U.S. patent application number 13/177555 was filed with the patent office on 2012-07-05 for methods for enhanced root nodulation in legumes.
This patent application is currently assigned to ADVANCED BIOCATALYTICS CORPORATION. Invention is credited to John W. Baldridge, Michael G. Goldfeld, Andrew H. Michalow, Carl W. Podella.
Application Number | 20120172219 13/177555 |
Document ID | / |
Family ID | 45441786 |
Filed Date | 2012-07-05 |
United States Patent
Application |
20120172219 |
Kind Code |
A1 |
Podella; Carl W. ; et
al. |
July 5, 2012 |
METHODS FOR ENHANCED ROOT NODULATION IN LEGUMES
Abstract
Disclosed herein are methods of increasing, enhancing, or
accelerating root nodulation in a plant, accelerating growth of
nitrogen fixing bacteria in nodules of a plant, increasing protein
content in a plant, increasing yield of a plant, improving water
retention of a plant, or reducing water use of a plant, the method
comprising identifying a plant in need of root nodulation, and
applying to the plant a composition comprising a protein component
comprising yeast stress proteins resulting from subjecting a
mixture obtained from the yeast fermentation to stress.
Inventors: |
Podella; Carl W.; (Irvine,
CA) ; Baldridge; John W.; (Newport Beach, CA)
; Michalow; Andrew H.; (Mission Viejo, CA) ;
Goldfeld; Michael G.; (Irvine, CA) |
Assignee: |
ADVANCED BIOCATALYTICS
CORPORATION
Irvine
CA
|
Family ID: |
45441786 |
Appl. No.: |
13/177555 |
Filed: |
July 7, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61399095 |
Jul 7, 2010 |
|
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Current U.S.
Class: |
504/117 |
Current CPC
Class: |
A01N 63/30 20200101;
Y02A 40/10 20180101; Y02A 40/143 20180101 |
Class at
Publication: |
504/117 |
International
Class: |
A01N 63/02 20060101
A01N063/02; A01P 21/00 20060101 A01P021/00 |
Claims
1. A method of increasing, enhancing, or accelerating root
nodulation in a plant, accelerating growth of nitrogen fixing
bacteria in nodules of a plant, increasing protein content in a
plant, increasing yield of a plant, improving water retention of a
plant, or reducing water use of a plant, the method comprising
identifying a plant in need of root nodulation, and applying to the
plant a composition comprising a protein component comprising yeast
stress proteins resulting from subjecting a mixture obtained from
the yeast fermentation to stress.
2. The method of claim 1, wherein the composition is applied to the
soil near the plant.
3. The method of claim 1, wherein the composition is applied
through irrigation.
4. The method of claim 3, wherein the irrigation is spray
irrigation or drip irrigation.
5. The method of claim 1, wherein the composition is applied with
every watering cycle or intermittent basis.
6. The method of claim 1, wherein the protein component is from
aerobic fermentation of yeast.
7. The method of claim 1, wherein the protein component comprises
proteins obtained from exposing a product obtained from the
fermentation of yeast to additional procedures that increase the
yield of proteins produced from the fermentation.
8. The method of claim 1, wherein the stress is selected from the
group consisting of heat shock of the fermentation product,
physical and/or chemical disruption of the yeast cells to release
additional polypeptides, and lysing of the yeast cells.
9. The method of claim 1, wherein the stress comprises exposing a
product obtained from the fermentation of yeast to heat shock
conditions.
10. The method of claim 1, wherein the stress comprises physically
or chemically disrupting the yeast after the fermentation of the
yeast.
11. The method of claim 1, wherein the stress comprises lysing the
yeast after the fermentation of the yeast.
12. The method of claim 1, further comprising mixing the protein
component with additional nutrients prior to the application to the
plant.
13. The method of claim 1, wherein the composition further
comprises one or more of an anionic surfactant, a non-ionic
surfactant, a cationic surfactant, and amphoteric surfactant.
14. The method of claim 1, wherein the yeast is selected from the
group consisting of Saccharomyces cerevisiae, Kluyveromyces
marxianus, Kluyveromyces lactis, Candida utilis (Torula yeast),
Zygosaccharomyces, Pichia pastoris, and Hansanula polymorpha.
