U.S. patent application number 15/880173 was filed with the patent office on 2018-06-14 for lipo-chitooligosaccharides combination compositions for enhanced plant growth and yield.
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 Robert Martin Osburn, Raymond Stewart Smith.
Application Number | 20180160685 15/880173 |
Document ID | / |
Family ID | 39609001 |
Filed Date | 2018-06-14 |
United States Patent
Application |
20180160685 |
Kind Code |
A1 |
Smith; Raymond Stewart ; et
al. |
June 14, 2018 |
Lipo-Chitooligosaccharides combination compositions for enhanced
plant growth and yield
Abstract
Compositions and methods for enhancing plant growth and crop
yield in legumes and non-legumes are described. The compositions
include lipo-chitooligosaccharides in combination with
chitins/chitosans or in combination with flavonoid compounds or in
combination with a herbicide. The method includes applying the
compositions to seeds and/or plants either concomitantly or
sequentially.
Inventors: |
Smith; Raymond Stewart;
(Pewaukee, WI) ; Osburn; Robert Martin; (Mequon,
WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NOVOZYMES BIOAG A/S |
Bagsvaerd |
|
DK |
|
|
Assignee: |
NOVOZYMES BIOAG A/S
Bagsvaerd
DK
|
Family ID: |
39609001 |
Appl. No.: |
15/880173 |
Filed: |
January 25, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15005478 |
Jan 25, 2016 |
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15880173 |
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13714556 |
Dec 14, 2012 |
9253989 |
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15005478 |
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12521375 |
Jun 26, 2009 |
8357631 |
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PCT/US2008/000235 |
Jan 8, 2008 |
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13714556 |
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60879436 |
Jan 9, 2007 |
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60980287 |
Oct 16, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01N 39/04 20130101;
A01N 43/88 20130101; A01N 63/10 20200101; A01N 2300/00 20130101;
Y02A 40/10 20180101; A01N 41/06 20130101; A01N 43/50 20130101; A01N
43/40 20130101; A01N 35/10 20130101; A01N 43/16 20130101; A01N
41/10 20130101; Y02A 40/143 20180101; A01N 37/48 20130101; A01N
43/80 20130101; A01N 43/16 20130101; A01N 35/10 20130101; A01N
37/48 20130101; A01N 39/04 20130101; A01N 41/06 20130101; A01N
41/10 20130101; A01N 43/16 20130101; A01N 43/40 20130101; A01N
43/50 20130101; A01N 43/80 20130101; A01N 43/88 20130101; A01N
47/36 20130101; A01N 57/20 20130101; A01N 63/10 20200101; A01N
35/10 20130101; A01N 37/48 20130101; A01N 39/04 20130101; A01N
41/06 20130101; A01N 41/10 20130101; A01N 43/16 20130101; A01N
43/40 20130101; A01N 43/50 20130101; A01N 43/80 20130101; A01N
43/88 20130101; A01N 47/36 20130101; A01N 57/20 20130101; A01N
43/16 20130101; A01N 2300/00 20130101; A01N 63/10 20200101; A01N
2300/00 20130101; A01N 63/10 20200101; A01N 35/10 20130101; A01N
37/48 20130101; A01N 39/04 20130101; A01N 41/06 20130101; A01N
41/10 20130101; A01N 43/16 20130101; A01N 43/40 20130101; A01N
43/50 20130101; A01N 43/80 20130101; A01N 43/88 20130101; A01N
47/36 20130101; A01N 57/20 20130101; A01N 63/10 20200101; A01N
2300/00 20130101 |
International
Class: |
A01N 43/16 20060101
A01N043/16; A01N 63/02 20060101 A01N063/02; A01N 35/10 20060101
A01N035/10; A01N 37/48 20060101 A01N037/48; A01N 39/04 20060101
A01N039/04; A01N 41/06 20060101 A01N041/06; A01N 41/10 20060101
A01N041/10; A01N 43/40 20060101 A01N043/40; A01N 43/50 20060101
A01N043/50; A01N 43/80 20060101 A01N043/80; A01N 43/88 20060101
A01N043/88; A01N 47/36 20060101 A01N047/36; A01N 57/20 20060101
A01N057/20 |
Claims
1. A method comprising spraying corn plants with a composition
comprising at least one flavonoid in an amount effective to
increase the yield of said corn plants.
2. The method of claim 1, wherein said at least one flavonoid
comprises genistein.
3. The method of claim 1, wherein said at least one flavonoid
comprises daidzein.
4. The method of claim 1, wherein said at least one flavonoid
comprises genistein and daidzein.
5. The method of claim 1, wherein said at least one flavonoid is
present in said composition at a concentration of between about 20
.mu.M to about 800 .mu.M.
6. The method of claim 1, wherein said at least one flavonoid is
present in said composition at a concentration of between about 100
.mu.M to about 500 .mu.M.
7. The method of claim 1, wherein said at least one flavonoid is
present in said composition at a concentration of about 10 mM.
8. The method of claim 1, further comprising applying at least one
lipo-chitooligosaccharide to said corn plants.
9. The method of claim 1, wherein said composition further
comprises at least one lipo-chitooligosaccharide.
10. The method of claim 9, wherein said at least one
lipo-chitooligosaccharide is present in said composition at a
concentration of between about 10.sup.-14 M to about 10.sup.-5
M.
11. The method of claim 9, wherein said at least one
lipo-chitooligosaccharide is present in said composition at a
concentration of between about 10.sup.-10 M to about 10.sup.-6
M.
12. The method of claim 9, wherein said at least one
lipo-chitooligosaccharide is present in said composition at a
concentration of about 10.sup.-8 M.
13. The method of claim 1, wherein said composition is sprayed on
said corn plants in an amount effective to increase the yield of
said corn plants by at least 3 bushels per acre, as compared to
untreated control plants.
14. A method comprising spraying corn plants with a composition
comprising genistein and daidzein in an amount effective to
increase the yield of said corn plants.
15. The method of claim 1, wherein said genistein and daidzein are
present in said composition at a genistein:daidzein ratio of about
8:2.
16. The method of claim 1, wherein said genistein and daidzein are
present in said composition at a concentration of about 10 mM.
17. The method of claim 12, further comprising applying at least
one lipo-chitooligosaccharide to said corn plants.
18. The method of claim 12, wherein said composition further
comprises at least one lipo-chitooligosaccharide.
19. The method of claim 16, wherein said at least one
lipo-chitooligosaccharide is present in said composition at a
concentration of about 10.sup.-8 M.
20. The method of claim 14, wherein said composition is sprayed on
said corn plants in an amount effective to increase the yield of
said corn plants by at least 3 bushels per acre, as compared to
untreated control plants.
Description
BACKGROUND OF THE INVENTION
[0001] Nitrogen fixation plays a vital role in agricultural
production by making atmospheric nitrogen available in a form that
can be used by plants. In plants of the Leguminoseae family, the
symbiotic interaction between the plants and nitrogen-fixing
bacteria of the Rhizobiaceae family ("rhizobia") enhances plant
growth and crop yield. The symbiotic interaction is initiated when
a plant releases flavonoid compounds that stimulate rhizobial
bacteria in the soil to produce "Nod-factors." Nod-factors are
signaling compounds that induce the early stages of nodulation in
plant roots, which lead to the formation of root nodules containing
the nitrogen-fixing rhizobial bacteria. Although this process
occurs naturally over time in legumes, agricultural procedures have
been developed to begin the process earlier. These procedures
include providing nitrogen-fixing bacteria to seeds or soil and
applying Nod factors directly to seeds or soil prior to or at
planting.
