US20090192040A1 - Plant support formulation, vehicle for the delivery and translocation of phytologically benefical substances and compositions containing same - Google Patents

Plant support formulation, vehicle for the delivery and translocation of phytologically benefical substances and compositions containing same Download PDF

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US20090192040A1
US20090192040A1 US12/280,880 US28088007A US2009192040A1 US 20090192040 A1 US20090192040 A1 US 20090192040A1 US 28088007 A US28088007 A US 28088007A US 2009192040 A1 US2009192040 A1 US 2009192040A1
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plant
plants
elementol
acid
oil
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Anne Frederica Grobler
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North West University
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North West University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/64—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with three nitrogen atoms as the only ring hetero atoms
    • A01N43/647—Triazoles; Hydrogenated triazoles
    • A01N43/653—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/02—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing liquids as carriers, diluents or solvents
    • A01N25/04—Dispersions, emulsions, suspoemulsions, suspension concentrates or gels
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/02—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/06—Unsaturated carboxylic acids or thio analogues thereof; Derivatives thereof

Definitions

  • 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”].
  • 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.
  • 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”.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the shuttle ligand 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. 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.
  • the dispersion is further also characterized in that the micro-emulsion has a zeta potential of between ⁇ 35 mV and 60 mV.
  • 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 ⁇ 3], decosahexaenoic acid [C22:6 ⁇ 3], and ricinoleic acid, and derivatives thereof selected from the group consisting of the C 1 to C 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.
  • 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.
  • 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:
  • the fatty acid component may alternatively include or consist of the long chain fatty acids known as eicosapentaenoic acid [C20:5 ⁇ 3] and decosahexaenoic acid [C22:6 ⁇ 3].
  • eicosapentaenoic acid [C20:5 ⁇ 3]
  • decosahexaenoic acid [C22:6 ⁇ 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.
  • 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.
  • 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.
  • 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.
  • the gas is preferably selected from the group consisting of nitrous oxide, carbon oxysulfide and carbon dioxide.
  • a method for producing a plant supporting formulation or delivery vehicle 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.
  • the mixing of the fatty acid component is preferably effected with heating and stirring, preferably by means of a high speed shearer.
  • 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.
  • 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.
  • 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.
  • the gas is preferably selected from the group consisting of nitrous oxide, carbon oxy sulfide and carbon dioxide.
  • 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.
  • a plant pesticide including a herbicide, fungicide, bactericide, insecticide, anti-plant virus agent
  • a plant growth regulator e.g., a plant immune modulator
  • biostimulant e.g., a biostimulant
  • 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.
  • the invention accordingly also provides a plant nutrient composition
  • 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.
  • 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.
  • 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.
  • 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)—a chiral oxadiazine insecticide, insect extracts, isocyanate, lauryl sulfate, lime sulfur,
  • the invention also provides for a herbicidal composition
  • 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 conserveancy, Tu et al.).
  • 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.
  • 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
  • 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.
  • 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-lyl 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 X S21, Bradyrhizobium
  • viracide composition comprising a viracidally effective concentration of at least one viracide in the delivery vehicle described above.
  • the viracide may be selected from the viracides known to be suitable for use on plants to combat viruses that infect plants.
  • the invention further provides a plant growth regulator composition
  • 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.
  • 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;
  • the invention also provides for a method of enhancing the structural and functional integrity of plants or parts of plants.
  • 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.
  • the invention also provides for a method of supporting the local defence and acquired resistance of plants according to the mechanism described below by simultaneously supplying precursors for defence signalling molecules, anti-oxidants, ethylene, oleic acid and hexadecatrienoic acid.
  • SA salicylic acid
  • SAR systemic acquired resistance
  • ⁇ -DOX1 oxidizes 16-C and 18-C fatty acids, the last of which is a component of the formulation of the invention.
  • 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).
  • 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. 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.
  • C16:3 hexadecatrienoic acid
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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.
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • FIG. 26 is a graph that shows the effect of ComCat® (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;
  • FIG. 27 is a graph that shows the total accumulative fruit mass observed from plants treated with ComCat® that is entrapped in Elementol R as compared to the increase observed with Elementol R or ComCat® individually as described in Example 18;
  • 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;
  • 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® and combination treatment as described in Example 18;
  • 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;
  • 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 4 as background;
  • FIG. 32 is a photograph of germinating radishes on germination paper in the in vitro study described in Example 19;
  • 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;
  • 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;
  • 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.
