U.S. patent application number 15/207700 was filed with the patent office on 2017-01-12 for use of synergistic microorganisms and nutrients to produce signals that facilitate the germination and plant root colonization of mycorrhizal fungi in phosphorus rich environments.
This patent application is currently assigned to NOVOZYMES BIOAG A/S. The applicant listed for this patent is NOVOZYMES BIOAG A/S. Invention is credited to Thomas D. Johnson.
Application Number | 20170008815 15/207700 |
Document ID | / |
Family ID | 49378852 |
Filed Date | 2017-01-12 |
United States Patent
Application |
20170008815 |
Kind Code |
A1 |
Johnson; Thomas D. |
January 12, 2017 |
Use of synergistic microorganisms and nutrients to produce signals
that facilitate the germination and plant root colonization of
mycorrhizal fungi in phosphorus rich environments
Abstract
A composition of matter comprising: a combination of a phytate
and a plurality of microorganisms comprising a Trichoderma virens
fungus, a Bacillus amyloliquefaciens bacterium, and one or a
plurality of mycorrhizae fungi that is placed in the vicinity of a
plant root in a manner that allows the microorganisms in the
composition of matter to colonize said plant root; and a method for
increasing plant yield comprising: placing a combination of a
phytate and a plurality of microorganisms comprising a Trichoderma
virens fungus, a Bacillus amyloliquefaciens bacterium, and one or a
plurality of mycorrhizae fungi in the vicinity of a plant root in a
manner that allows the microorganisms in the composition of matter
to colonize said plant root.
Inventors: |
Johnson; Thomas D.;
(Buffalo, SD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NOVOZYMES BIOAG A/S |
Bagsvaerd |
|
DK |
|
|
Assignee: |
NOVOZYMES BIOAG A/S
Bagsvaerd
DK
|
Family ID: |
49378852 |
Appl. No.: |
15/207700 |
Filed: |
July 12, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14684592 |
Apr 13, 2015 |
9416061 |
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15207700 |
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13815856 |
Mar 15, 2013 |
9017442 |
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14684592 |
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61687210 |
Apr 20, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C05B 15/00 20130101;
C05F 11/08 20130101; C12N 1/14 20130101; C12N 1/20 20130101 |
International
Class: |
C05F 11/08 20060101
C05F011/08; C12N 1/20 20060101 C12N001/20; C12N 1/14 20060101
C12N001/14 |
Claims
1. A composition, comprising: a Trichoderma virens component; a
Bacillus amyloliquefaciens component comprising Bacillus
amyloliquefaciens TJ-1000, Bacillus amyloliquefaciens 1BE and/or
Bacillus amyloliquefaciens FZB24; a mycorrhizal fungus or
mycorrhizal fungi component; and a phytate or phytic acid
component.
2. The composition of claim 1, wherein said Trichoderma virens
component comprises Trichoderma virens G1-3.
3. The composition of claim 1, wherein said Trichoderma virens
component comprises about 1.times.10.sup.11 to about
1.times.10.sup.7 colony forming units per gram of Trichoderma
virens G1-3.
4. The composition of claim 1, wherein said Trichoderma virens
component comprises about 4.2.times.10.sup.9 to about
6.75.times.10.sup.8 colony forming units per gram of Trichoderma
virens G1-3.
5. The composition of claim 1, wherein said Trichoderma virens
component comprises about 5.times.10.sup.8 colony forming units per
gram of Trichoderma virens G1-3.
6. The composition of claim 1, wherein said Trichoderma virens
component comprises Trichoderma virens G1-21.
7. The composition of claim 1, wherein said Trichoderma virens
component comprises about 1.times.10.sup.11 to about
1.times.10.sup.7 colony forming units per gram of Trichoderma
virens G1-21.
8. The composition of claim 1, wherein said Trichoderma virens
component comprises about 4.2.times.10.sup.9 to about
6.75.times.10.sup.8 colony forming units per gram of Trichoderma
virens G1-21.
9. The composition of claim 1, wherein said Trichoderma virens
component comprises about 5.times.10.sup.8 colony forming units per
gram of Trichoderma virens G1-3.
10. The composition of claim 1, wherein said Bacillus
amyloliquefaciens component comprises Bacillus amyloliquefaciens
TJ1000.
11. The composition of claim 1, wherein said Bacillus
amyloliquefaciens component comprises about 5.times.10.sup.11 to
about 1.times.10.sup.7 colony forming units per gram of Bacillus
amyloliquefaciens TJ1000.
12. The composition of claim 1, wherein said Bacillus
amyloliquefaciens component comprises about 1.35.times.10.sup.10 to
about 8.40.times.10.sup.10 colony forming units per gram of
Bacillus amyloliquefaciens TJ1000.
13. The composition of claim 1, wherein said Bacillus
amyloliquefaciens component comprises Bacillus amyloliquefaciens
1BE.
14. The composition of claim 1, wherein said Bacillus
amyloliquefaciens component comprises about 5.times.10.sup.11 to
about 1.times.10.sup.7 colony forming units per gram of Bacillus
amyloliquefaciens 1BE.
15. The composition of claim 1, wherein said Bacillus
amyloliquefaciens component comprises about 1.35.times.10.sup.10 to
about 8.40.times.10.sup.10 colony forming units per gram of
Bacillus amyloliquefaciens 1BE.
16. The composition of claim 1, wherein said Bacillus
amyloliquefaciens component comprises Bacillus amyloliquefaciens
FZB24.
17. The composition of claim 1, wherein said Bacillus
amyloliquefaciens component comprises about 5.times.10.sup.11 to
about 1.times.10.sup.7 colony forming units per gram of Bacillus
amyloliquefaciens FZB24.
18. The composition of claim 1, wherein said Bacillus
amyloliquefaciens component comprises about 1.35.times.10.sup.10 to
about 8.40.times.10.sup.10 colony forming units per gram of
Bacillus amyloliquefaciens FZB24.
19. A method, comprising applying the composition of claim 1 to a
soil.
20. The method of claim 19, wherein the composition of claim 1 is
applied to the soil at a rate of about 1.35 grams per acre.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 61/687,210, filed Apr. 20, 2012, the
disclosure of which patent application is incorporated by reference
as if fully set forth herein.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not Applicable
THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT
[0003] Not Applicable
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT
DISC
[0004] Not Applicable
BACKGROUND OF THE INVENTION
[0005] This invention relates to combining synergistic
microorganisms to produce the signals that are necessary to
facilitate germination and plant root colonization of mycorrhizae
fungi. The colonization of the plant root by mycorrhizal fungi
results in the increase of the availability of nutrients to plants,
control of pathogens, and improved soil structure and/or soil
quality. In particular, an illustrative embodiment of the invention
relates to combining phytate as a nutrient source with a
combination of Trichoderma virens, Bacillus amyloliquefaciens, and
mycorrhizal fungi, including the following known species: Glomus
intraradices, Glomus etunicatum, Glomus aggregatum, Glomus mosseae,
for the purpose of replacing, duplicating, or enhancing the effect
of standard phosphorus-containing fertilizer compounds, such as a
10-34-0, 9-18-9, 3-18-18 fertilizer or other NPK fertilizer
combinations.
[0006] Phytic acid [known as inositol hexakisphosphate (IP6) or
phytate when in salt form] is the principal storage form of
phosphorus in many plant tissues, especially bran and seeds.
Phytate is also a major form of organic phosphorus within the soil
profile, typically constituting 20%-50% of soil organic
phosphorus.
[0007] Trichoderma is a genus of fungi that contains about 20
species. Synonyms for the genus name include Aleurisma and
Sporoderma. Trichoderma virens, 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, Trichoderma
(Gliocladium) virens G1-21 is known and commercially available at a
reasonable price, and is being marketed under the trademark
SoilGuard.RTM. 12G (EPA Registration Number: 70051-3 and EPA
Establishment Number: 067250-IL-001). It is manufactured by Thermo
Trilogy Corporation of Columbia, Md. Other known and commercially
available Trichoderma virens strains include those having the
following ATCC accession numbers: 10043, 10044, 10045, 13213,
13362, 204067, 204443, 204444, 204445, 20903, 20904, 20906, 24290,
42955, 44327, 44734, 48179, 52045, 52199, 58676, 58677, 58678,
62399, 64271, 74180, 9645, MYA-297, MYA-298, MYA-649 and
MYA-650.
[0008] 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, 23845, 31592, 49763, 53495 and BAA-390 (Int.
J. Sys. Bacteriol. 37:69-71, 1987; J. Bacteriol. 94:1124-1130,
1967).
[0009] In the past, before the name was officially changed to
recognize that the microorganism was a new species, Bacillus
amyloliquefaciens was also called Bacillus subtilis var.
amyloliquefaciens by some investigators. A protease produced from
Bacillus subtilis var. amyloliquefaciens is commonly used as a
tenderizer for raw meat products. According to the U.S.
Environmental Protection Agency (EPA), Bacillus subtilis var.
amyloliquefaciens strain FZB24 is a naturally-occurring
microorganism and widespread in the environment. Bacillus subtilis
var. amyloliquefaciens FZB24 (EPA Registration Number: 72098-5 and
EPA Establishment Number: 73386-DEU-001) is known and commercially
available at a reasonable price, being marketed under the trademark
Taegro.RTM. by Novozymes, Inc. of Brookfield, Wis.
[0010] An arbuscular mycorrhiza fungus is a type of mycorrhiza in
which the fungus penetrates the cortical cells of the roots of a
vascular plant. Arbuscular mycorrhizae fungi help plants to capture
nutrients such as phosphorus, sulfur, nitrogen and micronutrients
from the soil. It is believed that the development of the
arbuscular mycorrhizal symbiosis played a crucial role in the
initial colonization of land by plants and in the evolution of the
vascular plants.