[0002] Nod factors have recently been shown to also enhance the
germination, growth and yield of legumes and non-legumes through
processes other than nodulation (U.S. Pat. No. 6,979,664;
Prithivaraj et al., Planta 216: 437-445, 2003). Although the
effects of Nod factors on nodulation have been widely studied and
reviewed, e.g., Ferguson and Mathesius, J. Plant Growth Regulation
22: 47-72, 2003, the mechanisms for Nod factor effects independent
of nodulation are not well understood. Application of Nod factors
to seeds of legumes and non-legumes stimulates germination,
seedling emergence, plant growth and yield in crop and
horticultural plant species, e.g., as described in U.S. Pat. No.
6,979,664 and U.S. Pat. No. 5,922,316. Nod factors have also been
shown to enhance root development (Olah, et al., The Plant Journal
44:195-207, 2005). Foliar application of Nod factors has also been
demonstrated to increase photosynthesis (U.S. Pat. No. 7,250,068),
and fruiting and flowering (WO 04/093,542) In crop and
horticultural plant species.
[0003] Nod factors are lipo-chitooligosaccharide compounds (LCO's).
They consist of an oligomeric backbone of .beta.-1,4-linked
N-acetyl-D-glucosamine ("GlcNAc") residues with an N-linked fatty
acyl chain at the nonreducing end. LCO's differ in the number of
GlcNAc residues in the backbone, in the length and degree of
saturation of the fatty acyl chain, and in the substitutions of
reducing and nonreducing sugar residues. LCO structure is
characteristic for each rhizobial species, and each strain may
produce multiple LCO's with different structures. LCO's are the
primary determinants of host specificity in legume symbiosis (Diaz,
Spaink, and Kljne, Mol. Plant-Microbe Interactions 13: 268-276,
2000).
[0004] LCO synthesis can be stimulated by adding the appropriate
flavonoid, for a given genus and species of rhizobium during growth
of the bacteria. The flavonoid molecules bind to the rhizobium and
turn on bacterial genes for the production of specific LCO's which
are released into the fermentation medium. In nature, leguminous
plants release the appropriate flavonoid, which binds to soil
rhizobia, turning on genes for LCO production. These LCO's are
released by bacteria into the soil, bind to the roots of leguminous
plants, and initiate a cascade of plant gene expression that
stimulates formation of nitrogen-fixing nodule structures on legume
roots. Alternatively, modified and synthetic LCO molecules can be
produced through genetic engineering or chemical synthesis.
Synthetic LCO's of the same molecular structure interact with
plants and stimulate nodulatlon in the same manner as naturally
produced molecules.
[0005] Chitins and chitosans, which are major components of the
cell walls of fungi and the exoskeletons of insects and
crustaceans, are also composed of GlcNAc residues. These
compositions have been applied to seeds, roots, or foliage of a
broad spectrum of crop and horticultural plants. Chitin and
chitosan compositions enhance protection against plant pathogens,
in part, by stimulating plants to produce chitinases, enzymes that
degrade chitin (Collinge, et al., The Plant Journal 3: 31-40,
1993).
[0006] Flavonoids are phenolic compounds having the general
structure of two aromatic rings connected by a three carbon bridge.
Flavonoids are produced by plants and have many functions, e.g., as
beneficial signaling molecules, and as protection against insects,
animals, fungi and bacteria. Classes of flavonoids include
chalcones, anthocyanidins, coumarins, flavones, flavanols,
flavonols, flavanones, and isoflavones. (Jain and Nainawatee, J.
Plant Biochem. & Biotechnol. 11: 1-10, 2002; Shaw, et al.,
Environmental Microbiol. 11: 1867-1880, 2006.)
SUMMARY OF THE INVENTION
[0007] The invention includes methods and compositions for
increasing plant growth and crop yield. An exemplary composition
comprises at least one lipo-chitooligosaccharide and at least one
chitinous compound. Another exemplary composition comprises at
least one lipo-chitooligosaccharide and at least one flavonoid
compound selected from the group consisting of flavones, flavanols,
flavonols, flavanones, and isoflavones. A further exemplary
composition comprises at least one lipo-chitooligosaccharide and at
least one herbicide. An exemplary method comprises administering a
composition according to the invention to a plant or seed in an
effective amount for enhancing plant growth or crop yield. In
another embodiment, the method comprises sequentially treating a
plant or a seed with at least one lipo-chitooligosaccharide and at
least one chitinous compound or at least one flavonoid compound
selected from the group consisting of flavones, flavanols,
flavonols, flavanones, and isoflavones.
DETAILED DESCRIPTION OF THE INVENTION
[0008] The invention provides compositions and methods for
enhancing plant growth and crop yield, and arises from the results
of experiments, reported herein, that reveal improved effects of
lipo-chitooligosaccharide in combination with chitin/chitosan,
flavonoid compounds, or herbicidal compounds on plant growth and
crop yield when applied to seeds and/or foliage.
[0009] For the purposes of this invention, a
"lipo-chitooligosaccharide" ("LCO") is a compound having the
general LCO structure, i.e., an oligomeric backbone of
.beta.-1,4-linked N-acetyl-D-glucosamine residues with an N-linked
fatty acyl chain at the nonreducing end, as described in U.S. Pat.
No. 5,549,718; U.S. Pat. No. 5,646,018; U.S. Pat. No. 5,175,149;
and U.S. Pat. No. 5,321,011. This basic structure may contain
modifications or substitutions found in naturally occurring LCO's,
such as those described in Spaink, Critical Reviews in Plant
Sciences 54: 257-288, 2000; D'Haeze and Holsters, Glycobiology 12:
79R-105R, 2002. Also encompassed by the invention are synthetic LCO
compounds, such as those described in WO2005/063784, and LCO's
produced through genetic engineering. Precursor oligosaccharide
molecules for the construction of LCOs may also be synthesized by
genetically engineered organisms, e.g., as in Samain et al.,
Carbohydrate Research 302: 35-42, 1997.
[0010] LCO's used in embodiments of the Invention may be recovered
from Rhizobiaceae bacterial strains that produce LCO's, such as
strains of Azorhizobium, Bradyrhizobium (Including B. japonicum),
Mesorhizobium, Rhizobium (including R. leguminosarum),
Sinorhizobium (including S. meliloti), and bacterial strains
genetically engineered to produce LCO's. These methods are known in
the art and have been described, for example, in U.S. Pat. Nos.
5,549,718 and 5,646,018, which are incorporated herein by
reference. Commercial products containing LCO's are available, such
as OPTIMIZE.RTM. (EMD Crop BioScience).
[0011] LCO's may be utilized in various forms of purity and may be
used alone or with rhizobia. Methods to provide only LCO's include
simply removing the rhizobial cells from a mixture of LCOs and
rhizobia, or continuing to isolate and purify the LCO molecules
thru LCO solvent phase separation followed by HPLC chromatography
as described by Lerouge, et. al (U.S. Pat. No. 5,549,718).
Purification can be enhanced by repeated HPLC, and the purified LCO
molecules can be freeze-dried for long-term storage. This method is
acceptable for the production of LCO's from all genera and species
of the Rhizobiaceae.
[0012] Within the legume family, specific genera and species of
rhizobium develop a symbiotic nitrogen-fixing relationship with a
specific legume host. These plant host:rhizobia combinations are
described in Hungria and Stacey, Soil Biol. Biochem. 29: 819-830,
1997, which also lists the effective flavonoid Nod gene inducers of
the rhizobial species, and the specific LCO structures that are
produced by the different rhizobial species. However, LCO
specificity is only required to establish nodulation in legumes. It
is not necessary to match LCO's and plant species to stimulate
plant growth and/or crop yield when treating seeds or foliage of a
legume or non-legume with LCO's.