  • a formulation according to the invention may be made up as follows:
  • Stable particles of fairly homogeneous sizes ranging from 50 nm to 50 ⁇ 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.
  • 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 2 water.
  • the CaCl 2 water was prepared 48 hours in advance.
  • the purpose for the advance dissolution of the CaCl 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.
  • control strips were treated identically to the trial strips, but excluded the Elementol B.
  • 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.
  • Planting of Sugar beans on a 120 Ha plot was done on seedbeds measuring 910 mm apart (old 3 feet spacing).
  • the experimental spray, per hectare comprised of the following:
  • control area comprised of 10 hectares on the same block.
  • Sampling the pods was done by hand.
  • the sampling method used was 10 ⁇ 10 metre random rows. This method was also used to sample the control.
  • 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%.
  • the experimental spray, per hectare comprised of the following:
  • the spraying was done under the following conditions:
  • 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.
  • 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.
  • 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.
  • the experimental spray, per hectare comprised of the following:
  • 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
  • Dicla plastic-covered tunnels (2 um thick plastic with inherent UV-protection for plants) with 2 ⁇ 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).
  • 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.
  • 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.
  • 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° C. inside the tunnels.
  • 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 is a plant beneficial delivery system, called Elementol R. It was hypothesized that this system may increase
  • test product was administered by root irrigation.
  • Elementol R was mixed with the nutrient of the tank that supplied irrigation to the test tunnel.
  • the nutrient mixture for irrigation was as follows:
  • Plant length 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 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • cucumbers were classified as medium to large (up to 37 cm). However, by the end of the 4 th week and up to the 20 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.
  • Table 2 shows the total difference as well as % difference between the yields in cucumbers from the two tunnels.
  • 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.
  • FIG. 6 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.
  • Table 3 shows the total yield and % difference in yield per tunnel.
  • 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.
  • Elementol R 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 R to the plant nutrients mixture resulted in statistically significant increases of yield of harvestable fruit in both plant species.
  • Elementol B consists mainly of a function-specific number and combination of unsaturated fatty acids and nitrous oxide.
  • 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 ⁇ /1.4 Oil DIC H; Plan Apo 60 ⁇ /1.4 Oil DIC H; and a Plan Fluor/0.75 DIC M.
  • Confocal images 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.
  • Plant 1 In this micrograph, no Elementol was administered to the plant. Material is visualized because of autofluorescence.
  • 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).
  • 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.
  • 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.
  • 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.
  • study 2 a basic hydroponic nutrient mixture was entrapped in Elementol vesicles and growth of the plants was monitored.
  • Group 1 received 5 ml of H 2 O
  • Group 2 received 5 ml of hydroponic medium diluted in H 2 O to the stipulated concentration
  • Group 3 received 5 ml of hydroponic medium mixed with a low concentration Elementol B (1.98%)
  • 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 2 O to the same concentrations used for the other groups.
  • 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.
  • 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 2 O or N 2 O—H 2 O but no Elementol B are much on a par, with the leaves of group that received N 2 O—H 2 O slightly longer than the plants that received water only.
  • the group 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.
  • Elementol R as Delivery Vehicle for Foliar Nutrient (Calcium) Administration on Strawberries
  • 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%.
  • the experimental spray, per hectare comprised of the following:
  • 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 2 ” complex.
  • the percentage calcium in both trial and control was the same.
  • the leaf calcium levels in the trial blocks were determined 21 days after application and found to be as follows:
  • the leaf calcium levels in the control blocks were determined 21 days after application and found to be as follows:
  • 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.
  • the fertilisation 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.
  • peak picking 4 tons/ha may be harvested every 10 days.
  • the experimental spray, per hectare comprised of the following:
  • control area comprised a small area on the same block and received no Elementol R.
  • 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.
  • the yield of fruit harvested was increased by 15% due to Elementol R administration
  • the colouring of the Elementol-treated plants is “aggressive”.
  • 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.
  • the trial plants were sprayed with the following:
  • the spray mixtures were made up in a mixing tank car and application was by aerial spraying.
  • control was sprayed with the same mixture, excluding Elementol B.
  • 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.
  • the diameter of the treated vine stems were significantly thickened and foliar index dramatically increased.
  • the yield of fruit was also higher.
  • 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.