15. The method of claim 1, wherein the plant is a legume.
16. The method of claim 12, wherein the legume is selected from the
group consisting of alfalfa, clover, peas, beans, lentils, lupins,
mesquite, carob, soy, peanuts, locust trees (Gleditsia or Robinia),
wisteria, and the Kentucky coffeetree (Gymnocladus dioicus).
17. A soil mixture comprising soil and a composition comprising a
protein component comprising yeast stress proteins resulting from
subjecting a mixture obtained from the yeast fermentation to
stress.
18. A method of improving water retention of a legume, reducing
water use of a legume, accelerating root nodulation in a legume,
accelerating nitrogen fixation by a legume, accelerating growth of
nitrogen fixing bacteria in nodules of a legume, increasing protein
content in a legume, or increasing yield of a legume, the method
comprising: identifying a legume in need thereof, and applying to
the legume a soil mixture of claim 17.
Description
RELATED APPLICATION
[0001] The present application claims priority to the U.S.
Provisional Application Ser. No. 61/399,095, filed Jul. 7, 2010,
and entitled "Methods for Enhanced Root Nodulation in Legumes," the
entire disclosure of which is incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The present invention relates to enhancement of plant growth
and crop yield by applying compositions of fermentation liquids and
surfactant that improve root nodulation by rhizobacteria, e.g.,
Rhizobium, Bradyrhizobium, Sinorhizobium, etc.
BACKGROUND OF THE DISCLOSURE
[0003] Legumes are plants, such as alfalfa, clover, peas, beans,
lentils, lupins, mesquite, carob, soy, peanuts, locust trees
(Gleditsia or Robinia), wisteria, and the Kentucky coffeetree
(Gymnocladus dioicus), that form a symbiotic relationship between
their roots and bacteria, specifically of the family Rhizobiaceae.
The bacteria penetrate the plant root hairs, and then induce the
formation of nodules. The plant provides the bacteria both
sustenance and an energy source in the form of adenosine
triphosphate (ATP) that is generated by photosynthesis. In return,
the bacteria are able to fix elemental nitrogen from the atmosphere
into ammonia, a usable form of nitrogen that is digestible by
plants thereby providing a rich nitrogen source to the plant. This
process is called nitrogen fixation. Nitrogen is the nutrient that
is most frequently a limiting item to the growth of green plants
and optimizing its application is a key to optimizing plant yield.
The term yield will be referred to as the crop which is being
grown, be it peas, soybeans, or other legume.
[0004] Legumes are generally higher in protein content than other
plant families due to the availability of nitrogen from nitrogen
fixation. The high protein content makes legumes one of the most
important food crops for both human consumption and animal feed.
Further, legumes are used in crop rotation practice to increase the
nitrogen content of soils, through nitrogen fixation, for future
growth seasons and to reduce the amount of fertilizer that needs to
be applied. This has cost benefits to the grower and can reduce
nitrogen runoff.
[0005] Each plant species requires a particular strain of Rhizobia
species for nodulation to form. Native rhizobial populations are
not typically optimized for a particular plant species unless the
crop grown previously was the specific legume to be planted. To
optimize the effectiveness of nodule formation, appropriate species
of Rhizobia can be inoculated into a crop. There are three methods
of inoculation, each with its own advantages and limitations;
solid, liquid and freeze-dried. Solid peat-based inoculants can be
applied to seed or directly to the soil. Liquid inoculants are
mixed with water and applied to the seed furrow at the time of
planting. To maintain viability of the live bacteria, liquid
inoculants must be kept frozen or refrigerated when stored and
during shipment. The handling requirements increase costs and
further limit their availability through standard distribution.
Seed-applied inoculants are the most commonly used and precautions
in handling need to be employed to preserve the live bacteria. A
key limitation with inoculating legumes to maximize yield is that
even under the best storage conditions, rhizobial populations will
decline over time.
[0006] The nitrogen fixation process is a transfer of electrons by
oxidizing hydrogen and reducing elemental nitrogen to form ammonia.
The reduction of nitrogen is an energy intensive process, and, to
fix elemental nitrogen, the rhizobial bacteria gets its energy from
the plant that it infects. The chemical process involves a two-part
enzyme system known as nitrogenase. The system contains iron and is
highly susceptible to being inactivated in the presence of oxygen.