[0011] The development of arbuscular mycorrhizal fungi prior to
root colonization, known as presymbiosis, comprises three stages:
propagule germination, hyphal growth, and host recognition and
appressorium formation. Propagule are thick-walled multi-nucleate
resting structures. Arbuscular mycorrhizal fungi propagules may
germinate given suitable conditions of the soil matrix,
temperature, carbon dioxide concentration, pH, and phosphorus
concentration. The germination of the propagule is not thought to
be under direct control of the plant as propagules have been
germinated under experimental conditions in the absence of plants
both in vitro and in soil. However, the rate of propagule
germination can be increased by plant host root exudates.
[0012] The growth of arbuscular mycorrhizal hyphae through the soil
is controlled by host root exudates and the soil phosphorus
concentration. Arbuscular mycorrhizal fungi colonization is higher
in nutrient poor soils and decreased with the addition of phosphate
fertilizer. Low soil phosphorus concentrations increase hyphal
growth and branching as well as induce plant exudation of compounds
which control hyphal branching intensity. Arbuscular mycorrhizal
fungi also have chemotaxic abilities which enable hyphal growth
toward the roots of a potential host plant.
[0013] A major challenge for the mycorrhizologist is to understand
the extremely harmonious arbuscular mycorrhizal fungus host
signaling mechanisms and the colonization process. This harmonious
symbiotic relationship is reflected in the obligate biotrophic
nature of the fungi, which cannot be cultured in the absence of a
host. While success in achieving effective mycorrhizal associations
with crop plants growing in sterilized soil has been achieved, the
ultimate success for agricultural use of vesicular-arbuscular
mycorrhizal (VAM) fungi will occur when they can be used dependably
to improve performance of crops grown in nonfumigated soil.
[0014] This invention provides a signal that produces propagule
germination and subsequent root colonization of mycorrhizae in a
very surprising way. It has long been known that seeds store
phosphorus as phytate (IP6) and that a germinating seed produces
the enzyme phytase to break down the phytate into plant-useable
forms to provide nutrients for the seedling. It has also been known
that the breakdown of phytate (a six phosphorus molecule) by the
enzyme phytase releases three moles of inorganic phosphorus
(orthophosphate) and myo-inositol triphosphate (IP3). Plants need
phosphorus in an inorganic form, primarily orthophosphate, to take
the nutrient into the root. Plants use very little organic
phosphorus as they do not possess an effective method to break down
phytate. Myo-inostitol triphosphate (IP3) is known as a second
messenger that can facilitate communications and/or responses
between organisms. The release of myo-inositol that occurs through
hydrolysis of phytate with B. amyloliquefaciens phytase has an
impact on plant-microbe interactions and specifically interactions
between plants and N fixing bacteria. The signal that is
responsible for the germination of mycorrhizae and the subsequent
colonization of the plant root by mycorrrhizal fungi is unknown. In
addition, the IP3 signal has not been suggested in the literature
as having any link to mycorrhizal fungi response, propagule
germination, or root colonization. In fact, it is well known that
mycorrhizae root colonization can be achieved in low phosphorus
soil conditions but it is extremely difficult to produce
mycorrhizal germination and root colonization in high phosphorus
soil conditions or high phosphorus rhizosphere environment. It is,
therefore, also a fact that the literature teaches away from the
notion that using a phytase enzyme to reduce phytate and release
readily-plant-available phosphorus in the rhizosphere would result
in a signal that facilitates germination and subsequent
colonization of plant roots by mycorrhizal fungi.
[0015] It is likely that additional study of this invention will
produce dual and perhaps multiple signal mechanisms as it is known
that germination of mycorrhizae propagules can occur in the absence
of the plant root; however, the propagule germination is more
likely when the root is present. This suggests an unknown signal
response.
[0016] The background art is characterized by U.S. Pat. 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; 6,103,228; and 7,339,091; the
disclosures of which patents are incorporated by reference as if
fully set forth herein.
BRIEF SUMMARY OF THE INVENTION
[0017] One aspect of the invention is that it creates an effective
association of mycorrhizal fungi with the root of a host plant in a
phosphorous rich environment. Other aspects of the invention are
the germination of mycorrhizae propagules and subsequent root
colonization triggered by a signal molecule. Yet another aspect of
the invention is the presence of the IP3 signal molecule. Yet
another aspect of the invention is the use of a bacterium that
produces a phytase enzyme in a high phosphorous environment because
of the presence of the Phy C gene. Yet another aspect of the
invention is the use of a microbial component, a Trichoderma
fungus, that produces phosphatase enzymes that can break the bonds
on stable compounds such as tricalium phosphate and release Ca++
ions.
[0018] As used herein, the following terms and variations thereof
have the meanings given below, unless a different meaning is
clearly intended by the context in which such term is used:
[0019] "A," "an" and "the" and similar referents used herein are to
be construed to cover both the singular and the plural unless their
usage in context indicates otherwise.
[0020] "About" means within one percent of a recited parameter or
measurement, and preferably within 0.1 percent of such parameter or
measurement.
[0021] "Comprise" and variations of the term, such as "comprising"
and "comprises," are not intended to exclude other additives,
components, integers or steps.
[0022] "E" means "times 10 to the power of." "Exemplary,"
"illustrative," and "preferred" mean "another."
[0023] An illustrative embodiment of the invention comprises a
combination of major nutrients, micronutrients, fungi, and bacteria
that increases the yield of plants in a manner that is comparable
to or superior to the application of a standard fertilizer
chemistry.
[0024] In use, an illustrative embodiment of the invention creates
a synergism that occurs when a proper type and proper ratios of
nutrients, fungi, and bacteria are brought together in such a way
as to provide plants with available nutrients that increase plant
yield or seed yield.
[0025] An illustrative embodiment of the invention comprises: a
plurality of nutrients, that may or may not be plant-available,
combined with a plurality of fungi, and a plurality of bacteria in
the presence of a seed, a plant, or a root.
[0026] An illustrative embodiment of the invention is a method
comprising the following steps: producing a mixture that is
comprised of nutrients, fungi, and bacteria in such a way as to
produce a synergy between the components; applying the mixture to a
seed or applying the mixture in a band in contact with seed or near
to the seed or in a soil mixture wherein said fungi and said
bacteria reduce said nutrients into a plant-available form, thereby
allowing mycorrhizal fungi to colonize the root in the presence of
the plant-available nutrients and increasing plant yield or seed
yield.
[0027] In an illustrative embodiment, the invention is a
composition of matter comprising: a combination of phytate and a
plurality of microorganisms comprising a Trichoderma virens fungus
or another calcium solubilizing fungus or a soluble calcium
component, a Bacillus amyloliquefaciens bacterium or another
bacterium that produces a phytase enzyme or a phytase enzyme, and a
mycorrhiza fungus or a plurality of mycorrhizae fungi that is
placed in the vicinity of a plant root in a manner that allows said
plurality of microorganisms in the composition of matter to
colonize said plant root.
[0028] In another illustrative embodiment, the invention is a
method for increasing plant yield comprising: placing a combination
of phytate and a plurality of microorganisms comprising a
Trichoderma virens fungus or another calcium solubilizing fungus or
a soluble calcium component, a Bacillus amyloliquefaciens bacterium
or another bacterium that produces a phytase enzyme or a phytase
enzyme and a mycorrhiza fungus or a plurality of mycorrhizae fungi
in the vicinity of a plant root in a manner that allows said
microorganisms to colonize said plant root. Preferably, said
composition is placed in the vicinity of said plant root by
application to a preplanted seed, by in-furrow applications as a
seed is being planted, or by broadcast over a seed row.
[0029] In yet another preferred embodiment, the invention is a
method for improving soil aggregation and soil quality by placing a
Trichoderma virens fungus, a Bacillus amyloliquefaciens bacterium
or another bacterium that produces a phytase enzyme, and a
mycorrhiza fungus or a plurality of mycorrhizae fungi in the
vicinity of a plant root in a manner that allows said
microorganisms to colonize said plant root.
[0030] In another illustrative embodiment, the invention is a
composition of matter comprising: about 30,000 propagules of
Mycorrhizae in about five gallons of water; about one gallon of an
about 15 to about 40 percent aqueous solution of a phytate; about
6.75E8 to about 4.20E9 colony forming units of Trichoderma virens;
and about 1.35E10 to about 8.40E10 colony forming units Bacillus
amyloliquefaciens.
[0031] In another illustrative embodiment, the invention is a
composition of matter comprising: a combination of a phytate and a
plurality of microorganisms comprising a Trichoderma virens fungus
or another calcium solubilizing fungus, a Bacillus
amyloliquefaciens bacterium or another bacterium that produces a
phytase enzyme, and a mycorrhiza fungus or a plurality of
mycorrhizae fungi; wherein said combination is operative to enable
said plurality of microorganisms to colonize a plant root when said
combination is placed in the vicinity of said plant root.
[0032] In yet another illustrative embodiment, the invention is a
composition of matter comprising: a phytate or phytic acid; means
for producing a soluble calcium or a soluble calcium; and means for
producing a phytase enzyme or a phytase enzyme.
[0033] In a further illustrative embodiment, the invention is a
composition of matter comprising:
[0034] a plurality of nutrients comprising an organic phosphorus
(e.g., a phytate or phytic acid), combined with a plurality of
fungi, and a plurality of bacteria in the presence of a seed, a
plant, or a root.
