WO2023209388A1

WO2023209388A1 - Seed coating

Info

Publication number: WO2023209388A1
Application number: PCT/GB2023/051134
Authority: WO
Inventors: Laurence BERMAN; Lutz GLANDORF
Original assignee: BioLevel Limited
Priority date: 2022-04-29
Filing date: 2023-04-28
Publication date: 2023-11-02
Also published as: GB2618149A; GB202206317D0

Abstract

Provided is a coating composition for seeds comprising a mixture suspended in a non-polar liquid, the mixture comprising: a consortium of bacteria; and a bacterial carrier powder. The bacteria in the consortium are: saprophytic bacteria; and/or Nitrogen-fixing and Phosphorus-solubilizing bacteria. The non-polar liquid is not bactericidal against the consortium bacteria, and optionally, wherein the seed coating is a liquid at 20 degrees C.

Description

SEED COATING Background Seed coatings are used to grow commercial crops such as corn, wheat, barley and so forth. Coatings provide the seeds with surface protection, may have pesticidal properties, and can include nutrients and other beneficial agents. Various approaches have been tried, including applying liquid microbial products which require refrigeration to maintain viability to seed directly before planting to ensure the then non-refrigerated microbial products is applied via the seed to the soil in a timely manner to avoid loss of microbial viability, or the blending of microbial products with liquid biostimulants to increase the product viability on seed. The problem with the existing products on the market or otherwise known is that they cannot provide a stable bacterial colony without refrigeration. Another issue is the existing compositions may clog the apparatus when sprayed onto the seeds or result in the seeds sticking together. Surprisingly, we have found that providing a mixture, of beneficial bacteria and a carrier, as a suspension in a non-polar liquid, such as an oil, has a number of advantages. Brief Description of the Invention Provided is a coating composition for seeds comprising a mixture suspended in a non-polar liquid, the mixture comprising: a. a consortium of bacteria; and b. a bacterial carrier powder; wherein bacteria in the consortium are: i. saprophytic bacteria; and/or ii. Nitrogen-fixing and Phosphorus-solubilizing bacteria; wherein the non-polar liquid is not bactericidal against the consortium bacteria, and optionally, wherein the seed coating is a liquid at 20 degrees C. In some embodiments, the non-polar liquid is inert and so is not reactive with the coating composition components or the intended seeds. In some embodiments, non-polar liquid is an oil. In some embodiments, the oil is: a. vegetable oil, including but not limited to Rapeseed Oil; Sunflower Oil; Safflower Oil; Soybean Oil; corn Oil; b. is mineral oil; or c. is a mixture of: i. one or more vegetable oils; ii. one or more mineral oils; or iii. a mixture of one or more vegetable oils and one of more mineral oils. In some embodiments, the bacterial carrier is a powder, optionally particles with a diameter in the range of 100 micrometres (μm) to 300 micrometres (μm), optionally 200 micrometres (μm). In some embodiments, the bacterial carrier is a an organic or inorganic powder, optionally: calcified seaweed, talcum, graphite, corn starch, a monosaccharide, disaccharide or polysaccharide powder, optionally dextrose monohydrate. In some embodiments, the non- polar liquid is sprayable at 20 degrees C. In some embodiments, the composition has viscosity of between 35 centipoise at 40 degrees centigrade or 40 centipoise at 35 degrees centigrade. In some embodiments, the consortium comprises: c. at least one Pseudomonas species; d. at least one Bacillus species; and/or e. at least one Paenibacillus species; and, optionally f. at least one Azospirillum species. In some embodiments, the consortium comprises: a) Pseudomonas putida b) Bacillus subtilis c)Bacillus licheniformis d) Bacillus amyloliquefaciens e) Bacillus megaterium f) Bacillus pumilus; and g) Paenibacillus polymyxa In some embodiments, consortium further comprises h) Azospirillum brasilense In some embodiments, the consortium comprises i. Pseudomonas putida at a minimum concentration of 1x10^8 CFU/g ii. Bacillus subtilis at a minimum concentration of 1x10^8 CFU/g iii. Bacillus licheniformis at a minimum concentration of 1x10^8 CFU/g iv. Bacillus amyloliquefaciens at a minimum concentration of 1x10^8 CFU/g v. Bacillus megaterium at a minimum concentration of 1x10^8 CFU/g vi. Bacillus pumilus at a minimum concentration of 1x10^8 CFU/g vii. Paenibacillus polymyxa at a minimum concentration of 1x10^8 CFU/g viii. Azospirillum brasilense at a minimum concentration of 5x10^7 CFU/g In some embodiments, the coating composition further comprises a dye or colourant. Also provided is a coated seed comprising the coating composition. In some embodiments, the coted seed is a seed from: a. member of the grass family, Graminacea (a.k.a. Poaceae); or b. member of the Leguminaceae family. In some embodiments, the coated seed is a seed selected from: c. Corn/maize; d. Alfalfa; e. Sorgum; f. Soya; g. Wheat; h. Barley; i. Oats; j. Rice; k. Sugar Cane; or l. a mixture, optionally a forage mixture, of any two or more of the above. In some embodiments, the coated seed is provided in a forage mixture. Thus, also provided is a forage mixture comprising a mixture of the present coated seeds. Also provided is a coated granule, wherein the coating is a coating composition as provide herein. In some embodiments, the granule is a fertilizer, a liming product, a soil amendment product or shell particles (optionally crushed oyster shell). In some embodiments, the granule is an agricultural liming product or agricultural liming material selected from: a. limestone (calcitic or dolomitic); b. burned lime; slaked lime; c. marl; d. shells; and e. industrial by-products for example sugar beet lime and sludge from water treatment plants. In some embodiments, the granule is a particle in the range of 2 to 4 mm in diameter, for example: poly4 ® Here, the oil based product is sprayed on granules as a carrier. Also provides is a mixture or suspension comprising: a. the present coating composition, coated seed or coated granule; and b. other seed coatings or applications (such as starter fertilizers , such as ENTEC ). Here, in some embodiments, one blends granulated fertilizer and seeds for planting. Also provided is a vessel for storing or transporting the present coating composition, coated seed, coated granule or mixture, suspension or emulsion. Also provided is a method of use for the present coating composition, comprising: a. mixing seeds, granules or providing a mixture, suspension or emulsion with the composition; and b. sowing the coated seed or coated granule or mixture or suspension in a suitable environment such as soil or growth medium that supports a rhizosphere. Also provided is a method of use for the present coated seed, coated granule or mixture or suspension, comprising sowing the coated seed, coated granule or mixture or suspension in a suitable environment such as soil or growth medium that supports a rhizosphere. Also provided is a method of manufacture of the coated seed, coated granule or mixture or suspension. Figure Legends Fig 1 shows the trial field layout for Example 4, measurements are in metres. Detailed Description of the Invention We have found that providing the mixture (of the beneficial bacteria and the carrier) in a non- polar liquid has a number of advantages. The use of an oil as the non-polar liquid is preferred, in some embodiments. The mixture is provided as a suspension in the non-polar liquid. The present composition and related aspects comprises a concentrated consortium of microorganisms designed to solubilise Phosphate reserves in the soil, increase Nitrogen, K and/or micronutrient uptake or fixation in the rhizosphere, as well as free up essential micronutrients in the soil environment. Without being bound by theory, it is thought that enzymes and other organic chemicals produced by the provided microorganisms (beneficial bacteria) break P bonds between Al, Ca, and Fe. This process results in a plant-accessible form of P. The synergistic relationship whereby roots supply the microorganisms in the form of root exudates and the beneficial bacteria efficiently provide accessible nutrients to the plant has evolved over millions of years. Most soils have plenty of Phosphorus but lack the micro- organisms to disassociate this P into a form the plant can use. The present composition is a microorganism consortium and, in some embodiments, not a single-species product enabling it to be suited to a range of soil types and climatic conditions. In some embodiments, the present composition contains beneficial microorganisms at a concentration 1BB CFU/g (1 billion CFU/gram: this refers to the total CFU count in the product across individual strains. By way of example, 5x10^7 CFU/g would be the CFU per gram or ml for an individual strain in the composition. In some embodiments, the present composition comprises bacteria that are GM free (i.e. are not genetically modified according to the EU’s definition of this) and/or certified for use in organic farming (according to the EU’s definition of this). In some embodiments, the total bacterial count or concentration is approximately 2 billion (2 x 10⁹) CFU/ml or non-polar liquid. In some embodiments, the present composition is able to produce 200 billion (2 x 10¹¹) CFU per hectare in recommended applications, for example when applied to corn (maize) seeds. In some embodiments, the present composition has a viscosity that allows the composition to be added to seeds without clogging up (agglutinating inside) the spraying device nozzle and/or binding the sprayed and coated seeds or granules together such that they cannot be individually separated and sown by a seed applicator machine for sowing seeds into the earth in a field. In some embodiments, the composition is sprayed or sprayable, optionally by an MPL Pump or a Procam pump, optionally at up to 2 bar. In some embodiments, this does not clog the nozzle to thereby significantly impact delivery. In some embodiments, the sprayed composition provides an even and/or sufficient coating to the seed to achieve benefits to the formation, development or maintenance of the rhizosphere. In some embodiments, the viscosity of the composition is 35-40 centrepoise at varying temperatures. These are further discussed below. In some embodiments, the present composition comprises an oil with a mixture of dextrose and the bacterial consortium suspended with the oil, and has a viscosity of 35-40 centrepoise at varying temperatures. In