U.S. patent application number 15/381006 was filed with the patent office on 2017-06-08 for protein compositions for plant treatment.
The applicant listed for this patent is ADVANCED BIOCATALYTICS CORPORATION. Invention is credited to John W. BALDRIDGE, Michael G. GOLDFELD, Andrew H. MICHALOW, Carl W. PODELLA.
Application Number | 20170156343 15/381006 |
Document ID | / |
Family ID | 42243101 |
Filed Date | 2017-06-08 |
United States Patent
Application |
20170156343 |
Kind Code |
A1 |
MICHALOW; Andrew H. ; et
al. |
June 8, 2017 |
PROTEIN COMPOSITIONS FOR PLANT TREATMENT
Abstract
Disclosed herein are methods of accelerating root growth in a
plant, the method comprising applying to the plant root a
composition comprising a) a mixture of proteins and polypeptides,
and b) a surfactant, whereby root growth is accelerated as compared
to an untreated plant. Also disclosed herein are methods of
improving the foliar uptake of a biologically active compound by a
plant, the method comprising applying to the plant foliage a
composition comprising a) a mixture of proteins and polypeptides,
and b) a surfactant, whereby root growth is accelerated as compared
to an untreated plant.
Inventors: |
MICHALOW; Andrew H.;
(Irvine, CA) ; PODELLA; Carl W.; (Irvine, CA)
; BALDRIDGE; John W.; (Irvine, CA) ; GOLDFELD;
Michael G.; (Irvine, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ADVANCED BIOCATALYTICS CORPORATION |
Irvine |
CA |
US |
|
|
Family ID: |
42243101 |
Appl. No.: |
15/381006 |
Filed: |
December 15, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14524119 |
Oct 27, 2014 |
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15381006 |
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13140830 |
Feb 14, 2012 |
8871682 |
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PCT/US2009/067779 |
Dec 11, 2009 |
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14524119 |
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61201600 |
Dec 12, 2008 |
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61201661 |
Dec 12, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01N 63/10 20200101;
C05F 11/10 20130101; C05G 3/70 20200201; C05F 11/10 20130101; A01N
25/30 20130101; A01N 37/46 20130101; C05F 11/10 20130101; C05G 3/70
20200201; C05G 3/70 20200201 |
International
Class: |
A01N 63/02 20060101
A01N063/02; A01N 37/46 20060101 A01N037/46; C05G 3/06 20060101
C05G003/06; A01N 25/30 20060101 A01N025/30 |
Claims
1-19. (canceled)
20. A method of improving the foliar uptake of a biologically
active compound by a plant, the method comprising applying to the
plant foliage a composition comprising a) a mixture of proteins and
polypeptides, obtained from fermentation of yeast, and b) a
surfactant, wherein the surfactant is non-ionic, anionic, an
anionic/non-ionic surfactant blend or combinations thereof, whereby
protein/surfactant composition enhances the efficacy of the
biologically active compound.
21-30. (canceled)
31. The method of claim 20, wherein the biologically active
compound is selected from the group consisting of pesticides,
insecticide, nutrients, fertilizers, growth regulators, herbicides,
fungicides, defoliants, anti-parasitics, anti-pathogenics and
combinations thereof.
32. The method of claim 20, wherein the penetration of the
biologically active compound into the plant, plant leaf, plant
foliage or combination thereof is/are improved.
33-34. (canceled)
35. The method of claim 20, wherein the translocation of the
biologically active compound into the plant, or the sticking of the
biologically active compound to exterior surface of the plant, is
improved.
36. (canceled)
37. The method of claim 36, wherein the exterior surface of the
plant is selected from the group consisting of leaves, needles, and
vegetation.
38. A method of reducing plugging within an irrigation system,
comprising a) dosing the irrigation system with an aqueous
composition comprising a) a mixture of proteins and polypeptides,
obtained from fermentation of yeast, and b) a surfactant, wherein
the surfactant is non-ionic, anionic, an anionic/non-ionic
surfactant blend, or combinations thereof.
39. The method of claim 38, further comprising a biologically
active compound selected from the group consisting of pesticides,
insecticide, nutrients, fertilizers, growth regulators, herbicides,
fungicides, defoliants, anti-parasitics, anti-pathogenics and
combinations thereof.
40. The method of claim 38, wherein the dosing is performed
continuously for between about 5 hours to 12 hours at a
concentration of at least about 0.1 ppm.
41. The method of claim 43, wherein the biofouling is provided with
biofilms within the irrigation system.
42. A method of improving the wetting of soil, growth media, or a
combination thereof, the method comprising applying to the soil,
growth media, or a combination thereof an aqueous composition,
comprising a) a mixture of proteins and polypeptides, obtained from
fermentation of yeast, and b) a surfactant, wherein the surfactant
is non-ionic, anionic, an anionic/non-ionic surfactant blend, or
combinations thereof.
43. The method of claim 46, further comprising a biologically
active compound selected from the group consisting of pesticides,
insecticide, nutrients, fertilizers, growth regulators, herbicides,
fungicides, defoliants, anti-parasitics, anti-pathogenics and
combinations thereof.
44. The method of claim 46, wherein the composition is applied
through a spray irrigation system, drip irrigation system or
combination thereof.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/524,119, filed Oct. 27, 2014, which claims
priority to U.S. patent application Ser. No. 13/140,830, filed Feb.
14, 2012, now U.S. Pat. No. 8,871,682, issued Oct. 28, 2014, which
is the National Stage of International Application No.
PCT/US2009/067779 filed Dec. 11, 2009, which claims priority to the
U.S. Provisional Application No. 61/201,600, filed Dec. 12, 2008,
and to U.S. Provisional Application No. 61/201,661, filed Dec. 12,
2008, each of which is hereby incorporated in its entirety
including all tables, figures and claims.
FIELD OF THE INVENTION
[0002] The invention belongs to the field of agricultural
chemicals, more specifically to the materials which improve the
transfer to, penetration into, and uptake by the foliage and roots
of agricultural plants of water and water-soluble chemicals, or
herbicides by the shoots or roots of weeds. The invention also
relates to the field of adjuvants used to increase the foliar
uptake of biologically active compounds.
BACKGROUND OF THE DISCLOSURE
[0003] The dimensions of root systems vary dramatically between
plant species, and differ within the same species depending on
soil/growth conditions. Roots provide the key mechanism of uptake
of water and nutrients for plants by developing a complex network
structure within soil. They maintain a symbiotic relationship with
microbes by secreting compounds that feed microbes, which then
break down organic nutrients into forms that can be more readily
absorbed by the roots and utilized by the plant. The robustness of
the root system is a key factor to the yield that a plant can
produce. It is desirable to have a more dense root system, as
increased in root density translates into a faster growth for the
plant, which is agriculturally and economically useful.
[0004] Further, many biologically active compounds, such as
pesticides, are sprayed on the leaves of plants for foliar uptake.
Plant leaves are generally waxy with small pores, which make it
difficult for the organic molecules to penetrate the leaf.
Solutions containing the active compounds typically contain
adjuvants that help with the foliar uptake. However, the adjuvants
currently used are not very efficient and most of the biologically
active compounds do not reach their targets.
