AU2016207759B2

AU2016207759B2 - Agricultural composition

Info

Publication number: AU2016207759B2
Application number: AU2016207759A
Authority: AU
Inventors: Warren John BANKS; Craig Robert Bunt; Trevor Anthony Jackson; Per John WESSMAN; David Anthony WRIGHT
Original assignee: AgResearch Ltd
Current assignee: AgResearch Ltd
Priority date: 2015-01-13
Filing date: 2016-01-12
Publication date: 2019-05-16
Anticipated expiration: 2036-01-12
Also published as: WO2016113666A1; AU2016207759A1

Abstract

The present invention provides granules comprising a core, a binder and an exterior coating that at least partially coats the core and that reduces the water permeability of the granule wherein the exterior coating comprises a polymeric compound, in addition to methods of manufacturing and using such granules, for example, for controlling pests and improving plant health and production, such as treating the soil to reduce pests or to increase pasture production.

Description

AGRICULTURAL COMPOSITION

FIELD OF THE INVENTION [0001] The present invention relates to granules comprising a core comprising a substrate, a first coating comprising biological material, a second coating comprising one or 5 more particulate densifiers and one or more disintegrants, and an exterior coating that at least partially coats the core and that reduces the water permeability of the granule wherein the exterior coating comprises a polymeric compound, methods of preparing such granules, and application of such granules into or on to soil.

BACKGROUND TO THE INVENTION [0002] Biological materials, such as beneficial microorganisms including bacteria, are useful alternatives to chemical agents for the improvement and/or maintenance of soil and plant health, including the control of pests.

[0003] Biological materials are sensitive to environmental exposure, particularly desiccation and UV radiation. The utility of biological materials, for example, microbes, for 15 application into or on to soil is limited by the sensitivity of such materials to environmental conditions on the soil surface prior to entry into the soil and during storage of compositions comprising the biological material, such as granules.

[0004] There remains a need for granules comprising biological material that are suitable for application on to or into the soil and that adequately protect the biological 20 material in the granule until the biological material is released from the granule and enters the soil profile.

[0005] It is an object of the present invention to provide granules, an agricultural composition comprising granules, a method of making a granule or a method of distributing granules on to or into soil to control a pest that meet one or more of these needs, or to at 25 least provide the public with a useful choice.

SUMMARY OF THE INVENTION [0006] Described herein are granules comprising a core, the core comprising

a) a substrate,

b) a first coating that at least partially coats the substrate, the first coating comprising biological material, preferably one or more microorganisms, and optionally a biodegradable polymer and/or a non-cytotoxic oil, and

2016207759 24 Apr 2019

c) a second coating that at least partially coats the first coating, the second coating comprising one or more particulate densifiers, and one or more disintegrants, preferably a pre-gelatinised starch, and a binder, the granules further comprising an exterior coating that at least partially coats the core and 5 that reduces the water permeability of the granule, the exterior coating comprising a polymeric compound.

[0007] In a first aspect the invention provides granules comprising a core, the core comprising

a) a substrate selected from the group comprising a clay granule, a silicate mineral, an aluminosilicate mineral, vermiculite, a fertiliser granule, or a combination of any two or more thereof,

b) a first coating that at least partially coats the substrate, the first coating comprising biological material selected from one or more bacteria, one or more yeasts, one or more fungi, or one or more endophytes, or a combination of any two or more thereof, a biodegradable polymer and a non-cytotoxic oil, and

c) a second coating that at least partially coats the first coating, the second coating comprising one or more particulate densifiers, one or more disintegrants, and a binder, the granules further comprising an exterior coating that at least partially coats the core and 20 that reduces the water permeability of the granule, the exterior coating comprising a polymeric compound, the granules comprising about 10 to about 25 % by weight moisture.

[0008] Described herein is an agricultural composition comprising granules of the present invention.

[0009] In a second aspect the invention provides an agricultural composition comprising 25 granules of the first aspect.

[0010] Described herein is a method of producing a granule, the method comprising

a) providing a core comprising

i. a substrate, ii. a first coating that at least partially coats the substrate, the first coating comprising biological material, preferably one or more microorganisms, and optionally a biodegradable polymer and/or a non-cytotoxic oil, and

2016207759 24 Apr 2019 iii. a second coating that at least partially coats the first coating, the second coating comprising one or more one particulate densifiers, one or more disintegrants, and a binder, and

b) applying an exterior coating to the core to at least partially coat the core, the exterior coating comprising a polymeric compound.

[0011] In a third aspect the invention provides a method of making a granule, the method comprising

a) providing a core comprising

i. a substrate selected from the group comprising a clay granule, a silicate mineral, an aluminosilicate mineral, vermiculite, a fertiliser granule, or a combination of any two or more thereof, ii. a first coating that at least partially coats the substrate, the first coating comprising an aqueous concentrate of biological material selected from one or more bacteria, one or more yeasts, one or more fungi, or one or more endophytes, or a combination of any two or more thereof, a biodegradable polymer and a non-cytotoxic oil, and iii. a second coating that at least partially coats the first coating, the second coating comprising one or more particulate densifiers, one or more disintegrants, and a binder,

b) applying an exterior coating to the core to at least partially coat the core, the exterior coating comprising a polymeric compound, and

c) drying the granule to about 10 to about 25 % moisture by weight.

[0012] In a fourth aspect the invention provides a method of distributing granules on to or into soil to control a pest, preferably an insect pest, the method comprising

a) supplying granules of the first aspect to a distribution device, and

b) applying the granules on to or into soil using the distribution device.

[0013] Described herein is a method of distributing granules or a composition of the invention into or on to soil, the method comprising

a) supplying granules or a composition of the invention to a distribution device, and

b) applying the granules or composition into or on to soil using the distribution device.

[0014] Described herein is a method of treating the soil to control a pest, preferably an insect pest, comprising applying granules or a composition of the invention into or on to soil.

2016207759 24 Apr 2019 [0015] Described herein is a method of treating soil to maintain or increase plant growth, the method comprising applying granules or a composition of the invention into or on to soil.

[0016] Described herein is a method of treating soil of pastoral land to maintain or increase pasture production, the method comprising applying granules or a composition of the invention into or on to soil.

[0017] Described herein is use of granules or a composition of the invention to control pests, preferably insect pests.

[0018] Described herein is use of granules or a composition of the invention to maintain 10 or increase plant growth.

[0019] Described herein is use of granules or a composition of the invention to maintain or increase pasture production.

[0020] Any one or more of the following embodiments may relate to any of the aspects described herein or any combination thereof.

Core [0021] In various embodiments the first coating may coat at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% of the surface of the substrate, and suitable ranges may be selected between any of these values (for example, about 50 to about 100, about 60 to about 100, about 70 to about 100, about 80 to about

100, or about 90 to about 100%). In one embodiment the first coating coats at least about

90% of the surface of the substrate. In another embodiment the first coating fully coats the substrate.

[0022] In various embodiments the second coating may coat at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% of the first coating, and suitable ranges may be selected between any of these values (for example, about 50 to about 100, about 60 to about 100, about 70 to about 100, about 80 to about 100, or about 90 to about 100%). In one embodiment the second coating coats at least about 90% of the first coating. In another embodiment the second coating fully coats the first coating.

[0023] In various embodiments the substrate is selected from the group comprising a clay, a clay mineral, a seed, a pelletised grain, a granulate or an extruded granule, or a combination of any two or more thereof. In a preferred embodiment the substrate is

2016207759 24 Apr 2019 selected from the group comprising a clay, a clay mineral, a pelletised grain, a granulate or an extruded granule, or a combination of any two or more thereof.

[0024] In various embodiments the substrate is selected from the group comprising a clay granule, a silicate mineral, an aluminosilicate mineral, for example, zeolite, diatomaceous earth or perlite, vermiculite, a seed, for example, a vegetable seed, a fertiliser granule, or a combination of any two or more thereof. In various embodiments the substrate may comprise a seed, for example an angiosperm, vegetable, legume, cereal or conifer seed. In various embodiments the seed comprises a spinach, carrot, onion, soybean, lucerne, plantain, brassica, maize rye grass, canola or clover seed. In various embodiments the substrate may comprise wheat, barley, bran, maize, rye, rice, sorghum, millet, oats, forage brassica, canola or triticale, or a combination of any two or more thereof. In various embodiments the substrate may comprise a granule comprising a fertiliser, for example, a granule of urea, superphosphate, monoammonium phosphate (MAP), diammonium phosphate (DAP, single superphosphate (SSP), triple superphosphate (TSP), calcium ammonium nitrate (CAN) or muriate of potash (MOP).

[0025] In one embodiment the biodegradable polymer may comprise a polysaccharide. In one embodiment the biodegradable polymer may comprise an exopolysaccharide produced by a microorganism, for example, an exopolysaccharide produced by Achromobacter, Acetobacter, Acinetobacter, Agrobacterium, Alcaligenes, Aspergillus,

Aureobasidium, Aureomonas, Azotobacter, Bacillus, Beijerinckia, Lactobacillus, Lentinus, Leuconostoc, Mucorales, Pantoea stewartii, Pseudomonas, Rhizobium, Schizophylum, Sclerotium, Serratia, Sinorhizobium, Sphingomonas, Streptococcus, Xanthomonas, Zooglea, or Zymomonas spp.

[0026] In one embodiment the biodegradable polymer may comprise a gum such as a plant gum. In various embodiments the biodegradable polymer may be selected from the group comprising xanthan gum, agar, alginate, cassia, dammar, pectin, beta-glucan, glucomannan, mastic, chicle, psyllium, spruce gum, gellan gum, acacia gum, guar gum, locust bean gum, carrageenans, gum arabic, karaya gum, ghatti gum, tragacanth gum, konjac gum, tara gum, pullulan or a combination of any two or more thereof.

[0027] In one embodiment the biodegradable polymer may comprise a synthetic polysaccharide, for example a synthetic polymer of sucrose. In one embodiment the polysaccharide may comprise Ficoll®.

[0028] In various embodiments the first coating may comprise about 10, 15, 20, 25, 30, 25, 40, 45 or about 50% by weight of the biodegradable polymer, and useful ranges

2016207759 24 Apr 2019 may be selected between any of these values, for example, from about 10% to about 50% by weight.

[0029] In one embodiment the first coating may comprise a non-cytotoxic oil. In various embodiments the non-cytotoxic oil may comprise a plant, animal, paraffin or mineral oil.

For example, in one embodiment the non-cytotoxic oil may be selected from the group comprising canola, olive, sunflower seed, palm, coconut, corn, peanut, safflower, sesame, soybean, peanut, wheat germ, rice bran, flaxseed, palm or grapeseed oil, hydrolyzed oils, or a combination of any two or more thereof. In another embodiment, the non-cytotoxic oil may comprise a marine oil, for example a fish, seal, krill or seaweed oil. In a further embodiment the non-cytotoxic oil may comprise Excel® Oil, Excel® Organic Oil, Organic JMS Stylet-Oil or Syn Oil.

[0030] In various embodiments the first coating may comprise about 10, 15, 20, 25, 30, 25, 40, 45 or about 50% by weight of the non-cytotoxic oil, and useful ranges may be selected between any of these values, for example, from about 10% to about 50% by weight.

[0031] In various embodiments the biological material may comprise one or more microorganisms, hormones, pheromones, spores, proteins ora combination of any two or more thereof. In various embodiments the biological material may comprise bacteria, yeast, fungi, or an endophyte. In various embodiments the biological material is agriculturally and/or horticulturally useful, for example, the biological material is pesticidal and/or insecticidal, and/or supports plant growth and/or development, or any combination thereof.

[0032] In various embodiments the biological material may comprise Serratia (for example, Serratia entomophila or Serratia proteomaculans), Xanthamonas, Pseudomonas, Rhizobium, Beauveria, Penicillium, Metarhizium, Bifidobacterium, Lactobacillus,

Streptococcus (Enterococcus), Yersinia (for example, Yersinia entomophaga) Trichoderma, Pseudomonas, Bacillus, Pasteuria, Azobacter, Enterobacter, Azospirillum, Cyanobacteria, Gliocladium, Coniotherium, Verticillium, Paecilomyces, Streptomycetes, Chromobacterium, Rhanella, Burkholderia, Paenibacillus, Collimonas, Sinorhizobium, Pantoea, Lecanicillum, Erwinia, Pediococus, Sclerotinia, Leuconostoc, Aeromonas, Neptunomonas, Klebsiella,

Ponchonia, Brevibacillus, Acinetobacter or a combination of any two or more thereof. In one embodiment the biological material may comprise one or more bacteria and one or more fungi. In one exemplary embodiment the biological material comprises Serratia entomophila and Metarhizium anisopliae. In various embodiments the protein may comprise a metabolite produced by a microorganism, for example a toxin produced by a

Yersinia, Bacillus or Metarhizium species. In a preferred embodiment the biological material

2016207759 24 Apr 2019 may comprise Serratia entomophila, Pseudomonas chloraphis, Burkholderia terricola, Yersinia entomophaga, Serratia proteomaculans, Penicillium janczewskii, Beauveria bassiana, Metarhizium anisopliae, Bacillus subtilis, or a combination of any two or more thereof.

[0033] In various embodiments the first coating may comprise about 10, 15, 20, 25,

30, 25, 40, 45, 50, 55, 60, 65, 70, 75 or about 80% by weight of a composition comprising the biological material, and useful ranges may be selected between any of these values, for example, from about 10% to about 80% by weight.

[0034] In one embodiment the first coating may further comprise one or more layers of 10 one or more particulate densifiers as described below.

[0035] In one embodiment the granules may further comprise an agricultural agent, that is, an agent that is beneficial for agricultural and/or horticultural purposes. In various embodiments the granules may comprise one or more fertilisers, one or more trace elements, one or more nitrification inhibitors, one or more urease inhibitors, one or more plant growth promoters, such as hormones, or one or more pesticides, for example fungicides. For example, in one embodiment the agricultural agent may comprise a NPK fertiliser. In various embodiments the agricultural agent may comprise one or more plant hormones selected from the group comprising gibberellins, auxins, cytokinins, abscisic acid, ethylene, salicylic acid, brassinosteroids, jasmonates, peptide hormones, polyamines, nitric oxide, and strigolactones, or a combination of any two or more thereof.

[0036] In various embodiments the granules may further comprise a surfactant. In various embodiments the surfactant is selected from the group comprising nonionic surfactants, anionic surfactants, cationic surfactants and/or amphoteric surfactants. Examples of surfactants include but are not limited to Tween™ and Triton™ (Rohm and Hass

Company), Fortune®, Pulse, C. Daxoil®, Codacide oil®, D-C. Tate®, Supamet Oil, Bond®, Penetrant, Giowelt® and Freeway, Citowett®, Fortune Plus™, Fortune Plus Lite, Fruimec, Fruimec lite, alkali metal, alkaline earth metal and ammonium salts of aromatic sulfonic acids, e.g., ligninsulfonic acid, phenolsulfonic acid, naphthalenesulfonic acid and dibutylnaphthalenesulfonic acid, and of fatty acids, alkyl and alkylaryl sulfonates, and alkyl, lauryl ether and fatty alcohol sulfates, and salts of sulfated hexadecanols, heptadecanols, and octadecanols, salts of fatty alcohol glycol ethers, condensation products of sulfonated naphthalene and naphthalene derivatives with formaldehyde, condensation products of naphthalene or naphthalenesulfonic acids with phenol and formaldehyde, polyoxyethylene octylphenol ethers, ethoxylated isooctylphenol, ethoxylated octylphenol and ethoxylated nonylphenol, alkylphenol polyglycol ethers, tributylphenyl polyglycol ethers, alkylaryl

2016207759 24 Apr 2019 polyether alcohols, isotridecyl alcohol, fatty alcohol ethylene oxide condensates, ethoxylated castor oil, polyoxyethylene alkyl ethers, ethoxylated polyoxypropylene, lauryl alcohol polyglycol ether acetal, sorbitol esters, lignin-sulfite waste liquors and methyl cellulose.

[0037] In various embodiments the granules may further comprise a lubricant. In various embodiments the lubricant is selected from the group comprising a lipid such as a fat or oil, for example magnesium stearate, vegetable stearin, or stearic acid, or a mineral, for example talc or silica.

[0038] In various embodiments the particulate densifier may be selected from the group comprising a clay, a silicate mineral, a calcium salt, or a volcanic glass. In various embodiments the particulate densifier may be selected from the group comprising a sulphate mineral, an aluminosilicate, a phyllosilicate, a clay mineral, a silicate, or a combination of any two or more thereof. In various embodiments the particulate densifier may be selected from the group comprising bentonite, montmorillonite, gypsum, zeolite, kaolin, calcium carbonate, calcium phosphate, perlite, celite, diatomaceous earth, talc, silicon dioxide, or a combination of any two or more thereof. In a preferred embodiment the particulate densifier comprises bentonite, gypsum, or bentonite and gypsum. In a particularly contemplated embodiment the particulate densifier comprises bentonite and gypsum.

[0039] In various embodiments the disintegrant may comprise a starch, preferably a pre-gelatinised starch, a cross-linked polymer, or an effervescent agent. In various embodiments the disintegrant may comprise potato starch, peat, rice powder, corn starch, sodium starch glycolate, cellulose, methylcellulose, sodium alginate, polyvinylpyrrolidone, croscarmellose sodium, alginic acid, citric acid, sodium bicarbonate or tartaric acid or a combination of any two or more thereof.

