U.S. patent application number 13/441332 was filed with the patent office on 2012-11-29 for plant support formulation, vehicle for the delivery and translocation of phytologically beneficial substances and compositions containing same.
This patent application is currently assigned to NORTH-WEST UNIVERSITY. Invention is credited to Anne Frederica GROBLER.
Application Number | 20120302442 13/441332 |
Document ID | / |
Family ID | 38284003 |
Filed Date | 2012-11-29 |
United States Patent
Application |
20120302442 |
Kind Code |
A1 |
GROBLER; Anne Frederica |
November 29, 2012 |
PLANT SUPPORT FORMULATION, VEHICLE FOR THE DELIVERY AND
TRANSLOCATION OF PHYTOLOGICALLY BENEFICIAL SUBSTANCES AND
COMPOSITIONS CONTAINING SAME
Abstract
A plant supporting formulation which is also suitable for use as
a delivery vehicle, or a component of a delivery vehicle, for the
delivery of one or more phytologically beneficial substances to a
plant, and for enhancing the translocation of such delivered
substance(s) in or on the plant, the formulation comprising a
micro-emulsion constituted by a dispersion of vesicles or
microsponges of a fatty acid based component in an aqueous carrier,
the fatty acid based component comprising at least one long chain
fatty acid based substance selected from the group consisting of
free fatty acids and derivatives of free fatty acids. The
dispersion is preferably characterized in that at least 50% of the
vesicles or microsponges are of a diametrical size of between 50 nm
and 5 micrometer. The dispersion is further also characterized in
that the micro-emulsion has a zeta potential of between -25 mV and
-60 mV.
Inventors: |
GROBLER; Anne Frederica;
(Potchefstroom, ZA) |
Assignee: |
NORTH-WEST UNIVERSITY
Potchefstroom
ZA
|
Family ID: |
38284003 |
Appl. No.: |
13/441332 |
Filed: |
April 6, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12280880 |
Oct 30, 2008 |
|
|
|
PCT/IB2007/050580 |
Feb 23, 2007 |
|
|
|
13441332 |
|
|
|
|
Current U.S.
Class: |
504/127 ;
504/130; 504/136; 504/142; 504/313 |
Current CPC
Class: |
A01N 37/06 20130101;
A01N 43/653 20130101; A01N 37/02 20130101; A01N 25/04 20130101;
A01N 37/06 20130101; A01N 2300/00 20130101; A01N 37/02 20130101;
A01N 2300/00 20130101 |
Class at
Publication: |
504/127 ;
504/313; 504/142; 504/136; 504/130 |
International
Class: |
A01N 37/06 20060101
A01N037/06; A01N 43/54 20060101 A01N043/54; A01N 43/40 20060101
A01N043/40; A01P 7/04 20060101 A01P007/04; A01P 13/00 20060101
A01P013/00; A01P 3/00 20060101 A01P003/00; A01P 1/00 20060101
A01P001/00; A01N 57/12 20060101 A01N057/12; A01P 21/00 20060101
A01P021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 27, 2006 |
ZA |
2006/01725 |
Claims
1. A plant supporting formulation which is physiologically
beneficial comprising a micro-emulsion constituted by a dispersion
of vesicles or microsponges of a fatty acid based component in an
aqueous carrier, the fatty acid based component comprising at least
one long chain fatty acid based substance selected from the group
consisting of oleic acid, linoleic acid, alpha-linolenic acid,
gamma-linolenic acid, arachidonic acid, eicosapentaenoic acid
[C20:5.omega.3], decosahexaenoic acid [C22:6.omega.3], and
ricinoleic acid, and derivatives thereof selected from the group
consisting of C.sub.1 to C.sub.5 alkyl esters thereof,
glyceropolyethylene glycol esters thereof, and reaction products of
hydrogenated and unhydrogenated natural oils composed largely of
ricinoleic acid based oils with ethylene oxide and which
incorporates a gas dissolved in the fatty acid mixture.
2. A plant supporting formulation according to claim 1, wherein
said ricinoleic acid is castor oil.
3. A plant supporting formulation according to claim 1 wherein the
dispersion is characterized in that at least 95% of the vesicles or
microsponges are of a diametrical size of between 50 nm and 5
micrometer.
4. A plant supporting formulation according to claim 1
characterized in that the micro-emulsion has a zeta potential of
between -35 mV and -60 mV.
5. A plant supporting formulation according to claim 1 wherein the
fatty acid component of the micro-emulsion includes a mixture of
esterified fatty acids.
6. A plant supporting formulation according to claim 5 wherein the
fatty acid component of the micro-emulsion consists of the product
known as Vitamin F Ethyl Ester.
7. A plant supporting formulation according to claim 1 wherein the
fatty acid component of the micro-emulsion includes the long chain
fatty acids known as eicosapentaenoic acid [C20:5.omega.3] and
decosahexaenoic acid [C22:6.omega.3].
8. A plant supporting formulation according to claim 5 wherein the
fatty acid component of the micro-emulsion in addition includes the
reaction product of hydrogenated natural oils composed largely of
ricinoleic acid based oils with ethylene oxide.
9. A plant supporting formulation according to claim 8 wherein the
reaction product of hydrogenated natural oils composed largely of
ricinoleic acid based oils with ethylene oxide is produced from
castor oil.
10. A plant supporting formulation according to claim 1 wherein the
gas is selected from the group consisting of nitrous oxide, carbon
oxysulfide and carbon dioxide.
11. A method for producing a plant supporting formulation
comprising the steps of mixing a fatty acid based component with
water to obtain a micro-emulsion, and introducing a gas selected
from the group consisting of nitrous oxide, carbon oxysulfide and
carbon dioxide into the mixture, to impart a size distribution of
vesicles or microsponges so that at least 95% of the vesicles or
microsponges are of a diametrical size of between 50 nm and 5
micrometer or a zeta potential of between -35 mV and -60 mV to the
micro-emulsion, and wherein the fatty acid based component
comprises at least one long chain fatty acid based substance
selected from the group consisting of oleic acid, linoleic acid,
alpha-linolenic acid, gammalinolenic acid, arachidonic acid,
eicosapentaenoic acid [C20:5.omega.3], decosahexaenoic acid
[C22:6.omega.3] ricinoleic acid, and derivatives thereof selected
from the group consisting of C.sub.1 to C.sub.5 alkyl esters
thereof, glycerol-polyethylene glycol esters thereof, and reaction
products of hydrogenated and unhydrogenated natural oils composed
largely of ricinoleic acid based oils with ethylene oxide.
12. A method according to claim 11, wherein the reaction product of
hydrogenated natural oils composed largely of ricinoleic acid based
oils with ethylene oxide is produced from castor oil.
13. A method according to claim 11 wherein the mixing of the fatty
acid component is effected with heating and stirring.
14. A method according to claim 13, wherein the mixing of the fatty
acid component is effected with heating and stirring by means of a
high speed shearer.
15. A method for producing a plant supporting formulation
comprising the steps of introducing a gas into water, and
subsequently mixing a fatty acid based component with said water to
obtain a micro-emulsion, wherein said gas is selected from the
group consisting of nitrous oxide, carbon oxysulfide and carbon
dioxide, to impart a size distribution of vesicles or microsponges
so that at least 95% of the vesicles or microsponges are of a
diametrical size of between 50 nm and 5 micrometer or a zeta
potential of between -35 mV and -60 mV to the micro-emulsion, and
wherein the fatty acid based component comprises at least one long
chain fatty acid based substance selected from the group consisting
of oleic acid, linoleic acid, alpha-linolenic acid, gammalinolenic
acid, arachidonic acid, eicosapentaenoic acid [C20:5.omega.3],
decosahexaenoic acid [C22:6.omega.3] ricinoleic acid, and
derivatives thereof selected from the group consisting of C.sub.1
to C.sub.5 alkyl esters thereof, glycerol-polyethylene glycol
esters thereof, and reaction products of hydrogenated and
unhydrogenated natural oils composed largely of ricinoleic acid
based oils with ethylene oxide.
16. A method according to claim 15, wherein the reaction product of
hydrogenated natural oils composed largely of ricinoleic acid based
oils with ethylene oxide is produced from castor oil.
17. A method according to claim 15 wherein the gas is dissolved in
the water to obtain a saturated solution of the gas in water, and
the saturated solution of the gas is thereafter mixed with the
fatty acid component of the micro-emulsion being prepared.
18. A method according to claim 17 wherein the saturated solution
of the gas in water is prepared by sparging the water with the gas,
or by exposing the water to the gas at a pressure in excess of
atmospheric pressure for a period of time in excess of the time
required for the water to become saturated with the gas.
19. A method according to claim 11 wherein an emulsion of the fatty
acid component in water is first prepared and is thereafter gassed
by exposing the emulsion to the gas.
20. A method according to claim 19 wherein the emulsion is gassed
by sparging.
21. A formulation for the use in the treatment of a plant
comprising a carrier vehicle constituted by a plant supporting
formulation according to claim 1 and which further includes at
least one phytologically beneficial substance selected from the
group consisting of substances known to be useful as a plant
nutrient; a plant pesticide; a plant growth regulator, a plant
immune modulator; and a biostimulant.
22. A formulation according to claim 21, wherein said plant
pesticide is selected from the group consisting of an herbicide, a
fungicide, a bactericide, an insecticide, and an antiplant virus
agent.
23. A formulation according to claim 21 characterized in that it is
in a form suitable for spraying onto plants as a liquid and which
incorporates further additives that enhance effectiveness,
stability, or ease of application.
24. A formulation according to claim 21 comprising at least one
plant nutrient which is a source of at least one element selected
from the group of elements consisting of carbon, hydrogen, oxygen,
nitrogen, phosphorus, potassium, calcium, magnesium, sulphur, iron,
manganese, zinc, copper, boron, molybdenum and chlorine.
25. A formulation according to claim 21 comprising a pesticidally
effective concentration of at least one plant pesticide selected
from the group consisting of insecticides, herbicides, fungicides,
plant regulators, defoliants, and desiccants.
26. A formulation according to claim 21 wherein the pesticide is
selected from chemical and biological pesticides, synthetic
arsenic, Bt liquid w/zylene, Bt liquid-no Xylene, Bt wettable
powder, beneficial organisms, biodynamic preparations, bordeaux
mixes--copper, hydroxide/fixed copper, boric acid, carbamates,
chlorinated hydrocarbons, chromate ions, citric acid, copper
hydroxide, copper sulfate, herbal preparations selected from
cinnamon, cloves, garlic, mint, peppermint, rosemary, thyme, and
white pepper, herbicides, hydrated lime, a neonicotinoid
insecticide, a chiral oxadiazine insecticide, insect extracts,
isocyanate, lauryl sulfate, lime sulfur, malathion, malic acid,
methyl bromide, methyl sulfoxide, milky spore disease, popillae,
nematocides, nematodes, nicotine, oils selected from carrot oil,
castor Oil (U.S.P. or equivalent), cedar oil, cinnamon oil,
citronella oil, citrus oil, clove oil, peppermint oil, rosemary
oil, sesame oil, soybean oil, summer oils, thyme oil and weed oils,
organophosphates selected from acephate, azinphosmethyl, bensufide
cadusafos, chlorethoxyphos, chlofenvinpos, chlorpyrifos,
chlorpyrifos-methyl, chlothiophos, coumaphos, ddvp (dichlorvos),
dialifor, diazinon, dicotophos, dimethoate, dioxathion, disulfoton,
ethion, ethoprop, ethyl parathion, fenamiphos, fenitrothion,
fenthion, fonofos, isazophos, malathion, methamidophos,
methidathioln, methyl pharathion, mevinphos, monocrotophoa, naled,
oxydemeton-methyl, phorate phosalone, phosmet, phosphamidon,
phostebupirim, pirimiphos-methyl, profenofoe, propetamphos,
sulfotapp, sulprofos, temephos, terbufos, tetrachiovinphos,
tribufos (def) and trichiorfon, pentachlorophenol, pesticides,
petroleum distillates, petroleum oil spray ajuvants, 2-phenethyl
propionate (2-phenylethyl propionate), pheromones, piperonyl
butoxide, plant extracts selected from hellebore, pyrethrum,
quassia, sabadilla, citronella, sesame, eugenol and geraniol,
potassium sorbate, putrescent whole egg solids, pyrethroids, rock
salt, rotenone, ryania, see animal wastes, soap based herbicides,
sodium chloride, sodium lauryl sulfate, soil fumigants,
streptomycin, sulfur, virus sprays, or Zinc Metal Strips consisting
solely of zinc metal and impurities.
27. A formulation according to claim 21, wherein said chemical and
biological pesticides are organic pesticides.
28. A formulation according to claim 21, wherein said herbicides
are synthetic herbicides.
29. A formulation according to claim 26, wherein said neonicotinoid
insecticide is imidacloprid.
30. A formulation according to claim 26, wherein said chiral
oxadiazine insecticide is indoxacarb (p).
31. A formulation according to claim 26, wherein said milky spore
disease is milky spore disease B.
32. A formulation according to claim 26, wherein said nematocides
are synthetic nematocides.
33. A formulation according to claim 26, wherein said pesticides
are synthetic pesticides.
34. A formulation according to claim 26, wherein said pyrethroids
are synthetic pyrethroids.
35. A formulation according to claim 21 including a herbicidally
effective concentration of at least one herbicide having a mode of
action selected from the group consisting of auxin mimics, mitosis
inhibitors, photosynthesis inhibitors, amino acid synthesis
Inhibitors and lipid biosynthesis inhibitors.
36. A formulation according to claim 35 including a herbicide
selected from the group consisting of 2,4-d (2,4-dimethylphenol),
Clopyralid, Fluazifop-p-buty, a triazolopyrimidine herbicide,
Fosamine Ammonium, Glyphosate, Hexazinone, Imazapic, Imazapyr,
Picioram, Sethoxydim, Triclopyr.
37. A formulation according to claim 36, wherein said
triazolopyrimidine herbicide is flumetsulam.
38. A formulation according to claim 21 including at least one
fungicide selected from the group consisting of 1,3
dichloropropene, 2,5-dichlorobenzoic acid methyl ester, 8
hydroxyquinoline, acibenzolar-S-methyl, Agrobacterium radiobacter,
ammonium phosphite, ascorbic acid, azoxystrobin, bacillus subtilis
DB 101, bacillus subtitle D8 102, Bacillus subtilis isolate B248,
Bardac, Benalaxyl, Benomyl, Bifenthin, Bitertanol, Borax, boric
acid equivalent, boscalid, bromuconazole, bupirimate, captab,
carbendazim, Carboxin, chlorine dioxide, chloropicrin,
chlorothalonil, chlorpyrifos, copper ammonium acetate, copper
ammonium carbonate, copper hydroxide, copper oxychloride, cupric
hydroxide, cymoxanil, cyproconazole, cyprodinil, Dazomet,
Deltamethrin, Dichlarophen, dicloran, didesyl dimethyl ammonium
chloride, difanaconazole, dinocap, diphenylamine, disulfoton,
dithianon, dodemorph, dodine, epoxiconazole, famoxadone, alcohols,
anti-oxidants, Fenemidone, Fenarimol, Fenbuconazole, Fenhexamid,
Fludioxonil, Flusilazofe, Flutriafo, Folpet, fosetyl-Al, furalaxyl,
furfural, guazatine, hexaconazole, hydroxyquinoline sulphate,
imazalil, iprodione, iprovalicarb, kresoxim-methyl, lime, lindane,
mancozeb, maneb, mefenoxam, Mercaptothion, Metalaxyl, metalaxyl-M
(mefenoxam), metam-sodium, methyl bromide, metiram, mineral oil,
mono potassium phosphate, myclobutanil, octhilinone, oxycarboxin,
paraffinic complex, penconazole, pencycuron, phosphorous acid,
polysulphide sulphur, potassium phosphate, potassium phosphonate,
prochlorax zinc complex, prochloraz, prochloraz manganese chloride
complex, prochloraz zinc complex, procymidone, profenofos,
propaconazole, propamocarb HCl, propiconazole, propineb,
pseudomonas resinovonans, pyraclostrobin, pyrimethanil, QAC,
Quazatine, Quinoxyfen, Quintozene, salicylic acid, silthiopharn,
sodium-o-phenol phenate(Na salt), spiroxamine, sulphur, TBTO,
Tebuconazole, Thiabendazole, Thiabendazole, thiophanate methyl,
thiram, tolclofos-methyl, triadimefon, triadimenol, tributyltin
oxide, Trichoderma harzianum, Tridemorph, Trifloxystrobin,
Triflumuron, Triforine, Triticonazole, Vinclozolin, zinc oxide,
Zineb and Zoxamide.
39. A formulation according to claim 38, wherein said paraffinic
complex is light mineral oil.
40. A formulation according to claim 21 including a bactericidally
effective concentration of at least one bactericide selected from
the bactericides known to be suitable for use on plants to combat
bacteria infecting plant.
41. A formulation according to claim 21 including an insecticidally
effective concentration of at least one insecticide selected from
the group consisting of (E)-7-dodecenyl acetate, (E,E)-8,10
dodecadien-1-ol, 1,3 dichloropropene, 3(s)
ethyl-6-isopropenyl-9-docadien-1yl acetate, Allium sativum,
Bacillus thuringiensis Serotype H-7, Bacillus thuringiensis subsp
laraelensis, Bacillus thuringiensis var aiziwal kurstaki, Bacillus
thuringiensis var kurstaki, Beauveria bassiana, Bradyrhizobium
japonicum, Bradyrhizobium japonicum WB 74, Bradyrhizobium sp Luinus
VK, Bradyrhizobium sp.times.S21, Bradyrhizobium apum, Chlorpyrifos,
Dimilin, E8,E10-dodecadlenol, EDB, Metarhizium anisopliae var
acridium isolate IMI 330 189, Paecilumyces Illacinus strain 251,
Rhizobium leguminosarum blovar phaseoli, Rhizobium leguminosarum
viciaeTJ 9, Rhizobium meliloti, Spinosad, Sulfur, Trichoderma
harzianum, Z-8-dodecenylacetate, Abarnectin, abamectin, acephate,
acetamiprid, acrinathrin, aldicarb, alpha-cypermethrin, aluminum
phosphide, amltraz, azadlrachtin, azinphos-methyl, benfuracarb,
beta-cyfluthrin, beta-cypermethrin, bifenthrin, borax, brodifacorn,
bromopropylate, buprofenzin, burpfezin, cadusafos, carbaryl,
carbofuran, carbosulfan, cartap hyrochloride, chlorphenapyr,
chlorpyrifos, citronella oil, clofentezine, codimone
(E,E-8,10-dodecadiene-1-01), copper, coumatetralyl, cryptophlebia
leucotreta, cyanophos, cyfluthrin, cyhexatin, Cypermethin,
cyromazine, d-allethrin, dazomet, deltamethrin, demeton-S-methyl,
diazinon, dichlorvos, dicofol, difenacourn, diflubenzuron,
imethoate, disulfoton, emamectin, endosulfan, esfenvalerate,
ethoprophos, ethoprophos, ethylene dibromide, etoxazole,
fenamiphos, fenamiphos, fenazaquin, fenbutatin, fenbutatin oxide,
fenitrothion, fenoxycarb, fenpropathrin, fenpyroximate, fenthion,
fenvalerate, ferric sodium EDTA, pronil, fipronil, flufenoxuron,
flumethrin, fosthiazate, fumagillin, furfural, gamma-BHC, garlic
extract, hydramethylnon, imidacloprid, indoxacarb,
lambda-cyhalothrin, lavandulyl, senecioate, lufenuron, magnesium
phosphide, mancozeb, maple lactone, mercaptothion, metaldehyde,
metham-sodium, methamidophos, methidathion, methlocarb, methomyl,
methyl bromide, methyl-parathion, mevinphos, milbernectin, mineral
oil, novaluron, omethoate, orth-phenylphenol, oxamyl,
oxydemeton-methyl, parafinic complex, parathion, permethrin,
phenothoate, phorate, phosmet, phoxim, pirimicarb, polysulphide
sulphur, potassium salts of fatty acids, profenofos, propargite,
propoxur, protein hydrolysate, prothiofos, pyrethrins,
pyriproxyfen, quinalphos, rape oil, silicon based repellent, sodium
fluosilicate, spinosad, spirodiclofen, sulfur, tartar emetic,
tau-fluvalinate, tebufenozide, temephos, terbufos,
tetrachlorvinphos, tetradecenyl acetate, tetradifon, thiacloprid,
thiamethoxam, thiodicarb, thiram, trichlorfon, triflumuron,
trimediure, zeta-cypermethrin, zinc phosphide.
42. A formulation according to claim 41, wherein said paraffinic
complex is mineral oil.
43. A formulation according to claim 21 including a viracidally
effective concentration of at least one viracide selected from the
viracides known to be suitable for use on plants to combat viruses
that infect plants.