This is not a problem with anaerobic bacteria. However, nitrogen
fixing aerobic bacteria, such as Rhizobium in the soil can overcome
the problem of oxygen because they contain oxygen scavenging
molecules called leghaemoglobin. In nodules, leghaemoglobin may
regulate oxygen in a similar way as hemoglobin does in mammalian
tissues. Nodules that are actively fixing nitrogen will appear
reddish or pink, which can be evident from the exterior or when the
nodule is cut open. In extreme cases the reddish color will extend
into the roots themselves. Tan colored nodules are not actively
fixing bacteria and white, grey or green colored nodules are doing
little nitrogen fixing or could be dying.
[0007] To optimize legume crop yield it is important to maintain
the proper soil fertility, high nodulation and high level of
nitrogen fixing activity are the keys to maintaining nitrogen
levels in legumes. Depending on the particular legume and soil
conditions, the plant might obtain a small percentage or a majority
of its nitrogen from fixation. Adding nitrogen fertilizer can be
detrimental because some legumes don't respond to nitrogen
fertilizer. In other cases, because the nitrogen fixation process
is energy intensive the plant may not expend energy for nodulation
if it can absorb nitrogen directly from the added fertilizer using
less energy. This process uptakes less nitrogen than by fixation
and yields can be compromised.
[0008] The legume is efficient in using the nitrogen that is fixed
by its partner bacteria. Almost all nitrogen that is fixed is used
by the plant. Higher levels of nitrogen fixation translate to
higher yield. But the higher rate of nodule formation does not
always translate into higher yield. Typically, only a small amount
might leak to neighboring plants. Only when the plant dies does it
return nitrogen to the soil and relative to the amount of biomass
of stems, leaves and roots that is turned into the soil.
[0009] Inoculation methods are numerous and revolve around delivery
methods and specific strains of bacteria to legume crops to
increase nodulation. A key element in activating the rhizobial
nodulation (nod) genes is chemical signal that is sent by the host
plant through its root hairs. Infection can happen only when root
hairs are present. Flavonoid compounds, such as LCO, are known to
activate rhizobial nod genes. U.S. Pat. No. 7,250,068 describes
methods of improving yield of a legume by treating with the
addition of lipo chitooligosaccharide (LCO), where, "an LCO which
can increase the photosynthetic rate, and/or growth, and/or yield
of a legume, in to acting as a trigger to initiate legume symbiotic
nitrogen fixation."
[0010] U.S. Pat. No. 6,855,536 states that, "Unfortunately, for
most of the United States, inoculation has been shown to be
ineffective. Therefore, the inoculant industry remains relatively
small (approximately $20-$30 million per year.)."
[0011] Therefore, there is a need to provide a simple, broad based
treatment to improve nodulation of legumes and eliminate or reduce
the need for inoculation.
SUMMARY OF THE INVENTION
[0012] Disclosed herein are methods of increasing, enhancing, or
accelerating root nodulation in a plant, accelerating growth of
nitrogen fixing bacteria in nodules of a plant, increasing protein
content in a plant, increasing yield of a plant, improving water
retention of a plant, or reducing water use of a plant, the method
comprising identifying a plant in need of root nodulation, and
applying to the plant a composition comprising a protein component
comprising yeast stress proteins resulting from subjecting a
mixture obtained from the yeast fermentation to stress.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0013] The treatment compositions disclosed herein are based on a
fermentation product that is mixed with a surfactant, where the
fermentation, based on yeast, is either aerobic or anaerobic, and
preferably incorporates a mechanism to stress the yeast cells to
yield essentially stress proteins. The proteins and surfactant form
a complex to be termed the protein/surfactant complex, or PSC. The
process and compositions are described in U.S. Pat. Nos. 7,476,529
and 7,658,848 and U.S. Patent Application Nos. 20080167445, all of
which are incorporated by reference herein in their entirety,
especially the passages describing the fermentation processes and
the stress steps following the fermentation processes.
[0014] In certain embodiments, the protein component of the
compositions disclosed herein are derived from the fermentation of
yeast. In some embodiments, the fermentation is an aerobic
fermentation, while in other embodiments the fermentation is an
anaerobic fermentation. In some embodiments, the protein systems
disclosed herein are derived from an aerobic fermentation of
Saccharomyces cerevisiae, which, when blended with surface active
agents or surfactants, enhance multiple chemical functions, at
ambient conditions, or during and after exposure to the extreme
conditions. The protein systems disclosed herein can also be
derived from the fermentation of other yeast species, for example,
kluyveromyces marxianus, kluyveromyces lactis, candida utilis,
zygosaccharomyces, pichia, or hansanula.
