton,SD Rate 140.2 a
0.5x rate 50/50 3 Groton,SD Rate 153.6 be
1 x rate 50/50 2 G roton,SD Rate 156.2 be
1.5x rate 50/50 1 Groton,SD Rate 161.1 c
2x rate 50/50 4 Groton,SD Rate 152.07 b
CV% 5.31
LSD (0.05%) 8.61

Liquid Biocontrol Preparations Working Example

Materials and Methods: Field trials were conducted using the corn varieties NK
3030 and
NK 3030Bt at a location in Brookings, SD and NK 303OBt and NK2555 at a
location in Groton,
SD. The purpose of the trial was to compare pathogen control of liquid
biocontrol preparations
to a control treated with only water. The results of the trial were quantified
in yield of corn in
bushels per acre. The water was applied to the control at a 10 gallon per acre
rate. Biocontrol
treatments were prepared by adding 1X108 CFU per gram of a wettable powder
(Mycotech, Inc.).

Two and one half grams of the wettable powder was added per one gallon of
water and soil
applied in the seed furrow at a rate of 10 gallons per acre. The seed was
Maxim (Maxim is a
trademark of Syngenta Crop Protection) treated and was planted at a seeding
rate of 25,000 seeds
per acre in 30-inch rows in a randomized, replicated block. Each entry was
replicated four times.
The pathogen levels were natural populations at each location. The entries
were as follows:

Control -- Water

TJ 1000 -Bacillus amyloliquefaciens TJ 1000 or 1BE
43


CA 02471555 2009-06-11
TJ 0300 - Trichoderma virens G1-3

TJ 1300 - 50/50 combination of B. amyloliquefaciens TJ1000 or 1BE and T.
virens G1-3
TJ 1310 - Coculture 30/70 combination of B. amyloliquefaciens TJ1000 or 1BE
and T.
vixens G1-3

TJ 66/300 - 50/50 combination of Bacillus lentimorbus and T. virens G1-3

Results: Table 5 shows a significant yield increase to the biocontrol
treatments of TJ
1000, TJ1300, and TJ 66/300. All of the biocontrol treatments showed a
numerical yield
increase.

Table 6 shows a significant yield increase to the biocontrol treatments of
TJ1000,
TJ0300, and TJ1300. Again, all of the biocontrol treatments showed a numerical
yield increase.
Table 7 shows no significance in the yield between the treatments and the
control,

however, the yield of TJ0300 was numerically less than the control by over 10
bushels per acre
and is significantly less than the yields of the TJ1000 and TJ 1310
bacterial/fungal combination.
This table demonstrates the strength of the disclosed bacterial/fungal
combinations over the
fungal control alone.

Table 8 shows the treatments of TJ 1000 and TJ 66/300 with significantly less
yield than
the control while the treatments of TJ0300, TJ1300, and TJ1310 having no
significant difference.
In this trial, it was the bacterial entry of TJ1000 alone that shows weakness
in pathogen control.
This table demonstrates the strength of disclosed bacterial/fungal
combinations over the bacterial
treatment alone.

44


CA 02471555 2004-06-18

Conclusion: The bacterial/fungal combination of entries TJ 1300 and TJ 1310
produce
consistent pathogen control and/or yield response, while the bacteria entry of
TJ 1000 alone and
fungal entry of TJ 0300 alone produce inconsistent pathogen control and/or
yield response.


Table 5. Liquid Drench Treatment on Corn Variety NK3030 at Brookings, SD
Location
Treatment Variety Ratio Rank Location Trial Yield
Control NK3030 0/0 6 Brookins, SD Liquid 162.2 a
TJI000 NK3030 100/0 1 Brookings, SD Liquid 179.7 b
TJ0300 NK3030 0/100 5 Brookings, SD Liquid 170.7ab
TJ1300 NK3030 50/50 2 Brookins, SD Liquid 177.9 b
TJ1310 NK3030 30/70 4 Brookings, SD Liquid 172.8ab
TJ66/300 NK3030 50/50 3 Brookin s, SD Liquid 175.0 b
CV% 7.38
LSD 0..20% 12.36

15

Table 6. Liquid Drench Treatment on Corn Variety NK2555 at Groton, SD Location
Treatment Variety Ratio Rank Location Trial Yield
Control NK2555 0/0 6 Groton, SD Liquid 136.2 a
TJ1000 NK2555 100/0 1 Groton, SD Liquid 147.7 c
TJ0300 NK2555 0/100 2 Groton, SD Liquid 145.Obc
TJ1300 NK2555 50/50 3 Groton, SD Liquid 142.5bc
TJ1310 NK2555 30/70 4 Groton SD Liquid 141.5abc
TJ66/300 NK2555 50/50 5 Groton, SD Liquid 138.5abc
CV% 10.92
LSD (0.20%) 8.42


Table 7. Liquid Drench Treatment on Corn Variety NK 3030Bt at Brookings, SD
Location