[0035] In another illustrative embodiment, the invention is a
composition of matter comprising: a Trichoderma virens component; a
Bacillus amyloliquefaciens component; a mycorrhizal fungus or
mycorrhizal fungi component; and a phytate or phytic acid
component.
[0036] In yet another illustrative embodiment, the invention is a
method comprising: producing a mixture that is comprised of a
nutrient component, a fungus component, and a bacterium component
that is operative to produce a synergy among the components;
applying said mixture to a seed or applying said mixture in a
growth medium in contact with said seed or near to said seed or in
a soil mixture wherein, said fungus component and said bacterium
component reduce said nutrient component into a plant-available
form; allowing a mycorrhizal fungus to colonize a root produced by
said seed in the presence of said plant-available form and
increasing plant yield or seed yield.
[0037] In a further illustrative embodiment, the invention is a
method for increasing plant yield comprising: placing a combination
of phytate and a plurality of microorganisms comprising a
Trichoderma virens fungus or another calcium solubilizing fungal
component, a Bacillus amyloliquefaciens bacterium or another
bacterial phytase enzyme producing bacterial component and a
mycorrhiza fungus or a plurality of mycorrhizae fungi in the
vicinity of a plant root in a manner that allows said
microorganisms to colonize said plant root. In another embodiment,
the method further comprises: applying said Trichoderma virens
fungus or another calcium solubilizing fungal component in a
concentration ranging from about 1.0E7 to about 1.0E11 colony
forming unit per gram (cfu/g) or colony forming unit per milliliter
(cfu/ml) of viable Trichoderma virens G1-3 or Trichoderma virens
strain G1-21 spores per gram of said Trichoderma virens fungus or
another calcium solubilizing fungal component and at an application
rate of about 1.35 g per acre or 1.35 ml per acre. In another
embodiment, the method further comprises: applying said Bacillus
amyloliquefaciens bacterium or another phytase enzyme producing
bacterial component in a concentration ranging from about 1E7 to
about 5E11 cfu/g or cfu/ml of viable Bacillus amyloliquefaciens
strain BAA-3 or Bacillus amyloliquefaciens strain FZB24 spores per
gram of Bacillus amyloliquefaciens bacterium or another phytase
enzyme producing bacterial component and at an application rate of
about 1.35 g or 1.35 ml per acre. In another embodiment, said
combination is placed in the vicinity of said plant root by
application to a pre-planted seed, by in-furrow application as a
seed is being planted, or by broadcasting over a seed row.
[0038] In a further illustrative embodiment, the invention is a
method for improving soil aggregation and soil quality comprising:
placing a Trichoderma virens fungus or another calcium solubilizing
fungus, a Bacillus amyloliquefaciens bacterium or another bacterium
that produces a phytase enzyme, and a mycorrhiza fungus or a
plurality of mycorrhizae fungi in the vicinity of a plant root in a
manner that allows said fungi and bacterium to colonize said plant
root.
[0039] In another illustrative embodiment, the invention is a
method for increasing the yield of a plant, said method comprising:
using a phytase enzyme to reduce phytate and release
readily-plant-available phosphorus in a rhizosphere in which said
plant is growing; and producing a signal molecule that facilitates
germination of a a mycorrhizal fungus and subsequent colonization
of the roots of the plant by said mycorrhizal fungus. In another
embodiment, said signal molecule is myo-inostitol triphosphate. In
another embodiment, the method further comprises: using a Bacillus
amyloliquefaciens bacterium to produce said phytase enzyme in said
rhizosphere. In another embodiment, the method further comprises:
using a microorganism to produce said phytase enzyme. In another
embodiment, said germination and root colonization occurs in a high
phosphorus environment created by applying a fertilizer comprising
phosphorus to said rhizosphere. In another embodiment, the method
further comprises: using a Trichoderma fungus to produce a
phosphatase enzyme that is operative to break the bonds on
tricalium phosphate and release calcium ions. In another
embodiment, the Trichoderma fungus is Trichoderma virens G1-3.
[0040] In a further illustrative embodiment, the invention is a
method for increasing the yield of a crop, said method comprising:
applying a composition of matter comprising the following
components to each acre of cropland: about one quart of an about 40
percent (by weight) phytate solution; about one gallon to five
gallons of water or water plus a standard NPK fertilizer; about
1.35 g of a B. amyloliquefaciens TJ1000 spore composition (at a
concentration of about 1E10 cfu/g); about 1.35 g of a T virens G1-3
spore composition (at a concentration of about 5.0E8 cfu/g); and
about 0.136 g of a Mycorrhizae propagule composition (at a
concentration of about 220,000 propagules/g). In another
embodiment, the method further comprises: mixing the composition of
matter in a fertilizer applicator tank; and applying it to the
cropland in a furrow or a band in close proximity to a seed furrow
or a plant root.
[0041] In another illustrative embodiment, the invention is a
method for increasing the yield of a crop, said method comprising:
applying a composition of matter to each acre of cropland; wherein
said composition of matter comprises: about 30,000 propagules of
Mycorrhizae in about five gallons of water, about one gallon of an
about 15 to about 40 percent aqueous solution of a phytate, about
6.75E8 to about 4.20E9 colony forming units of Trichoderma virens,
and about 1.35E10 to about 8.40E10 colony forming units Bacillus
amyloliquefaciens.
[0042] Further aspects of the invention will become apparent from
consideration of the drawings and the ensuing description of
exemplary 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 invention can be modified in a number
of respects, all without departing from the concept. Thus, the
following drawings and description are to be regarded as
illustrative in nature and not restrictive.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0043] The features of the invention will be better understood by
reference to the accompanying drawings which illustrate exemplary
embodiments of the invention. In the drawings:
[0044] FIG. 1 is a table that presents yield data from a field
trial of an illustrative embodiment of the invention that was
conducted near Watertown, S. Dak. in 2011. The crop grown in the
trial was a 95 Relative Maturity hybrid corn. In FIG. 1, CHK stands
for Check and represent the untreated control within the trial,
Myco stands for mycorrhizal fungi, IF stands for in furrow
application, T.V stands for Trichoderma vixens, B.A. stands for
Bacillus amyloliquefaciens, LSD stands for least significant
difference and CV stands for coefficient of variation. In the
footnotes, "a" denotes the corresponding value is significantly
different from any other value that does not contain the letter
"a"; "ab" denotes the corresponding value is significantly
different from any other value that does not contain the letters
"ab"; "bcd" denotes the corresponding value is significantly
different from any other value that does not contain the letters
"bcd"; "cd" denotes the corresponding value is significantly
different from any other value that does not contain the letters
"cd"; "ef" denotes the corresponding value is significantly
different from any other value that does not contain the letters
"ef"; "f" denotes the corresponding value is significantly
different from any other value that does not contain the letter
"f"; "cde" denotes the corresponding value is significantly
different from any other value that does not contain the letters
"cde"; "g" denotes the corresponding value is significantly
different from any other value that does not contain the letter
"g".
[0045] FIG. 2 is a table that shows root scan data from a corn
trial conducted in a grow room. In the footnotes, a denotes the
corresponding value is significantly different from any other value
that does not contain the letter "a"; "ab" denotes the
corresponding value is significantly different from any other value
that does not contain the letters "ab"; "b" denotes the
corresponding value is significantly different from any other value
that does not contain the letter "b"; "c" denotes the corresponding
value is significantly different from any other value that does not
contain the letters "c".
[0046] FIG. 3 is a schematic diagram illustrating the Bacillus
amyloliquefaciens production of the enzyme phytase and the reaction
of phytase on phytate (IP6) and the subsequent breakdown into 3
moles of inorganic Phosphorous and the signal component Myo
Inositol triphosphate (IP3).
[0047] FIG. 4 is a photograph produced by staining with trypan blue
a corn root embodying an embodiment of the invention.
[0048] FIG. 5 is a photograph of roots of a control corn plant (on
the left) and a corn plant treated with an illustrative embodiment
of the invention (on the right), respectively.
[0049] FIG. 6 is a photograph showing the mycorrhizal hyphae
structure colonizing inside a root of a plant treated with an
illustrative embodiment of the invention.
[0050] FIG. 7 is a chart that presents data that were collected at
Carmi, Ill. with treatments on hybrid corn during the 2012 growing
season.
[0051] FIG. 8 is a chart that shows that the interaction of the
components of an illustrative embodiment of the invention.
[0052] FIG. 9 is a chart that presents data that were collected at
Ashkum, Ill. with treatments on hybrid corn during the 2012 growing
season.
[0053] FIG. 10 is another chart that shows that the interaction of
the components of an illustrative embodiment of the invention.
[0054] FIG. 11 is a chart that presents data that were collected at
Olivia, Minn. with treatments on hybrid corn during the 2012
growing season.
[0055] FIG. 12 is another chart that shows that the interaction of
the components of an illustrative embodiment of the invention.
[0056] FIG. 13 is a chart presenting yield data on soybeans that
were grown in a field trial at the Irrigation Research Foundation,
Yuma, CO in the growing season 2012.
DETAILED DESCRIPTION OF THE INVENTION
[0057] This disclosure incorporates by reference the disclosures of
U.S. Pat. Nos. 6,808,917, 7,429,477, and 8,148,138 and U.S. Patent
Application Publication No. US 2005-0096225 A1 as if fully set
forth herein.
[0058] In an illustrative embodiment, the invention comprises four
components: a Trichoderma virens component, a Bacillus
amyloliquefaciens component, a mycorrhizal fungi component, and a
phytate or phytic acid component.
[0059] In an illustrative embodiment of the invention, the
Trichoderma virens component (e.g., a composition comprising
Trichoderma virens G1-3) is grown using solid substrate techniques.