44. A formulation according to claim 21 including a plant growth
regulating effective concentration of at least one plant growth
regulator selected from the products in the group consisting of
dl-alpha-tocopherol or its physiologically active isomer,
2-(1-2-methylnaphthyl)acetamide; 2-(1-2-methylnaphthyl)acetic acid;
2-(1-naphthyl)acetamide; 2-(1-naphthyl)acetic avid; 2,4-D; 3,5,6
TPA; 4-indol-3-ylbutyric acid; 6-benzyl adenine; alkoxylated fatty
alkylamine polymer; alkylamine polymer; aminoethoxyvinylglycine
hydrochloride; ammoniated nitrates; auxins; calcium arsenate;
carbaryl; chlormequat chloride; chlorpropharn; chlorthal-dimethyl;
cloprop; cyanamide; daminozide; decan-1-ol; dichlorprop;
dichlorprop 2-butoxyethyl ester; dimethipin; dinocap; diquat
dibromide; diuron; ethephon; fluazifop-p-butyl; gibberellins;
glyphosphate-isopropylamine; glyphosphate-trimesium;
haloxyfop-P-methyl; indolylacetic acid; maleic hydrazide; mepiquat
chloride, methylcyclopropene; mineral oil; n-decanol; octan-1-ol;
paclobutrazole; paraquat dichloride; pendimethalin;
prohexadione-calcium; salicylic acid; sodium chlorate; thidiazuron;
trinexapac-ethyl; and uniconazole.
45. A formulation according to claim 44 wherein said 2,4-D is the
sodium salt of 2,4-D.
46. A formulation according to claim 21 including a biostimulatory
effective concentration of at least one biostimulant
phytohormone,
47. A formulation according to claim 46 wherein the phytohormone is
a brassinosteriod.
48. A method of administering a plant support formulation as
claimed in claim 1 to a plant comprising the step of applying the
formulation by means of aerial or surface application, by
incorporation in water borne irrigation system, or by trunk
injection where appropriate.
49. A method of administering a phytologicaly beneficial substance
to a plant, comprising the step of applying a formulation as
claimed in claim 21 to the plant by means of aerial or surface
application, by incorporation in water borne irrigation system, or
by trunk injection where appropriate.
50. A method of stimulating at least one of the growth stages of a
plant, or of improving the production or yield of crop by the
plant, or the appearance of the plant or of enhancing disease
resistance in the plant comprising the step of administering to the
plant a plant support formulation as claimed in claim 1.
51. A method of providing a plant nutrient to a plant comprising
the step of applying to the plant a formulation as claimed in claim
24 to the plant, or where appropriate, the locus thereof.
52. A method of combating plant pests comprising the step of
applying a formulation as claimed in claim 25 to the plant or,
where appropriate, the locos thereof.
53. A method of stimulating growth or yield of a plant comprising
the step of applying a formulation as claimed in claim 46 to the
plant.
Description
[0001] This application is a continuation of U.S. Ser. No.
12/280,880 filed on Oct. 30, 2008 which is a 35 U.S.C. 371 National
Phase Entry Application from PCT/IB2007/050580, filed Feb. 23,
2007, which claims the benefit of South African Patent Application
No. 2006/01725 filed on Feb. 27, 2006, the disclosure of which is
incorporated herein in its entirety by reference.
FIELD OF THE INVENTION
[0002] This invention relates to a plant supporting formulation
which in itself is phytologically beneficial and which is also
suitable for use as a delivery vehicle, or a component of a
delivery vehicle, for use in delivering to a plant, and for
distributing or translocating in a plant, a variety of
phytologically beneficial substances in the form of molecules,
compounds, biologicals or chemicals that have a phytologically
beneficial effect to plants [herein collectively referred to as
"phytologically beneficial substances"]. The expression "plant
supporting" is used herein to signify that the formulation has the
property, without the addition of other phytologically beneficial
substances for which it may serve as a delivery vehicle, to have a
growth stimulatory effect on plants in at least one of the growth
stages of a plant, to improve the production or yield of crop by
the plant, or to improve appearance of the plant or to enhance
disease resistance in the plant. It also relates to methods of
producing the plant supporting formulation and delivery vehicle,
and to the preparation of various formulations incorporating the
formulation as a delivery vehicle and any one or more of a variety
of phytologically beneficial substances and to methods of
administering such phytologically beneficial substances to a plant
involving the use of the delivery vehicle of the invention which
then also serves to effect the translocation or distribution of the
phytologically beneficial substances in or on the plant. It will be
appreciated or become apparent that reference to "beneficial
effects" as it applies to a plant, is to be understood from a human
perspective in that phytotoxic substances, such as substances used
as herbicides in the control of undesirable plants, are intended to
be included within the group of substances herein referred to as
"phytologically beneficial substances".
BACKGROUND TO THE INVENTION
[0003] Vast quantities of a great variety of substances are applied
to plants for the purpose of enhancing the growth of the plants in
order to improve the production (in the case of crop and field
plants) or appearance (in the case of ornamentals) of the plants.
Such substances include the group defined above as phytologically
beneficial substances. It includes fertilizers, both of the macro-
and micro-nutrient variety, growth stimulants or regulators, and
pesticides, including fungicides, insecticides and herbicides. As
used herein the word "plant" is intended to cover land and water
plants, including sea plants, and "ornamentals" are intended to
cover all plants that are not intended to produce a crop having
economic value.
[0004] The application of phytologically beneficial substances is
generally regarded as an art that is in need of improvement as a
large percentage of the applied substances are not absorbed by or
retained on the plants to which it is applied. Apart from the
consequential wastage of expensive material and hence the
unnecessary increase in production cost brought about by such
wastage, the unutilized substances also give rise to pollution of
the soil and water resources.
[0005] There appears to be no reference in the literature to the
use of a designed biological delivery system to address the
enhanced administration of specific nutrients or growth regulators
to plants and/or the systemic translocation of such nutrients or
growth regulators throughout the plants. It is known in the
agricultural field that nutrients and other phytologically
beneficial substances may be formulated with so-called chelating
agents or adjuvants. Unlike the present invention the chelating
agents are a clearly distinguishable group with no reference to a
delivery system and are used as micro-nutrient sources that are
formed by combining a chelating agent with a metal through
coordinate bonding. Stability of the metal-chelate bond affects the
availability to plants of the micronutrient metals--copper, iron,
manganese, and zinc. An effective chelate is one in which the rate
of substitution of the chelated micronutrient for other cations in
the soil is quite low, thus maintaining the applied micronutrient
in chelated form. Chelates are generally only applicable to
cationic substances. A chelating agent, such as EDTA, is thought to
have a negative impact on the environment.
[0006] According to prescriptions for chelates in the Preliminary
Organic Materials List by the California Departments of Food and
Agriculture, natural chelates are allowed but synthetic chelating
agents are restricted for use only with micronutrient sprays for a
documented deficiency. All other uses of synthetic chelates are
prohibited. EDTA, lignin sulfonates and lignosulfonic acids are
considered to be synthetic chelating agents. Recently, a shuttle
system for the delivery of cations was announced. The shuttle
system consists of long chain polysaccharides which can complex
with cationic nutrients in clusters (nanoclusters), thus rendering
the nutrient-chelate complex neutral. The chelators (shuttle
ligand) then envelop the enclustered nutrients and shuttle them to
the cell wall where they deliver their nutrients. The delivery are
thought to take place through a random process whereby the pores on
the plant and the shuttle ligand both contract and expand as a
result of a thermal vibration, a natural phenomenon. It is thought
that when contraction of the chelator and expansion of the pore
synchronize, the nutrient is delivered. Upon unloading the mineral,
the shuttle ligand is repulsed from the plant surface, and is
attracted back to the nanocluster where it can repeat the process
again and again. The shuttle chelating system may extend to other
dormant cations in the soil. However, the system is still based on
the use of chelates, can complex only to cationic compounds and do
not penetrate the plant tissue.
[0007] Cloak Spray oil, marketed in South Africa by Nutri-Tech
Solutions, is an organic blend of emulsified, cold press canola oil
and omega-3 fish oil. Cloak oil is thought to be a high quality
spreader, sticker synergist (see below) which is claimed to improve
the performance of all foliar fertilizers. However, no claims are
made regarding either the translocation of substances within the
plant or the delivery of other substances or fertilization by the
root system of the plant.
[0008] The most established method of introducing material or
substances into plant cells is by spraying of the substance in the
presence of a wetting agent, spreader or sticker. By this technique
material is sprayed onto leaves of plants in the presence of a
wetting agent which would cause the material to adhere to the waxy
outer layer of leaves, thereby increasing contact time between the
material to be absorbed by the plant and the plant leaf itself.
While some of the material gets taken up, the wetting agent, which
usually contains an adherent, cause the leaves to become sticky and
attract dust, which in turn may lead to occlusion of the stomata.
Carriers for the agricultural sector have been described but relate
to methods of application and not to the enhancement of the action
of the active compound due to increased delivery to the target cell
or organism. The closest approximation to a delivery system that
may be used to overcome barriers to entry in plants are to be found
in the use of adjuvants for enhancing the activity of some active
compounds in the herbicide and hormone classes.
[0009] While these techniques work adequately in the appropriate
environment on some compounds that are easily absorbed by leaves,
they are not regarded as being generally suitable for the effective
delivery of a number of macro- and micro-nutrients, as well as a
large number of pesticides and growth regulators. There has thus
been a long-felt need for an appropriate process by which compounds
may be introduced selectively into plant cells there to enhance
growth or to treat plant diseases or deficiencies.
[0010] Adjuvants are chemically and biologically active (not
chemically inert) compounds and may be classified according to
their function (activator or utility), their chemistry (such as
organosilicones), or source (vegetable or petroleum oils). They
produce pronounced effects. Most adjuvants are incompatible with
some materials and conditions and may result in toxic effects in
plants and animals, and some adjuvants have the potential to be
mobile and pollute surface or groundwater sources. The use of
adjuvants may be problematic near water, as adverse effects may
occur in some aquatic species.
OBJECT OF THE INVENTION
[0011] It is an object of the invention to provide a plant
supporting formulation which by itself has beneficial effects in
terms of the growth, appearance, production and/or yield of plants
to which it is applied in use, and which formulation is also
suitable for use as a delivery vehicle, or a component of a
delivery vehicle, for the delivery of one or more phytologically
beneficial substances to a plant, and distributing or translocating
phytologically beneficial substances in plants, to provide for
formulations incorporating such vehicles with or without at least
one phytologically beneficial substance whereby at least some of
the disadvantages of existing formulations may at least be reduced,
to provide a method for producing such vehicles and a method of
preparing formulations incorporating such vehicles and at least one
phytologically beneficial substance, and to provide a method of
administering such phytologically beneficial substances to a plant
involving the use of the delivery vehicles of the invention which
then also serves to effect the translocation or distribution of the
phytologically beneficial substances in or on the plant.
GENERAL DESCRIPTION OF THE INVENTION
[0012] According to the present invention there is provided a plant
supporting formulation which is phytologically beneficial and
suitable for use as a delivery vehicle, or a component of a
delivery vehicle, for the delivery of one or more phytologically
beneficial substances to a plant, and for enhancing the
translocation of such delivered substance(s) in or on the plant,
the formulation comprising a micro-emulsion constituted by a
dispersion of vesicles or microsponges of a fatty acid based
component in an aqueous carrier, the fatty acid based component
comprising at least one long chain fatty acid based substance
selected from the group consisting of free fatty acids and
derivatives of free fatty acids.
[0013] The dispersion is preferably characterized in that at least
95% of the vesicles or microsponges are of a diametrical size of
between 50 nm and 5 micrometer. It will be understood that the
vesicles or microsponges in the dispersion are elastic and not
necessarily of perfectly spherical shape and accordingly the term
"diametrical size" is not to be understood as a term of geometric
precision.
[0014] It is further to be understood that it is not practicable to
determine such diametrical size in three dimensions without the use
of highly sophisticated instrumentation. It is accordingly to be
determined in two dimensions by means of microscopic observation
and thus refers to the maximum measurement across observed vesicles
or microsponges as seen in two dimensions.
[0015] The dispersion is further also characterized in that the
micro-emulsion has a zeta potential of between -35 mV and -60
mV.
[0016] The fatty acid based component may be selected from the
group consisting of oleic acid, linoleic acid, alpha-linolenic
acid, gamma-linolenic acid, arachidonic acid, eicosapentaenoic acid
[C20:5.omega.3], decosahexaenoic acid [C22:6.omega.3], and
ricinoleic acid, and derivatives thereof selected from the group
consisting of the C.sub.1 to C.sub.6 alkyl esters thereof, the
glycerol-polyethylene glycol esters thereof, and the reaction
product of hydrogenated and unhydrogenated natural oils composed
largely of ricinoleic acid based oils, such as castor oil, with
ethylene oxide.
[0017] In one form of the invention the fatty acid component of the
micro-emulsion may consist or include a mixture of esterified fatty
acids, and in this regard it is preferred to make use of the
product known as Vitamin F Ethyl Ester. This product is
commercially available under the trade description of Vitamin F
Ethyl Ester CLR 110 000 Sh.L. U./g from CLR Chemicals Laboratorium
Dr. Kurt Richter GmbH of Berlin, Germany. The typical fatty acid
distribution of this product is as follows:
<C.sub.16: 0
C.sub.16.0: 8.3%
C.sub.18.0: 3.5%
C.sub.18.1: 21.7%
C.sub.18.2: 34.8%
C.sub.18:3: 28.0%
>C.sub.18: 1.6%
[0018] unknown: 2.1%
[0019] The fatty acid component may alternatively include or
consist of the long chain fatty acids known as eicosapentaenoic
acid [C20:5.omega.3] and decosahexaenoic acid [C22:6.omega.3]. Such
a product combination is available from Roche Lipid Technology
under the trade name "Ropufa `30` n-3 oil". It has been found
useful to incorporate these acids where a hydrophobic substance is
desired to be delivered to the plant. An alternative product that
may be used for this purpose is one of the group of Incromega
products available from BASF.
[0020] The fatty acid component may in addition to the
aforementioned substances or mixtures of substances also include
the reaction product of hydrogenated natural oils composed largely
of ricinoleic acid based oils with ethylene oxide. It is preferable
for this substance to be produced from castor oil of which the
fatty acid content is known to be predominantly composed of
ricinoleic acid. This product may be modified as to the extent of
hydrogenation, ethylation and the addition of groups such as
polyethylene glycol. A range of such products is being marketed by
BASF under the trade description of Cremophor of various grades.
According to a preferred form of the invention for certain
applications there is provided a delivery vehicle in which the
Cremophor grade, or other composition of modified ricinoleic acid
used, is one in which the ricinoleic acid molecules are modified by
the addition thereto of polyethylene glycol groups which comprise
between 35 and 45 ethylene oxide units.
[0021] The vehicle may incorporate a suitable gas dissolved in the
fatty acid mixture, the gas being selected to be suitable to impart
the requisite size distribution of vesicles and the requisite zeta
potential to the micro-emulsion.
[0022] The gas is preferably selected from the group consisting of
nitrous oxide, carbon oxysulfide and carbon dioxide.
[0023] According to another aspect of the invention there is
provided a method for producing a plant supporting formulation or
delivery vehicle according to the present invention as defined
above, comprising the steps of mixing the fatty acid based
component with water to obtain a micro-emulsion, and introducing a
suitable gas into the mixture, the gas being selected to be
suitable to impart the requisite size distribution of vesicles and
the requisite zeta potential to the micro-emulsion.
[0024] The mixing of the fatty acid component is preferably
effected with heating and stirring, preferably by means of a high
speed shearer.
[0025] The gas may be introduced into the water either before or
after the fatty acid based component of the micro-emulsion is mixed
with the water. Thus in one form of the invention the gas may be
dissolved in the water to obtain a saturated solution of the gas in
water, and the saturated solution of the gas is thereafter mixed
with the fatty acid component of the micro-emulsion being prepared.
The saturated solution of the gas in water may be prepared by
sparging the water with the gas, or by exposing the water to the
gas at a pressure in excess of atmospheric pressure for a period of
time in excess of the time required for the water to become
saturated with the gas. In an alternative form of this aspect of
the invention an emulsion of the fatty acid component in water may
first be prepared and may thereafter be gassed by exposing the
emulsion to the gas. This is preferably done by sparging.
[0026] The gas is preferably selected from the group consisting of
nitrous oxide, carbon oxy sulfide and carbon dioxide.
[0027] The phytologically beneficial substance that may be
delivered to a plant by means of the delivery vehicle according to
the present invention may be any one or more of the substances
known to be useful as a plant nutrient; a plant pesticide including
a herbicide, fungicide, bactericide, insecticide, anti-plant virus
agent; a plant growth regulator; a plant immune modulator; a
biostimulant; or genetic material for the transformation of the
plant to allow the incorporation of a new characteristic or
property in the plant. Such property may inter alia consist of
drought resistance, pest resistance and enhanced fruit
production.
[0028] A formulation is typically available in forms that can be
sprayed on as liquids. It includes the active ingredient(s) of
substance(s) as listed in the present invention, any additives that
further enhance effectiveness, stability, or ease of application
such as surfactants and other adjuvants, and any other ingredients
including solvents, carriers, or dyes. The application method and
species to be treated determine which formulation is
preferable.
[0029] The invention accordingly also provides a plant nutrient
composition comprising at least one plant nutrient in the delivery
vehicle described above. Plant growth in its germination,
vegetative or productive phases may be stimulated by enhancing the
delivery of nutrients, including nutrients in the gas phase. The
plant nutrients may be selected from the group of elements
consisting of carbon, hydrogen, oxygen, nitrogen, phosphorus,
potassium, calcium, magnesium, sulphur, iron, manganese, zinc,
copper, boron, molybdenum and chlorine.
[0030] The invention further provides a plant pesticide composition
comprising a pesticidally effective concentration of at least one
plant pesticide in the delivery vehicle described above. A
pesticide is any substance or mixture of substances intended for
preventing, destroying, repelling, or mitigating any pest.
[0031] Pesticides do not only refer to insecticides, but also to
herbicides, fungicides, and various other substances used to
control pests. Under United States law, a pesticide is also any
substance or mixture of substances intended for use as a plant
regulator, defoliant, or desiccant. It is intended to use the term
in this broad meaning thereof in this specification.
[0032] It is accordingly within the ambit of this application to
provide a vehicle for, and to provide formulations that include any
one or more phytologically beneficial substances in the form of
pesticides selected from the group consisting of the following
chemical and biological (organic) pesticides synthetic arsenic, Bt
liquid w/xylene, Bt liquid-no xylene, Bt wettable powder,
beneficial organisms, biodynamic preparations, bordeaux
mixes--copper, hydroxide/fixed copper, boric acid, carbamates,
chlorinated hydrocarbons, chromate ions, citric acid, copper
hydroxide, copper sulfate, herbal preparations selected from
cinnamon, cloves, garlic, mint, peppermint, rosemary, thyme, and
white pepper, herbicides--synthetic, hydrated lime, imidacloprid--a
neonicotinoid insecticide, indoxacarb (p) 13 a chiral oxadiazine
insecticide, insect extracts, isocyanate, lauryl sulfate, lime
sulfur, malathion, malic acid, methyl bromide, methyl sulfoxide,
milky spore disease--B. popillae, nematocides-synthetic, nematodes,
nicotine, oils selected from carrot oil, castor Oil (U.S.P. or
equivalent), cedar oil, cinnamon oil, citronella oil, citrus oil,
clove oil, corn oil, cottonseed oil, dormant oils, garlic oil,
geranium oil, lemon grass oil, linseed oil, mint oil, peppermint
oil, rosemary oil, sesame oil, soybean oil, summer oils, thyme oil
and weed oils, organophosphates selected from acephate,
azinphos-methyl, bensulide, cadusafos, chlorethoxyphos,
chlorfenvinphos, chlorpyrifos, chlorpyrifos-methyl, chlorthiophos,
coumaphos, ddvp (dichlorvos), dialifor, diazinon, dicrotophos,
dimethoate, dioxathion, disulfoton, ethion, ethoprop, ethyl
parathion, fenamiphos, fenitrothion, fenthion, fonofos, isazophos,
malathion, methamidophos, methidathion, methyl parathion,
mevinphos, monocrotophos, naled, oxydemeton-methyl, phorate,
phosalone, phosmet, phosphamidon, phostebupirim, pirimiphos-methyl,
profenofos, propetamphos, sulfotepp, sulprofos, temephos, terbufos,
tetrachlorvinphos, tribufos (def) and trichlorfon,
pentachlorophenol, pesticides--synthetic, petroleum distillates,
petroleum oil spray adjuvants, 2-phenethyl propionate
(2-phenylethyl propionate), pheromones, piperonyl butoxide, plant
extracts selected from hellebore, pyrethrum, quassia, sabadilla,
citronella, sesame (includes ground sesame plant stalks), eugenol
and geraniol, potassium sorbate, putrescent whole egg solids,
pyrethroids--synthetic, rock salt--weed control, rotenone, ryania,
sea animal wastes, soap based herbicides, sodium chloride, sodium
lauryl sulfate, soil fumigants, streptomycin, strychnine, sulfur,
virus sprays, and Zinc Metal Strips (consisting solely of zinc
metal and impurities).