[0015] After the aerobic fermentation process a fermentation
mixture is obtained, which comprises the fermented yeast cells and
the proteins and peptides secreted therefrom. In some embodiments,
the fermentation mixture can be subjected to additional stress,
such as overheating, starvation, oxidative stress, or mechanical or
chemical stress, to obtain a post-fermentation mixture. The
post-fermentation stress causes additional proteins to be expressed
by the yeast cells and released into the fermentation mixture to
form the stress protein mixture. These additional proteins are not
normally present during a simple fermentation process. The
additional proteins are known as "stress proteins," and are
sometimes referred to as "heat shock proteins". Once the
post-fermentation mixture is centrifuged, the resulting supernatant
comprises both the stress proteins and proteins normally obtained
during fermentation. The compositions described herein comprise
stress proteins.
[0016] Several, rather low molecular weight proteins can be
produced by Saccharomyces cerevisiae during fermentation as
practiced by those familiar in the art. These proteins appear when
the yeast cells have been placed under stress conditions during or
near the end of the fermentation process. Although referred to as
"heat shock proteins," the stress conditions can occur during
periods of very low food to mass concentrations, or as the result
of heat shock or pH shock conditions as described in U.S. Pat. No.
6,033,875, Bussineau, et al., incorporated by reference herein in
its entirety. In addition, chemical stress, oxidative stress,
ultrasonic vibration and other stress conditions can cause the
yeast to express the formation of heat shock proteins, more
accurately termed, "stress proteins."
[0017] Conditions for the post-fermentation procedures that produce
the "heat shock proteins" are described in above-incorporated U.S.
patents and publications. As is clear from the passages in the '414
publication, and the passages below, the regular fermentation steps
do not generate heat shock proteins. Steps that generate heat shock
proteins are administered after the fermentation step. It is
necessary for the generation of heat shock proteins to cause shock
to the fermented yeasts. This shock includes, for example, rapid
increase in the temperature, rapid change in the pH of the
fermentation broth, rapid physical stress, and the like.
[0018] As used herein, the term "protein component" refers to a
mixture of proteins that includes a number of proteins having a
molecular weight of between about 100 and about 450,000 daltons,
and most preferably between about 500 and about 50,000 daltons, and
which, when combined with one or more surfactants, enhances the
surface-active properties of the surfactants. In some embodiments,
the protein component comprises a mixture of multiple intracellular
proteins and compounds, where at least a portion of the mixture
includes yeast polypeptides obtained from fermenting yeast and
yeast stress proteins resulting from subjecting a mixture obtained
from the yeast fermentation to stress. The "multiple intracellular
proteins and compounds" includes proteins, small proteins,
polypeptides, protein fragments, and the like, that are not
normally expressed by yeast cells during the fermentation process.
These proteins and compounds are only expressed when the yeast
cells are subjected to stress and shock following the fermentation
process.
[0019] In a first example, the protein component comprises the
supernatant recovered from an aerobic yeast fermentation process.
Yeast fermentation processes are generally known to those of skill
in the art, and are described, for example, in the chapter entitled
"Baker's Yeast Production" in Nagodawithana T. W. and Reed G.,
Nutritional Requirements of Commercially Important Microorganisms,
Esteekay Associates, Milwaukee, Wis., pp 90-112 (1998), which is
hereby incorporated by reference. Briefly, the aerobic yeast
fermentation process is conducted within a reactor having aeration
and agitation mechanisms, such as aeration tubes and/or mechanical
agitators. The starting materials (e.g., liquid growth medium,
yeast, a sugar or other nutrient source such as molasses, corn
syrup, or soy beans, diastatic malt, and other additives) are added
to the fermentation reactor and the fermentation is conducted as a
batch process.
[0020] After fermentation, the fermentation product may be
subjected to additional procedures intended to increase the yield
of the protein component produced from the process. Several
examples of post-fermentation procedures are described in more
detail below. Other processes for increasing yield of protein
component from the fermentation process may be recognized by those
of ordinary skill in the art.
[0021] The supernatant is obtained when the fermentation broth is
centrifuged and the cellular debris is separated from liquid broth.
While in some embodiments, as discussed above, the supernatant of
the fermentation process is used in preparing the compositions
described herein, in other embodiments, the fermentation broth is
used without any processing. Therefore, in these embodiments, the
mixture used in preparing the compositions described herein is the
fermentation broth containing excreted proteins and polypeptides
and cellular debris, and whole yeasts.