CA 02471555 2004-06-18

Treatment Variety Ratio Rank Location Trial Yield
Control NK303OBt 0/0 4 Brookings, SD Liquid 181.5 ab
TJI000 NK303OBt 100/0 2 Brookings, SD Liquid 185.5 b
TJ0300 NK303OBt 0/100 6 Brookin s, SD Liquid 171.3 a
TJ1300 NK303OBt 50/50 5 Brookings, SD Liquid 180.7ab
TJ1310 NK303OBt 30/70 1 Brookings, SD Liquid 185.8 b
TJ66/300 NK303OBt 50/50 3 Brookins, SD Liquid 181.6 ab
CV% 6.32
LSD (0.20%) 11.40

Table 8. Liquid Drench Treatment on Corn Variety 303OBt at Groton, SD Location
Treatment Varie Ratio Rank Location Trial Yield
Control NK303OBt 0/0 2 Groton, SD Liquid 173.9 c
TJ1000 NK303OBt 100/0 6 Groton, SD Liquid 164.1 a
TJ0300 NK3030Bt 0/100 4 Groton, SD Liquid 171.3abc
TJ1300 NK3030Bt 50/50 3 Groton, SD Liquid 171.5abc
TJ1310 NK303OBt 30/70 1 Groton, SD Liquid 176.3 c
TJ66/300 NK303OBt 50/50 5 Groton, SD Liquid 164.4 ab
CV% 10.92
LSD 0.20%. 8.42


Compatibility with Dry Granule Micro-Nutrient Fertilizer Working Example

Materials and Methods: A field trial was conducted using the corn variety NK
3030Bt at a
location in Groton, SD. The purpose of the trial was to compare the
compatibility and yield
benefit of the biocontrol preparation TJ1300 in combination with a dry granule
micro-nutrient
fertilizer vs. the micro-nutrient fertilizer alone vs. a control with no micro-
nutrient fertilizer. The
micro-nutrient fertilizer is sold commercially by the applicant under the
trademark TJ

Micromixtm. Biocontrol treatments were prepared by adding 1X106 CFU per seed.
The control
46


CA 02471555 2009-06-11

seed was Maxim (Maxim is a trademark of Syngenta Crop Protection) treated with
the biocontrol
treatments applied in addition to the Maxim. The seed was planted at a seeding
rate of 25,000
seeds per acre in 30-inch rows in a randomized, replicated block. TJ
Micromixt" was applied at a
rate of 20 pounds per acre. Each entry was replicated four times. The pathogen
levels were

natural populations at each location. The entries were as follows:
Control: Maxim

TJ Micromix

TJ Micromix + TJ 1300 - 50/50 combination of B. amyloliquefaciens TJ1000 or
1BE and
T. vixens G1-3


Results: In this trial, as shown in Table 9, the Granular TJ Micromix produced
a non-
significant yield increase compared to the control. When the seed-applied
biocontrol treatment
TJ1300 was applied in combination with the TJ Micromix, the treatment resulted
in a significant
increase in yield.

Conclusion: The trial shows that TJ 1300 is compatible with micro-nutrient
applications
and the combination produces a significant yield response.

Table 9. Effect of TJ Micromix and TJ Micromix + TJ 1300 on Corn Variety NK
3030Bt
Treatment Variety Rank Location Trial Yield
Control NK3030Bt 3 Groton, SD Fertilizer 157.0 a
TJ Micromix NK3030Bt 2 Groton, SD Fertilizer 163.3 ab
TJ Micromix
+ TJ 1300 NK303OBt 1 Groton, SD Fertilizer 175.5 b
CV% 9.04
LSD (0.05%) 5.64


47


CA 02471555 2009-06-11

Compatibility with Liquid Chelate Micro-Nutrient Fertilizer Working Example
Materials and Methods: A field trial was conducted using the corn variety NK
3030Bt at a
location in Groton, SD. The purpose of the trial was to compare the
compatibility and yield
benefit of the biocontrol preparation TJ1300 in combination with a liquid
chelate micro-nutrient
fertilizer vs. the liquid chelate micro-nutrient fertilizer alone. The liquid
chelate micro-nutrient

fertilizer is sold commercially under the Trademark TJ Micromixtm - Cornmix.
Biocontrol
treatments were prepared by adding 1X106 CFU per seed. The control seed was
Maxim (Maxim
is a trademark of Syngenta Crop Protection) treated with the biocontrol
treatments applied in
addition to the Maxim. The seed was planted at a seeding rate of 25,000 seeds
per acre in 30-inch
rows in a randomized, replicated block. TJ Micromixt' - Cornmix was applied at
a rate of 1.5

quarts per acre. Each entry was replicated four times. The pathogen levels
were natural
populations at the location. The entries were as follows:

Control: Maxim + Liquid Chelate TJ Micromix

TJ Micro + TJ1000: Liquid Chelate TJ Micromix plus TJ 1000 - B.
amyloliquefaciens
TJ1000 or 1BE