[0033] The invention also provides for a herbicidal composition
comprising a herbicidally effective concentration of at least one
herbicide in the delivery vehicle described above irrespective of
its mode of action and hence includes herbicidal formulations in
which the mode of action is any one of the group having the
following modes of action, namely:
Auxin mimics (2,4-D, clopyralid, picloram, and triclopyr), which
mimic the plant growth hormone auxin causing uncontrolled and
disorganized growth in susceptible plant species; Mitosis
inhibitors (fosamine), which prevent re-budding in spring and new
growth in summer (also known as dormancy enforcers); Photosynthesis
inhibitors (hexazinone), which block specific reactions in
photosynthesis leading to cell breakdown; Amino acid synthesis
inhibitors (glyphosate, imazapyr and imazapic), which prevent the
synthesis of amino acids required for construction of proteins;
Lipid biosynthesis inhibitors (fluazifop-p-butyl and sethoxydim),
that prevent the synthesis of lipids required for growth and
maintenance of cell membranes (Weed Control Methods Handbook, The
Nature Conservancy, Tu et al.).
[0034] It is accordingly within the ambit of this application to
provide a vehicle for, and to provide formulations that include any
one or more phytologically beneficial substances in the form of
herbicides selected from the group consisting of the following:
2,4-D (2,4-dimethylphenol), Clopyralid, Fluazifop-p-butyl,
Flumetsulam--a triazolopyrimidine herbicide, Fosamine Ammonium,
Glyphosate, Hexazinone, Imazapic, Imazapyr, Picloram, Sethoxydim,
Triclopyr.
[0035] It also provides for a fungicide composition comprising a
fungicidally effective concentration of at least one fungicide in
the delivery vehicle described above. The fungicide may be selected
from the group consisting of: 1,3 dichloropropene,
2,5-dichlorobenzoic acid methyl ester, 8 hydroxyquinoline,
acibenzolar-S-methyl, Agrobacterium radiobacter, ammonium
phosphite, ascorbic acid, azoxystrobin, bacillus subtilis DB 101,
bacillus subtilis DB 102, Bacillus subtilis isolate B246, Bardac,
Benalaxyl, Benomyl, Bifenthin, Bitertanol, Borax, boric acid
equivalent, boscalid, bromuconazole, bupirimate, captab,
carbendazim, Carboxin, chlorine dioxide, chloropicrin,
chlorothalonil, chlorpyrifos, copper ammonium acetate, copper
ammonium carbonate, copper hydroxide, copper oxychloride, cupric
hydroxide, cymoxanil, cyproconazole, cyprodinil, Dazomet,
Deltamethrin, Dichlorophen, Dicloran, didesyl dimethyl ammonium
chloride, difenaconazole, dinocap, diphenylamine, disulfoton,
dithianon, dodemorph, dodine, epoxiconazole, famoxadone, alcohols,
anti-oxidants, Fenamidone, Fenarimol, Fenbuconazole, Fenhexamid,
Fludioxonil, Flusilazole, Flutriafol, Folpet, fosetyl-Al,
furalaxyl, furfural, guazatine, hexaconazole, hydroxyquinoline
sulphate, imazalil, iprodione, iprovalicarb, kresoxim-methyl, lime,
lindane, mancozeb, maneb, mefenoxam, Mercaptothion, Metalaxyl,
metalaxyl-M (mefenoxam), metam-sodium, methyl bromide, metiram,
mineral oil, mono potassium phosphate, myclobutanil, octhilinone,
oxycarboxin, paraffinic complex (light mineral oil), penconazole,
pencycuron, phosphorous acid, polysulphide sulphur, potassium
phosphite, potassium phosphonate, prochlorax zinc complex,
prochloraz, prochloraz manganese chloride complex, prochloraz zinc
complex, procymidone, profenofos, propaconazole, propamocarb HCl,
propiconazole, propineb, pseudomonas resinovorans, pyraclostrobin,
pyrimethanil, QAC, Quazatine, Quinoxyfen, Quintozene, salicylic
acid, silthiopham, sodium-o-phenol phenate(Na salt), spiroxamine,
sulphur, TBTO, Tebuconazole, Thiabendazole, Thiabendazole,
thiophanate methyl, thiram, tolclofos-methyl, triadimefon,
triadimenol, tributyltin oxide, Trichoderma harzianum, Tridemorph,
Trifloxystrobin, Triflumuron, Triforine, Triticonazole,
Vinclozolin, zinc oxide, Zineb and Zoxamide
[0036] It also provides for a bactericidal composition comprising a
bactericidally effective concentration of at least one bactericide
in the delivery vehicle described above. The bactericide may be
selected from the bactericides known to be suitable for use on
plants to combat bacteria infecting plants.
[0037] It also provides for an insecticide composition comprising
an insecticidally effective concentration of at least one
insecticide in the delivery vehicle described above. The
insecticide may be selected from the group consisting of
(E)-7-dodecenyl acetate, (E,E)-8,10 dodecadien-1-ol, 1,3
dichloropropene, 3(S) ethyl-6-isopropenyl-9-docadien-1yl acetate,
Allium sativum, Bacillus thuringiensis Serotype H-7, Bacillus
thuringiensis subsp israelensis, Bacillus thuringiensis var aiziwai
kurstaki, Bacillus thuringiensis var kurstaki, Beauveria bassiana,
Bradyrhizobium japonicum, Bradyrhizobium japonicum WB 74,
Bradyrhizobium sp Luinus VK, Bradyrhizobium sp.times.S21,
Bradyrhizobium spum, Chlorpyrifos, Dimilin, E8,E10-dodecadienol,
EDB, Metarhizium anisopliae var acridium isolate IMI 330 189,
Paecilomyces lilacinus strain 251, Rhizobium leguminosarum biovar
phaseoli, Rhizobium leguminosarum viciaeTJ 9 Rhizobium meliloti,
Spinosad, Sulfur, Trichoderma harzianum, Z-8-dodecenylacetate,
Abamectin, abamectin, acephate, acetamiprid, acrinathrin, aldicarb,
alpha-cypermethrin, aluminum phosphide, amitraz, azadirachtin,
azinphos-methyl, benfuracarb, beta-cyfluthrin, beta-cypermethrin,
bifenthrin, borax, brodifacoum, bromopropylate, buprofenzin,
buprofezin, cadusafos, carbaryl, carbofuran, carbosulfan, cartap
hyrochloride, chlorphenapyr, chlorpyrifos, citronella oil,
clofentezine, codlimone (E,E-8,10-dodecadiene-1-01), copper,
coumatetralyl, cryptophlebia leucotreta, cyanophos, cyfluthrin,
cyhexatin, Cypermethin, cyromazine, d-allethrin, dazomet,
deltamethrin, demeton-S-methyl, diazinon, dichlorvos, dicofol,
difenacoum, diflubenzuron, imethoate, disulfoton, emamectin,
endosulfan, esfenvalerate, ethoprophos, ethoprophos, ethylene
dibromide, etoxazole, fenamiphos, fenamiphos, fenazaquin,
fenbutatin, fenbutatin oxide, fenitrothion, fenoxycarb,
fenpropathrin, fenpyroximate, fenthion, fenvalerate, ferric sodium
EDTA, pronil, fipronil, flufenoxuron, flumethrin, fosthiazate,
fumagillin, furfural, gamma-BHC, garlic extract, hydramethylnon,
imidacloprid, indoxacarb, lambda-cyhalothrin, lavandulyl,
senecioate, lufenuron, magnesium phosphide, mancozeb, maple
lactone, mercaptothion, metaldehyde, metham-sodium, methamidophos,
methidathion, methiocarb, methomyl, methyl bromide,
methyl-parathion, mevinphos, milbemectin, mineral oil, novaluron,
omethoate, ortho-phenylphenol, oxamyl, oxydemeton-methyl, parafinic
complex (mineral oil), parathion, permethrin, phenothoate, phorate,
phosmet, phoxim, pirimicarb, polysulphide sulphur, potassium salts
of fatty acids, profenofos, propargite, propoxur, protein
hydrolysate, prothiofos, pyrethrins, pyriproxyfen, quinalphos, rape
oil, rotenone, silicon based repellent, sodium fluosilicate,
spinosad, spirodiclofen, sulfur, tartar emetic, tau-fluvalinate,
tebufenozide, temephos, terbufos, tetrachlorvinphos, tetradecenyl
acetate, tetradifon, thiacloprid, thiamethoxam, thiodicarb, thiram,
trichlorfon, triflumuron, trimedlure, zeta-cypermethrin, zinc
phosphide.
[0038] It also provides for a viracide composition comprising a
viracidally effective concentration of at least one viracide in the
delivery vehicle described above.
[0039] The viracide may be selected from the viracides known to be
suitable for use on plants to combat viruses that infect
plants.
[0040] The invention further provides a plant growth regulator
composition comprising a plant growth regulating effective
concentration of at least one plant growth regulator in the
delivery vehicle described above. The plant growth regulator may
preferably be dl-alpha-tocopherol, or the plant physiologically
active isomer thereof, which product is also known as Vitamin E,
which presence is particularly useful in regulating the onset of
the reproductive phase of plants, i.e. may be used to regulate the
onset of the flowering of the plant and hence to advance the fruit
bearing phase of the plant. More generally however the delivery
vehicle may be used to deliver to a plant any one or more of the
products in the group consisting of:
2-(1-2-methylnaphthyl)acetamide; 2-(1-2-methylnaphthyl)acetic acid;
2-(1-naphthyl)acetamide; 2-(1-naphthyl)acetic acid; 2,4-D (sodium
salt); 3,5,6 TPA; 4-indol-3-ylbutyric acid; 6-benzyl adenine;
alkoxylated fatty alkylamine polymer; alkylamine polymer;
aminoethoxyvinylglycine hydrochloride; ammoniated nitrates; auxins;
calcium arsenate; carbaryl; chlormequat chloride; chlorpropham;
chlorthal-dimethyl; cloprop; cyanamide; daminozide; decan-1-ol;
dichlorprop; dichlorprop (2-butoxyethyl ester); dimethipin;
dinocap; diquat dibromide; diuron; ethephon; fluazifop-p-butyl;
gibberellins; glyphosate-isopropylamine; glyphosate-trimesium;
haloxyfop-P-methyl; indolylacetic acid; maleic hydrazide; mepiquat
chloride; methylcyclopropene; mineral oil; n-decanol; octan-1-ol;
paclobutrazole; paraquat dichloride; pendimethalin;
prohexadione-calcium; salicylic acid, sodium chlorate; thidiazuron;
trinexapac-ethyl; and uniconazole.
[0041] The invention also provides for a method of enhancing the
structural and functional integrity of plants or parts of
plants.
[0042] The invention also provides for a method of administering a
phytologically beneficial substance to a plant, comprising the step
of formulating the substance in a delivery vehicle according to the
invention and as described herein, and applying the formulated
product to the plant. The application may be by means of aerial or
surface application, either mechanical or by manual spraying, by
incorporation in water borne irrigation system, or by trunk
injection where appropriate.
[0043] The invention also provides for a method of supporting the
local defense and acquired resistance of plants according to the
mechanism described below by simultaneously supplying precursors
for defense signalling molecules, anti-oxidants, ethylene, oleic
acid and hexadecatrienoic acid.
[0044] The involvement of salicylic acid (SA) as a signal molecule
in local defenses and in systemic acquired resistance (SAR) is well
known. SA synthesis is activated by exposure to pathogens or
ultraviolet light. Salicylic-acid signaling is mediated by at least
two mechanisms, with feedback loops to modulate the effect. These
feedback loops may also provide a point for integrating
developmental, environmental and other defense-associated signals,
and thus fine-tune the defense responses of plants. (Jyoti Shah The
salicylic acid loop in plant defense. Current Opinion in Plant
Biology 2003, 6:365-371)
[0045] Studies had suggested a role for lipid peroxidation in the
SA-activated expression of resistance genes. SA activates the
expression of .alpha.-dioxygenase (.alpha.-DOX1). .alpha.-DOX1
oxidizes 16-C and 18-C fatty acids, the last of which is a
component of the formulation of the invention. In addition, fatty
acids 16:3 and 18:3 are precursors for the synthesis of oxylipins,
which are potent defense signaling molecules. Various research
findings thus indicate that fatty-acid-derived signal(s) are
involved in modulating SA-signaling in plant defense (Jyoti Shah
The salicylic acid loop in plant defense. Current Opinion in Plant
Biology 2003, 6:365-371).
[0046] Multiple stimuli can activate SA synthesis/signaling.
Chloroplasts/plastids in plants may be the source of signals that
affect responses to pathogens. Chloroplast/plastid
function/integrity is important for the outcome of plant--pathogen
interactions. Chloroplasts/plastids are also important for lipid
metabolism and the generation of lipid-derived signals. A lipid
signal is required for the activation of at least one of the
pathways by salicylic acid.
[0047] Ethylene, which contributes to fruit ripening and colouring,
potentiates signaling through this pathway. Studies show that the
presence of oleic acid--a component of the invention--is necessary
for the lipid derived signal(s) in both resistance pathways.
Furthermore, the genetic suppression of resistance is associated
with a lowered content of hexadecatrienoic acid (C16:3). The
delivery of the 16:3 by an exogenous source should therefore
contribute to plant resistance.
EXAMPLES OF THE INVENTION
[0048] The invention will now be illustrated, purely by way of
examples with reference to the following non-limiting description
of Preparations, Examples and Figures in which
[0049] FIG. 1 is a graph illustrating the increase in number of
nodes on cucumber plants treated by use of the plant support
formulation of the invention as described in Example 5;
[0050] FIG. 2 is a graph illustrating the increase in leaf size of
cucumber plants treated by use of the plant support formulation of
the invention as described in Example 5;
[0051] FIG. 3 is a graph showing the numbers of medium to large
cucumbers harvested at different times from plants treated with a
plant support formulation according to the invention compared to
untreated control plants as described in Example 5;
[0052] FIG. 4 is a graph showing the numbers of extra large
cucumbers harvested at different times from plants treated with a
plant support formulation according to the invention compared to
untreated control plants as described in Example 5;
[0053] FIG. 5 is a graph showing the total numbers of cucumbers
harvested at different times from plants treated with a plant
support formulation according to the invention compared to
untreated control plants as described in Example 5;
[0054] FIG. 6 is a graph showing the numbers of green peppers
harvested at different times from plants treated with a plant
support formulation according to the invention compared to
untreated control plants as described in Example 5;
[0055] FIGS. 7, 8, 9 and 10 are micrographs of sections of baby
marrow plants treated with plant support formulations according to
the invention as described in Study 1 of Example 6;
[0056] FIGS. 11 and 12 are graphs illustrating the growth of Clivia
plants treated with different plant support formulations according
to the invention as described in Study 2 of Example 6;
[0057] FIG. 13 is a graph showing the average head diameter of
Elementol R-treated lettuce plants versus control plants over a 12
week period after transplantation as described in Example 16;
[0058] FIG. 14 is a graph showing the average comparative growth in
plant height of Elementol R-treated lettuce plants versus control
plants over a 12 week period after transplantation as described in
Example 16;
[0059] FIG. 15 is a graph showing an example of a plant by plant
comparison of Elementol R-treated lettuce plants versus control
plants as described in Example 16, using plants with a similar
number of leaves at 1st treatment;
[0060] FIG. 16 is a graph that illustrates the average %
enhancement in Fm:Dm ratios during the trial period caused by
Elementol R-treatment of the lettuce plants versus control plants
as described in Example 16.
[0061] FIG. 17 is a graph that illustrates the difference in the
Elementol R-treated lettuce plants and control plants in terms of
the % moisture as described in Example 16;
[0062] FIG. 18 is a graph that illustrates the respiration rate per
mg protein for the study period in the Elementol R-treated lettuce
plants and control plants as described in Example 16;
[0063] FIG. 19 are two graphs showing a comparison of the average
chlorophyll A and B contents per mg of protein per fresh mass
between Elementol R-treated lettuce plants and control plants for
the period of the study as described in Example 16;
[0064] FIG. 20 is a graph that reflects the chlorophyll A:B ratios
obtained from the chlorophyll corrected for mg of protein and fresh
mass as described in Example 16;
[0065] FIG. 21 is a graph showing the changes in average number of
flower buds formed during the first few weeks after transplantation
(WAT) in Elementol R treated and control tomato plants as described
in Example 17;
[0066] FIG. 22 is a graph showing the average % enhancement in
flower bud production of Elementol R treated and control tomato
plants as described in Example 17;
[0067] FIG. 23 is a graph that shows the linear increase of
accumulative average yield for 3 tomato plants over the period of
the study as described in Example 17;
[0068] FIG. 24 is a graph that shows the average accumulative fruit
to average accumulative bud ratio of tomato plants treated as
described in Example 17;
[0069] FIG. 25 is a graph that shows the average % of moisture
found in the fruit of Elementol R treated tomato plants versus
control plants as described in Example 17;
[0070] FIG. 26 is a graph that shows the effect of ComCat.RTM.
(CC), Elementol R (E) and combinations thereof on changes in
accumulative number of fruit harvested from 3 plants per group over
a period of 13 weeks as described in Example 18;
[0071] FIG. 27 is a graph that shows the total accumulative fruit
mass observed from plants treated with ComCat.RTM. that is
entrapped in Elementol R as compared to the increase observed with
Elementol R or ComCat.RTM. individually as described in Example
18;
[0072] FIG. 28 is a graph that shows the increase in fresh fruit
mass by the combination of Elementol R and CC as described in
Example 18;
[0073] FIG. 29 is a graph that shows the respiration rate per
protein content after the first administration (week 5) and the
second administration (week 9) of the Elementol R, Comcat.RTM. and
combination treatment as described in Example 18;
[0074] FIG. 30 is a graph that illustrates the comparative amounts
of chlorophyll B per mg of protein as determined in week 13 of the
trial described in Example 18;
[0075] FIG. 31 is a graph that shows the comparative Brix readings
in week 13 for Elementol R treated, CC treated and the combination
treated plants described in Example 18 with HClO.sub.4 as
background;
[0076] FIG. 32 is a photograph of germinating radishes on
germination paper in the in vitro study described in Example
19;
[0077] FIG. 33 is a graph that illustrates the comparative average
length measured for coleoptiles of wheat for the fertilizer
control, and the various dosages of Elementol R described in
Example 19;
[0078] FIG. 34 is a graph that shows the enhancement in the yield
of grain from wheat by a single administration of Elementol R
cultivated in field trials as described in Example 19;
[0079] FIG. 35 is a graph that shows the average comparative plant,
root and leaf weights of maize plants cultivated from seeds treated
with the fungicide Captan, with a combination of Captan and
Elementol R or with untreated seeds as described in Example 19.
PREPARATION 1
Preparation of Plant Supporting Formulation Suitable for Use as a
Delivery Vehicle for Use in Delivering a Phytologically Beneficial
Substance to Plants
[0080] A formulation according to the invention may be made up as
follows: [0081] Step 1: A desired volume of water is saturated with
the indicated gas (in this example nitrous oxide but the same
general procedure with minor modifications is used when employing
carbon dioxide) at ambient pressure using a pressure vessel and
sparger. The vessel is connected to a supply of nitrous oxide via a
flow control valve and pressure regulator. The closed vessel is
supplied with nitrous oxide at a pressure of 2 bar for a period of
96 hours, it having been determined that at the aforementioned
temperature the water is saturated with nitrous oxide over such
period of time under the above-mentioned pressure. In the case of
the preparation of the basic or stock formulation (herein referred
to as Elementol B) to be used on its own, or when it is to be used
as a delivery vehicle for nutrients or the majority of synthetic
organic pesticides unchlorinated water is used. Where the stock
formulation is intended to be used as a delivery vehicle for
peptides or biocatalisators to plants the water is phosphate
buffered to a pH of 5.8. [0082] Step 2: The following fatty acid
based compositions was made up: First, Vitamin F Ethyl Ester CLR
110 000 Sh.L. U./g obtained from CLR Chemicals Laboratorium Dr.
Kurt Richter GmbH of Berlin, Germany which is composed mainly of
21% oleic acid, 34% linolenic acid, and 28% linoleic acid that are
modified by esterification with an ethylene group of the carboxy
terminal, was heated to 75.degree. C. Secondly, pegylated,
hydrogenated fatty acid, ricinoleic acid (also known by the INCI
name as PEG-n-Hydrogenated Castor Oil), was heated to 80.degree. C.
and mixed with the first group of fatty acid based Vitamin F Ethyl
Ester at 70.degree. C. The ratio of the first group of fatty acids
to the latter fatty acid was generally 3:1 for foliar application.