SUMMARY OF THE INVENTION
[0005] Disclosed herein are methods of accelerating root growth in
a plant, the method comprising applying to the plant root a
composition comprising a) a mixture of proteins and polypeptides,
and b) a surfactant, whereby root growth is accelerated as compared
to an untreated plant. Also disclosed herein are methods of
improving the foliar uptake of a biologically active compound by a
plant, the method comprising applying to the plant foliage a
composition comprising a) a mixture of proteins and polypeptides,
and b) a surfactant, whereby root growth is accelerated as compared
to an untreated plant.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0006] Most plant absorption is believed to take place in the fine
roots, less than two millimeters in diameter, and root hair at the
tip of the root, forming big surface area. For roots to be able to
uptake nutrients or other chemicals, they must come into contact
with those chemicals. To "feed" the roots, it is therefore
essential that the mass flow of water, macro- and micro-nutrients,
herbicides, various growth treatments, collectively to be called
"effector," is directed toward the immediate vicinity of the roots.
Then diffusion driven by the concentration gradients results in
uptaking the effector by the root.
[0007] In most cases, such as turf, agriculture, ornamentals, etc.,
where harvesting is a goal, it is important to develop adequately
large root systems and as quickly as possible to optimize yield
within a growing period. In plug propagation root maturity can
range from three weeks for Colesia species to ten weeks for
Ranunculus, to over fourteen weeks for Cyclamen. Faster root growth
reduces time to maturity when plants are ready for transplant,
which can cut costs substantially at plug nurseries. In some
species of plants, the initial shoot growth far outpaces root
growth, which makes transplanting more difficult and may require
use of shoot growth inhibitors. Many herbs such as tarragon,
oregano and savory have a difficult time developing good root
systems. In agricultural production of tarragon, for example, the
root systems are typically thin and delicate. Abrupt changes in
environmental conditions can be detrimental to a crop reducing
yields by 70% in some cases. Tarragon can be harvested numerous
times from a single plant, being cut back multiple times.
[0008] Plant's growth cycle after seeding typically starts with a
foundation of a tap root that extends down with side branching
following. At some point of root maturity, the embryonic first
leaves penetrate through the soil of the seedling, the cotyledon
stage. It has been observed that the protein-surfactant complexes
(PSC) disclosed herein stimulate root growth once roots start to
appear, and at this early growth stage appears to focus the plant's
energy production toward root growth, which takes away somewhat
from shoot growth. At a certain point in root maturity the more
dense roots then have the capacity to accelerate shoot growth
relative to root density for a particular plant species, as would
be expected with more dense root systems.
[0009] In plug nurseries, the goal is to allow the plants to mature
to a point where the roots can be easily transplanted. This is done
by pulling plugs to determine whether the entire root mass and soil
remain intact when pulled. In some plant species the shoots grow
disproportionately fast as compared to the roots, which means that
the plants could be fragile when transplanting. In such species,
plant growth regulators are used to reduce the shoot growth rate at
early stages of plant growth. One of the features of the PSCs
disclosed herein is that they show the ability in early stages of a
plant's growth cycle to enhance the root growth, while at the same
time curtailing the shoot growth. This provides many benefits for a
nursery. The application of a plant growth regulator (PGR) for
species such as Vinca vine, is typically done manually. Both the
cost of the PGR and the labor for application are significant. The
second disadvantage for growers is that the PGR has to be applied
before the excess shoot growth. The PSC, on the other hand can be
fed at a low dose continuously with each watering cycle, including
nutrients as needed. The cost of the PSC is relatively low and
there is no secondary, manual dosing application required.
[0010] PGRs are generally plant hormones, chemicals that regulate
plant growth in various aspects. They are produced within plants
and occur in extremely low concentrations, 10.sup.-5 to 10.sup.-6
mol/L. There are several classes of PGRs: abscisic acid, auxins,
cytokinins, ethylene generators, gibberellins, brassinolides. The
PSC does not fall under any of these classes. PGRs are typically
dosed in limited applications at a very specific period of a
plant's growth cycle to elicit a desired response. For example,
auxins are used to promote root growth by dipping cuttings into an
auxin based solution in one step. PGRs in general can be phytotoxic
in higher concentrations.
[0011] PGRs are well known and regularly used in agriculture. The
purpose of a PGR is to manipulate the growth rate of a plant to
accelerate or inhibit certain aspects of plant growth--roots,
shoots, vegetation, sprouting, ripening, side branching, canopy,
etc. Many agents are synthetic chemicals.
[0012] The PSC effects as a plant growth regulator have been
limited in growth effects. In early stages of plug propagation, it
was observed that plants accelerated root growth at the expense of
shoot growth. This was not observed in all species, but is likely
due to different growth cycles. The heights of Vinca, broccoli,
Gerbera Daisies, Tarragon were reduced, while root production was
either enhanced or the same as controls. All of the species were
treated with, more or less, continuous dosing with water
cycles.
[0013] From a safety and environmental perspective, a key
difference between the PSC and other PGR's, drip line treatments,
adjuvants systems and agricultural chemical treatments is that the
PSC is classified as a "food" when appropriate surfactants, like
sodium lauryl sulfate, Sorbitans and other food additive
surfactants are used. The PSC is environmentally and
toxicologically benign, again, when appropriate surfactants are
used. A range of such surfactants exist that can be chosen from to
optimize performance for a particular set of conditions and desired
effects. From a regulatory standpoint, this means that the PSCs
disclosed herein have minimal regulatory hurdles. As an example to
distinguish the safety benefits of the PSC, if typical PGR's were
accidentally misapplied, environment effects could be detrimental.
The PSC can be applied at a minimum concentration of 12 ppm and
with, more or less, continuous dosing the PSC's can accumulate in
the short-term when root systems are constrained, as in pot
growth.
[0014] Further, the protein component has been shown to be
environmentally benign. Using OECD (Office of Environmental
Compliance and Documentation) Method 301B (CO.sub.2 Evolution Test)
the proteins were shown to be "readily biodegradable" according to
Group of Experts on Scientific Aspects of Marine Environmental
Protection (GESAMP) Hazard Evaluation procedure and was not
inhibitory to the degradation of the reference compound, which is
the surfactant component. In aquatic toxicity analysis, at up to
10,000 ppm (1%) of the proteins, survival of sheepshead minnow was
100%. The tests followed protocol and guidelines followed by the
U.S. Environmental Protection Agency (USEPA) Ecological Effects
Tests Guidelines (OECD Guideline for Testing Of Chemicals 203),
supplemented by the USEPA Acute guidelines for Whole Effluent
Toxicity testing. Finally, the GESAMP hazard profile rating was
concluded to be zero and that the hydrophilic proteins are
"estimated to have no potential to bioaccumulate based on the
GESAMP hazard profile rating scheme (Section 4.1.1.2, Table 3 of
GESAMP Procedure Document). Accidental spillage or misapplication
of the PSC should not cause any harm either to the environment or
to a grower or the grower's crops.
[0015] Accidental spillage or misapplication of the PSC should not
cause any harm either to the environment or to a grower or the
grower's crops.
[0016] In a growth area with adequate weather, where watering and
feeding are not limiting factors in root development, the
soil/growth media conditions become the determining factor in how
well roots, and then the desired plant features, will grow. Growth
media, in addition to soil, can range from porous ceramics
materials, to highly organic composts and peat, clay pebbles,
vermiculite, coconut fibers, perlite and others. The purpose of
soil or any growth media is to support the plant roots, retain
moisture, allow space for good air flow for oxygen to get to the
roots and act as a nutrient delivery system.