[0022] Saccharomyces cerevisiae is a preferred yeast starting
material, although several other yeast strains may be useful to
produce yeast ferment materials used in the compositions and
methods described herein. Additional yeast strains that may be used
instead of or in addition to Saccharomyces cerevisiae include
Kluyveromyces marxianus, Kluyveromyces lactis, Candida utilis
(Torula yeast), Zygosaccharomyces, Pichia pastoris, and Hansanula
polymorpha, and others known to those skilled in the art.
[0023] In the first embodiment, Saccharomyces cerevisiae is grown
under aerobic conditions familiar to those skilled in the art,
using a sugar, preferably molasses or corn syrup, soy beans, or
some other alternative material (generally known to one of skill in
the art) as the primary nutrient source. Additional nutrients may
include, but are not limited to, diastatic malt, diammonium
phosphate, magnesium sulfate, ammonium sulfate zinc sulfate, and
ammonia. The yeast is preferably propagated under continuous
aeration and agitation between 30 to 35.degree. C. and at a pH of
4.0 to 6.0. The process takes between 10 and 25 hours and ends when
the fermentation broth contains between 4 and 8% dry yeast solids,
(alternative fermentation procedures may yield up to 15-16% yeast
solids), which are then subjected to low food-to-mass stress
conditions for 2 to 24 hours. Afterward, the yeast fermentation
product is centrifuged to remove the cells, cell walls, and cell
fragments. It is worth noting that the yeast cells, cell walls, and
cell fragments will also contain a number of useful proteins
suitable for inclusion in the protein component described
herein.
[0024] In an alternative embodiment, the yeast fermentation process
is allowed to proceed until the desired level of yeast has been
produced. Prior to centrifugation, the yeast in the fermentation
product is subjected to heat-stress conditions by increasing the
heat to between 40 and 60.degree. C., for 2 to 24 hours, followed
by cooling to less than 25.degree. C. The yeast fermentation
product is then centrifuged to remove the yeast cell debris and the
supernatant, which contains the protein component, is
recovered.
[0025] In a further alternative embodiment, the fermentation
process is allowed to proceed until the desired level of yeast has
been produced. Prior to centrifugation, the yeast in the
fermentation product is subjected to physical disruption of the
yeast cell walls through the use of a French Press, ball mill,
high-pressure homogenization, or other mechanical or chemical means
familiar to those skilled in the art, to aid the release of
intracellular, polypeptides and other intracellular materials. It
is preferable to conduct the cell disruption process following a
heat shock, pH shock, or autolysis stage. The fermentation product
is then centrifuged to remove the yeast cell debris and the
supernatant is recovered.
[0026] In a still further alternative embodiment, the fermentation
process is allowed to proceed until the desired level of yeast has
been produced. Following the fermentation process, the yeast cells
are separated out by centrifugation. The yeast cells are then
partially lysed by adding 2.5% to 10% of a surfactant to the
separated yeast cell suspension (10%-20% solids). In order to
diminish the protease activity in the yeast cells, 1 mM EDTA is
added to the mixture. The cell suspension and surfactants are
gently agitated at a temperature of about 25.degree. to about
35.degree. C. for approximately one hour to cause partial lysis of
the yeast cells. Cell lysis leads to an increased release of
intracellular proteins and other intracellular materials. After the
partial lysis, the partially lysed cell suspension is blended back
into the ferment and cellular solids are again removed by
centrifugation. The supernatant, containing the protein component,
is then recovered.
[0027] In a still further alternative embodiment, fresh live
Saccharomyces cerevisiae is added to a jacketed reaction vessel
containing methanol-denatured alcohol. The mixture is gently
agitated and heated for two hours at 60.degree. C. The hot slurry
is filtered and the filtrate is treated with charcoal and stirred
for 1 hour at ambient temperature, and filtered. The alcohol is
removed under vacuum and the filtrate is further concentrated to
yield an aqueous solution containing the protein component.
[0028] The compositions described herein include one or more
surfactants at a wide range of concentration levels. Some examples
of surfactants that are suitable for use in the detergent
compositions described herein include the following:
[0029] Anionic: Sodium linear alkylbenzene sulphonate (LABS);
sodium lauryl sulphate; sodium lauryl ether sulphates; petroleum
sulphonates; linosulphonates; naphthalene sulphonates, branched
alkylbenzene sulphonates; linear alkylbenzene sulphonates; alcohol
sulphates; PO and/or PO/EO sulfated alcohols.