  • 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.
  • a concentration of 0.6% Roundup Turbo and 40 ml Elementol B was diluted to 401 and applied to 1 ha. A field of 80 ha were sprayed with this mixture.
  • 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 (80 ha).
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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 stool beds received the same treatment except that no Elementol R was added.
  • 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.
  • 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.
  • 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.
  • Plant Lettuce or cos, romaine ( Lactuca sativa ) of the family: Asteraceae/Compositae (aster/daisy family).
  • Cultivar Lettuce ( Lactuca sativa L.), cultivar Red Poem, was used and was well established (approximately six weeks old) when purchased from a local nursery.
  • 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.
  • 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 ⁇ 1 ). Non-circulation of the nutrient medium may curb the spread of diseases in the system from infected plants to uninfected plants.
  • 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.
  • 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.
  • 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.
  • 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.
  • the plants were also placed in random order each week to ensure they receive equal amounts of sunlight, heat, water etc.
  • the temperature in the glass house was regulated by an air conditioner.
  • the temperature was regulated at maximum 24° C. and minimum 15.
  • the maximum temperature was 28° C. and the lowest temperature was 4° C.
  • the maximum and minimum temperature was obtained by using a thermohydrograph and both a daytime and night temperature was taken.
  • 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).
  • 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 ⁇ E.m ⁇ 2 sec ⁇ 1 .
  • the lowest light intensity at 12 h00 was 850 ⁇ E.m ⁇ 2 sec ⁇ 1 .
  • 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.
  • 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.
  • 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.)
  • 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 ⁇ 100
  • 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.
  • FIG. 15 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%.
  • 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° 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.
  • 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%.
  • 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.
  • 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.
  • 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.
  • 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 3 mM Tris-HCl buffer (pH 6.8) containing 2 mM EDTA, 14 mM ⁇ -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.
  • the protein concentrations of the treated plants and controlled plants were determined weekly and showed no significant difference.
  • the O 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 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. Respiration was measured in dark conditions, whilst both photosynthesis and respiration was measured in conditions of constant light intensity.
  • R 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 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 2 consumed and synthesized.
  • the rate of respiration and photosynthesis is obtained by: the following formulas:
  • 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.
  • 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.
  • 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 ).
  • 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.
  • Photosystem I Photosystem II
  • PS II Photosystem II
  • 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.
  • C 3 photosynthesis most plants
  • C 4 photosynthesis most grasses
  • CAM Crassulacean Acid Metabolism
  • 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 ⁇ . 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.
  • Chlorophylls were determined as follows (MacKinney (1941)):
  • Chlorophyll b (mg/g) [22.9( A 645) ⁇ 4.68( A 663) ⁇ ( V ⁇ (1000 ⁇ W ))]
  • 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.
  • FIG. 20 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.).
  • 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.
  • Brix index is a measure of the percent soluble solids content (SSC) in a solution.
  • SSC percent soluble solids content
  • Each degree of Brix is equivalent to 1 gram of sugar and other SSC per 100 grams of juice.
  • 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).
  • 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.
  • 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.
  • 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
  • the Brix refractometer was calibrated at room temperature using a 10% sucrose solution with a Brix reading of 1.3475. Neutralized HClO 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.
  • 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 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 4 .
  • Each system consisted of 2 rectangular asbestos trays (90 cm ⁇ 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 ⁇ 1.35 cm 2 per plant, resulting in 9 plants/1.22 m 2
  • the temperature in the greenhouse was partially controlled by an air conditioner. Average night and day temperatures ranged from 16° C. to 25° 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 th week after transplant (WAT) until termination in the 25 th WAT.
  • WAT 14 th week after transplant
  • 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 th week after transplantation, a drop to as low as 50% is noticed (24 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.
  • 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 vigour and transpiration rate ceases naturally as the harvesting period comes to an end.
  • 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.
  • 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.
  • 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.
  • 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.
  • Elementol R 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.
  • 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 th and 7 th week after transplant ( FIG. 21 ).
  • 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 ).
  • Elementol R Elementol R to yield could not be determined in Example 16, where leaf and plant growth were the relevant parameters.
  • 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 ).
  • 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 analyse 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.
  • the average moisture content would thus give an indication as to the quality of the tomato.
  • 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° 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.
  • 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.)
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 2 O. The test tube was shaken for 30 minutes, followed by vigorous Vortexing, and then quickly poured into a small measuring cup.