[0017] Most agriculture is grown, however in some type of soil.
Soil conditions are characterized by a number of features including
its bulk density, porosity, pore size, aggregate structure and soil
chemistry. For example, soil particles can be hydrophobic
(lipophilic), or water resistant, and this condition reduces water
permeability into the soil and reduces water retention by
preventing wetting of soil particles. Consistent wetting of soil
promotes dense root growth and is necessary for healthy root
growth. The PSC compositions of the current invention improve
wetting of soil and penetration of aqueous solutions into
soils.
[0018] A key benefit to the embodiments of the current invention is
that water usage can be significantly reduced both directly through
improved soil/growth media penetration and retention, and
indirectly by improving productive yield. Worldwide, agriculture is
responsible for 70% of water use by humans and fresh water
resources are diminishing in many areas. Though 60% of food is
produced using rain-fed systems, supplemental irrigation can
increase yields substantially.
[0019] One of the most efficient ways to deliver water and aqueous
compounds to plants is the use of drip irrigation systems. Drip
systems can, for example, improve banana yields by 52% with 45%
less water. But drip systems are prone to plugging from particles
in water, scaling or biofouling in the form of biofilms, or their
combination. Biofilms are particularly problematic as they
exacerbate the scaling problem and act to trap particulates both of
which enhance the plugging tendency. The PSC does not exhibit
direct scale inhibiting characteristics and it is surmised, though
not a limitation to the current invention, that the biofilm control
mechanism is helping to achieve scale inhibition, where the
formation of a biofilm acts as a propagation site and forms a
composite structure with the scale-causing molecules.
[0020] In a field study with water typically above 1,000 ppm total
dissolved solids, a good portion of which is calcium and magnesium
hardness, and iron, a PSC content of under 10 ppm would not control
scale if it was directly interacting with scale-causing molecular
species. A 1 ppm dose was used to control scale. The low dose rate
adds to benign nature of the PSC for scale and fouling control and,
further, reduces costs, which makes its use practical and cost
effective in both low value and high value plant species. To
control drip irrigation. To control drip irrigation line and nozzle
tip plugging, filtering techniques, and chemical cleaners and
treatments have traditionally been used.
[0021] Chlorination is one method to control biological growth, but
chlorine is environmentally hazardous and forms chloramines with
ammonia in effluents used in irrigation. Chloramines are 80 times
less effective than chlorine in controlling biological growth.
Further, the PSC is generally nonreactive with nutrients, such as
N, P, K, etc., and in this regard can be fed in conjunction with
the nutrients. This would simplify the dosing process by
eliminating a second operation to add cleaning chemicals to a drip
system.
[0022] In some instances copper salts have been used with the dual
purpose of being antimicrobial agents to control bacterial growth
in drip irrigation lines, and then act as micronutrients once in
the soil. However, copper salts can be toxic and pose risks to farm
workers. In addition, the copper solution would have to be
continuously dosed to prevent biofilm formation. Once biofilms
form, the copper would not be able to penetrate or remove them.
Other cleaning systems use phosphonic acids and organic phosphonic
acids or fatty acids to clean lines. These are typically below pH
of 2 to clean lines and then the phosphorus acts as nutrient in the
soil. The low pH can be hazardous to the farm worker and equipment.
Continuous feed to maintain clean lines could be costly and
overfeeding of phosphorus could lead to run-off of phosphorus,
which can exacerbate eutrophication of ecosystems. Many of these
need to be metered in and this adds to the workload of the farm
worker. In the current invention the PSC composition is designed to
be supplied continuously, intermittently, typically between 1 to 10
ppm, simplifying application and keeping costs low.
[0023] As discussed herein below, the protein enhanced surfactants
can be effective in both foliar and root uptake. This could
simplify the end users' needs when it comes to inventory of
adjuvants and when foliar application is used, the overspray is not
wasted but has efficacy in soil or other growth media
treatment.
Root Growth Applications
[0024] The prototype formulae consist of two primary
components;
[0025] 1) surfactants that facilitate the penetration of the water
into the soil or other growth media, and increase bioavailability
to the plant's roots, and
[0026] 2) proteins derived from the yeast fermentation, which
increase the functionality of the surfactants by further reducing
the interfacial tension of the surfactant solution, and reducing
the critical micelle concentration of the surfactant, thus allowing
a reduction in the amount of surfactant required to achieve good
penetration of the water.
Surfactants
[0027] Surfactants that are useful in the protein/surfactant
complex may be either nonionic, anionic, amphoteric or cationic, or
a combination of any of the above, depending on the application.
Suitable nonionic surfactants include ethoxylated amines and/or
amides, alkanolamides, 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, glycol
esters, polymeric polysaccharides, and certain polymeric
surfactants. Suitable anionic surfactants include sulfates and
sulfonates of ethoxylated alkylphenols, sulfosuccinates and
derivatives, sulfates of ethoxylated alcohols, sulfates of
alcohols, sulfonates and sulfonic acid derivatives, phosphate
esters, and certain polymeric surfactants. Suitable amphoteric
surfactants include amphoacetates, sulfobetaines, betaines and
amine oxides as well as derivatives thereof. Suitable cationic
surfactants include alkyl quaternary ammonium derivatives and
certain amine based surfactants. Those skilled in the art will
recognize that other and further surfactants are potentially useful
in the PSC composition. Some examples of surfactants that may be
applicable for use in the soil penetration and root uptake
compositions described herein include the following:
[0028] Anionic: Sodium linear alkylbenzene sulfonate (LABS); sodium
lauryl sulfate; sodium lauryl ether sulfates; sodium dioctyl
sulfosuccinates; petroleum sulfonates; linosulfonates; naphthalene
sulfonates, branched alkylbenzene sulfonates; linear alkylbenzene
sulfonates; fatty acid alkylolamide sulfosuccinate; alcohol
sulfates; ethosulfate compounds.
[0029] Cationic: Stearalkonium chloride; ammonium compounds, such
as benzalkonium chloride; quaternary ammonium compounds; amine
compounds
[0030] Non-ionic: coco diethanol-amides; alcohol ethoxylates;
linear primary alcohol polyethoxylate; branched primary and
secondary alcohol ethoxylates, alkyl phenol ethoxylates; EO/PO
polyol block polymers; alkykl EO/PO block copolymers; polyethylene
glycol esters; fatty acid alkanolamides.
[0031] Amphoteric: Dodecyl dimethylamine oxide;
Cocoamphocarboxyglycinate; cocamidopropyl betaine; betaine
derivatives; imidazoline derivatives, sulfobetaines and derivatives
thereof; amphoacetates and derivatives thereof.
[0032] 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.
[0033] It should be understood that these surfactants and the
surfactant classes described above are identified only as preferred
materials and that this list is neither exclusive nor limiting of
the compositions and methods described herein.
Fermentation
[0034] There have been numerous attempts in the past to utilize
chemicals from fermentation of yeast. The current patent is
differentiated from these in subtle, yet critical and relevant,
ways. Fermentation of yeast is used for applications including the
production of beer, sake and enzymes. The specifics of the present
invention's fermentation process form the basis for its uniqueness
and divergence from these other processes. These have been
discussed in previous patent applications (See U.S. Pat. Appn. Nos.
20050245414, 20040180411 and 20080167445, all of which are
incorporated herein by reference in their entirety).