some embodiments, the present composition can be stored at RTP, so approximately 20 degrees C without loss of function for at least several weeks. It can therefore remain functional for several weeks before and after application. Importantly, at this temperature, no refrigeration is required to prevent large proportions of the bacteria form multiplying prior to deployment. Example 2 shows that the CFU count is stable after plating after several weeks of storage at 20 degrees C. The present composition can allow seeds to be coated with a low volume of material compared to other types of liquid coating. The present composition can allow even coating on the seed and prevents product loss and/or wastage from pooling of the applied product. The present composition can allow the seed coating to deliver the microbial consortium directly to the rhizosphere of the growing plant. The present composition can reduce the amount of product required with other application methods. The present composition can accelerate the establishment of symbiotic plant-microbial relationships. The present composition can allow or improve solubilization of Phosphorus in the soil. The present composition can allow or improve fixation of atmospheric Nitrogen in the soil. The present composition can allow or improve fixation of atmospheric Potassium and/or other micronutrients in the soil. The present composition can allow or improve colonization of the plant rhizosphere, to thereby increase the availability of both soil-based and applied macro and micro nutrients to the plant. This increased availability of nutrients, especially on a continuous basis, can lead to increased plant growth and thus yield. The present composition can allow or improve essential NPK (Nitrogen, Phosphorus and Potassium (K)) nutrients to be made available to the plant. The present composition can allow or improve the release of locked-up soil nutrients (Phosphorus, Potassium and/or other micronutrients) for plants when applied to the soil. The present composition can allow or improve conversion of atmospheric Nitrogen into usable plant nutrients. The present composition can allow or improve crop yields & food quality. Micronutrients as referred to herein may include, in some embodiments, zinc, iron, manganese, and/or calcium. The present composition can allow or improve the cycle efficiency of such N, P and/or K and thus reduces fertilizer use. Excessive application of fertilizer and chemical adversely effects soil biology and may contaminate the environment. The present composition can allow or improve sustainability, for example through reduced fertilizer use or increased fertilizer use efficiency. The present composition can provide broad compatibility with plant protection products, such as pesticides. In some embodiments, the present composition may further comprise plant protection products, such as pesticides. Many microbial nutritional products in the market are simple nitrogen fixers or limited in being single strain products. The present composition can, in some embodiments, offer a complete nutrition package by adding phosphorus and potassium solubilizing microbes, as well as micronutrients, to our nitrogen fixers. Plant-associated rhizosphere bacteria have an important role in establishing and improving plant growth on different soil types, since they affect the availability of essential elements and provide plants with extra Nitrogen. Rhizosphere formation, development or improvement is an important aim of the present invention and is thus provided in some embodiments. Various methods are typically used to add beneficial bacteria to the soil so as to form a rhizosphere: Addition to, and distribution via, a slurry, for example an animal waste slurry; spraying onto the soil; seed coating; foliar application and application to granulated fertilizers. In respect of foliar application, in some embodiments, endophytic microbial products (meaning they go into the plant cells) are used and, in some embodiments, sprayed as a foliar application. In respect of granulated fertilizer, in some embodiments, the microbes are coated onto a standard granulated fertilizer. We have shown here (see Example 1) that the consortium are able to create a rhizosphere via slurry delivery. However, there are disadvantages with slurry (for example that dosing the microbe in slurry is difficult and can require specialty mechanical equipment). Thus, seed coatings are preferred in the present invention. In some embodiments, pesticides, including fungicides and insecticides can also be added (see for example Example 3, where Coozer/Afron Star were added without compromising the composition). In some embodiments, fertilizers can also be added, in particular micronutrients. This is complementary to one of the benefits of the present invention, which is that less Nitrogen and/or Phosphorus-containing fertilizers are necessary. In some embodiments, a typical application rate of the present composition to seeds, in particular, corn seeds, is 4 litres of composition per tonne of seed. Seed The seed to which the present composition may be applied is a plant seed, for example a seed of an agricultural crop, a vegetable seed, a herb seed, a wildflower. In some embodiments, the seed is a seed of an agricultural crop. In some embodiments, the seed may be of the class of Monocotyledoneae or of the class of Dicotyledoneae. In some embodiments, the seed is a seed of: ● soybean, ● cotton, ● corn or maize ● peanut, ● barley, ● oat, ● wheat, ● rye, ● riticale, ● mustard, ● oil seed rape (or canola), ● sunflower, ● sugar beet, ● safflower, ● millet, ● chicory, ● flax, ● rapeseed, ● buckwheat, ● tobacco, ● hemp seed, ● alfalfa, ● signal grass, ● clover, ● sorghum, ● chick pea, ● beans, ● peas, ● vetch, ● rice, ● sugar cane, and/or ● linseed. Consortium A consortium or community, may be two or more bacterial or microbial groups living synergistically. In some embodiments, two or more bacterial or microbial groups may live symbiotically. Numerous consortia are known. Examples include US20180235235A1 which relates to a specific consortia of bacteria referred to as Microbial Consortium A1006 and deposited under ATCC Patent Deposit Designation PTA-121755. WO2021146209A1 relates to a consortia of microbes that are functionally optimized for nitrogen fixation and deliver such to plants in a targeted, efficient, and environmentally sustainable manner. The microbes within the consortium differ in nutrient utilization, temporal occupation, oxygen adaptability, and/or spatial occupation, which enables the microbes to deliver nitrogen to a cereal plant in a spatially targeted (e.g. rhizospheric) and temporally targeted (e.g. during advantageous stages of plant's life cycle) manner. US2014352376A1 relates to a binder such as Sealmaster which is a starch that pulls together granules of biosolid particles, the particles themselves holding the bacteria. The consortium of bacteria is, in some embodiments, dried prior to, or as part of, mixing with the bacterial carrier powder. Thus, in some embodiments, the composition comprises a dried consortium of bacteria. In some embodiments, the methods and processes of the present invention comprise a step of drying the consortium consortium of bacteria. The drying may be by vacuum drying or freeze-drying, or other methods known in the art. In some embodiments, therefore , the composition comprises freeze-dried or vacuum-dried consortium consortium of bacteria. The consortium may comprise a wide range of different microbial combinations. These bacterial species are typically described as naturally occurring, often saprophytic bacteria. Thus, in some embodiments, bacteria in the consortium are saprophytic. They may also be, in some embodiments, naturally occurring, or in other embodiments the bacteria may have been genetically modified, gene edited or contain a trans gene (i.e. comprise polynucleotides encoding a trans gene). In some embodiments, bacteria in the consortium may have the European federation of Biotechnologies Class 1 classification or a global equivalent. This classification is defined as: “Commonly occurring saprophytes, never been known to cause disease in man” or alternatively “naturally occurring micro-organisms that have never been identified as causative agents of disease in man and that offer no threat to the environment.” Such terms may apply, in some embodiments, to the present invention. The bacteria of the consortium are, in some embodiments, beneficial microbes (i.e. beneficial bacteria). The definitions are largely used interchangeably, although saprophytic refers to the mode of nutrition and saprotroph refers to an organism that utilises the saprophytic mode of nutrition. The bacteria may be, in some embodiments, saprophytic (or saprophytes). These may also be referred to as saprotrophs and therefore saprotrophic bacteria. In some embodiments, the bacteria of the consortium are dormant , at least at the time of application to the seed. In the consortium, the bacteria are encouraged to sporulate, in some embodiments. They may, in some embodiments, be partially dehydrated to induce a state of reduced metabolism in the microbes. Bacterial Species Bacteria in the consortium may be, in some embodiments, Nitrogen-fixing bacteria. Example species include: Paenibacillus polymyxa Azospirillum brasilense Bacteria in the consortium may be, in some embodiments, be both Nitrogen-fixing and Phosphorus-solubilizing bacteria, i.e. can perform both functions. Example species include: Paenibacillus polymyxa In some embodiments, bacteria in the consortium may be saprophytic, Nitrogen-fixing and Phosphorus-solubilizing bacteria. Example species include: Paenibacillus polymyxa Nitrogen-fixing bacteria are bacteria capable of transforming (“fixing”) atmospheric Nitrogen into solid or liquid nutrients or compounds for use by, for example, plants, as part of the Nitrogen cycle. Bacteria in the consortium may be, in some embodiments, Phosphorus-solubilizing bacteria, for example Phosphate Solubilizing Microorganisms (PSM). Phosphorus-solubilizing bacteria are bacteria capable of solubilizing Phosphorus in the soil. Plants acquire phosphorus from soil solution in the form of phosphate anion. It is the least mobile element in plant and soil in comparison to other macronutrients. It remains in a precipitated form in the soil as mono or orthophosphate or is absorbed by Fe or Al oxides through ligand exchange. Phosphate Solubilizing Microorganisms (PSM) play a very important role in phosphorus nutrition by exchanging its availability to plants through release from inorganic and organic soil phosphorus pools