WO2025003663A1 - Enrobage de semences - Google Patents

Enrobage de semences Download PDF

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Publication number
WO2025003663A1
WO2025003663A1 PCT/GB2024/051637 GB2024051637W WO2025003663A1 WO 2025003663 A1 WO2025003663 A1 WO 2025003663A1 GB 2024051637 W GB2024051637 W GB 2024051637W WO 2025003663 A1 WO2025003663 A1 WO 2025003663A1
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WO
WIPO (PCT)
Prior art keywords
seed
cfu
mixture
coated
coating
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/GB2024/051637
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English (en)
Inventor
Laurence BERMAN
Lutz GLANDORF
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Biolevel Ltd
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Biolevel Ltd
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Filing date
Publication date
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Publication of WO2025003663A1 publication Critical patent/WO2025003663A1/fr
Anticipated expiration legal-status Critical
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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P21/00—Plant growth regulators
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01C—PLANTING; SOWING; FERTILISING
    • A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
    • A01C1/06—Coating or dressing seed
    • 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
    • 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
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • 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
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • 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
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/27—Pseudomonas
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G5/00—Fertilisers characterised by their form
    • C05G5/30—Layered or coated, e.g. dust-preventing coatings

Definitions

  • Seed coatings are used to grow commercial crops such as corn, wheat, barley and so forth. Coatings provide the seeds with surface protection, may have pesticidal properties, and can include nutrients and other beneficial agents.
  • Various approaches have been tried, including applying liquid microbial products which require refrigeration to maintain viability to seed directly before planting to ensure the then non-refrigerated microbial products is applied via the seed to the soil in a timely manner to avoid loss of microbial viability, or the blending of microbial products with liquid biostimulants to increase the product viability on seed.
  • the problem with the existing products on the market or otherwise known is that they cannot provide a stable bacterial colony without refrigeration.
  • a liquid-free composition for coating seeds comprising a mixture, the mixture comprising: a. a consortium of bacteria; and b. a bacterial carrier substrate, optionally a powder; wherein bacteria in the consortium are: i. saprophytic bacteria; and/or ii.
  • the bacterial carrier substrate functions to reduce, or substantially eliminate, surface moisture on the seed.
  • the bacterial carrier may also carry organisms other than bacterial, for example fungi, e.g. fungal spores.
  • the bacterial carrier substrate is a polysaccharide powder, a drying agent, glidant or seed lubricant.
  • the polysaccharide powder is a starch.
  • peat may be used, but this is generally not preferred due to environmental considerations.
  • the polysaccharide powder or starch is or comprises corn starch (maize starch or cornflour).
  • the polysaccharide powder or starch is selected from or comprises: corn flour, cornflour, masa flour, modified starch, potato starch, semolina, tapioca starch, wheat flour, arrowroot, rice flour, and combinations thereof.
  • the bacterial carrier comprises seaweed, including flaked seaweed.
  • the seaweed and especially flaked seaweed is used to coat grass seeds. Coated grass seeds are provided. Coating grass seeds with a coating composition wherein the carrier comprises seaweed and particularly flaked seaweed is effective as grass seed germinates relatively quickly.
  • the seaweed meal is Ascophyllum sp., especially Ascophyllum nodosum. This may be harvested mainly from the Scottish Land and/or Irish coastline.
  • the processing of the material is washing, drying, then milling.
  • the dried flaked seaweed may then be inoculated with the biological consortia
  • the biology and substrate is applied to the dried seaweed material by vacuum drying it to the surface of the meal.
  • the inoculated seaweed is both slightly dusty to the touch but also embedded with biology (consortia).
  • the biological infused combined product has a slight dusty and electrostatic characteristic, which further coats the grass seed with a more even biological inoculation when mixed in a bag.
  • the bacterial carrier is a powder, optionally particles with a diameter in the range of 100 micrometres ( ⁇ m) to 300 micrometres ( ⁇ m), optionally 200 micrometres ( ⁇ m).
  • the consortium comprises: ⁇ at least one Pseudomonas species; ⁇ at least one Bacillus species; and/or ⁇ at least one Paenibacillus species; and, optionally ⁇ at least one Azospirillum species.
  • the consortium comprises: a) Pseudomonas putida b) Bacillus subtilis c) Bacillus licheniformis d) Bacillus amyloliquefaciens e) Bacillus megaterium f) Bacillus pumilus; and g) Paenibacillus polymyxa
  • the consortium further comprises h) Azospirillum brasilense
  • the consortium comprises i.
  • Pseudomonas putida at a minimum concentration of 1 or 2x10 ⁇ 8 CFU/g ii.
  • Bacillus subtilis at a minimum concentration of 1 or 2x10 ⁇ 8 CFU/g iii.
  • Bacillus licheniformis at a minimum concentration of 1 or 2x10 ⁇ 8 CFU/g 2024.06.26 Seed Coating II Specification - PCT - P000587WO iv.
  • Bacillus amyloliquefaciens at a minimum concentration of 1 or 2x10 ⁇ 8 CFU/g
  • Bacillus megaterium at a minimum concentration of 1 or 2x10 ⁇ 8 CFU/g vi.
  • the seed is a seed from: ⁇ member of the grass family, Graminacea (a.k.a. Poaceae); or ⁇ member of the Leguminaceae family.
  • the seed is a seed selected from: ⁇ Corn/maize; ⁇ Alfalfa; ⁇ Sorgum; ⁇ Wheat; ⁇ Barley; ⁇ Oats; ⁇ Rice; ⁇ Sugar Cane; ⁇ Grass Seed; ⁇ pumpkin/squash or ⁇ a mixture, optionally a forage mixture, of any two or more of the above. 2024.06.26 Seed Coating II Specification - PCT - P000587WO Also provided is coated seed comprising the coating composition described herein and one or more seeds. Also provided is a coated granule, wherein the coating is a coating composition as described herein.
  • the granule is a fertilizer, a liming product, a soil amendment product or shell particles (optionally crushed oyster shell).
  • the granule is an agricultural liming product or agricultural liming material selected from: ⁇ limestone (calcitic or dolomitic); ⁇ burned lime; slaked lime; ⁇ marl; ⁇ shells; and ⁇ industrial by-products for example sugar beet lime and sludge from water treatment plants.
  • the granule is a particle in the range of 2 to 4 mm in diameter, for example: poly4 ®
  • a mixture comprising: ⁇ the coating composition, coated seed or coated granule according to any one of the preceding claims; and ⁇ other seed coatings or applications (such as starter fertilizers , such as ENTEC®.
  • the composition may be a coating for seeds, hence may also be referred to a coating composition.
  • the function of the composition is not necessarily to protect it, although that can be an advantage. Instead, the primary purpose of the of the seed is that it is a conveyor or transporter to allow a simple means to introduce the composition into the soil (or environment or microenvironment around the seed).