[0035] The present inventor obtained low molecular weight protein
factor from yeast fermentation, preferably aerobic, processes
which, when coupled with surfactants, reduce the critical micelle
concentration of surfactants, surface tension and interfacial
tension of surfactant solutions, with reductions in the critical
micelle concentration, surface tension, and interfacial tension as
compared to the surfactants taken alone, and further reduction of
the same parameters observed after exposure to grease and oil. This
factor was found in the yeast fermentation-derived polypeptide
fractions ranging in molecular weights between about 6,000 and
17,000 daltons by the results of polyacrylamide gel
electrophoresis.
[0036] The compositions disclosed herein comprise a yeast aerobic
fermentation supernatant, surface-active agents and stabilizing
agents. Saccharomyces cerevisiae is grown under aerobic conditions
familiar to those skilled in the art, using a sugar source, such as
molasses, or soybean, or corn, or cane sugar, as the primary
nutrient source. Alternative types of yeast that can be utilized in
the fermentation process may include: Kluyeromyces maxianus,
Kluyeromyces lactus, Candida utilis (Torula yeast),
Zygosaccharomyces, Pichia and Hansanula. Those skilled in the art
will recognize that other and further yeast strains are potentially
useful in the fermentation and production of the low molecular
weight proteins, "the protein system." It should be understood that
these yeasts and the yeast classes described above are identified
only as preferred materials and that this list is neither exclusive
nor limiting of the compositions and methods described herein.
[0037] The proteins of the disclosed compositions comprise
proteins, protein fragments, peptides, and stress proteins having a
size less than 30 kDa. In some embodiments, the size range is from
about 0.5 kDa to about 30 kDa. Throughout the present disclosure,
the protein mixture used in the PSC compositions disclosed herein
is referred to as the "protein system."
[0038] The word "peptide" includes long chain polypeptides, such as
proteins, as well as short chain peptides, such as dimers, trimers,
oligomers, and protein fragments. In some embodiments, the words
"polypeptide" and "protein" are interchangeable.
[0039] In some embodiments, the protein systems disclosed herein
are derived from a fermentation of Saccharomyces cerevisiae, which,
when blended with surface active agents or surfactants, enhance
multiple chemical functions. 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. In a preferred
embodiment, the fermentation process is aerobic.
[0040] 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, overfeeding, oxidative stress, or
mechanical or chemical stress, to obtain a post-fermentation
mixture. The additional stress causes additional proteins ("stress
proteins") to be expressed by the yeast cells and released into the
fermentation mixture. These additional proteins are not normally
present in significant quantity during a simple fermentation
process. Once the post-fermentation mixture is centrifuged, the
resulting supernatant comprises both the stress proteins and
proteins normally obtained during fermentation. The
post-fermentation mixture may then be stabilized to prevent
degradation or bacterial contamination through the use of
antimicrobial agents, preservatives and/or pH adjustment. The
compositions described herein comprise stress proteins.
[0041] The preferred embodiments of fermentation processes used for
the current invention are defined in previous patent applications
(See U.S. Pat. Appn. Nos. 20050245414, 20040180411 and 20080167445,
all of which are incorporated herein by reference in their
entirety). In addition, the ratio of fermentation supernatant to
surfactant is optimally in the range of 1 to 3, but in instances
where emulsifying characteristics are not important, it has been
found that interfacial tension can be reduced with higher protein
(supernatant) ratio relative to the amount of surfactant.
Alternatively, the protein ratio might be much less than 1. The
broad range of functionality gives the formulator flexibility in
optimizing products for specific end uses because the range of
different types of biologically active compounds (BAC's) may be
more or less compatible with the protein based adjuvant.
[0042] The application of stress proteins to improve the
performance of surface active agents has been previously introduced
in other fields by the Assignee of the current invention and these
were limited to application on inanimate surfaces. It was well
documented in the previous patents and patent applications that
yeast proteins considerably enhance surface activity of a broad
range of synthetic detergents, reducing surface and interfacial
tension, dynamic surface tension, interfacial tension and critical
micelle concentration in their solutions, and enhance their
performance in such areas as control and prevention of biofilm
formation, waste water treatment, cleaning, soil remediation, odor
control, etc. These fundamental aspects are now applied in the
current invention for application in the field of agricultural
chemicals, or adjuvants for active biological compounds.
[0043] Plant Growth Tests: Treatments included a fermentation-based
protein system, two surfactant systems, and combinations thereof
and an untreated control (fertilizer only). The Examples are
describes as follows:
EXAMPLE 1
[0044] A fermentation mixture that is derived from the fermentation
of Saccharomyces cerevisiae 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. prior to
centrifugation. Upon removal of the yeast cells by centrifugation,
the pH is adjusted to 4.0 and, 0.42% sodium benzoate and 21.10%
propylene glycol is incorporated to provide stability.
TABLE-US-00001 Component Composition (%) by Weight Stabilized
Fermentation Mixture 70.00% Water 30.00% Total 100.00%
EXAMPLE 2
[0045] A fermentation mixture that is derived from the fermentation
of Saccharomyces cerevisiae 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. prior to
centrifugation. Upon removal of the yeast cells by centrifugation,
the pH is adjusted to 4.0 and 0.42% sodium benzoate is incorporated
to provide stability.
TABLE-US-00002 Component Composition (%) by Weight Stabilized
Fermentation Mixture 62.50% Water 37.50% Total 100.00%
EXAMPLE 3
TABLE-US-00003 [0046] Component Composition (%) by Weight Linear
Primary Alcohol (C.sub.12-C.sub.15) 22.50% 7 mole Ethoxylate Sodium
Lauryl Ether (3 mole) 7.50% Sulfate (60%) Water 70.00% Total
100.00%
EXAMPLE 4
TABLE-US-00004 [0047] Component Composition (%) by Weight Dioctyl
Sulfosuccinate (75%) 25.00% Hexylene Glycol 12.50% Water 62.50%
Total 100.00%
EXAMPLE 5
TABLE-US-00005 [0048] Component Composition (%) by Weight Linear
Primary Alcohol (C.sub.12-C.sub.15) 22.50% 7 mole Ethoxylate Sodium
Lauryl Ether (3 mole) 7.50% Sulfate (60%) Stabilized Fermentation
Mixture 70.00% (Example 1) Total 100.00%
EXAMPLE 6
TABLE-US-00006 [0049] Component Composition (%) by Weight Dioctyl
Sulfosuccinate (75%) 25.00% Hexylene Glycol 12.50% Stabilized
Fermentation Mixture 62.50% (Example 2) Total 100.00%
[0050] The experimental compounds were tested on spinach beginning
in the cotyledon stage to 35 days after treatment initiation. A
destructive sample of treated plants was taken on days 7, 14, 24,
and 35, and root and shoot mass were determined by drying samples
and weighing them on a precision balance. At 35 days after
treatment initiation, Example 5 and Example 6 promoted
statistically greater root mass compared to the untreated control
plants, the surfactant only treated plants, and the ferment only
treated plants. In addition, fitted lines representing the
progression of growth during the trial indicated that Example 5 and
Example 6 had significantly greater slopes than all other
treatments, which suggests that Example 5 and Example 6 cause
greater root mass production than normal growing practices. Shoot
mass growth trends suggested that the larger root mass shows
absolute higher values of shoot mass in the PSC treatments.