by solubilization and mineralization. The main mechanism in the soil for mineral phosphate solubilization is by lowering the soil pH by the microbial production of organic acids and mineralization of organic phosphorus by acid phosphates. To fulfill the phosphorous demand of plant, an additional source of phosphorous is applied to plants in the form of chemical fertilizers (Phosphate Solubilizing Microbes: An effective and alternative approach as Biofertilizers (Kumar Anand, Baby Kumari1, M. A. Mallick, Int J Pharm Pharm Sci, Vol 8, Issue 2, 37-40, 2016)). Example species include species of Pseudomonas, Bacillus, Micrococcus, Flavobacterium, Aspergillus, Penicillium, Fusarium, and/or Sclerotium. Strains from bacterial genera Pseudomonas, Bacillus, Rhizobium and Enterobacter along with Penicillium and Aspergillus fungi are the most powerful P solubilizers. Bacillus megaterium, B. circulans, B. subtilis, B. polymyxa, B. sircalmous, Pseudomonas striata, and Enterobacter are generally thought of as the most important strains (Kumar Anand, Baby Kumari1, M. A. Mallick, Int J Pharm Pharm Sci, Vol 8, Issue 2, 37-40, 2016)). Phosphorus-solubilizing bacteria in the consortium or additional PSM microbes provided in the composition may, in some embodiments, include any one or more of: ● Pseudomonas, ● Bacillus, ● Micrococcus, ● Flavobacterium, ● Aspergillus, ● Penicillium, ● Rhizobium, ● Enterobacter and/or Thus, Phosphorus-solubilizing bacteria in the consortium or additional PSM microbes provided in the composition may, in some embodiments, include any one or more of: ● Bacillus megaterium, ● B. circulans, ● B. subtilis, ● B. polymyxa, ● B. sircalmous, ● Pseudomonas striata, and/or ● Enterobacter. In some embodiments, the consortium additionally comprises the pathogens Fusarium and/or Sclerotium. In some embodiments, the consortium comprises at least one (saprophytic, Nitrogen-fixing and/or Phosphorus-solubilizing) Pseudomonas, preferably Pseudomonas putida. In some embodiments, this is present at a minimum concentration of 1x10^8 CFU/g of the mixture. In some embodiments, the consortium comprises at least one (saprophytic, Nitrogen-fixing and/or Phosphorus-solubilizing) Bacillus sp. In some embodiments, this is present at a minimum concentration of 1x10^8 CFU/g of the mixture. In some embodiments, the Bacillus is Bacillus subtilis. In some embodiments, this is present at a minimum concentration of, or at least, 1x10^8 CFU/g of the mixture. In some embodiments, the Bacillus is Bacillus licheniformis. In some embodiments, this is present at a minimum concentration of, or at least, 1x10^8 CFU/g of the mixture. In some embodiments, the Bacillus is Bacillus amyloliquefaciens. In some embodiments, this is present at a minimum concentration of, or at least, 1x10^8 CFU/g of the mixture. In some embodiments, the Bacillus is Bacillus megaterium. In some embodiments, this is present at a minimum concentration of, or at least, 1x10^8 CFU/g of the mixture. In some embodiments, the Bacillus is Bacillus pumilus. In some embodiments, this is present at a minimum concentration of, or at least, 1x10^8 CFU/g of the mixture. In some embodiments, the consortium comprises at least one (saprophytic, Nitrogen-fixing and/or Phosphorus-solubilizing) Paenibacillus, preferably Paenibacillus polymyxa. In some embodiments, this is present at a minimum concentration of, or at least, 1x10^8 CFU/g of the mixture. In some embodiments, the consortium comprises at least one (saprophytic, Nitrogen-fixing and/or Phosphorus-solubilizing) Azospirillum, preferably Azospirillum brasilense. In some embodiments, this is present at a minimum concentration of, or at least, 5x10^7 CFU/g. Each of the bacteria at the genera, species or strain level may have one or more of the above properties (e.g are saprophytic, Nitrogen-fixing and/or Phosphorus-solubilizing). However, not every individual organism within the consortium needs to be active or capable of such functions. As long as the consortium is able, together, to provide these functions at the population (not individual) level. In particular, in some embodiments, the consortium is capable of forming and/or contributing to a rhizosphere. The rhizosphere is the microbially active area of the nutrient exchange around the root area of a plant. The genera, species and relative proportions of bacteria vary per crop. For example, in some embodiments, the consortium may comprise a combination of Bacillus, Paenibacillus, Pseudomonas & Azospirillum. This combination is ideal for use on plants that are members of the grass family, Graminacea (a.k.a. Poaceae). Therefore, in some embodiments, the composition may comprise Bacillus, Paenibacillus, Pseudomonas & Azospirillum and is for use in coating seeds from members of the grass family, Graminacea (a.k.a. Poaceae). Corresponding coated seeds are also provided. In some embodiments, the consortium may comprise a combination of Bacillus, Paenibacillus, and Pseudomonas. This combination is ideal for use on plants that are members of the Leguminaceae family. Therefore, in some embodiments, the composition may comprise Bacillus, Paenibacillus, Pseudomonas (and in some embodiments, not Azospirillum) and is for use in coating seeds from members of the Leguminaceae family. Corresponding coated seeds are also provided. In some embodiments, the present composition comprises a consortium of synergistic, naturally-occurring soil bacteria. In some embodiments, bacteria in the consortium are rhizobacteria. In some embodiments, the seed coating composition is a bacterial, or bacterial-promoting, seed coating composition. This is because the non-polar liquid may be, in some embodiments, non-bactericidal. The coating may also comprise, in some embodiments, other microbes, especially beneficial microbes or beneficial bacteria to assist the growth of the plant. For example, the coating could also include, in some embodiments, fungi or fungal spores. Examples may include Trichoderma sp.. Concentrations of Bacteria in the Mixture Bacteria of one species or another can, in some embodiments, be present at a minimum concentration of 5x10^7 CFU/g or 1x10^8 CFU/g. Upper limits may be 1x10^9 CFU/g. In some embodiments, they can be present in concentrations of: at least 1x10^4 CFU/g; at least 1x10^5 CFU/g; at least 1x10^6 CFU/g; at least 5x10^6 CFU/g; at least 1x10^7 CFU/g; at least 5x10^7 CFU/g; at least 1x10^8 CFU/g; at least 5x10^8 CFU/g; at least 1x10^9 CFU/g; at least 5x10^9 CFU/g; or even a as high as at least 1x10^10 CFU/g. In some embodiments, the soluble powder contains approx. 750 million CFU/g to 1 billion CFU and the liquid seed coating will be approx. double that value. All concentrations values provided herein are in CFU per gram. The gram refers to per gram dry weight of the mixture of the bacteria and the bacterial carrier powder. This is the dried bacterial carrier powder. Other equivalent measurements are envisioned. Mixture The bacteria are preferably dried and preferably dormant, in stasis or sporulated as discussed herein. They are mixed with a bacterial carrier powder to form the mixture, which is then suspended in the non-polar liquid. The purpose of the bacterial carrier powder is to provide a substrate for the bacteria in the sense of a material to carry and allow transport and physical manipulation of the bacteria. In other words, the carrier is a medium that can convey or hold the bacteria. Preferably, the carrier conveys or holds the bacteria in sufficient quantities and keeps them viable at room temperature (approx.20 degrees C) and through the seed coating and sowing process. The bacterial carrier powder, in some embodiments, includes a saccharide. In some embodiments, the carrier powder is a carbohydrate-based powder. In some embodiments, the bacterial carrier powder is, or includes, a monosaccharide. In some embodiments, the monosaccharide is glucose or dextrose or a mixture of monosaccharides, for example a mixture of glucose and dextrose. Various hydrates of the monosaccharide, especially dextrose, may be used, although monohydrates are preferred, especially dextrose monohydrate. In some embodiments, the monosaccharide is fructose, and again mixtures with glucose and /or other monosaccharides such as dextrose are preferred in some embodiments. In some embodiments, the bacterial carrier powder is, or includes, a disaccharide, such as dextrose. In some embodiments, the bacterial carrier powder is, or includes, a polysaccharide, such as a starch. In some embodiments, the bacterial carrier powder is not a saccharide, and so examples may include organic or inorganic substrates, zeolites calcified seaweed and/or bentonites. The mixture may, in some embodiments, be referred to as an inoculant. In some embodiments, the bacterial carrier powder is replaced by a bacterial carrier which is not a powder, but a gel. The bacteria are preferably dried and mixed in the bacterial carrier powder. Alternatively, they may be dried onto the powder, for example by using a using a vacuum or freeze drying method. Suitable methods are known in the art, but may include spray drying or heat drying. Once the mixture is formed of the bacterial carrier powder and the consortium, then it may be blended to encourage equal distribution of the consortium with the powder. The mixture may comprise or may be formed exclusively of the bacterial carrier powder and the consortium. There are a range of monosaccharide, disaccharide and polysaccharide powders, of various levels of hydration that can be used as an alternative substrate for the microbial consortium. Dextrose monohydrate is a preferred carried powder. In some embodiments, it is present in the mixture at 97.6% w/w. In some embodiments, it is present in the mixture in at least 80% w/w; at least 85% w/w; at least 90% w/w; at least 95% w/w; at least 97% w/w; at least 98% w/w; at least 99% w/w; at least 99.5% w/w. In some embodiments, it is present in the mixture: in a range of 80%-100% w/w; in a range of 80%-98% w/w; in a range of 90%-100% w/w; in a range of 90%-98% w/w; in a range of 95%-98% w/w; in a range of 96%-97.8% w/w; or in a range of 96%-98% w/w. The powder may, in some embodiments, have a particle diameter in the range of 100 -300 micrometres. In some embodiments, the particle diameter is approximately 200 micrometres (+/- 10 or 20%). Non-Polar Liquid The mixture is suspended in the non-polar liquid. It thus forms a suspension, preferably not a solution. Emulsions are typically avoided. In some embodiments, the microbes are dried on the powder and are added to the carrier. In some embodiments, the carrier forms a suspension in the non-polar liquid. The non-polar liquid