  • 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.
  • Table 3 shows the comparative glucose, fructose and sucrose content for the harvested fruit in week 13 of the study.
  • the Elementol R-treated tomatoes showed a considerable increase in fructose and sucrose content, resulting in sweeter tomatoes, which are preferred by the consumer.
  • 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 pipeffe. 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).
  • 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.
  • 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®, 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®, on hydroponically grown lettuce and tomatoes.
  • Example 16 and 17 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.
  • ComCat® an eco-friendly plant strengthening agent, contains one of a group of phytohormones, called brassinosteroids (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®. Brassinosteroids, as pure phytohormones, have been reported to not only increase crop yields but also crop quality (Prusakova et al., 1999). ComCat® contains high-quality, biochemical active substances which have been extracted from synecologically active wild plants.
  • ComCat® 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® is the first of its kind to have succeeded in catalyzing this activation of the plant's own ability of defence in an optimum way. Plants develop induced resistance that increases the plant's ability to resist pathogens.
  • 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.
  • 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.
  • ComCat® application resulted in a slightly reduced growth rate.
  • ComCat® is applied together with Elementol of either concentration (CC/E and 0.5CC/E)
  • this reduction in vegetative growth is alleviated in a dose-dependent fashion, but growth is still significantly below that of Elementol R alone.
  • Elementol R alone, as well as ComCat® (CC), and combination treatments showed a marked increase in flower buds, especially between 5 th and 7 th week after transplant. No clear difference was measured between these treatments, although CC showed the least increase.
  • Elementol R stimulated the yield of tomatoes significantly (Example 17).
  • ComCat® is mixed with Pheroids, both in full (CC/E) and half (0.5CC/E) strength markedly stimulated fruit production (See FIG. 26 which is a graph that shows the effect of ComCat® (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® that is entrapped in Elementol R as compared to the increase observed with Elementol R or ComCat® individually.).
  • the % enhancement in terms of yield was calculated as 99% and 81% CC/E and 0.5CC/E respectively and total harvested mass as 199% and 204% for CC/E and 0.5CC/E respectively when compared with that obtained with CC.
  • the enhancement of 33% and 21% for CC/E and 0.5CC/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® molecules. It would also indicate that Elementol R enhanced the uptake of ComCat ⁇ to exert its bio-stimulatory effect. A synergistic effect of these two products may also come into play.
  • CC alone showed a higher average fresh fruit mass than E alone.
  • 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 0.5CC/E, except for week 13 and as the standard deviation on Fm is quite large, it may not be significant.
  • 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.
  • respiration rate 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 ).
  • the combination of E and CC stimulates respiration rate per mg of protein.
  • the E plants 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.
  • 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.
  • dilution of the CC concentration led to an increase in chlorophyll B/mg protein.
  • the dosage of the CC should be decreased when entrapped in Elementol R.
  • 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 ⁇ 4CC/E combination and 1 ⁇ 2CC/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.
  • 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.
  • the enhancement in Brix readings by the combination is indicative of the higher uptake and translocation of CC by the Elementol carrier.
  • C3 and C4 plants To investigate the effect of Elementol R on germination and seedling growth in both C3 and C4 plants.
  • CO 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 3 path involves the Calvin cycle, whereas the C 4 path uses a cycle where 3-phosphoglyceric acid is not the first product.
  • C 4 photosynthesis provides a mechanism for high rates of carbon assimilation and is more resistant to the process of photo respiration.
  • 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.
  • the growing conditions in terms of temperature and relative humidity were relatively constant. Plants were planted in earth and irrigated by drip irrigation.
  • 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 suppliers 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.
  • 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.
  • the response in coleoptile growth is specifically due to the administration of a specific dose of Elementol R.
  • the cultivar was PAN 3377. Wheat was cultivated according to normal farming practices in the Central Free State, South Africa.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • Elementol C was prepared as described in Preparation 1 for Elementol B but CO 2 was used as gas during the preparation procedure.
  • the size of the vesicles was determined to range between 300 nm and 2 ⁇ m.
  • the z-potential was measured as ⁇ 44 mV, using a Malvern Z-sizer.
  • the vesicles dispersed in the CO 2 containing Elementol C was labelled fluorescently with Nile red to a final concentration of 1 ⁇ M.
  • 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.
  • 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 2 containing Elementol C.

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