[0051] Host plants: Spinach, (cv. Avon Hybrid). Two seeds were sown
on 7 Jun. 2008 in one-gallon pots (6 inch diameter, approx. 3.8L
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 were visible above ground. The
resulting plants were culled to one plant per pot at the cotyledon
(seed leaf) stage. Treatment applications commenced following
culling.
[0052] Host Plant Care: Plants were placed on raised greenhouse
benches for study. Plants were watered every other day with exactly
50 ml of solution containing fertilizer and 25-ppm of each protein
depending on the treatment above. Chemigation (treatment
applications and fertilization) will occur simultaneously. The
plants were fertilized with Miracle Grow.RTM. fertilizer at 100 ppm
nitrogen.
[0053] Treatment Applications: Host plant media was fully wetted
prior to the start of the trial. Treatment applications were
prepared in volume at the appropriate ppm and applied by volumetric
cylinder at 50-ml pot to avoid leaching and to maintain consistency
in treatment application. Early on, when the plants were small and
did not need as many applications, treatments were applied on day
1, 4, 7, 11, and 13 and approximately every third or fourth day
thereafter.
TABLE-US-00007 Treatments Rate PPM Frequency 1. Example 1 25 During
Watering Cycles 2. Example 2 25 During Watering Cycles 3. Example 3
25 During Watering Cycles 4. Example 4 25 During Watering Cycles 5.
Example 5 25 During Watering Cycles 6. Example 6 25 During Watering
Cycles 7. Untreated Control Fertilizer During Watering Cycles
only
[0054] Experimental Design: A randomized complete block design was
used in the trial. Five plants per treatment per week were used for
sampling, i.e. 5 plants.times.7 Treatments.times.5 weeks=175 plants
total. Plants treated with selected formulas will be compared to
plants treated with water and fertilizer alone.
[0055] Sampling: Three replicates/treatment were removed from the
trial prior to the application of selected formulas to compare
plants prior to the start of the trial. Thereafter and beginning
two weeks after the initial application, five replicates/treatment
were removed from the trial at 7, 14, 24, and 35 days to determine
if treatments have an effect on plant growth. These plants were
photographed to compare plant volumes between treatments. In
addition, the plants were dried in an oven, and weighed for dry
root and shoot mass on a Jennings Precision 20 brand scale with a
0.002-gram resolution.
[0056] Analysis: Data were analyzed using analysis of variance.
Where appropriate, data were transformed log (x+0.5) prior to
analysis to satisfy the assumptions of the analysis of
variance.
Results and Observations
[0057] Root Mass: Statistical differences between treatments were
not observed until Day 35 following initiation of treatment
applications (F=9.2; df=6.27; P <0.0001). A significantly
greater root mass was observed in plants treated with Example 5 and
Example 6 than the Control, 35 days after treatment initiation
(Table 2). There appears to be a trend in plants treated with both
Example 3 and Example 4 (Surfactants alone) where root mass was
beginning to increase but not significantly different than the
control. This is most likely due to the ability of the products to
wet the soil or other growth media and retain moisture thereby
benefiting the plant in the long run. Root mass in plants treated
with Example 4 was not significantly different than the Example 5
or Example 6, due to the limited number of plots, but absolute
figures show a double the root mass of Example 5 and Example 6
compared to Example 3 and Example 4.
[0058] Linear growth trends are as follows. Root mass growth
appeared exponential after 30 days of growth under the selected
treatments. Example 5 and Example 6 performed best compared to the
control and there were no statistical differences in slope between
the Control and all other treatments. There is a significant
difference between the slopes of the lines of Example 5 and Example
6 and the Control, i.e. there is a significant increase in growth
due to the Example 5 and Example 6 compared to the Control.
[0059] Shoot Mass: Statistical differences between treatments were
not observed until Day 24 (Table 4. F=2.87; df=6.28; P =0.0262)
following initiation of treatment applications and continued on Day
35 (Table 4. F=3.88; df=6.27; P =0.0064). There was a significantly
different shoot mass observed in treated plants both 24 and 35 Days
after treatment initiation.
[0060] Linear growth trends in shoot mass appeared exponential
after approximately 30 days of growth under the selected
treatments. Linear parameters of shoot mass growth over time are
presented in Table 5. It is generally understood that root mass
promotes a healthier and larger shoot mass given time to grow, as
increase root mass increases the ability of the plant to feed the
shoots.
[0061] The best examples of the treated plants from each sample
date were washed free of potting media and photographed. The
progression of root and shoot mass and a comparison of the
treatments are presented in the photographs. The surfactants
Example 3 and Example 4 acted as soil or media wetting agents, and
often a single strand of the roots followed the column of wet soil
to the bottom of the pots and proliferated, especially early on in
the trial. This is contrasted with an analogous effect with Example
5 and Example 6, the key difference with the PSC compositions being
that the root structure was significantly larger throughout the
depth of its growth, suggesting an effect more than merely improved
wetting of the soil. The larger root mass with the PSC solutions
indicated that the combination has a synergistic effect on
stimulating the growth of the roots and the subsequent trend toward
larger shoot mass. The mechanism has not been studied and is not
imperative for the purposes of the current invention, but is it
anticipated that the PSC mixtures stimulate symbiotic bacterial
activity between roots, bacteria and nutrients bound up organically
in the soil. This is important as it suggests that the current
invention has fundamental effects that would benefit a broad range
of plant species.
[0062] There is a trend in the data that is not observed in the
statistical analysis that shows that the `protein only` treatments
may have an antagonistic effect, i.e. there was less root and shoot
mass in plants treated with ferment only. The lack of positive
effects of the protein mixture absent added surfactants is
consistent with other work done by the Assignee of the current
invention, in that the proteins alone have little practical
benefit, but that they multiply the benefits of surfactants when
the both ingredients, proteins and surfactants, are formulated
together.
[0063] It is also important to note that the beneficial effects are
seen at very low ppm' s compared to previous attempts at using
surfactant systems to improve root and plant growth. This provides
a major cost benefit and return on investment for the grower.
[0064] Further noted was the observation that the Example 5 and
Example 6 treated soil maintained a black, "wet" appearance longer
than the untreated, and more so than the surfactant only pots.
Untreated soil tended to return to a crumby brown appearance more
quickly. This is consistent with what would be expected with the
other results of the trial. Improved wetting of the soil should
improve water retention and be beneficial to root and plant
growth.
[0065] Finally, it is important to note that the different
fermentation and stress shock techniques yielded consistent and
positive results. This result indicates that the protein
manufacturer has a ranee of processes to consider vis-a-vis its
process and its cost of production.