[0013] Chitinous compounds include chitin, (IUPAC:
N-[5-[[3-acetylamino-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]methoxymeth-
yl]-2-[[5-acetylamino-4,6-dihydroxy-2-(hydroxymethyl)oxan-3-yl]
methoxymethyl]-4-hydroxy-6-(hydroxymethyl)oxan-3-yl]ethanamide),
and chitosan, (IUPAC:
5-amino-6-[5-amino-6-[5-amino-4,6-dihydroxy-2-(hydroxymethyl)oxan-3-yl]ox-
y-4-hydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-2-(hydroxymethyl)oxane-3,4-diol-
). These compounds may be obtained commercially, e.g., from
Sigma-Aldrich, or prepared from insects, crustacean shells, or
fungal cell walls. Methods for the preparation of chitin and
chitosan are known in the art, and have been described, for
example, in U.S. Pat. No. 4,536,207 (preparation from crustacean
shells), Pochanavanich and Suntornsuk, Lett. Appl. Microbiol. 35:
17-21, 2002 (preparation from fungal cell walls), and U.S. Pat. No.
5,965,545 (preparation from crab shells and hydrolysis of
commercial chitosan). Deacetylated chitins and chitosans may be
obtained that range from less than 35% to greater than 90%
deacetylation, and cover a broad spectrum of molecular weights,
e.g., low molecular weight chitosan oligomers of less than 15 kD
and chitin oligomers of 0.5 to 2 kD; "practical grade" chitosan
with a molecular weight of about 150 kD; and high molecular weight
chitosan of up to 700 kD. Chitin and chitosan compositions
formulated for plant and soil treatment are also commercially
available. Commercial products include, for example,
ELEXA.RTM.-4PDB (Plant Defense Boosters, Inc.) and BEYOND.TM.
(Agrihouse, Inc.).
[0014] LCO's and chitins/chitosans are structurally related. Chitin
and chitosan can stimulate the production of chitinases by plants,
and it has been shown that plant chitinases may inactivate and
degrade LCO's as well as chitinous compounds (Staehelin, et al.,
P.N.A.S. USA 91: 2196-2200, 1994; Ferguson and Mathesius, J. Plant
Growth Regulation 22: 47-72, 2003)). In addition, commercially
available chitosan formulations often contain heavy metals that are
toxic to rhizobial bacteria and so prevent the production of LCOs.
For these reasons, the use of rhizobial bacteria in combination
with chitins/chitosans was previously contraindicated. However, as
shown in the examples below, it is now demonstrated that
application of an LCO compound and chitin/chitosan, either
sequentially or simultaneously, to a plant or seed induces
beneficial responses in plant growth and yield. While the mechanism
for this effect is not proven, one hypothesis is that the LCO
compounds bind to specific receptors on the plant or seed and
initiate these beneficial responses before LCO degradation by
chitinases can occur. Furthermore, this novel treatment method
obviates the effects of heavy metals on LCO production by rhizobial
bacteria.
[0015] In one embodiment of the invention, the composition may be
prepared by mixing chitosan, and one or more LCO in an
agriculturally appropriate solvent. In a second embodiment, the
composition may also contain chitin. Chitosan concentration may
range from 0.1 to 15% w/v, preferably from 3 to 12%. Chitin may be
included at from 0 to 4% w/v. The LCO concentration may range from
10.sup.-5M to 10.sup.-14M, preferably from 10.sup.-6M to
10.sup.-10M. The LCO component may consist of purified or partly
purified LCO, or a mixture of the LCO and the rhizobia that
produced the LCO. The agriculturally appropriate solvent is
preferably an aqueous solvent, such as water.
[0016] Appropriate flavonoids include compounds from the classes of
flavones, flavanols, flavonols, flavanones, and isoflavones. Such
compounds may Include, but are not limited to, genistein, daidzein,
formononetin, naringenin, hesperetin, luteolin, and apigenin.
Flavonoid compounds are commercially available, e.g., from Natland
International Corp., Research Triangle Park, N.C.; MP Biomedicals,
Irvine, Calif.; LC Laboratories, Woburn Mass. Flavonoid compounds
may be Isolated from plants or seeds, e.g., as described in U.S.
Pat. No. 5,702,752; U.S. Pat. No. 5,990,291; U.S. Pat. No.
6,146,668. Flavonoid compounds may also be produced by genetically
engineered organisms, such as yeast, as described in Ralston, et
al., Plant Physiology 137: 1375-1388, 2005.
[0017] In one embodiment of the invention, the composition may be
prepared by combining one or more flavonoid and one or more LCO in
an agriculturally appropriate solvent. An "effective amount" of the
composition is an amount that increases plant growth or crop yield
when compared with the growth or crop yield of plants or seeds that
have not been treated with the composition. For example, flavonoid
concentration in the composition may range from 20-800 .mu.m,
preferably 100-500 .mu.m. LCO concentration in the composition may
range from 10.sup.-5 M to 10.sup.-14 M, preferably from 10.sup.-6 M
to 10.sup.-10 M. The LCO component may consist of purified or
partly purified LCO, or a mixture of the LCO and the rhizobia that
produce the LCO. The agriculturally appropriate solvent Is
preferably an aqueous solvent, such as water.
[0018] Although it is efficient and convenient to combine and apply
the flavonoid or chitin/chitosan and LCO components in a single
mixture, in one embodiment of the invention the flavonoid or
chitin/chitosan component and the LCO component may be applied
separately and sequentially in either order. Other additives that
may be applied either simultaneously or sequentially include
fertilizers (e.g., calcium, nitrogen, potassium, phosphorous),
micronutrients (e.g., copper, aluminum, magnesium, manganese, and
zinc ions), and pesticides (e.g., fungicides, insecticides,
herbicides, and nematicides).
[0019] In one embodiment of the invention, a composition comprising
at least one LCO and at least one herbicide Is applied to the
foliage of a plant to Improve plant growth or crop yield. Suitable
herbicides include, but are not limited to bentazon, acifluorfen,
chlorimuron, lactofen, clomazone, fluazifop, glufosinate,
glyphosate, sethoxydim, imazethapyr, imazamox, fomesafe,
flumiclorac, imazaquin, and clethodim. Commercial products
containing each of these compounds are readily available. Herbicide
concentration in the composition will generally correspond to the
labeled use rate for a particular herbicide. LCO concentration in
the composition may range from 10.sup.-5 M to 10.sup.-14 M,
preferably from 10.sup.-6 M to 10.sup.-10 M. The agriculturally
appropriate solvent used in applying the composition is preferably
an aqueous solvent, such as water. The composition is generally
applied to the plant at any time appropriate for weed control,
preferably post-emergence.
[0020] In one embodiment, the composition comprises at least one
LCO with a glyphosate-based herbicide, and treatment comprises
application of this composition to plants that have been
genetically modified for resistance to glyphosate.
[0021] The term "plant" as used herein includes tubers, roots,
stems, leaves, flowers, and fruits. The composition may be applied
directly to seeds or plants or may be placed in soil in the
vicinity of a seed or plant prior to or at the time of planting. In
a preferred embodiment, the composition is sprayed on seeds,
tubers, or foliage. Seedlings, as well as more mature plants, may
be treated. Flowers and fruits may also be treated by spraying.
Roots of transplants may be sprayed or dipped in the composition
prior to planting.
[0022] An "effective amount" of the composition is an amount that
increases plant growth or crop yield when compared with the growth
or crop yield of plants or seeds that have not been treated with
the composition.
[0023] The composition may be applied to monocot or dicot plants,
and to legumes and non-legumes. In one embodiment, the composition
is applied to field-grown plants. In another embodiment, the
composition is applied to greenhouse-grown plants. For example, the
composition may be applied to seeds or foliage of legumes, such as
soybeans, peas, chickpeas, dry beans, peanuts, clover, alfalfa, and
of non-legumes such as corn, cotton, rice, tomatoes, canola, wheat,
barley, sugar beet, and grass. In general, for seed treatment, the
composition is applied to seeds in a single application, and the
seeds may be planted immediately or stored before planting. The
composition may be applied to foliage. Foliar application generally
consists of spraying the composition on the plant foliage one or
more times during the growing period. In addition, if the flavonoid
compound and LCO are applied sequentially, the flavonoid compound
may be applied to seeds and the LCO to foliage.