In the case of the addition of the preparation to large containers
supplying plants by drip irrigation in controlled environments on a
continuous basis, the ratio was 5:1 to 6:1. [0083] Step 3:
dl-.alpha.-Tocopherol of varying percentages (final concentration
of between 0.1% when used as general anti-oxidant (Elementol B) and
0.25% v/v when used as regulator of plant reproductive phase or for
synchronization (Elementol R) was added to the heated fatty acids
mixture above, either as anti-oxidant or as growth modulator.
[0084] Step 4: The water or buffered water was heated to 73.degree.
C. and mixed with the fatty acid mix with the aid of a high speed
shearer to a final concentration of between 3.2 and 4%, depending
on the specific use of the preparation. This fatty acid mixture
constituted the basic preparation that contains vesicles of sizes
in the nanometer range as determined by particle size analysis on a
Malvern sizer. [0085] Step 5: To the basic preparation may be added
additional ethylated fatty acids DHA (decahexonoic acid) and EPA
(eicosapentanoic acid). The preferable amount of the two fatty
acids for this invention was 0.5%. The addition of these fatty
acids results in die formation of microsponges rather than
vesicles, with particles between 2-5 .mu.m in size, as determined
by particle size analysis on a Malvern sizer. [0086] Step 6: This
basic preparation is diluted with water for administration to the
plants. The dilutions were generally 1:1 for stem application, 1:10
for ornamentals in open settings, 1:200 for stool beds, 1:600 and
1:800 for orchards, 1:1000 for open field crops and controlled
environments, 1:1500 for colouring of fruit, and 1:5000 in
hydroponic systems depending on the method of administration, the
type of cultivation (e.g. drip irrigation, foliar spraying by hand,
tractor or plane).
[0087] Stable particles of fairly homogeneous sizes ranging from 50
nm to 50 .mu.m can be manufactured with ease on a large scale. The
size and shape of the particles can be reproducibly controlled. The
Zeta potential of the Elementol B and Elementol R prepared as
described above were determined by means of and found to be -46 mV
and -38 mV respectively. Variations in the particle size of the
micro emulsions may be effected by varying the composition and
variations in the Zeta potential of the emulsion may likewise be
effected by varying the composition.
PREPARATION 2
Typical Preparation of a Formulation Containing a Phytologically
Beneficial Substance in the Plant Supporting Formulation According
to the Invention as a Component of a Delivery Vehicle.
[0088] Step 1: One or more phytologically beneficial substances may
be entrapped in the basic Elementol or buffered Elementol
preparations described above, by thorough mixing of the desired
substance into the Elementol formulation at room or field
temperature before dilution for administration as described in step
6 of Preparation 1. Mixing may occur by shaking or stirring. After
mixing, preparations are generally allowed to `cure` for at least
30 minutes, but not more than 3 hours, before dilution with water
for administration. In the case of substances with large molecular
weights such as peptides, the preparations are left overnight at
4.degree. C.
Example 1
Use of Elementol as Delivery Vehicle for Foliar Nutrient
Administration on Watermelon
Introduction:
[0089] Contrary to previous watermelon crops on a selected 160 Ha
plot, the watermelon crop of this study had a low yield potential
even though there were no changes when compared with previous
practices. The following was observed during January 2005: [0090]
1.) Premature senescence occurring during January of 2005. It was a
scattered phenomenon. [0091] 2.) The latter was mainly ascribed to
nematodes resulting in the reduction of root efficiency. This
resulted in many fruits becoming deformed and suffering "blossom
end rot". [0092] 3.) Foliar fungal infections were common,
irrespective of the pro-active application of fungicides on a 10
day basis. The fungicides were alternated to reduce the risk of
resistance by the fungi.
Trial:
[0093] The decision was made to maintain the fungicide program, but
to introduce a nutrient application as a foliar spray.
[0094] The experimental spray, per hectare, contained the
following:
5 kg CaCl.sub.2 dissolved in 26.0 litres of water. 1.0 litres of
"amino acid complexed Calcium" (100 g/litre Ca) 0.5 litres of
"amino acid complexed Copper" (75 g/litre Cu)
6 ml Elementol B
[0095] The concept had the following as objectives: [0096] 1.) To
boost the plants' internal resistance to the fungal infection with
the copper and Elementol B and [0097] 2.) To have calcium available
at the "meristem", to improve "cell wall integrity" during any
future foliar and root development, resulting potentially, in
additional fungal resistance and improved foliar and root
efficiency.
[0098] The Elementol B was added to the amino acids and the blend
was allowed to "cure" for 15 minutes before dilution. The dilution
was done by adding 28.5 litres of the CaCl.sub.2 water. The
CaCl.sub.2 water was prepared 48 hours in advance.
[0099] The purpose for the advance dissolution of the CaCl.sub.2
was to subject the chlorine to "UV" hoping to have a reduced effect
of this element during the trial. The 1.56 litre "amino
acid/Elementol blend", along with the 28.5 litres "Ca-enriched"
water resulted in a total of some 30 litres of the preparation
being applied per hectare. Application was by aerial foliar
spray.
[0100] The same application was repeated 10 days later, having
increased the Elementol B in the preparation to 12 ml/ha.
Control:
[0101] The control strips were treated identically to the trial
strips, but excluded the Elementol B.
Repetition:
[0102] Since both the trial and the control received two aerial
applications, repetition integrity was obtained by using a SATLOC
differential global positioning system (DGPS). This instrument was
mounted on the aircraft as standard equipment. Each "spray run"
during the first application was saved. This allowed for the second
application to be applied with less than 0.5 metre deviation from
the first application.
Observations:
[0103] Within 48 hours of the first application, there was a visual
difference between the treated strips and those of the control. The
trial strips showed signs of "rejuvenation". The treated plants
showed up a much darker shade of green compared to the control. At
the same time these plants were showing an observable increase in
flowering compared to the control. This phenomenon prompted the
grower to request a second application with an increased Elementol
B component (12 ml/ha).
[0104] Both applications were done during January 2005.
[0105] The Elementol B treated watermelons, irrespective of the
very low applied volumes (6 ml & 12 ml respectively), senesced
well after the control. This delay in senescence varied between 2
to 5 weeks. Although deforming amongst fruit was not reduced by
this treatment, it did significantly reduce the blossom end
rot.
[0106] Due to the scattered occurrence, across the field, of the
initial problem, only observations were made.
Example 2
Use of Elementol B as Delivery Vehicle for Foliar Administration of
Fungicide on Sugar Beans
Introduction:
[0107] Planting of Sugar beans on a 120 Ha plot was done on
seedbeds measuring 910 mm apart (old 3 feet spacing).
Trial:
[0108] This trial had the following as objective:
Spraying Elementol B as a foliar application together with a
fungicide, by tractor, to observe any reaction by the plants with
regards to flowering/yield.
[0109] For the trial, an area of 10 hectares was demarcated, using
GPS technology and ground markers.
[0110] The experimental spray, per hectare, comprised of the
following:
200 litres of water
40 ml of Elementol Basic
250 ml Punch.RTM. C
Control:
[0111] The control area comprised of 10 hectares on the same block.
A buffer area of 30 metres separated trial and control. The spray
applied here contained no Elementol.
Repetition:
[0112] Provision was made for repetition by demarcating both trial
and control blocks using GPS technology and ground markers. Two
sprays were administered.
Observations:
[0113] Sampling the pods was done by hand. The sampling method used
was 10.times.10 metre random rows. This method was also used to
sample the control.
Conclusion:
[0114] The sampling result was as follows:
Punch.RTM. C with Elementol B: 2,390 kg/ha Punch.RTM., no
Elementol: 2,180 kg/ha Subsequent studies showed that Elementol B
contributed to the antifungal effect, as well as to the yield
improvement.
Example 3
Determination of Phytotoxicity and Beneficial Effects of Elementol
R by Foliar Administration on Strawberries
Introduction:
[0115] The planting of the strawberries on the 12 ha trial plot
commenced during early April 2005. The plant material is all first
generation. The planted blocks slope down in a westerly direction
and the elevation is roughly 100 metres above mean sea level. The
soil has a clay content of less than 5% and an organic carbon
content of 0.5%.
Trial:
[0116] This trial had the following as objective:
Spraying Elementol R as a foliar application, by tractor, to
observe any reaction of the plants with regards to flowering.
[0117] The experimental spray, per hectare, comprised of the
following:
200 litres of water
250 ml of Elementol R
[0118] The spraying was done under the following conditions:
Temperature: 23.degree. Celsius (The .DELTA. between wet and dry
bulb: <5.degree. C.)
Humidity: 28%
[0119] Droplet distribution: averaging 15/cm.sup.2 Treated blocks:
Blocks 6 & 7 Control block: Block 5 [0120] Physiology: Spraying
commenced only once 20% of the plants initiated flowering.
Control:
[0121] Closing the control tunnel #5 during the application of the
Elementol R to blocks 6 & 7 prevented contamination by
drift.
Observations:
[0122] The two treated blocks, by random sampling, yielded in
access of 100% more flowers than the control block. This
observation was made 21 days after application. No signs of
phytotoxicity were observed.
Example 4
Use of Elementol B as Delivery Vehicle for Foliar Boric Acid
Administration on citrus (navel var. Lina)
Introduction:
[0123] The trial orchard was a 15 Ha orchard on which the trees are
about 12 years old, meaning that the trees are mature. The plant
population per hectare is 617 trees/ha. Lina navels is an early
variety. Getting these to the market first has great financial
advantages to the grower.
[0124] High levels of gibberelic acid, within fruit bearing plants,
results in delayed colouring of fruit. Field experience indicated
that the vegetative growth rate of most plants may be reduced by
applying, as a foliar spray, a calculated volume of Boron. The
Boron source generally used was boric acid (H.sub.3B0.sub.3).
[0125] At the same grower, during the trial season, Boric acid was
applied, in a calculated fashion, to lemons that have been
over-nitrified. Over-nitrification of lemons leads to vigorous
growth with a reduction in fruit formation. Harnessing this growth
phenomenon was achieved using boric acid.
Trial:
[0126] Having achieved the inhibition of vigorous growth with boric
acid on the lemons, it was assumed that such an application in
combination with Elementol B may result in early colouring of
Navels on the trees thus saving on de-greening with ethylene in a
controlled atmosphere chamber.
[0127] This trial was set out on Navels, variety Lina. The surface
area was 15 hectares. The objective was early colouring on the
trees. No controls were demarcated within the trial area. Orchards
of growers adjacent to the trial were monitored as a possible
control.
[0128] The experimental spray, per hectare, comprised of the
following:
2000 litres of water
130 ml of Elementol B
[0129] 1 kg Boric acid
[0130] The boron was dissolved/suspended in water prior to adding
the Elementol. A curing time of 30 minutes was allowed before the
water was added for final dilution.
Observations:
[0131] The treated Linas changed colour on the trees approximately
2 weeks earlier than the adjacent controls. These navels were
picked a week earlier than any other in the vicinity
Example 5
Controlled Environment Investigations into the Impact of Elementol
R on Cucumber Plant Yield
Materials and Methods:
Materials
[0132] Dicla plastic-covered tunnels (2 um thick plastic with
inherent UV-protection for plants) with 2.times.50001 tanks and
pumps, saw dust growth medium, 15 litre plastic bags, seedlings
(cucumber) from Dicla, South Africa, Green pepper seedlings from
King Athur, Stihl mistblower, calcium nitrate from Ocean or Omnia
(South Africa), NutriVeg (Omnia) or HydroGro (Ocean), nitric acid
(Ocean), potassium sulphate (Ocean).
[0133] Methods:
[0134] General set-up: One tunnel and tank each were allocated to
the test product, and one tunnel and tank each was used as control.
The tunnels were cooled by air cooling with opening and closing of
flaps. Flaps and doors were usually closed at between 18:00 and
19:00 for the night, and opened at between 06:00 and 08:00 every
morning, depending on temperature. The orientation of the tunnels
was north to south, catering for the prevailing wind direction to
assist with cooling. No artificial heating or cooling system was
used in the tunnels.
Plants:
[0135] Cucumbers: 720 Cucumber seedlings of 3 weeks old were
transplanted from seedling trays to plastic bags containing saw
dust in each of the tunnels at the start of summer. Planting were
done in 6 rows of 120 plants per row. The strongest plants were
selected for the control tunnel.
[0136] Green peppers: 500 King Arthur seedlings were planted in 10
litre plastic bags filled with saw dust in the test tunnel, while
504 similar seedlings were planted in 15 litre plastic bags filled
with saw dust. The plants were grown outside the tunnels for the
first 2 months without any addition of Elementol R, and then moved
to the tunnels, for their pepper-bearing season. Addition of
Elementol R to test plants was started two weeks after the transfer
of the plants from the outside to the tunnels. A significant
difference in yield of green peppers was observed in the test. The
possibility was investigated that plants may just be happier inside
the test tunnel for reasons other than the treatment with Elementol
R. To control for this possibility, Elementol R treatment was
interrupted for a 10 day period (day 120-130), after which it was
resumed.
Irrigation:
[0137] Cucumbers: Small plants received 15 minutes of
drip-irrigation 3 times a day through 4 litre/hour drippers, thus a
total of 3 litres/day. The irrigation was increased to 30-40
minutes/day (>4 litres/day) after 6 weeks, when plants started
bearing fruit that could be harvested and to accommodate the high
summer temperatures of up to 45.degree. C. inside the tunnels.
[0138] Peppers: Treatment of small plants were similar to that of
the cucumbers, but the volume of irrigation was increased after 8
weeks to >5 liters/day/plant.
Test Product:
[0139] The test product is a plant beneficial delivery system,
called Elementol R. It was hypothesized that this system may
increase
a) the solubility and b) the absorption of nutrients, and more
specifically calcium.
[0140] The test product was administered by root irrigation.
Elementol R was mixed with the nutrient of the tank that supplied
irrigation to the test tunnel.
[0141] The nutrient mixture for irrigation was as follows:
[0142] To each tank filled with 5000 l of borehole water, 500 ml
nitric acid was added to lower the pH to 6.0, after which 2 kg of
nutrient mix and 2 kg Calcium nitrate were pre-mixed with water and
added to the tank in that order. For the test tank and tunnel,
pre-mixing was with 1 l of Elementol and water. In the case of the
green peppers, 500 g of the calcium nitrate was replaced with 500 g
of potassium sulphate when the plants started bearing fruit. Every
two weeks, 100 ml of a disinfectant such as Prasine, were added to
the full tank to prevent growth of algae. Every 4.sup.th day, the
plants were flushed with borehole water only, after which nutrient
feeding continued.
Analysis:
Cucumbers:
[0143] The following parameters were investigated during the
various phases of plant growth: [0144] i) Plant length [0145] ii)
Leaf length [0146] iii) Nr. of nodes [0147] iv) Cucumber yield
Plant Length:
[0148] During the initial growth period it is possible to measure
plant length. Twenty randomly selected plants of each row (120
plants for each tunnel) were measured for length from the level of
the saw dust to the highest branching from stem. The plastic bags
of the plants measured were marked with lime, to prevent repeated
measurement of the same plants. The average length of the plants in
each row was calculated and used for comparison. Leaf length of the
bottom two leaves of a plant were determined, using a similar
number of plants and selection and calculation procedure as
described for plant length.
Number of Internodes:
[0149] The number of branches formed was counted, using a similar
number of plants and selection and calculation procedure as
described for plant length.
Cucumber Yield:
[0150] The cucumbers were harvested. Only those cucumbers fit for
sale in an upmarket chain store were counted and weighed. Cucumbers
that were bent, yellow or of which the general appearance were not
according to sales requirements, were not taken into account.
Green Peppers:
[0151] The green pepper experiment was stopped due to the approach
of winter. An electrical heating system installed in the tunnels
proved to be insufficient and plants were exposed to temperatures
below 2.degree. C. Only the saleable yield was determined for the
green peppers.
Results and Discussion:
Cucumbers:
[0152] Plant length was determined for 120 randomly selected
seedlings at ages of 4, 5, and 6 weeks after transplantation. The
average length, representing average growth for each tunnel was
calculated. Table 1 illustrates the average weekly growth of the
seedlings. Whereas the average control plants were initially taller
(week 4) than the plants of the test tunnel, the plants that were
irrigated with the added Elementol R, grew faster than that of the
control tunnel as determined two weeks after the start of the
Elementol R treatment.
TABLE-US-00001 TABLE 1 Average growth in length (cm) Weeks
Elementol Control 4 4.08 4.5 5 6.33 6.45 6 13.04 12.9
[0153] FIG. 1 illustrates the increase in number of nodes by the
addition of Elementol R to the nutrient mix 3 weeks after
transplantation of the seedlings and initiation of treatment. The
nodes were determined for 20 randomly selected plants in each of
the 6 rows, taking care that different plants were used than for
the length determination. In each row, the plants treated with
Elementol R contained more nodes after 3 weeks of treatment,
although the increase was less than 1 (0.73) node per plant when
averaged. The standard error is smaller for the plants that were
irrigated by the Elementol-nutrient mixture, indicating a
synchronizing effect on plant growth.
[0154] When an increase of 0.73 nodes per 3 weeks of treatment are
projected to a total growth period of 18 weeks, the average
difference in number of nodes/plant as a result of Elementol R
administration is 4.4 nodes/plant, which is statistically
significant. The importance of increased nodes is that it indicates
the number of both leaves and fruit-bearing buds that the plant
will develop.
[0155] FIG. 2 illustrates the increase in leaf size by Elementol R
root administration. Leaf length was determined for 120 plants in
each tunnel; 20 plants per row three weeks after the start of
Elementol R administration. As is the case with plant length, the
sizes of the leaves of the plants in the test tunnel were slightly
smaller than that of the control plants before Elementol
administration was started. The difference in leaf size caused by
Elementol treatment is significant and is important in the
development of the plant, since the leaves are responsible for the
photosynthesis. Once again, the standard error was smaller for the
plants that received Elementol R.
[0156] It is generally accepted that the period of yield for
cucumbers is 12 weeks, although some producers harvest fruit for a
period of 16 weeks. In FIGS. 3 and 4 the yield of the plants over a
12 week period is illustrated, thus plant age as illustrated below
is the summation of:
3 weeks from seeding to seedling growth (untreated)+3 weeks of a
pre-harvesting Elementol R treated growth+12 weeks of harvesting
with Elementol R treatment. Although plants were still producing
flowers at week 20, the investigation was stopped at that point,
due to a heavy white fly infestation in the absence of a formal
pesticide program.
[0157] At the start of harvesting, cucumbers were classified as
medium to large (up to 37 cm). However, by the end of the 4.sup.th
week and up to the 20.sup.th week of harvesting, the cucumbers
harvested were between 41 to 47 cm in length, resulting in a lower
number of cucumbers, but a better harvest in terms of weight. For
that reason, the results on yield are separated for the two time
periods.
[0158] It is necessary to remark that harvesting of the two tunnels
occurred simultaneously, and therefore the yield is linked to
specific days of the week. This may be slightly artificial, as
harvesting of the control tunnel 3 days later than the test tunnel,
may have given a more equal distribution of cucumber yield for
weeks 9 to 13. During week 14, a breakdown of the electrical supply
to the irrigation and pumps over a 48 h period caused a significant
decrease in yield in both control and Elementol-treated plants. The
stress caused by non-irrigation seemed to be better tolerated by
the Elementol-treated plants, as can be seen from FIG. 4.
[0159] Table 2 shows the total difference as well as % difference
between the yields in cucumbers from the two tunnels.
TABLE-US-00002 TABLE 2 Difference in yield Experimental Control Sum
7797 5941 % of total 56.75498617 43.24501 Ratio 0.761959728 % diff
31.24053 Nr/month 3898.5 2970.5 Nr/plant/mnth 5.414583333 4.125694
Fruit/plant 16.24375 12.37708
Green Peppers:
[0160] FIG. 6 illustrates the yield of the green peppers over a 70
day period. Harvesting was started 3 months (90 days) after
planting, whilst treatment with Elementol R started two weeks
pre-harvesting. After day 160, plants were exposed to such low
temperatures that the experiment was stopped, although the plants
were still producing harvestable fruit.
[0161] The impact of Elementol R on the yield of green peppers is
illustrated in FIG. 6. The first arrow indicates the start of the
10 day interruption of treatment with Elementol, whereas the second
arrow indicates when Elementol R treatment was resumed. Each point
indicates the combined harvest for that tunnel over a ten day
period. A decrease in yield is immediately observable after
interruption of Elementol R treatment in the test tunnel. The yield
decreased and stabilized at a level similar to that of the control
tunnel, indicating that the increased yield can be specifically
ascribed to the presence of the Elementol R.
[0162] Table 3 shows the total yield and % difference in yield per
tunnel.
TABLE-US-00003 TABLE 3 Difference in yield Experimental Control
Total 3003 2458 % of total 54.98993 45.01007142 % difference over
total period 22.1725% % difference before treatment 42.35294%
interruption
[0163] The determination of the % difference between the two groups
can in reality only be made for the time period before the
interruption of treatment, since it is difficult to estimate the
long-term effect of such an interruption.