TJ Micro + TJ0300: Liquid Chelate TJ Micromix plus TJ 0300 - T. virens G1-3

TJ Micro + TJ1300: Liquid Chelate TJ Micromix + TJ 1300 - 50/50 combination of
B.
amyloliquefaciens TJ1000 or 1BE and T. virens G1-3

TJ Micro + TJ1310: Liquid Chelate TJ Micromix + TJ 1310 - Coculture 30/70
combination of B. amyloliquefaciens TJ1000 or 1BE and T virens G1-3

48


CA 02471555 2009-06-11

TJ Micro + TJ66/300: Liquid Chelate TJ Micromix + TJ 66/300 - 50/50
combination of
Bacillus lentimorbus and T virens GI-3

Results: As shown in Table 10, the biocontrol treatments TJ1000, 66/300, and
1300

combined with the liquid chelate TJ Micromix resulted in a significant
increase in yield over the
control of TJ Micromix alone. The other biocontrol entries showed numerical
but non-
significant increases in yield. The conclusion was that the biocontrol agents
used in this study
are compatible with liquid chelate micro-nutrient applications. This
biocontrol/liquid chelate
micro-nutrient fertilizer combination is a viable means to significantly
increase the yield of corn.

Table 10. Effect of TJ Micromix Liquid Chelate and TJ Micromix Liquid Chelate
+ TJ 1300
on Yield of Corn Variety NK303OBt

Treatment Variet Ratio Rank Location Trial Yield
Liquid TJ
Control NK3030Bt 0/0 6 Groton, SD Micromix 161.0 a
TJ Micro + Liquid TJ
TJ 1000 NK303OBt 100/0 3 Groton, SD Micromix 173.0 be
TJ Micro + Liquid TJ
TJ 0300 NK3030Bt 0/100 5 Groton, SD Micromix 163.0 ab
TJ Micro + Liquid TJ
TJ1300 NK3030Bt 50/50 1 Groton, SD Micromix 183.7 c
TJ Micro + Liquid TJ
TJ 1310 NK3030Bt 30/70 4 Groton, SD Micromix 172.0 ab
TJ Micro + Liquid TJ
TJ 66/300 NK3030Bt 50/50 2 Groton, SD Micromix 173.2 be
CV% 11.2
LSD (0.05%) 12.36


Sunflower Dry Granule Micro-Nutrient Fertilizer Working Example
49


CA 02471555 2009-06-11

Materials and Methods: A field trial was conducted using the sunflower variety
Pioneer
63M80 NuSun at a location in Hazelton, ND. The purpose of the trial was to
compare the
compatibility and yield benefit of the biocontrol preparation TJ1300 in
combination with a dry
granule micro-nutrient fertilizer vs. the micro-nutrient fertilizer alone vs.
a control with no

micro-nutrient fertilizer. Analyzing yield of sunflower is a function of seed
yield in pounds per
acre and the amount of oil in the seed which is expressed as a percentage. The
micro-nutrient
fertilizer is sold commercially under the Trademark TJ Micromixt. Biocontrol
treatments were
prepared by adding lX106 CFU per seed. The control seed was Maxim (Maxim is a
trademark
of Syngenta Crop Protection) treated with the biocontrol treatments applied in
addition to the

Maxim. The seed was planted at a seeding rate of 22,000 seeds per acre in 30-
inch rows in a
randomized, replicated block. TJ Micromixtm was applied at a rate of 20 pounds
per acre. Each
entry was replicated four times. The pathogen levels were natural populations
at the location.
The entries were as follows:

Control: Maxim
TJ Micromix

TJ 1300 - 50/50 combination of B. amyloliquefaciens TJ1000 or 1BE and T.
virens G1-3
TJ Micromix + TJ 1300 - 50/50 combination of B. amyloliquefaciens TJ1000 or
1BE and
T. virens Gl-3

Results: As shown in Table 11, in this trial, the Granular TJ Micromix
produced a
significant yield increase and a significant oil percentage increase compared
to the control.
When the seed-applied biocontrol treatment TJ1300 was applied in combination
with the TJ
Micromix, the treatment resulted in a significant increase in yield as
compared to the control but
not significantly different from the TJ Micromix application alone. The yield
of the TJ 1300 +



CA 02471555 2004-06-18

TJ Micromix was numerically higher in yield. The conclusion was that TJ 1300
is compatible
with micro-nutrient applications and may be a viable tool to increase the
yield of sunflower.