The fungal culture is first grown using submerged culture liquid
fermentation. The culture is evaluated for contaminants using
hemacytometer counts and serial dilution on potato dextrose agar
(PDA) and is then incubated at 20 degrees Centigrade (C) for 48
hours and on tryptic soy agar (TSA) and is then incubated at 37
degrees C. for 24 and 48 hours to check for contaminants in the
culture. Pure liquid culture is then sprayed evenly on fermentation
beds containing a solid substrate (e.g., barley, rice hulls, wheat
bran, or an organic nutrient substrate, such as paper pulp
supplemented with inorganic nutrient sources). Penicillin is added
at this stage to insure that no bacterial contaminants are present.
After incubation on the fermentation beds, the culture is evaluated
for contaminates using previously described methods, and once the
culture is determined not to have contaminants, it is dried to 15
percent moisture by weight. Spores are then separated from the
substrate using vibratory separators and evaluated for titer
concentration and contamination using serial dilution grown on PDA
and TSA as well as hemacytometer counts. The culture is then
standardized using dextrose to 1.times.10e9 colony forming units
per gram (cfu/g) and final titer concentration and contaminate
evaluation using serial dilution onto PDA is done to confirm
product integrity.
[0060] The Trichoderma virens (e.g., Trichoderma virens G1-3)
component of the invention has a preferred application
concentration of 5E8 cfu/g (if a liquid suspension, cfu/ml) and is
preferably applied at 1.35 g (1.35 ml) per acre. An acceptable
concentration range is from 1.0E7 to 1.0E11 cfu/g of viable
Trichoderma virens G1-3 spores per gram of Trichoderma virens
component. In an illustrative embodiment, the percent by weight of
this component may range between 1 percent and 99 percent of the
total weight of Trichoderma virens spores and Bacillus
amyloliquefaciens spores.
[0061] An illustrative embodiment of the invention comprises the
fungus Trichoderma virens isolate G1-3 (ATCC 58678) or other
isolates. These microorganisms may be obtained from the American
Type Culture Collection (ATCC), 10801 University Blvd., Manassas,
Va. 20110 and other culture collections or isolated from
nature.
[0062] Another illustrative embodiment of the invention comprises
Trichoderma (Gliocladium) virens isolate G1-21 which is being
marketed under the trademark SoilGuard.RTM. 12G by Certis USA
L.L.C., 9145 Guilford Road, Suite 175, Columbia, Md. 21046.
[0063] In addition to Trichoderma virens strain G1-3 and
Trichoderma virens strain G1-21, other strains that may be used to
make this invention include the following: Trichoderma virens T-1
(ATCC 9645), Trichoderma virens NCTC 7057 (ATCC 11043), Trichoderma
virens NCTC 7056 (ATCC 10044), Trichoderma virens NCTC 7055 (ATCC
10045), Trichoderma virens 167 (ATCC 13213), Trichoderma virens
UCLA 230 (ATCC 13362), Trichoderma virens 031 (ATCC 20903),
Trichoderma virens 035 (ATCC 20904), Trichoderma virens 035 (ATCC
20904), Trichoderma virens 41 (ATCC 20906), Trichoderma virens ANA
215 (ATCC 24290), Trichoderma virens IFO 8349 (ATCC 44734),
Trichoderma virens NRRL 1828 (ATCC 44734), Trichoderma virens ATCC
48179, Trichoderma virens GV-P (ATCC 52045) Trichoderma virens
290-4 (ATCC 52199), Trichoderma virens s G1-17 (ATCC 58676),
Trichoderma virens G1-9 (ATCC 58677), Trichoderma virens TUB F-109
(ATCC 62399), Trichoderma virens PREM 47610 (ATCC 64271)
Trichoderma virens MF5783 (ATCC 74180), Trichoderma virens ATCC
204067, Trichoderma virens s IBT 7706 (ATCC 20443), Trichoderma
virens IBT 9354 (ATCC 20444), Trichoderma virens s IBT 9355 (ATCC
204445), Trichoderma virens G-4 (MYA-297), Trichoderma virens G-6
(MYA-298), and Trichoderma virens GJS 95-194 (MYA-1298).
[0064] Because Trichoderma virens is widespread in the soil
environment, new strains may be isolated in the future and used in
other embodiments of the invention. Trichoderma virens may be
isolated by sampling soil or plant tissue and utilizing serial
dilutions to plate out samples onto PDA plates plus penicillin (to
reduce bacterial contamination). The colonies are then further
isolated. Identity confirmation is performed through DNA
sequencing. Other Trichoderma species that have similar
characteristics to T. virens and that may be used in the invention
include T. viride, T. harzanium, T. asperellum and T. gamsii and
others. Additionally, since the calcium ion is vital to the
stabilization of B. amyloliquefaciens phytase, an alternative
embodiment involves adding a soluble calcium component or another
calcium solubilizing fungus to the soil. Alternatively, any fungus
that releases free calcium through a novel enzymatic process that
does not require acidification of the calcium molecule may be used.
Calcium-solubilizing fungi may be obtained through culture
collections (i.e. ATCC; NRRL). Species known to be tri-calcium
phosphate solubilizers include T virens, T. viride, and T.
harzanium. A soluble form of calcium may be obtained from a number
of agricultural fertilizer dealers in forms such as calcium sulfate
or calcium EDTA. In addition, granular calcium (99.0%) may be
obtained from chemical suppliers including Sigma-Aldrich.
[0065] In an illustrative embodiment of the invention, the Bacillus
amyloliquefaciens component (e.g., Bacillus amyloliquefaciens
TJ-1000) is grown using submerged culture liquid fermentation. The
cultures are quality control tested for contamination using serial
dilution to 10E12 and grown on PDA and TSA plates. The plates are
incubated at 37 degrees C. and evaluated for contamination at 24
and 48 hours. If plate inspection does not reveal any
contamination, the culture is then concentrated to remove most of
the spent fermentation media. The remaining pellets are then
lyophilized. After lyophilization, titer concentration is
determined by suspending 10 grams of lyophilized spore powder into
90 ml sterile water containing a surfactant. Serial dilution is
used to determine titer concentration of the viable spores and to
check for contamination. The culture is then blended with dextrose
to standardize the spore powder to 1.0E11 cfu/g.
[0066] A preferred application rate for the Bacillus
amyloliquefaciens component is a solid application rate of 1.35 g
per acre at a concentration of 1E10 cfu/g of Bacillus
amyloliquefaciens or a liquid application rate of 1.35 ml per acre
at a concentration of 1E10 cfu/ml. The concentration range may vary
from 1E7 cfu/g to 5E11 cfu/g. In an illustrative embodiment, the
percentage by weight of this component ranges from 1 percent to 99
percent of the total weight of Trichoderma virens spores and
Bacillus amyloliquefaciens spores.
[0067] A further illustrative embodiment of the invention comprises
the bacterium Bacillus amyloliquefaciens TJ1000 or 1BE. This
microorganism was deposited with the ATTC on Oct. 31, 2001, and was
assigned accession number ATCC BAA-390. Alternative embodiments of
the invention comprise other Bacillus amyloliquefaciens strains
which may be isolated from nature or obtained from ATCC or other
culture collections.
[0068] Another illustrative embodiment of the invention comprises
Bacillus amyloliquefaciens strain FZB24 which is marketed under the
trademark Taegro.RTM. by Earth Bioscience, Inc., 26 Sherman Court,
PO Box 764, Fairfield, Conn. 06430.
[0069] Other Bacillus amyloliquefaciens strains that may be used to
make the invention include B. amyloliquefaciens B-543 (NRRL), B.
amyloliquefaciens B-644 (NRRL), B. amyloliquefaciens B-645 (NRRL),
B. amyloliquefaciens B-942 (NRRL), B. amyloliquefaciens NRS-763
(NRRL), B. amyloliquefaciens IFO 15535 (ATTC 23350), B.
amyloliquefaciens T (ATCC 23842), B. amyloliquefaciens SB-1 (ATCC
23844), B. amyloliquefaciens P (ATCC 23844), B. amyloliquefaciens N
(ATCC 23845), B. amyloliquefaciens K49 (ATCC 27505), B.
amyloliquefaciens RUB 500H (ATCC 31592), B. amyloliquefaciens RUB
500 (ATCC 49763), B. amyloliquefaciens H (ATCC 53495), and B.
amyloliquefaciens 3002 (ATCC 700385). B. amyloliquefaciens is a
naturally occurring soil/plant bacteria and future strains may be
isolated or redesignated as B. amyloliquefaciens and used to make
the invention. B. amyloliquefaciens may be obtained from soil,
plant tissue and silage and isolated using serial dilutions and may
be classified using DNA sequencing (16S RNA).
[0070] Furthermore, in an alternative embodiment, the phyC gene
which produces the stable, calcium-dependent phytase enzyme may be
produced by another bacterium that is genetically engineered to
produce the enzyme, for example, see U.S. Pat. No. 7,339,091 which
is incorporated by reference as if fully set forth herein. For this
reason, the applicant believes that any bacterium that expresses
the phyC gene may be used in the invention to encourage mycorrhizal
colonization. The transfer of a gene to another organism can be
accomplished by purifying the DNA of a Bacillus amyloliquefaciens,
designing primers corresponding to the phyC gene sequence, and
amplifying the sequence through Polymerase Chain Reaction (PCR).
The amplified fragments can then be purified and transferred to
another organism by use of a cloning vector. Confirmation that the
gene has been inserted by PCR amplification may be accomplished by
means of hybridization utilizing the primers used for the initial
PCR amplification. A bacterium producing a phytase enzyme may be
obtained through culture collections (i.e., ATTC, NRRL). Species
known to be phytase producers include B. amyloliquefaciens and B.
subtilis. A phytase enzyme may also be obtained through commercial
chemical suppliers such as Sigma-Aldrich. Additionally, many
phytase enzymes are used as feed additives for swine and chickens.