is, in some embodiments, non-aqueous. Mono-ols, diols, tiols (such as glycerine/glycerol) and polyols are therefore preferably not used as these are polar. Nonpolar liquids may include, in some embodiments, oils such as mineral oil or vegetable oils. In some embodiments, the carrier forms a suspension in an oil. In other words, the non-polar liquid is an oil. The non-polar liquid is not detrimental to the bacteria of the consortium. In some embodiments, the non-polar liquid is non-bactericidal. In some embodiments, the non-polar liquid is a non-bactericidal oil. The non-polar liquid, preferably an oil, has, in some embodiments, a low freezing point, i.e. a freezing point below 20 degrees C. This ensures that the non-polar liquid, preferably an oil, is a liquid at this temperature. There are a range of vegetable oils with low levels of saturated fats and high levels of mono and polyunsaturated fats that have a low freezing point (thus they remain liquid in a wider range of temperatures) and are non-bactericidal which could be used in the present invention. In some embodiments, the non-polar liquid may be or comprise an oil, such as vegetable oil. In some embodiments, the non-polar liquid may be or comprise Rapeseed Oil; Sunflower Oil; Safflower Oil; Soybean Oil; or Corn Oil. In some embodiments, the non-polar liquid may be or comprise a mineral oil. The mineral oil preferably does not contain a bactericide preservative, so in some embodiments, coating oils may be used. Mixtures of any of the non-polar liquids described herein are also envisaged. Therefore, reference herein to a non-polar liquid being a certain oil, also includes that the non-polar liquid may comprise that oil. In some embodiments, the non-polar liquid, preferably an oil, does not contain a bactericide. In some embodiments, the non-polar liquid, preferably an oil, does not contain Neem, Eucalyptus and Citrus oils. In some embodiments, the non-polar liquid, preferably an oil, has a high freezing point, i.e. are a wax or solid at 20 degrees C. Lower freezing points may be appropriate in colder climates or higher in warmer climates, so the non-polar liquid may, in some embodiments, have a freezing point: at or below 5 degrees C; at or below 10 degrees C; at or below 15 degrees C; at or below 20 degrees C; at or below 25 degrees C; or even at or below 30 degrees C. The non-polar liquid, preferably an oil, is in some embodiments present in at least 95% by weight of the composition. In this instance, the carrier powder makes up 4.8% with the remaining 0.2% the consortium and any other additives. In other embodiments, the non-polar liquid, preferably an oil, is present in at least 85% by weight of the composition. In some embodiments, the non-polar liquid, preferably an oil, is present in at least 85% by weight of the composition. In some embodiments, the non-polar liquid, preferably an oil, is present in at least 90% by weight of the composition. In some embodiments, the non-polar liquid, preferably an oil, is present in at least 95% by weight of the composition. In some embodiments, the non-polar liquid, preferably an oil, is present in at least 96% by weight of the composition. In some embodiments, the non-polar liquid, preferably an oil, is present in at least 97% by weight of the composition. In some embodiments, the non-polar liquid, preferably an oil, is present in at least 98% by weight of the composition. In some embodiments, the non-polar liquid, preferably an oil, is present in at least 99% by weight of the composition. In some embodiments, the non-polar liquid, preferably an oil, is present in at least 99.5% by weight of the composition. In some embodiments, the non-polar liquid, preferably an oil, has a certain viscosity. A preferred viscosity (dynamic (absolute) viscosity rather than kinematic viscosity) with one centipoise = 1 millipascal second, a preferred viscosity for the present composition, in some embodiments, is: 35 centipoise at 40 degrees centigrade; to 40 centipoise at 35 degrees centigrade. In some embodiments, the preferred viscosity for the present composition, is: 30 centipoise at 40 degrees centigrade; to 45 centipoise at 35 degrees centigrade. In some embodiments, the preferred viscosity for the present composition, is: 25 centipoise at 40 degrees centigrade; to 45 centipoise at 35 degrees centigrade. In some embodiments, the preferred viscosity for the present composition, is: 25 centipoise at 40 degrees centigrade; to 35 centipoise at 35 degrees centigrade. In some embodiments, the preferred viscosity for the present composition, is: 30 centipoise at 40 degrees centigrade; to 50 centipoise at 35 degrees centigrade. In some embodiments, the preferred viscosity for the present composition, is: 30 centipoise at 40 degrees centigrade; to 38 centipoise at 35 degrees centigrade. The composition may also be referred to as a seedcoat. In some embodiments, the non-polar liquid is inert. In some embodiments, the non-polar liquid is not reactive with the coating composition components or the intended seeds Dye In some embodiments, the composition comprises a dye. This dye may be a dye visible to humans once the composition has been added to the seeds. This allows the user, such as the farmer to see that the composition has effectively been added to the seed, prior to sowing. It may also allow some quantification ‘by eye’ if exact measurement of volume per weight of seed is not possible. In some embodiments, the dye is a red or orange dye (once added to the seed). In a preferred embodiment, the dye is an orange dye, for example a paprika-based dye. This is used as it does not interfere significantly with the colour of other standard red-dyed additives that have already been or will be added to the seed. Manufacturing Process: One example of a method of manufacturing or process for providing a composition of the present invention is described below. ● The microbial consortium is dried onto dextrose monohydrate. ● Rapeseed oil is used as the carrier and all manufacturing processes are carried out at ambient temperature. ● To 100 litres of rapeseed oil, 110ml of orange, paprika-based food safe colourant is added to the oil and mixed with a high shear mixer. ● 0.8kg of bacterial consortium is slowly added to the coloured rapeseed oil, whilst being mixed with a high shear mixer. ● After mixing, any frothing from the mixing process is allowed to subside. ● Quality control checks are carried out to ensure that the total microbial count of the product is 2billion cfu/ml. ● Product is bottled or placed in another suitable vessel and sealed. Use One example of a method of use is provided below ● Directions for use. Shake in bottle. Apply at a rate of 4 litres per Ton of seed. Apply directly to the seed on its own, or it can be used in combination with other seed applications. Avoid temperatures over 50 degrees C of extreme freezing cold (below zero degrees C?). ● Apply to Corn seeds (maize); ● Dry planter box treatment: o Single application per growing season is recommended at an application rate of 250 gram per Hectare. o Dry product can be used by tossing it into the seed bag and shaking it or by stirring it into the seed in the planter box o Dextrose monohydrate powder 97.6% w/w o Minimum components, always present (*): ^ Pseudomonas putida (minimum) 1x10^8 CFU/g ^ Bacillus subtilis (minimum) 1x10^8 CFU/g ^ Bacillus licheniformis (minimum) 1x10^8 CFU/g ^ Bacillus amyloliquefaciens (minimum) 1x10^8 CFU/g ^ Bacillus megaterium (minimum) 1x10^8 CFU/g ^ Bacillus pumilus (minimum) 1x10^8 CFU/g ^ Paenibacillus polymyxa (minimum) 1x10^8 CFU/g ^ Azospirillum brasilense (minimum) 5x10^7 CFU/g Result: in a UK trial, Grain Yield and Dry Matter Yield was maintained at a 20% reduction in Nitrogen fertilizer use. Examples Example 1 – field testing of the consortium shows that increased yield and/or lower fertilizer requirements This shows that the consortium, in a mixture with the bacterial carrier powder, increase yield. Although the mixture was not suspended in a non-polar liquid, such as oil, it is plausible it would also work with oil. In other words, the significance of these results is that we have shown that the consortium will also have benefits when delivered in a suspension in a non- polar liquid such as an oil. Example 1a :Test in fields Materials and Methods In 2021, Wageningen University & Research, Open Teelten was commissioned by ForFarmers to test the application of a fertilizer additive, referred to as MaizeNP, in the crop maize. The additive is added to the basic fertilization with 35m³ cattle slurry before sowing maize. As row fertilization during sowing, a granulated NS fertilizer 38 kg nitrogen per ha was applied. Also included is a completely unfertilized object and an object with only cattle slurry without additive. The trial was conducted in Vredepeel on moderately humous, loamy sandy soil. The test subjects are shown in Table S1. The total effective N application applied from slurry and fertilizer was almost equal to the N application standard for maize on southern sandy soil in 2021 of 112 kg N per ha. The experiment was set up in four replicates. The cultivation method and crop care took place according to practice. MaizeNP is a highly concentrated consortium of microorganisms designed to solubilise Phosphate reserves in the soil and increase Nitrogen fixation in the rhizosphere. Enzymes produced by the MaizeNP microorganisms break P (Phosphorus) bonds between Al, Ca, and Fe. This process results in a plant accessible form of P. The synergistic relationship whereby roots supply the microorganisms in the form of root exudates and the beneficial bacteria efficiently provide accessible nutrients to the plant has evolved over millions of years. Most soils have plenty of P but lack the micro-organisms to disassociate this P into a form the plant can use. MaizeNP is a microorganism consortium and not a single species product enabling it to be suited to all soil types. MaizeNP contains beneficial micro-organisms at a concentration 1BB CFU/g, is GM free and certified for use in organic farming. Biolevel MaizeNP as a soluble powder contains approx.1 billion CFU, as a liquid seed coating it contains approx.2 billion CFU. In the table below, if one sums up the individual strain CFU count guaranteed per strain, the numbers for MaizeNP powder add up to 750 million CFU/g and the MaizeNP liquid to 1.5 billion CFU/ml. The work in this Example was done with the powdered product which shows in sum 750 million CFU. Thus, in some embodiments, the soluble powder contains approx.750 million CFU/g to 1 billion CFU and the liquid seed coating will be approx. double that value.