  • the mixture is dry and/or liquid-free.
  • Liquid-free can mean, in some embodiments, a solid, for example a powder, especially a dry powder. Liquid-free may also mean, in some embodiments, devoid of liquid. In some other embodiments, the composition may comprise some water vapour, but it is still a dry powder, rather than a liquid preparation or composition.
  • the coating composition is not intended to be a suspension (of the mixture of the consortia and carrier) in a liquid.
  • the coating composition is liquid-free, although in some embodiments it may be that the mixture of the consortia and the carrier are the liquid-free component and other parts are added to that liquid-free mixture.
  • the mixture of the coating composition comprises only of, or consists of, the consortia and one or more carrier powders as well as optional additives such as a stabilising agent and/or powders having electrostatic properties that improve adherence of the composition to seeds.
  • the bacterial carrier substrate, optionally a powder is a polysaccharide, powder, talc, drying agent or glidant and this may also have electrostatic properties that improve adherence of the composition to seeds.
  • the composition is a seed coating or is for use as a seed coating.
  • the composition consists of the consortium of bacteria; and the bacterial carrier substrate, optionally a powder,; and optionally at least one of the stabilising agent and/or powders having electrostatic properties that improve adherence of the composition to seeds.
  • the mixture may, in some embodiments, comprise one or more, i.e. a blend of, carrier powders.
  • the one or more additional carrier powders may be any suitable carrier powders, 2024.06.26 Seed Coating II Specification - PCT - P000587WO suitable for the use of the consortia as a seed coating, provided that the blend does comprise the required polysaccharide powder.
  • the bacterial carrier powder is peat.
  • Peat is used as a carrier for fungi as an active ingredient, where peat is required for the biological survival of the fungi. In general, though, peat is not preferred where environmental considerations apply.
  • the bacterial carrier powder is a glidant, for example known to improve the flowability of seed by reduction of friction. Graphite powder is preferred as such a glidant.
  • the bacterial carrier substrate functions to reduce, or substantially eliminate, surface moisture on the seed. The surface moisture on the seed may be present prior to application of the coating composition (e.g. by admixture). In some embodiments, this occurs via adsorption. In some embodiments, the moisture reduction is by greater than 70%, preferably 80-90% reduction in surface moisture on the seed.
  • the seed is therefore dried so as to prevent clogging of for example the dry planter box.
  • the bacterial carrier substrate is a seed lubricant. These are known to reduce the moisture content of the seed surface via absorption.
  • the bacterial carrier substrate is a drying agent, for example a talc powder. These are known to be used as drying agents.
  • the bacterial carrier substrate is chosen based on its characteristics to adhere to the seed, for example an adhesive carrier.
  • a preferred example of an adhesive carrier is a protein powder.
  • the bacterial carrier substrate is a combination of graphite.
  • the carrier is a dextrose monohydrate powder which adheres to seed via static action and when in contact with surface moisture on the seed from condensation or liquid seed coatings. 2024.06.26 Seed Coating II Specification - PCT - P000587WO
  • the composition is suitable for use in a dry planter box.
  • a planter box preferably a dry planter box, comprising the present composition.
  • the mixture is not a suspension.
  • the mixture is not suspended in a non-polar liquid.
  • that excluded non-polar liquid is an oil.
  • the oil is: a.
  • the bacterial carrier is a powder, optionally particles with a diameter in the range of 100 micrometres ( ⁇ m) to 300 micrometres ( ⁇ m), optionally 200 micrometres ( ⁇ m).
  • the polysaccharide powder is a starch powder.
  • Starch powders may include corn starch or alternatives to corn starch. In some embodiments, the starch is corn starch.
  • Corn starch, maize starch, or cornflour is the starch derived from corn (maize) grain.
  • the corn starch may be obtained from the endosperm of the kernel and extracted by wet milling.
  • Corn starch is also referred to as maize starch or cornflour.
  • Alternatives to corn starch include corn flour which is a term used in the USA, for example, as referring to cornmeal that is very finely milled; or, to masa flour.
  • Cornflour is a reference, used in the UK for example, to corn starch.
  • Further alternatives to corn starch include cornmeal, masa flour, modified starch, potato starch, semolina, tapioca starch, wheat flour, arrowroot, rice flour, and combinations thereof.
  • the consortium comprises two or more species or strains of bacteria. In some embodiments, the consortium comprises two or more, three or more, four or more, five or more, 10 or more, 15 or more, 20 or more, 25 or more, or 30 or more species or strains of bacteria. In some embodiments, the consortium comprises: a) at least one Pseudomonas species; b) at least one Bacillus species; and/or c) at least one Paenibacillus species; and, optionally d) at least one Azospirillum species.
  • the consortium comprises: e) Pseudomonas putida f) Bacillus subtilis g) Bacillus licheniformis h) Bacillus amyloliquefaciens i) Bacillus megaterium j) Bacillus pumilus; and k) Paenibacillus polymyxa
  • consortium further comprises i) Azospirillum brasilense
  • the consortium comprises ix. Pseudomonas putida at a minimum concentration of 1 or 2x10 ⁇ 8 CFU/g 2024.06.26 Seed Coating II Specification - PCT - P000587WO x.
  • Bacillus subtilis at a minimum concentration of 1 or 2x10 ⁇ 8 CFU/g xi.
  • Bacillus licheniformis at a minimum concentration of 1 or 2x10 ⁇ 8 CFU/g xii.
  • Bacillus amyloliquefaciens at a minimum concentration of 1 or 2x10 ⁇ 8 CFU/g xiii.
  • Bacillus megaterium at a minimum concentration of 1 or 2x10 ⁇ 8 CFU/g xiv.
  • Bacillus pumilus at a minimum concentration of 1 or 2x10 ⁇ 8 CFU/g xv.
  • Paenibacillus polymyxa at a minimum concentration of 1 or 2x10 ⁇ 8 CFU/g xvi.
  • the composition may be add to granulated fertilisers, MAP or DAP fertilisers, ureas to provide a combination product or composition. This is especially the case where, in some embodiments, cornstarch is used as the polysaccharide bacterial carrier.
  • a coated seed comprising the coating composition.
  • the coted seed is a seed from: a. member of the grass family, Graminacea (a.k.a. Poaceae), for example grass seed; or b. member of the Leguminaceae family.
  • the coated seed is a seed selected from: c. Corn/maize; d. Soy; e. Alfalfa; f. Sorgum; g. Wheat; h. Barley; 2024.06.26 Seed Coating II Specification - PCT - P000587WO i. Oats; j. Rice; k. Sugar Cane; l. Grass seed; m. Pumkin/Squash; or n. a mixture, optionally a forage mixture, of any two or more of the above.