One such phytase enzymes is Ronozyme.RTM., a product of DSM.
[0071] In another alternative embodiment, B. amyloliquefaciens is
grown in a broth and the phytase produced is combined with
Mycorrhizae, T. virens, and phytate, thereby producing a
combination having the same effect as incorporation of a Bacillus
amyloliquefaciens bacterium in the invention. For the production of
the phytase enzyme, a simple growth media (e.g., a glucose media)
is supplemented with phytate and calcium. A B. amyloliquefaciens or
a genetically modified organism containing the phyC gene is
inoculated into the growth media. The phytase enzyme is produced in
adequate quantities due to the availability of phytate and calcium.
The vegetative cells are removed from the media through a process
of centrifugation and the phytase growth media is used to produce
an illustrative embodiment of the invention.
[0072] The mycorrhizal fungi component of the invention may be
cultivated through the following steps. Corn seeds are surface
sterilized (most crop seeds may be used but fibrous root crops tend
to produce more hyphal branching) and pre-germinated on germination
paper. A low phosphorus medium for growing corn plants is
sterilized and propagule inoculums of G. intraradices, G.
etunicatum, G. aggregatum, and G. mossae are obtained from a
culture collection. In this embodiment, four separate batches of
medium containing one part by volume mycorrhizal inoculums to 20
parts by volume of growing medium are prepared (one batch for each
Glomus species). The inoculated media are added to 6-10 inch pots.
Four to six corn seedlings are planted per pot and allowed growth
for 14-16 weeks. The plants are watered daily and are fertilized
every week with a low phosphorus fertilizer.
[0073] The plants are harvested by removing the roots from the pots
and cutting them into small fragments of 1 cm to 2 cm in length.
The root fragments from the four Glomus species are then preferably
mixed together and are used as the mycorrhizal fungi component of
the invention. The preferred application rate of the four Glomus
species is 30,000 total propagules per acre (or 0.136 g at 220,000
propagules/gram). The range of propagules per gram may vary from 50
propagules to 220,000 propagules per gram. This component
preferably comprises one percent to 99 percent of the weight of the
other biological components of the invention
[0074] Another illustrative embodiment of the invention comprises a
mycorrhizal fungi component which is marketed under the trademark
MycoApply.RTM. by Mycorrhizal Applications Inc., 810 NW E St.,
Grants Pass, Oreg. 97526.
[0075] In addition to Glomus intraradices, Glomus etunicatum,
Glomus aggregatum, and Glomus mossae, other Glomus species may be
used to make mycorrhizal fungi component of the invention,
including the following: G. albidum, G. caledonium, C. claroideum,
G. clarum, G. clavispora, G. constrictum, G. coronatum, G.
deserticola, G. diaphanum, G. eburneum, G. fragilistratum, G.
gerosporum, G. globiferum, G. hadleyi, G. hyalinum, G. insculptum,
G. lamellosum, G. luteum, G. macrocarpum, G. manihot, G.
microaggregatum, G. mirificum, G. monosporum, G. pustulatum, G.
sinuosum, G. spurucum, G. tortuosum, G. verruculosum, G.
versiforme, and G. viscosum (available from INVAM-West Virginia
University). The following endomycorrhizal species may also be used
to make the mycorrhizal fungi component of the invention:
Ambisporaceae spp.; Archaeosporaceae spp. [Ar. leptoticha, Ar.
gerdemannii, and A. trappei (available from INVAM-West Virginia
University)] Geosiphonaceae spp., Acaulosporaceae spp. [A.
colossica, A. delicatta, A. denticulate, A. foveata, A. koskei, A.
lacunosa, A. laevis, A. longula, A. mellea, A. morrowiae, A.
rehmii, A. scrobiculata, A. spinosa, and A. tuberculata (available
from INVAM-West Virginia University)]; Enterophosporaceae spp. (E.
colombiana, E. contigua, E. infrequens, E. kentinesis),
Dicersisporaeceae spp, Gigasporaceae spp. [including Gi. albida,
Gi. decipiens, Gi. gigantea, Gi. margarita, and Gi. rosea)
(available from INVAM, West Virginia University)]; Paraglomus spp.
(P. brasilianum and P. occultum (available from INVAM-West Virginia
University)]; and Scutellospora spp (S. calospora, S. cerradensis,
S. coralloidea, S. dipurpurascens, S. erythropa, S. fulgida, S.
gregaria, S. heterogama, S. pellucida, S. persica, S. reticulate,
S. rubra, S. scutata, and S. verruscosa (available from INVAM-West
Virginia University)]. Arbuscular mycorrhizal fungi are naturally
occurring soil fungi, and new strains and species may be discovered
in the future and used to make the invention.
[0076] Another illustrative embodiment of the invention comprises a
phytate or phytic acid component which may be obtained from
Northwest Scientific, Inc., PO Box 1811 Billings, Mont. 59103. The
preferred application rate of phytate is one quart per acre of a 40
percent phytate solution. Because high amounts of phytate do not
inhibit mycorrhizal or plant growth, higher application rates may
be used. The concentration of phytate in solution may range from
one percent to 99 percent weight of phytate as a percentage of the
weight of the solution or standard fertilizer chemistry, e.g., a
solution containing 9-18-9 or 10-34-0 Nitrogen (N)-Phosphorus
(P)-Potassium (K). Thus, water or a solution having a standard
fertilizer chemistry, i.e., 10N-34P-0K may be used as the delivery
liquid for the phytate/biological composition. Phytate is usually a
byproduct of agricultural crop processing or a byproduct of manure
treatment/bioreactor facilities. Thus, phytate/phytic acid may be
obtained from corn, soybeans, wheat, rice, manure, etc.
[0077] In an illustrative embodiment, the invention is a
composition of matter comprising: a combination of phytate and a
plurality of microorganisms comprising a Trichoderma virens fungus,
a Bacillus amyloliquefaciens bacterium, and one mycorrhiza fungus
or a plurality of mycorrhizae fungi that is placed in the vicinity
of a plant root in a manner that allows said plurality of
microorganisms in the composition of matter to colonize said plant
root.
[0078] In another embodiment, the invention is a method for
increasing plant yield comprising: placing a combination of phytate
and a plurality of microorganisms comprising a Trichoderma virens
fungus, a Bacillus amyloliquefaciens bacterium, and a plurality of
mycorrhizae fungi in the vicinity of a plant root in a manner that
allows said microorganisms to colonize said plant root. In another
embodiment, said composition is placed in the vicinity of said
plant root by application to a preplanted seed, by in-furrow
application as a seed is being planted, or by broadcast over a seed
row.
[0079] In an illustrative embodiment, the invention is a method
comprising applying the following composition of matter to each
acre of cropland: about one quart of an about 40 percent (by
weight) phytate solution; about one gallon to five gallons of water
or water plus a standard NPK fertilizer; 1.35 g of a B.
amyloliquefaciens TJ1000 spore composition (at a concentration of
about 1E10 cfu/g); 1.35 g of a T. vixens G1-3 spore composition (at
a concentration of about 5.0E8 cfu/g); and 0.136 g of a Mycorrhizae
propagule composition (at a concentration of about 220,000
propagules/g). The biological components are preferably mixed in a
fertilizer applicator tank and applied in furrow or banded in close
proximity to the seed furrow or plant root.
[0080] Regarding the Bacillus amyloliquefaciens component of the
invention, a preferred dry, lyophilized formulation has a spore
count of about 1E10 cfu/g of Bacillus amyloliquefaciens TJ1000
spores and is applied at a preferred rate of 1.35 grams of Bacillus
amyloliquefaciens component per acre. A preferred liquid
application rate is about 1.35 ml of Bacillus amyloliquefaciens
component per acre at a concentration of about 1E10 cfu/ml of
Bacillus amyloliquefaciens TJ1000 spores. An acceptable
concentration range for the solid formulation is from about 1E7
cfu/g to about 5E11 cfu/g. In a preferred embodiment of the
invention, the Bacillus amyloliquefaciens component comprises
between about one percent to about 99 percent of the combined
weight of combined weight of the combined weight of the Trichoderma
virens component and the Bacillus amyloliquefaciens component.
These values also be used with other Bacillus amyloliquefaciens
strains.
[0081] With respect to the Trichoderma virens component of the
invention, the preferred application rate for this component of the
invention is about 1.35 gram per acre at a concentration of about
5E8 cfu/gram, with a preferred concentration range of from about
1E7 cfu to about 1E11 cfu of viable Trichoderma virens G1-3 spores
per gram of Trichoderma virens component. This component as a
percent by weight may vary between about one percent to about 99 of
the combined weight of the Trichoderma virens component and the
Bacillus amyloliquefaciens component. These values may also be used
with other Trichoderma virens strains.
[0082] With respect to the mycorrhizae fungi component of the
invention, the preferred application rate of the four Glomus
species is about 30,000 propagules per acre (about 0.136 gram of
the mycorrhizae fungi component at a concentration of about 220,000
propagules/gram). The range of propagules per gram may vary from
about 50 propagules per gram to about 220,000 propagules per gram.
This component preferably comprises from about one percent to about
99 percent of the combined weight of the Trichoderma virens
component and the Bacillus amyloliquefaciens component.