Method Location : Vredepeel Repetitions : 4 Design : Randomized block test Period : 2021 Treatment reference : 35m³ slurry + GM 25-0 Treatment 2 : 35m³ slurry + NPMaize + GM 25-0 Fertilization NP Maize : 333 gram /ha, dissolved in slurry Slurry : 30 m³ per ha Mineral fertilzier : 38 kg N uit GroMaize 25-0 Harvesting of the maize occurred

• MaizeNP contributed 2% to extra dry matter yield, compared to the reference. In absolute numbers this means 370 kg per ha. Usually 1 kg of dry matter is valued at €0.15. The added value would be €55 when applying this rule of thumb. However, we see that the concentration of starch in the dry matter has increased. For this reason, applying this rule of thumb is not fair. • MaizeNP contributed 3% to extra VEM yield, compared to the reference. In absolute numbers this means 650 VEM per ha. At a VEM price of 22.3 cents (reference date Sept 2021), this justifies an additional yield of €145 per ha. • MaizeNP contributed 11% to extra starch yield, compared to the reference. In absolute numbers this means 843 kg of starch per ha. With this additional starch yield, savings can be made on the purchase of by-products or concentrates. Its value is ration dependent. Discussion 1. Adding 333 grams of MaizeNP over hectares requires a carrier, which ensures a homogeneous distribution over the maize land. In this test, slurry was used as a carrier. In practice, there is no technique available (yet) that can accurately dose a powder in animal manure. For the application of MNP in practice, a solution must be sought for this. 2. No two growing seasons are the same, as is the interaction between crop growth and soil. For this reason, this research requires a repetition.