Table 11. Effect of TJ1300 Liquid Biological Treatment Plus Dry Granular TJ
Micromix on
Yield of Nu-sun Sunflower Variety 63M80

Treatment Rank Location Trial Yield Oil
Control Hazelton, ND TJ Micro 1709.7 a 44.8 a
TJ Micromix Hazelton, ND TJ Micro 1857.3 be 47.2 b
TJ 1300 Hazelton, ND TJ Micro 1734.7ab 45.5 a
TJ 1300 + TJ
Micromix Hazelton, ND MM 1864.7 be 44.9 a
CV h 7.48 4.67
LSD (0.20) 132.8 1.5

Sunflower Liquid Chelate Micro-Nutrient Working Example

Materials and Methods: Field trial was conducted using the sunflower variety
Pioneer
63M80 NuSun at 3 locations: Hazelton, ND; Kensal, ND; and Selby, SD. The
purpose of each
trial was to compare the compatibility and yield benefit of the biocontrol
preparation TJ1300 in
combination with a liquid chelate micro-nutrient fertilizer vs. an untreated
control. Analyzing

yield of sunflower is a function of seed yield in pounds per acre and the
amount of oil in the seed
which is expressed as a percentage. The liquid chelate micro-nutrient
fertilizer is sold
commercially under the Trademark TJ Micromix". Biocontrol treatments were
prepared by
adding 1X108 CFU per gram to a wettable powder (Mycotech, Inc). 25 grams of
the wettable
powder was then added to 1.5 quarts of liquid chelate TJ Micromix and the
combination applied

51


CA 02471555 2009-06-11

in the seed furrow at a rate of 1.5 quarts per acre. The control seed was
Maxim (Maxim is a
trademark of Syngenta Crop Protection) treated with the biocontrol treatments
applied in addition
to the Maxim. The seed was planted at a seeding rate of 22,000 seeds per acre
in 30- inch rows in
a randomized, replicated block. Each entry was replicated four times. The
pathogen levels were
natural populations at each location. The entries were as follows:

Control - no treatment

TJ 1300 - 50/50 combination of B. amyloliquefaciens GL-3 and T vixens G1-3
TJ1300 + TJ Micromix - Liquid chelate TJ Micromix + 50/50 combination of B.
amyloliquefaciens and T. virens


Result: As shown in Table 12, TJ Micromix liquid and the combination of TJ
Micromix
plus TJ 1300 both gave sunflower a significant increase in yield. TJ 1300 + TJ
Micromix
produced an additional numerical increase in yield over the TJ Micromix alone.

Conclusion: TJ 1300 + TJ Micromix is a viable means of biocontrol delivery on
sunflower and is a viable means of increasing the seed yield of sunflower.

Table 12. Effect of TJ1300 Biological Liquid Plus Liquid TJ Micromix
Fertilizer on Yield
of Nu-sun Sunflower Variety 63M80

Treatment Ratio Location Trial Yield Oil
Liquid TJ
Control 0/0 Hazelton, ND Micro 1709.7 44.8
Liquid TJ
TJ 1300 50/50 Hazelton, ND Micro 1765.0 45.5
TJ1300+TJ Liquid TJ
Micromix 50/50 Hazelton, ND Micro 1992.3 45.9
Liquid TJ
Control 0/0 Kensal, ND Micro 2000.3 N/a
52


CA 02471555 2004-06-18

Liquid TJ
TJ1300 50/50 Kensal, ND Micro 2159.0 N/a
TJ1300+TJ Liquid TJ
Micromix 50/50 Kensal, ND Micro 2329.0 N/a
Liquid TJ
Control 0/0 Selby, SD Micro 2225.0 43.2
Liquid TJ
TJ 1300 50/50 Selby, SD Micro 2324.0 44
TJ1300 + TJ Liquid TJ
Micromix 50/50 Selby, SD Micro 2228.5 44
Control
Average 1978.3 a 44 a
TJ 1300 2082.8 b 44.75 a
TJ 1300 + TJ
Micromix 2173.3 b 45.5 a
CV=A 10.58 4.67
LSD (0.05) 104.1 NS

Soybean Liquid Chelate Micro Nutrient Fertilizer Working Example

Materials and Methods: A field trial was conducted using the soybean variety
Pioneer
91B52 a location near Groton, SD. The purpose of the trial was to compare the
compatibility and
yield benefit of the biocontrol preparation TJ1300 in combination with a
liquid chelate micro-
nutrient fertilizer vs. the liquid chelate alone vs. an untreated control.
Yield in bushels per acre
was used as the measure of the treatment response. The liquid chelate micro-
nutrient fertilizer is

sold commercially under the Trademark TJ Micromix". Biocontrol treatments were
prepared by
adding 1X108 CFU per gram to a wettable powder (Mycotech, Inc). Twenty-five
grams of the
wettable powder was then added to 10 gallons of water and applied in the seed
furrow at a rate of
10 gallons per acre to establish treatment TJ1300. Twenty-five grams of the
wettable powder
was added to 1.5 quarts of liquid chelate TJ Micromix and the combination
added to water to

form a 10 gallon solution and applied in the seed furrow at a rate of 10
gallons per acre. The
53


CA 02471555 2009-06-11

seed was planted at a seeding rate of 175,000 seeds per acre in 30-inch rows
in a randomized,
replicated block. Each entry was replicated four times. The pathogen levels
were natural
populations at the location. The entries were as follows:

Control - no treatment

TJ 1300 - 50/50 combination of B. amyloliquefaciens TJ1000 or 1BE and T virens
G1-3
TJ1300 + TJ Micromix - Liquid chelate TJ Micromix + 50/50 combination of B.
amyloliquefaciens TJ1000 or 1BE and T virens G1-3

Result: As shown in Table 13, TJ Micromix liquid and the combination of TJ
Micromix
plus TJ 1300 both gave soybean a significant increase in yield. TJ 1300 + TJ
Micromix
produced an additional numerical but non significant increase in yield over
the TJ Micromix
alone.