[0083] With respect to the phytate component of the invention, a
preferred application rate of phytate is about one quart per acre
of an about 40 percent phytate solution. Because high amounts of
phytate will not inhibit mycorrhizal or plant growth, a larger
amount may be used. The concentration of the solution may range
from one percent to 99 percent w/w phytate/water or a NPK
fertilizer solution. Conventional farming practices have been
making use of NPK starter or pop up fertilizers which are applied
either banded or in furrow at the time of planting. The components
in this invention can be applied by themselves, or mixed with water
or with NPK fertilizers.
Working Examples
[0084] In the working examples, the four Mycorrhizae are Glomus
aggregatum, Glomus etunicatum, Glomus intraradices, and Glomus
mossae. The Bacillus amyloliquefaciens strain is Bacillus
amyloliquefaciens TJ-1000 and the Trichoderma virens strain is
Trichoderma virens G1-3. As was noted above, other Mycorrhizae
species or strains and other Trichoderma virens strains and other
Bacillus amyloliquefaciens strains may be used in practicing the
invention.
[0085] Referring to FIG. 1, the results of South Dakota (SD) corn
field trial are presented. In this field trial, corn was planted in
two blocks: one block with no fertilizer applied at planting and
the other block had 4 gallons of an industry standard liquid
phosphorus fertilizer with an analysis of 10 percent nitrogen and
34 percent phosphorus. The treatments within the blocks were
identical and are as follows:
[0086] Treatment 1--CHK: This was the untreated check that
consisted of corn seed that was treated with the industry standard
fungicide/insecticide seed treatment. This seed treatment was
consistent throughout the other treatments and blocks.
[0087] Treatment 2--Myco IF: This treatment was an in-furrow
application of mycorrhizae propagule applied at a rate of 30,000
propagules per acre with 5 gallons of water per acre as a
carrier.
[0088] Treatment 3--Myco+T.V.+B.A.IF: This treatment was an
in-furrow application of Mycorrhizae propagules applied at a rate
of 30,000 propagules per acre plus Trichoderma virens spore applied
at a rate of 4.05E08 per acre and a Bacillus amyloliquefaciens
spore applied at a rate of 1.35E10 per acre.
[0089] Treatment 4--Phytate IF: This treatment was an in-furrow
application of a 15 percent phytate solution applied at 1 gallon
per acre plus 4 gallons of water for carrier in the No Starter
Block or 4 gallons of liquid fertilizer for the 10-34-0 Block.
[0090] Treatment 5--Phytate+Myco IF: This treatment was an
in-furrow application of Mycorrhizae propagules at a rate of 30,000
propagules per acre in a solution of 15 percent phytate applied at
a rate of 1 gallon per acre plus 4 gallons of water for carrier in
the No Starter Block or 4 gallons of liquid fertilizer for the
10-34-0 Block.
[0091] Treatment 6--Phytate+T.V.+B.A. IF: This treatment was an
in-furrow application of a combination of Trichoderma virens spore
applied at a rate of 4.05E08 per acre and Bacillus
amyloliquefaciens applied at a rate of 1.35E10 per acre in a 15
percent solution of phytate at a rate of 1 gallon per acre plus 4
gallons of water for carrier in the No Starter Block or 4 gallons
of liquid fertilizer for the 10-34-0 Block.
[0092] Treatment 7--Phytate+Myco+T.V.+B.A. IF: This treatment was
an in-furrow application of a combination of Mycorrhizae propagules
at a rate of 30,000 per acre plus Trichoderma virens at a rate of
4.05E08 per acre plus Bacillus amyloliquefaciens applied at a rate
of 1.35E10 per acre in a 15 percent solution of phytate at a rate
of 1 gallon per acre plus 4 gallons of water for a carrier in the
No Starter Block or 4 gallons of liquid fertilizer for the 10-34-0
block.
[0093] Trial Conclusion: The application of Mycorrhizae propagules
alone (Treatment 2) produced a significant increase in yield in the
No Starter Block but produced no significant yield in the high
phosphorus environment in the 10-34-0 Block over the CHK (Treatment
1). This was an expected response as it was expected that
Mycorrhizae would experience difficulty producing a yield response
in a high phosphorus environment.
[0094] When mycorrhizae, Trichoderma virens, and Bacillus
amyloliquefaciens were applied together (Treatment 3) or when
phytate was added by itself (Treatment 4) there was no significant
yield increase from Treatment 2 in either the No Starter or the
10-34-0 Blocks.
[0095] Phytate plus Mycorrhizae (Treatment 5) significantly
increased yield when compared to mycorrhizae alone (Treatment 2) in
the No Starter Block but did not significantly impact yield in the
10-34-0 Block in that high phosphorus environment.
[0096] Trichoderma virens and Bacillus amyloliquefaciens plus
phytate (Treatment 6) significantly increased yield performance
over phytate alone (Treatment 4) in the No Starter Block but did
not increase the yield in the 10-34-0 Block.
[0097] Mycorrhizae, Trichoderma virens, Bacillus amyloliquefaciens,
plus phytate (Treatment 7) significantly increased yield over all
of the other treatment entries (Treatments 1 through 6) in both the
No Starter Block and the 10-34-0 Block with the high phosphorus
environment. Thig' result was indeed a surprising breakthrough and
a confirmation that Treatment 7 is both novel and surprising.
[0098] In summary, the data in FIG. 1 show that a mycorrhizal fungi
composition (Treatment 2) applied in the seed furrow increased
yield in low phosphorus conditions. However, when a mycorrhizal
fungi composition was applied in the same manner in high phosphorus
conditions (i.e., with four gallons of a 10-34-0 fertilizer) there
was no yield response. This is an expected response.
[0099] When phytate was applied in combination with a mycorrhizal
fungi combination plus a Trichoderma virens fungus plus a Bacillus
amyloliquefaciens bacterium with no starter fertilizer (Treatment
7), corn yield was increased to levels greater than the application
of a 10-34-0 fertilizer alone. In addition, the same combination
increased corn yield to greater levels even under high phosphorus
conditions and was the only treatment that produced significant
yield response with the application of a 10-34-0 fertilizer. Both
of the results produced by this treatment are surprising. It is
well know that mycorrhizal fungi are inhibited from germinating and
colonizing the roots of plants in high phosphorus environments or
in the presence of an applied phosphorus fertilizer, such as a
10-34-0 fertilizer.
[0100] Treatment 7 combined a Trichoderma virens fungus and
Bacillus amyloliquefaciens bacterium with phytate (IP6) and a
mychorrizal fungi composition to produce composition that is
surprisingly effective at increasing corn yield. The fact that this
composition increased yield more effectively that a 10-34-0
fertilizer establishes that a long-sought-after solution for
increasing plant yield was discovered by the applicant. The
invention allows replacement of a standard chemical fertilizer with
one that is microbial in its mode of action. Treatment 7 also
provided a mechanism to establish mycorrhizae fungi on the roots of
plants in the presence of phosphorus.
[0101] Referring to FIG. 2, the results of a corn grow room
experiment are presented. Corn was planted in Deepots.TM. planting
cones in a 2:1 sand:soil mixture and randomly replicated within the
trays. The treatments were placed 1 inch to the side and 1 inch
below where the seeds were planted. The plants were grown under
1000 W dual High Pressure Sodium and Metal Halide lights with a 12
hours on/12 hours off growth cycle. Plants were watered daily with
the equivalent of 1 inch water. Corn plants were harvested 28 days
after planting.
[0102] Treatment 1--Check: The CHK treatment was fungicide seed
treatment (FST) and insecticide seed treatment (IST) and this seed
treatment was consistent across the trial. The equivalent of 5
gallons of water was placed 1 inch to the side and 1 inch below the
seed.
[0103] Treatment 2--Phytate: The equivalent of 15 percent phytate
at a rate of 1 gallon per acre and water at a rate of 4 gallons per
acre were placed 1 inch to the side and 1 inch below the seed.
[0104] Treatment 3--Phytate+T.V.+B.A.: The equivalent of
Trichoderma virens spores at 4.05E08 per acre plus Bacillus
amyloliquefaciens spores at 1.35E10 per acre plus 15 percent
phytate at a rate of 1 gallon per acre and water at 4 gallons per
acre.
[0105] Treatment 3--Phytate+T.V.+B.A.: The equivalent of
Trichoderma virens spores at 4.05E08 per acre plus Bacillus
amyloliquefaciens spores at 1.35E10 per acre plus 15 percent
phytate at a rate of 1 gallon per acre and water at 4 gallons per
acre.
[0106] Treatment 4--Phytate+Myco: The equivalent of Mycorrizhae
propagules at 30,000 per acre plus 15 percent phytate at a rate of
1 gallon per acre and water at 4 gallons per acre.
[0107] Treatment 5--Phytate+Myco+T.V.+B.A.: The equivalent of
Mycorrhizae propagules at 30,000 per acre plus Trichoderma virens
spores at 4.05E8 per acre plus Bacillus amyloliquefaciens spores at
1.35E10 per acre plus 15 percent phytate at 1 gallon per acre and
water at a rate of 4 gallons per acre.
[0108] The addition of phytate alone (Treatment 2) caused an
anti-nutrient property, decreasing plant growth. The addition of
phytate+T.V.+B.A. (Treatment 3) and phytate+Myco (Treatment 4)
brought increased plant growth. Phytate+Myco+T.V.+B.A. (Treatment
5) brought the greatest yield to plant growth, and this provides
evidence that the addition of phytate, Trichoderma virens and
Bacillus amyloliquefaciens can speed up the germination and
colonization of Mycorrhizae propagules over a control and over what
mycorrhizae can do alone. This trial is further confirmation of the
field results presented in FIG. 1.