[0030] Cationic: Stearalkonium chloride; benzalkonium chloride;
quaternary ammonium compounds; amine compounds.
[0031] Non-ionic: Dodecyl dimethylamine oxide; coco diethanol-amide
alcohol ethoxylates; linear primary alcohol polyethoxylate;
alkylphenol ethoxylates; alcohol ethoxylates;
[0032] EO/PO polyol block polymers; polyethylene glycol esters;
fatty acid alkanolamides.
[0033] Amphoteric: Cocoamphocarboxyglycinate;
cocamidopropylbetaine; betaines; imidazolines.
[0034] In addition to those listed above, suitable nonionic
surfactants include alkanolamides, amine oxides, block polymers,
ethoxylated primary and secondary alcohols, ethoxylated
alkylphenols, ethoxylated fatty esters, sorbitan derivatives,
glycerol esters, propoxylated and ethoxylated fatty acids,
alcohols, and alkyl phenols, alkyl glucoside glycol esters,
polymeric polysaccharides, sulfates and sulfonates of ethoxylated
alkylphenols, and polymeric surfactants. Suitable anionic
surfactants include ethoxylated amines and/or amides,
sulfosuccinates and derivatives, sulfates of ethoxylated alcohols,
sulfates of alcohols, sulfonates and sulfonic acid derivatives,
phosphate esters, and polymeric surfactants. Suitable amphoteric
surfactants include betaine derivatives. Suitable cationic
surfactants--include amine surfactants. Those skilled in the art
will recognize that other and further surfactants are potentially
useful in the compositions depending on the particular detergent
application.
[0035] Preferred anionic surfactants used in some detergent
compositions include CalFoam.RTM. ES 603, a sodium alcohol ether
sulfate surfactant manufactured by Pilot Chemicals Co., and
Steol.RTM. CS 460, a sodium salt of an alkyl ether sulfate
manufactured by Stepan Company. Preferred nonionic surfactants
include Neodol.RTM. 25-7 or Neodol.RTM. 25-9, which are C12-C15
linear primary alcohol ethoxylates manufactured by Shell Chemical
Co., and Genapol.RTM. 26 L-60, which is a C12-C16 natural linear
alcohol ethoxylated to 60E C cloud point (approx. 7.3 mol),
manufactured by Hoechst Celanese Corp.
[0036] Several of the known surfactants are non-petroleum based.
For example, several surfactants are derived from naturally
occurring sources, such as vegetable sources (coconuts, palm,
castor beans, etc.). These naturally derived surfactants may offer
additional benefits such as biodegradability.
[0037] One of the features of the PSC is the ability to accelerate
uptake of nutrients and accelerate metabolic processes of aerobic
bacteria based on a mechanism called uncoupling of oxidative
phosphorylation. And it has been shown that the uncoupling effect
and its benefits are observed at low temperature as well as at
ambient temperatures. An effect of this feature is to limit the
amount of biomass being formed, including the amount of biofilm
being developed, typically based on polysaccharides. The uncoupling
effect uncouples the microbe's ability to form complex proteins.
Nitrogenase is a complex protein and it would be expected that the
level of nitrogenase would be reduced with the uncoupling factor of
the PSC. To protect the needed nitrogenase system in the nodulation
process when aerobic bacteria are present, species like Azotobacter
and Rhizosium (The Microbial World: The Nirogen cycle and Nitrogen
fixation, Jim Deacon, University of Edinburgh) produce large
amounts of extracellular polysaccharide to limit the rate of
diffusion of oxygen into cells. The PSC treatment would appear to
be detrimental to the nitrogenase based on these phenomena. The
results of the tests, however, show otherwise.
[0038] The Rhizobia bacteria infect a plant's roots through its
root hairs. We have observed that PSC treated plants had a
substantially greater amount of fine root hair.