Conclusion: TJ 1300 + TJ Micromix is a viable means of biocontrol deliver on
soybean
and is a viable means of increasing the yield of soybean.


Table 13. Effect of TJ1300 Liquid Biological Treatment Plus Liquid TJ Micromix
Fertilizer
on Yield of Soybean Variety 91B52

Treatment Ratio Location Trial Yield
Liquid TJ
Control 0/0 Groton, SD Micromix 54.2 a
Liquid TJ
TJ 1300 50/50 Groton, SD Micromix 60.8 b
TJ1300 + TJ Liquid TJ
Micromix 50/50 Groton, SD Micromix 61.8 b
CV% 8.92
LSD (0.05) 4.19

54


CA 02471555 2009-06-11

Soybean Dry Granule Micro-Nutrient Working Example

Materials and Methods: A field trial was conducted using the soybean variety
Pioneer

91B52 at a location near Groton, SD. The purpose of the trial was to compare
the compatibility
and yield benefit of the biocontrol preparation TJ1300 in combination with a
dry granule micro-
nutrient fertilizer vs. the micro-nutrient fertilizer alone vs. a control with
no micro-nutrient
fertilizer. Soybean yield in bushels per acre was used to measure the
treatment response. The
micro-nutrient fertilizer is sold commercially under the Trademark TJ
Micromixtm. Biocontrol

treatments were prepared by adding 1X105 CFU per seed. The seed was planted at
a seeding rate
of 175,000 seeds per acre in 30-inch rows in a randomized, replicated block.
TJ Micromixtm was
applied at a rate of 20 pounds per acre. Each entry was replicated four times.
The pathogen
levels were natural populations at each location. The entries were as follows:

Control: Maxim
TJ Micromix

TJ 1300 - 50/50 combination of B. amyloliquefaciens TJ1000 or 1BE and T.
virens G1-3
TJ Micromix + TJ 1300 - 50/50 combination of B. amyloliquefaciens TJ1000 or
1BE and
T. virens G1-3

Results: As shown in Table 14, in this trial, the Granular TJ Micromix
produced a
significant yield increase compared to the control. When the seed-applied
biocontrol treatment
TJ1300 was applied in combination with the TJ Micromix, the treatment resulted
in a significant



CA 02471555 2004-06-18

increase in yield as compared to the control but not significantly different
from the TJ Micromix
application alone. The yield of the TJ 1300 + TJ Micromix was numerically
higher.

Conclusion: TJ 1300 is compatible with micro-nutrient applications and is a
viable tool to
increase the yield of soybean.

Table 14. Effect of TJ1300 Biological Seed Treatment Plus Dry Granule TJ
Micromix
Fertilizer on Yield of Soybean Variety 91B52

Treatment Ratio Location Trial Yield
Control 0/0 Groton, SD TJ Micro 54.2 a
TJ Micromix
Granule 0/0 Groton, SD TJ Micro 61.6 b
TJ 1300 50/50 Groton, SD TJ Micro 62.5 b
TJ 1300 + TJ
Micromix 50/50 Groton, SD TJ Micro 63.3 b
CV% 8.92
LSD (0.05) 4.19
Spring Wheat Working Example

Materials and Methods: A field trial was conducted using Russ Spring wheat at
a
location near Kensal, ND. The purpose of the trial was to test biocontrol TJ
1300 on spring
wheat against an untreated control. The biocontrol TJ 1300 was applied to the
seed so as to
achieve an application rate of 2.5X109 CFU per acre. The plot was planted in a
randomized,
replicated block design with each entry replicated three times.

Result: As shown in Table 15, the entry TJ 1300 produced a non-significant
yield
increase. The conclusion was that TJ 1300 may be of value as a seed treatment
on wheat.

Table 15. Effect of TJ1300 Biological Seed Treatment Plus Fertilizer on Russ
Spring Wheat
56


CA 02471555 2004-06-18

Treatment Ratio Location Trial Yield
Kensal,
Control 0/0 ND MM 43.8
Kensal,
1300 50/50 ND MM 44.0
CV% 7.52
LSD (0.05) NS
Field Peas Working Example


Materials and Methods: A field trial was conducted to compare the biocontrol
treatment
TJ 1300 to a non-treated control on field peas. The seed was treated with the
biocontrol agent to
achieve an application of 2.5X109 CFU per acre. Yield response was measured as
pounds per
acre.


Results: As shown in Table 16, the entry TJ 1300 produced a non-significant
yield
increase in field peas. The conclusion was that TJ 1300 may be an effective
tool to increase the
yield of field peas.