  • the pumpkin is species or variety in the genus Cucurbita and may be used interchangeably with "squash” or "winter squash", for example cultivars of Cucurbita argyrosperma, Cucurbita ficifolia, Cucurbita maxima, Cucurbita moschata, and Cucurbita pepo.
  • the coated seed is provided in a forage mixture.
  • a forage mixture comprising a mixture of the present coated seeds.
  • a coated granule wherein the coating is a coating composition as provide herein.
  • the granule is a fertilizer, a liming product, a soil amendment product or shell particles (optionally crushed oyster shell).
  • the granule is an agricultural liming product or agricultural liming material selected from: a. limestone (calcitic or dolomitic); b. burned lime; slaked lime; c. marl; d. shells; and e. industrial by-products for example sugar beet lime and sludge from water treatment plants.
  • the granule is a particle in the range of 2 to 4 mm in diameter, for example: poly4 ®
  • the product is applied to granules as a carrier.
  • a mixture or suspension comprising: a. the present coating composition, coated seed or coated granule; and b. other seed coatings or applications (such as starter fertilizers , such as ENTEC).
  • a vessel for storing or transporting the present coating composition, coated seed, coated granule or mixture, suspension or emulsion such as a box, container, bottle or drum.
  • a method of use for the present coating composition comprising: a. mixing seeds, granules or providing a mixture, suspension or emulsion with the composition; and b. sowing the coated seed or coated granule or mixture or suspension in a suitable environment such as soil or growth medium that supports a rhizosphere.
  • a method of coating seeds comprising: 1) add the seed, preferably corn/maize seed, into the or each box (hopper) on a planter; 2) adding the present seed coating, preferably using cornstarch as the carrier, preferably at the equivalent powder to seed ratio of volume (US Fluid ounces or mL of powder) per unit of seed (80,000 seeds
  • Fig 1 shows the trial field layout for Example 2, measurements are in metres.
  • Figures 2 and 3 show field trial layouts with lanes of untreated, and BioLevel MaizeNP and Biolevel MaizeNP – 20% Nitrogen treated lanes.
  • Detailed Description of the Invention Data provided herein shows that bacterial consortia provided as a seed coating to seeds produces beneficial results including good growth and/or reduced fertilizer requirements.
  • US2018235235A1 relates to s composition comprising five or more of certain bacterial species and methods of mixing them with a chitin-containing biological source, fermenting the mixture and separating the mixture in solid, aqueous and lipid fractions.
  • WO2021146209A1 (Pivot Bio) relates to the use of peat, but this is generally thought of as ecologically and environmentally unfavourable.
  • US2014352376A1 (Carpenter; Bio Wish) relates to coating fertiliser ingredients, not seeds.
  • the mixture is not provided as a suspension in the non-polar liquid.
  • the polysaccharide, ideally a starch, is a carrier for the bacterial consortia. What we have found is that this carrier adheres to seeds with waxy or smooth outer surfaces or seed coats.
  • a preferred polysaccharide carrier is a starch such as Corn starch. Corn starch has good coverage and/or adherence on grass seeds. Some powder separation can be seen, but the distribution of the carrier tends to stay relatively even across the surface of the grass seed. Thus the coated seed is planted with its coating substantially intact.
  • the planted seed preferably has at least 70%, more preferably at least 80%, at least 90 or at least 95% of its seed coating intact.
  • the carrier can be sufficiently fine to adhere to the seeds evenly.
  • the diameter or particle size range of the substrate is the range of: 1.0 microns to 1000 microns.
  • the powder may, in some embodiments, have a particle diameter in the range of 100 -300 micrometres. In some embodiments, the particle diameter is approximately 200- 500 micrometres (+/- 10 or 20%). In some embodiments, the particle diameter is approximately 1-300 micrometres (+/- 10 or 20%).
  • the particle diameter is approximately 1-1000 micrometres (+/- 10 or 20%). In some embodiments, the particle diameter is approximately 500-1000 micrometres (+/- 10 or 20%). In some embodiments, the particle diameter is greater than approximately 200 or 500 micrometres (+/- 10 or 20%). In some embodiments, the particle diameter is approximately 1-10 micrometres (+/- 10 or 20%).
  • the carrier can have electromagnetic or electrostatic properties that reduce clogging in planting machinery. The clogging is usually caused by electrostatic or electromagnetic interactions with the metal machinery parts. Thus, carriers with low electrostatic properties are preferred so as to reduce adherence to the planter box or other seed delivery mechanisms.
  • WO2021146209A1 relates to a consortia of microbes that are functionally optimized for nitrogen fixation and deliver such to plants in a targeted, efficient, and environmentally sustainable manner.
  • the microbes within the consortium differ in nutrient utilization, temporal occupation, oxygen adaptability, and/or spatial occupation, which enables the microbes to deliver nitrogen to a cereal plant in a 2024.06.26 Seed Coating II Specification - PCT - P000587WO spatially targeted (e.g. rhizospheric) and temporally targeted (e.g. during advantageous stages of plant's life cycle) manner.
  • US2014352376A1 relates to a binder such as Sealmaster which is a starch that pulls together granules of biosolid particles, the particles themselves holding the bacteria.
  • a binder such as Sealmaster which is a starch that pulls together granules of biosolid particles, the particles themselves holding the bacteria.
  • the focus is on the specific makeup of the consortia, not how they are delivered a seed coating.
  • the present composition and related aspects comprises a concentrated consortium of microorganisms designed to solubilise Potassium (K) and/or Phosphate reserves in the soil, increase Nitrogen, K and/or micronutrient uptake or fixation in the rhizosphere, as well as free up essential micronutrients in the soil environment.
  • K solubilise Potassium
  • Phosphate reserves in the soil, increase Nitrogen, K and/or micronutrient uptake or fixation in the rhizosphere, as well as free up essential micronutrients in the soil environment.
  • enzymes and other organic chemicals produced by the provided microorganisms break P bonds between Al, Ca, and Fe. This process results in a plant-accessible form of P.
  • the synergistic relationship whereby roots supply the microorganisms in the form of root exudates and the beneficial bacteria efficiently provide accessible nutrients to the plant has evolved over millions of years.
  • the present composition is a microorganism consortium and, in some embodiments, not a single-species product enabling it to be suited to a range of soil types and climatic conditions.
  • the present composition contains beneficial microorganisms at a concentration 1BB CFU/g (1 billion CFU/gram: this refers to the total CFU count in the product across individual strains. By way of example, 5x10 ⁇ 7 CFU/g would be the CFU per gram or ml for an individual strain in the composition.
  • the present composition comprises bacteria that are GM free (i.e.
  • the total bacterial count or concentration is approximately 2 billion (2 x 10 9 ) CFU/ml or non-polar liquid.
  • the present composition is able to produce 200 billion (2 x 10 11 ) CFU per hectare in recommended applications, for example when applied to corn (maize) seeds.