Example 1b : Biolevel AD Maize Trial report 2021 Trial: Compare Biolevel MaizeNP at normal and reduced nitrogen rates with standard farm practice. Aim and design of the trial was to assess the effect Biolevel MaizeNP has at normal and reduced rates of Nitrogen by twenty percent (including the effects of the treatment on the availability of Nitrogen and Phosphorus) on plant development, yield, and quality. Materials and Methods Trial Report: Biolevel MaizeNP (AD Maize) Field Name: CL Polo Ground Product: Bio-level Maize NP Crop: Maize Group: AD Soil Type: Loamy Sand Date Planted: 5th May 2021 Application date: 5th May 2021 Field Area: 7.55ha Area Treated: 3.7 ha Application method: Earliest spray Table 1: Field Trial information and product application Treatments: Farm standard Biolevel MaizeNP@ 250g/ha Biolevel MaizeNP @ 250g/ha -20% N Trial Layout:

Initial Assessments: 20% N Based on assessments made at the 5-6 leaf stage both Biolevel MaizeNP treatments has increased root and tops weight compared to the farm standard. Biolevel MaizeNP had the largest increase (9.3%) in root weight and Tops weight (10.5%) followed by Biolevel MaizeNP @ 250g/ha -20% N which also had an increase in root weight (2.3%) and Tops weight (2.8%) compared to the farm standard.