[0039] It is a hypothesis, but not a limitation of the current
invention, that the mechanism for the enhanced nodulation when
treated by the PSC is due to the following factors. The increased
uptake of nutrient by bacteria in soil treated by the PSC
accelerates the growth rate of appropriate nitrogen fixing bacteria
in the plant and the plant responds in kind by increasing the
amount of leghaemoglobin it produces in the nodules. The effect is
noted by the intensity of the reddish color observed in the treated
nodules, which can extend up into the roots. Further, since the PSC
has been shown to accelerate the growth of fine root hairs, then
this is believed to be an additional embodiment of the current
invention that improves nodulation of legumes. The increased uptake
of nutrient, as in nitrogen and Nod factors, is hypothesized to be
a factor in the higher rate of nitrogen fixation, which is noted by
the reddish color of the nodules.
[0040] Thus, in one aspect, disclosed herein are methods of
increasing, enhancing, or accelerating root nodulation in a plant,
the method comprising identifying a plant in need of root
nodulation, and applying to the plant a composition comprising a
protein component comprising yeast stress proteins resulting from
subjecting a mixture obtained from the yeast fermentation to
stress.
[0041] In another aspect, disclosed herein are methods of
accelerating growth of nitrogen fixing bacteria in nodules of a
legume, the method comprising identifying a legume in need thereof,
and applying to the legume a composition comprising a protein
component comprising yeast stress proteins resulting from
subjecting a mixture obtained from the yeast fermentation to
stress.
[0042] In another aspect, disclosed herein are methods of
increasing protein content in a legume, the method comprising
identifying a legume in need thereof, and applying to the legume a
composition comprising a protein component comprising yeast stress
proteins resulting from subjecting a mixture obtained from the
yeast fermentation to stress.
[0043] In another aspect, disclosed herein are methods of
increasing yield of a legume, the method comprising identifying a
legume in need thereof, and applying to the legume a composition
comprising a protein component comprising yeast stress proteins
resulting from subjecting a mixture obtained from the yeast
fermentation to stress.
[0044] In some embodiments of the above methods, the protein
component is obtained through the processes described above. In
some embodiments, the plant in need of such methods is a plant
being used to increase the nitrogen content of soil during crop
rotation, or a plant required to provide higher yield, or higher
nutrition content, or a plant required to have reduced water
use.
[0045] In some embodiments of the above methods, the composition is
applied to the soil near the plant. In some of these embodiments,
the composition is applied through irrigation, which can be spray
irrigation or drip irrigation. In certain embodiments, the
composition is applied with every watering cycle or, in other
embodiments, on an intermittent basis.
[0046] In some embodiments of the above methods, the protein
component is from aerobic fermentation of yeast. In some of these
embodiments, the protein component comprises proteins obtained from
exposing a product obtained from the fermentation of yeast to
additional procedures that increase the yield of proteins produced
from the fermentation. In certain embodiments, the stress is
selected from the group consisting of heat shock of the
fermentation product, physical and/or chemical disruption of the
yeast cells to release additional polypeptides, and lysing of the
yeast cells. In further embodiments, the stress comprises exposing
a product obtained from the fermentation of yeast to heat shock
conditions. In some embodiments, the stress comprises physically
disrupting the yeast after the fermentation of the yeast, while in
other embodiments, the stress comprises chemically disrupting the
yeast after the fermentation of the yeast. In some embodiments, the
stress comprises lysing the yeast after the fermentation of the
yeast.
[0047] In some embodiments of the above methods, the methods
further comprise mixing the protein component with additional
nutrients prior to the application to the plant. The additional
nutrients include, but are not limited to, fertilizers, sources of
phosphate, minerals, herbicides, and insecticides.
[0048] In some embodiments of the above methods, the composition
further comprises one or more of an anionic surfactant, a non-ionic
surfactant, a cationic surfactant, and amphoteric surfactant, as
described elsewhere herein.
[0049] In some embodiments of the above methods, the yeast is
selected from the group consisting of Saccharomyces cerevisiae,
Kluyveromyces marxianus, Kluyveromyces lactis, Candida utilis
(Torula yeast), Zygosaccharomyces, Pichia pastoris, and Hansanula
polymorpha.
[0050] In some embodiments of the above methods, the plant is a
legume. In certain embodiments, the legume is selected from the
group consisting of alfalfa, clover, peas, beans, lentils, lupins,
mesquite, carob, soy, peanuts, locust trees (Gleditsia or Robinia),
wisteria, and the Kentucky coffeetree (Gymnocladus dioicus).
[0051] In other embodiments of the above methods, a volume of soil
is premixed with the above composition to form a mixture, and then
the mixture is applied to the plant. Thus, in another aspect,
disclosed herein is a soil mixture comprising soil and a
composition comprising a protein component comprising yeast stress
proteins resulting from subjecting a mixture obtained from the
yeast fermentation to stress, as described above.