  • the present composition has properties such as viscosity that allows the composition to be added to seeds without clogging up (agglutinating inside) the planting or application device such that they cannot be individually separated and sown by a seed applicator machine for sowing seeds into the earth in a field. In some embodiments, this does not clog the device to thereby significantly impact delivery. In some embodiments, the composition provides an even and/or sufficient coating to the seed to achieve benefits to the formation, development or maintenance of the rhizosphere. In some embodiments, the present composition can be stored at RTP, so approximately 20 degrees C without loss of function for at least several weeks.
  • Example 2 shows that the CFU count is stable after plating after several weeks of storage at 20 degrees C.
  • the present composition can allow seeds to be coated with a low volume of material compared to other types of coating, especially liquid coatings.
  • the present composition can allow even coating on the seed and prevents product loss and/or wastage from pooling of the applied product.
  • the present composition can allow the seed coating to deliver the microbial consortium directly to the rhizosphere of the growing plant.
  • the present composition can reduce the amount of product required with other application methods.
  • the present composition can accelerate the establishment of symbiotic plant-microbial relationships.
  • the present composition can allow or improve solubilization of Phosphorus in the soil.
  • the present composition can allow or improve fixation of atmospheric Nitrogen in the soil.
  • the present composition can allow or improve solubilisation of Potassium and/or other micronutrients in the soil.
  • the present composition can allow or improve colonization of the plant rhizosphere, to thereby increase the availability of both soil-based and applied macro and micro nutrients to the plant. This increased availability of nutrients, especially on a 2024.06.26 Seed Coating II Specification - PCT - P000587WO continuous basis, can lead to increased plant growth and thus yield.
  • the present composition can allow or improve essential NPK (Nitrogen, Phosphorus and Potassium (K)) nutrients to be made available to the plant.
  • the present composition can allow or improve the release of locked-up soil nutrients (Phosphorus, Potassium and/or other micronutrients) for plants when applied to the soil.
  • the present composition can allow or improve conversion of atmospheric Nitrogen into usable plant nutrients.
  • the present composition can allow or improve crop yields & food quality.
  • Micronutrients as referred to herein may include, in some embodiments, zinc, iron, manganese, and/or calcium.
  • the present composition can allow or improve the cycle efficiency of such N, P and/or K and thus reduces fertilizer use. Excessive application of fertilizer and chemical adversely effects soil biology and may contaminate the environment.
  • the present composition can allow or improve sustainability, for example through reduced fertilizer use or increased fertilizer use efficiency.
  • the present composition can provide broad compatibility with plant protection products, such as pesticides.
  • the present composition may further comprise plant protection products, such as pesticides.
  • Many microbial nutritional products in the market are simple nitrogen fixers or limited in being single strain products.
  • the present composition can, in some embodiments, offer a complete nutrition package by adding phosphorus and potassium solubilizing microbes, as well as micronutrients, to our nitrogen fixers.
  • Plant-associated rhizosphere bacteria have an important role in establishing and improving plant growth on different soil types, since they affect the availability of essential elements and provide plants with extra Nitrogen. Rhizosphere formation, development or improvement is an important aim of the present invention and is thus provided in some embodiments.
  • Various methods are typically used to add beneficial bacteria to the soil so as to form a rhizosphere: Addition to, and distribution via, a slurry, for example an animal waste slurry; spraying onto the soil; seed coating; foliar application and application to granulated fertilizers. 2024.06.26 Seed Coating II Specification - PCT - P000587WO
  • foliar application in some embodiments, endophytic microbial products (meaning they go into the plant cells) are used and, in some embodiments, sprayed as a foliar application.
  • the microbes are coated onto a standard granulated fertilizer.
  • the Example herein show that that the application of the biology (bacterial consortia) to the seed prior to sowing is effective at increasing yield and/or reducing fertiliser use.
  • the use of a polysaccharide carrier contributes to this and also reduces clogging in the planter machines.
  • Polysaccharides also known as polycarbohydrates, are the most abundant carbohydrates found in food. They are long-chain polymeric carbohydrates composed of monosaccharide units bound together by glycosidic linkages.
  • the carrier of the present invention in some embodiments comprises, or consists of, a polysaccharide, for example 3 or more monomers linked by 2 or more glycosidic linkages.
  • the polysaccharide has 10 or more, 20 or more, 50 or more or 100 or more such monomers.
  • the polysaccharide may, for example be a starch where there are thousands or hundreds of thousands of monomers.
  • We have shown here (see Example 1) that the consortium are able to create a rhizosphere via slurry delivery.
  • seed coatings are preferred in the present invention.
  • pesticides including fungicides and insecticides can also be added (see for example in Example 3, where Coozer/Afron Star were added without compromising the composition).
  • fertilizers can also be added, in particular micronutrients. This is complementary to one of the benefits of the present invention, which is that less Nitrogen and/or Phosphorus-containing fertilizers are necessary.
  • a typical application rate of the present composition to seeds, in particular, corn seeds is 4 litres of composition per tonne of seed. 2024.06.26 Seed Coating II Specification - PCT - P000587WO Seed
  • the seed to which the present composition may be applied is a plant seed, for example a seed of an agricultural crop, a vegetable seed, a herb seed, a wildflower. In some embodiments, the seed is a seed of an agricultural crop.
  • the seed may be of the class of Monocotyledoneae or of the class of Dicotyledoneae.
  • the seed is a seed of: ⁇ corn or maize, ⁇ grass seed, ⁇ pumpkin/squash, ⁇ cotton, ⁇ peanut, ⁇ barley, ⁇ oat, ⁇ wheat, ⁇ rye, ⁇ riticale, ⁇ mustard, ⁇ oil seed rape (or canola), ⁇ sunflower, ⁇ sugar beet, ⁇ safflower, ⁇ soy, ⁇ millet, ⁇ chicory, ⁇ flax, ⁇ rapeseed, ⁇ buckwheat, ⁇ tobacco, ⁇ hemp seed, ⁇ alfalfa, ⁇ signal grass, 2024.06.26 Seed Coating II Specification - PCT - P000587WO ⁇ clover, ⁇ sorghum, ⁇ chick pea, ⁇ beans, ⁇ peas, ⁇ vetch,
  • consortium A consortium or community may be two or more bacterial or microbial groups living synergistically. In some embodiments, two or more bacterial or microbial groups may live symbiotically.
  • the consortium of bacteria is, in some embodiments, dried prior to, or as part of, mixing with the bacterial carrier substrate, optionally a powder.
  • the composition comprises a dried consortium of bacteria.
  • the methods and processes of the present invention comprise a step of drying the consortium of bacteria. The drying may be by vacuum drying or freeze-drying, or other methods known in the art. In some embodiments, therefore, the composition comprises freeze-dried or vacuum-dried consortium of bacteria.