Examples
[0024] 1. Soybean (Northrup King S24-k4) Foliar Treatment with
LCO+Chitin/Chitosan
[0025] A soybean field trial was conducted to evaluate the effects
of an LCO and two commercial chitosan products on grain yield when
applied to foliage alone or in combination. The two commercial
chitosan products utilized in the trial were BEYOND.TM. (Agri-House
Inc., 307 Welch Ave, Berthoud, Colo.), and ELEXA.RTM.-4PDB (Plant
Defense Boosters, 235 Harrison St, Syracuse, N.Y.). The exact
chitin/chitosan concentration in BEYOND.TM. is unknown, but is
estimated to be in the range of 6-12% w/v chitosan and 0-3% w/v
chitin, based on U.S. Pat. No. 6,193,988. The chitosan
concentration in ELEXA.RTM.-4PDB is 4% w/v. ELEXA.RTM.-4PDB does
not contain chitin. The chitosan concentration in ELEXA.RTM.-4PDB
is 4% w/v. The LCO product was produced by Rhizobium leguminosarum
by viceae and contained approximately 1.times.10.sup.-8 M LCO. The
field trial was located near Whitewater, Wis. at a site
characterized by Milford silty clay loam soil. The soil had a pH of
6.6, an organic matter content of 4.8%, and phosphorus and
potassium contents of 41 ppm and 131 ppm, respectively.
[0026] The soybean seed used in the study was Northrup King variety
S24-K4. The LCO treatment was applied by spraying onto foliage at
the V4 growth stage (see Soybean Growth and Development, Iowa State
University Extension Bulletin PM 1945, May 2004), at a rate of 1
quart/acre in 25 gallons of water. BEYOND.TM. was diluted to a
concentration of 0.132% w/v and ELEXA.RTM.-4PDB to 2.5% w/v in
water. Each product was applied by spraying onto foliage at a rate
of 1 quart/acre in 25 gallons of water. When the
LCO-chitin/chitosan combination was applied, the same
concentrations of LCO and chitin/chitosan products were used as
when each product was applied alone.
[0027] The study was conducted in a randomized complete block
design, with a plot size of 10 feet by 50 feet, 30 inch row
spacing. Four replications were performed. Seeds were planted at a
depth of 1 Inch and a seeding rate of 175,000 seeds per acre using
a John Deere 750 NT grain drill.
[0028] Results of this study are shown in Table 1. The LCO,
BEYOND.TM., and ELEXA.RTM.-4PDB products each significantly
Increased grain yield by 3.5, 6.6, and 5.0 bu/acre, respectively,
when applied to foliage as stand-alone treatments (p=0.1).
Application of ELEXA.RTM.-4PDB In combination with LCO
statistically Increased yield by 6.2 bu/acre over LCO alone and 4.7
bu/acre over ELEXA.RTM.-4PDB alone. Application of BEYOND.TM. in
combination with LCO statistically increased yield by 5.3 bu/acre
over LCO alone, and numerically increased yield by 2.2 bu/acre over
BEYOND.TM. alone.
[0029] Treatment with LCO+ELEXA.RTM.-4PDB Increased yield compared
to the control by 9.7 bu/acre, showing an unexpected synergistic
effect of the combination compared with LCO or ELEXA.RTM.-4PDB
treatment alone.
TABLE-US-00001 TABLE 1 Treatment Grain yield (bu/acre) Control -
non-treated 56.2 LCO 59.7 BEYOND .TM. 62.8 ELEXA .RTM.-4PDB4 PDB
61.2 LCO + BEYOND .TM. 65.0 LCO + ELEXA .RTM.-4PDB 65.9 Probability
% <0.1 LSD 10% 2.6 CV % 3.5
2. Soybean (Dairyland DSR 2300SR) Foliar Treatment with
LCO+Chitosan
[0030] A soybean field trial was conducted to evaluate the effects
of an LCO and a commercial chitosan product on grain yield when
applied to foliage alone or in combination. The LCO product was the
same as that used in Example 1. The commercial chitosan product
utilized in the trial was ELEXA.RTM.-4PDB. The field trial was
located near Whitewater, Wis. at a site characterized by Milford
silty clay loam soil. The soil had a pH of 6.8, an organic matter
content of 4.8%, and phosphorus and potassium contents of 46 ppm
and 144 ppm, respectively.
[0031] The soybean seed used in the study was Dairyland variety DSR
2300RR. The study was conducted in a randomized complete block
design, with a plot size of 10 feet by 50 feet and 15 inch row
spacing. Four replications were performed. Seeds were planted at a
depth of 1 inch at a seeding rate of 185,000 seeds per acre using a
John Deere 750 NT grain drill.
[0032] Both LCO and ELEXA.RTM.-4PDB treatments were applied by
spraying onto foliage at the V4 growth stage (see Soybean Growth
and Development, Iowa State University Extension Bulletin PM 1945,
May 2004), at a rate of 1 quart/acre in 25 gallons of water using a
International Harvester Cub plot sprayer at a ground speed of 2.5
mph. When the LCO-chitosan combination was applied, the same
concentrations of LCO and chitosan products were used as when each
product was applied alone.
[0033] Results of this study are shown in Table 2. The LCO and
ELEXA.RTM.-4PDB products numerically increased grain yield by 1.7
and 0.6 bu/acre, respectively, when applied to foliage as
stand-alone treatments (p=0.1). Application of ELEXA.RTM.-4PDB in
combination with LCO numerically Increased yield by 0.8 bu/acre
over LCO alone and 1.9 bu/acre over ELEXA.RTM.-4PDB alone. The 2.5
bu/acre increase with the combined LCO and ELEXA.RTM.-4PDB exceeded
the combined benefit of the individual products alone, showing an
unexpected synergistic effect of the combination.
TABLE-US-00002 TABLE 2 Treatment Grain yield (bu/acre) Control -
nontreated 63.2 LCO 64.9 ELEXA .RTM.-4PDB4 PDB 63.8 LCO + ELEXA
.RTM.-4PDB 65.7 Probability % <0.1 LSD 10% 3.9 CV % 5.3
3. Soybean Seed (Dairyland DSR 234RR) Treatment with
LCO+Chitin/Chitosan
[0034] A soybean field trial was conducted to evaluate the effect
of an LCO and two different commercial chitin/chitosan products on
grain yield when applied on seed either alone or in combination.
The field trial site was located near Whitewater, Wis. and
characterized by Milford silty clay loam soil. Soil testing showed
a soil pH of 6.8, an organic matter content of 5.1%, and phosphorus
and potassium contents of 37 ppm and 136 ppm, respectively.
[0035] The LCO product used in the trial (OPTIMIZE.RTM., EMD Crop
BioScience) was produced by Bradyrhizobium japonicum and contained
approximately 1.times.10.sup.-9 M LCO. The two commercial chitosan
products utilized in the trial were the same as those used in
Example 1. The soybean seed used in the study was Dairyland variety
DSR 234RR. The LCO product was sprayed onto seeds without dilution
at a rate of 4.25 fl oz/cwt. BEYOND.TM. was diluted to 0.132% w/v
and ELEXA.RTM.-4PDB to 2.5% w/v with water. Each was applied on
seed at the rate of 4.25 fl oz/cwt. When the LCO-chitin/chitosan
combination was applied, the same concentrations of LCO and
chitin/chitosan products were used as when each product was applied
alone. The combined composition was applied at 4.25 fl oz/cwt.
[0036] The study was conducted in a randomized complete block
design, with a plot size of 10 feet by 50 feet, 7.5 inch row
spacing. Four replications were conducted. Seeds were treated just
prior to planting and were planted at a depth of 1 inch and a
seeding rate of 225,000 seeds per acre using a John Deere 750 NT
grain drill.