[0052] In another aspect, disclosed herein are methods of improving
water retention of a legume, reducing water use of a legume,
accelerating root nodulation in a legume, accelerating nitrogen
fixation by a legume, accelerating growth of nitrogen fixing
bacteria in nodules of a legume, increasing protein content in a
legume, or increasing yield of a legume, the method comprising
identifying a legume in need thereof, and applying to the legume a
soil mixture as described above.
[0053] In some embodiments of the above methods, the methods
further comprises inoculating the soil with bacteria prior to the
application of the composition. In other embodiments, the methods
are practiced without inoculation of the soil with any
bacteria.
[0054] In yet another aspect, disclosed herein are methods of
increasing the tolerance of a legume to colder climates, the method
comprising identifying a legume in need thereof, and applying to
the legume a protein component comprising yeast stress proteins
resulting from subjecting a mixture obtained from the yeast
fermentation to stress, as described above.
Example 1
[0055] A fermentation mixture derived from the fermentation of
Saccharomyces cerevisieae in which the yeast cells are stressed by
raising the temperature to at least 35.degree. C. for at least two
hours, then cooling to <30.degree. C. centrifugation. Upon
removal of the yeast cells by centrifugation the pH is adjusted to
4.0 and sodium benzoate and 21.1% propylene glycol is incorporated
to provide stability.
TABLE-US-00001 PSC Linear Primary Alcohol (C12-C15), 7 mole
Ethoxylate 7.5% Sodium Lauryl Ether (3 mole) Sulfate (60%) 2.5%
Stabilized Fermentation Mixture 23% Water 67% TOTAL 100%
[0056] Host plants: Peas, Pisum sativum. Two seeds were sown in 6
inch diam pots (approx. 3500 ml volume) filled with UC Mix II
(Matkin and Chandler 1957). Soil Mix II is formulated with plaster
sand, bark, peat moss, Dolomite, limestone flour, triple super
phosphate, potassium nitrate, muriate of potash, ferrous sulfate,
copper sulfate, magnesium sulfate, zinc sulfate, and manganese
sulfate. Once sown, the pots were watered on a daily basis until
the plant was visible above ground. The resulting plants were
culled to one plant per pot at the cotyledon (seed leaf) stage.
Treatment applications commenced following culling.
[0057] Host Plant Care: Plants were placed on raised greenhouse
benches for study. The plants were fertilized with Miracle-Gro
(with minors) general-purpose fertilizer at 200 ppm nitrogen.
Fertigation protocol was such that each plant in the experiment
received the same amount of fertilizer throughout the
experiment.
[0058] Applications: A control treatment of water only was used in
this experiment. Protein Surfactant Combination PSC was applied by
hand at 75 ppm three times a week. Product was added to water at
the appropriate concentration such that each plant received 90 ml
of solution at every application. Water at 90 ml was added to each
control plant when treatment applications were performed.
[0059] Experimental Design: There were eight plants that were
treated and the number of treatments of control pots that had no
PSC treatment.
[0060] Sampling: A destructive sample was taken at 30 and 60 days
after treatment initiation. Root weight and rhizome production were
determined.
Results
[0061] Mean (grams.+-.SE) dry weight of roots and the number and
dry weight of rhizomes of sweet peas treated with selected protein
surfactant combinations.
TABLE-US-00002 Mean root Mean no. of Mean dry weight Treatment dry
weight.sup.1 rhizomes.sup.2 of rhizomes.sup.1 PSC 2.16 .+-. 0.15a
49.2 .+-. 12.7a 0.086 .+-. 0.014 Control 1.93 .+-. 0.14a 31.6 .+-.
12.6a 0.040 .+-. 0.014 .sup.1Means followed by different letters
are significantly different, LSD (p = 0.05) .sup.2Means are
significantly different at p = 0.01, ChiSq = 5.11, df = 2, P =
0.0775.
Discussion
[0062] The PSC nodules were more than twice the weight of the
Control and there were 56% more nodules than the Control. Further,
the PSC treated nodules were reddish in color, indicating a high
level of nitrogen fixation. The Control nodules had a brownish
color, indicating little nitrogen fixation. This suggest a higher
protein content and higher crop yield.
* * * * *