[0040] In various embodiments the granules may comprise a particulate densifier in an amount of about 0.1, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8 fold relative to the mass of the core, and useful ranges may be selected between any of these values, for example, from about 0.25 to about 8, about 0.25 to about 5, about 0.25 to about 3, about 0.5 to about 8, about 0.5 to about 5, about 0.5 to about 3, about 1 to about

8, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 3 to about 8, about 3 to about 7, about 3 to about 6, about 3 to about 5, about 4 to about 8, about 4 to about 7, about 4 to about 6, about 5 to about 8, about 5 to about 7, or about 6 to about 8 fold. In a particularly contemplated embodiment the granules comprise particulate densifier in an amount of from about 1 fold to about 4 fold relative to the mass of the core.

2016207759 24 Apr 2019 [0041] In various embodiments the second coating may comprise at least about 50, 60, 70, 75, 80, 85, 90, 95 or about 99% by weight of particulate densifier, and useful ranges may be selected between any of these values, for example, from about 50 to about 99, about 50 to about 95, about 50 to about 90, about 50 to about 85, about 50 to about 80, about 50 to about 75, about 60 to about 99, about 60 to about 95, about 60 to about90, about 60 to about 85, about 70 to about 99, about 70 to about 95, about 70 to about90, about 70 to about 85, about 80 to about 99, about 80 to about 95, about 80 to about90, about 90 to about 99, or about 90 to about 99% by weight of particulate densifier.

[0042] In various embodiments the second coating may comprise about 0.1, 0.5, 1, 2, 10 3, 4, 5, 6, 7, 8, 9, 10, 12.5, 13, 14 or about 15% by weight of disintegrant, and useful ranges may be selected between any of these values, for example, from about 0.1 to about

15, about 0.1 to about 12.5, about 0.1 to about 10, about 0.1 to about 8, about 0.1 to about 6, about 0.5 to about 15, about 0.5 to about 12.5, about 0.5 to about 10, about 0.5 to about 8, about 0.5 to about 6, about 1 to about 15, about 1 to about 12.5, about 1 to about 10, about 1 to about 9, about 1 to about 8, about 1 to about 7, about 2 to about 15, about 2 to about 12.5, about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 3 to about 15, about 3 to about 12.5, about 3 to about 10, about 3 to about 9, about 3 to about 8, about 3 to about 7, about 4 to about 15, about 4 to about 12.5, about 4 to about 10, about 4 to about 9, about 4 to about 8, about 4 to about 7, about 5 to about 15, about 5 to about 12.5, about 5 to about 10, about 5 to about 9, about 5 to about 8, about 5 to about 7%, by weight of disintegrant.

[0043] In various embodiments the mass of the second coating applied to the granules may be about 0.1:1, 0.25:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1 or 8:1 relative to the combined mass of the substrate and first coating. Useful ranges may be selected between 25 any of these values, for example, from about 0.1:1 to about 8:1, about 0.1:1 to about 6:1, about 0.1:1 to about 5:1, about 0.1:1 to about 4:1, about 0.5:1 to about 8:1, about 0.5:1 to about 7:1, about 0.5:1 to about 6:1, about 0.5:1 to about 5:1, about 0.5:1 to about 4:1, about 1:1 to about 8:1, about 1:1 to about 8:1, about 1:1 to about 7:1, about 1:1 to about 6:1, about 1:1 to about 5:1, about 1:1 to about 4:1, about 2:1 to about 8:1, about 2:1 to about 6:1, about 2:1 to about 5:1, or about 2:1 to about 4:1 relative to the mass of the substrate and first coating.

[0044] In one embodiment the granules may comprise at least one layer of the second coating. In one embodiment the granules may comprise two or more layers of the second coating comprising alternating layers of the binder and the second coating.

2016207759 24 Apr 2019 [0045] In various embodiments the binder may comprise one or more polymeric compounds, as described below in relation to the exterior coating. In various embodiments the binder may comprise one or more polyhydroxyl compounds as described below.

[0046] In various embodiments the polymeric compound may be a polyhydroxyl compound, a polymer or copolymer of a C1-C6 alkyl substituted with one or more groups selected from the group comprising lactams or 2-pyrollidone, a shellac resin, a disaccharide, a polyelectrolyte, a polyethylene glycol, a polyethylene oxide, a polyacrylamide, a polyester, a cellulose, a lignin, a biodegradable gum, an oligopeptide, a polypeptide or a latex.

[0047] In various embodiments the binder may comprise a polymer or copolymer of a

C1-C6 alkyl substituted with one or more groups selected from the group comprising lactams, preferably γ-lactam, or 2-pyrollidone. In one embodiment the binder may comprise a polyvinylpyrrolidone.

[0048] In various embodiments the binder may comprise shellac resin, a disaccharide, an oligosaccharide, a polysaccharide, a cellulose ora starch.

[0049] In various embodiments the binder may be agriculturally and/or horticulturally acceptable, non-cytotoxic, food grade and/or biodegradable.

[0050] In various embodiments the granules may comprise about 0.01, 0.025, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9 or 1% of the binder relative to the mass of the core of the granules, and useful ranges may be selected between any of these values, for example, from about 0.01 to about 0.1, 0.05 to about 0.4, about 0.05 to about 0.35, about 0.05 to about 0.3, about 0.05 to about 0.25, about 0.05 to about 0.2, about 0.15 to about 1, about 0.1 to about 0.4, about 0.1 to about 0.35, about 0.1 to about 0.3, about 0.1 to about 0.25, about 0.1 to about 0.2, about 0.15 to about 1, about 0.15 to about 0.4, about 0.15 to about 0.35, about 0.15 to about 0.3, about 0.15 to about 0.25, about 0.15 to about 0.2, about 0.2 to about 1, about 0.1 to about 0.4, about 0.1 to about 0.35, about 0.1 to about 0.3, about 0.1 to about 0.25, about 0.1 to about 0.2, about 0.2 to about 1, about 0.2 to about 0.4, about 0.2 to about 0.35, about 0.2 to about 0.3, about 0.25 to about 1, about 0.25 to about 0.4, about 0.25 to about 0.35, about 0.25 to about 0.3, about 0.3 to about 1, about 0.3 to about 0.4, or about 0.4 to about 1% binder relative to the mass of the granules.

Exterior coating [0051] In various embodiments the exterior coating may be agriculturally and/or horticulturally acceptable, non-cytotoxic, food grade and/or biodegradable.

2016207759 24 Apr 2019 [0052] In one embodiment the exterior coating may comprise one or more or two or more polymeric compounds.

[0053] In various embodiments the polymeric compound may be a polyhydroxyl compound, a polymer or copolymer of a C1-C6 alkyl substituted with one or more groups selected from the group comprising lactams or 2-pyrollidone, a shellac resin, a disaccharide, a polyelectrolyte, a polyethylene glycol, a polyethylene oxide, a polyacrylamide, a polyester, a cellulose, a lignin, a biodegradable gum, an oligopeptide, a polypeptide or a latex.

[0054] In various embodiments the polymeric compound may be a polyhydroxyl compound, a polymer or copolymer of a C1-C6 alkyl substituted with one or more groups 10 selected from the group comprising lactams or 2-pyrollidone, for example, polyvinylpyrollidone, or a latex.

[0055] In various embodiments the polyhydroxyl compound may be selected from the group comprising a polyvinyl alcohol, a polyvinyl alcohol co-polymer, a polysaccharide, or an oligosaccharide. In one embodiment the polyhydroxyl compound may be a polyvinyl 15 alcohol-polyethylene glycol co-polymer (e.g. Kollicoat® Protect). In a particularly preferred embodiment the polyhydroxyl compound is a polyvinyl alcohol.

[0056] In various embodiments the polyhydroxyl compound may be selected from the group comprising a polyvinyl alcohol, a polyethylene oxide, methyl cellulose, a polyvinyl alcohol-polyethylene glycol co-polymer (e.g. Kollicoat® Protect) or gum arabic. In a particularly preferred embodiment the polyhydroxyl compound is a polyvinyl alcohol.

[0057] In various embodiments the polysaccharide may be selected from the group comprising a starch, a dextrin, chitosan, a gum or a synthetic polysaccharide. In various embodiments the starch may be potato starch or maize starch. In one embodiment the starch is a pre-gelatinised starch. In one embodiment the dextrin may be maltodextrin. In 25 various embodiments the gum may be xanthan gum or gum arabic. In one embodiment the synthetic polysaccharide may be Ficoll®.

[0058] In one embodiment the polyhydroxyl compound may be a polymer or copolymer of (1) a C2-C6 hydroxyalkyl, or (2) a C2-C10 hydroxyalkyl, optionally substituted with acyl and/or optionally comprising an ether moiety, for example, methacrylic acid-ethyl acrylate copolymer (Kollicoat® MAE100).

2016207759 24 Apr 2019 [0059] In various embodiments the polyelectrolyte may be polyacrylic acid or xanthan gum.

[0060] In one embodiment the polymer or copolymer of a Οι-Οβ alkyl may be substituted with one or more γ-lactam groups. In one embodiment the polymer or copolymer of a Οι-Οβ alkyl substituted with one or more groups selected from the group comprising lactams or 2-pyrollidone is polyvinylpyrollidone.

[0061] In one embodiment the polyester is an aliphatic polyester. In one embodiment the polyester is polylactic acid. In various embodiments the cellulose may be selected from the group comprising methyl cellulose, carboxymethylcellulose, hydroxypropyl methylcellulose and hydroxyethyl cellulose. In various embodiments the biodegradable gum may be an exopolysaccharide produced by a microorganism or a plant gum as described above. In various embodiments the biodegradable gum may be xanthan gum or gum arabic. In various embodiments the polypeptide is gluten or gelatin. In one embodiment the latex is EC 23009.

[0062] In various embodiments the polymeric compound may be a compound selected from the group comprising copolymers of vinyl pyrrolidone, and vinyl acetate, poly (methyl vinyl ether), maleic anhydride copolymers, free acids of the copolymer of methyl vinyl ether and maleic anhydride, vinyl pyrrolidone/styrene copolymers, vinyl acetate/butyl acrylate copolymers, vinyl acetate homopolymers, acrylic copolymers, styrene/acrylic ester copolymers, vinyl acetate/ethylene copolymers and polyvinyl acetate, or a combination of any two or more thereof.

[0063] In one embodiment the exterior coating and/or the polymeric compound may be a latex polymer, for example, EC 23009.

[0064] In various embodiments the polyhydroxyl compound may comprise at least 2, 3,

4, 5, 10, 20, 50, 100, 200, 300, 400, 500, 750, 800, 1,000, 1,200, about 1,500, about

2,000, about 3,000, about 4,000 or at least about 5,000 hydroxyl groups, and useful ranges may be selected between any of these values, for example, from about 2 to about 5,000, about 2 to about 3,000, about 2 to about 1,500, about 2 to about 1,000, about 2 to about 800, about 2 to about 500, about 2 to about 300, about 2 to about 50, about 10 to about

5,000, about 10 to about 3,000, about 10 to about 1,500, about 10 to about 1,000, about to about 500, about 10 to about 300, about 10 to about 100, about 10 to about 50, about 50 to about 5,000, about 50 to about 4,000, about 50 to about 3,000, about 50 to about 1,500, about 50 to about 1,000, about 50 to about 800, about 50 to about 500, about 50 to about 200, about 100 to about 5,000, about 100 to about 3,000, about 100 to about

1,500, about 100 to about 1,000, about 100 to about 800, about 100 to about 500, about

2016207759 24 Apr 2019

300 to about 5,000, about 300 to about 3,000, about 300 to about 1,500, about 300 to about 1,000, about 300 to about 800, about 300 to about 500, about 500 to about 5,000, about 500 to about 4,000, about 500 to about 3,000, about 500 to about 1,500, about 500 to about 1,000, about 800 to about 5,000, about 800 to about 4,000, about 800 to about

3,000, about 800 to about 1,500, about 800 to about 1,000, about 1,000 to about 5,000, about 1,000 to about 4,000, about 1,000 to about 3,000, about 1,000 to about 2,000, about 1,500 to about 5,000, about 1,500 to about 4,000, or from about 1,500 to about 3,000 hydroxyl groups.

[0065] In various embodiments the polymeric compound may comprise a polymer of a monomer comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hydroxyl groups, and useful ranges may be selected between any of these values, for example, from about 1 to about 10, about 1 to about 8, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 2 to about 10, about 2 to about 8, about 2 to about 6, about 2 to about 4, about 3 to about 10, about 3 to about 8, about 3 to about 6, about 3 to about 5, about 4 to about 10, about 4 to about 8, about 4 to about 7, about 4 to about 6, about 5 to about 10, about 5 to about 9, about 5 to about 8, about 5 to about 7, about 6 to about 10, about 6 to about 9, about 6 to about 8, about 7 to about 10, about 7 to about 9, or about 8 to about 10 hydroxyl groups.

[0066] In various embodiments the polymeric compound may have an average molecular weight of about 1, 2, 5, 10, 20, 22.5, 24.5, 25, 30, 40, 50, 75, 80, 90, 100, 118, 120, 125, 126, 130, 140, 150, 200, 250, 300 or about 350 kDa, and useful ranges may be selected between any of these values, for example, from about 1 to about 350, 5 to about 350, about 5 to about 250, about 5 to about 200, about 5 to about 150, about 5 to about 125, about 5 to about 100, about 5 to about 75, about 5 to about 50, about 10 to about

350, about 10 to about 250, about 10 to about 200, about 10 to about 150, about 10 to about 100, about 10 to about 75, about 10 to about 50, about 25 to about 350, about 25 to about 250, about 25 to about 200, about 25 to about 150, about 25 to about 125, about 25 to about 100, about 25 to about 75, about 25 to about 150, about 25 to about 125, about 25 to about 100, about 25 to about 75, about 50 to about 350, about 50 to about 250, about 50 to about 200, about 50 to about 150, about 50 to about 150, about 50 to about 100, about 75 to about 350, about 75 to about 250, about 75 to about 200, about 75 to about 150, about 75 to about 100, about 100 to about 350, about 100 to about 250, about 100 to about 200, about 100 to about 150, or from about 100 to about 150 kDa.

[0067] In various embodiments the polyhydroxyl compound may have a degree of hydrolysis of at least about 80, 82.5, 85, 87.5, 90, 92.5, 95 or 98 mol%, and useful ranges may be selected between any of these values, for example, from about 80 to about 100,

2016207759 24 Apr 2019 about 80 to about 98, about 80 to about 95, about 80 to about 90, about 85 to about 100, about 85 to about 98, about 85 to about 95, about 85 to about 90, about 90 to about 100, about 90 to about 98, or from about 90 to about 95 mol%.

[0068] In various embodiments the exterior coating and/or the binder may further comprise a plasticiser, for example, an acetylated monoglyceride or alkyl citrate, a polyethylene glycol or monopropylene glycol.

[0069] In various embodiments, the binder, exterior coating and/or second coating may comprise a dye, for example, Rhodamine B500, Methyl Violet, Blue 2313, Eosine Y, Sunset Yellow, Magenta, Blue 23123, Pigment Green 7, Tartrazine, Malachite Green,

Auramine 0, Oil Yellow 21756, Green 19102 and Methylene Blue 2B, or titanium dioxidecoated micas (lustres) such as 100 Silver Pearl, 120 Lustre Pearl, 235 Green Pearl, 300 Gold Pearl, 500 Bronze Pearl and 504 Red Pearl.

[0070] In various embodiments the granules may comprise about 0.01, 0.025, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9 or 1% of the exterior coating relative to the mass of the core of the granules, and useful ranges may be selected between any of these values, for example, from about 0.01 to about 0.1, 0.05 to about 1, 0.05 to about 0.4, about 0.05 to about 0.35, about 0.05 to about 0.3, about 0.05 to about 0.25, about 0.05 to about 0.2, about 0.15 to about 1, about 0.1 to about 0.4, about 0.1 to about 0.35, about 0.1 to about 0.3, about 0.1 to about 0.25, about 0.1 to about 0.2, about

0.15 to about 1, about 0.15 to about 0.4, about 0.15 to about 0.35, about 0.15 to about

0.3, about 0.15 to about 0.25, about 0.15 to about 0.2, about 0.2 to about 1, about 0.1 to about 0.4, about 0.1 to about 0.35, about 0.1 to about 0.3, about 0.1 to about 0.25, about 0.1 to about 0.2, about 0.2 to about 1, about 0.2 to about 0.4, about 0.2 to about 0.35, about 0.2 to about 0.3, about 0.25 to about 1, about 0.25 to about 0.4, about 0.25 to about

0.35, about 0.25 to about 0.3, about 0.3 to about 1, about 0.3 to about 0.4, or about 0.4 to about 1% exterior coating relative to the mass of the core of the granules.

[0071] In one embodiment the exterior coating may further comprise a particulate material, the particulate material comprising particles having a mean size of from about 0.1 pm to about 50 pm.