[0037] Results of the study are shown in Table 3, below. The LCO
treatment numerically increased grain yield by 2.0 bu/acre relative
to the non-treated control group (p=0.1). The chitosan products,
BEYOND.TM. and ELEXA.RTM.-4PDB, each provided statistically
significant increases of 2.5 and 3.4 bu/acre, respectively, over
the non-treated control group. The combination of LCO and
BEYOND.TM. significantly increased yield by 2.3 bu/acre relative to
the LCO treatment alone, and numerically increased yield by 1.8
bu/acre compared to the BEYOND.TM. treatment alone. Treatment with
a combination of LCO and ELEXA.RTM.-4PDB significantly increased
yield by 2.3 bu/acre compared to the LCO treatment alone and
numerically increased yield by 0.9 bu/acre relative to
ELEXA.RTM.-4PDB treatment alone.
TABLE-US-00003 TABLE 3 Treatment Grain yield (bu/acre) Control -
non-treated 55.5 LCO 57.5 BEYOND .TM. 58.0 ELEXA .RTM.-4PDB4 PDB
58.9 LCO + BEYOND .TM. 59.8 LCO + ELEXA .RTM.-4PDB 59.8 Probability
% 9.6 LSD 10% 2.3 CV % 3.3
4. Corn Seed (Shur Grow SG-686-RR) Treatment with
LCO+Chitin/Chitosan
[0038] A corn field trial was conducted to evaluate the effects of
an LCO and commercial chitosan product on grain yield when applied
on seed either alone or in combination. The field trial site was
located near Marysville, Ohio and characterized by Blount silt loam
soil. Soil testing showed a soil pH of 6.2 and an organic matter
content of 2.7%. The field was disk cultivated in the spring prior
to planting.
[0039] The LCO product used in the trial was the same as that used
in Example 1. The commercial chitosan product utilized in the trial
was ELEXA.RTM.-4PDB.
[0040] The corn seed used in the study was Shur Grow hybrid
SG-686-RR. The seed was commercially treated with a combination of
Maxim XL (0.167 fl oz/cwt, Apron XL (0.32 fl oz/cwt) and Actellic
(0.03 fl oz/cwt). When used alone, the LCO product was sprayed on
seed without dilution at a rate of 15 fl oz/cwt. The use rate for
the chitosan product was 0.375 ft oz/cwt. The product was diluted
with water and applied on seed at a slurry rate of 15 fl oz/cwt.
When applied in combination, the LCO was applied at 1/10.sup.th
rate of 1.5 ft oz/cwt and the chitosan at a rate of 0.375 fl
oz/cwt. The combined products were diluted with water and applied
on seed at a slurry rate of 15 fl oz/cwt.
[0041] The study was conducted in a randomized complete block
design, with four replications and a plot size of 10 feet by 20
feet, and 30 Inch row spacing. Seeds were treated Just prior to
planting and planted at a depth of 1.5 inch and a seeding rate of
28,000 seeds per acre.
[0042] Results of the study are shown in Table 4. The LCO and
chitosan treatments significantly increased yield 18.6 and 16.9
bu/acre, respectively, relative to the non-treated control group
(p=0.1). In contrast, the combined LCO+chitosan treatment
significantly increased yield by 40.0 bu/acre. This increase in
yield was significantly greater than the individual treatments, and
exceeded the combined benefit of the of the individual LCO and
chitosan treatments.
TABLE-US-00004 TABLE 4 Treatment Grain yield (bu/acre) Control -
nontreated 116.9 LCO 135.5 ELEXA .RTM.-4PDB4 PDB 133.8 LCO + ELEXA
.RTM.-4PDB 156.9 Probability % 0.0001 LSD 10% 9.3 CV % 5.3
5. Corn Seed (Dairyland DSR-8194) Treatment with
LCO+Chitin/Chitosan
[0043] A corn field trial was conducted to evaluate the effects of
the Rhizobium leguminosarum by viceae-based LCO and the two
chitosan products referenced in Example 1 on grain yield when
applied on corn seed alone or In combination. The field trial was
conducted at a location near Whitewater, Wis., characterized by
Milford silty day loam soil. The soil had a pH of 6.5, an organic
matter content of 4.5%, and phosphorus and potassium contents of 40
and 142 ppm, respectively.
[0044] Dairyland variety DSR 8194 YGPL corn seed was used in the
study. The LCO product was applied without dilution on seed at a
rate of 15.3 fl oz/cwt. BEYOND.TM. was diluted to a concentration
of 0.132% w/v and ELEXA.RTM.-4PDB 2.5% w/v with water. Each was
applied by spraying on seed at the rate of 15.3 fl oz/cwt. When the
LCO-chitin/chitosan combination was applied, the same
concentrations of LCO and chitin/chitosan products were used as
when each of these products was applied alone.
[0045] The study was conducted in a randomized complete block
design, with a plot size of 15 feet by 50 feet, 30 inch row
spacing. Four replications were performed. Seeds were treated just
prior to planting and were planted at a depth of 2'' at a seeding
rate of 33,000 seeds per acre. Seeds were planted with a John Deere
Max Emerge II NT 6-row corn planter. Starter fertilizer (7-21-7)
was applied at a rate of 200 lb/acre, with a subsequent application
of 160 units nitrogen as 28% nitrogen.
[0046] The results are shown in Table 5. LCO treatment
significantly increased grain yield by 4.6 bu/acre relative to the
non-treated control group (p=0.1). Seeds treated with the
BEYOND.TM. product alone showed a numerical yield increase of 3.7
bu/acre, while seed treatment with ELEXA.RTM.-4PDB alone showed no
effect on grain yield. Combined treatment with LCO and BEYOND.RTM.
numerically increased grain yield by 2.1 bu/acre over LCO alone and
3.0 bu/acre over BEYOND.TM. alone.
[0047] Combined treatment with LCO and ELEXA.RTM.-4PDB
significantly increased grain yield by 8.4 bu/acre compared with
ELEXA.RTM.-4PDB treatment alone, and numerically increased grain
yield by 3.7 bu/acre compared with LCO treatment alone. The LCO and
ELEXA.RTM.-4PDB combination increased yield to a greater extent
than the additive effects of LCO or ELEXA.RTM.-4PDB treatment
alone, showing a synergistic effect of the combined treatment.
TABLE-US-00005 TABLE 5 Treatment Grain yield (bu/acre) Control -
non-treated 162.1 LCO 166.7 BEYOND .TM. 165.8 ELEXA .RTM.-4PDB4 PDB
162.0 LCO + BEYOND .TM. 168.8 LCO + ELEXA .RTM.-4PDB 170.4
Probability % <0.1 LSD 10% 3.9 CV % 2.0
6. Corn (Jung 6573RR/YGPL) Foliar Treatment with
LCO+Chitin/Chitosan
[0048] A corn field trial was conducted evaluating the effect of
the Rhizobium leguminosarum by viceae-based LCO and the two
chitosan products described in Example 1 on grain yield when
applied as a foliar application alone or in combination. The field
trial was located near Whitewater, Wis. at a site with Milford
silty clay loam soil. The soil had a pH of 6.5, and soil test
results showed an organic matter content of 4.5%, and phosphorus
and potassium contents of 40 and 142 ppm, respectively.
[0049] The corn seed used in the study was Jung variety 6573RR/YGPL
The LCO product was applied on the foliage at the V4 growth stage
at a rate of 1 quart/acre in 25 gallons of water. BEYOND.TM. and
ELEXA.RTM.-4PDB were diluted to concentrations of 0.132% w/v and
2.5% w/v, respectively, in water and applied on foliage at a rate
of 25 gallons/acre. When the LCO-chitin/chitosan combination was
applied, the same concentrations of LCO and chitin/chitosan
products were used as when each of these products was applied
alone.