  • the consortium may comprise a wide range of different microbial combinations. These bacterial species are typically described as naturally occurring, often saprophytic bacteria. Thus, in some embodiments, bacteria in the consortium are saprophytic. They may also be, in some embodiments, naturally occurring, or in other embodiments the bacteria may have been genetically modified, gene edited or contain a trans gene (i.e. comprise polynucleotides encoding a trans gene). In some embodiments, bacteria in the consortium may have the European federation of Biotechnologies Class 1 classification or a global equivalent.
  • This classification is defined as: “Commonly occurring saprophytes, never been known to cause 2024.06.26 Seed Coating II Specification - PCT - P000587WO disease in man” or alternatively “naturally occurring micro-organisms that have never been identified as causative agents of disease in man and that offer no threat to the environment.” Such terms may apply, in some embodiments, to the present invention.
  • the bacteria of the consortium are, in some embodiments, beneficial microbes (i.e. beneficial bacteria).
  • beneficial microbes i.e. beneficial bacteria.
  • the definitions are largely used interchangeably, although saprophytic refers to the mode of nutrition and saprotroph refers to an organism that utilises the saprophytic mode of nutrition.
  • the bacteria may be, in some embodiments, saprophytic (or saprophytes).
  • the bacteria of the consortium are dormant , at least at the time of application to the seed. In the consortium, the bacteria are encouraged to sporulate, in some embodiments. They may, in some embodiments, be partially dehydrated to induce a state of reduced metabolism in the microbes.
  • Bacterial Species Bacteria in the consortium may be, in some embodiments, Nitrogen-fixing bacteria.
  • Example species include: Paenibacillus polymyxa Azospirillum brasilense Bacteria in the consortium may be, in some embodiments, be both Nitrogen-fixing and Phosphorus-solubilizing bacteria, i.e. can perform both functions.
  • Example species include: Paenibacillus polymyxa
  • bacteria in the consortium may be saprophytic, Nitrogen-fixing and Phosphorus-solubilizing bacteria.
  • Example species include: Paenibacillus polymyxa
  • PSM Phosphate Solubilizing Microorganisms
  • Phosphorus-solubilizing bacteria are bacteria capable of solubilizing Phosphorus in the soil. Plants acquire phosphorus from soil solution in the form of phosphate anion. It is the least mobile element in plant and soil in comparison to other macronutrients. It remains in a precipitated form in the soil as mono or orthophosphate or is absorbed by Fe or Al oxides through ligand exchange. Phosphate Solubilizing Microorganisms (PSM) play a very important role in phosphorus nutrition by exchanging its availability to plants through release from inorganic and organic soil phosphorus pools by solubilization and mineralization.
  • PSM Phosphate Solubilizing Microorganisms
  • the main mechanism in the soil for mineral phosphate solubilization is by lowering the soil pH by the microbial production of organic acids and mineralization of organic phosphorus by acid phosphates.
  • an additional source of phosphorous is applied to plants in the form of chemical fertilizers (Phosphate Solubilizing Microbes: An effective and alternative approach as Biofertilizers (Kumar Anand, Baby Kumari1, M. A. Mallick, Int J Pharm Pharm Sci, Vol 8, Issue 2, 37-40, 2016)).
  • Example species include species of Pseudomonas, Bacillus, Micrococcus, Flavobacterium, Aspergillus, Penicillium, Fusarium, and/or Sclerotium.
  • Phosphorus-solubilizing bacteria in the consortium or additional PSM microbes provided in the composition may, in some embodiments, include any one or more of: ⁇ Pseudomonas, ⁇ Bacillus, ⁇ Micrococcus, ⁇ Flavobacterium, ⁇ Aspergillus, ⁇ Penicillium, ⁇ Rhizobium, 2024.06.26 Seed Coating II Specification - PCT - P000587WO ⁇ Enterobacter and/or
  • Phosphorus-solubilizing bacteria in the consortium or additional PSM microbes provided in the composition may, in some embodiments, include any one or more of: ⁇ Bacillus megaterium, ⁇ B. circulans, ⁇ B. subtilis, ⁇ B.
  • the consortium additionally comprises the pathogens Fusarium and/or Sclerotium.
  • the consortium comprises at least one (saprophytic, Nitrogen-fixing and/or Phosphorus-solubilizing) Pseudomonas, preferably Pseudomonas putida. In some embodiments, this is present at a minimum concentration of 1x10 ⁇ 8 CFU/g of the mixture.
  • the consortium comprises at least one (saprophytic, Nitrogen-fixing and/or Phosphorus-solubilizing) Bacillus sp.
  • this is present at a minimum concentration of 1x10 ⁇ 8 CFU/g of the mixture.
  • the Bacillus is Bacillus subtilis. In some embodiments, this is present at a minimum concentration of, or at least, 1x10 ⁇ 8 CFU/g of the mixture.
  • the Bacillus is Bacillus licheniformis. In some embodiments, this is present at a minimum concentration of, or at least, 1x10 ⁇ 8 CFU/g of the mixture.
  • the Bacillus is Bacillus amyloliquefaciens. In some embodiments, this is present at a minimum concentration of, or at least, 1x10 ⁇ 8 CFU/g of the mixture.
  • the Bacillus is Bacillus megaterium. In some embodiments, this is present at a minimum concentration of, or at least, 1x10 ⁇ 8 CFU/g of the mixture. In some embodiments, the Bacillus is Bacillus pumilus. In some embodiments, this is present at a minimum concentration of, or at least, 1x10 ⁇ 8 CFU/g of the mixture. 2024.06.26 Seed Coating II Specification - PCT - P000587WO In some embodiments, the consortium comprises at least one (saprophytic, Nitrogen-fixing and/or Phosphorus-solubilizing) Paenibacillus, preferably Paenibacillus polymyxa.
  • the consortium comprises at least one (saprophytic, Nitrogen-fixing and/or Phosphorus-solubilizing) Azospirillum, preferably Azospirillum brasilense. In some embodiments, this is present at a minimum concentration of, or at least, 1, 2 , 3, 4 or 5x10 ⁇ 7 CFU/g or at least 2x10 ⁇ 8 CFU/g.
  • Each of the bacteria at the genera, species or strain level may have one or more of the above properties (e.g. are saprophytic, Nitrogen-fixing and/or Phosphorus-solubilizing).
  • the consortium is capable of forming and/or contributing to a rhizosphere.
  • the rhizosphere is the microbially active area of the nutrient exchange around the root area of a plant.
  • the genera, species and relative proportions of bacteria vary per crop.
  • the consortium may comprise a combination of Bacillus, Paenibacillus, Pseudomonas & Azospirillum. This combination is ideal for use on plants that are members of the grass family, Graminacea (a.k.a. Poaceae).