TABLE-US-00008 TABLE 2 Root Mass, gram Control Day Mean SE 7 0.01
0.00 14 0.02 0.01 24 0.09 0.03 35 1.19 b 0.22 Day Mean SE Mean SE
Mean SE Example 5 Example 3 Example 1 7 0.02 0.01 0.02 0.00 0.01
0.00 14 0.01 0.00 0.01 0.00 0.02 0.00 24 0.21 0.07 0.17 0.05 0.05
0.02 35 4.61 a 0.77 2.06 b 0.50 1.32 b 0.32 Example 6 Example 4
Example 2 7 0.03 0.01 0.03 0.01 0.02 0.00 14 0.02 0.01 0.01 0.00
0.02 0.01 24 0.16 0.04 0.16 0.05 0.27 0.06 35 4.34 a 0.83 2.45 ab
0.40 0.58 b 0.11 Shoot Mass, gram Control Day Mean SE 7 0.01 0.00
14 0.03 0.01 24 0.27 ab 0.06 35 1.34 ab 0.20 Day Mean SE Mean SE
Mean SE Example 5 Example 3 Example 1 7 0.02 0.00 0.03 0.01 0.01
0.00 14 0.03 0.01 0.03 0.01 0.03 0.00 24 0.52 a 0.10 0.39 ab 0.11
0.18 b 0.06 35 1.87 a 0.29 1.49 a 0.30 0.97ab 0.19 Example 6
Example 4 Example 2 7 0.02 0.01 0.02 0.00 0.02 0.00 14 0.02 0.01
0.03 0.01 0.05 0.01 24 0.46 ab 0.05 0.36 ab 0.03 0.56 a 0.10 35
1.95 a 0.34 1.19 ab 0.23 0.57 b 0.11
TABLE-US-00009 TABLE 4 Line parameters for dry weight root mass in
response to selected treatments. Root Mass, gram Treatment Slope
(SE) RSQ Y intercept Control 0.04 .+-. 0.01 0.60 (-0.48) .+-. 0.17
Example 5 0.16 .+-. 0.03 0.61 (-1.91) .+-. 0.67 Example 6 0.15 .+-.
0.03 0.58 (-1.79) .+-. 0.66 Equality of F = 6.94 df = 2.54 P =
0.0021 Slopes
TABLE-US-00010 TABLE 5 Line parameters for dry weight shoot mass in
response to selected treatments. Shoot Mass Treatment Slope (SE)
RSQ Y intercept Control 0.05 .+-. 0.01 0.72 (-0.85) .+-. 0.19
Example 5 0.07 .+-. 0.01 0.74 (-1.16) .+-. 0.28 Example 6 0.07 .+-.
0.01 0.70 (-1.26) .+-. 0.26 Equality of F = 1.78 df = 2.54 P =
0.1781 Slopes
[0066] In a second growth trial of spinach, the treatment methods
were the same but the number of pots was increased to ten per data
point. Results are shown in Table 6. Again, Day 40 refers to 40
days of treatment, commenced at the cotelydon stage, which was
about 14 days after seeding. Day 40 in Table 6 shows a dry root
mass five times larger in the PSC treated pots than the control and
a shoot mass 2.3 times larger in the PSC treated pots than the
control. For the baby, fresh spinach market a typical growth cycle
if 50 to 55 days and the results show significant, potential
commercial benefits. By Day 60 below the treated pots exhibited a
root bound condition and the growth of shoots in the control were
able to "catch up" to the treated pots.
TABLE-US-00011 TABLE 6 Mean, standard error and N (number of
experimental units) of root and shoot dry mass of spinach plants
treated with 25 ppm of Example 5. Control PSC Mean SE N Mean SE N
Dry Root Mass Day 20 0.0 0.0 9 0.1 0.0 10 Day 40 0.4 0.1 10 2.0 0.6
9 Day 60 17.2 2.9 9 42.7 8.7 7 Dry Shoot Mass Day 20 0.1 0.0 9 0.1
0.0 10 Day 40 0.3 0.1 10 0.7 0.1 9 Day 60 18.1 2.6 9 19.9 2.0 7
Plug Growth Data.
[0067] Plugs were treated from seeding at 25 ppm Example 5, watered
approximately 2 mL per watering with boom sprayer, about every
other day in a commercial plug growing facility using production
techniques to water and treat plugs. Each data point represents 10
plugs randomly pulled from trays. The PSC had positive effects on
root growth on both slow and fast maturing roots. Soil was an
organic peat moss blend with wetting agents and nutrients added to
the mix.
Ranunculus--Analyzed at 10 Weeks from Seeding
TABLE-US-00012 average average average average Average shoots, gr
root, gr dry shoots dry roots Height, mm Control 0.294 0.263 0.040
0.0334 42.971 Treated 0.437 0.496 0.051 0.0512 59.354
Coleus--5 Weeks from Seeding
TABLE-US-00013 average average average average shoots, gr roots, gr
dry shoots dry roots Control 0.622 0.410 0.0352 0.0418 Treated
0.729 0.805 0.0462 0.0924
Petunia--5 Weeks from Seeding
TABLE-US-00014 average average average average shoots, gr roots, gr
dry shoots dry roots control 0.857 0.299 0.0442 0.0229 treated
0.931 0.446 0.0484 0.052
Celosia--3 Weeks from Seeding
TABLE-US-00015 average average average average shoots, gr roots, gr
dry shoots dry roots control 0.281 0.1522 0.0196 0.0166 treated
0.297 0.1972 0.0182 0.0353
Fine Root Hairs and Secondary Roots.
[0068] A phenomenon that was not quantified but a significant
observation was that the PSC treated plants had a substantially
greater amount of fine root hairs and secondary roots than on the
untreated control. The phenomena was not observed in
surfactant-only or protein-only tests. It was observed in every
plant tested, whether treatment was (a) initiated immediately at
seeding, (b) initiated at the cotelydon stage as in the spinach
trials or (c) initiated in treatment of transplanted, previously
untreated plugs. This observation suggests that the root promotion
is a fundamental effect that, with proper optimization of treatment
protocols, should benefit a wide range of cultivars.
[0069] As this relates to PGRs, the PCS treatment is distinctly
different in that, (a) it is less specific in terms of when the
treatment could be dosed and (b) it is effective in a broad range
of plant species. Further, due to a required continuous stimulation
of the roots required for benefits to occur, coupled with the fact
that the PSC does not bio-accumulate in the environment, is
toxic-free (albeit with appropriate surfactant candidates of which
there are numerous) and can even be a food product, it poses
virtually no risk to the grower, the growers crop or to the
environment, even if accidentally misapplied.
Plant Growth Regulator Effects.
[0070] It was noted that several species of PSC treated plants
exhibited a reduction in shoot height, while at the same time root
weights were higher as shown in Tables 7 and 8.
TABLE-US-00016 TABLE 7 Tarragon - 8 weeks form seeding average
average average average Average shoots, gr roots, gr dry shoots dry
roots Height, mm Control 0.672 0.375 0.100 0.0464 54.16 PSC treat
0.637 0.433 0.093 0.0656 41.01
TABLE-US-00017 TABLE 8 Gerberas - 8 weeks from seeding average
average average average Average shoots, gr roots, gr dry shoots dry
roots Height, Mm Control 2.233 0.688 0.2274 0.072 102.219 PSC treat
2.354 1.0838 0.246 0.119 94.12
Scale and Fouling Inhibition
[0071] In a 3.5 acre field of larkspur flowers, irrigation starts
with spraying the field with sprinklers. After the cotyledon stage
is achieved, irrigation is done using drip tape. The well water
that is used in this field has a high TDS up to 2,000 ppm, always
above 1,000 ppm and a relatively high amount of iron, noted by the
orange scale color. The high level of solids in the water leads to
plugging of holes in the drip tape. This requires laborers to walk
the fields and manually dislodge the scaling where water has
stopped percolating. Manual cleaning is a costly process. In
addition, the life of the tape is limited due to the build-up of
scaling with multiple uses.