[0072] In various embodiments the particulate material may be selected from the group comprising a phyllosilicate, for example, attapulgite, an aluminosilicate, for example zeolite, a clay mineral, for example kaolin or kaolin/TiCb, a sulphate mineral, for example, gypsum, a silicate, for example talc, silica, microsilica, or a combination of any two or more thereof. In one embodiment the particulate material is selected from the group comprising hydrophilic fumed silica, amorphous silica, microsilica and kaolin/TiCD. In a preferred

2016207759 24 Apr 2019 embodiment the particulate material is selected from the group comprising hydrophilic fumed silica, amorphous silica and microsilica. In a particularly contemplated embodiment the particulate material is microsilica.

[0073] In various embodiments the particulate material may comprise particles having a mean size of about 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5 or about 50 pm, and useful ranges may be selected between any of these values, for example, from about 0.1 to about 50 pm, about 0.1 to about 40, about 0.1 to about 30, about 0.1 to about 20, about 0.1 to about 10, about 0.5 to about 50, about 0.5 to about 40, about 0.5 to about 30, about 0.5 to about 20, about 0.5 to about

100, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, about 5 to about 50, about 5 to about 40, about 5 to about 30, about 5 to about 20, about 5 to about 10, about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, about 20 to about 50, about 20 to about 40, about 20 to about 30, about 30 to about 50, about 30 to about 40, or about 40 to about 50 pm.

[0074] In various embodiments the particulate material may comprise particles having a D90% of about 0.1, 0.5, 1, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5 30, 32.5, 35,

37.5, 40, 42.5, 45, 47.5 or about 50 pm, and useful ranges may be selected between any of these values, for example, from about 0.1 to about 50 pm, about 2 to about 50, about 2 to about 20, about 2 to about 15, about 2 to about 10, about 5 to about 50, about 5 to about

20, about 5 to about 15 or about 5 to about 10 pm.

[0075] In various embodiments the particulate material may comprise particles having a D50% of about 0.1, 0.5, 1, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5 30, 32.5, 35,

37.5, 40, 42.5, 45, 47.5 or about 50 pm, and useful ranges may be selected between any of these values, for example, from about 0.1 to about 50 pm, about 0.1 to about 20, about 0.1 to about 5 about 0.1 to about 2, about 0.5 to about 50 pm, about 0.5 to about 20, about

0.5 to about 5 about 0.5 to about 2, about 1 to about 50 pm, about 1 to about 20, about 0 to about 5 or about 0.1 to about 2.

[0076] In various embodiments the granules may comprise about 0.5, 1, 1.5, 2, 2.5, 3,

3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8% of particulate material relative to the mass of the granules, and useful ranges may be selected between any of these values, for example, from about 0.5 to about 8, about 0.5 to about 7, about 0.5 to about 6, about 0.5 to about 5, about 0.5 to about 4, about 0.5 to about 3, about 1 to about 8, about 1 to about 8% by weight. In a particularly contemplated embodiment the granules comprise from about 2 to about 5% particulate material.

2016207759 24 Apr 2019 [0077] In various embodiments, the particulate material may coat at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% of the granule surface, and suitable ranges may be selected between any of these values. For example, the particulate material may coat at least about 10 to about 95, about 10 to about 90, about 10 to about 80, about 20 to about 95, about 20 to about 90, about 20 to about 80, about 30 to about 95, about 30 to about 90, about 30 to about 80, about 40 to about 95, about40 to about 90, about 40 to about 80, about 50 to about 95, about 50 to about 90, about50 to about 80, about 60 to about 95, about 60 to about 90, about 70 to about 95, about70 to about 90, about 70 to about 80, about 80 to about 95, or about 80 to about 90, about 50 to about 100, about 60 to about 100, about 70 to about 100, about 80 to about 100, or about to about 100% of the granule surface. In one particularly contemplated embodiment the particulate material coats at least about 90% of the granule surface. In another embodiment the particulate material fully coats the granule surface.

Granule properties [0078] In various embodiments the granules may comprise up to about 5, 6, 7, 8, 9,

10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or about 25% by weight moisture and useful ranges may be selected between any of these values. For example, the granules comprises from about 5 to about 25, 5 to about 20, about 5 to about 17, about 5 to about 16, about 5 to about 15, about 5 to about 14, about 5 to about 13, about 5 to about 12, about 5 to about 11, about 5 to about 10, 8 to about 25, about 8 to about 20, about 8 to about 17, about 8 to about 16, about 8 to about 15, about 8 to about 14, about 8 to about

13, about 8 to about 12, about 8 to about 11, about 8 to about 10, 10 to about 25, about to about 20, about 10 to about 17, about 10 to about 16, about 10 to about 15, about 10 to about 14, about 10 to about 13, about 10 to about 12, about 10 to about 11, 11 to about 25, about 11 to about 20, about 11 to about 19, about 11 to about 18, about 11 to about 17, about 11 to about 16, about 11 to about 15, 12 to about 25, about 12 to about 20, about 12 to about 17, about 12 to about 16, about 12 to about 12, about 12 to about

14, about 12 to about 13% moisture. In a particularly contemplated embodiment the granules comprise up to about 14% by weight moisture.

[0079] In various embodiments the granules have a water activity of about 0.5, 0.55,

0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 0.98, or about 0.99, and useful ranges may be selected between any of these values. For example, the water activity of the granules is about 0.5 to about 0.99, 0.5 to about 0.98, 0.5 to about 0.95, 0.5 to about 0.9, 0.5 to about 0.85, 0.5 to about 0.8, 0.5 to about 0.75, 0.5 to about 0.7, 0.6 to about 0.99, 0.6 to about 0.98, 0.6 to about 0.95, 0.6 to about 0.9, 0.6 to about 0.85, 0.6 to about 0.8, 0.6 to about 0.75, 0.6 to about 0.7, 0.65 to about 0.99, 0.65 to about 0.98, 0.65 to about 0.95,

2016207759 24 Apr 2019

0.65 to about 0.9, 0.65 to about 0.85, 0.65 to about 0.8, 0.65 to about 0.75, 0.7 to about

0.99, 0.7 to about 0.98, 0.7 to about 0.95, 0.7 to about 0.9, 0.7 to about 0.85, 0.7 to about 0.8, 0.75 to about 0.99, 0.75 to about 0.98, 0.75 to about 0.95, 0.75 to about 0.9, 0.75 to about 0.85, 0.75 to about 0.8, 0.8 to about 0.99, 0.8 to about 0.98, 0.8 to about 0.95, 0.8 to about 0.9, 0.85 to about 0.99, 0.85 to about 0.98, or about 0.85 to about 0.95.

[0080] In various embodiments the granules may comprise at least about 1 x 10⁵, 2.5 x 10⁵, 5 x 10⁵, 7.5 x 10⁵, 1 x 10⁶, 2 x 10⁶, 2.5 x 10⁶, 5 x 10⁶, 7.5 x 10⁵, 1 x 10⁷, 2 x 10⁷, 2.5 x 10⁷, 5 x 10⁷, 7.5 x 10⁷, 1 x 10⁸, 2 x 10⁸, 2.5 x 10⁸, 5 x 10⁸, 7.5 x 10⁸, 1 x 10⁹, 2 x 10⁹, 2.5 x 10⁹, 5 x 10⁹, 7.5 x 10⁹ or about 1 x 10¹⁰ cfu (colony forming units) of the biological material per gram of granules after 1, 2, 3, 4, 5, 6, 7 days, or 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 24 months storage at ambient temperature, and useful ranges may be selected between any of these values, for example, from about 1 x 10⁵ to about 1 x 10¹⁰, about 1 x 10⁵ to about 7.5 x 10⁹, about 1 x 10⁵ to about 5 x 10⁹, about 1 x 10⁵ to about 1 x 10⁹, about 1 x 10⁵ to about 5 x 10⁸, about 1 x 10⁵ to about 1 x 10⁸, about 1 x 10⁵ to about 5 x 10⁷, about 1 x 10⁵ to about 1 x 10⁷, about 1 x

10⁵ to about 5 x 10^s, about 1 x 10⁵ to about 1 x 10⁵, about 5 x 10⁵ to about 1 x 10¹⁰, about x 10⁵ to about 1 x 10⁹, about 5 x 10⁵ to about 1 x 10⁸, about 5 x 10⁵ to about 1 x 10⁷, about 1 x 10⁶ to about 1 x 10¹⁰, about 1 x 10⁶ to about 1 x 10⁹, about 1 x 10⁶ to about 1 x 10⁸, about 1 x 10⁶ to about 1 x 10⁷, about 1 x 10⁷ to about 1 x 10¹⁰, about 1 x 10⁷ to about lx 10⁹, about 1 x 10⁷ to about 1 x 10⁸, about 1 x 10⁸ to about 1 x 10¹⁰, or about 1 x 10⁸ to about 1 x 10⁹ cfu.

[0081] In various embodiments the biological material retains at least about 0.1, 0.2,

0.5, 0.75, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99 or 100% viability after 1, 2, 3, 4, 5, 6, 7 days, or 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or 3, 4, 5, 6, 25 7, 8, 9, 10, 11, 12, 15, 18, 24 months storage of the granules at ambient temperature, and useful ranges may be selected between any of these values for example, about 0.1 to about

99, about 0.1 to about 98, about 0.1 to about 95, about 0.1 to about 90, about 2 to about

99, about 2 to about 98, about 2 to about 95, about 2 to about 90, about 2 to about 85, about 2 to about 80, about 2 to about 70, about 2 to about 60, about 2 to about 50, about 30 5 to about 99, about 5 to about 98, about 5 to about 95, about 5 to about 90, about 5 to about 85, about 5 to about 80, about 5 to about 70, about 5 to about 60, about 10 to about

99, about 10 to about 98, about 10 to about 95, about 10 to about 90, about 10 to about

85, about 10 to about 80, about 10 to about 70, about 20 to about 99, about 20 to about

98, about 20 to about 95, about 20 to about 90, about 20 to about 85, about 20 to about

80, about 20 to about 70, about 20 to about 60, about 30 to about 99, about 30 to about

98, about 30 to about 95, about 30 to about 90, about 30 to about 85, about 30 to about

80, about 40 to about 99, about 40 to about 98, about 40 to about 95, about 40 to about

2016207759 24 Apr 2019

90, about 40 to about 80, about 40 to about 70, about 40 to about 60, about 50 to about

99, about 50 to about 98, about 50 to about 95, about 50 to about 90, about 50 to about

85, about 50 to about 80, about 50 to about 70, about 50 to about 60, about 60 to about

99, about 60 to about 98, about 60 to about 95, about 60 to about 90, about 60 to about

85, about 60 to about 80, about 60 to about 70, about 70 to about 99, about 70 to about

98, about 70 to about 95, about 70 to about 90, about 70 to about 80, about 80 to about

99, about 80 to about 98, about 80 to about 95, about 80 to about 90, about 85 to about

99, about 85 to about 98, about 85 to about 95, about 85 to about 90, about 90 to about

99, about 90 to about 98, about 90 to about 95, about 95 to about 99, or from about 95 to about 98%.

[0082] In various embodiments the colony forming units (cfu) of the biological material per gram of granules after at least about 1, 2, 3, 4, 5, 6, 7 days, or 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks storage, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 24 months storage at ambient temperature is at least about 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80,

85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200%, 250%, 300%, 350%,

400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or at least about 1000% of the cfu of biological material per gram of freshly prepared granules, and useful ranges may be selected between any of these values, for example from about 2 to about 1000, about 2 to about 500, about 2 to about 100, about 2 to about 50, about 2 to about 25, about 5 to about 200, about 5 to about 100, about 5 to about 50, about 5 to about 25, about 10 to about 1000, about 10 to about 500, about 10 to about 200, about 10 to about 180, about 10 to about 160, about 10 to about 150, about 10 to about 140, about 10 to about 120, about 10 to about 100, about 10 to about 80, about 10 to about 60, about 10 to about 50, about 10 to about 40, about 25 to about 1000, about 25 to about 500, about 25 to about 200, about 25 to about 180, about 25 to about 150, about 25 to about 140, about 25 to about 120, about 25 to about 100, about 25 to about 80, about 25 to about 60, about 25 to about 50, about 40 to about 200, about 40 to about 180, about 40 to about 160, about 40 to about 150, about 40 to about 140, about 40 to about 120, about 40 to about 100, about 40 to about 80, about 40 to about 60, about 50 to about 1000, about

50 to about 500, about 40 to about 50, about 50 to about 200, about 50 to about 180, about 50 to about 160, about 50 to about 150, about 50 to about 140, about 50 to about

120, about 50 to about 100, about 50 to about 80, about 50 to about 60, about 75 to about

200, about 75 to about 180, about 75 to about 160, about 75 to about 150, about 75 to about 140, about 75 to about 120, about 75 to about 100, about 90 to about 200, about 90 to about 180, about 90 to about 160, about 90 to about 150, about 90 to about 140, about to about 120, about 90 to about 100, about 100 to about 1000, about 100 to about 500, about 100 to about 200, about 100 to about 180, about 100 to about 160, about 100 to

2016207759 24 Apr 2019 about 150, about 100 to about 140, about 100 to about 120, about 120 to about 200, about 120 to about 180, about 120 to about 160, about 120 to about 150, about 120 to about 140, about 140 to about 200, about 140 to about 180, about 140 to about 150, about 150 to about 200, about 150 to about 180, about 150 to about 160, about 160 to about 200, about 160 to about 180, about 180 to about 200%, about 200 to about 1000, or about 200 to about 500.

[0083] In various embodiments the number of colony forming units (cfu) of biological material in the granules increases by at least about 1.2, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,

5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or at least about 10 fold after at least about 1, 2, 3, 4, 5,

6, 7 days, or 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 24 months storage at ambient temperature, and useful ranges may be selected between any of these values, for example from about 1.2 to about 10 fold, about 2 to about 10 fold, about 3 to about 10 fold, or about 5 to about 10 fold.

[0084] In various embodiments the number of colony forming units (cfu) of the biological material is reduced by less than about 1 log, 2 log or 3 log after about 1, 2, 3, 4,

5, 6, 7 days, or 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 24 months storage at ambient temperature, and useful ranges may be selected between any of these values, for example from about 1 log to about 3 log or from about 1 log to about 2 log.

[0085] In various embodiments the viability of the biological material in the granules

0.1, 0.5, 1, 2, 3, 4, 5, 6, or 7 days after application to the soil may be at least about 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99 or 100% of the viability of the biological material in freshly prepared granules, and useful ranges may be selected between any of these values for example, about 2 to about 99, about 2 to about

98, about 2 to about 95, about 2 to about 90, about 2 to about 85, about 2 to about 80, about 2 to about 70, about 2 to about 60, about 2 to about 50, about 5 to about 99, about 5 to about 98, about 5 to about 95, about 5 to about 90, about 5 to about 85, about 5 to about 80, about 5 to about 70, about 5 to about 60, about 10 to about 99, about 10 to about 98, about 10 to about 95, about 10 to about 90, about 10 to about 85, about 10 to about 80, about 10 to about 70, about 20 to about 99, about 20 to about 98, about 20 to about 95, about 20 to about 90, about 20 to about 85, about 20 to about 80, about 20 to about 70, about 20 to about 60, about 30 to about 99, about 30 to about 98, about 30 to about 95, about 30 to about 90, about 30 to about 85, about 30 to about 80, about 40 to about 99, about 40 to about 98, about 40 to about 95, about 40 to about 90, about 40 to about 80, about 40 to about 70, about 40 to about 60, about 50 to about 99, about 50 to about 98, about 50 to about 95, about 50 to about 90, about 50 to about 85, about 50 to

2016207759 24 Apr 2019 about 80, about 50 to about 70, about 50 to about 60, about 60 to about 99, about 60 to about 98, about 60 to about 95, about 60 to about 90, about 60 to about 85, about 60 to about 80, about 60 to about 70, about 70 to about 99, about 70 to about 98, about 70 to about 95, about 70 to about 90, about 70 to about 80, about 80 to about 99, about 80 to about 98, about 80 to about 95, about 80 to about 90, about 85 to about 99, about 85 to about 98, about 85 to about 95, about 85 to about 90, about 90 to about 99, about 90 to about 98, about 90 to about 95, about 95 to about 99, or from about 95 to about 98%.

[0086] In one embodiment the granules may increase in size by at least about 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99 or 100% after 1, 2, 3,

4, 5, 6, 7 days, or 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks post-application to the soil, and useful ranges may be selected between any of these values for example, about 2 to about 99, about 2 to about 98, about 2 to about 95, about 2 to about 90, about 2 to about 85, about 2 to about 80, about 2 to about 70, about 2 to about 60, about 2 to about 50, about 5 to about 99, about 5 to about 98, about 5 to about 95, about 5 to about 90, about 5 to about

85, about 5 to about 80, about 5 to about 70, about 5 to about 60, about 10 to about 99, about 10 to about 98, about 10 to about 95, about 10 to about 90, about 10 to about 85, about 10 to about 80, about 10 to about 70, about 20 to about 99, about 20 to about 98, about 20 to about 95, about 20 to about 90, about 20 to about 85, about 20 to about 80, about 20 to about 70, about 20 to about 60, about 30 to about 99, about 30 to about 98, about 30 to about 95, about 30 to about 90, about 30 to about 85, about 30 to about 80, about 40 to about 99, about 40 to about 98, about 40 to about 95, about 40 to about 90, about 40 to about 80, about 40 to about 70, about 40 to about 60, about 50 to about 99, about 50 to about 98, about 50 to about 95, about 50 to about 90, about 50 to about 85, about 50 to about 80, about 50 to about 70, about 50 to about 60, about 60 to about 99, about 60 to about 98, about 60 to about 95, about 60 to about 90, about 60 to about 85, about 60 to about 80, about 60 to about 70, about 70 to about 99, about 70 to about 98, about 70 to about 95, about 70 to about 90, about 70 to about 80, about 80 to about 99, about 80 to about 98, about 80 to about 95, about 80 to about 90, about 85 to about 99, about 85 to about 98, about 85 to about 95, about 85 to about 90, about 90 to about 99, about 90 to about 98, about 90 to about 95, about 95 to about 99, or from about 95 to about 98%.