Conclusion
[0164] The impact of Elementol R on the yield of fruit of two
different plant species was investigated--that of cucumbers and
green peppers. The addition of Elementol
[0165] R to the plant nutrients mixture resulted in statistically
significant increases of yield of harvestable fruit in both plant
species.
Example 6
Penetration and Distribution in Dicothyl Plants--Investigation into
the Potential of Elementol B Technology for Agricultural
Applications:
[0166] The background to the projects is as follows:
Background to the Study
[0167] Elementol B consists mainly of a function-specific number
and combination of unsaturated fatty acids and nitrous oxide.
Preliminary Studies were Undertaken to Determine [0168] 1) the
permeation/penetration of Elementol B into plants and the
translocation of Elementol B in the plants over time and [0169] 2)
the possible contribution of Elementol B to the delivery of plant
nutrients to plants.
Methods and Materials:
Elementol Preparation:
[0170] 45 g Basic Elementol medium was diluted with 225 g nitrous
oxide saturated purified water (N.sub.2O--H.sub.2O) at room
temperature. The mixture was shaken vigorously and 1250 .mu.l of
the fluorescent marker Nile Red (1.6 .mu.g/.mu.l; Molecular Probes,
Holland) was added.
Study 1
Test Subjects:
[0171] Hydroponically cultivated (n=3) baby marrow plants
(dicothyl) in bloom stage planted in bags containing wood chips
(support medium) were obtained from a nursery for this pilot study.
Plants were allocated as follows: [0172] Plant 1: Control--Nothing
administered. [0173] Plant 2: Addition of 100 ml prepared Elementol
mixture to the support medium bag with wood chip to investigate
root application. [0174] Plant 3: The whole plant was sprayed with
the Elementol mixture except for one leaf which was covered with
plastic before spraying.
[0175] After the administration of the Elementol mixture as
described above, the plants received no further nutrients but were
watered daily. After 3 weeks, harvested baby marrows were compared
in terms of size and weight.
Permeation/Penetration and Translocation Visualization:
[0176] Leaves were dissected to obtain plant tissue from locations
devoid of prominent veins as well as crosscuts from prominent
veins. Root dissections were performed along the length of the
superior root. The absorption and translocation of the
fluorescently labelled Elementol were visualized by Confocal Laser
Scanning Microscopy on a Nikon PCM2000 with an inverted Nikon
Eclipse 300 microscope, equipped with Spectra Physics Krypton/Argon
and Helium/Neon lasers. The following objectives were used--Plan
Apo 100.times./1.4 Oil DIC H; Plan Apo 60.times./1.4 Oil DIC H; and
a Plan Fluor/0.75 DIC M. Confocal images (micrographs) were
digitally captured via fluorescence detectors and photomultupliers.
Real time micro-imaging was done with a Nikon DMX video camera
system. Depth studies were obtained using a 3D scanning head in
combination with a depth z-step drive.
Results:
[0177] The results are illustrated in the micrographs obtained by
confocal laser scanning microscopy.
[0178] Plant 1: In this micrograph, no Elementol was administered
to the plant. Material is visualized because of
autofluorescence.
[0179] Plant 2: Elementol R (pre-labeled with the red fluorescent
marker Nile Red) were absorbed by the plant through the leaves and
is visible in cross sections of prominent veins of both the covered
as well as the treated parts and in dissections of the leaves. In
this micrograph, nearly all vesicles of the Elementol have
permeated the cells of leaf itself, with few of the Elementol
vesicles remaining in prominent veins of the plant. Leaf
penetration and translocation throughout the leaves occurred in
less than 60 minutes (average time approximately 20 minutes).
[0180] Plant 3: Vesicles of Elementol B penetrated the plant
through the roots and are visualised in the root segments as well
as the cross sections of prominent veins. Root permeation and
translocation were observed in less than 60 minutes.
[0181] The weights found for the first baby marrows harvested are
given below:
Plant 1: Although several flowers were observed on this plant, no
baby marrows were present on the date of harvesting, while plants 2
and 3 produced fruit from a single application of Elementol B and
water.
Plant 2: 64.95 g
Plant 3: 28.38 g
[0182] The study was not continued.
Study 2
[0183] Enhanced Uptake and/or Delivery of Nutrients in
Hydroponically-Cultivated Clivias
[0184] The uptake of some of the minerals and trace elements from
supplied hydroponic media is sometimes problematic. Study 1 showed
in a very small number of plants that Elementol vesicles are taken
up by plants and may even contribute to their growth. In study 2, a
basic hydroponic nutrient mixture was entrapped in Elementol
vesicles and growth of the plants was monitored.
Test Subjects:
[0185] 5 groups of 6 Clivia seeds each were planted in wooden chips
in carton plant holders. The groups were treated daily as described
below: Group 1 received 5 ml of H.sub.2O Group 2 received 5 ml of
hydroponic medium diluted in H.sub.2O to the stipulated
concentration Group 3 received 5 ml of hydroponic medium mixed with
a low concentration Elementol B (1.98%) to the stipulated
concentration Group 4 received 5 ml of hydroponic medium mixed with
a high concentration Elementol B (4%) to the same concentration
used in Groups 3 and 4 Group 5 received 5 ml of hydroponic medium
diluted with nitrous oxide saturated H.sub.2O to the same
concentrations used for the other groups.
Results:
Bulb Formation:
[0186] After 5 weeks bulbs were investigated with the following
results:
Group 3 showed significant bulb formation with 2 of the seeds
showing the formation of multiple bulbs from a single seed, whereas
group 5 showed bulb formation but the bulbs seemed soft and slimy.
Group 1 showed poor small bulb formation. Group 2 showed bulb
formation, but bulbs weighed only 38% of the bulbs of group 3.
Vegetative Growth:
[0187] The vegetative growth was determined by measuring the length
of the longest leaf of the plant after the indicated time periods,
as indicated in FIGS. 11 and 12, which illustrate growth over time
and a comparison of growth after 5 weeks. The growth of the 2
groups containing hydroponic nutrients dissolved in H.sub.2O or
N.sub.2O--H.sub.2O but no Elementol B are much on a par, with the
leaves of group that received N.sub.2O--H.sub.2O slightly longer
than the plants that received water only. Of the groups that
received hydroponic nutrients mixed with Elementol B, the group
that received the low Elementol concentration showed the best
growth of all groups, whereas the group that received the high
Elementol concentration showed the worst growth. The plants were
grown in carton plant holders and problems with drainage were clear
from the mold growth in the wooden chips and on the cartons of
plant holders that received the high Elementol concentration, as
well as from the sliminess of the bulbs of this group. At this
stage no conclusions can be drawn from this group. An Elementol
dilution series will have to be investigated.
Example 7
Use of Elementol R as Delivery Vehicle for Foliar Nutrient
(Calcium) Administration on Strawberries
Introduction:
[0188] The planting of the strawberries on the 12 ha trial plot
commenced during early
[0189] April 2005. The plant material is all first generation. The
planted blocks slope down in a westerly direction and the elevation
is roughly 100 metres above mean sea level. The soil has a clay
content of less than 5% and an organic carbon content of 0.5%.
Trial:
[0190] This Trial Had the Following as Objective:
[0191] Spraying Elementol B and calcium as a foliar application, by
tractor, to observe any reaction by the plants with regards to
improved calcium levels in the leafs.
The experimental spray, per hectare, comprised of the following:
250 litres of water 250 ml of Elementolid
5 kg CaCl.sub.2
Trial and Control:
[0192] The trial blocks were numbers 5, 6 & 7, whilst the
control blocks were 1, 2, 3 & 4. The trial blocks were treated
with the mentioned combination, whilst the control blocks were
treated using a commercial "fulvic acid/CaCl.sub.2" complex. The
percentage calcium in both trial and control was the same.
Observations:
[0193] The leaf calcium levels in the trial blocks were determined
21 days after application and found to be as follows:
TABLE-US-00004 Block Pre treatment % Ca Post treatment % Ca % gain
5 0.86 1.00 16.28 6 0.85 1.01 18.52 7 0.88 1.07 21.59
[0194] The leaf calcium levels in the control blocks were
determined 21 days after application and found to be as
follows:
TABLE-US-00005 Block Pre treatment % Ca Post treatment % Ca % loss
1 0.86 0.85 1.16 2 1.15 0.84 26.95 3 1.08 0.80 25.93 4 1.03 0.84
18.45
Conclusion:
[0195] It is clear from the results that there is a definite
improvement in the leaf calcium levels when CaCl.sub.2, in
combination with Elementol R is applied to strawberries.
Example 8
Use of Elementol R in Foliar Administration to Determine Effects on
Cherry Bell Peppers
Introduction:
[0196] Planting was done on a 1.2 ha test plot using seedlings from
the nursery. The plants were drip irrigated. Spacing within the row
left the plants 300 mm apart, whilst the rows were double rows
measuring 450 mm apart. Plant population per hectare was
30,000.
[0197] The fertilization approach was to supply some 300 kg/ha of
nitrogen, mainly in the form of calcium nitrate and potassium
nitrate. The yield objective was 30 ton/ha. Flowering occurs during
December and continues, while harvesting starts in late February
and continues to the end of June. Prime picking is from mid March
to mid May after which the volumes started to taper off. During
peak picking 4 tons/ha may be harvested every 10 days.
Trial:
[0198] This trial had the following as objective:
Spraying Elementol R as a foliar application to observe the effect
on "increased flowering" as well as early colouring towards the
harvesting period.
[0199] The experimental spray, per hectare, comprised of the
following:
200 litres of water
200 ml of Elementol R
Control:
[0200] The control area comprised a small area on the same block
and received no Elementol R.
Observations:
[0201] More flowers were observed in the trial compared to the
control towards the end of December, but no counts were made.
[0202] Towards the end of January, fruit in the trial showed signs
of advanced colouring compared to the control, but observation was
made difficult due to high temperatures resulting in colouring on
the control too. The feel is, however, that there was a better
colouring on the trial compared to the control.
Conclusion:
[0203] It is unclear whether the Elementol did in actual fact
contribute significantly to the advanced colouring of the cherry
bells since other factors, such as the temperatures, fertilization
distribution, etc. may have influenced the result. The grower did
however feel that there was a difference.
[0204] The real significance is that the grower yielded 29 ton/ha
over the harvest period of which 24 ton were of commercial value.
This yield is substantially better, compared to the area
average.
[0205] Due to the grower's observations, he increased the
application of Elementol R to 250 ml/ha for 4 consecutive weeks
when plants start flowering with the following results:
Plants were larger with better leaf coverage; The yield of fruit
harvested was increased by 15% due to Elementol R administration;
The colouring of the Elementol-treated plants is "aggressive".
[0206] The grower found that at least 3 treatments were necessary
before maximum impact of Elementol R was observed.
Example 9
Use of Elementol B as Delivery Vehicle for Foliar Nutrient
Administration on Sun Flower
Introduction:
[0207] Planting was done in seedbeds measuring 910 mm apart (old 3
feet spacing). The plant population at planting was calculated at
40,000 seeds per hectare with an expected emergence of between
35,000 and 38,000 plants.
Action (Trial):
[0208] Two fields about 1 Km apart were involved, not because they
were destined or prepared for a trial, but simply because they were
in close proximity to each other and one could serve as a control
for the other. The trial plot was about 95 ha in extent and the
control plot about 200 ha.
[0209] The trial plants were sprayed with the following:
1 litre/ha "AminoPotas" (100 g/l "K" complexed or chelated with
amino acid) 1/2 litre/ha "Aminocalcium" (100 g/l "Ca" complexed or
chelated with amino acid) 5 kg/ha urea (2.3 kg "N" as NH4) 50 ml/ha
Elementol B 27 litre/ha water
[0210] The spray mixtures were made up in a mixing tank car and
application was by aerial spraying.
Control:
[0211] The control was sprayed with the same mixture, excluding
Elementol B.
Observations:
[0212] Measurements made to ascertain the difference in yield
between the trial and control was done by the separate "weigh in"
of the combine harvester's hopper (the bin into which the seed
flows once separated from the flower bowl).
Conclusion:
[0213] The sampling result was as follows:
Trial: 2,735 kg/ha Control: 1,650 kg/ha Difference: 1,085 kg/ha
Average enhancement: 65.8%
Example 10
Use of Elementol R in Degreening Apples
[0214] Elementol R was applied by hand spray at the start of fruit
formation in a trial row of an orchard, while other rows in the
orchard received no treatment. The Elementol R sprayed apples
degreened substantially before the untreated apples.
[0215] Similar results were obtained with Cherry Bell peppers with
aggressive colouring due to Elementol treatment. (4 applications)
application rate 1 l/ha (see Example 8). What makes the colouring
results of the apples, citrus and cherry bell pepper significant is
the fact that these results show that the administration of
Elementol R had the same impact on C3 and C4 plants, on annuals and
perennials, on controlled environment and open field trials.
Example 11
Effect of Elementol Foliar Application on Vines
[0216] Two vines in the same vineyard were selected to compare the
effect of a single application of Elementol B to the whole vine,
including the stems with handspray, but excluding the roots.
[0217] The diameter of the treated vine stems were significantly
thickened and foliar index dramatically increased. The yield of
fruit was also higher.
Example 12
Fungal Protection by Elementol and Increase of Shelf Life of Roses
with Elementol B
[0218] Red Success roses known to be highly susceptible to white
rust infestation were treated with Dithane made up and applied
according to the manufacturer's specification. Trial plants were
sprayed with similar Dithane formulations to which Elementol B was
added to obtain a 1 in 10 dilution.
[0219] It was found that the Dithane/Elementol B treated plants had
no sign of white rust when plants all around it became infected,
and moreover seemed to last for a very long time after picking
before it started wilting.
Example 13
A Comparative Study of the Enhancement of the Efficacy of Round-Up
by Elementol
[0220] Aim: The eradication of steenboksuring. [0221] Weed:
Steenboksuring, a hardy and stubborn weed that is nearly impossible
to eradicate with any treatment.
Treatment:
[0222] Roundup Turbo was used as herbicide in the following manner.
Reference control plots were treated and evaluated in the same
manner as the treatment plots with respect to added herbicide and
culturing practices. Various treatment plots were allocated. The
treatment is described in more detail below.
Test Treatment:
[0223] A concentration of 0.6% Roundup Turbo and 40 ml Elementol B
was diluted to 401 and applied to 1 ha. A field of 80ha were
sprayed with this mixture.
Reference Treatment:
[0224] Roundup Turbo was used as herbicide in the following manner:
The herbicide was diluted to a final concentration of 2.8% of
Roundup Turbo without the addition of Elementol B. A similar volume
was applied per hectare to a similar acreage (80ha).
[0225] Control plot: The treatment plots were set out in strips
within a bigger field planted with Smutsvinger grass. The untreated
areas of this field were used as control plot.
Method of Application:
[0226] The method of application was exactly the same for both test
and reference treatment in terms of dosage rates and application
equipment (nozzle with pressure). The herbicide was applied by
spraying with tractor and spraying apparatus. The herbicide was
applied once only, during the mid-winter. No wetting agent or
adjuvant was added to either of the test or reference
treatments
Results and observations: [0227] a) One week after application, the
grass or steenboksuring showed wilting in the test but not
reference plants. [0228] b) After two weeks, the test treated
plants showed typical phytotoxic symptoms i.e. a yellowing of the
leaves (chlorosis), which was followed by necrosis. [0229] c) One
and a half month after application, most of the steenboksuring
showed severe phytotoxicity while all of the grasses were dead.
[0230] d) Observations reported include all variations, either
inhibitory or stimulatory, between the treated and the untreated
(control) plants. Such variations may be formative (leaf and stem
deformation) effects, and/or growth and development rates.
Conclusion
[0231] Despite using 79% less Roundup Turbo in the test treatment,
the resultant death of the weed was enhanced in the presence of
Elementol B.
Example 14
A Comparative Study of the Enhancement of Apple Stool Beds and
Nursery Trees by Elementol R (2005/2006)
[0232] Stool beds: This is a conglomerate of stems cultivated from
a specific rootstock, examples of which are M7 or M9. The purpose
of this cultivation is to produce a large quantity of "stems" onto
which apple varieties of choice may be grafted. Such varieties may
be Gala, Royal Gala, Brae burn, Oregon Red Spur etc. During such
cultivation, success is measured by the amount of stems available
for grafting from any conglomerate. Stem thickness is the main
criteria whilst root quality and volume is secondary. Stems that
are too thin do not allow for grafting.
[0233] Nursery trees: This is rootstock that has been grafted prior
to being transplanted for initial growth. The ideal is to have
these to grow to at least 1.5 meters in height before it is
considered ready for commercial transplanting.
Trial Objective
[0234] The primary objective was to introduce Elementol R with the
purpose to establish the effect it has on the improvement on stem
thickness in a nursery environment. This effect was first noticed
on randomly treated oak trees. The secondary objective was to
enhance the growth of the grafted trees for commercial
transplantation.
Method
[0235] The application method was as a foliar spray along with some
foliar applied nutrient spray. 80 Stool beds were treated with 100
ml Elementol R/20 litre water, meaning 1.25 ml Elementol R was
applied along with nutrients per stool bed. This application
started during November 2005 and was repeated every 10 days. The
programme was maintained until the present.
Control
[0236] The control stool beds received the same treatment except
that no Elementol R was added.
Result
[0237] Results obtained during the first week of February 2006: The
treated beds yielded 63/100 (63%) graftable stems, whilst the
control yielded only 34/100 (34%). The average stem thickness was
11 mm.
[0238] Results obtained during the second week of February 2006:
The trees grafted from rootstock stems that are on the Elementol R
programme are on average 2 m tall, while those cultivated without
Elementol are on average 1.5 m tall. The Elementol R treated trees
have started to feather, i.e. side shoots have developed, whereas
feathering is completely absent in the trees where Elementol R was
not applied.
[0239] Cognizance must be taken that approximately 6 weeks of
development remains for both control and trial. Though it is
anticipated that the control may improve, it is unlikely to match
the trial with Elementol R. Many variations of the invention may be
devised without thereby departing from the spirit of the invention
as formulated in the above statements of the invention.
Example 15
A Comparative Study to Determine to Effect of Elementol R on the
Germination of Hardscaled Seeds
[0240] Arrow Leaf clover seed is known to be a hard scaled seed
that lacks consistency in germination. The Elementol formulation
according to the invention was shown to be beneficial with regards
to the germination of these seeds by soaking quantities of the seed
in clean water, undiluted Elementol R and in a 5% solution of
Elementol in water for 24 and then packing the soaked seeds on seed
beds, and observing the germination thereof. It was found that the
seeds that had been soaked for 24 hours in the 5% solution of
Elementol in water had a 30% better germination rate than the two
other groups of seeds.
Example 16
The Biostimulatory Effect of Elementol R: Effect of Elementol
Foliar Administration on the Growth and Development of Lettuce
1. Material, Plant Growth and Treatment
[0241] Plant: Lettuce or cos, romaine (Lactuca sativa) of the
family: Asteraceae/Compositae (aster/daisy family).
[0242] Cultivar: Lettuce (Lactuca sativa L.), cultivar Red Poem,
was used and was well established (approximately six weeks old)
when purchased from a local nursery.
1.1 Culturing Method: Non-Circulating Hydroponic "Drip" System
[0243] PVC pipes with holes to fit the pots were used and connected
to a reservoir and an aquarium pump to supply the plants with equal
amounts of water and nutrients via the PVC pipe. Leaks were sealed
to ensure that no water leaks from the system. A reservoir that
contains the nutrient solutions were placed under the pipes and an
aquarium pump supplied the plants with water and nutrients. The
pump was connected to a timer to control the amount of water and
nutrients supplied to the plants. The runoff was caught in a
separate reservoir thus non-circulating the system and was
discarded.
[0244] To control the amount of water for each plant, drippers were
used to regulate pressure in the system and supply equal amounts of
water (.+-.9 ml four times a day) to each plant. The
non-circulating drip system ensured that the plants received
optimal water supply and the nutrient medium pH and EC (electrical
conductivity) were constant. The EC of nutrients in the supplying
reservoir as well as the runoff reservoir was measured, which
enabled a determination of the amount of nutrients supplied versus
the amount discarded. The amount of nutrients used by the plant or
retained by the support medium can thus be calculated. Thus when
the EC drops or increases too much, the nutrients could be added or
retained from the nutrient solution supplied to the plants
accordingly. A PW 9526 Digital Conductivity meter was used to
measure the EC in milliSiemens per centimeter (mS.cm.sup.-1).
Non-circulation of the nutrient medium may curb the spread of
diseases in the system from infected plants to uninfected
plants.
1.2 Growth Medium, Nutrients and Transplantation
[0245] Coconut fibre was used as support medium in the hydroponic
system. It is an inert medium with the ability to retain enough
water and air for good root development and good water
retention.