Table 2: Plant population. Biomass and NPK concentrations

Nitrogen, Phosphorus and Potassium levels all appeared greater in the Biolevel Maize NP and Biolevel MaizeNP -20% N than the farm standard. As expected, there was little difference in plant populations between treatments. Harvesting: The harvesting data for each treatment were recorded on the maize harvester computer and downloaded onto the main computer. The Maize yield results were averaged for each treatment. Samples from each treatment were taken for analysis. Maize Analysis Summary:

Based on the Maize analysis carried out there was no difference in the ME’s, Crude Protein, Acid Load or pH between treatments. Starch levels (key energy source) are greater in the Biolevel MaizeNP and Biolevel MaizeNP @ 250g/ha -20% N compared to the farm standard. Maize Yield Results:

The Biolevel MaizeNP treatments resulted in an increase in fresh weight yield, dry matter content and dry matter yield compared to the farm standard. As the trial results shows, reducing Nitrogen by 20% and applying Biolevel MaizeNP does not have a negative impact on yield and in fact has a slight increase. Conclusion: In conclusion the results from this trial indicate that the application of Biolevel MaizeNP with field standard inputs has a positive impact on yield, early growth of roots and foliage, Nutrient uptake of Nitrogen, Phosphorous and Potassium and starch compared to the field standard. The Biolevel MaizeNP -20% Nitrogen treatment has demonstrated that there isn’t a reduction in yield, early growth (root, tops) or nutrient uptake compared to the field standard and has actually shown a slight increase. Based on these trial results, it indicates that Biolevel MaizeNP has achieved its role of making Nitrogen and Phosphorus more available to the plant which is allowing the key nutrients to carry out their function within the Maize crop of vegetative growth, root development and resulting in healthy yields. It wasn’t expected that Potassium would be made more available to the plant which is a key nutrient requirement in Maize for maturity, lodging and cob formation, so further trials and evidence of this would be of benefit. Overall, this trial has demonstrated the potential of increasing or maintaining Maize yields and quality with the input of Biolevel MaizeNP. As it looks increasingly likely that inorganic (potentially organic) fertilisers will need to be reduced on crops due to environmental and cost reasons, products such as Biolevel MaizeNP could have a key role to play in bridging the potential nutrient requirement of crops to maintain yields and quality. Example 1c : Biolevel Forage Maize Trial report 2021 Trial Report: Biolevel MaizeNP (Forage Maize) Materials and Methods Field Name: Top Field Product: Bio-level Maize NP Crop: Maize Group: Forage Soil Type: Sandy clay loam Date Planted: 20th May 2021 Application date: 20th May 2021 Field Area: 12.2 ha Trial Area: 8 ha Application rate: 250g in 200lt/ha water Table 1: Field Trial information and product application Treatments: Farm standard Biolevel @ 250g/ha Biolevel @ 250g/ha -20% N Trial: Compare Biolevel MaizeNP at normal and reduced nitrogen rates with standard farm practice. Aim and design of the trial was to assess the effect Biolevel MaizeNP has at normal and reduced rates of Nitrogen (including the effects of the treatment on the availability of Nitrogen and Phosphorus) on plant development, yield and quality. Trial Layout:

Initial Assessments: Based on assessments made at the 5-6 leaf stage both Biolevel MaizeNP treatments had increased root and tops weight compared to the farm standard. Biolevel MaizeNP had the largest increase (29.5%) in root weight and Tops weight (18.7%) followed by Biolevel @ 250g/ha -20% N which also had an increase in root weight

There were minor differences in plant populations between the treatments although Biomass levels did appear greater in Biolevel Maize NP and Biolevel MaizeNP -20%N than the field standard. Nitrogen, Phosphorus and Potassium levels all appeared greater in the Biolevel Maize NP and Biolevel MaizeNP -20% N than the farm standard. Harvesting/Yield Digs: The yield was assessed and calculated by filling individual trailers and weighing over a weighbridge and then divided over the area harvested. Samples from each treatment were taken and sent for quality analysis. Maize Analysis Summary:

Based on the Maize analysis carried out there was no difference in the DM%, ME’s, Crude Protein, Acid Load or pH between treatments. Starch levels (key energy source) are greater in the Biolevel MaizeNP and Biolevel MaizeNP @ 250g/ha -20% N compared to the farm standard.

Table 4 Maize Yield Results The Biolevel MaizeNP treatments resulted in an increase in yield compared to the farm standard. As the trial results shows, reducing Nitrogen by 20% and applying Biolevel MaizeNP does not have a negative impact on yield and in fact has a slight increase. Conclusion: In conclusion, the trial to Compare Biolevel MaizeNP (applied pre-drilling) at normal and reduced nitrogen rates with standard farm practice has demonstrated that the Biolevel MaizeNP treatment has increased the early growth of roots and foliage, increased Nutrient uptake of Nitrogen, Phosphorous and Potassium and increased starch and overall yield while Biolevel MaizeNP -20N treatment has demonstrated that there is no reduction in plant growth, yield or quality compared to the farm standard. The indication from this trial is that Nitrogen, Phosphorus and Potassium is being made more available to the plant from the application of Biolevel MaizeNP, which is critical to the growing of maize as Nitrogen is important for vegetative growth and grain production, phosphorus is essential for vigorous root development which improves establishment, cob weight and dry matter yield and Potassium demand from Maize is huge therefore inadequate take up can show symptoms such as irregular cob formation, late maturity and risk of lodging. Based on this trial, Biolevel MaizeNP has a place as part of the Integrated Crop Management of forage maize production now and certainly in the future. Due to the financial cost and the environment impacts of inorganic fertilisers and still the need to maintain economic yields on farm, products such as MaizeNP that can maintain yields and quality with reduced inorganic fertiliser applications will have important role to play. Example 1d : 2020 Forage Maize Trial. Materials and Methods tional products

Results

Discussion The addition of Biolevel MaizeNP did increase all of the relevant yield measures starch, dry matter yield and net energy for lactation (VEM).