[0072] In our study, half the field was treated as follows and
results compared to the control. The dose rate was 1 ppm of PSC
formulation Example 5. The irrigation cycle was set for 10 hours
once per week, more or less. Total irrigation water used was
approximately 1,000,000 gallons at a flow rate of 270 gpm. The drip
tape had been previously used and already had some scaling from
past use. After 10 weeks of treatment it was noted and photographed
from 9 each of 4 foot sections of tape, randomly cut out from both
PSC treated and control, that there was significant reduction in
scale in the PSC treated as compared the control. It is believed
that one of the mechanisms of action of scale formation is biofilm
formation that acts as a foundation for scale to form. The
detergency properties as well as the uncoupling effects of the PSC,
which as shown in previous patent applications of the Assignee are
believed to be the basis for keeping the lines clean. In some holes
that had already been plugged, the PSC actually cleaned previously
plugged holes where percolation of water was observed after a
couple weeks of treatment. The benefits provided by the current
invention are that, in a drip irrigation system, a multi-purpose
function can be achieved. The PSC can keep drip lines clean, while
at the same time treating plants, increasing the cost effectiveness
of the product due to its multifunctional characteristics.
Foliar Applications
[0073] Also disclosed herein are methods of using the
above-described PSC such that the proteins improve the function of
surfactants, where the protein/surfactant compositions are
adjuvants that enhance the performance of plant treatments in above
ground application, largely through improved foliar wetting and
uptake. Examples of plant treatments utilizing foliar uptake may
include, but are not limited to, herbicides and nutrients, with
additional foliar applications including fungicides, insecticides
and the like. Plant treatment application areas include
agricultural, forestry, turf, ornamental, industrial, home,
aquatic, and others where such treatments are used. Finally, the
use of renewable, naturally derived adjuvant systems, such as the
proteins, are desirable because they are environmentally benign
with the possibility of being acceptable to being used on food
crops certified as "organic."
[0074] The improved penetration of agricultural chemical adjuvant
systems based on the protein/surfactant systems disclosed herein,
and specifically as related to foliar uptake, was a surprise. The
waxy surface of many leaves presents a tough barrier for the
penetration of plant chemical treatment solutions. The upper leaf
surface is coated with a waxy film, the cuticle, which prevents
moisture loss and acts as a barrier to insects and chemicals that
might harm the plant. A formulation using biologically active
compounds (BAC's) and adjuvants ultimately needs to penetrate the
plant's cuticle. It has generally been thought that smaller
molecules, given the same chemical properties, have a better chance
of penetrating the cuticle, or passing through defects in the
cuticle, than large ones. The diffusion rate of both lipophilic and
hydrophilic compounds across isolated leaf cuticles is thought to
be negatively correlated with their molecular weight (MW), or
molecular size. That is, lower MW molecules are understood to
diffuse through the cuticle and pass through defects in the cuticle
more readily than large ones. It was therefore a surprise to have
found that the relatively large protein/surfactant complexes, where
the proteins are orders of magnitude larger than surfactants, would
improve penetration through foliage. Studies of the
protein/surfactant mixture indicated that it forms a tight complex
that is difficult to break apart, such as when tested by way of
dialysis. The proteins themselves are several orders of magnitude
larger than surfactant molecules.
[0075] Adjuvants, most of which are based on surfactants, are
widely used in arable agriculture. They are materials added to
spray solutions to improve the performance of BAC's, which are
applied to either enhance, inhibit, protect or otherwise affect the
growth and development of plants. Examples include compatibility
agents (used to aid mixing e.g. two or more pesticides in a common
spray solution), drift retardants (used to decrease the potential
for pesticide drift), suspension aids (used to mix and suspend
pesticide formulations with lower solubility ingredients), spray
buffers (used to change the spray solution acidity), and especially
surfactants that improve wettability of plant tissues, and enhance
absorption and penetration of active ingredients into plant
tissues. The biologically active ingredients may directly affect
plant metabolism as nutrients, fertilizers, growth regulators,
herbicides, fungicides, defoliants, or indirectly, by targeting
parasitic, pathogenic, or symbiotic species.
[0076] In an example regarding pesticides, it is estimated that in
the worldwide use of agricultural chemicals only 0.1% of the total
amount of pesticides applied actually reach their targeted
destinations where their desired effects are utilized. The
remainder is lost to run-off of plants before having a chance to be
absorbed, spray drift, off-target spraying, environmental
degradation, and the like. Spray drift and targeting of the spray
are limited by logistics of the density of plants in a field and
mechanical spray systems. An embodiment of the current invention is
to reduce the amount of pesticides by improving both the rate of,
and degree of, penetration into plants, which would reduce costs
and environmental impact of residual pesticides.
[0077] The efficiency of a plant's uptake of BAC's, in most cases,
determines the optimal dose rates. Improving this efficiency would
reduce costs of BAC's, which are in many instances more expensive
than the cost of application or the cost of the adjuvants. Many
BAC's are cidal agents and are toxic. Minimizing their dose rate
would benefit the environment and exposure to farm and nursery
laborers. Environmental benefits are gained by reducing dosage
rates of micronutrients and fertilizers that can leach as runoff
into streams and rivers. In a further embodiment, since
phytotoxicity issues of surfactants are related to dosage, a
reduced dosage could allow the use of surfactants that would
otherwise be phytotoxic at higher dose levels than when formulated
with the protein mixture.
[0078] Among pesticidal adjuvants, nonylphenol ethoxylates (NPE)
surfactants are commercially available and broadly applied due to
their low cost and high level of efficacy. However, the wide use of
NPE's is currently questioned, because of their slow
biodegradation, transfer and bioaccumulation along the food chain
and their environmental impacts as endocrine mimickers. Another
embodiment of the current invention is the replacement of NPE
surfactants and improvement of their performance as pesticidal
adjuvants with the protein system of the current invention.
[0079] Other approaches to improve on environmental objectives in
the chemical treatment of crops include replacing environmentally
undesirable synthetic adjuvants with botanically derived, or
"green" products that also could meet the requirements of "organic"
farming. As an example, U.S. Pat. No 5,385,750 teaches that
polyglycosides are excellent surfactants for solubilizing
water-insoluble compounds, but that polyglycosides are poor at
wetting and reducing the interfacial tension of solutions and that,
"increase in wetting rate and spreading on an oily or waxy surface
is important for agricultural pesticide material, which must be
spread on the surface of a leaf." The addition of long-chain
alcohols, to a certain extent, compensate for the polyglycoside
wetting deficiencies. But fatty alcohols have the disadvantages of
reducing detergency and having odor problems. Contrary to that,
yeast proteins increase surface activity of a variety of synthetic
detergents, additionally reduce interfacial tension as compared to
those detergents applied alone, without any odor issues, and
without creating any environmental or toxicological
complications.
Absorption and Contact Angle (Wetting) Measurements
[0080] The kinetics of the leaf penetration of an aqueous solution
was recorded by measurements of the contact angle of a droplet on
the surface of the leaves of two test plants: cabbage and tomato.
Cabbage leaves provide a model to show the effectiveness of wetting
agents using contact angle, and penetration using absorption
measurements, for the genus of waxy leaf plants called brassica and
similar ones. Tomato leaves are used as a model to show chemical
absorption effects for broadleaf weeds, which includes a large
number of invading plant species.