[0246] A Hydrotech nutrient solution with the following composition
was used: Macro elements: Nitrogen (N) 68 g/kg, Potassium (K) 208
g/kg, Phosphorous (P) 42 g/kg, Magnesium (Mg) 30 g/kg, Sulphur (S)
64 g/kg. Microelements: Iron (Fe) 1254 mg/kg, Copper (Cu) 22 mg/kg,
Zinc (Zn) 149 mg/kg, Manganese (Mn) 299 mg/kg, Boron (B) 373 mg/kg
and Molybdenum (Mo) 37 mg/kg.
[0247] Nutrients consisted of a mixture of Hygrotech nutrient
solution and Calcium nitrate nutrient solution in equal amounts: 36
g of Hygrotech and 36 g of Calcium nitrate were dissolved in 2 L of
water and then added to a reservoir containing 38 L of water. The
pH and electrical conductivity of the nutrient solution are an
indication of the dissolved ions present in the nutrient solutions
and were monitored.
[0248] The lettuce were transplanted from the original containers
into the hydroponic containers containing coconut fibre as well as
course gravel in the bottom of the container to ensure adequate
drainage of water and aeration to the roots. Before the lettuce was
transplanted they were rinsed of any additional soil that might
still be around the roots. The plants were weighed. After
transplantation the plants were placed in the system and left to
acclimatize for one week before experimentation began.
[0249] The plants were also placed in random order each week to
ensure they receive equal amounts of sunlight, heat, water etc.
1.3 Glass House Conditions
[0250] The study was done inside a glass house to ensure optimum
temperature as well as humidity levels to the plants in the
hydroponic system. Most of the atmospheric conditions could be
controlled effectively and the risk of diseases was minimized. The
temperature of the glass house was measured on a weekly basis at
twelve in the afternoon right above the hydroponic system with a
thermograph.
[0251] The temperature in the glass house was regulated by an air
conditioner. The temperature was regulated at maximum 24.degree. C.
and minimum 15. The maximum temperature was 28.degree. C. and the
lowest temperature was 4.degree. C. The maximum and minimum
temperature was obtained by using a thermohydrograph and both a
daytime and night temperature was taken.
[0252] The relative humidity (RH) was measured by using a swirl
thermohydrograph and both daytime and night time humidity was taken
into consideration. The relative humidity could be determined in
percentage of maximum humidity of the atmosphere, % RH. The highest
RH % was 98% and the lowest RH % was 29% (26 Mar. 2006).
1.4 Light Intensity
[0253] Light intensity inside the glass house was measured with a
Quantum/radio/photometer. Light intensity was determined at twelve
daily right above the hydroponic system. Clouds and overcast
conditions influenced the light intensity. The changing of the
season also affected the light intensity. During the winter months
the light intensity was lower than those taken during the warmer
months. The maximum light intensity at 12 h00 was 4600
.mu.E.m.sup.-2sec.sup.-1. The lowest light intensity at 12 h00 was
850 .mu.E.m.sup.-2sec.sup.-1.
[0254] Care was taken to expose all plants to equal amounts of
sunlight and other a-biotic factors. Plants were moved into
different arrangements every week.
1.5 Plant Treatment
[0255] Control plants (C) received no treatment at all. Treatment
with Elementol R as described above was prepared as follows:
3 ml Elementol R was mixed with 250 ml H.sub.2O
[0256] Leaf treatment of the test plants consisted of spraying the
Elementol R mix onto the leaves until saturation state but just
before drip status. The plants were sprayed with spray bottles and
care was taken not to contaminate the system or the support medium.
The plants were treated every four weeks (week 1, 5 and 9) till the
end of the study. For every two plants used as control, 3 plants
were treated with the Elementol R mix. By treating two or more than
two plants with the same treatment, a good average could be
obtained per treatment.
1.6 Treatment of Diseases
[0257] Various diseases occur on lettuce. Fungal diseases were
treated systemically with Funginex.RTM.. The plants were treated
whenever fungal disease was noted by applying diluted Funginex.RTM.
(3 ml of fungicide added to 500 ml of H.sub.2O) onto the
leaves.
2. Measurement of Growth and Development Related Parameters
[0258] Before transplantation of the young lettuce plants they were
weighed and thereafter they were weighed weekly with a Mettler PJ
3000 balance. The weight of the non-plant material and pot was
determined and was subtracted from the total mass to determine the
plant weight after each week's growth.
2.1 Growth and Development
[0259] The growth of the lettuce heads were measured on a weekly
basis. The average head diameter values were calculated from three
diameter values. The plant height was measured from the top of the
coconut fibre to the top of the tallest leaf. The average head
diameter and height for each treatment was then calculated.
[0260] Treatment with Elementol enhanced the average growth of the
plants as determined by head diameter by an average of 11% over the
trial period (see FIG. 13 which is a graph showing the average head
diameter of Elementol R-treated lettuce plants versus control
plants over a 12 week period after transplantation.) The asterisks
indicate the time of treatment. Three treatments with Elementol
were given during the trial period.)
[0261] The % enhancement was calculated according to the following
formula:
% Enhancement = ave head diameter of test plant - ave head diameter
of control plants ave head diameter of control plants .times. 100
##EQU00001##
[0262] The average comparative growth in plant height of the plants
was very similar for the treated and control plants until week 11
when the plants reached maturity (See FIG. 14 which is a graph
showing the average comparative growth in plant height of Elementol
R-treated lettuce plants versus control plants over a 12 week
period after transplantation.) Note the dramatic increase in growth
in week 11. The enhancement in growth correlated with
flowering--the Elementol R treated plants were the first to flower,
suggesting that Elementol R might shorten development time.
[0263] Another measurement of the enhancement of plant development
is to compare the number of leaves of the treated and control plant
(See FIG. 15 which is a graph showing a plant by plant comparison
of Elementol R-treated lettuce plants versus control plants using
plants with a similar number of leaves at 1st treatment.) The
asterisks indicate the weeks of treatment (week 1 and 5.) The
average enhancement over the 5 week period was calculated to be
20.7%.
2.2. Fresh and Dry Mass (Fm:Dm), Fm:Dm Ratio and % Water
[0264] This ratio indicates the amount of water and dry mass
present for each gram of plant material. Dry mass is the amount of
dry material left after all water has been removed and is an
indication of the effectiveness of growth. The fresh and dry mass
of the plants was measured every two weeks. To determine the fresh
mass ten cylindrical disks of exactly the same size were cut from
fresh leaves and the mass of each disc was determined. The disk was
placed in a Labotec oven at 72.degree. C. for 72 hours. The dry
mass was then determined. The fresh mass to dry mass ratio was
obtained by dividing the fresh mass by the dry mass.
[0265] The total average % enhancement in Fm:Dm ratios caused by
Elementol R treatment over the trial period was calculated to be
39.5% (see FIG. 16 which is a graph that illustrates the average %
enhancement in Fm:Dm ratios during the trial period caused by
Elementol R-treatment of the lettuce plants versus control plants.)
The total average % enhancement over the trial period was
calculated to be 39.5%. See also FIG. 17 which is a graph that
illustrates the difference in the Elementol R-treated lettuce
plants and control plants in terms of the % moisture.
[0266] To determine the % of moisture in the leaves, the following
calculation was used:
% Moisture = ( Fresh mass - Dry mass ) Fresh mass .times. 100
##EQU00002##
[0267] The % moisture indicates the amount of water present in the
plant. The amount of water present in lettuce must be in
correlation with the dry mass of the lettuce. The moisture % was
relatively stable during the period of the trial, although the %
moisture of the Elementol-treated plants maintained a 5% moisture
content during the last 6 weeks of the trial (week 8 to week 14),
indicating that Elementol treatment results in some water retention
ability. The higher moisture content is not sufficient to explain
the much higher increase in Fm:Dm ratio.
3. Measurement of Physiological Related Parameters
[0268] Plant respiration, photosynthesis, chlorophyll, protein (12%
SDS PAGE) and sugar content were used as physiological parameters.
Besides reflecting the health of the plant, these parameters may
give an indication of reason for the enhancement in growth and
development by Elementol. Each of these parameters (except sugars)
was determined once a week for all plants.
3.1 Protein Content
[0269] Protein was measured on a two weekly basis from week one
onward according to the method described below. .+-.1 gram of fresh
mass was taken weekly to determine the protein concentration of
each plant. The fresh leaves were grounded in 5 cm.sup.3 mM
Tris-HCl buffer (pH 6.8) containing 2 mM EDTA, 14 mM
.beta.-2-Mercapto-etanol and 2 mM PMSF using a mortar and pestle.
The crude extract was centrifuged on a cooled bench centrifuge for
ten minutes at 12 000 rpm. The supernatant was removed and diluted
5 times. The protein concentration of the dilution was determined
according to the Bio-Rad method of Bradford (1976). The absorbency
of the dilution was determined at 595 nm with a Bio-Rad microplate
reader with bovine gamma globulin as standard with a concentration
of 0.5 mg/ml. By taking four readings per plant the protein
concentration could be determined reasonably accurately.
[0270] The protein concentrations of the treated plants and
controlled plants were determined weekly and showed no significant
difference.
3.2 Respiration and Photosynthesis
[0271] The O.sub.2 consumption rate for respiration as well as the
rate of photosynthesis could be determined by means of pressure
manometry, using a submersible differential Gilson respirometer.
Readings, expressed in nmol O.sub.2 per hour per gram of fresh
mass, were taken every few minutes. This method was adapted from
Stauffer (1972). A steady state of gas exchange method was
followed.
[0272] Respiration was measured in dark conditions, whilst both
photosynthesis and respiration was measured in conditions of
constant light intensity.
[0273] Ten leaf disks per plant were cut from fresh leaves with
approximately 1.5 cm diameter. The disks were removed at random
from random leaves to ensure well-representative results for each
plant. The disks were weighed, then placed into a Warburg reaction
vessel with 500 .mu.l distilled H.sub.2O. 300 .mu.l 12% KOH was
added to the centre well along with folded filter paper to enlarge
the absorption area for CO.sub.2 from the inter vessel atmosphere.
KOH absorbs CO.sub.2 to form bicarbonate and ensures that only the
amount of O.sub.2 consumption and synthesis is measured. Each
vessel was attached to the apparatus and left to equilibrate in the
dark for the required period. Equilibration took place while the
machine was oscillating at 25.degree. C. in a water bath. After
equilibration the atmospheric and manomertric valves were closed to
ensure an air tight system. Readings (R) were taken at
pre-determined time intervals: R.sub.1 is the manometer reading
difference between 10 and 20 minutes in the dark. P&R is the
manometric reading difference between 40 and 50 minutes in the
light. R.sub.2 is the manometric reading difference between 65 and
75 minutes in the dark. The manometric readings correspond with a
change in gas volume, which equals the amount of O.sub.2 consumed
and synthesized. The rate of respiration and photosynthesis is
obtained by: the following formulas:
Respiration : l O 2 conserved = O 2 R 1 Minutes R 1 + O 2 R 2
Minutes R 2 / 2 ##EQU00003## Photosynthesis : ##EQU00003.2## l O 2
produced = O 2 P & R Minutes P & R + O 2 R 1 Minutes R 1 +
O 2 R 2 ? Minutes R 2 / 2 ##EQU00003.3## ? indicates text missing
or illegible when filed ##EQU00003.4##
[0274] The rate of .mu.l O.sub.2/minute was converted to:
.mu.l O.sub.2/h/g Fm.fwdarw.(.DELTA..mu.l/min.times.60 minutes)/g
Fresh mass
[0275] The gas exchange values were corrected according to the
method of Gregory and Purvis (1965) using the following
equation:
X = .DELTA. Vg .times. ( T ' ) ( Pb - 3 - Pw ) ( T + 273 ) ( P ' )
##EQU00004##
[0276] Where:
[0277] X=Total volume of gas measured (mm.sup.3) at standard
temperature and pressure (STP)
.DELTA.Vg=Volume change on respirometer T'=Standard temperature,
273.degree. K T=Temperature of warm bath, 25.degree. C.
Pb=Prevailing atmospheric pressure, mm Hg Pw=Vapor pressure of
water at the prevailing temperature at which the experiment was
conducted
[0278] P'=Standard pressure, 760 mm Hg
If : 1 l volume .times. 273 [ 645 mm Hg ( BFN ) - 3 - 23.756 ] ( 25
.degree. C . + 273 ) ( 760 ) = 273 ( 618.244 ) ( 298 ) ( 760 ) =
0.745234 l at 25 .degree. C . ##EQU00005##
[0279] Thus 1 .mu.l=0.745234 .mu.l real volume in Bloemfontein
(BFN).
[0280] [O.sub.2] in atmosphere=.+-.21% [0281] 1 mol O.sub.2=22.414
dm.sup.3 (liter)=22.414 liters (dm.sup.3)=1 mol O.sub.2
[0282] If: 1 liter=0.0446149 mol O.sub.2
[0283] At sea level 1 .mu.l=0.0446149 .mu.mol O.sub.2
[0284] At BFN: 1 .mu.l=0.745234 .mu.l=0.0332485 .mu.mol O.sub.2
[0285] To convert .mu.l O.sub.2 to .mu.mol O.sub.2: [0286] .mu.l
O.sub.2/h/g Fm.fwdarw..DELTA. .mu.l .mu.l O.sub.2/h/g
Fm.times.0.0332485 .mu.mol O.sub.2
[0287] Respiration and photosynthetic rates were determined every
week and by applying the above mentioned formula, the values are
corrected to compensate for difference in air pressures at sea
level or at higher altitudes. The respiration and photosynthesis
rates as well as the photosynthesis: respiration ratios were
relatively constant and comparable over the 13 week period of this
trial. However, when the respiration rate is corrected for the
protein content, enhancement of the respiration rate are found in
the Elementol treated plants.
[0288] The respiration and photosynthesis rate were measured and
placed in correlation with each other. Photosynthesis rate must
always exceed the respiration rate because the gain of carbon must
exceed the usage of carbon or else there will be a net loss of
carbons. The higher the photosynthesis: respiration ratio, the
better the growth rate, as there is a higher net profit of carbon
when ratios are high. The ratio was relatively constant over the 13
weeks of the trial.
[0289] Photosynthesis, like respiration, shows a "U" shape; when
the lettuce was planted the plants were very green and had a high
chlorophyll content. The rate of both photosynthesis and
respiration was high during the initial growth period as the high
metabolism of young plants also requires a high photosynthesis rate
to supply the plant with adequate amounts of sugars which is
respired. Photosynthesis and respiration then decreased after which
photosynthesis rate increased again. Photosynthesis rate must
always exceed than respiration rate to supply the plant with enough
sugars for primary metabolism and to supply the plant with sugars
during secondary metabolism as well as to store additional
compounds for later usage. The photosynthesis rate increased during
the last few weeks to accompany the rise in respiration rate. A
higher photosynthesis is also due to more chlorophyll present in
the last few weeks. Higher chlorophyll content results in better
photosynthesis ability.
[0290] The respiration rate of the Elementol treated plants is
generally slightly higher than that of the controls, but the
differences are not statistically significant, except in week 5
directly after the second Elementol treatment (FIG. 18).
3.3 Chlorophyll Content
[0291] The synthesis of new living material requires an input of
energy which is obtained from the sun through the process of
photosynthesis. Chlorophyll is an essential component in
photosynthesis. Chlorophyll is the main light absorbing pigment.
Chlorophyll molecules are specifically arranged in and around
pigment protein complexes called photosystems, which are embedded
in the thylakoid membranes of chloroplasts. A few different forms
of chlorophyll occur naturally, including chlorophyll a,
chlorophyll b. Protecting pigments are also formed by many plants.
Some of these accessory pigments, particularly the carotenoids,
serve to absorb and dissipate excess light energy, or work as
antioxidants. Other pigments such as caretenoids play a role in
light absorption at different wavelengths.
[0292] The overall reaction of photosynthesis is shown in the
following equation (producing one hexose sugar) (Stern, 2003).
6CO.sub.2+12H.sub.2O+light.fwdarw..sub.Chlorophyll.fwdarw.C.sub.6H.sub.1-
2O.sub.6+6O.sub.2
[0293] During photosynthesis two light reactions are involved which
include Photosystem I (PS I) and Photosystem II (PS II). These
harvest light at different wavelengths for maximum efficiency.
These two systems have to work co-operatively in order to be
efficient. Systems can by light dependent or light independent. A
major reaction during photosynthesis involves the transport of
electrons from water to NADP, possibly through the mechanism known
as the Z scheme. The rate of photosynthesis can be measured by
determining the amount of carbon dioxide consumed or amount of
oxygen released by using manometric techniques. Different types of
photosynthesis occur and are termed O.sub.3 photosynthesis (most
plants), C.sub.4 photosynthesis, most grasses, and CAM
(Crassulacean Acid Metabolism) photosynthesis, which occur in most
of the succulent plants. Factors influencing photosynthesis include
light intensity and amount, availability of water, adaptation to
sun and shady areas, availability of CO.sub.2, temperature, leaf
age, and carbohydrate translocation.
[0294] Chlorophyll content was determined weekly by using the
extraction method of MacKinney (1941) by cutting 10 equal size
disks at random from random leaves of the plant. The disks were
grinded in 80% acetone in a mortar with a pestle on ice and the
homogenate were centrifuged in a cooled bench centrifuge for 10
minutes at 12 000 rpm. The supernatant was diluted 5.times.. The
absorbance values of each dilution were determined by using a Pye
unicam SP8-400 uv/vis spectrophotometer. Absorbance values were
measured at 663 nm as well as 645 nm in a 1 cm glass cuvette.
[0295] The concentrations of Chlorophylls were determined as
follows (MacKinney (1941)):
Chlorophyll
a(mg/g)=[12.7(A663)-2.69(A645).times.(V/(1000.times.W))]
Chlorophyll
b(mg/g)=[22.9(A645)-4.68(A663).times.(V/(1000.times.W))]
[0296] Where: [0297] A=Absorbency of the dilution at the given
wavelength [0298] V=Final volume of extract [0299] W=Fresh mass of
disks used
[0300] When a comparison is undertaken between the amount of
chlorophyll in the experimental and control plants, one should
correct for the amount of protein and fresh mass, as these has been
shown to differ between the two groups.
[0301] The Elementol R treated plants show an average increase in
both chlorophyll a and b when compared to the control plants (FIG.
19).
[0302] Interestingly, the enhancement in especially chlorophyll a
but to some extent also in chlorophyll b reflects a similar
enhancement in Elementol-treated plants as that observed in plant
height, number of leaves and amount of protein. An average
enhancement of 14% and 20% over the total study period was observed
for chlorophyll a and b respectively, while an average enhancement
of 42% and 34% was observed during the last 4 weeks (week 9 to 13)
of the study for chlorophyll a and b respectively. The combined
results strongly suggest that the increase in chlorophyll content
caused by Elementol treatment is directly responsible for the
bio-stimulatory effect of Elementol R.
[0303] Despite the difference in relative enhancement of
chlorophyll A and B, a comparison between the corrected chlorophyll
a to b ratios in the Elementol-treated and control plants showed no
difference (see FIG. 20 which is a graph that reflects the
chlorophyll A:B ratios obtained from the chlorophyll corrected for
mg of protein and fresh mass. The nearly identical curves confirm
the absence of any phytotoxic effect on the photosynthesis
apparatuses of the plants.).
3.4 Sugars Content
[0304] The amount of sugar present is a direct result of the amount
of nutrients available. Increasing the N and P rates gradually
increased glucose content in lettuce but decreased the shelf life
(www.ars.usda.gov). The respiration rate as well as photosynthesis
rate has an effect on the amount of available sugars. The UV method
of Boehringer Mannheim (Kit nr. 10 716 260 035) was used to
determine sucrose, fructose and glucose concentrations present in
lettuce leaves. Sucrose is present in much higher concentrations
than glucose. A statistically significant but small increase in the
amount of sucrose was found in control plants compared to Elementol
R treated plants. Glucose on the other hand was slightly higher in
the treated than in the control plants.
3.5 Brix
[0305] Plant phloem sap contains many substances which supply the
plant with energy. One of the terms used in reference to quality is
called Brix index and this concept was introduced by a 19.sup.th
century German chemist, A.F.W. Brix. The Brix value is a measure of
the percent soluble solids content (SSC) in a solution. Although
Brix is often expressed as the percentage of sucrose, it is
important to realise that the "sucrose" here is actually a
summation of sucrose, fructose, vitamins, amino acids, protein,
hormones and other solids (www1.agric.gov.ab.ca). The main storage
form of carbohydrates in plants, namely starch, is insoluble and
therefore does not contribute directly to the Brix value.
[0306] Each degree of Brix is equivalent to 1 gram of sugar and
other SSC per 100 grams of juice. Generally, the higher the Brix,
the higher sugar content, especially increased sucrose and glucose
levels (Baxter et al., 2005) and this normally results in better
taste (Baxter et al., 2005; www1.agric.gov.ab.ca). High Brix, high
EC and low pH are generally associated with high fruit quality
(www.cals.ncsu.edu).