[0087] In one embodiment the moisture content of the granules may increase by at least about 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99 or 100% after 1, 2, 3, 4, 5, 6, 7 days, or 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks post-application to the soil, and useful ranges may be selected between any of these values for example, about 2 to about 99, about 2 to about 50, about 5 to about 99, about 5 to about 60, about 10 to about 99, about 10 to about 70, about 20 to about 99, about 20 to about 60, about 30 to

2016207759 24 Apr 2019 about 99, about 30 to about 80, about 40 to about 99, about 40 to about 60, about50 to about 99, about 50 to about 60, about 60 to about 99, about 60 to about 70, about70 to about 99, about 70 to about 80, about 80 to about 99, about 80 to about 90, about85 to about 99, or from about 95 to about 98%.

[0088] In one embodiment 10 g granules placed on paper towels wetted with 100 mL water absorbs at least about 1, 1.5, 2, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5,

5.5, 6, 6.5 or at least about 7 g water, and useful ranges may be selected between any of these values for example, from about 1 to about 7 g, about 1 to about 6 g, about 1 to about 5 g, about 2 to about 5 g, about 2 to about 4 g, about 2 to about 3.5 g, about 2.5 to about

5 g, about 2.5 to about 4.5 g, or about 2.5 to about 4 g.

[0089] In various embodiments the granules may be substantially resistant to deformation upon application of a pressure of up to about 0.5,1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 kg/cm², and useful ranges may be selected between any of these values, for example from about 0.5 to about 5, about 0.5 to about 4.5, about 0.5 to about 4, about 0.5 to about

3.5, about 0.5 to about 3, about 1 to about 5, about 1 to about 4.5, about 1 to about 4, about 1 to about 3.5, about 1 to about 3, about 1.5 to about 5, about 1.5 to about 4.5, about 1.5 to about 4, about 1.5 to about 3.5, about 1.5 to about 3, about 2 to about 5, about 2 to about 4.5, about 2 to about 4, about 2 to about 3.5, about 2 to about 3, about 2.5 to about 5, about 2.5 to about 4.5, about 2.5 to about 4, about 2.5 to about 3.5, about

2.5 to about 3, about 3 to about 5, about 3 to about 4.5, about 3 to about 4 kg, about 3.5 to about 5, about 3.5 to about 4, or about 4 to about 5 kg/cm².

[0090] In various embodiments, the granules may have a hardness of from about 0.5,

1, 1.5, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 15, 20, 30, 40 or about 50 N, and useful ranges may be selected between any of these values, for example, from about 1 to about 50, 2 to about

50, about 2 to about 30, about 2 to about 20, about 2 to about 10, about 2 to about 9, about 2 to about 5, about 3 to about 50, about 3 to about 30, about 3 to about 20, about 3 to about 10, about 3 to about 9, about 3 to about 5, about 4 to about 50, about 4 to about 30, about 4 to about 20, about 4 to about 10, about 4 to about 9, or about 4 to about 5 N.

[0091] In various embodiments that granules may have a mean size of about 0.5, 1,

1.5, 2, 2.5, 3, 4, 5, 6, 7.5 or about 10 mm, and useful ranges may be selected between any of these values, for example, from about 0.5 to about 10, about 0.5 to about 7.5, about 0.5 to about 6, about 0.5 to about 5, 1 to about 10, about 1 to about 7.5, about 1 to about 6, about 1 to about 5, about 2 to about 10, about 2 to about 7.5, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 2.5 to about 10, about 2.5 to about 7.5, about 2.5 to about 6, about 2.5 to about 5, or about 2.5 to about 4 mm.

2016207759 24 Apr 2019 [0092] In one embodiment the agricultural composition comprises one or more agricultural agents. In a particularly contemplated embodiment the agricultural agent is urea.

Method of manufacture [0093] In one embodiment the core may be formed by (a) providing a substrate, (b) coating the substrate with a first coating comprising biological material, preferably one or more microorganisms, and optionally a biodegradable polymer and/or a non-cytotoxic oil to form a first-coated material, (c) coating the first-coated material with a second coating comprising one or more one particulate densifiers, one or more disintegrants, and with a binder to form the core.

[0094] In one embodiment the granule is formed by applying an exterior coating to the core to at least partially coat the core, the exterior coating comprising a polymeric compound, and optionally drying the granule.

[0095] In various embodiments the substrate may be coated with a first coating comprising biological material, and optionally a biodegradable polymer and/or a noncytotoxic oil, followed by one or more coatings comprising one or more particulate densifiers and one or more disintegrants.

[0096] In various embodiments the first coated material may be coated with a second coating comprising at least one particulate densifier and with a binder to form a core. In one embodiment the first-coated material may be coated with alternating layers of the second coating and the binder. In one embodiment the first-coated material may be coated with two or more layers of the second coating.

[0097] In one embodiment the first coated material may be coated with the second coating and with a binder and then coated with an exterior coating. In one embodiment the exterior coating may be less soluble in water than the binder.

[0098] In various embodiments the binder may be soluble in water to form a 1, 1.5, 2,

2.5, 3, 3.5, 4, 4.5 or 5% solution at a temperature of less than about 25, 30, 35, 40, 45,

50, 55, 60, 65 or about 70°C, and useful ranges may be selected between any of these values, for example, from about 25 to about 70, about 25 to about 65, about 25 to about 60, about 25 to about 55, about 25 to about 50, about 25 to about 45, about 30 to about

2016207759 24 Apr 2019

70, about 30 to about 65, about 30 to about 60, about 30 to about 55, about 30 to about

50, about 35 to about 70, about 35 to about 65, about 35 to about 60, about 35 to about

55, about 35 to about 50, about 35 to about 45, about 40 to about 70, about 40 to about

65, about 40 to about 60, about 40 to about 55, about 40 to about 50, about 45 to about

70, about 45 to about 65, about 45 to about 60, about 45 to about 55, about 45 to about

50, or from about 50 to about 70°C.

[0099] In one embodiment the binder completely dissolves in water in less than 2 hours. For example, the binder completely dissolves in water in less than 2 hours at a temperature of 55 °C to form a 2% solution.

[OO1OO] In various embodiments the exterior coating may be soluble in water to form a 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5% solution at a temperature of greater than about 50, 55, 60, 65, 70, 75, 80, 85, 90 or about 95°C, and useful ranges may be selected between any of these values, for example, from about 50 to about 95, about 50 to about 90, about 50 to about 85, about 50 to about 80, about 50 to about 75, about 50 to about 70, about 55 to about 95, about 55 to about 90, about 55 to about 85, about 55 to about 80, about 55to about 75, about 55 to about 70, about 60 to about 95, about 60 to about 90, about 60to about 85, about 60 to about 80, about 60 to about 75, about 60 to about 70, about 65to about 95, about 65 to about 90, about 65 to about 85, about 65 to about 80, about 65to about 75, or about 65 to about 70°C.

[OO1O1] In one embodiment the exterior coating completely dissolves in water in less than 2 hours. For example, the exterior coating completely dissolves in water in less than 2 hours at a temperature of 70 °C to form a 2% solution.

[00102] In various embodiments the binder and/or the exterior coating may have a pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7 or about pH 8, and useful ranges may be selected between any of these values, for example, from about pH 4 to about pH 8, about pH 4 to about pH 7, about pH 4 to about pH 6, about pH 4 to about pH 5, about pH 5 to about pH 8, about pH 5 to about pH 7, about pH 5 to about pH 6, about pH 6 to about pH 8, about pH 6 to about pH 7, or about pH 7 to about pH 8.

[00103] In various embodiments the binder and/or the exterior coating may have a viscosity of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 22, 25, 27, 30, 35,

40, 45, 50, 55, 60, 70, 75, 80, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275 or 300 cP, and useful ranges may be selected between any of these values, for example, about 1 to about 300, about 1 to about 250, about 1 to about 200, about 1 to about 150, about 1 to about 100, about 1 to about 70, about 1 to about 50, 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about

2016207759 24 Apr 2019 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, 2 to about 300, about 2 to about 200, about 2 to about 100, about 2 to about 70, about 1 to about 50, 2 to about 30, about 2 to about 25, about 2 to about 20, about 2 to about 15, about 2 to about 10, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 2 to about 3, 3 to about 300, about 3 to about 200, about 1 to about 100, about 3 to about 70, about 3 to about 50, about 3 to about 30, about 3 to about 10, about 3 to about 6, about 3 to about 5, about 3 to about 4, about 4 to about 300, about 4 to about 200, about 4 to about 100, about 4 to about 50, about 4 to about 20, about 4 to about 10, about 10 to about 300, about 10 to about 200, about 10 to about 100, about 10 to about 50, about 10 to about 20, about 11 to about 300, about 11 to about 200, about 11 to about 100, about 11 to about 70, about 11 to about 50, about 50 to about 300, about 50 to about 200, about 50 to about 100, about 70 to about 300, about 70 to about 200, or about 70 to about 100.

[00104] In various embodiments the binder and/or the exterior coating applied to the granule may comprise about 0.5, 1, 1.5, 2, 2.5, 3, 4 5, 6, 7, 7.5, 8, 9, 10, 15, 20, 25, 30,

35 or about 40% w/v of the polymeric compound, and useful ranges may be selected between any of these values, for example, about 0.5 to about 40, about 0.5 to about 30, about 0.5 to about 20, about 0.5 to about 10, about 0.5 to about 5, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 15, about 1 to about 10, about to about 5, about 1 to about 3, about 1.5 to about 40, about 1.5 to about 30, about 1.5 to about 20, about 1.5 to about 10, about 1.5 to about 5, about 2 to about 40, about 2 to about 30, about 2 to about 20, about 2 to about 10, about 2 to about 5, about 2.5 to about 40, about 2.5 to about 30, about 2.5 to about 20, about 2.5 to about 10, about 5 to about 40, about 5 to about 30, about 5 to about 20, about 5 to about 10, about 7.5 to about 40, about 7.5 to about 30, about 7.5 to about 20, about 10 to about 40, about 10 to about 30, about 10 to about 20% w/v of the polymeric compound.

[00105] In various embodiments about 0.1, 0.2, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or about 5 L binder may be applied per 10 kg of coated granules, and useful ranges may be selected between any of these values, for example, about 0.25 to about 5, about 0.25 to about 4, about 0.25 to about 3, about 0.25 to about 2.5, about 0.25 to about 2, about 0.5 to about 5, about 0.5 to about 4.5, about 0.5 to about 4, about 0.5 to about 3.5, about 0.5 to about 3, about 0.5 to about 2.5, about 0.5 to about 2, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1 to about 2.5, or about 1 to about 2 L.

[00106] In one embodiment the method may additionally comprise drying the granule.

[00107] In various embodiments the coated granules may be dried in a fluid bed dryer.

In one embodiment the coated granules may be dried until the granules have a moisture

2016207759 24 Apr 2019 content of about 10, 11, 12, 12.5, 13, 13.5, 14, 14.5, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24% moisture, and useful ranges may be selected between any of these values, for example, from about 10 to about 24, 10 to about 20, about 10 to about 19, about 10 to about 18, about 10 to about 17, about 10 to about 16, about 10 to about 15, about 10 to about 14, about 10 to about 13.5, about 10 to about 12.5, from about 11 to about 20, about 11 to about 19, about 11 to about 18, about 11 to about 17, about 11 to about 16, about 11 to about 15, about 11 to about 14, about 11 to about 13.5, about 11 to about

12.5, about 12 to about 24, about 12 to about 20, about 12 to about 19, about 12 to about 18, about 12 to about 17, about 12 to about 16, about 12 to about 15, about 12 to about

14, about 12 to about 13.5, about 12 to about 12.5, about 12.5 to about 20, about 12.5 to about 17.5, about 12.5 to about 15, about 12.5 to about 14.5, about 12.5 to about 14, or about 12.5 to about 13.5 moisture. In a particularly contemplated embodiment the coated granules are dried until the granules comprise from about 11 to about 19% moisture.

Application [00108] In one embodiment the distribution device may be a truck. In a further embodiment the distribution device may be a ground spreader. In one embodiment the distribution device may be a seed drill. For example, in various embodiments the distribution device may be a gravity drill or an air-speeder drill.

[00109] In various embodiments the granules or composition may be concurrently applied using the distribution device with a second granule or composition, for example, urea.

[OO11O] In various embodiments the granules or composition may be applied to substantially cover a locus, for example a crop field or pasture.

[OOlll] In various embodiments the granules or composition may be applied to the soil at a rate of at least about 5, 10, 20, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or about 200 kg per hectare, and useful ranges may be selected between any of these values, for example, from about 10 to about 200, about 10 to about 150, about 10 to about 120, about 10 to about 100, about 10 to about 50, about 30 to about 200, about 30 to about 150, about 30 to about 100, about 30 to about 75, about 40 to about 200, about 40 to about 180, about 40 to about 150, about 40 to about 100, about 40 to about 80, about 40 to about 75, about 50 to about 200, about 50 to about 180, about 50 to about 160, about 50 to about 150, about 50 to about 120, about 50 to about 100, about 50 to about 75, about 60 to about 200, about 60 to about 180, about 60 to about 150, about 60 to about 120, about 60 to about 100, about 60 to about 80, about 75 to about 200, about 75 to about 180, about 75 to about 160, about 75

2016207759 24 Apr 2019 to about 150, about 75 to about 120, about 75 to about 100, about 90 to about 200, about to about 180, about 90 to about 160, about 90 to about 150, about 90 to about 120, about 100 to about 200, about 100 to about 180, about 100 to about 160, about 100 to about 150, about 100 to about 120, about 120 to about 200, about 120 to about 180, about

120 to about 160, about 120 to about 150, or about 150 to about 200 kg.

[00112] In various embodiments the granules or composition may be applied to the soil at a rate of at least about 1 x 10⁹, 1 x 10¹⁰, 5 x 10¹⁰, 1 x 10¹¹, 5 x 10¹¹, 1 x 10¹², 2.5 x 10¹², 5 x 10¹², 7.5 x 10¹², 1 x 10¹³, 2.5 x 10¹³, 5 x 10¹³, 7.5 x 10¹³, 1 x 10¹⁴, 2.5 x 10¹⁴, 5 x 10¹⁴,

7.5 x 10¹⁴, or about 1 x 10¹⁵ cfu of biological material per hectare and useful ranges may be 10 selected between any of these values, for example, from about 1 x 10⁹ to about 1 x 10¹⁵, about 1 x 10⁹ to about 1 x 10¹⁴, about 1 x 10⁹ to about 1 x 10¹³, about 1 x 10⁹ to about 1 x 10¹², 1 x 10¹⁰ to about 1 x 10¹⁵, about 1 x 10¹⁰ to about 5 x 10¹⁴, about 1 x 10¹⁰ to about 1 x 10¹⁴, about 1 x 10¹⁰ to about 5 x 10¹³, about 1 x 10¹⁰ to about 1 x 10¹³, about 1 x 10¹⁰ to about 5 x 10¹², about 1 x 10¹⁰ to about 1 x 10¹², about 1 x 10¹¹ to about 1 x 10¹⁵, about 1 x

10¹¹ to about 5 x 10¹⁴, about 1 x 10¹¹ to about 1 x 10¹⁴, about 1 x 10¹¹ to about 5 x 10¹³, about 1 x 10¹¹ to about 1 x 10¹³, about 1 x 10¹¹ to about 5 x 10¹², about 1 x 10¹¹ to about 1 x 10¹², about 1 x 10¹² to about 1 x 10¹⁵, about 1 x 10¹² to about 5 x 10¹⁴, about 1 x 10¹² to about 1 x 10¹⁴, about 1 x 10¹² to about 5 x 10¹³, about 1 x 10¹² to about 1 x 10¹³, about 5 x

10¹² to about 1 x 10¹⁵, about 5 x 10¹² to about 5 x 10¹⁴, about 5 x 10¹² to about 1 x 10¹⁴, about 5 x 10¹² to about 7.5 x 10¹³, about 5 x 10¹² to about 5 x 10¹³, about 5 x 10¹² to about

2.5 x 10¹³, about 5 x 10¹² to about 1 x 10¹³, about 1 x 10¹³ to about 1 x 10¹⁵, about 1 x

10¹³ to about 5 x 10¹⁴, about 1 x 10¹³ to about 1 x 10¹⁴, about 1 x 10¹³ to about 7.5 x 10¹³, about 1 x 10¹³ to about 5 x 10¹³, about 2.5 x 10¹³ to about 1 x 10¹⁵, about 2.5 x 10¹³ to about 7.5 x 10¹⁴, about 2.5 x 10¹³ to about 5 x 10¹⁴, about 2.5 x 10¹³ to about 2.5 x 10¹⁴, about 2.5 x 10¹³ to about 1 x 10¹⁴, about 2.5 x 10¹³ to about 5 x 10¹⁴, about 5 x 10¹³ to about 1 x 10¹⁵, about 5 x 10¹³ to about 7.5 x 10¹⁴, about 7.5 x 10¹³ to about 5 x 10¹⁴, about x 10¹³ to about 2.5 x 10¹⁴, about 5 x 10¹³ to about 1 x 10¹⁴, about 7.5 x 10¹³ to about 1 x 10¹⁵, about 7.5 x 10¹³ to about 1 x 10¹⁴, or about 1 x 10¹³ to about 1 x 10¹⁵ cfu per hectare.