Table 16. Effect of TJ1300 Biological Seed Treatment on Yield of Integra Field
Pea
Treatment Ratio Rep Location Trial Yield Test weight
Carrington,
Control 0/0 Ave of 3 ND Pea 3590.0 62.9
Carrington,
1300 50/50 Ave of 3 ND Pea 3613.0 63.5
CV% 7 0.5
LSD 0.05 ns Ns

57


CA 02471555 2009-06-11

Increased Manganese Uptake Working Example

A surprising aspect of the subject invention is that plants that grow from
seeds treated
with the disclosed combination experience increased uptake of manganese. The
protective nature
of increased manganese uptake is documented in Project S-269: Biological
Control and

Management of Soilborne Plant Pathogens for Sustainable Crop Production, 5th
International
Conference on the Biogeochemistry of Trace Elements. July 11-15 1999. Vienna,
Austria, p.
1086-1087. Dr. Don Huber of Purdue University has documented the connection
between an
imbalance in the ratio of nitrogen to manganese and the incidence of stalk rot
in corn. (Huber D.
2000. "Hidden Hunger" threatens many crops. Purdue News.


The disclosed combination of Trichoderma virens and Bacillus amyloliquefaciens
for the
purpose of plant pathogen control and increased plant yield thus has
unexpected characteristics.
The first is the fact that the combination produces an increase in yield, not
just plant protection

from the pathogen. Plant tissue analysis from test plots presented in Tables
17 and 18 below
show an unexpected trend toward higher nutrient intake of a nutrient,
manganese.

58


CA 02471555 2009-06-11

The treatments that produced the surprising results shown in Table 17 are
defined as
follows:

bs-unt-bt = Brookings, SD location - no treatment on the seed - Bt variety of
corn

bs-max-bt = Brookings, SD location -chemical fungicide Maxim on the seed -Bt
variety of corn
bs-1000-bt = Brookings, SD location -Bacillus amyloliquefaciens TJ 1000 on the
seed- Bt
variety

bs-0300-bt=Brookings, SD location-Trichoderma virens G1-3 on the seed - Bt
variety of corn
bs-1300-bt = Brookings, SD location- B. amyloliquefaciens TJ 1000 and T virens
G1-3 (1 to 1
ratio) on the seed - Bt variety of corn (one of the claimed combinations)

bs-1310-bt = Brookings, SD location - B. amyloliquefaciens TJ 1000 and T
virens G1-3 (7 to 3
ratio) on the seed - Bt variety of corn

bs-66/300-bt = Brookings, SD location - B. lentimorbus and T. virens G1-3 (1
to 1 ratio) on the
seed - Bt variety of corn

The term "Bt" is defined as: A corn hybrid that has been genetically modified
by the insertion of
a gene from the bacteria Bacillus thuringiensis. The inserted gene produces a
protein that will
kill European corn bore that feed on the plant tissue.

Table 17. Effects of Treatments on Plant Mineral Content on Bt Variety of Corn
at Brookings
SD Location

Concentration
Treatment N P K Mg Ca S Na Fe Mn B Cu Zn
bs-unt-bt 3.43 0.39 1.65 0.66 1.11 0.29 0.003 110 105 17 18 32

59


CA 02471555 2009-06-11

bs-max-bt 3.42 0.43 2.10 0.56 0.91 0.27 0.005 117 91 14 18 29
bs-1000-bt 3.44 0.40 2.10 0.52 0.86 0.24 0.004 96 91 12 13 25
bs-300-bt 3.38 0.41 2.02 0.58 1.00 0.27 0.004 97 98 12 14 25
bs-1300-bt 3.36 0.43 1.89 0.66 1.11 0.27 0.004 118 134 13 16 28
bs-1310-bt 3.45 0.41 1.69 0.59 1.02 0.25 0.004 182 106 16 15 27
bs-66/300-bt 3.30 0.42 2.19 0.58 1.04 0.27 0.004 112 107 16 15 29

The treatments that produced the surprising results in Table 18 are defined as
follows:
bl-unt-non = Brookings location - no treatment on the seed - non Bt variety of
corn (non Bt can
also be described as: non genetically modified)

bl-max-non = Brookings location - chemical fungicide Maxim on the seed - non
Bt variety of
corn

bl-1000-non = Brookings location - Bacillus amyloliquefaciens TJ 1000 on the
seed - non Bt
variety of corn

bl-300-non = Brookings location - Trichoderma virens G1-3 on the seed - non Bt
variety of corn
bl-1300-non = Brookings location - B. amyloliquefaciens TJ 1000 and T. vixens
G1-3 on the seed
(1 to 1 ratio) - non Bt variety of corn (one of the claimed combinations)

bl-1310-non = Brookings location - B. amyloliquefaciens TJ 1000 and T virens
G1-3 on the seed
(7 to 3 ratio) - non Bt variety of corn

bl-66/300-non = Brookings location - B. lentimorbus and T. virens G1-3 on the
seed (1 to 1
ratio) - non Bt variety of corn



CA 02471555 2004-06-18

Table 18. Effects of Treatments on Plant Mineral Content on Non Bt Variety of
Corn at
Brookings SD Location