[0109] In summary, in the grow room trial (FIG. 2), plant height,
shoot mass, and root mass of Treatment 5 were all significantly
different from the control treatment 1. Treatment 5 contains all of
the components (phytate, T. virens, B. amyloliquefaciens, and
mycorrhizae fungi) that produce surprising results. This trial
repeats the response of the field trial to further substantiate the
surprising results.
[0110] FIG. 3 represents the reaction of Bacillus amyloliquefaciens
production of phytase enzyme and the impact of the of the phytase
enzyme on phytate (IP6). This reaction results in the release of 3
moles of inorganic phosphorus plus Myo Inositol Triphosphate (IP3).
Myo Inositol Triphosphate is likely acting as a signal to promote
the germination and/or root colonization of mychorrhizae.
[0111] Use of illustrative embodiments of this invention is
advantageous for the growing of most agriculturally important
crops. Conventional farming (high phosphorus fertilizer
applications, crop rotation, no crop in the ground for extended
periods of time, chemical/biological fungicides) reduces native
mycorrhizal populations. Increasing the germination and
colonization of mycorrhizal fungi allows farmers to reduce input
costs and obtain greater yields by utilizing nutrients already
present in the soil while still maintaining adequate inorganic
nutrient fertilization. Arbuscular mycorrhizal fungi colonize 80
percent of plants--mostly green, leafy plants and commercially
produced plants. This invention finds utility in the growing of
agriculturally important crops including alfalfa, barley, beans
(all), corn, cotton, millet, rice, sorghum, soybeans, sunflower,
and wheat. This invention is also applicable to a host of other
commercially important crops including but not limited to: acacia,
agapanthus, alder, almond, apple, apricot, artichoke, ash,
asparagus, aspen, avocado, bamboo, banana, basil, bayberry, beech,
begonia, black cherry, blackberry, black locust, blue gramma, box
elder, boxwood, buckeye, cacao, cactus, camellia, carrisa, carrot,
cassava, ceanothus, cedar, celery, cherry, chrysanthemum, citrus
(all), clover, coconut, coffee, coral tree, cottonwood, cowpea,
crab tree, creosote, crytomeria, cucumber, currant, cypress,
dogwood, eggplant, elm, eucalyptus, euonymus, fern, fescue, fig,
flax, flowers (mostly all), forsythia, fuchsia, gardenia garlic,
geranium, grape (all), grasses (perennials), green ash, guayule,
gum, hackberry, hawthorn, hemp, herbs (all), hibiscus, holly,
hostas, impatiens, jatropha, jojoba, juniper, kiwi, leek, lettuce,
ligustrum, lily, locust, lychee, mahogany, magnolia, mahonia,
mango, maples (all), marigolds, mesquite, mimosa, morning glory,
mulberry, myrtle, nasturtium, okra, olive, onion, pacific yew,
palms (all), pampas grass, passion fruit, papaya, paw paw, peas,
peaches, peanuts, pear, peppers (all), pistachio, persimmon,
pittosporum, plum, podocarpus, poinsettia, poplar, potato, pumpkin,
raspberry, redwood, rice, rose, rubber, ryegrass, sagebrush,
saltbrush, serviceberry, sequoia, shallot, snapdragon, sourwood,
squash, star fruit, strawberry, succulents, sudan grass, sugar
cane, sumac, sweet gum, sweet potato, sycamore, taxus, tea,
tobacco, tomato, violets, yams, yucca, and willow.
[0112] Most modern planters have a fertilizer/insecticide
applicator tank. In illustrative embodiments of this invention, the
phytate/microorganisms solution is mixed with water and/or NPK
fertilizer and applied either in a band or in furrow application.
The solution may also be broadcast applied through a sprayer either
prior to or right after seed planting. Phytate may also be applied
as a dry powder, prilled, or coated onto a prill with the
Mycorrhizae, B. amyloliquefaciens, and T. virens. It may be applied
as a band, in furrow, or broadcast in a field. The combination may
also be applied to or mixed in soil and used in greenhouses. The
combination may be applied as a seed treatment where components are
applied either as a dry treatment to the seed or applied in a
liquid solution to the seed.
[0113] In another illustrative embodiment, the invention involves
combining phytate or phytic acid (Component D) with three
microorganisms at a range of possible cfu/g values. Three
microorganisms make up three components of the invention: Component
A, Component B, and Component C.
[0114] In this embodiment, Component A is a composition comprising
a Trichoderma virens fungus. This component preferably has a viable
Trichoderma virens concentration range of between about 1.0E6 to
about 1.0E11 cfu per gram of Component A. In a preferred embodiment
of the invention, Component A preferably comprises between about
one percent to about 99 percent of the combined weight of Component
A plus Component B (termed Combination AB). A preferred application
rate of Component A is about 1.35 gram (at a concentration of about
5.0E8 cfu/gram) per acre of cropland, wherein Component A comprises
about 50 percent of the weight of Combination AB.
[0115] In this embodiment, Component B is a composition comprising
a Bacillus amyloliquefaciens bacterium. This component preferably
has a viable Bacillus amyloliquefaciens concentration range of
between about 1.0E7 to about 5.0E11 cfu/gram of Component B. In a
preferred embodiment of the invention, Component B preferably
comprises between about one percent to about 99 percent of the
combined weight of Combination AB. A preferred application rate of
Component B is about 1.35 gram (at a concentration of about 1.0E10
cfu/gram) per acre of cropland, wherein Component B comprises about
50 percent of the weight of Combination AB.
[0116] In this embodiment, Component C is a composition comprising
Glomus spp. This component preferably has a viable Glomus spp.
Concentration range of between about 5000 to about 220,000
propagules/gram. Component C preferably comprises between about one
percent to about 99 percent of the combined weight of Combination
AB. For example, the biological part of an illustrative embodiment
of the invention comprises about 98 percent by weight of
Combination AB and about 2 percent by weight of Component C. A
preferred application rate of Component C is about 0.136 gram (at a
concentration of about 220,000 propagules/gram) or about 30,000
propagules per acre of cropland.
[0117] While there is a preferred lower limit of the Glomus
propagules application rate (e.g., 5,000 propagules per acre of
cropland), there is (other than economics) no upper limit to Glomus
propagule application per acre of cropland. The following chart
describes a preferred minimum and maximum ratios of Bacillus
amyloliquefaciens cfu and Trichoderma vixens cfu to Glomus
propagules.
TABLE-US-00001 Bacillus Bacillus Trichoderma Trichoderma
amyloliquefaciens amyloliquefaciens virens virens minimum Maximum
minimum maximum Per each Glomus 3.3E2 cfu 1.7E7 cfu 3.3E1 cfu 3.3E6
cfu propagule
[0118] The chart above shows the lower and upper limits of what the
applicant believes is a range of the effective numbers of Bacillus
spores and Trichoderma spores per each Glomus propagule. The upper
end of the range is believed to be appropriate for field (crop
land) environments but lower rates may be effective in more
controlled environments, e.g., in greenhouse settings.
[0119] In this embodiment, Component D is a composition comprising
phytate. A preferred application rate for Component D is about one
quart of an about 40 percent phytate solution per acre. The
solution may be with either water or water plus a standard
fertilizer (Nitrogen (N), Phosphorus (P), Potassium (K) (for
example, a 10-34-0 fertilizer). The phytate concentration may range
from about 1 percent to about 90 percent of the weight of the total
solution. Alternatively, the phytate component may also be applied
in salt form, i.e., as a 99 percent calcium phytate molecule.
[0120] Referring to FIG. 3, a schematic diagram illustrating the
Bacillus amyloliquefaciens reaction is presented. This figure
illustrates that using a phytase enzyme to reduce phytate and
release readily-plant-available phosphorus in the rhizosphere
results in a signal that facilitates germination and subsequent
colonization of plant roots by mycorrhizal fungi.
[0121] Referring to FIG. 4, a photograph is presented that was
produced by staining a corn root with trypan blue. The corn root
was taken from a field where the treatment applied was
Phytate+Mycorrhizae+T. virens+B. amyloliquefaciens and 3 gallons of
10-34-0 in furrow at planting. When the root sample was taken 18
days after emergence, the root was already showing healthy
mycorrhizal colonization both outside and inside the root. The
magnification of the root is 50 times (50.times.).
[0122] Referring to FIG. 5, a photograph is presented of the roots
of corn plants that are 28 days old. This photograph provides
empirical evidence that the illustrative embodiment of the
invention used is effective in producing signals that cause
Mycorrhizae propagules to germinate and colonize the root of corn
in a high phosphorus environment. The increase in root
proliferation is caused by mycorrhizae increasing root growth.
[0123] Referring to FIG. 6, a photograph is presented of a root
with Mycorrhizae stained blue with trypan blue and photographed
under a microscope at 50.times. magnification. This photograph
shows the mycorrhizal hyphae structure colonizing inside the
root.
[0124] Referring to FIG. 7, a chart is presented that shows data
that were collected at Carmi, Ill. with treatments on hybrid corn
during the 2012 growing season. The location was under significant
drought pressure as indicated by the low yield. The yield of corn
under average moisture conditions is expected to be 200+ bushels
per acre in this area. These data show that the illustrative
embodiment of the invention used was effective under very low
moisture conditions. The location has a standard application of
fertilizer for a 200+ bushel yield, which would provide a high
phosphorus environment. Use of the illustrative embodiment of the
invention clearly produced an improved yield response of 16+
bushels per acre that was provided by the Mycorrhizae root
colonization in spite of the high phosphorus conditions. These data
show the in-furrow application of Mycorrhizae+Phytate+Trichoderma
virens+Bacillus amyloliquefaciens increased yield in this high
phosphorus-low moisture environment. The treatments were as
follows:
[0125] Base=Industry standard fungicide and insecticide seed
treatment
[0126] Base+Myco+Phytate+T.V.+B.A.=Base seed treatment+an in-furrow
application of 5 gallons of a solution containing 4 strains of
Mychorrizae propagules at 30,000 propagules per acre+40% Phytate at
1 quart per acre+Trichoderma virens at 4.20E9 colony forming units
per acre, Bacillus amyloliquefaciens at 8.40E10 colony forming
units per acre.