[00113] In one embodiment the granules or composition may be applied when the soil is 30 wet, or after irrigation or rainfall.

[00114] In one embodiment application of the granules or composition may kill plant pests, such as grass grubs.

[00115] In one embodiment application of the granules or composition may reduce the grass grub population in the soil by at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,

60, 65, 70, 75, 80, 85, 90, 95 or 100% in the 12 months following application, and useful

2016207759 24 Apr 2019 ranges may be selected between any of these values, for example, from about 10 to about 100, about 10 to about 90, about 10 to about 75, about 10 to about 60, about 10 to about 50, about 10 to about 40, about 20 to about 100, about 20 to about 90, 20 to about 75, 20 to about 60, 20 to about 50, 20 to about 40, 40 to about 100, 40 to about 90, 40 to about

75,40 to about 60, 40 to about 50, 50 to about 100, 50 to about 90, 50 to about 75, 50 to about 60, 60 to about 100, 60 to about 90, 60 to about 75, 75 to about 100, 75 to about 90, or from about 75 to about 80%. In one particularly contemplated embodiment application of the granules or composition reduces the grass grub population in the soil by at least about 50% in the 12 months following application to soil.

[00116] In one embodiment application of the granules or composition may increase plant growth, such as pasture growth, or increases plant yield, such as pasture yield.

[00117] In various embodiments, application of the granules or composition may increase pasture dry matter production by at least about 2, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100%, and useful ranges may be selected 15 between any of these values, for example, from about 2 to about 100, about 2.5 to about

100, about 5 to about 100, about 10 to about 100, about 10 to about 90, about 10 to about 75, about 10 to about 60, about 10 to about 50, about 10 to about 40, about 20 to about 100, about 20 to about 90, 20 to about 75, 20 to about 60, 20 to about 50, 20 to about 40, 40 to about 100, 40 to about 90, 40 to about 75,40 to about 60, 40 to about 50, 50 to about 100, 50 to about 90, 50 to about 75, 50 to about 60, 60 to about 100, 60 to about 90, 60 to about 75, 75 to about 100, 75 to about 90, or from about 75 to about 80%.

[00118] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7).

[00119] This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in 30 the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[00120] In this specification, where reference has been made to external sources of information, including patent specifications and other documents, this is generally for the purpose of providing a context for discussing the features of the present invention. Unless 35 stated otherwise, reference to such sources of information is not to be construed, in any

2016207759 24 Apr 2019 jurisdiction, as an admission that such sources of information are prior art or form part of the common general knowledge in the art.

[00121] The term comprising as used in this specification means consisting at least in part of. When interpreting statements in this specification which include that term, the features, prefaced by that term in each statement, all need to be present but other features can also be present. Related terms such as comprise and comprised are to be interpreted in the same manner.

BRIEF DESCRIPTION OF THE DRAWINGS [00122] The invention will now be described by way of example only and with reference 10 to the drawings in which:

[00123] Figure 1 is a flowchart summarising methods of manufacturing granules of the invention.

[00124] Figure 2 shows the release of S. entomophila into soil six weeks after application of granules to the soil surface. The line indicates the target level of release to achieve acceptable grass grub control. The granules comprised the following disintegrants in the second coating: 10% starch, 5% starch, peat, or an effervescent formulation.

[00125] Figure 3 shows granules of the invention having a second coating comprising 2 part gypsum, 1 part bentonite, 1 part pregelatinised potato starch (right), compared with granules having a second coating comprising bentonite only (left).

[00126] Figure 4 shows the shelf life of granules stored in thick gas transferable (TGT) bags at 20 °C over 64 weeks. The granules comprised the following disintegrants in the second coating: 10% starch, 5% starch, peat, or an effervescent formulation.

[00127] Figure 5 compares the viability of granules of the invention coated with an exterior coating comprising (A) PVA (BF17W) and, (B) EC23009 seed coating. Granules that 25 had been dried for 4, 24 or 72 hours were stored in TGT bags at ambient temperature for up to 8 weeks.

[00128] Figure 6 shows S. entomophila loadings of A) Swellable granules; and B) Swell+ granules over 60 days storage at 4°C (), ambient temperature (♦), and 20°C (^A). Error bars show the standard error of the mean (SEM).

2016207759 24 Apr 2019 [00129] Figure 7 shows the water activity of Swellable and Swell+ granules at time of manufacture (0), and after 3, 7, 30 and 60 days storage at 4°C, ambient temperature, or 20°C. Error bars show the standard error of the mean (SEM).

[00130] Figure 8 shows the levels of S. entomophila in soil treated with Swellable (grey bars) or Swell+ (white bars) granules over six weeks. The theoretical background level of

S. entomophila in the soil and the target level for effective control of grass grubs are shown. Error bars show the standard error of the mean (SEM).

[00131] Figure 9 shows S. entomophila levels in soil treated in a pot trial with granules of the invention following four irrigation events at daily intervals of 20 mm/day (black bars) or 10 40 mm/day (white bars). Measurements were made five days after the final irrigation event. Error bars show the standard error of the mean (SEM).

[00132] Figure 10 shows the rate of amber disease in grass grubs recovered from A) a Central Plateau site; and B) Canterbury sites up to 8 or 9 weeks post application of large control granules, large granules of the invention, small granules of the invention, and no 15 granules (untreated).

[00133] Figure 11 shows S. entomophila levels in soil at two sites at 6 weeks (Canterbury) and 8 weeks (Southland) post application of no granules (0); large control granules (1, drill application); small granules of the invention (2, broadcast application) and large granules of the invention (3, broadcast application).

[00134] Figure 12 shows the viability of bacteria in granules of the invention stored at ambient temperature for 8 or 9 months, the granules comprising A) a 1:4; or B) a 1:1 ratio of first-coated material to second coating.

[00135] Figure 13 shows the viability of bacteria in granules of the invention (♦) and granules that do not comprise an exterior coating () following exposure to direct sunlight.

[00136] Figure 14 shows the moisture content of granules of the invention (♦) and granules that do not comprise an exterior coating () following exposure to direct sunlight.

[00137] Figure 15 shows the water activity of granules of the invention (♦) and granules that do not comprise an exterior coating () following exposure to direct sunlight.

DETAILED DESCRIPTION OF THE INVENTION [00138] The present invention relates to granules or an agricultural composition comprising granules, methods of preparing such granules, and application of such granules

2016207759 24 Apr 2019 into or on soil. The granules comprise a core, a binder and an exterior coating that at least partially coats the core and that reduces the water permeability of the granule wherein the exterior coating comprises a polymeric compound.

[00139] The present invention provides for the delivery of beneficial biological material to the soil in granules that protect the biological material during storage of the granules and after application to the soil before the biological material enters the soil profile. The granules of the invention are suitable for delivery of a wide range of biological materials that improve soil quality and control pests and disease in plants.

1. Granules [00140] As used herein the term granule includes granules, prills, pellets, small particles and grains. Granules have a mean particle size of, for example, at least about 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7.5 or about 10 mm, and useful ranges may be selected between any of these values, for example, from about 0.5 to about 10 mm.

[00141] The granules of the invention comprise

a) a substrate,

c) a second coating that at least partially coats the first coating, the second coating comprising one or more particulate densifiers, one or more disintegrants, preferably a pre-gelatinised starch, and a binder, and the granules further comprising an exterior coating that at least partially coats the core and that reduces the water permeability of the granule, the exterior coating comprising a polymeric compound.

[00142] In one embodiment the binder may comprise a polymeric compound, preferably a polyhydroxyl compound as described above.

[00143] In various embodiments the polymeric compound may be selected from the group comprising a polyvinyl alcohol, a polyethylene oxide (for example, POLYOX™ N10, POLYOX™ N750), methyl cellulose (for example, METHOCEL™ A4M) or gum arabic. In a particularly preferred embodiment the polyhydroxyl compound is a polyvinyl alcohol, for example, Poval® PVA 105, Sigma-80 PVA, Poval® PVA 405, Poval® PVA 205, Poval® PVA 205MB, BF17W, BF17 or PB17 polyvinyl alcohol.

2016207759 24 Apr 2019 [00144] In another embodiment the polymeric compound may comprise a latex polymer, for example, EC 23009, a shellac resin, or a polyvinylpyrrolidone.

[00145] In various embodiments the granules may comprise additional agents, for example, a dye, a plasticiser or an agricultural agent as described above. These additional agents may be incorporated into the first or second coatings.

[00146] In one embodiment the granules comprise two or more polymeric compounds, for example, the binder comprises a first polymeric compound and the exterior coating comprises a second polymeric compound. The granules may comprise alternating layers of the binder and the second coating. The exterior coating may bind particulate material to the granules.

[00147] It is desirable for the exterior coating and/or the binder of the granules of the invention to have the following properties.

[00148] Preferably, a solution comprising the exterior coating and/or the binder atomises finely to enable uniform application when sprayed on to the surface of the granules.

Preferably, the solution comprising the exterior coating and/or the binder has a low viscosity.

[00149] Preferably a solution comprising the exterior coating and/or the binder initially forms an adherent surface that at least partially coats the surface of the granules when first applied to the granules. The adherent surface enables adherence of the second coating and/or the particulate material to the granules.

[00150] Preferably, a solution comprising the exterior coating and/or the binder dries within about 15 seconds following application to the surface of the granules.

[00151] Preferably, the binder comprises a first polymeric compound that rapidly dissolves when contacted by moisture in the soil to enable rapid release of the biological material and/or other agricultural agents in the granules. Preferably, the exterior coating comprises a second polymeric compound that resists dissolution during storage of the granule, for example, if condensation forms in the bags in which the granules are stored. The exterior coating helps to protect the biological material in the granules from environmental conditions, such as UV radiation and desiccation when the granules are applied on to the soil. The exterior coating of the granules dissolves once the granules are applied into or on soil. The rate of dissolution of the exterior coating of the granules varies depending on the moisture content of the soil. For example, the exterior coating of the granules will dissolve more rapidly in a wet soil.

2016207759 24 Apr 2019 [00152] In various embodiments the exterior coating of the granules dissolves within about 1, 1.5, 2, 2.5 3, 3.5, 4, 4.5 or 5 hours of application to wet soil, and useful ranges may be selected from between any of these values, for example from about 1 to about 5, about 1 to about 4, about 1 to about 3, about 2 to about 5, about 2 to about 4, about 3 to about 4, or about 3 to about 5 hours.

[00153] In various embodiments the exterior coating of the granules dissolves within about 12, 24, 36, 48, 60, or about 72 hours of application to dry soil, and useful ranges may be selected from between any of these values, for example from about 12 to about 72, about 12 to about 60, about 12 to about 48, about 12 to about 36, about 12 to about 24, about 24 to about 72, or about 24 to about 48 hours.

[00154] In a particularly preferred embodiment, the binder may comprise a first polymeric compound that is soluble in water at a temperature of less than about 50°C to form a 2% solution of the first polymeric compound, and the exterior coating may comprise a second polymeric compound that is soluble in water at a temperature of at least about

70°C to form a 2% solution of the second polymeric compound. The second polymeric compound may have a higher degree of hydrolysis and/or a higher molecular weight than the binder.

[00155] Polymeric compounds for use in the invention must be compatible with, and nontoxic to, the biological material contained in the granules. Preferably, the binder and/or exterior coating comprise compounds that are agriculturally acceptable, non-toxic, biodegradable and/or food grade.

[00156] Preferably, the exterior coating and/or the binder resists fungal or mould formation on the surface of the granules.

[00157] Preferably, the polymeric compound is a film-forming compound. Preferably, the 25 polymeric compound forms a film on the granules that is uniform.

[00158] Preferably, a film formed on the surface of the granules by the polymeric compound is non-adhesive, so that adjacent granules do not adhere to each other. This property of the polymeric compound may be assessed by determining the cling tendency of films comprising the polymeric compound. A solution comprising the polymeric compound is prepared and applied to two glass sheets at the maximum temperature the granules will be exposed to during manufacture, for example, 35°C. Each of the films is peeled away from the glass and then slid across each other. The absence of any bonding between the films indicates that the polymeric compound is suitable for use in the invention.

2016207759 24 Apr 2019 [00159] Following application of the granules to soil, the exterior coating of the granules is initially impermeable to moisture and some gases in order to protect the biological material within the granule from degradation. Once the soil is sufficiently moist, for example following rainfall, the exterior coating dissolves, leading to swelling and degradation of the granule and release of the biological material into the soil.

[00160] The permeability of an exterior coating may be determined by preparing films comprising a polymeric compound and measuring diffusion of water or gases through the film.

[00161] In various embodiments the exterior coating of the granules may comprise particulate material that is embedded or compressed on to the surface of the granules to at least partially coat the surface of the granules. The void spaces on the surface of the granules are filled by the particulate material and the surface of the granules is smoothed to form a surface with a low coefficient of friction. Without wishing to be bound by any theory, the particulate material likely forms small fractures on the surface of the granules that allow ingress of moisture when the granules are applied to soil to facilitate break up of the granule and delivery of the biological material. Suitable particulate materials for use in the exterior coating are described above.

[00162] In relation to the particle size distribution of the particulate material, this can be defined by reference to the percentage of particles below a particular particle size. For example, a D90 defines the upper limit of the diameter of 90% of the particles, whereas a D50 defines the upper limit of the diameter of 50% of the particles. A D90% of 10 pm means that 90% of the particles have a diameter of less than 10 pm. A D50% of 2 pm means that 10% of the particles have a diameter of less than 2 pm.

[00163] The substrate is an inert, absorbent material that forms the inner core of the granules. In one embodiment the substrate is an edible and/or biodegradable solid or semisolid. Suitable substrates for use in the invention are described above.

[00164] The substrate is porous and adsorbs a substantial amount of the first coating applied to the granules as described above. For example, in one embodiment the substrate is zeolite that adsorbs at least about 20% of the first coating material. In another embodiment the substrate is a seed that adsorbs at least about 4% of the first coating, or from about 4% to about 15% of the first coating.

[00165] The granules of the invention are suitable for delivery of a wide range of biological materials to soil. In particular, the granules of the invention are suitable for delivery of microorganisms that are sensitive to the environment, for example,

2016207759 24 Apr 2019 microorganisms that are sensitive to desiccation or ultraviolet radiation. The granules of the invention preserve the viability of the biological material in the first coating during storage at ambient temperature, for example, storage for at least about 5 weeks. It will be appreciated that ambient temperature is a temperature of about 20°C. The granules of the invention rapidly degrade and release the biological material in the first coating of the granule after application of the granules to soil.

[00166] In one embodiment the first coating may comprise one or more biodegradable polymers and one or more non-cytotoxic oils. The biodegradable polymer/s and noncytotoxic oil/s may be combined to form a gel stock. The biological material is added to the gel stock before application to the granule to form the first coating of the granules. Water and the biodegradable polymer together form a gel that provides a suitable environment to support the viability of the biological material. The polymer particles in the gel are coated with the non-cytotoxic oil, which ensures that the particles disperse evenly in water volume before swelling.

[00167] Suitable biological materials for use in the invention are described above. The biological material may comprise a combination of any two or more biological materials, for example, a microorganism and a protein, or two microorganisms. In one embodiment the biological material may comprise a combination of a bacterium and a fungus.

[00168] In a particularly contemplated embodiment the biological material is Serratia entomophila.

[00169] In one embodiment the biodegradable polymer may be a polysaccharide. Preferably, the biodegradable polymer is of neutral or negative charge. In one embodiment the biopolymer may be an exopolysaccharide produced by a microorganism, or a gum. In an alternative embodiment the biodegradable polymer may be a synthetic polysaccharide, 25 for example a synthetic polymer of sucrose.

[00170] In one embodiment the first coating may comprise a non-cytotoxic oil that is compatible with, and non-toxic to, the biological material, particularly micrororganisms in the granule. In a preferred embodiment the non-cytotoxic oil is a plant or animal oil. In various embodiments, the non-cytotoxic oil is a marine oil, for example a fish or seaweed 30 oil, or a paraffin oil or a mineral oil.

[00171] The second coating of the granule comprises at least one particulate densifier and preferably a disintegrant as discussed above.

2016207759 24 Apr 2019 [00172] The particulate densifier increases the density of the granules to enable effective distribution, contributes to the hardness of the granules, protects the biological material from environmental conditions such as moisture and UV radiation, adsorbs and retains moisture, and regulates the rate of release of biological material from the granules.