[0050] The study was conducted in a randomized complete block
design with a plot size of 15 feet by 50 feet, 30 inch row spacing.
Four replications were performed. Seeds were planted at a depth of
2 inches and a seeding rate of 33,000 seeds per acre using a John
Deere Max Emerge II NT 6-row corn planter. Starter fertilizer
(7-21-7) was applied at a rate of 200 lb/acre, with a subsequent
application of 160 units nitrogen as 28% nitrogen.
[0051] Results of this study are shown in Table 6. The LCO,
BEYOND.TM., and ELEXA.RTM.4PDB products significantly increased
grain yield over the non-treated control group by 11.3, 8.8, and
7.4 bu/acre, respectively, when applied to foliage as stand-alone
treatments (p=0.1). Application of ELEXA.RTM.-4PDB in combination
with LCO further increased yield by 1.1 bu/acre compared with
ELEXA.RTM.-4PDB alone, and 5.0 bu/acre compared with LCO alone.
Application of BEYOND.TM. in combination with LCO further increased
yield by 2.3 bu/acre compared with LCO alone, and 4.8 bu/acre
compared with BEYOND.TM. alone.
TABLE-US-00006 TABLE 6 Treatment Grain yield (bu/acre) Control -
non-treated 162.6 LCO 173.9 BEYOND .TM. 171.4 ELEXA .RTM.-4PDB4 PDB
170.0 LCO + BEYOND .TM. 176.2 LCO + ELEXA .RTM.-4PDB 175.0
Probability % 0.3 LSD 10% 6.5 CV % 3.2
7. Corn Seed (Pioneer 38H52) Treatment with LCO+Flavonoid
[0052] A corn field trial was conducted evaluating the effect of
liquid formulations of LCO and flavonoid on grain yield when
applied alone or in combination on seed. The field trial was
conducted at a site near Whitewater, Wis. in a Piano silt loam
soil. The soil had a pH of 6.5 and soil test results showed an
organic matter content of 4.4% and phosphorus and potassium content
of 42 and 146 ppm, respectively. The field was previously planted
to soybeans. It was fall chisel plowed and field cultivated in the
spring prior to planting.
[0053] The LCO product used in the trial was the same as that used
in Example 1. The flavonoid product used (ReVV.RTM., EMD Crop
BioScience) had a 10 mM total flavonoid concentration comprising
genistein and daidzein.
[0054] The corn seed used in the trial was Pioneer variety 38H52.
The use rate for the LCO and flavonoid products were 1.5 and 0.184
fl oz/cwt, respectively. The products were each diluted with water
and applied on seed at a slurry rate of 15.3 fl oz/cwt. The
LCO/flavonoid combination was applied at the same concentration and
slurry rate as when applied alone. The study was conducted in a
randomized complete block design, with a plot size of 10 feet by 50
feet, with 30 inch row spacing, and four replications per
treatment. Seeds were planted at a depth of 2 inches at a seeding
rate of 33,000 seeds per acre. Planting was carried out using a
four row precision vacuum planter. One hundred and forty units of
nitrogen were applied as urea in advance of planting, and an
additional 150 lb of 7-21-7 starter fertilizer was applied at
planting.
[0055] Results of the study are shown in Table 7. The flavonoid
treatment statistically increased grain yield by 5.3 bu/acre, while
the LCO treatment numerically increased grain yield by 3.3 bu/acre.
Application of the two products in combination resulted in a
statistically significant increase in yield over each of the two
products administered as alone. The increase observed with the
combination treatment of 19.2 bu/acre unexpectedly exceeded the
combined effect of the individual products alone (8.6 bu/acre) by
more than two-fold, demonstrating a synergistic effect of the
combination treatment.
TABLE-US-00007 TABLE 7 Treatment Application Grain yield (bu/acre)
Control None 142.5 LCO Seed 145.8 Flavonoid Seed 147.8 Flavonoid +
LCO Seed 161.7 Probability % <0.1 LSD 10% 4.2 CV % 4.4
8. Corn Seed (DynaGro 51K74) Treatment with LCO+Flavonoid
[0056] A second corn trial was conducted as described in Example 7
at a location near Fergus Falls, Minn., in a nutrient rich loam
soil previously planted to soybeans. The LCO and flavonoid products
were applied alone or in combination on DynaGro variety 51K74 corn
seed. The study was conducted in a randomized complete block
design, with a plot size of 10 feet by 20 feet, with 30 inch row
spacing, and four replications per treatment.
[0057] Results of the study are shown in Table 8. The LCO and
flavonoid seed treatments numerically increased grain yield
compared to the non-treated control by 7.3 and 15.3 bu/acre,
respectively. Application of the two products in combination
statistically increased yield compared to the control by 24.0
bu/acre, and by 17.1 bu/acre compared to the LCO treatment. The
increase in yield observed with the combined treatment exceeded the
combined increase in yield from the individual products alone.
TABLE-US-00008 TABLE 8 Treatment Application Grain yield (bu/acre)
Control None 141.2 LCO Seed 148.5 Flavonoid Seed 156.5 Flavonoid +
LCO Seed 165.2 Probability % <0.1 LSD 5% 13.9 CV % 6.3
9. Corn (Dairyland DSR 4497) Seed, Furrow, and Foliage Treatment
with LCO+Flavonoid
[0058] A corn field trial was conducted at the same site described
above in Example 7 to evaluate the effect of flavonoid seed
treatment on grain yield compared to application of LCO either in
the seed furrow at planting or spray-applied as a foliar
application. These individual product treatments were additionally
compared to flavonoid seed treatment combined with in-furrow LCO
application and flavonoid seed treatment combined with foliar LCO
application. The LCO and flavonoid products were the same as those
used in the prior examples.
[0059] The corn seed used in the trial was Dairyland variety DSR
4497. The flavonoid product was applied on seed at the same use
rate of 0.184 fl oz/cwt and slurry rate in water of 15.3 fl oz/cwt
as in prior examples. The LCO product was applied at planting in
the seed furrow at a rate of 1 pint/acre in 5 gallons of water, or
spray-applied to foliar surfaces at a rate of 1 qt/acre in 25
gallons of water at the V4 stage of corn development. The
seed/furrow and seed/foliar applications were at the same rates for
the combination as when applied alone.
[0060] The study was conducted in a randomized complete block
design, with a plot size of 10 feet by 50 feet, with 30 inch row
spacing, and four replications per treatment. Seeds were planted at
a depth of 2 inches at a seeding rate of 33,000 seeds per acre.
Planting was carried out using a four row precision vacuum planter.
One hundred and forty units of nitrogen were applied as urea in
advance of planting, and an additional 150 lb of 7-21-7 starter
fertilizer was applied at planting.
[0061] Results of the study are shown in Table 9. Application of
flavonoid on seed and LCO in the seed furrow numerically increased
grain yield by 4.3 and 2.6 bu/acre, respectively, compared to the
control treatment. In contrast, combined application of the two
products on seed and in furrow statistically increased yield by 5.5
bu/acre.
[0062] Separate application of flavonoid on seed and LCO as a
foliar application resulted in a numerical increase in yield with
flavonoid seed treatment of 4.3 bu/acre and a statistically
significant increase of 7.4 bu/acre with LCO foliar application.
Combined flavonoid seed treatment and LCO foliar application
further increased yield by 9.2 bu/acre compared to the control
treatment.
TABLE-US-00009 TABLE 9 Treatment Application Grain yield (bu/acre)
Control None 173.6 Flavonoid Seed 177.9 LCO Furrow 176.0 LCO Foliar
181.0 Flavonoid/LCO Seed, furrow 179.1 Flavonoid/LCO Seed, foliar
182.8 Probability % <0.1 LSD 10% 4.9 CV % 5.3
10. Corn (Spangler 5775) Seed, Furrow, and Foliage Treatment with
LCO+Flavonoid
[0063] A parallel corn field trial was conducted at the same
location and with the same treatments and trial design as described
in Example 9, but differing in the variety of corn used (Spangler
5775).