[0307] When a crop is cultivated under favourable conditions, such
as hydroponic systems where there is unlimited supply of minerals
and other required nutrients, sufficient sunlight and temperature,
a higher Brix in the plants can be expected in those produce
(www1.agric.gov.ab.ca). Bisogni et al. (1976) found correlation
between SSC and sweetness, flavour and overall quality. Winsor
(1966) reported that the best quality of fruit were those high in
both sugars and organic acids. (www1.agric.gov.ab.ca).
[0308] Brix equals the % dissolved solids in the phloem sap. A high
Brix sap has a reduced water activity, with a corresponding
reduction in freezing point, as well as a proportionally greater
tendency to retain moisture.
[0309] Produce with higher Brix also have a longer shelf life, and
are more resistant to pest infestation and disease. While
temperature, pH, etc can influence if and how fast organisms will
grow, water activity may be the most important factor.
[0310] Water Activity is thus a critical factor in determining
shelf life as well as field success. Brix sap levels in excess of
12% also generally ensure against sap-sucking insect
infestations.
[0311] Most importantly, high Brix provides proportionally greater
nutritional content of the food and ensures good, true
nature-ripened flavour, especially where the refractometer shows a
diffuse or spread reading, indicating a variety of complex
dissolved plant proteins and flavour components in good
measure.
[0312] Brix is often used to determine the quality of some selected
foods. Brix readings are readings of all dissolved substances
present in the lettuce leaf and not only the sugar or sucrose
content. Brix is in fact used to determine quality of lettuce
[0313] The Brix refractometer was calibrated at room temperature
using a 10% sucrose solution with a Brix reading of 1.3475.
Neutralized HClO.sub.4 was used as standard. The reading was
subtracted from the Brix reading as well as the % sugars. After
calibration a sample was placed in the refractometer and the Brix
readings were taken in Brix readings as well as % sugar.
[0314] Another method was used to determine the Brix reading.
.+-.0.1 grams of fresh mass were grounded in 200 .mu.l water (Thus
the sample was diluted 4.times.) and 20 .mu.l of sample was placed
on the refractometer and the Brix readings were taken.
[0315] Despite the lower sucrose content, the Brix values indicate
a better quality lettuce obtained from the Elementol treated
plants. Since Brix reflects the insolubles in the lettuce, the
Elementol-treated lettuces are enriched in plant material other
than sucrose. The % enhancement in Brix by Elementol treatment
obtained with the HClO.sub.4 method was 15% and that with the water
method 12%. The 3% difference obtained with these two methods
should be the due to a higher presence of organic acids, hormones
or oil-based vitamins, as those are soluble in HClO.sub.4.
Example 17
The Biostimulatory Effect of Elementol R Administration on The
Yield and Quality of Fruit in a Controlled Environment
1. Material, Plant Growth and Treatment
[0316] Cultivar: Tomato Lycopersicon esculentum Mill of the family:
Solanaceae cv.
Seedlings: Floradade seedlings, approximately six to eight weeks
old, were purchased from a local nursery in Bloemfontein. Twelve of
these seedlings were transplanted to the prepared hydroponic system
in the glasshouse. This glasshouse was situated on the roof of the
Plant Science building of the University of the Free State.
1.1. Culturing Method:
[0317] Two identical recycling ebb and flow hydroponic systems were
set up. Each system consisted of 2 rectangular asbestos trays (90
cm.times.20 cm), filled with the support medium which consisted of
disinfected, medium size, silica gravel. Three seedlings per tray
were transplanted .+-.30 cm apart and rows .+-.42 cm apart. This
spacing allows .+-.0.135 cm.sup.2 per plant, resulting in 9
plants/1.22 m.sup.2
[0318] In order to limit algae and bacterial growth, black
non-translucent PVC piping, fittings and reservoirs were used to
construct the recycling systems. Each system had a separate 70
litre reservoir, with a small water pump inside. Both these pumps
were connected to a single digital timer, which regulated the
intervals of watering cycles. The watering time was synchronized in
order that the trays were filled up to a specific level, where
after the timer switches off, and the water drained into the
reservoir. The plants were flooded six times a day for 5 minutes,
ranging from 06:00 to 18:00.
1.2 Greenhouse Conditions
[0319] The temperature in the greenhouse was partially controlled
by an air conditioner. Average night and day temperatures ranged
from 16.degree. C. to 25.degree. C., respectively. Three
instruments, namely a thermometer, thermohygrograph and a swirl
hygrometer, were used to determine the temperature. The thermometer
was mounted on the eastern wall (facing north). The
thermohygrograph was placed strategically inside the greenhouse to
provide a 24 h record of the greenhouse conditions from Monday to
Friday. The thermohygrograph provide an indication of both the
temperature as well as the relative humidity. The light intensity
of three different locations was measured with an LI-185A model
photometer on a height of 2 m from floor level. Light intensity
varies considerably with latitude and time of the year. This is a
result of the inclination of the earth and rotation around the sun.
Mid-day light intensity (LI) decreased as the winter months
approached, followed by an increase from the 14.sup.th week after
transplant (WAT) until termination in the 25.sup.th WAT.
[0320] The temperature, relative humidity and the irradiance
intensity were measured following the same procedure as the weekly
measurements. The readings were taken every two hours from 8:00 to
16:00 for one day during May and July. The relative humidity (RH)
is the ratio between the weight of moisture actually present in the
air and the total moisture-holding capacity of a unit volume of air
at a specific temperature and pressure (Smith & Bartok, 2006).
The mid-day RH initially increased to 82%, but from the 18.sup.th
week after transplantation, a drop to as low as 50% is noticed
(24.sup.th WA). RH is temperature dependant, seeing that warm air
has a higher moisture-holding capacity than cooler air; therefore
as the temperature of air increases, the relative humidity
decreases even though the amount of water remains constant.
However, in this case the temperature remains relatively constant;
therefore the drop in RH might be a result of vigorous growth of
the plants, resulting in dense and high transpiration until
commencement of the harvesting period. The growing vigor and
transpiration rate ceases naturally as the harvesting period comes
to an end.
1.2. Nutrient Solution
[0321] The nutrient solution applied, namely Hygrotech Hygroponic,
is an optimized mixture of nutrients specifically developed for
hydroponic tomato production. This mixture initially consisted of
Hygroponic Mix and calcium nitrate. Potassium nitrate was added
from third flower truss to the end the trial. The combination of
the prescribed concentration of each component was dissolved in tap
water.
[0322] The reservoirs were filled with 70 litres of nutrient
solution and replenished as necessary. Every alternating week,
before refilling, the reservoirs were flushed with clean tap water
to dispose with any harmful substances that might have accumulated.
The pH and EC of the nutrient solution in each reservoir were
measured before and after refilling the reservoirs, using a PHM 85
Precision pH meter and a PW 9526 digital conductivity meter
respectively.
2.1.3. Treatments
[0323] During the second WAT, the plants were raked up with black
nylon twine in order to support the plants. During the 2.sup.nd
week, the first of six applications of applicable treatments were
applied. The treatments are summarized below:
TABLE-US-00006 Treatment Abbreviation Treatment composition Control
C no application Elementol R P 3 ml Elementol R/250 ml H2O
(2xdist)
[0324] The plants were specifically arranged in an effort to have
both sun and shade plants for each treatment. The only
differentiation between plants was therefore the particular foliar
treatment.
2. Physical Parameters: Growth, Development and Yield of Plants
2.1 Plant Height
[0325] The height of each plant (from the level of gravel to
highest tip) was determined with a measuring tape. As soon as the
plants reached the roof and the weight of the plant pulled the
plants down, this procedure were ended.
[0326] Plants of both treatments showed a linear increase in
height, with an average height for both the treated and control
plants ranging between 130 and 160 cm in week 10 after
transplantation.
2.2 Regenerative Development
[0327] The impact of Elementol R on the yield of plants was
evaluated firstly by counting the number of flower buds on the
plants. The development and growth of plants are directly related
to the formation of flower buds, flowers and fruit. Flower buds
were recorded as soon as a clearly distinguishable flower bud
appears, and flowers when a definite yellow colour is apparent. The
first flower buds appeared three weeks after transplant to reach an
average of approximately 25 buds for Control (C) plants at 7 weeks
after transplantation.
[0328] Although Elementol R (Er) treatments had no statistically
significant effect on plant height, treatment with Elementol R
resulted in a statistically significant increase in average number
of flower buds, especially between 5.sup.th and 7.sup.th week after
transplant (FIG. 21).
[0329] Compared to Control plants, the Elementol R treatment
stimulated bud formation significantly as from week 6. The %
enhancement was calculated according to the formula described in
Example 16, with an enhancement of 92% recorded, with an average
enhancement in flower buds of 44% from week 4, when clearly
distinguishable flower buds could be counted, to week 7 (table 1
below and FIG. 22).
TABLE-US-00007 TABLE 1 Average flower buds % WAT Er C enhancement 4
16.5 13 26.92308 5 21 16.5 27.27273 6 30 23 30.43478 7 48 25 92
Average % enhancement week 44.15765 4-7
[0330] To prevent damage to developing plants, and impracticality
of bud counting in densely populated hydroponics setup, it was
decided to terminate this procedure 7 weeks after transplant.
2.3 Yield
[0331] The contribution of Elementol R to yield could not be
determined in Example 16, where leaf and plant growth were the
relevant parameters. In the case of the tomato plants however, an
enhancement in flower buds should reflect an enhancement in the
yield of plants, if the nutrition given to the plants
hydroponically is sufficient. The fruit was therefore counted.
Fruit needed to reach 5 mm in diameter before its appearance was
recorded. The average accumulative yield of fruit during the study
period is recorded in table 2 (see also FIG. 23).
TABLE-US-00008 TABLE 2 Average accumulative yield (total n) WOH
Control E 1 0.0 0.0 2 1.5 6.0 3 13.5 12.0 4* 39.0 49.5 5* 49.5 63.0
6* 51.0 63.0 7* 64.5 96.0 8* 72.0 97.5 9* 81.0 114.0 10* 88.5 126.0
11 105.0 142.5 12 121.5 157.5 13 123.0 178.5
[0332] The weekly increase in yield for both the control and
treated plants is linear from week 3, with a lag phase from
transplantation to week 3. The Fisher t-test (1 tailed), which
returns the probability associated with a Student's t-Test and
determines whether two samples are likely to have come from the
same two underlying populations, was used to analyze the yield
data. The probability value was determined as 0.000261, meaning
that the probability that the yield series obtained for the
Elementol R treated fruit and control fruit is the same is less
than 1 in a 1000.
[0333] The average enhancement in yield calculated over the period
of the study, excluding week 1, again using the formula described
in example 1, was 53.7%.
[0334] The average accumulative yield per plant was calculated. As
expected, the % enhancement in fruit yield per plant was exactly
equal to that obtained for total accumulative yield (53.7%).
[0335] A calculation of the fruit to bud ratios for both groups
(table 3) show a progressive but similar decrease over the first 7
weeks, after which bud counting was terminated. In week 7, only 26
or 26 fruit are grown from every 100 buds (see FIG. 24). Thus is
probably due to insufficient nutrition for both groups in view of
the high yields obtained, despite the use of a nutrient mix
optimized for hydroponically grown tomatoes. The higher the yield,
the greater would be the impact of insufficient nutrition.
Therefore a greater enhancement in yield of Elementol R treated
plants compared to control plants could probably have been obtained
if the nutrition were to have been adjusted to the increased
yield.
2.4. Physical Parameters of Fruit
2.4.1. Moisture Content
[0336] Both total fruit yield and soluble solids content plays and
important role in the economic success in the processed tomatoes
market. For choice of tomatoes for processing purposes, specific
attention is paid to biochemical quality. Fruit with high soluble
solids content, for example, contain less water and are sweeter and
consequently require less processing and addition of sugar to
prepare pastes of proper texture (Baxter et al., 2005). In
addition, a number of organoleptic and nutritional parameters are
be used to define fruit quality. These quality parameters include
sugars, titratable acidity (TA), electrical conductivity (EC),
vitamin C and phenolic compound content, soluble solid content
(SSC) and firmness, to name but a few (Anza, Riga & Garbisu,
2006).
[0337] The average moisture content would thus give an indication
as to the quality of the tomato. To determine the moisture content,
a slice of each representative tomato fruit was placed in a Petri
dish (of which the weight was pre-determined) and weighed by means
of a Sauter RL 200 microscale. It was then placed into a labotech
oven at .+-.68.degree. C. for 7 days. After the dehydration period,
the Petri dish containing the tomato slice was weighed again. The
loss in weight represents the amount moisture present in the
tomato. On average, the Elementol R treated fruit contains slightly
less moisture than the control group although the difference is not
statistically significant (see FIG. 25 which shows the average % of
moisture found in the fruit of Elementol R treated tomato plants
versus control plants as described in Example 17. Elementol R
treated fruit generally had a lower moisture content relative to
total tomato mass, indicating a fruit with more insolubles, such as
sugars and protein, resulting in tomatoes of higher quality.)
[0338] The average % enhancement of dry mass (Dm) of Elementol
treated fruit is -1.05% over the study period, indicating that no
difference exist between the treated and control plants. However,
the comparative dry mass has a wide distribution. The T-test of
probability that the two ranges originated from the same group
(i.e. similarity) was calculated as 0.330525. A reverse pattern is
observed when the moisture mass: Dm ratios are compared. This may
indicate that the procedure used for this determination is not
accurate. A possible cause is that the organic acid and oil content
of the fruit is not taken into account.
3. Biochemical Parameters of Fruit
3.1. Electrical Conductivity (EC) and pH
[0339] Every second week, 15 fruit, representative of each
treatment, were objectively selected. A part of the fruit was
ground up in a test tube using a Polytron Homogenizer. The pH and
EC of the tissue were determined, by means of a PHM 85 Precision pH
meter and the PW 9526 digital conductivity meter, respectively.
[0340] A greater flow in electrical current implies a higher
concentration of dissolved ions in the fruit. Both total fruit
yield and soluble solids content plays and important role in the
economic success in the processed tomatoes market. For choice of
tomatoes for processing purposes, specific attention is paid to
biochemical quality. Fruit with high soluble solids content, for
example, contain less water and are sweeter and consequently
require less processing and addition of sugar to prepare pastes of
proper texture (Baxter et al., 2005).
[0341] The EC of the fruit showed a progressive increase. The
average EC determined for control plants over the study period was
3.395, while that for the Elementol R treated plants was 3.393. An
inverse relationship, although it be with a very moderate slope,
are evident when the relation between pH and EC values of the fruit
are compared.
[0342] The average pH of the control fruit for the period of the
study was determined to be 4.245, while a pH of 4.248 was found for
the fruit of the Elementol R treated plants. Therefore, despite the
greatly enhanced yield of the treated plants, no difference in the
quality of the fruit in terms of moisture, dry mass, EC or pH. The
close correlation in values also indicates the accuracy of the
measurements.
3.2. Carbohydrates
[0343] The fruit quality and yield of tomatoes are largely
determined by one of the biochemical components of fruit quality,
namely the amount of soluble sugar content (Damon et al., 1988;
Islam et al., 1996). The glucose and fructose concentrations in the
apoplast are present in a ratio of approximately 1:1 (Damon et al.,
1988), with the hexose concentrations at least four times greater
than the sucrose at all stages of development. Guan and Janes
(1991) found that sucrose levels are relatively low in tomato
fruit, are independent of light intensity and that it continues to
decline during development. The sucrose content of light- and
dark-grown fruit in their studies did not shown any significant
differences. The accumulation of carbohydrates may therefore be
driven by the metabolism of sucrose.
[0344] Preparation of samples for assaying the carbohydrate content
of the harvested tomatoes: Samples were prepared by adding 10 g of
representative fruit tissue to 5 ml twice distilled water in a test
tube. This mixture was homogenised for .+-.30 seconds with a
Polytron Homogeniser. The remaining material on the side of the
test tube was rinsed into the test tube with an additional 2 ml of
twice distilled H.sub.2O. The test tube was shaken for 30 minutes,
followed by vigorous Vortexing, and then quickly poured into a
small measuring cup. While the puree was being stirred on an
electronic stirrer, the pH was adjusted to .+-.8.00 by using 1M and
5M KOH, where after the solution (.+-.13-17 ml) was made up to a
final volume of 20 ml. An aliquot (.+-.1.5 ml in microfuge tubes)
of the solution was centrifuged at 12 000 rpm for 10 minutes. The
supernatant was collected with a Pasteur pipette and transferred to
a clean tube. Assay samples were stored at -20.degree. C. until
final analysis.
[0345] To determine the sugar content of the fruit, the
Sucrose/D-Glucose/D-Fructose--kit (10 716 260 035), manufactured by
Boehringer Mannheim/R--Biopharm was used. The prescribed procedure
was adapted to 1 ml volumes. Dilution factors were taken into
account when calculating the carbohydrate content.
[0346] Table 3 shows the comparative glucose, fructose and sucrose
content for the harvested fruit in week 13 of the study.
TABLE-US-00009 TABLE 3 Comparative sugar content mg/Fm Elementol R
Control Glucose 13.73 13.52 Fructose 14.45 13.32 Sucrose 30.11
28.04
[0347] The Elementol R-treated tomatoes showed a considerable
increase in fructose and sucrose content, resulting in sweeter
tomatoes, which are preferred by the consumer.
3.3 Brix
[0348] The Brix value is an indication of the percent total soluble
solids (TSS) in the fruit juice. Every second week, the Brix value
of the same puree of the 15 representative fruit used for pH and
EC, were determined. The procedure of grounding up a part of the
fruit in a test tube using a Polytron Homogenizer, are therefore
exactly the same as for determination of pH and EC of the fruit.
The puree container was then slightly tilted in order to collect a
clear juice sample with a pasteur pipette. The Brix value was
determined by means of a refractometer. High Brix, high EC and low
pH are associated with high quality (www.cals.ncsu.edu). Despite
the fact that no statistical difference between control tomatoes
and Elementol treated fruit was observed with regards to EC and low
pH or moisture content of the fruit observed during the 13.sup.th
week of harvest, fruit from Elementol treated plants with an
average Brix value of 8% outperformed the control plant, that had
an average Brix value of 7.4%. Both of the groups had a
significantly higher Brix value than the average published value
for tomato.
[0349] In conclusion, Elementol R treatment enhanced both the yield
of tomatoes as well as the quality of the harvested fruit in terms
of % moisture, insolubles and sugars.
Example 18
Enhancement of Uptake and Translocation of a Commercial
Bio-Stimulant by Means of Elementol R
[0350] 1. The Aim of this Study
[0351] The previous two examples showed that Elementol R on its own
can act as a bio-stimulant in terms of plant growth and yield. This
study investigates whether the pre-entrapment of a commercial
bio-stimulant, ComCat.RTM., into Elementol R can enhance the uptake
and translocation of this bio-stimulant, resulting in an increase
in plant growth and yield beyond that observed with Elementol R or
the known slight effect of ComCat.RTM., on hydroponically grown
lettuce and tomatoes.
2. Experimental Set-Up:
[0352] The experimental set-up was similar to that described in
Example 16 and 17, except that the bio-stimulant (alone and in
combination with Elementol R) was administered. The study was
executed in a similar fashion to those described in Examples 16 and
17 and will not be described again.
2.1 The Commercial Biostimulant ComCat.RTM.
[0353] ComCat.RTM., an eco-friendly plant strengthening agent,
contains one of a group of phytohormones, called brassinosteriods
(Schnabl, et al., 2001). Brassinosteroids is a growth-promoting
steroid found in higher plants. Brassinosteroids are thought to act
at low concentrations to affect the growth of plants, by enhancing
the elongation of stems and regulating gene expression in plants.
Improved seedling development, strong roots and shoots, optimum
flower development have been observed with the use ComCat.RTM..
Brassinosteroids, as pure phytohormones, have been reported to not
only increase crop yields but also crop quality (Prusakova et al.,
1999). ComCat.RTM. contains high-quality, biochemical active
substances which have been extracted from synecologically active
wild plants.
[0354] Due to interference from cultivators most cultivated plants
have lost access to defend themselves against pathogens.
ComCat.RTM. increases the resistance of plants to all types of
stress and pathogens. Brassinosteroids play a decisive part in
activating the plant's own resistance and tolerance mechanisms.
ComCat.RTM. is the first of its kind to have succeeded in
catalyzing this activation of the plant's own ability of defense in
an optimum way. Plants develop induced resistance that increases
the plant's ability to resist pathogens.
[0355] This bio-stimulant is a water-soluble powder, and when
applied to crops as a foliar spray or a seed treatment, it
increases root development, accelerates nutrient absorption,
intensifies nutrient assimilation, induces flower bud formation,
increases yields (Huster, 1999, Schnabl et al., 2001, Pretorius
quoted by Alam, 2004) and induces the natural resistance of plants
against pathogens and biotic stress (Agra Forum as quoted by Alam,
2004; Huster, 1999; Schnabl et al., 2001). Khripach et al. (2000)
also claimed that this newly discovered phytohormone has the
ability to regulate the uptake of ions into the plant cell.