  • the composition may comprise Bacillus, Paenibacillus, Pseudomonas & Azospirillum and is for use in coating seeds from members of the grass family, Graminacea (a.k.a. Poaceae). Corresponding coated seeds are also provided.
  • the consortium may comprise a combination of Bacillus, Paenibacillus, and Pseudomonas. This combination is ideal for use on plants that are members of the Leguminaceae family.
  • the composition may comprise Bacillus, Paenibacillus, Pseudomonas (and in some embodiments, not Azospirillum) and is for use in coating seeds from members of the Leguminaceae family. Corresponding coated seeds are also provided.
  • the present composition comprises a consortium of synergistic, naturally-occurring soil bacteria. 2024.06.26 Seed Coating II Specification - PCT - P000587WO
  • bacteria in the consortium are rhizobacteria.
  • the seed coating composition is a bacterial, or bacterial-promoting, seed coating composition. This is because the non-polar liquid may be, in some embodiments, non-bactericidal.
  • the coating may also comprise, in some embodiments, other microbes, especially beneficial microbes or beneficial bacteria to assist the growth of the plant.
  • the coating could also include, in some embodiments, fungi or fungal spores. Examples may include Trichoderma sp.. Concentrations of Bacteria in the Mixture Bacteria of one species or another can, in some embodiments, be present at a minimum concentration of at least 5x10 ⁇ 7 CFU/g, at least 1x10 ⁇ 8 CFU/g or at least 2x10 ⁇ 8 CFU/g. Upper limits may be 1x10 ⁇ 9 CFU/g.
  • they can be present in concentrations of: at least 1x10 ⁇ 4 CFU/g; at least 1x10 ⁇ 5 CFU/g; at least 1x10 ⁇ 6 CFU/g; at least 5x10 ⁇ 6 CFU/g; at least 1x10 ⁇ 7 CFU/g; at least 5x10 ⁇ 7 CFU/g; at least 1x10 ⁇ 8 CFU/g; at least 5x10 ⁇ 8 CFU/g; at least 1x10 ⁇ 9 CFU/g; at least 5x10 ⁇ 9 CFU/g; or even a as high as at least 1x10 ⁇ 10 CFU/g or at least 2x10 ⁇ 8 CFU/g, at least 3x10 ⁇ 8 CFU/g, or at least 4x10 ⁇ 8 CFU/g, or at least 5x10 ⁇ 8 CFU/g.
  • the soluble powder contains approx. 750 million CFU/g to 1 billion CFU and the liquid seed coating will be approx. double that value. All concentrations values provided herein are in CFU per gram.
  • the gram refers to per gram dry weight of the mixture of the bacteria and the bacterial carrier substrate, optionally a powder. This is the dried bacterial carrier substrate, optionally a powder. Other equivalent measurements are envisioned.
  • Mixture The bacteria are preferably dried and preferably dormant, in stasis or sporulated as discussed herein. They are mixed with a bacterial carrier substrate, optionally a powder, to form the mixture.
  • the bacterial carrier substrate optionally a powder
  • the carrier is a medium that can convey or hold the bacteria.
  • the carrier conveys or holds the bacteria in sufficient quantities and keeps them viable at room temperature (approx.20 degrees C) and through the seed coating and sowing process.
  • the bacterial carrier powder is a polysaccharide.
  • the carrier powder may be described as a carbohydrate-based powder.
  • the bacterial carrier powder is not a monosaccharide.
  • the bacterial carrier powder is not glucose or dextrose or a mixture of monosaccharides, for example a mixture of glucose and dextrose. This exclusion also covers various hydrates of the monosaccharide, especially dextrose. Also excluded in some embodiments, is fructose, and again mixtures with glucose and /or other monosaccharides such as dextrose are also excluded in some embodiments.
  • the mixture may, in some embodiments, be referred to as an inoculant.
  • the bacteria are preferably dried and mixed in the bacterial carrier substrate, optionally a powder. Alternatively, they may be dried onto the powder, for example by using a using a vacuum or freeze drying method.
  • Suitable methods are known in the art, but may include spray drying or heat drying.
  • the mixture may comprise or may be formed exclusively of the bacterial carrier substrate, optionally a powder, and the consortium.
  • the mixture may comprise or may be formed exclusively of the bacterial carrier substrate, optionally a powder, and the consortium.
  • monosaccharide, disaccharide and polysaccharide powders of various levels of hydration that can be used as an alternative substrate for the microbial consortium.
  • Dextrose monohydrate is a preferred carrier powder if provided in addition to the polysaccharide carrier.
  • the bacterial carrier substrate, optionally a powder is present in the mixture at 97.6% w/w.
  • it is present in the 2024.06.26 Seed Coating II Specification - PCT - P000587WO mixture in at least 80% w/w; at least 85% w/w; at least 90% w/w; at least 95% w/w; at least 97% w/w; at least 98% w/w; at least 99% w/w; at least 99.5% w/w.
  • the powder may, in some embodiments, have a particle diameter in the range of 100 -300 micrometres. In some embodiments, the particle diameter is approximately 200 micrometres (+/- 10 or 20%).
  • Corn Starch In some embodiments, the carrier is cornstarch (corn starch). Corn starch is a common food ingredient and industrial product.
  • corn starch Physical and Chemical Properties ⁇ Appearance: Fine, white to off-white powder ⁇ Odour: Odourless ⁇ Taste: Bland ⁇ pH (10% solution): 4.5 - 7.0 ⁇ Moisture Content: ⁇ 14% ⁇ Ash Content: ⁇ 0.2% ⁇ Protein Content: ⁇ 0.35% ⁇ Fat Content: ⁇ 0.15% ⁇ Whiteness: ⁇ 88% ⁇ Bulk Density: 0.55 - 0.75 g/cm3 Particle Size Distribution The particle size distribution of corn starch is a crucial aspect, especially for its application in various industries.
  • ⁇ D10 (10% of particles are smaller than this size): 3 - 5 microns
  • D50 Median particle size: 10 - 15 microns
  • D90 90% of particles are smaller than this size: 20 - 30 microns
  • Functional Properties ⁇ Gelatinization Temperature: 62 - 72°C ⁇
  • Viscosity Depends on specific type and application; measured using a viscometer
  • Solubility Insoluble in cold water; forms a paste in hot water 2024.06.26 Seed Coating II Specification - PCT - P000587WO Manufacturing Process: One example of a method of manufacturing or process for providing a composition of the present invention is described below.
  • ⁇ The microbial consortium is dried onto corn starch.