[0064] Results of the study are shown in Table 10. Application of
flavonoid on seed statistically increased grain yield by 7.4
bu/acre compared to the non-treated control, while LCO application
in the seed furrow numerically increased grain yield by 3.5
bu/acre. Combined flavonoid seed treatment and LCO furrow
application further increased yield by 9.7 bu/acre compared to the
control treatment.
[0065] Separate application of flavonoid on seed and LCO as a
foliar application resulted in a statistically significant increase
in yield with flavonoid seed treatment of 7.4 bu/acre (as stated
above) and a numerical increase of 1.1 bu/acre with LCO foliar
application. Application of the two products in combination
resulted in a statistically significant increase in yield greater
than that seen for each of the two products alone. Further, the
increase observed with the combination treatment (16.2 bu/acre)
exceeded the combined effect of the individual products alone (8.5
bu/acre), showing a synergistic effect of the combination
treatments.
TABLE-US-00010 TABLE 10 Treatment Application Grain yield (bu/acre)
Control None 160.7 Flavonoid Seed 168.1 LCO Furrow 164.2 LCO Foliar
161.8 Flavonoid/LCO Seed, furrow 170.4 Flavonoid/LCO Seed, foliar
176.9 Probability % <0.1 LSD 10% 5.6 CV % 4.8
11. LCO Foliar and Flavonoid Seed Treatment of Soybean (Dairyland
DSR 1701)
[0066] A soybean field trial was conducted to evaluate the effect
of flavonoid seed treatment on grain yield compared to the effect
of foliar application of LCO. The individual product treatments
were additionally compared to flavonoid seed treatment combined
with LCO foliar application. The LCO product, was the same as that
used in Example 1, and the flavonoid product was the same as that
used in prior examples.
[0067] The field trial was conducted at a site near Whitewater,
Wis. in a Milford silty clay loam soil. The soil had a pH of 6.5
and soil test results showed an organic matter content of 4.7% and
phosphorus and potassium content of 48 and 136 ppm, respectively.
The field was no-till and was previously planted to corn.
[0068] The soybean seed used in the trial was Dairyland variety DSR
1701. The flavonoid product was applied at a use rate of 0.184 fl
oz/cwt and slurry rate in water of 4.25 fl oz/cwt. The LCO product
was spray-applied to foliar surfaces at a rate of 1 qt/acre in 25
gallons of water at the V4 stage of soybean development. The
combined seed/foliar application was at the same rate as when
applied alone. The study was conducted in a randomized complete
block design, with a plot size of 10 feet by 50 feet, with 30 inch
row spacing, and four replications per treatment. Seeds were
planted at a depth of 1 Inch at a seeding rate of 160,000 seeds per
acre. Planting was carried out using a John Deere 750 NT grain
drill.
[0069] Results of the study are shown in Table 11. Application of
flavonoid on seed statistically increased grain yield by 3.2
bu/acre compared to the non-treated control, while LCO foliar
application numerically increased grain yield by 1.2 bu/acre.
Application of the two products in combination resulted in a
statistically significant Increase above each of the two products
alone, with the increase in yield (5.0 bu/acre) exceeding the
combined effect of the individual products alone (4.4 bu/acre),
showing a synergistic effect of the combination treatment.
TABLE-US-00011 TABLE 11 Treatment Application Grain yield (bu/acre)
Control None 47.8 Flavonoid Seed 51.0 LCO Foliar 49.0 Flavonoid/LCO
Seed/foliar 52.8 Probability % <0.1 LSD 10% 1.3 CV % 5.2
12. LCO Foliar and Flavonoid Seed Treatment of Soybean (Dairyland
DSR 2000)
[0070] A parallel soybean field trial was conducted at the same
location and with the same treatments and trial design as described
in Example 11, but differing in the variety of soybean used
(Dairyland variety DSR 2000).
[0071] Results of the study are shown in Table 12. Application of
flavonoid on seed and LCO as a foliar application statistically
increased grain yield by 2.6 and 4.5 bu/acre, respectively,
compared to the non-treated control. Combined flavonoid seed
treatment and LCO foliar application further increased yield by 7.1
bu/acre compared to the control treatment.
TABLE-US-00012 TABLE 12 Treatment Application Grain yield (bu/acre)
Control None 40.9 Flavonoid Seed 43.5 LCO Foliar 45.4 Flavonoid/LCO
Seed/foliar 48.0 Probability % <0.1 LSD 10% 1.8 CV % 4.5
13. Soybean Seed (Dairyland DSR 2300RR) Treatment with
LCO+Flavonoid
[0072] A soybean field trial was conducted to evaluate the effect
of LCO and flavonoid products on grain yield when applied on seed
either alone or in combination. The field trial site was located
near Whitewater, Wis. and characterized by Piano silt loam soil.
Soil testing showed a soil pH of 6.5, an organic matter content of
3.9%, and phosphorus and potassium contents of 40 ppm and 138 ppm,
respectively. The field was no-till and was previously planted to
corn.
[0073] The LCO product used in the trial (OPTIMIZE.RTM., EMD Crop
BIoScience) was produced by Bradyrhizobium japonicum and contained
approximately 1.times.10.sup.-9 M LCO. The flavonoid product used
(ReVV.RTM., EMD Crop BioScience) had a 10 mM total flavonoid
concentration comprising genistein and daidzein in a ratio of 8:2
w/w.
[0074] The soybean seed used in the study was Dairyland variety DSR
2300RR. The LCO and flavonoid products were sprayed onto seeds
alone or in combination at a rate of 4.25 and 0.184 fl oz/cwt,
respectively. The study was conducted in a randomized complete
block design, with four replications and a plot size of 10 feet by
50 feet, and 15 Inch row spacing. Seeds were treated just prior to
planting and planted at a depth of 1 inch and a seeding rate of
185,000 seeds per acre using a John Deere 750 NT grain drill.
[0075] Results of the study are shown in Table 13. The LCO and
flavonoid treatments numerically increased yield 2.9 and 4.0
bu/acre, respectively, relative to the non-treated control group
(p=0.1). In contrast, the combined LCO+flavonoid treatment
significantly increased yield by 7.0 bu/acre. This increase in
yield was greater than the combined benefit of the of the
individual LCO and flavonoid treatments.
TABLE-US-00013 TABLE 13 Treatment Grain yield (bu/acre) Control -
nontreated 54.1 LCO 57.0 Flavonoid 58.1 LCO + flavonoid 61.1
Probability % <0.1 LSD 10% 4.2 CV % 3.6
14. Corn (Pioneer Hybrid 34A17) Foliar Treatment with LCO+Flavonoid
or LCO+Chitosan
[0076] A corn field trial was conducted to evaluate the effects of
LCO/flavonoid, and LCO/chitosan products on grain yield when
applied to foliage alone or in combination. The LCO product was
produced by Rhizobium leguminosarum by viceae and contained
approximately 10.sup.-8 M LCO. The flavonoid product used had a 10
mM total flavonoid concentration comprising genistein and daidzein
in a ratio of 8:2 w/w. The chitosan product (ELEXA.RTM.-4PDB) was
the same as that used in the prior examples.
[0077] The field trial was located near York, Nebr. at a site
characterized by Hastings silt loam soil. The soil had a pH of 6
and an organic matter content of 3%. The site was conventionally
tilled, and the prior crop was soybeans. The corn seed used in the
study was Pioneer hybrid 34A17. The study was conducted in a
randomized complete block design, with a plot size of 10 feet by 30
feet and 30 inch row spacing. Four replications were performed.
Seeds were planted at a depth of 2 Inches at a seeding rate of
30,200 seeds per acre.