Concentration
Treatment I'T P K Mg Ca S Na Fe Mn B Cu Zn
bl-unt-non 3.33 0.39 1.93 0.55 0.85 0.21 0.005 76 103 12 13 24
bl-max-non 3.28 0.48 2.39 0.62 0.92 0.24 0.007 101 116 12 15 28
bl-1000-non 3.14 0.51 2.39 0.64 0.95 0.25 0.008 103 115 12 15 26
bl-300-non 3.19 0.48 2.21 0.65 0.93 0.24 0.009 95 99 15 15 24
bl-1300-non 3.38 0.48 2.43 0.60 0.96 0.25 0.006 111 137 13 15 26
bl-1310-non 3.21 0.46 2.18 0.68 1.03 0.26 0.007 108 117 18 16 25
bl-66/300-non 3.23 0.43 1.96 0.61 0.86 0.23 0.009 93 95 11 13 25

Manganese is known in the art as a disease prevention micronutrient. However,
if
manganese is added to fertilizer and applied to corn, the expected result is a
decrease in yield.
The significance of the subject invention is that it increases the manganese
content of the corn
plant while increasing yield. Furthermore, the increase in the manganese
content in the plant
does not occur with either organism alone or when the Trichoderma virens is
combined with a

different organism (e.g., treatment 66/300) or the formulation of the mixture
is altered (e.g.,
treatment 1310). This increase in manganese content of the plant tissue is
documented in tables
1 and 2 above on Bt (genetically modified) corn and conventional (non-
genetically modified)
corn. Tissue analysis of the corn in the charts above was done after the
silking and pollination of
the corn, documenting that this increase in manganese continues into the late
stages of growth.

61


CA 02471555 2009-06-11

Late season intake is significant because the lack of manganese in the plant
is implicated in mid
to late season stalk rot.

Data from disclosed combinations of the Trichoderma with other bacteria
strains show
that other combinations tested did not increase the manganese levels to the
level of the present
invention. It is surprising that neither organism alone increased the
manganese level in the tissue
of the corn. Only seed treatment with the claimed combination of the T virens
G1-3 fungus and
the B. amyloliquefaciens bacterium increase the manganese level in the tissue
of both the Bt and
non-Bt corn.


Consistency of Increased Yield Working Example

Another surprising aspect of the subject invention is unexpected consistency
of increased
yield: (1) consistency compared to either organism alone, in that our field
trial results show the
claimed combination to be significantly higher in yield over the control in
both individual

locations and multiple location and either organism alone did not produce a
significant yield
response over the control; (2) consistency across geography, in that the field
trial results show the
combination to be effective in a number of geographies from North Dakota to
Arizona; and (3)
consistency of higher yield in a more than one crop, in that the field data
collected on corn,

soybeans, sunflowers and wheat show significant increased in yield with the
claimed
combination. Field trial results are presented in the above working examples.
The results of
those field trials produced a surprisingly consistent yield response, and
consistency is what is
commercially important.

62


CA 02471555 2009-06-11

The disclosed combination of microorganisms gives more consistent yield
response than
either microorganism alone. The claimed combination produces a consistent
increase in yield
over a range of conditions while alone the microorganisms do not. The data in
the patent

application show this, but the data presented in Table 19 below that was
produced at the
experiment station in Carrington, ND show this effect.

Table 19. Consistency of Yield Response

X01 3) R
Quid 96, 87.7 110.2
dIIus 93 1 94 1127
T. wens 94 162 88 1151
Qi 106 156 90.4 117.1
131 89 151 8B 10aO
In Table 19, the treatments are defined as follows:

Control = chemical fungicide Maxim
Bacillus = B. amyloliquefaciens alone
T. virens = T virens G1-3 alone

Quick Root = QuickRootsTM is the product name of the claimed combination of T.
virens G1-3
and B. amyloliquefaciens

1310 = T. virens G1-3 and B. amyloliquefaciens at a 7:3 ratio.
63


CA 02471555 2004-06-18

The column headings in Table 19 denote the year of the trial with "3YR"
indicating the
average treatment response for the combined three years. Note that in 2000,
seed treatment with
the individual organisms alone (the individual components of the claimed
combination) produced
yields that were less than control. In 2001, seed treatment with individual
organisms both

produced yields that were greater than the control as did the claimed
combination. In 2002, seed
treatment with the individual organisms produced yields that were greater than
the control and
again the claimed combination increased yield as well.

The North Dakota data presented in Table 19 document consistency in two of
ways.
First, in reviewing year 2000 data, neither the Bacillus bacteria (1000) seed
treatment nor the
Trichoderma fungi (GI-3) seed treatment by themselves produced a positive
yield response; but
the claimed combination did produce a positive response. Two negative
responses added
together do not produce a positive. Synergism is what creates positive
response from two
negatives. In years 2001 and 2002, the performance of treatments with the
bacteria and the fungi

traded places as the top seat while the performance of the claimed combination
performed
between treatments with the individual components. Overall, the consistent
performance of the
claimed combination gave the largest yield advantage because of consistency of
response. These
data are from the same location; only weather changed from season to season.
The Bacillus
alone seed treatment did not perform well at all in the average and the
Trichoderma alone seed

treatment only averaged well because it had one great performance out of
three.