  • o Dry product can be used by tossing it into the seed bag and shaking it or by stirring it into the seed in the planter box o corn starch powder at least 97.6% w/w o Minimum components, always present (*): ⁇ Pseudomonas putida (minimum) 1x10 ⁇ 8 CFU/g ⁇ Bacillus subtilis (minimum) 1x10 ⁇ 8 CFU/g ⁇ Bacillus licheniformis (minimum) 1x10 ⁇ 8 CFU/g ⁇ Bacillus amyloliquefaciens (minimum) 1x10 ⁇ 8 CFU/g ⁇ Bacillus megaterium (minimum) 1x10 ⁇ 8 CFU/g ⁇ Bacillus pumilus (minimum) 1x10 ⁇ 8 CFU/g ⁇ Paenibacillus polymyxa (minimum) 1x10 ⁇ 8 CFU/g ⁇ Azospirillum brasilense (minimum) 2x10 ⁇ 8 C
  • Example 1a Test in fields Materials and Methods In 2021, Wageningen University & Research, Open Teelten was commissioned by ForFarmers to test the application of a fertilizer additive, referred to as MaizeNP, in the crop maize.
  • the additive is added to the basic fertilization with 35m3 cattle slurry before sowing maize.
  • a granulated NS fertilizer 38 kg nitrogen per ha was applied.
  • Also included is a completely unfertilized object and an object with only cattle slurry without additive.
  • the trial was conducted in Vredepeel on moderately humous, loamy sandy soil. The test subjects are shown in Table S1.
  • MaizeNP is a highly concentrated consortium of microorganisms designed to solubilise Phosphate reserves in the soil and increase Nitrogen fixation in the rhizosphere. Enzymes produced by the MaizeNP microorganisms break P (Phosphorus) bonds between Al, Ca, and Fe. This process results in a plant accessible form of P.
  • MaizeNP is a microorganism consortium and not a single species product enabling it to be suited to all soil types. MaizeNP contains beneficial micro-organisms at a concentration 1BB CFU/g, is GM free and certified for use in organic farming.
  • Biolevel MaizeNP as a soluble powder contains approx.1 billion CFU, as a liquid seed coating it contains approx.2 billion CFU.
  • the numbers for MaizeNP powder add up to 750 million CFU/g and the MaizeNP liquid to 1.5 billion CFU/ml.
  • the work in this Example was done with the powdered product which shows in sum 750 million CFU.
  • the soluble powder contains approx.750 million CFU/g to 1 billion CFU and the liquid seed coating will be approx. double that value.
  • Biolevel MaizeNP had the largest increase (9.3%) in root weight and Tops weight (10.5%) followed by Biolevel MaizeNP @ 250g/ha -20% N which also had an increase in root weight (2.3%) and Tops weight (2.8%) compared to the farm standard.
  • Maize yield results were averaged for each treatment. Samples from each treatment were taken for analysis. Maize Analysis Summary: Based on the Maize analysis carried out there was no difference in the ME’s, Crude Protein, Acid Load or pH between treatments. Starch levels (key energy source) are greater in the Biolevel MaizeNP and Biolevel MaizeNP @ 250g/ha -20% N compared to the farm standard. Maize Yield Results:
  • Biolevel MaizeNP -20% Nitrogen treatment has demonstrated that there isn’t a reduction in yield, early growth (root, tops) or nutrient uptake compared to the field standard and has actually shown a slight increase. Based on these trial results, it indicates that Biolevel MaizeNP has achieved its role of making Nitrogen and Phosphorus more available to the plant which is allowing the key nutrients to carry out their function within the Maize crop of vegetative growth, root development and resulting in healthy yields. It wasn’t expected that Potassium would be made more available to the plant which is a key nutrient requirement in Maize for maturity, lodging and cob formation, so further trials and evidence of this would be of benefit.
  • Example 1c Biolevel Forage Maize Trial report 2021 Trial Report: Biolevel MaizeNP (Forage Maize) Materials and Methods Field Name: Top Field Product: Bio-level Maize NP 2024.06.26 Seed Coating II Specification - PCT - P000587WO Crop: Maize Group: Forage Soil Type: Sandy clay loam Date Planted: 20th May 2021 Application date: 20th May 2021 Field Area: 12.2 ha Trial Area: 8 ha Application rate: 250g in 200lt/ha water Table 1: Field Trial information and product application Treatments: Farm standard Biolevel @ 250g/ha Biolevel @ 250g/ha -20% N Trial: Compare Biolevel MaizeNP at normal and reduced nitrogen rates with standard farm practice.
  • Biolevel MaizeNP is critical to the growing of maize as Nitrogen is important for vegetative growth and grain production, phosphorus is essential for vigorous root development which improves establishment, cob weight and dry matter yield and Potassium demand from Maize is huge therefore inadequate take up can show symptoms such as irregular cob formation, late maturity and risk of lodging.
  • Biolevel MaizeNP has a place as part of the Integrated Crop Management of forage maize production now and certainly in the future.
  • Example 1d 2020 Forage Maize Trial.
  • Example 1e 2021 Forage Maize Trial. Materials and Methods code during seeding product Leaf A 40 m3 CS - - boron fertilization B 40 m3 CS 130 kg MM 25-0 MM Entec 25-0 E 40 m3 CS 130 kg MM 25-0+ MaizeNPMM Entec 25-0 + MaizeNP brought onto granulate 2024.06.26 Seed Coating II Specification - PCT - P000587WO Results Discussion The addition of Biolevel MaizeNP did increase the relevant yield measures dry matter yield and net energy for lactation (VEM).****************** Example 2 – Composition provides a stable CFU count over time at room temperature, without refrigeration Materials and Methods A mixture of the consortium in a saccharide powder is made up.
  • the consortium is prepared as follows: The consortium had the following minimum components (*) 2024.06.26 Seed Coating II Specification - PCT - P000587WO ⁇ Saccharide powder 97.6% w/w ⁇ Minimum components, always present (*): o Pseudomonas putida (minimum) 1x10 ⁇ 8 CFU/g o Bacillus subtilis (minimum) 1x10 ⁇ 8 CFU/g o Bacillus licheniformis (minimum) 1x10 ⁇ 8 CFU/g o Bacillus amyloliquefaciens (minimum) 1x10 ⁇ 8 CFU/g o Bacillus megaterium (minimum) 1x10 ⁇ 8 CFU/g o Bacillus pumilus (minimum) 1x10 ⁇ 8 CFU/g o Paenibacillus polymyxa (minimum) 1x10 ⁇ 8 CFU/g o Azospirillum brasilense (minimum) 5
  • the composition is kept at Room Temperature and Pressure. At various time intervals, the composition is plated out onto agar and the number of CFU (Colony-Forming Units) measured as is standard in the art. Results & Discussion Stability in a High CFU Count is achieved using a non-polar liquid without refrigeration. A higher, but still stable over time, CFU count is required in Corn seed coatings compared to Small Grain seed coatings. For ease of use refrigeration should be avoided.
  • the typical application rate for corn seed coatings in the market is 4 liter per ton of seed (as opposed to 2 liter for small grain).