[0078] Treatments were applied by spraying onto foliage at the V5
growth stage. The LCO and ELEXA.RTM.-4PDB treatments were applied
at a rate of 1 quart/acre in 20 gallons of water using a small plot
sprayer at a ground speed of 2.3 mph. The flavonoid treatment was
initially diluted 25.times. in water, then applied at a rate of 1
quart/acre in 20 gallons of water. The LCO-chitosan combination
treatment was applied at a reduced rate of 3.2 fl oz/acre of LCO
and 12.8 fl oz chitosan in 20 gallons of water. For the
LCO-flavonoid combination, the flavonoid was first diluted
25.times. in water, then applied to similarly to the LCO-chitosan
combination at 3.2+12.8 fl oz/acre diluted in 20 gallons of
water.
[0079] Results of this study are shown in Table 14. The LCO,
flavonoid, and ELEXA.RTM.-4PDB treatments numerically increased
grain yield by 1.2, 3.5, and 1.5 bu/acre, respectively, when
applied to foliage as stand-alone treatments (p=0.1). Combined
application of LCO with flavonoid and LCO with ELEXA.RTM.-4PDB
significantly increased yield by 8.6 and 12.1 bu/acre compared to
the control treatment. In each case, the combined treatment
response exceeded the combined benefit of the Individual products
alone, demonstrating a synergistic effect of the combination
compositions. This occurred even though the combination products
were applied at reduced rates compared to when applied alone.
TABLE-US-00014 TABLE 14 Treatment Grain yield (bu/acre) Control -
nontreated 222.0 LCO 223.2 Flavonoid 225.5 ELEXA .RTM.-4PDB4 PDB
223.5 LCO + flavonoid 230.6 LCO + ELEXA .RTM.-4PDB 234.1
Probability % 0.0909 LSD 10% 6.5 CV % 2.4
15. Corn (Midwest Seed Genetics Hybrid 8463859 RR2) Foliar
Treatment with LCO+Flavonoid or LCO+Chitosan
[0080] A corn field trial was conducted similar to that of Example
14 to evaluate the effects of LCO/flavonoid, and LCO/chitosan
products on grain yield when applied to foliage alone or in
combination. The LCO, flavonoid, and chitosan products were the
same as that used in Example 14.
[0081] The field trial was located near Sparta, Ill. at a site
characterized by silt loam soil. The soil had a pH of 6.5 and an
organic matter content of 2.6%. The site was conventionally tilled,
and the prior crop was soybeans. The corn seed used in the study
was Midwest Seed Genetics hybrid 8463859 RR2. The study was
conducted in a randomized complete block design, with a plot size
of 10 feet by 40 feet and 30 inch row spacing. Four replications
were performed. Seeds were planted at a depth of 2 inches at a
seeding rate of 26,100 seeds per acre.
[0082] Treatments were applied by spraying onto foliage at the
V3-V4 growth stage. The individual and combined treatments were
applied at the rates described in Example 14 in 20 gallons of water
using a backpack sprayer at a ground speed of 3 mph.
[0083] Results of this study are shown in Table 15. The LCO,
flavonoid, and ELEXA.RTM.-4PDB treatments numerically increased
grain yield by 3.4, 7.1, and 3.3 bu/acre, respectively, when
applied to foliage as stand-alone treatments (p=0.1). Combined
application of LCO with flavonoid significantly increased yield by
16.5, while combined application of LCO with ELEXA.RTM.-4PDB
numerically increased yield by 10.5 bu/acre compared to the control
treatment. In each case, the combined treatment response exceeded
the combined benefit of the individual products alone,
demonstrating a synergistic effect of the combination compositions.
This occurred even though the combination products were applied at
reduced rate compared to when applied alone.
TABLE-US-00015 TABLE 15 Treatment Grain yield (bu/acre) Control -
nontreated 71.7 LCO 75.1 Flavonoid 78.8 ELEXA .RTM.-4PDB4 PDB 75.0
LCO + flavonoid 88.2 LCO + ELEXA .RTM.-4PDB 82.2 Probability %
0.6459 LSD 10% 13.8 CV % 14.6
16. Corn Foliar Treatment with LCO and Herbicide
[0084] Three corn field trials were conducted to evaluate the
effect of foliar application of LCO in combination with four
different herbicides. The LCO is the same as that used in prior
foliar application examples. The herbicides included glyphosate
(Roundup Original Max.RTM., Monsanto Company, St. Louis, Mo.),
glufosinate-ammonium (Liberty.RTM., Bayer CropScience LP, Research
Triangle Park, N.C.), mesotrione (Calisto.RTM., Syngenta Crop
Protection, Inc., Greensboro, N.C.), and nicosulfu/rimsulfuron
(Steadfast.RTM., E. I. du Pont de Nemours and Company, Wilmington,
Del.).
[0085] Two of the trials were located near Whitewater, Wis. at
sites characterized by Milford silty clay loam soil (fields F-5 and
P-1). The F-5 site was conventionally tilled with a prior crop of
corn, and the P-1 site was minimum tilled with soybean as the prior
crop. The corn seed used for both studies was Pioneer hybrid 36B05
HXX/RR/LL. The studies were conducted in a randomized complete
block design, with a plot size of 10 feet by 50 feet, 30 inch row
spacing, and four replications. Seeds were planted at a depth of 2
Inches at a seeding rate of 33,000 seeds per acre using a vacuum
precision plot planter.
[0086] The third field trial was located near York, Nebr. at a site
characterized by Hastings silt loam soil. The site was
conventionally tilled with soybean as the prior crop. The corn seed
used in the study was Pioneer hybrid 34A17. The study was conducted
in a randomized complete block design, with a plot size of 10 feet
by 30 feet, 30 inch row spacing, and four replications. Seeds were
planted at a depth of 2 inches at a seeding rate of 30,200 seeds
per acre.
[0087] Treatments at the two Whitewater, WO sites were applied by
spraying onto foliage at the V4 growth stage. The LCO treatment was
applied at a rate of 1 quart/acre; the herbicide products were
applied at label rate for each product. The herbicide and
LCO+herbicide treatments were foliar-applied in 25 gallons of water
using a small plot sprayer at a ground speed of 2.5 mph. Treatments
at the York, Nebr. site were applied at the V6 growth stage at the
same 1 quart/acre for the LCO and label rate for the herbicide
products in 20 gallons of water using a small plot sprayer at a
ground speed of 2.3 mph.
[0088] Results of this study are shown in Table 16. With the two
Whitewater, Wis. trials, application of LCO in combination with the
four different herbicides enhanced grain yield compared to the
herbicide alone with all LCO/herbicide combinations at the two
locations, with the exception of the LCO+Steadfast combination at
the P-1 site. At the York, Nebr. location, application of LCO In
combination with the four different herbicides enhanced grain yield
compared to the herbicide alone with each of the LCO/herbicide
combinations, with the exception of the LCO+Calisto treatment.
TABLE-US-00016 TABLE 16 LCO + LCO + LCO + LCO + Trial Round-
Round-Up Liberty Calisto Steadfast Steadfast location Up 1 qt/A
Liberty 1 qt/A Calisto 1 qt/A Steadfast 1 qt/A Whitewater. 157.5
161.9 152.1 156.9 156 158.8 140.6 141.2 WI Whitewater. 161.2 169.2
159.6 164.2 162.8 169.1 154.4 152.1 WI York, NE 195.8 204.6 201
208.9 202.8 202 194.3 201.3
[0089] Although preferred embodiments of the invention have been
shown and described herein, it will be understood that such
embodiments are provided by way of example only. Numerous
variations, changes and substitutions will occur to those skilled
in the art without departing from the spirit of the invention.
Accordingly, it is intended that the appended claims cover all such
variations as fall within the spirit and scope of the
invention.
* * * * *