[0127] Referring to FIG. 8, more data from Carmi, Ill. are
presented. This chart shows that the interaction of the components
of the illustrative embodiment of the invention in an in-furrow
application are not hindered by the T. virens+B. amyloliquefaciens
active ingredients applied to the seed. In fact, the combined Seed
Applied (SA) T. virens+B. amyloliquefaciens and the in furrow
application of the invention resulting a 23+ bushel per acre yield
advantage. The treatments were as follows:
[0128] Base=Industry standard fungicide and insecticide seed
treatment
[0129] Base+T.V.+B.A.-SA=Base+Trichoderma virens+Bacillus
amyloliquefaciens that is seed applied (SA).
[0130] Base+Myco+Phytate+T.V.+B.A.=Base seed treatment+an in-furrow
application of 5 gallons of a solution containing 4 strains of
Mychorrizae propagules at 30,000 propagules per acre+40% Phytate at
1 quart per acre+Trichoderma virens at 4.20E9 colony forming units
per acre, Bacillus amyloliquefaciens at 8.40E10 colony forming
units per acre.
[0131] Base+T.V+B.A-SA+Myco+Phytate+T.V+B.A.=Base+T.V.+B.A.(SA)
plus an in-furrow application of 5 gallons of a solution containing
4 strains of Mychorrizae propagules at 30,000 propagules per
acre+40% Phytate at 1 quart per acre+Trichoderma virens at 4.20E9
colony forming units per acre, Bacillus amyloliquefaciens at
8.40E10 colony forming units per acre.
[0132] Referring to FIG. 9, a chart is presented that shows data
collected at Ashkum, Ill. with treatments on hybrid corn during the
2012 growing season. The location was under significant drought
pressure as indicated by the low yield, however, drought pressure
was not a great as at the Carmi Ill. location. The yield of corn
under average moisture conditions is expected to be 200+ bushels
per acre in this area. These data show that the illustrative
embodiment of the invention used is effective under very low
moisture conditions. The location has a standard application of
fertilizer for a 200+ bushel yield, which would provide a high
phosphorus environment. The data clearly show the improved yield
response of 32+ bushels per acre provided by the Mycorrhizae root
colonization as a result of use of an illustrative embodiment of
the invention in spite of the high phosphorus conditions. The data
confirm in-furrow application of Mycorrhizae+Phytate+Trichoderma
virens+Bacillus amyloliquefaciens increased yield in this high
phosphorus-low to medium moisture environment. The treatments were
as follows:
[0133] Base=Industry standard fungicide and insecticide seed
treatment
[0134] Base+Myco+Phytate+T.V.+B.A.=Base seed treatment+an in-furrow
application of 5 gallons of a solution containing 4 strains of
Mychorrizae propagules at 30,000 propagules per acre+40% Phytate at
1 quart+Trichoderma virens at 4.20E9 colony forming units per acre,
Bacillus amyloliquefaciens at 8.40E10 colony forming units per
acre.
[0135] Referring to FIG. 10, data obtained at Ashkum, Ill. are
presented. The chart shows that the interaction of the components
of an illustrative embodiment of the invention in an in-furrow
application are not hindered by the T. virens+B. amyloliquefaciens
active ingredients applied to the seed. In fact, the combined Seed
Applied (SA) T. virens+B. amyloliquefaciens and the in furrow
application of the invention resulting a 38+ bushel per acre yield
advantage. The treatments were as follows:
[0136] Base=Industry standard fungicide and insecticide seed
treatment
[0137] Base+T.V.+B.A.-SA=Base+Trichoderma virens+Bacillus
amyloliquefaciens that is seed applied (SA).
[0138] Base+Myco+Phytate+T.V.+B.A.=Base seed treatment+an in-furrow
application of 5 gallons of a solution containing 4 strains of
Mychorrizae propagules at 30,000 propagules per acre+40% Phytate at
1 quart per acre+Trichoderma virens at 4.20E9 colony forming units
per acre, Bacillus amyloliquefaciens at 8.40E10 colony forming
units per acre.
[0139] Base+T.V+B.A-SA+Myco+Phytate+T.V+B.A.=Base+T.V.+B.A.(SA)
plus an in-furrow application of 5 gallons of a solution containing
4 strains of Mychorrizae propagules at 30,000 propagules per
acre+40% Phytate at 1 quart per acre+Trichoderma virens at 4.20E9
colony forming units per acre, Bacillus amyloliquefaciens at
8.40E10 colony forming units per acre.
[0140] Referring to FIG. 11, a chart is presented that shows data
collected at Olivia, Minn. with treatments on hybrid corn during
the 2012 growing season. This location had normal precipitation
unlike the locations at Carmi and Ashkum, Ill. The yield of corn
under average moisture conditions are expected to be 200+ bushels
per acre in this area. These data show that the illustrative
embodiment of the invention used is effective under adequate or
normal moisture conditions. The location has a standard application
of fertilizer for a 200+ bushel yield, which would provide a high
phosphorus environment. Use of the illustrative embodiment of the
invention clearly produced an improved yield response of 33+
bushels per acre provided by the Mycorrhizae root colonization in
spite of the high phosphorus conditions. The data show the
in-furrow application of Mycorrhizae+Phytate+Trichoderma
virens+Bacillus amyloliquefaciens increased yield in this high
phosphorus-adequate moisture environment. The treatments were as
follows:
[0141] Base=Industry standard fungicide and insecticide seed
treatment
[0142] Base+Myco+Phytate+T.V.+B.A.=Base seed treatment+an in-furrow
application of 5 gallons of a solution containing 4 strains of
Mychorrizae propagules at 30,000 propagules per acre+40% Phytate at
1 quart per acre+Trichoderma virens at 4.20E9 colony forming units
per acre, Bacillus amyloliquefaciens at 8.40E10 colony forming
units per acre.
[0143] Referring to FIG. 12, a chart is presented that shows data
from Olivia, Minn. The chart shows that the interaction of the
components of the illustrative embodiment of the invention used in
an in-furrow application are not hindered by the T. virens+B.
amyloliquefaciens active ingredients applied to the seed. In fact,
the combined Seed Applied (SA) T. virens+B. amyloliquefaciens and
the in furrow application of the invention resulting a 44+ bushel
per acre yield advantage. The treatments were as follows:
[0144] Base=Industry standard fungicide and insecticide seed
treatment
[0145] Base+T.V.+B.A.-SA=Base+Trichoderma virens+Bacillus
amyloliquefaciens that is seed applied (SA).
[0146] Base+Myco+Phytate+T.V.+B.A.=Base seed treatment+an in-furrow
application of 5 gallons of a solution containing 4 strains of
Mychorrizae propagules at 30,000 propagules per acre+40% Phytate at
1 quart per acre+Trichoderma virens at 4.20E9 colony forming units
per acre, Bacillus amyloliquefaciens at 8.40E10 colony forming
units per acre.
[0147] Base+T.V+B.A-SA+Myco+Phytate+T.V+B.A.=Base+T.V.+B.A.(SA)
plus an in-furrow application of 5 gallons of a solution containing
4 strains of Mychorrizae propagules at 30,000 propagules per
acre+40% Phytate at 1 quart+Trichoderma virens at 4.20E9 colony
forming units per acre, Bacillus amyloliquefaciens at 8.40E10
colony forming units per acre.
[0148] Referring to FIG. 13, a chart is presented that shows yield
data on soybeans that were grown in a field trial at the Irrigation
Research Foundation, Yuma, CO in the growing season 2012. At this
trial location, the soybeans were irrigated and a fertilizer with
the analysis of 15-20-0-2s-.027ZN was applied in furrow at a rate
of 4.5 gallons per acre. The data again confirm that an
illustrative embodiment of the invention performs in a similar
manner on a dicot legume, in this case soybeans, just as it does on
a monocot, corn in the presence of high phosphorus. The data also
confirm that the use of a seed applied (SA) T virens+B.
amyloliquefaciens continued to allow the embodiment of the
invention used to produce increased yield. The overall increase of
the combined treatments was 6.7 bushel of soybeans per acre. The
treatments were as follows:
[0149] Base=Industry standard fungicide and insecticide seed
treatment
[0150] Base+T.V.+B.A.-SA=Base+Trichoderma virens+Bacillus
amyloliquefaciens that is seed applied (SA).
[0151] Base+T.V+B.A-SA+Myco+Phytate+T.V+B.A.=Base+T.V.+B.A.(SA)
plus an in-furrow application of 5 gallons of a solution containing
4 strains of Mychorrizae propagules at 30,000 propagules per
acre+40% Phytate at 1 quart per acre+Trichoderma virens at 4.20E9
colony forming units per acre, Bacillus amyloliquefaciens at
8.40E10 colony forming units per acre.
[0152] Many variations of the invention will occur to those skilled
in the art. Some variations include liquid formulations. Other
variations call for solid formulations. All such variations are
intended to be within the scope and spirit of the invention.
[0153] Although some embodiments are shown to include certain
features or steps, the applicant specifically contemplates that any
feature or step disclosed herein may be used together or in
combination with any other feature or step in any embodiment of the
invention. It is also contemplated that any feature or step may be
specifically excluded from any embodiment of the invention.
* * * * *