[0081] The principle of the method of measuring the contact angle
and absorption by the leaf is as follows. A droplet is placed on
the surface of the leaf, its evolution is recorded. For each data
point, five 1.0 microliter drops of each solution were placed on
each type of leaf and allowed to spread and penetrate into the leaf
over time. The profile of the drop against the surface of the leaf
was recorded and measured optically. The absorption by the leaf is
essentially the area of the drop profile at a point in time
relative to the area at initial condition. The second measured
parameter was the contact angle, i.e. the angle between the leaf
surface plane and the tangent to the droplet surface at the
crossing point. The contact angle decreased in the course of the
exposure, while the droplet spread over the leaf surface. The
contact angle is a standard measure of the interfacial tension
between the surface and the solution. Surfactants typically
facilitate the spread of the liquid on the solid surface,
accelerating the decrease in contact angle.
[0082] Data in Tables 9 and 10 show that with cabbage leaves, which
are especially hydrophobic, creating a high hurdle for any attempt
to penetrate the leaves due to their tenacious wax coating, the
absorption of protein/surfactant compositions were significantly
more pronounced than that of the standard commercial NPE adjuvant.
Tomato leaves are more open to water penetration, but there, too,
the protein composition was significantly more efficient than the
NPE based adjuvant.
EXAMPLE 7
[0083] R-11 (nonylphenol (NPE) based adjuvant)--Commercially
available adjuvant from Wilbur-Ellis Company
EXAMPLE 8
TABLE-US-00018 [0084] Component Composition (%) by Weight Dioctyl
sulfosuccinate (75%) 25.00% Hexylene glycol 12.50%
Post-fermentation stress protein mixture 62.50% Total 100.00%
TABLE-US-00019 TABLE 9 Approximate Equilibrium Equilibrium Time to
Complete Non-penetrated Contact Droplet Penetration Drop Volume
Angle Solution Leaf (seconds) (microliters) (degrees) Example 7 @
Tomato 50 0.0 0.degree. 0.25% Example 8 @ Tomato 20 0.0 0.degree.
0.25% Example 7 @ Cabbage 400 + (.infin.) 0.4 37.degree. 0.25%
Example 8 @ Cabbage 140 0.0 0.degree. 0.25%
[0085] Further comparisons of the protein/surfactant system
compared to the surfactant only, indicated that the proteins, when
added to the surfactant, accelerated the rate of absorption and the
wetting characteristics in both cabbage and tomato leaves, Table
10.
EXAMPLE 9
TABLE-US-00020 [0086] Component Composition (%) by Weight Dioctyl
sulfosuccinate (75%) 25.00% Hexylene glycol 12.50% Water 62.50%
Total 100.00%
TABLE-US-00021 TABLE 10 Rates of sorption into the leaves vary with
a general summary being as follows: Approximate Equilibrium
Equilibrium Time to Complete Non-penetrated Contact Droplet
Penetration Drop Volume Angle Solution Leaf (seconds) (microliters)
(degrees) Example 7 @ Tomato 50 0.0 0.degree. 0.25% Example 9 @
Tomato 30 0.0 0.degree. 0.25% Example 8 @ Tomato 20 0.0 0.degree.
0.25% Example 7 @ Cabbage 400 + (.infin.) 0.40 37.degree. 0.25%
Example 9 @ Cabbage 400 + (.infin.) 0.25 22.degree. 0.25% Example 8
@ Cabbage 140 0.00 0.degree. 0.25%
[0087] In Table 11 a proprietary surfactant that meets label
requirements for "organic" farming was evaluated. Organic
surfactants have restricted manufacturing processes and typically
do not perform as well as non-organic adjuvants in terms of wetting
and effectiveness as penetrants. To compensate for some of the
difference, higher concentrations are used. In the example of Table
11, the concentration was 1%. As in the case with the NPE based
adjuvant, the protein/surfactant system accelerated the
penetration, or the absorption, into the cabbage leaf surface
compared to the surfactant alone.
EXAMPLE 10
TABLE-US-00022 [0088] Component Composition (%) by Weight
Polyethylene glycol lauryl ether (HLB 10) 25.00% Water 75.00% Total
100.00%
EXAMPLE 11
TABLE-US-00023 [0089] Component Composition (%) by Weight
Polyethylene glycol lauryl ether (HLB 10) 25.00% Post-fermentation
stress protein mixture 25.00% Water 50.00% Total 100.00%
EXAMPLE 12
TABLE-US-00024 [0090] Component Composition (%) by Weight
Polyethylene glycol lauryl ether (HLB 10) 25.00% Post-fermentation
stress protein mixture 75.00% Total 100.00%
TABLE-US-00025 TABLE 11 Average Approximate Equilibrium Equilibrium
Time to Complete Non-penetrated Contact Droplet Penetration Drop
Volume Angle Solution Leaf (seconds) (microliters) (degrees)
Example 10 @ Cabbage 400 + (.infin.) 0.17 18.degree. 1% Example 11
@ Cabbage 199 0.00 0.degree. 1% Example 12 @ Cabbage 121 0.00
0.degree. 1%
[0091] Three additional surfactants were compared to use with and
without the addition of proteins. In all cases, the addition of the
protein mixture improved the wetting (contact angle) and absorption
(penetration) characteristics compared to surfactant alone. The
results are shown in Table 12.
EXAMPLE 13
TABLE-US-00026 [0092] Component Composition (%) by Weight Toximul
8364 Surfactant Proprietary 10.00% Non-ionic Blend (Stepan) Water
90.00% Total 100.00%
EXAMPLE 14
TABLE-US-00027 [0093] Component Composition (%) by Weight Toximul
8364 Surfactant Proprietary 10.00% Non-ionic Blend (Stepan)
Post-fermentation stress protein mixture 20.00% Water 70.00% Total
100.00%
EXAMPLE 15
TABLE-US-00028 [0094] Component Composition (%) by Weight Agent
3109-6 Surfactant Proprietary 10.00% Non-ionic Blend (Stepan) Water
90.00% Total 100.00%
EXAMPLE 16
TABLE-US-00029 [0095] Component Composition (%) by Weight Agent
3109-6 Surfactant Proprietary 10.00% Non-ionic Blend (Stepan)
Post-fermentation stress protein mixture 20.00% Water 70.00% Total
100.00%
EXAMPLE 17
TABLE-US-00030 [0096] Component Composition (%) by Weight Agent X-1
Surfactant Proprietary 10.00% Cationic Blend (Stepan) Water 90.00%
Total 100.00%
EXAMPLE 18
TABLE-US-00031 [0097] Component Composition (%) by Weight Agent X-1
Surfactant Proprietary 10.00% Cationic Blend (Stepan)
Post-fermentation stress protein mixture 20.00% Water 70.00% Total
100.00%
TABLE-US-00032 TABLE 12 A general summary of the results is as
follows: Average Approximate Equilibrium Equilibrium Time to
Complete Non-Penetrated Contact 80:1 Dilution Droplet Penetration
Drop Volume Angle in Water of Leaf (seconds) (microliters)
(degrees) Example 13 Cabbage 600 + (.infin.) 0.49 36.6 Example 14
Cabbage 600 + (.infin.) 0.10 12.9 Example 15 Cabbage 600 +
(.infin.) 0.27 23.1 Example 16 Cabbage 189 0.00 0.0 Example 17
Cabbage 600 + (.infin.) 0.15 14.8 Example 18 Cabbage 141 0.00
0.0
* * * * *