  • the CFU loading of the seed coating can be sometimes as high as 2 billion CFU per ml.
  • a lower CFU count could mean that too much liquid would go onto the seed, which would not be desirable.
  • the powdered product can have a much higher CFU count when the mixture is suspended in a non-polar liquid such as an oil.
  • This finished product achieves stability while bringing the CFU count/ml or per gram to the desired level, all the while not requiring refrigeration.
  • the conclusion that is reached is that the CFU count is stable without the need for refrigeration of the composition. This is an improvement on other compositions which do require refrigeration in order to preserve CFU count over time.
  • Example 2 Mechanical Testing of the Composition applied to Seeds Materials and Methods
  • These are added into the funnel of a seed applicator (the Arktos model from Momesso (BR) from 2021) with a rotary mixer, where the seeds are spun with a centrifugal force whilst the liquid is added. Approximate spinning times were in the region of 10-15 mins.
  • the seeds are removed and inspected. They were allowed 10 to 20 mins to dry, although there was little aqueous moisture. Results Approximately 80-90% of the seeds are at least partially coated with the composition. This is expected to be sufficient when sown as the consortium bacteria will populate the rhizosphere of the coated seeds and also spread or at least provide nutrients (N and solubilized-P) to nearby seeds over time, especially as the rhizospheres develop and the bacteria multiply.
  • the coated seeds do not stick together or agglutinate in such a way that would prevent sowing in the soil by a mechanical seed applicator or sowing apparatus common in the field. Croozer/Afron Star insecticides are added to the coated seeds.
  • the trial also investigates the least effective rate in combination with a reduced fertilizer program (75%) that will be considered based on the growth, quality and yield of Maize compared to other nutrition programs.
  • Application of Biolevel NP at the rate of 250g and 500g in addition to 75% of the recommended fertilizer program significantly increased the growth, quality and yield components of maize across the four sites in Kenya.
  • Materials and Methods Study sites Trials were conducted at the Powa Agriconsult Trial Fields in Kithini-Machakos County, Waruhiu ATC-Kiambu County, Karii-Kirinyaga County and Maragua-Murang’a County.
  • Biolevel Maize was evaluated in 1 cropping season for maize, which was cultivated in open field and irrigated by drip system.
  • Seed dressing was done to the equivalent of product in 1L of water and the seeds soaked into the solution for 3 hours. Treatment application Treatments were done at sowing as per the application rate recommended by the manufacturer. Applications was done by seed dressing.
  • NB Conventional fertilizers were applied according to recommended/standard application practices i.e. NPK 23:23 at 5g/plant at planting and as CAN at 5g/plant as a top dress in split top-dress application with Ammonium Sulphate at tasselling at the rate of 5g per plant. The application of fertilizers was reduced by 25% on all the Biolevel Maize treatments i.e.
  • Results Assessment Schedule Assessment was done on growth, yield and quality parameters as shown in Table 5 during the trial periods in all the four sites due to treatment application. 2024.06.26 Seed Coating II Specification - PCT - P000587WO Table 5.
  • Activity, treatment application and assessment schedule NB The dates are for the Kiambu site which was set first followed by Kirinyaga, Machakos and lastly Murang’a at an interval of 3 days apart Effect of treatments on the growth parameters of maize There were significant differences between the growth and quality parameters of maize due to treatment application in Kiambu (Table 6). In the treatments where Biolevel NP was applied there was significant increase on the maize height, root zone diameter and the stand count which was comparable to the reference product. Also, the quality parameters of maize viz number of cobs per plant and the ear length were significantly increased on the Biolevel NP treatments except where it was used solely.
  • Table 7 Influence of treatments on the stand count, plant height, number of cobs per plant, ear length and root diameter of maize at Kirinyaga Treatments with the same letter along the columns are not significantly different according to DMRT at P ⁇ 0.05.
  • RFP-Recommended Fertilizer Program Significant differences (P ⁇ 0.05) were observed in Machakos on the Biolevel NP trial where the treatments of the combined test product with the conventional fertilizer program was 2024.06.26 Seed Coating II Specification - PCT - P000587WO comparable to the reference product in increasing the growth parameters i.e. the plant height and the root zone diameter and improving the stand establishment with over 98% (Table 8).
  • Biolevel NP Alone had lower effect compared to the integrated treatment probably because of the nutritional composition compared to what the full program will add to the soil.
  • the positive influence compared to the untreated control shows the efficacy of Biolevel NP and if used judiciously with the other nutritional soil supplements there would be greater fertility and nutritional improvement.
  • Table 8 Effect of test treatments on the plant height, root zone diameter, stand count, number of cobs per plant and the ear length of maize in Machakos Treatments with the same letter along the columns are not significantly different according to DMRT at P ⁇ 0.05.
  • Biolevel NP Average number of cobs per plant, ear length, green cob yield, fodder yield, cob weight and grain yield as influenced by treatments in Kiambu Treatments with the same letter along the columns are not significantly different according to DMRT at P ⁇ 0.05.
  • Table 13 Mean number of cobs per plant, ear length, weight of cobs, total green cob yield and grain yield as influenced by treatments in Murang’a Treatments with the same letter along the columns are not significantly different according to DMRT at P ⁇ 0.05.
  • RFP-Recommended Fertilizer Program Discussion Application of Biolevel NP at the rate of 250g and 500g in addition to 75% of the recommended fertilizer program significantly increased the growth, quality and yield components of maize across the four sites in Kenya. 2024.06.26 Seed Coating II Specification - PCT - P000587WO Example 5- Planter Box example using Corn starch Product We used the cornstarch as a dry planter box treatment for corn (maize).
  • the cornstarch adheres well to the seed, even if the seed is untreated (non-sticky surface).
  • the farmers often have planters with individual boxes for each row planted.
  • the cornstarch may also act as a glidant. It can remove any potential surface moisture from the seeds. The seed may then glide better in the apparatus. farmers typically apply talcum for that purpose. They can now switch from talcum to our planterbox treatment, meaning they get the desired action the talcum would provide + they coat the seed with our biology (bacteria consortia). A 5kg bucket of the present product is used to treat corn for sowing on a 125 acre site. The ratio between cornstarch and biology is such that both the desired amount of cornstarch (as a talc replacement glidant) and the desired amount of biology is going on the seed.

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Abstract

L'invention concerne une composition d'enrobage exempte de liquide ou en poudre sèche pour les semences, comprenant un mélange, le mélange comprenant : un consortium de bactéries ; et un substrat de support bactérien, facultativement une poudre. Le substrat de support bactérien, facultativement une poudre, est une poudre de polysaccharide, par exemple de l'amidon de maïs. Les bactéries du consortium sont des bactéries saprophytes et/ou des bactéries fixant l'azote et solubilisant le phosphore. Le mélange n'est pas en suspension dans un liquide non polaire.
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