Presented in Table 20 is a compilation of data from three years of field
trials, 63 entries,
at 12 locations. The test plots were located at North Dakota State University,
University of

64


CA 02471555 2004-06-18

Arizona, and Colorado State University. This compilation clearly shows the
50/50 combination
of B. amyloliquefaciens + T. virens (one of the claimed combinations) produces
a significantly
higher yield than the control and than either organism alone. It should be
noted that while the
individual components show a numerical increase in yield, it is a non-
significant increase at a

0.05 rejection level while the claimed combination is significant at a 0.05
rejection level.
Table 20. QuickRootsTM Effect on Corn Yield in Replicated Field Trials.

3 Year Average Evaluating QuickRootsTM/Maxim vs. Maxim
Treatment Moisture Yield Pricing Advantage
Control 17.5 154.77 $300.25

B. amyloliquefaciens alone 17.5 158.7 $307.88 $7.62
T virens alone 17.4 158.81 $308.57 $8.31
B. amyloliquefaciens + T. virens

combined 50/50 17.5 161.62 $313.54 $13.29
Mean 17.5 158.88 $307.56

CV (%) 23.3 21.7
LSD (0.05) .19(NS) 5.05

Many variations of the invention will occur to those skilled in the art. Some
variations
include non-competitive culturing of the biocontrol organisms. Other
variations call for



CA 02471555 2004-06-18

competitive culturing. All such variations are intended to be within the scope
and spirit of the
invention.

66

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Administrative Status

Title Date
Forecasted Issue Date 2011-05-17
(22) Filed 2004-06-18
Examination Requested 2004-10-01
(41) Open to Public Inspection 2005-12-18
(45) Issued 2011-05-17

Abandonment History

There is no abandonment history.

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $400.00 2004-06-18
Request for Examination $800.00 2004-10-01
Maintenance Fee - Application - New Act 2 2006-06-19 $100.00 2006-06-16
Maintenance Fee - Application - New Act 3 2007-06-18 $100.00 2007-05-28
Maintenance Fee - Application - New Act 4 2008-06-18 $100.00 2008-06-05
Maintenance Fee - Application - New Act 5 2009-06-18 $200.00 2009-06-04
Maintenance Fee - Application - New Act 6 2010-06-18 $200.00 2010-05-07
Final Fee $300.00 2011-03-03
Maintenance Fee - Patent - New Act 7 2011-06-20 $200.00 2011-05-31
Maintenance Fee - Patent - New Act 8 2012-06-18 $200.00 2012-06-12
Maintenance Fee - Patent - New Act 9 2013-06-18 $200.00 2013-06-03
Registration of a document - section 124 $100.00 2013-06-20
Registration of a document - section 124 $100.00 2013-10-09
Maintenance Fee - Patent - New Act 10 2014-06-18 $250.00 2014-05-15
Maintenance Fee - Patent - New Act 11 2015-06-18 $250.00 2015-05-29
Maintenance Fee - Patent - New Act 12 2016-06-20 $250.00 2016-05-25
Maintenance Fee - Patent - New Act 13 2017-06-19 $250.00 2017-05-24
Maintenance Fee - Patent - New Act 14 2018-06-18 $250.00 2018-06-11
Maintenance Fee - Patent - New Act 15 2019-06-18 $450.00 2019-06-14
Maintenance Fee - Patent - New Act 16 2020-06-18 $450.00 2020-06-12
Maintenance Fee - Patent - New Act 17 2021-06-18 $459.00 2021-06-11
Maintenance Fee - Patent - New Act 18 2022-06-20 $458.08 2022-05-18
Maintenance Fee - Patent - New Act 19 2023-06-19 $473.65 2023-05-17
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
NOVOZYMES BIOAG A/S
Past Owners on Record
JOHNSON, THOMAS D.
TJ TECHNOLOGIES, INC.
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
Documents

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Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Abstract 2004-06-18 1 16
Description 2004-06-18 66 2,651
Claims 2004-06-18 12 343
Drawings 2004-06-18 2 86
Cover Page 2011-04-18 1 29
Cover Page 2005-11-29 1 27
Description 2009-06-11 66 2,489
Claims 2009-06-11 8 196
Assignment 2004-06-18 2 63
Prosecution-Amendment 2004-10-01 1 34
Prosecution-Amendment 2008-12-12 5 240
Fees 2006-06-16 1 41
Fees 2007-05-28 1 54
Fees 2008-06-05 1 43
Prosecution-Amendment 2009-06-11 52 1,899
Correspondence 2011-03-03 2 64
Assignment 2013-06-20 7 216
Fees 2012-06-12 1 163
Fees 2013-06-03 1 163
Assignment 2013-10-09 5 146