2.2 Foliar Administration Schedule
2.2.1 Lettuce
[0356] The treatments for the different groups of plants were
prepared as follows:
[0357] According to ComCat.RTM. dosage directions: ComCat.RTM.=2
g/L [0358] Thus:=0.5 g/250 ml
i) ComCat.RTM. (CC)
0.5 g CC+250 ml H.sub.2O
ii) Elementol R (E)
3 ml E+250 ml H.sub.2O
[0359] iii) Full strength ComCat.RTM. and Elementol combination
(CC/E)
0.5 g CC+3 ml E+250 ml H.sub.2O
[0360] iv) Half strength ComCat.RTM. and Elementol combination (1/2
CC/E)
0.25 g CC+3 ml E+250 ml H.sub.2O
[0361] v) Quarter strength ComCat.RTM. and Elementol combination
(1/4 CC/E)
0.125 g CC+3 ml E+250 ml H.sub.2O
2.2.2. Tomatoes
TABLE-US-00010 [0362] Treatment name Abbreviation Treatment
composition Elementol R PE 3 ml Elementol R/250 ml H.sub.2O
(2xdist) ComCat CC 0.5 g Comcat/250 ml H.sub.2O (2xdist) ComCat
& CC/E 0.5 g Comcat + 3 ml Elementol R/250 ml Elementol
H.sub.2O (2xdist) 0.5 Comcat & 0.5 CC/E 0.25 g Comcat + 3 ml
Elementol R/250 ml Elementol R H.sub.2O (2xdist)
3. Results
3.1 Growth and Development and Head Diameter
3.1.1. Lettuce
[0363] Pre-entrapment of CC in E did not greatly influence plant
head diameter of plant height. Some of the plants did not increase
100% which means that they did not double in size. Some plants that
were treated with CC and E individually performed the best of the
treated plants but differences were not statistically significant,
except from week 11 onwards, when Elementol R treated plants
outperformed all other treatments. Some of these combinations may
have an inhibitory effect on the plants, whereas E and CC
individually both had a stimulatory effect.
[0364] The plants reached a maximal head diameter during the first
7 to 8 weeks, after which the head diameter decreases, probably
because the plants were constantly pruned to obtain leaf material
to do physiological experiments.
3.1.2 Tomatoes
[0365] ComCat.RTM. application resulted in a slightly reduced
growth rate. However, when ComCat.RTM. is applied together with
Elementol of either concentration (CC/E and 0.5 CC/E), this
reduction in vegetative growth is alleviated in a dose-dependent
fashion, but growth is still significantly below that of Elementol
R alone.
3.2 Average Flower Buds of Tomatoes
[0366] Elementol R alone, as well as ComCat.RTM. (CC), and
combination treatments showed a marked increase in flower buds,
especially between 5.sup.th and 7.sup.th week after transplant. No
clear difference was measured between these treatments, although CC
showed the least increase.
3.3 Average Tomato Yield
[0367] No clear differences were observable for fruit size and mass
between all treatments. ComCat.RTM. (CC) application failed, as
bio-stimulant, to enhance both fruit size and mass in
hydroponically grown tomatoes. Full strength ComCom.RTM. with
Elementol R application had no effect on changes in fruit size and
mass, but CC/E combination application resulted in higher fruit
size and individual fruit diameter and fresh mass (see 3.2.2
below). This suggests that this low ComCat.RTM./Elementol
concentration decelerate the decrease in fruit mass observed for
the whole harvesting period which implies better physical yield for
harvesting period. The table below reflects the average
yield/plant:
TABLE-US-00011 Average no of fruit/plant Control E CC CC/E Avg Avg
Avg Avg 0.0 0.0 0.0 0.0 0.5 2.0 0.5 1.7 4.5 4.0 2.0 5.0 13.0 16.5
6.5 15.3 16.5 21.0 12.5 27.0 17.0 21.0 12.5 28.7 21.5 32.0 23.5
39.3 24.0 32.5 24.5 43.7 27.0 38.0 29.0 55.0 29.5 42.0 30.0 59.7
35.0 47.5 33.0 64.7 40.5 52.5 35.0 73.3 41.0 59.5 37.0 78.0
[0368] Elementol R stimulated the yield of tomatoes significantly
(Example 17). However, when ComCat.RTM. is mixed with Pheroids,
both in full (CC/E) and half (0.5 CC/E) strength markedly
stimulated fruit production (See FIG. 26 which is a graph that
shows the effect of ComCat.RTM. (CC), Elementol R (E) and
combinations thereof on changes in accumulative number of fruit
harvested from 3 plants per group over a period of 13 weeks) and
subsequent mass of fruit harvested (see FIG. 27 which is a graph
that shows a dramatic increase in total accumulative fruit mass
observed when plants are treated with ComCat.RTM. that is entrapped
in Elementol R as compared to the increase observed with Elementol
R or ComCat.RTM. individually).
TABLE-US-00012 Yield in terms of total fruit mass (avg acc
mass/plant) WOH Control P CC CC/P 1 Avg Avg Avg Avg 2 0 0 0 0 3
61.0 156.2 90.5 115.5 4 459.1 315.3 250.1 518.5 5 1083.4 1093.9
639.9 1424.9 6 1329.9 1331.4 974.8 2137.5 7 1361.0 1331.4 974.8
2221.6 8 1608.7 1888.7 1669.7 2844.7 9 1758.4 1928.6 1704.9 3092.5
10 1925.9 2152.9 1977.7 3808.5 11 2072.0 2261.9 2014.1 4109.5 12
2337.1 2498.0 2121.4 4385.5 13 2562.9 2682.0 2260.5 4818.5 Average
2589.1 2908.5 2358.2 5041.2
[0369] The % enhancement in terms of yield was calculated as 99%
and 81% CC/E and 0.5 CC/E respectively and total harvested mass as
199% and 204% for CC/E and 0.5 CC/E respectively when compared with
that obtained with CC. The enhancement of 33% and 21% for CC/E and
0.5 CC/E respectively is far less when compared to Elementol, which
on its own caused an increase in fruit yield and mass (FIGS. 26 and
27). Elementol as novel carrier molecule was demonstrated to be an
efficient translocator of ComCat.RTM. molecules. It would also
indicate that Elementol R enhanced the uptake of ComCat.COPYRGT. to
exert its bio-stimulatory effect. A synergistic effect of these two
products may also come into play.
3.2 Moisture % and Fresh and Dry Mass (Fm:Dm) Ratios
3.2.1 Lettuce
[0370] All treatments had a stimulatory effect on the plant Fm:Dm
ratios.
3.2.2 Tomatoes
[0371] CC alone showed a higher average fresh fruit mass than E
alone. However, pre-entrapment of CC into E increased the average
fresh mass of the tomatoes still further (see FIG. 28). No
significant difference was observed between CC/E and 0.5 CC/E,
except for week 13 and as the standard deviation on Fm is quite
large, it may not be significant.
4. Physiological Related Parameters in Lettuce
[0372] 4.1 Protein Content: Measured One Week after Each
Treatment
[0373] Protein content was highest in week 2 and showed a decrease
over the 12 weeks of the trial for all treatments. From weeks 4 to
12 CC had on average the least amount of proteins. In the final
week all plants had relatively the same amount of proteins. The
CC/E combination had the best stimulatory effect on proteins.
4.2 Respiration Rate
[0374] All plant treatments showed relatively the same respiration
rate. In week 9 the CC/E treated plants had the best respiration
rate. Respiration rate decreases until week 9 except for CC/E
combination and increases again the last 4 weeks. All plant
treatments show this "U" shape, due to higher energy requirements
during early growth and flowering. The CC/E combination is the only
treatment to show an increase in respiration rate (FIG. 29). Thus
in week 9, the CC/E combination treatment had a stimulatory effect
on the plants. All treatments involving E had a higher respiration
rate during this week than CC alone.
[0375] When the respiration rate is expressed in terms of the
amount of protein a fluctuation is observed. The respiration per
amount of protein for the CC/E treated plants show an increase
every time after the plants had been treated (week 5 and week 9;
see FIG. 29). Thus the combination of E and CC stimulates
respiration rate per mg of protein. At the end of week 13 the E
plants had the highest respiration rate per mg protein, probably
because the Elementol R treated plants flowered before plants
treated with CC or combinations of CC and E, requiring a high
respiration rate to supply adequate amounts of energy for
flowering.
4.3 Photosynthesis Rate
[0376] Again during week 9 the photosynthesis rate for CC/E was
very high. In week 11 the photosynthesis rate dropped considerably
indicating that the stimulation caused by CC/E may be of short
duration. At the end of week 13 the 1/4 CC/P combination group
showed the highest photosynthesis indicating that the 1/4 CC/P
combination stimulates photosynthesis for longer. Expressing
photosynthesis rate in terms of the amount of protein present
results in roughly the same result as respiration per mg protein,
except that the 1/4 CC/P treated plants show the highest
photosynthesis rate at the end of week 13, indicating that this
treatment may have a longer lasting effect on photosynthesis rate
per mg protein.
[0377] Photosynthesis must always exceed respiration rate. The
higher the gain of photosynthesis on respiration, the higher the
accumulation of carbons, resulting in the synthesis of more sugars.
More sugars can be respired and thus the gain of energy is better.
This energy acts as "fuel" for metabolic pathways. Bigger ratios
result in better growth. Again the 1/4 CC/P combination shows an
increase in photosynthesis: respiration ratio from week 5 to week
13. This combination has the best ratio at the end of week 13.
4.4 Chlorophyll Content
[0378] Despite fluctuations an overall increase in chlorophyll a
can be seen. By placing the amount of chlorophyll a in correlation
with the amount of protein present in the plant shows the
following. The E treatment has the most chlorophyll a per mg of
protein for week 13, followed firstly by 1/4 CC/E, secondly by 1/2
CC/E, and thirdly by CC/E, then by CC. Thus the least amount of CC
in combination with E stimulates chlorophyll A the most (see FIG.
30 which is a graph that illustrates the comparative amounts of
chlorophyll B per mg of protein as determined in week 13 of the
trial.) CC had an inhibitory effect on the amount of chlorophyll B
and this inhibitory effect is enhanced by the entrapment of CC in
Elementol R vesicles. However, dilution of the CC concentration led
to an increase in chlorophyll B/mg protein. Thus the dosage of the
CC should be decreased when entrapped in Elementol R.
[0379] Chlorophyll B showed a similar pattern. In the case of
chlorophyll B, an overall increase is observed. In FIG. 32 the
amount of chlorophyll B per mg of protein is shown. Here the 1/4
CC/E combination and 1/2 CC/E combination also shows the best
chlorophyll B concentration per mg of protein. E also has a high
concentration of chlorophyll B per mg of protein. Thus lower
amounts of CC used with E stimulated both chlorophyll A and B
synthesis. CC inhibited chlorophyll B content, but the combination
of CC/E inhibited the amount of chlorophyll B dramatically,
illustrating that pre-entrapment in E enhanced the uptake and
translocation of CC. The dilution of CC by 75% seemed to have
negated the inhibitory effect of the CC. For this inhibitory effect
to take effect, the entrapment of the CC in E had to have resulted
in a dose-dependent uptake and translocation of the CC by E, as can
be observed in FIG. 30.
4.5 Sugar Content
[0380] Both glucose and sucrose content is stimulated by the
entrapment of CC in E. The sugar content of the plants are similar
for CC and E, but the combination of CC/E increased the sucrose
content by an average of 91% and that of glucose by an average of
64%. Again an increase of both sucrose and glucose concentration is
found as the strength of the ComCat.RTM. decreases.
4.6 Brix
[0381] In the table below the Brix measurements with HClO.sub.4 as
background is presented. Brix values measures all dissolved
substances present in the lettuce leaf and not only the sugar or
sucrose content. Brix is in fact used to determine quality of
lettuce. A high Brix reading indicates many dissolved substances as
well as many sugars which indicate a good quality and healthy leaf.
This may have contributed to low growth rates and poorly developed
plants.
Average Brix Readings for Treated Plants with HClO.sub.4 (See Also
FIG. 31)
TABLE-US-00013 Treatment Brix reading (%) E 4.4261 .+-. 0.2867 CC
4.7652 .+-. 0.3586 CC/E 6.6760 .+-. 0.5235
[0382] The enhancement in Brix readings by the combination is
indicative of the higher uptake and translocation of CC by the
Elementol carrier.
Example 19
In Vitro and In Vivo Effect of Elementol R on Seedling Growth
1. Aims of the Study
[0383] To investigate the effect of Elementol R on germination and
seedling growth in both C3 and C4 plants. In the process of
photosynthesis, CO.sub.2 and water are substrates and carbohydrates
and oxygen are the products (Jakob and Heber 1996). Plants are
classified as C3, C4 or CAM according to their mechanism of
photosynthesis. The C.sub.3 path involves the Calvin cycle, whereas
the C.sub.4 path uses a cycle where 3-phosphoglyceric acid is not
the first product. C.sub.4 photosynthesis provides a mechanism for
high rates of carbon assimilation and is more resistant to the
process of photo respiration.
[0384] The inherent effect of Elementol R on its own and mixed with
an antifungal (see maize field trials below) were investigated.
2. In Vitro Effect of Elementol R on Seedling Growth
[0385] The conditions in terms of humidity and temperature were
controlled as described in Examples 16 to 18. Three groups of
radish seed were treated as follow:
TABLE-US-00014 Elementol Elementol Group Control 125 250 Dosage 20
l Water/ha 125 ml/ 250 ml/20 l/ha 20 l/ha Abbreviation C E125
E250
[0386] Seeds were soaked in the above treatments overnight and then
exposed to germination paper. The effect of the different
treatments was measured with regards to its influence on radish
root length (see FIG. 32 which is a photograph of germinating
radishes on germination paper in the in vitro study described in
Example 19. The increased root length on both sides of the short
control seedlings is due to both faster germination and growth). An
enhancement in root length above control of 53.3 and 52.6% was
observed for Ep125 and 250 respectively.
3. In Vivo Effect of Elementol R on Seedling Growth in Glass House
Trials
[0387] The following study was done on wheat in glass house
trials:
Cultivar: Wheat Kariega
[0388] The growing conditions in terms of temperature and relative
humidity were relatively constant. Plants were planted in earth and
irrigated by drip irrigation.
[0389] The treatments consisted of two groups: a reference group
(RG) receiving fertilizer and a test (E) group receiving Elementol
R. Seeds of the reference group were planted with fertilizer
(3:1:0) according to supplier's instructions. Plants were treated
with Elementol R at the three leave stage with similar
concentrations than that described for the in vitro trial above,
but with 20 ml E/100 L/ha at both the flag leave and just before
flowering. Treatment was administered through foliar application.
The trial outlay consisted of a randomized block design and ran for
3 and half months.
[0390] The following parameters were investigated weekly:
Any signs of phytotoxicity, Differences in seedling size and height
[0391] Wheat coleoptile's average growth (mm)
TABLE-US-00015 [0391] Ep Ep Control Ep 125 250 500 22 24 27 28
[0392] The table above illustrates the early response in small
seedlings, but is representative of the general response. The
growth response varied proportionately with the amount of dose of
Elementol. The administration of Elementol R resulted in a linear
dose response in terms of wheat coleoptile growth (see FIG. 33
which is a graph that illustrates the comparative average length
measured for coleoptiles of wheat for the fertilizer control, and
the various dosages of Elementol R.) The standard deviation from
the linear dose response is exceptionally small, indicating a high
confidence level in the data. Such a linear dose response can be
used to indicate that a specific intervention on a biological
system results in a specific response. Thus the response in
coleoptile growth is specifically due to the administration of a
specific dose of Elementol R. FIG. 33 shows that the maximum dose
has not been reached and that further enhancement in growth may be
possible with a higher dose. The enhancement in growth, using a
dose of 500 ml/ha Elementol R was calculated to be 27.3%. No signs
of toxicity (leaf burn, necrosis etc.) were observed.
4. Field Trials
4.1 In Vivo Effect of Elementol R in Wheat Field Trials
[0393] The cultivar was PAN 3377. Wheat was cultivated according to
normal farming practices in the Central Free State, South
Africa.
[0394] As in the glass house trials, the two groups consisted of a
fertilizer control (3:2:1) and Elementol R at dosage of 500 ml/100
L water/ha). Treatment was limited to a single application at the
three leave stage. The trial outlay was a randomized block design.
The trial lasted 7 months.
[0395] The yield was determined and is presented in FIG. 34. An
average increase of 108 kg in yield per hectare was observed with
the Elementol R treated group as compared to the reference
fertilizer group. No phytotoxicity was observed.
4.2 In Vivo Effect of Elementol R in Pea Field Trials
[0396] Peas were cultivated according to normal farming practices
on the farm Koedoesfontein in the Northern Free State, South
Africa, with the following exception: 100 dry peas each were soaked
overnight in either 500 ml borehole water (control group) of 5%
Elementol R. The diluent was water from the same source. While peas
from the control group absorbed all water during soaking, peas from
the Elementol group absorbed only 300 ml of the 5% Elementol R.
Peas were planted in two separate blocks to prevent any possible
contamination between the two groups. The plants were irrigated by
daily sprinkling.
[0397] Germination and seedling growth was observed from day 7. On
day 10 a comparison was made of the number of seedlings that
measured at least 300 mm in height in each block. In the block
where the seeds were soaked in Elementol R, 57 seedlings were
counted on day 10, whereas 18 seedlings were present in the control
group. This represents an enhancement in germination and seedling
growth of 3.1 times. Furthermore, the germination of the Elementol
R group needed only 0.6 times as much water as the control group.
This aspect may prove to very valuable in dry regions.
4.3 In Vivo Effect of Elementol R in Dry Maize Field Trials
[0398] A genetically modified cultivar, supplied by a large seed
producing company was used. One bag of treated seed was split and
one portion of the seeds in the bag was treated with Captan, while
another portion was treated with Captan mixed with Elementol R in
the following manner. Captan is a broad-spectrum contact fungicide
that has been used on corn seed since the 1950s. It is usually dyed
pink and leaves a pink dust in the seed bag and planter box. It is
very effective against a broad range of soil fungi. The prescribed
amount of Captan was mixed directly with the seeds (Captan
reference group). For the test group, seeds were mixed with a
similar amount of Captan in 2% Elementol R. The seeds of both
groups were briefly mixed or stirred with their individual
treatment and then left to dry. Seeds were planted in blocks of 3
or 5 rows stretching the length of the maize field with untreated
block s on both sides of each of the treatment groups in the North
West Province, South Africa. Culturing was done according to
general farming practices with no irrigation.
[0399] Plants of each of the untreated, the reference Captan group
and the Elementol R/Captan group were collected by pulling up every
fifth plant in a row. Plant collection started 5 m into the field
and continued towards the centre of the field until fifty plants of
each group were collected.
[0400] The total plant mass, the root mass and the leaf mass of
each plant were determined. FIG. 35 shows the comparative average
masses for each of the group. Untreated seeds acted as control.
Treatment of the seeds with Captan alone did not result in any
change of growth of the plant leaves, and only slightly enhanced
root mass, whereas seeds treated with the 2% Elementol R/Captan mix
showed increases in leaf mass, root mass and therefore total plant
mass.
[0401] Many variations of the invention may be devised without
thereby departing from the spirit of the invention as formulated in
the above statements of the invention.
Example 20
Translocation of Elementol Vesicles Prepared with CO.sub.2 instead
of N.sub.2O
[0402] Elementol C was prepared as described in Preparation 1 for
Elementol B but CO.sub.2 was used as gas during the preparation
procedure. The size of the vesicles was determined to range between
300 nm and 2 .mu.m. The z-potential was measured as -44 mV, using a
Malvern Z-sizer.
[0403] The vesicles dispersed in the CO.sub.2 containing Elementol
C was labelled fluorescently with Nile red to a final concentration
of 1 .mu.M. Using a brush, a leaf of an ivy plant was painted with
this mixture. A control of water was painted on the leaf of a
second ivy plant. After 30 minutes, the leaves on the opposite side
of the painted leaves were collected and investigated for the
presence of fluorescence, using confocal laser scanning microscopy
as described in Example 6, Study 1. Fluorescent vesicles were
present in the collected leaf of the plant painted with the
fluorescently labelled Elementol C, whereas no such fluorescence
was found in the leaf collected from the plant painted with water.
The fluorescence did not correspond to the auto fluorescence
observed for chloroplasts or thylakoid membranes. The fluorescence
observed in the test leaf was thus shown to be the result of
translocation from one leaf to the opposite leaf by the CO.sub.2
containing Elementol C.
[0404] Molecular modelling indicates that the relevant properties
of nitrous oxide and carbon dioxide in the preparation of Elementol
vesicles and microsponges are shared by carbon oxy sulphide.
* * * * *