[00173] The disintegrant enhances swelling and breakdown of the granules to enable the release of the biological material from the granules.

[00174] In one embodiment the granules may comprise an agricultural agent, for example, one or more fertilizers, one or more trace elements, one or more nitrification inhibitors, one or more urease inhibitors, pesticides, for example fungicides, or other 10 agents. In various embodiments the agricultural agent may be included in the first coating, the second coating, the binder or the exterior coating.

[00175] In one embodiment the granules may further comprise a dye. In one embodiment the dye may be included in the second coating. In another embodiment the dye may be included in the binder or the exterior coating.

[00176] The granules of the invention may be combined with an agricultural agent to form an agricultural composition for application to soil. Alternatively, the granules may be applied to the soil in a distribution device concurrently with another agricultural agent. For example, the granules may be combined with, or applied concurrently with, urea.

[00177] Frequently, when granules are combined with urea, moisture migrates from the 20 interior to the surface of the granules causing degradation of the granules and surface wetting of the urea. Wetted urea granules adhere together and smear preventing application of the urea and granules using distribution equipment. The exterior coating formed by the particulate material on the surface of the granules of the invention provides a barrier to prevent the egress of moisture from the granules, enabling the granules to be 25 combined or co-administered with urea.

[00178] Exemplary granules of the invention comprise a core, the core comprising

a) zeolite,

b) a first coating that at least partially coats the substrate, the first coating comprising a microorganism, xanthan gum and a non-cytotoxic oil, and

c) a second coating that at least partially coats the first coating, the second coating comprising bentonite, gypsum, potato starch, and a polyethylene oxide (for example, POLYOX™ N750),

2016207759 24 Apr 2019 the granules further comprising an exterior coating that at least partially coats the core, the exterior coating comprising polyvinyl alcohol (for example, BF17W).

Properties of the granules [00179] The granules of the invention are free-flowing, non-adherent, non-bridging and 5 substantially resistant to deformation.

[00180] It is desirable that the exterior coating of the granules provide maximum slip or minimum adhesion between the granules and hard surfaces, such as those of a distribution device. Further, it is desirable that the granules are substantially resistant to softening when exposed to condensation and dampness, for example during storage of the granules.

[00181] The exterior coating helps to maintain the moisture content inside the granules protecting the biological material from desiccating conditions during storage of the granules, or following application of the granules to dry soil. The exterior coating protects the biological material in the granules from degradation upon exposure to UV radiation, desiccation and/or other harsh environmental conditions.

[00182] In various embodiments that granules may have a mean size of from about 0.5 to about 10 mm, preferably from about 2 to about 4 mm.

[00183] The granules of the invention exhibit favourable flowability characteristics. Flowability refers to the ease with which the granules will flow under a specified set of conditions.

[00184] The flow properties and flowability of the granules may be determined using standard powder and/or particle flow tests known in the art.

[00185] For example, the flowability of the granules may be measured by pouring granules through a cone or at a tilted angle onto a flat surface to form a cone of granules. The flat surface may have a fixed base size. The maximum angle that the plane of powder 25 makes with the horizontal surface (the angle of repose) is measured. The angle of repose may be a static or kinetic angle of repose. In various embodiments the angle of repose of the granules is about 12, 14, 15, 16, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55 or about 60 degrees (for example, about 12 to about 60 degrees). In a particularly preferred embodiment the angle of repose of the granules is about 19 degrees.

[00186] Application of biological material to the soil using the granules of the invention provides prolonged benefits that are not achieved using other methods, such as sprays.

Without wishing to be bound by any theory, it is believed that the improved granules of the

2016207759 24 Apr 2019 invention enhance the availability of the biological material to target pests, for example, grass grubs, enabling the biological material to subsist for longer periods in the soil.

2. Manufacture of the granules [00187] Processes for the preparation of coated granules for use in agricultural applications are known, and will be apparent to a person skilled in the art on reading the present specification.

[00188] In one aspect the invention relates to a method of producing a granule, the method comprising providing a core comprising

a) a substrate,

b)

c) a first coating that at least partially coats the substrate, the first coating comprising biological material, preferably one or more microorganisms, and a biodegradable polymer and/or a non-cytotoxic oil, and a second coating that at least partially coats the first coating, the second coating comprising one or more one particulate densifiers, one or more disintegrants, and a binder, and applying an exterior coating that at least partially coats the core and that reduces the water permeability of the granule, the exterior coating comprises a polymeric compound.

[00189] Referring to the process diagram shown in Figure 1, the granules may be prepared as follows.

[00190] A substrate 1 is provided, and coated with a first coating 2a comprising biological material to form a first-coated material 2. Preferably, the first coating comprises an aqueous concentrate comprising the biological material. The first coating may be applied to the substrate using a blender, seed coater or other suitable mixing device.

[00191] In one exemplary embodiment the first coating is prepared by combining a biodegradable polymer and a non-cytotoxic oil with water to form a gel stock 2b, mixing the gel stock with the biological material to form the first coating 2a, and applying the first coating 2a to the substrate to form the first coated material 2. When the biological material is a microorganism, the biological material may be provided in a broth.

[00192] In one embodiment the first coating further comprises applying one or more layers comprising one or more particulate densifiers 2c. For example, following applications of the first coating 2a, the core is dusted with a first layer comprising two particulate densifiers and a second layer comprising one particulate densifier. In an exemplary

2016207759 24 Apr 2019 embodiment the first layer comprises bentonite and talc and the second layer comprises talc.

[00193] The first coated material 2 is coated with a second coating 3a comprising at least one particulate densifier and at least one disintegrant to form a core 3.

[00194] The second coating may be applied by combining the first coated material 2 and the second coating 3a in a coating device and circulating the coating device to form the core

3. Suitable coating devices for use in applying the second coating to the first coated material include a seed coater or a pan granulator.

[00195] The second coating preferably comprises at least two particulate densifiers. Most 10 preferably, the second coating comprises bentonite and gypsum.

[00196] In one exemplary embodiment the second coating is prepared by combining the one or more particulate densifiers, with a disintegrant, for example potato starch, using a blender until the second coating is visibly uniform.

[00197] Preferably, a binder 3b is applied to the first coated material 2 concurrently with the second coating 3a to facilitate adherence of the second coating to the first coated material. Preferably, binder 3b and second coating 3a are added alternately to the first coated material 2 in a seed coater to form the core 3. Binder 3b is sprayed into the coating device to ensure uniform coating of the granules as layers of the second coating 3a are built up on the first coated material.

[00198] When the first coated material 2 is stored for more than 24 hours before application of the second coating 3a, the surface of the first coated material may require remoistening before the second coating is applied. For example, the first coated material 2 is coated with binder 3c in a coating device immediately before the second coating 2 is applied.

[00199] In one embodiment a 1:1 ratio w/w of the first coated material 2 to second coating 3a is used. In another embodiment the w/w ratio of the first coated material 2 to second coating 3a is 1:4.

[00200] The core 3 is coated with the exterior coating 4a to form a coated granule 4 using a coating device as described above.

[00201] Optionally, the granule is further coated with a particulate material 4b. To apply the particulate material 4b, the core 3 is circulated in the coating device until substantially all of the surface of the core is coated with the particulate material 4b.

2016207759 24 Apr 2019 [00202] The coated granules 4 are dried to form the granules of the invention 5. The coated granules may be dried using devices well known in the art, for example a fluidised bed dryer. Alternatively, the coated granules may be spread out on trays to dry at ambient temperature.

[00203] In one embodiment the coated granules are dried until the granules have a moisture content of from about 11% to about 19%. The moisture content of the granules may be measured using a Berthold moisture meter or using other methods well known in the art.

3. Use of the granules [00204] The granules of the present invention are suitable for broadcast application on to the surface of the soil using distribution devices such as ground spreaders, trucks, or by aerial application.

[00205] Granules of the invention having an exterior coating comprising a particulate material are also suitable for application using distribution devices used for subsurface application that subject the granules to high mechanical forces. For example, the granules are suitable for application using a seed drill, for example, a gravity seed drill, a mechanical drill, or a pneumatic drill.

[00206] The granules of the invention preserve or increase the viability of the biological material in the granules and resist softening when exposed to condensation during storage.

[00207] The invention consists in the foregoing and also envisages constructions of which the following gives examples only and in no way limit the scope thereof.

EXAMPLES

EXAMPLE 1 [00208] This example describes the preparation of granules comprising the bacterium

Serratia entomophila. Infection of larvae with S. entomophila (amber disease) is an effective method for biological control of grass grubs. The performance of granules comprising various densifiers and disintegrants in the second coating of the granule was tested.

1. Granulation methods [00209] Prototype formulations were prepared by pan granulation using a 400mm

Erweka coating pan (45° angle, 200rpm). lOOg of the first-coated material was added to the pan and granules were built up by alternating addition of binder and the second coating

2016207759 24 Apr 2019 (400g). Binder (2% POLYOX™ WSR N10) was applied using a craft spray gun at 4 bar pressure. If clumping occurred, unheated forced air was applied by hairdryer to break clumps. 2% Methocel® A4M was applied using a craft spray gun at 4bar pressure.

[00210] Granules were also produced using a large pan granulator (3.6 m diameter).

2. Substrate material [00211] Both 1-2 mm and 2-4 mm zeolite chips (Zl-BDW grade, Blue Pacific Minerals, Tokoroa, NZ) were found to be suitable for use as the substrate material in the granule. Similar bacterial loadings were achieved on both 2-4 mm and 1-2 mm zeolite chips, and no significant difference in cell mortality was observed during a 24 hour ambient drying step.

3. First coating [00212] 10 g xanthan gum was mixed with 10 g canola oil, and stirred with an electric hand stirrer. 230 ml of Serratia entomophila strain SE626 (5xl0⁹ cfu/ml) broth was mixed in vigorously to product the first coating mix.

[00213] 650 g zeolite was coated with the first coating mix. The coated zeolite was subsequently dusted with 50 g 1:1 bentonite:talc followed by 50 g talc.

4. Second coating

Densifiers [00214] The use of bentonite, gypsum and talc in the second coating of the granule was investigated.

[00215] 200 g bentonite was bound on to 100 g 2-4 mm zeolite chips using approximately 100 mL of a 2% (w/v) POLYOX™ N10 solution as a binder. A small quantity of talc was added at the end to act as a free flow agent. A final coat of 2% Methocel A4M was applied as an exterior coating.

[00216] This granule showed good stability on dry soil and good release under optimal conditions. However, the physical integrity of the granules and release in the field was poor. The granules bridged during pouring and smeared when passed through rotating equipment.

[00217] Addition of gypsum to the bentonite improved both the hardness and density of the granules and different ratios of gypsum/bentonite/talc were trialled. Straight gypsum failed to bind. A 1:1 ratio of gypsum:talc formed very hard granules that failed to dissolve. A 2:1 ratio of gypsum:bentonite bound sufficiently to the core while achieving optimal hardness of the granule.

2016207759 24 Apr 2019

Disintegra nts [00218] The addition of a disintegrant to the second coating was investigated.

Disintegrants tested included pre-gelatinised starch, peat, an effervescent formulation comprising citric acid and sodium bicarbonate, and a superabsorbent polymer (Crystal

Rain™). The granule core was coated with a second coating comprising 2 parts gypsum to part bentonite and 1 part disintegrant.

[00219] Release of bacteria from the granules into the soil six weeks after application to the soil surface was measured as shown in Figure 2.

[00220] Granules formulated with 10% pre-gelatinised starch in the second coating exhibited increased swelling and breakdown of the granule in response to moisture compared with a granule comprising bentonite only in the second coating as shown in Figure 3.

[00221] Shelf life at ambient temperature over 64 weeks storage in thick gas transferable bags (TGT) bags was examined by measuring the viability of the bacteria as shown in Figure 15 4.

[00222] It proved difficult to granulate granules comprising Crystal Rain™ in the second coating as the Crystal Rain™ separated from the other ingredients upon wetting.

[00223] This example demonstrates suitable materials for use in the second coating of the granules of the invention.

EXAMPLE 2 [00224] This example investigates the suitability of various polymeric compounds in the exterior coating of the granules of the invention.

1. Preparation of granules for performance testing of exterior coatings [00225] 650 g zeolite (1-2 mm) was added to the bowl of a CIMBRIA CC-Lab seed coating machine followed by the biopolymer gel prepared as described for Example 1.

Granules were formed by alternate additions of pre-blended powder (560 g gypsum, 310 g bentonite, 125 g Passeli pre-gelatinised potato starch) and a binder solution comprising 1%

POLYOX™ N750. The granules coated with the second coating were coated with the exterior coating material. 500 g samples were collected within 4 hours of production and sealed in 30 TGT bags, and the remaining granules were spread out into an approximately 10 cm layer onto plastic trays and allowed to dry at 20°C. Samples were collected after 24 and 72 hours drying and sealed in TGT bags. All samples were stored at 20° C.

2016207759 24 Apr 2019 [00226] Other granules were prepared using the same method using a large pan granulator.

[00227] The following coating materials dissolved in water were tested: 5% PEG 2000, 2% Methocel® A4M, EC 23009 (a proprietary binder used for seed coating applications),

10% Gum Arabic, polyvinyl acetate glue, 2-10% polyvinyl alcohols (PVAs) (MW 9-10 kDa and 89 kDa), 10% Kollicoat® MAE100, 10% Kollicoat® Protect (a proprietary pharmaceutical copolymer of polyethylene glycol and polyvinyl alcohol).

[00228] Coatings comprising the following materials in an ethanol solvent were also tested: Methocel® A4M, shellac latex and Kollicoat® MAE100.

1. Performance testing [00229] The viability of bacteria in granules formulated with different exterior coating materials is shown in Table 1.

[00230] All the exterior coating materials tested resulted in a similar water activity in the granule after 24 hours drying as shown in Table 2.

Treatment	CFU initial	CFU 24 hour drying	CFU 48 hour drying
PEG 2000	1.07xl0⁸/g	9.78xl0⁵/g	0.00
Methocel A4M	9.33xl0⁷/g	9.96xl0⁵/g	0.00
Gum Arabic	7.65xl0⁸/g	9.68xl0⁵/g	1.44xl0^s/g
Polyvinyl acetate	0.00	0.00	0.00
Kollicoat MAE100	8.96xl0⁷/g	9.77xl0⁵/g	4.34xl0⁶/g
Kollicoat Protect	4.26 xl0⁷/g	2.00xl0⁷/g	Not tested

Table 1: Bacterial viability after 48 hours drying post-coating.

Treatment	Aw initial	Aw 24 hour drying
PEG 2000	0.888	0.665
Methocel A4M	0.958	0.677
Gum Arabic	0.952	0.670
Polyvinyl acetate	0.970	0.786
Kollicoat MAE100	0.864	0.647
Kollicoat Protect	0.885	0.661

Table 2: Water activity in granules after drying for 24 hours measured using a dew point reader (AquaLab Decagon).

[00231] Ethanol-solvent based film coats did not harden any faster than water-based film coats (data not shown). There was no immediate deleterious effect of ethanol on cell

2016207759 24 Apr 2019 viability and over 24 hours the loss of viability was similar to that observed in water based films.

[00232] Bacterial viability and water activity of granules coated with 9-10k PVA was also measured. The results are shown in Table 3. These results indicate that an exterior coating 5 comprising PVA provided good protection for bacteria in the granules against desiccating conditions for up to 120 hours.

Treatment	Initial	24 hours	48 hours	120 hours
CFU	4.57xl0⁸/9	1.69xl0⁸/g	7.90xl0⁷/g	5.00xl0⁶/g
Aw	0.967	0.694	0.728	0.584

Table 3: Bacterial survival and water activity (Aw) after drying of granules coated with 9-10k PVA.

[00233] Preliminary studies indicate that prototype granule formulations packaged in thick gas transferable (TGT) bags had no reduction in cell viability for a number of weeks and that granules broadcast onto a moist soil surface had little loss in viable bacteria for at least seven days (data not shown). This data suggests that PVA coated granules provide protection against desiccating conditions for a number of days.

[00234] This example demonstrates that granules of the invention coated with an exterior coating comprising various polymeric compound retain bacterial viability and have acceptable water activity.

EXAMPLE 3 [00235] This example describes further performance testing of granules of the invention having an exterior coating comprising a polymeric compound.

1. Preparation of granules [00236] Granules comprising a 1:1 core:second coating ratio were prepared. The performance of granules coated with an exterior coating comprising PVA or a commercially available seed coating polymer EC23009 was compared.

[00237] 10 g xanthan gum was mixed with 10 g cooking oil, then 230 ml of Serratia entomophila strain SE626 (5xl0⁹ cfu/ml) was mixed in vigorously to produce a biopolymer gel.

[00238] 650 g zeolite (1-2 mm) was added to the bowl of a CIMBRIA CC-Lab seed coating machine followed by the biopolymer gel. Granules were formed by alternate additions of pre-blended powder (560 g gypsum, 310 g bentonite, 125 g Passeli potato

2016207759 24 Apr 2019 starch) and either 1% POLYOX™ N750 or EC23009. Finished granules were coated with either 10% BF17W PVA (Molecular weight of 7782 kDa, 95-97 mol% hydrolysis) or EC23009 (a proprietary binder used for seed coating applications).

2. Viability testing results [00239] 500 g samples were collected within 4 hours of production and sealed in thick gas transferable bags (TGT). The remaining granules were spread out on plastic trays in a layer of approximately 10 cm and allowed to dry at 20°C. Samples were collected after 24 and 72 hours drying and sealed in TGT bags. All samples were stored at 20°C.

[00240] The viability of the bacteria in the granules was tested after 1, 2, 3, 4, and 8 10 weeks as shown in Figure 5.

[00241] This example demonstrates that granules of the invention coated with an exterior coating comprising a polymeric compound retain bacterial viability during storage.

EXAMPLE 4 [00242] This example assesses the efficacy of broadcast application of granules of the invention on to soil to increase Serratia entomophila levels in the soil.

1. Methods

Preparation of granules [00243] Second coating ingredients were pre-blended. The pre-blend for the Swellable granules comprised 2 kg gypsum (Soil life, Winstone Gypsum), 1 kg bentonite (Calben,

Transform minerals) and 1 kg potato starch (Paselli PC, National Starch). The pre-blend for the Swell+ granules further comprised 2% (100 g) magnesium stearate (Sigma).

[00244] The substrate and first coating material (1 kg; comprising 650 g zeolite, 230 ml

S. entomophila fermentation broth, 10 g xanthan gum, 10 g salad and cooking oil, and 50 g bentonite/talc, 50 g talc) was added to a seed coating machine (CIMBRIA CC-Lab). The second coating pre-blend (4 kg) and 200 ml 2% POLYOX™ N10 solution to bind were added alternately to produce granules. Granules were finally coated with 200 ml 2% PVA (89k) then air dried overnight.

[00245] Prototype granules were prepared with 1 kg of the substrate/first coating material and 4 kg bentonite added alternately with 200ml 2% POLYOX™ N10 to form the second coating on the granule. The second-coated material was then coated with 200 mL 2% PVA (89k), and air dried overnight.

2016207759 24 Apr 2019 [00246] All granule formulations were packaged in TGT bags and stored at 4 °C.

[00247] Prototype, Swellable and Swell+ granules had bacterial loadings of 3.8 χ 10⁵cfu/g, 4.6 χ 10⁷ cfu/g and 2.1 x 10⁷cfu/g, respectively.

[00248] Granules were enumerated using standard serial dilution and spread plating techniques immediately prior to application in the field.

Site selection, set up and application [00249] Trials were conducted on three farms located at Winchmore, Lincoln and Hororata in Canterbury, New Zealand. On each site, a series of 5 m x 5 m plots were established in a randomised block design each with four replicates and four treatments 10 (swellable, swell+, prototype and control). Control plots were untreated and provided a measure of background Serratia levels. Treatments were applied at a rate of 120 kg/ha using multiple passes with a hand held spreader to ensure even granule distribution.

Sample collection and analysis ofS. entomophila in soil [00250] Six weeks post treatment, 40 random soil cores (2.5 cm x 8.0 cm) were 15 collected from each plot and pooled into a single sample. Pooled soil samples were mixed thoroughly and sorted to remove grass grub larvae and large pieces of debris.

Approximately 200 g soil was accurately weighed into a tumbler container and well mixed with 1.8 litres of 0.1% Tween buffer (2.2 mM Na4P2C>7, 1.0 ml/litre Tween 80). The soil solution was dilution-plated on to a selective media comprising caprylate thallous agar. Agar 20 plates were incubated at 30°C for 4-5 days. Serratia colonies appear as round, white, smooth edged colonies. To differentiate S. entomophila from other Serratia species, 25 representative colonies per sample were transferred to the following selective media: DNase (Remel), adonitol and itaconate agars. Colonies that gave a positive reaction on all three media were identified as S. entomophila. S. entomophila colonies were further differentiated 25 by bacteriophage profiling against five phage types to separate the applied strain from field strains.

2. Results [00251] The Lincoln and Hororata sites did not have detectable background levels of S. entomophila in the soil as shown in Table 4. High levels of S. entomophila were recorded at 30 these sites in plots treated with the Swellable and Swell+ granules but not with the prototype granules.

Log io S.

entomophila 154 S. entomophila

Treatment (cfu/g)154 (cfu/g)

Winchmore

2016207759 24 Apr 2019

Swellable	4.914	8.2 x 10⁴
Prototype	3.331	2.14 x 10³
Swell +	4.260	1.82 x 10⁴
Control	3.358	2.28 x 10³
LSD 5%	0.917
LSD 1 %	1.318
Lincoln
Swellable	4.331	2.14 x 10⁴
Prototype	-
Swell +	4.685	4.84 x 10⁴
Control	-
LSD 5%	1.558
LSD 1 %	2.860
Hororata
Swellable	2.786	6.10 x 10²
Prototype	-
Swell +	3.071	1.18 x 10³
Control	-
LSD 5%	1.458
LSD 1 %	2.676

Table 4: Mean cfu per gram in soil samples at trial sites six weeks post treatment.

[00252] A threshold level of 10⁴ S. entomophila cfu per gram of soil is recommended as an indicator of successful bacterial establishment for the development of amber disease in grass grubs. These levels were achieved on the Winchmore and Lincoln sites with both the 5 Swellable and Swell+ granules.

[00253] This example demonstrates that that the granules of the invention are suitable for the effective delivery of biological material to soil.

EXAMPLE 5 [00254] This example assesses the properties, storage stability and efficacy of two 10 formulations of the granules of the invention.

1. Methods [00255] Swellable and Swell+ granules were prepared as described for Example 4.

2016207759 24 Apr 2019

Size and shape [00256] Granules were observed at 7x magnification using an Olympus SZX12 binocular microscope. Images were captured using Olysia Bioreport software (v 5.0) and 100 random granules were measured across the widest point using the arbitrary distance function of the 5 software.

Hardness [00257] Hardness of at least 20 granules from each batch was determined using an Instron model 4204 with third party computer interface and 100 Newton load cell. Granules were observed during testing and the point where the outer core split was recorded as the crush point. A compression modulus calculated from the slope of force applied vs compression distance was also determined.

Bacterial load and water activity [00258] Triplicate samples (1 g) collected from TGT bags were dilution plated on LB agar in duplicate to determine the number of viable bacteria present. Water activity was measured using an Aqualab series 3TE water activity meter.

Field trial [00259] A 3 x 6 set of 25 m² plots was established at a research farm having a low grass grub population. Granules were applied sequentially to the plots at weekly intervals at a rate of 120 kg/ha.

[00260] At 4, 5 and 6 weeks post-application, 20 random soil cores (2.5 cm diameter x

8.0 cm depth) were collected from each plot and pooled into a single sample. Analysis of Serratia entomophila levels in the samples was conducted as described for Example 4.

Data analysis [00261] The observed soil bacteria counts are counted values that exhibit large variance.

Therefore, negative binomial (NB) distributions were used for modelling treatment-specific distributions of these counts. In addition, because measurements were taken from each plot repeatedly (i.e. 4, 5 and 6 weeks after application), a generalised estimating equations (GEE) approach was used to account for correlations within the three repeated measurements taken from the same plot.

2016207759 24 Apr 2019

2. Results

Hardness [00262] Three batches of each of the Swellable and Swell+ granule formulations were manufactured. The mean size of the granules in each batch was 3.57-4.44 mm.

	Crush point (kg)	Compression modulus (kg/mm)
Swellable
Batch 1	2.56	1.37
Batch 2	2.19	1.47
Batch 3	2.33	1.34
Mean (+/- SEM)	2.36 +/- 0.11	2.84 +/- 0.34
Swell +
Batch 1	3.1	2.91
Batch 2	2.17	2.28
Batch 3	3.24	2.86
Mean (+/- SEM)	1.39 +/- 0.04	2.68 +/- 0.20

Table 5: Hardness testing of granules [00263] The compression modulus for the Swellable and Swell+ granules was not significantly different (p = 0.73) as shown in Table 5. However, the crush point was lower (p = 0.001) for the Swell+ granules compared to the Swellable granules (Table 5).

Storage stability [00264] The average initial loading of S. entomophila for the Swellable and Swell + granule batches was 2.0 x 10⁸ and 4.3 x 10⁸ cfu/g, respectively. The Swell+ granules had an initial water activity of 0.969, compared with 0.954 for the Swellable granules.

[00265] Granules stored in TGT bags at ambient, 4°C and 20°C were monitored for physical appearance, water activity and total viable cells (bacterial loading) over 60 days. After 60 days storage, both granule formulations at all temperatures showed no signs of contamination and the granules remained free-flowing.

[00266] The viability of the bacteria in the Swellable and Swell+ granules and water activity of the granules over 60 days storage is shown in Figures 6 and 7, respectively.

Water activity did not fall below 0.95 for either granule formulation at all storage temperatures tested.

2016207759 24 Apr 2019

Field trial [00267] S. entomophila levels in soil treated with Swellable or Swell+ granules was significantly increased over untreated soil (control) four and five weeks post-application as shown in Table 6.

Week Control

Swellable

Swell +

6.08X10³ ± 3.47Χ10³

4.23Χ10³ ± 1.85Χ10³

6.69Χ10³ ± 4.36Χ10³

5.49xl0⁴ ± 3.39Χ10⁴**

1.83Χ10⁴ ± 1.01Χ10⁴*

1.26Χ10⁴ ± 6.42Χ10³

3.56Χ10⁴ ± 1.48Χ10⁴*

3.18xl0⁴ ± 1.16Χ10⁴***

1.87Χ10⁴ ± 1.19Χ10⁴

Table 6: Soil S. entomophila counts (cfu/g soil) measured 4, 5 and 6 weeks postapplication of granules to soil, p-values relative to control: *p<0.05; **p<0.01; ***p<0.001.

[00268] Repeated, weekly applications of the Swellable granule to soil maintained S. entomophila levels on or above the target level of 1 x 10⁴ cfu/g required to control grass 10 grubs in soil as shown in Figure 8. Repeated application of the Swell+ formulation maintained soil S. entomophila levels at or above the target for weeks 1 to 4, but soil bacteria levels dropped below the target at weeks 5 and 6. This may have been due to drier soil conditions at weeks 5 and 6 of the trial.

[00269] This example demonstrates the size, hardness and storage stability of the granules of the invention. The example also demonstrates the efficacy of the granules of the invention for establishing target levels of biological material in soil when applied on to the soil.

EXAMPLE 6 [00270] This example assesses the storage stability of the granules of the invention, and 20 efficacy when applied to soil by broadcast application.

1. Methods

Granule manufacture [00271] Granules were prepared using the method described above for Example 4.

Component	Large granule (120 kg/ha)	Small granule (60 kg/ha)	Large control granule	Small control granule
Zeolite substrate 1-4 mm	650 g		650 g
Zeolite substrate 1-2 mm		650 g		650 g
First coating:	250 g	250 g	250 g	250 g

2016207759 24 Apr 2019 g xanthan gum g cooking oil

230 mL broth

S. entomophila loading (cfu/g)	7 x 10⁹ cfu/g	1.4 x 10¹⁰ cfu/g	7 x 10⁹ cfu/g	1.4 x 10¹⁰ cfu/g
Second coating	4 kg (2.375 kg gypsum 1.375 kg bentonite 250 g potato starch)	4 kg (2.375 kg gypsum 1.375 kg bentonite 250 g potato sta rch)	50g (1:1 bentonite:talc) 50g talc	50g (1:1 bentonite:talc) 50g talc
Binder: 1% POLYOX™ N10	200 mL	200 mL	No binder	No binder
Exterior coating: 2% 89 K PVA	200 mL	200 mL	No exterior coating	No exterior coating

Table 7: Composition of granules [00272] The composition of the granules is shown in Table 7. Samples (1 kg) of each granule formulation were packaged in TGT bags and stored at ambient temperature to assess storage stability. Large and small formulations of the granules of the invention were 5 prepared for broadcast application on to the soil, as well as large and small positive control granule formulations for application into the soil by drilling.

Pot trials [00273] Intact soil cores (100 mm diameter; 200 mm deep) were collected using a tractor-mounted soil corer from pasture with low background S. entomophila levels at the 10 AgResearch Lincoln research farm. The cores were placed into PVC piping and cut to about 5 mm below the top of the pipe. Soil was packed around the core/pipe interface to prevent water movement down the edge of the core. Cores were placed into an automatically watered glasshouse for 12 weeks to allow settling. Two weeks prior to the trial, pots were moved to a glasshouse that was not irrigated and vegetation was cut to the soil surface to 15 simulate grazing. Ten large granules were placed on the surface of each pot. Irrigation (20 mm or 40 mm) was applied daily for four consecutive days via a garden sprinkler over approximately 20 or 40 minutes. Five days after the final irrigation, the cores were dissected into three zones: 0-1 mm, 1-20 mm or 20-40 mm, and the number of S entomophila present in each zone was estimated using the protocol described for Example 20 4.

Field trials [00274] Field trials were conducted at three sites in New Zealand: Rangitaiki, Central Plateau; Hororata, Canterbury; and Heddon Bush, Southland. The Rangitaiki site consisted of sheep and beef grazing 4-5-year-old ryegrass/white clover pasture on volcanic pumice/ash soil. The Canterbury site was moderately open 2-year-old ryegrass/white clover, dairy pasture on a Lismore stony/very stony silt loam. The Southland site was a 351

2016207759 24 Apr 2019 year-old ryegrass/white clover pasture on a Braxton soil grazed by sheep and beef cattle. S. entomophila was undetectable at all three sites, however, relatively high levels of S. proteamaculans was measured at all three sites (data not shown).

[00275] At all sites, 200 m² plots were set up in a randomised block design with four treatments (untreated, large control granules (drilled, 30 kg/ha), small granules (60 kg/ha) and large granules (120 kg/ha)). There were five replicates of each treatment. The large control granules were applied at 30 kg/ha by direct drill (Canterbury and Southland - Agrow JMD experimental direct drill; Central Plateau - Duncan triple disc, Pritchard Agric.

Contracting) to a depth of approximately 30 mm. Surface granules were applied at either 60 10 or 120 kg/ha using a hand spreader (Solo 421-S portable spreader).

[00276] At 4, 6 and 9 weeks (Central Plateau), 6 and 8 weeks (Canterbury), and 8 weeks (Southland) post treatment, 30-40 grass grub larvae were collected from each plot and assessed for amber disease. At 4 and 6 weeks post-treatment (Canterbury, Central Plateau) and 8 weeks (Southland), 40 random soil cores (2.5 cm diameter x 8.0 cm depth) were collected from each plot and pooled into a single sample for estimation of S. entomophila levels using the soil enumeration method described in Example 4.

2. Results

Storage stability [00277] The viability of S. entomophila after four months storage at ambient temperature 20 is shown in Table 8.

	Small control granule	Small granule	Large control granule	Large granule
Initial (cfu/g)	2.00 x 10⁹	3.76 x 10⁸	3.8 x 10⁹	2.9 x 10⁷
4 months (cfu/g)	2.30 x 10⁹	1.20 x 10⁸	3.5 x 10⁸	2.8 x 10⁷

Table 8: Viability of S. entomophila in freshly prepared granules (initial) and after four months storage in TGT bags at ambient temperature.

Pot trial [00278] The granules retained their integrity after multiple irrigation events. S.

entomophila was released from granules and delivered to the target zone (20-40 mm) as shown in Figure 9.

Field trials [00279] Amber disease levels at all three sites 8 or 9 weeks after broadcast application on to the soil is shown in Table 9. Amber disease increased over time in grass grubs

2016207759 24 Apr 2019 recovered at the Central Plateau (Figure 10A) and Canterbury (Figure 10B) sites as shown in Figure 10.

[00280] S. entomophila levels in soil treated with the large and small granules of the invention at the Canterbury and Southland sites 6 weeks and 8 weeks post-application, 5 respectively, is shown in Figure 11.

Treatment	Central Plateau	Site Canterbury	Southland
Control	7.8 ± 2.0	2.5 ± 1.2	4.9 ± 1.7
Large control granule	40.3 ± 3.6*	19.2 ± 3.1*	18.6 ± 3.1*
(drilled)
Large granule	10.6 ± 2.3*	7.2 ± 2.1*	11.5 ± 2.5*
Small granule	15.9 ± 2.7*	4.9 ± 1.7	9.9 ± 2.4

Table 9: Amber disease in grass grub larvae recovered from three sites 8 weeks (Canterbury and Southland) or 9 weeks (Central Plateau) post-application, pvalues: * p<0.005.

[00281] This example demonstrates the efficacy of the granules of the invention for establishing target levels of biological material and establishing pest disease in soil when applied by broadcast application.

EXAMPLE 7 [00282] This example assesses the long term stability of granules of the invention.

[00283] Two granule formulations were prepared at scale using the method described for the Swellable granules in Example 4. The second coating pre-blend (1 kg and 4 kg for the 1:1 and 1:4 granules, respectively, per 1 kg of the core coated with the first coating material) was applied using PVA (80% hydrolysed, Sigma Aldrich) as a binder. The exterior coating comprised PVA (80% hydrolysed, Sigma Aldrich).

[00284] Six 15 kg bags of each granule formulation were stored at ambient temperature for 8 months. Bacterial viability was measured at the time of manufacture and at monthly intervals during storage. Water activity and moisture content were also measured.

1. Bacterial viability [00285] The mean bacterial load of the 1:4 granules at manufacture was 6 x 10⁷ cfu/g.

Bacterial viability over nine months storage is shown in Figure 12A.

2016207759 24 Apr 2019 [00286] The mean bacterial load of the 1:1 granules at manufacture was 1.55 x 10⁹cfu/g. Bacterial viability over eight months storage is shown in Figure 12B.

2. Water activity and moisture content [00287] The water activity of the granules over three months storage is shown in Table 5 10.

Granule	1	Months storage 2	3
1:1	0.9848 +/- 0.00323	0.9872 +/- 0.00097	0.9856 +/-	0.00206
1:4	0.9922 +/- 0.00058	0.9882 +/- 0.00092	0.988 +/-	0.00114

Table 10: Water activity of granules of the invention over three months storage at ambient temperature (+/- SEM).

[00288] The moisture content of the 1:1 granule was approximately 14-15% over 6 months storage as shown in Table 11. The 1:4 granule had a higher initial moisture content of about 18%, which decreased after two months storage, but remained relatively stable over the following four months as shown in Table 11.

Granule	1	2	Months 3	storage 4	5	6
1:1	15.02%	13.8%	14.04%	14.62%	14.92%	16.2%
1:4	18.26%	11.76%	12.58%	13.42%	14.02%	15.7%

Table 11: Moisture content of granules of the invention over six months storage at ambient temperature (+/- SEM).

[00289] This example demonstrates that the granules of the invention are shelf-stable for long periods at ambient temperature.

EXAMPLE 8 [00290] This example assesses the efficacy of granules of the invention for establishing amber disease in grass grub larvae in soil when applied by broadcast and drill methods.

2016207759 24 Apr 2019

1. Methods

Granule preparation [00291] Granules were prepared as described above using the method described for the Swellable granules in Example 4.

[00292] To form granules for drill application, 25 kg of the core coated with the first coating was coated with 25 kg of the second coating material (1:1 core:second coating). To form granules for broadcast application on to soil, 10 kg of the core coated with the first coating was coated with 40 kg second coating (1:4 core:second coating). The second coating was applied using a binder comprising 2% PVA (Poval® 105).

[00293] A final coating comprising 2% PVA (Poval® 105) was applied to form an exterior coating on the granules.

Field trial [00294] A site in Canterbury, New Zealand having a uniform grass grub population and pasture cover, and low background levels of S. entomophila were selected for field trials.

[00295] Six replicates of each treatment were arranged in a randomised block design at the site. Broadcast granules (1:4 ratio) were applied manually using a Solo 421 granule spreader in both directions (3.3 m swaths) at rates of 60, 120, 300 and 600 kg/ha. Drill treatments were applied using a proprietary gravity feed triple disc drill utilising a Duncan metering system and fitted with press wheels at a rate of 30 kg/ha.

[00296] To measure the rate of amber disease in grass grub larvae in each plot, at least visually intact grass grub larvae were collected from random spade sampling in each plot. The number of live and dead grubs in each plot was counted. Live grubs were subjected to standard visual and feeding assays to determine amber disease levels. Briefly, larvae were separated in individual cells of a tray, each cell comprising a 2x2x2 cube of carrot. Trays were sealed and incubated at 15°C. Grass grubs were considered positive for amber disease if they ceased feeding within 3-7 days.

2. Results [00297] The level of amber disease in larvae recovered from the soil was significantly increased in plots treated with broadcast granules at an equivalent rate of 60, 120, 300 or 30 600 kg/ha, or the drill granule as shown in Table 12.

2016207759 24 Apr 2019

Granule	Rate	Amber disease (% larvae +/- SEM)	p-value (to control)
Broadcast	60 kg/ha	9.4 +/- 1.8	<0.05
Broadcast	120 kg/ha	19.6 +/- 2.2	<0.001
Broadcast	300 kg/ha	19.0 +/- 2.0	<0.001
Broadcast	600 kg/ha	27.1 +/- 3.0	<0.001
Drill	30 kg/ha	20.4 +/- 1.9	<0.001
Control (untreated)		4.0 +/- 0.7

Table 12: Amber disease in larvae recovered from soil 6 weeks post application of broadcast or drill granules.

[00298] This example demonstrates efficacy of the granules of the invention for establishing amber disease in grass grub larvae when applied to soil.

EXAMPLE 9 [00299] This example demonstrates granules of the invention comprising various biological materials. This example also assesses the viability of the biological materials in the granules of the invention after storage.

[00300] Granules suitable for broadcast application comprising various biological materials were prepared as follows. 5 g xanthan gum was mixed with 5 g canola oil and 115 g broth comprising the biological material to form a gel (first coating). 325g zeolite was placed into a cake mixer and blended with the first coating to form 500 g first-coated material. The first-coated material was transferred to a CIMBRIA CC-Lab seed coater and 2 kg second coating (comprising 1.375 kg gypsum, 500 g bentonite and 125 g potato starch) was added with approximately 50 mL 2% PVA (grade BF17W) as binder to form the secondcoated material. An additional 10 mL 2% PVA (grade BF17W) was added to form the exterior coating on the granules.

[00301] The granules were stored in a 20°C incubator for 5 weeks.

[00302] The viability of the bacteria in the granules was measured at the time of manufacture and after 5 weeks storage. The results are shown in Table 13.

Species of	Description of	Viability	Viability after	%
biological	biological	after	5 weeks at	survival
material	material	manufacture	20 °C (cfu per
		(cfu per g)	9)

2016207759 24 Apr 2019

Pseudomonas chloraphis	Gram negative Non spore forming	1.44X10⁷	2.30x10^s	16%
Burkholderia terricola	Gram negative Non spore forming	4.97X10⁷	9.9x10^s	20%
Serratia proteomaculans	Gram negative Non spore forming	8.5x10^s	2.15 x 10^s	24.29%
Yersina entomophaga	Gram negative Non spore forming	4.9x10^s	2.65X10⁷	5.41%
Penicillium janczewskii	Fungi Spore forming	3.35 x 10⁷	6.30X10⁷	188%
Beauveria bassiana	Fungi Spore forming	9.30X10⁷	3.80X10⁷	41%
Metarhizium anisopliae	Fungi Spore forming	2.25x10^s	9.40x10^s	42%
Bacillus subtilis	Gram positive Spore forming	3.00x10^s	2.40X10⁷	800%
Metarhizium anisopliae/Serrati a entomophila combination	Combination	SE: 3.80x10^s MA: 1.00x10^s	SE: 2.5x10^s MA:1.60x10^s	0.66% 16000%

Table 13: Viability of microorganisms in granules of the invention at manufacture and after 5 weeks storage at 20°C.

[00303] This example demonstrates that the biological material in granules of the invention retains and/or increases viability during storage.

EXAMPLE 10 [00304] This example describes the preparation of granules comprising Serratia entomophila. The performance of granules prepared using various binders was tested and compared to prior art granules lacking an exterior coating.

[00305] Granules suitable for broadcast application were prepared according to the method described for Example 9. The binder was varied as shown in Tables 14 and 15. The exterior coating of the granules comprised PVA (BF17W).

[00306] Prior art granules that did not have an exterior coating were prepared according to the method described in Example 8 (formulation 9) of WO 2009061221. Briefly, a first 15 coating was prepared comprising 15 g xanthan gum, 15 g salad and cooking oils and 230 mL S. entomophila broth to form a gel. 650 g zeolite was coated with the gel to form a gel57

2016207759 24 Apr 2019 coated material. 50 g of bentonite and talc mixed at a 1:1 ratio was coated on the gel coated material. A further 50 g talc was added to the granules.

Physical properties [00307] The granules were tested for hardness, compressibility and swellability. The results are shown in Table 14.

[00308] Hardness of at least 20 granules from each batch was determined using an Instron model 4204 with third party computer interface and 100 Newton load cell. Granules were observed during testing and the point where the outer core split was recorded as the crush point.

[00309] A compression modulus calculated from the slope of force applied vs compression distance was also determined. The compression modulus refers to the slope of the straight line section of the compression curve prior to the crush point.

[00310] Swellability was determined by measuring adsorption of moisture by the granules. A lOcmxlOcm fine mesh bag was weighed and lOg granules added to the bag.

The bags were placed onto the surface of 2 paper towels wetted with 100ml water. Bags were collected after 2 hours, blotted dry and re-weighed. The weight of water absorbed by the granules was calculated as the difference between the dry weight and weight of the granules minus the weight of the bag.

Exterior coating	Binder	Type of binder	Hardness (N)	Compression Modulus (N/mm)	Swellability (mg water)	Water activity
PVA BF17W	Xanthan gum	Polyelectrolyte Polysaccharide	5.88	33.88	4940	0.971
PVA BF17W	Maltodextrin	Polysaccharide	7.01	37.95	4462	0.971
PVA BF17W	Carboxy methycellulose	Cellulose	8.65	37.04	4152	0.971
PVA BF17W	Pre-gel potato starch	Starch	7.92	36.51	5265	0.970
PVA BF17W	PVAIcohol	Polyvinyl alcohol	6.25	68.97	3977	0.955
PVA BF17W	Gelatin	Protein	7.87	64.90	3879	0.968
None	None	-	7.399	124.08	-	0.993

2016207759 24 Apr 2019

Table 14: Performance testing of granules of the invention compared with granules lacking an exterior coating.

Viability [00311] The granules were stored in a 20°C incubator for 5 weeks.

[00312] The viability of the bacteria in the granules was measured at the time of manufacture and after 5 weeks storage. The results are shown in Table 15.

Exterior coating	Binder	Type of binder	Viability after manufacture (cfu per g)	Viability after 5 weeks at 20 °C (cfu per g)	% survival
PVA BF17W	Xanthan gum	Polyelectrolyte Polysaccharide	i.6xio⁹/g	4.4xl0⁸/g	28
PVA BF17W	Maltodextrin	Polysaccharide	1.3xl0⁹/g	2.0xl0⁹/g	154
PVA BF17W	Carboxy methycellulo se	Cellulose	1.3xl0⁹/g	1.9xl0⁹/g	146
PVA BF17W	Pre-gel Potato starch	Starch	1.3xl0⁹/g	1.8xl0⁹/g	138
PVA BF17W	PVAIcohol	Polyvinyl alcohol	l.lxl0⁹/g	4.8xl0⁹/g	436
PVA BF17W	Gelatin	Protein	1.5xl0⁹/g	1.7xl0⁹/g	113
PVA BF17W	Polylactic acid	Polyester	5.3xl0⁸/g	4.0xl0⁸/g	75

Table 15: Viability of microorganisms in granules of the invention at manufacture and after 5 weeks storage at 20°C.

[00313] This example describes the preparation of granules of the invention comprising a range of different binders and the physical properties of the granules. The example also demonstrates that the biological material in granules of the invention retains and/or increases viability during storage.

EXAMPLE 11 [00314] This example describes the preparation of granules comprising Serratia entomophila. The performance of the granules was tested and compared to prior art granules lacking an exterior coating.

2016207759 24 Apr 2019 [00315] Granules suitable for broadcast application comprising Serratia entomophila as the biological material were prepared at laboratory scale according to the method described for Example 9.

[00316] 20 g aliquots of granules were placed inside fine mesh bags and laid on to a grass surface exposed to direct sunlight. Data loggers were placed next to the bags to measure temperature and relative humidity. The bags were subjected to diurnal fluctuations in temperature and relative humidity with day time surface temperatures exceeding 50 °C.

[00317] Bags were collected after 2, 4, 8, 24 and 48 hours and the amount of S. entomophila remaining was estimated using standard dilution plating methods. The results 10 are shown in Figure 13. Measures of percent moisture and water activity of the granules were also made. The results are shown in Figures 14 and 15.

[00318] This example demonstrates the stability of the granules of the invention following prolonged exposure to direct sunlight and humidity.

[00319] In the description in this specification reference may be made to subject matter 15 which is not within the scope of the claims of the current application. That subject matter should be readily identifiable by a person skilled in the art and may assist in putting into practice the invention as defined in the claims of this application

Claims

1. Granules comprising a core, the core comprising

c) a second coating that at least partially coats the first coating, the second coating comprising one or more particulate densifiers, one or more disintegrants, and a binder, the granules further comprising an exterior coating that at least partially coats the core and that reduces the water permeability of the granule, the exterior coating comprising a polymeric compound, the granules comprising about 10 to about 25 % by weight moisture.

2. Granules according to claim 1 wherein the biodegradable polymer is a polysaccharide.

3. Granules according to claim 1 or claim 2 wherein the biological material comprises one or more microorganisms, or one or more microorganisms and a protein.

4. Granules according to any one of claims 1 to 3 wherein the particulate densifier is selected from the group comprising a phyllosilicate, a sulphate mineral, an aluminosilicate, a clay mineral, a calcium salt, a volcanic glass, a silicate, or a combination of any two or more thereof.

5. Granules according to any one of claims 1 to 4 wherein the particulate densifier comprises bentonite, gypsum, or bentonite and gypsum.

6. Granules according to any one of claims 1 to 5 wherein the disintegrant is a pregelatinised starch, preferably potato starch.

7. Granules according to any one of claims 1 to 6 wherein the substrate is an aluminosilicate mineral.

8. Granules according to any one of claims 1 to 7 wherein the binder comprises a first polymeric compound and the exterior coating comprises a second polymeric compound.

2016207759 24 Apr 2019

9. Granules according to any one of claims 1 to 8 wherein the binder and/or the exterior coating comprise a polymeric compound selected from the group comprising a polyhydroxyl compound, a polymer or copolymer of a Οι-Οβ alkyl substituted with one or more groups selected from the group comprising lactams or 2-pyrollidone, shellac resin, a disaccharide, a polyelectrolyte, a polyethylene glycol, a polyethylene oxide, a polyacrylamide, a polyester, a cellulose, a lignin, a biodegradable gum, an oligopeptide, a polypeptide or a latex.

10. Granules according to any one of claims 1 to 9 wherein the polymeric compound has an average molecular weight of from about 50 kDa to about 200 kDa, preferably from about 50 kDa to about 100 kDa, most preferably from about 70 to about 90 kDa.

11. Granules according to claim 9 or claim 10 wherein the polymeric compound is a polyhydroxyl compound.

12. Granules according to any one of claims 9 to claim 11 wherein the polyhydroxyl compound has a degree of hydrolysis of from about 80 mol% to about 100 mol%, preferably from about 90mol% to about 100mol%.

13. Granules according to any one of claims 9 to 12 wherein the polyhydroxyl compound is polyvinyl alcohol.

14. Granules according to any one of claims 9 to 12 wherein the polyhydroxyl compound is a polysaccharide selected from the group comprising a starch, a dextrin, chitosan, a gum or a synthetic polysaccharide.

15. Granules according to any one of claims 1 to 10 wherein the polymeric compound is a polymer or copolymer of

a) a C2-C6 hydroxyalkyl, and

b) a C2-C10 hydroxyalkyl, optionally substituted with acyl and/or optionally comprising an ether moiety, for example, methacrylic acid-ethyl acrylate copolymer.

16. Granules according to any one or claims 1 to 10 wherein the polymeric compound is a polymer or copolymer of a Οι-Οβ alkyl substituted with one or more groups selected from the group comprising lactams, preferably γ-lactam, or 2-pyrollidone.

17. Granules according to claim 16 wherein the polymeric compound is polyvinylpyrollidone.

18. Granules according to any one of claims 1 to 17 wherein the biological material retains at least about 5% viability after five weeks storage at ambient temperature.

2016207759 24 Apr 2019

19. Granules according to any one of claims 1 to 18 wherein the exterior coating comprises a particulate material, the particulate material comprising particles having a mean size of from about 0.1 pm to about 50 pm.

20. An agricultural composition comprising granules of any one of claims 1 to 19.

21. A method of making a granule, the method comprising

a) providing a core comprising

c) drying the granule to about 10 to about 25 % moisture by weight.

22. A method according to claim 21 wherein the core is formed by

a) providing a substrate,

b) coating the substrate with a first coating comprising biological material, preferably one or more microorganisms, and optionally a biodegradable polymer and/or a noncytotoxic oil to form a first-coated material,

c) coating the first-coated material with a second coating comprising one or more one particulate densifiers and one or more disintegrants, and with a binder to form the core.

23. A method according to claim 22 wherein the first coated material is coated with alternating layers of the second coating and the binder.

24. A method according to any one of claims 21 to 23 wherein the exterior coating is less soluble in water than the binder.

25. A method according to any one of claims 21 to 24 wherein the binder and/or exterior coating has a viscosity of about 1 to about 300 cP.

2016207759 24 Apr 2019

26. A method according to any one of claims 21 to 25 wherein the binder and/or the exterior coating has a pH of from about pH 4 to about pH 6.

27. A method according to any one of claim 21 to 26 wherein the binder and/or the exterior coating comprise about 0.5% to about 5% w/v of a polymeric compound, preferably a polyhydroxyl compound, most preferably a polyvinyl alcohol.

28. A method of distributing granules on to or into soil to control a pest, preferably an insect pest, the method comprising

a) supplying granules of any one of claims 1 to 19 to a distribution device, and

b) applying the granules on to or into soil using the distribution device.