Example 1e: 2021 Forage Maize Trial. Material s and Methods

Results

Discussion The addition of Biolevel MaizeNP did increase the relevant yield measures dry matter yield and net energy for lactation (VEM). Example 2 – Composition provides a stable CFU count over time at room temperature, without refrigeration Materials and Methods A mixture of the consortium in dextrose monohydrate was made up. The consortium was prepared as follows: The consortium had the following minimum components (*) ● Dextrose monohydrate powder 97.6% w/w ● Minimum components, always present (*): o Pseudomonas putida (minimum) 1x10^8 CFU/g o Bacillus subtilis (minimum) 1x10^8 CFU/g o Bacillus licheniformis (minimum) 1x10^8 CFU/g o Bacillus amyloliquefaciens (minimum) 1x10^8 CFU/g o Bacillus megaterium (minimum) 1x10^8 CFU/g o Bacillus pumilus (minimum) 1x10^8 CFU/g o Paenibacillus polymyxa (minimum) 1x10^8 CFU/g o Azospirillum brasilense (minimum) 5x10^7 CFU/g The consortium was freeze dried under standard conditions. This mixture was suspended in a vegetable oil. The composition (comprising the mixture suspended in the oil) was kept at Room Temperature and Pressure. At various time intervals, the composition was plated out onto agar and the number of CFU (Colony-Forming Units) measured as is standard in the art. Results & Discussion Stability in a High CFU Count is achieved using a non-polar liquid without refrigeration. A higher, but still stable over time, CFU count is required in Corn seed coatings compared to Small Grain seed coatings. For ease of use refrigeration should be avoided. The typical application rate for corn seed coatings in the market is 4 liter per ton of seed (as opposed to 2 liter for small grain). To bring our desired 200 billion CFU to a hectare (less than the 250 billion CFU used for small grain because of larger plant spacing and less soil coverage), considering that 40 hectares per 4 liter, we need 2 billion CFU/ml for the liquid product. The current state of the art does not include bacterial products which are liquid without cooling requirements. Small grains require one ton of seed to typically go onto 5-6 hectare of land. In contrast, corn require a ton of seed which typically goes on 40 hectare of land. Having the goal in mind to bring the same amount of bacteria to a hectare, a corn seed coating must have a much higher bacterial count (5.5 vs 40 hectare). The market standard for seed coatings in maize is about twice as much liquid per ton of seed than in small grain (4 litre per ton of corn seed vs 2 litre per ton of small grain seed) and the higher application rate thus does not make up for the lower seeding rate. Using a liquid product with the same bacterial count as used in small grains would thus bring far too little bacteria to the land. To meet a corn field required CFU count per hectare, the CFU loading of the seed coating can be sometimes as high as 2 billion CFU per ml. A lower CFU count could mean that too much liquid would go onto the seed, which would not be desirable.

If we were to take a polar liquid seed coatings with typically 700 million CFU/ml and try to raise this to 2 billion CFU/ml, we would expect to run into viability problems. However, the powdered product can have a much higher CFU count when the mixture is suspended in a non-polar liquid such as an oil. This finished product achieves stability while bringing the CFU count/ml or per gram to the desired level, all the while not requiring refrigeration. The conclusion that was reached was that the CFU count is stable without the need for refrigeration of the composition. This is an improvement on water-based compositions which do require refrigeration in order to preserve CFU count over time.

Example 3 – Mechanical Testing of the Composition applied to Seeds Materials and Methods The composition of Example 2, provided as a Liquid Seed Coating (in sum 1.5 billion CFU/ml to 2 billion CFU/ml) was added to 50kg of maize seed, sufficient to give a 4 litres of composition per tonne of seed application rate, i.e.4 x 50/1000 = 0.2 Litres of composition. This is the application rate required by industry, but of course can vary. These were added into the funnel of a seed applicator (the Arktos model from Momesso (BR) from 2021) with a rotary mixer, where the seeds are spun with a centrifugal force whilst the liquid is added. Approximate spinning times were in the region f 10-15 mins. The seeds were removed and inspected. They were allowed 10 to 20 mins to dry, although there was little aqueous moisture. Results Approximately 80-90% of the seeds were at least partially coated with the composition. This is expected to be sufficient when sown as the consortium bacteria will populate the rhizosphere of the coated seeds and also spread or at least provide nutrients (N and solubilized-P) to nearby seeds over time, especially as the rhizospheres develop and the bacteria multiply. The coated seeds did not stick together or agglutinate in such a way that would prevent sowing in the soil by a mechanical seed applicator or sowing apparatus common in the field. Croozer/Afron Star insecticides were added to the coated seeds. This is the most convenient time to do so, i.e., prior to sowing the seeds. No adverse effect were seen wrt the composition, especially in respect of the tested properties of agglutination. Discussion In summary, this data tested if: ● the composition agglomerated/agglutinated – it did not ; and if ● the composition could be sprayed onto the seed or stirred onto the seed depending on the machinery to be use – it could, in both instances Example 4 – Field Testing in Kenya of the Composition applied as a Seed Coating to Seeds The aim of this experiment is to evaluate the efficacy of Biolevel NP on growth, quality and yield of Maize. The trial also investigates the least effective rate in combination with a reduced fertilizer program (75%) that will be considered based on the growth, quality and yield of Maize compared to other nutrition programs. Application of Biolevel NP at the rate of 250g and 500g in addition to 75% of the recommended fertilizer program significantly increased the growth, quality and yield components of maize across the four sites in Kenya. Materials and Methods Study sites Trials were conducted at the Powa Agriconsult Trial Fields in Kithini-Machakos County, Waruhiu ATC-Kiambu County, Karii-Kirinyaga County and Maragua-Murang’a County. Biolevel Maize was evaluated in 1 cropping season for maize, which was cultivated in open field and irrigated by drip system. Experimental Design This evaluation was conducted in open field grown maize variety Haraka WH 101 from Western Seed Company, Kenya. The experiments were laid out in a Randomized Complete Block Design (RCBD), and treatments replicated 3 times (Figure 1). Experimental plots measured 4m by 3m = 12 m2 each and the spacing between plots was 1.0 m and 1.0 m between replicates. In total, 18 experimental plots of each crop were raised as per the guidelines in table 2. The rest of the cultural practices were done as per the standard operating practice of the specific crop (Ceretis paribus).

NB: Seed dressing was done to the equivalent of product in 1L of water and the seeds soaked into the solution for 3 hours.

Treatment application Treatments were done at sowing as per the application rate recommended by the manufacturer. Applications was done by seed dressing. NB: Conventional fertilizers were applied according to recommended/standard application practices i.e. NPK 23:23 at 5g/plant at planting and as CAN at 5g/plant as a top dress in split top-dress application with Ammonium Sulphate at tasselling at the rate of 5g per plant. The application of fertilizers was reduced by 25% on all the Biolevel Maize treatments i.e. 75 kg/acre of NPK 23:23:0; 75kg/acre of CAN in two-splits and no Ammonium Sulphate at tasselling as done on the conventional practice treatments where 100 kg/acre of NPK 23:23:0, CAN and AS was applied at planting, top-dressing and at tasselling respectively. Data collection Five plants for Maize were sampled and tagged per plot for data collection. The following data on growth and yield of the crop will be collected:

Harvesting Harvesting of the experimental crops was done as provided in the guidelines in table below.

Yield will be translated into tons/ha and tabulated to obtain mean yield per treatment Data Analysis and Management The plant data were subjected to Analysis of variance (ANOVA) to help determine differences among treatments based on growth and yield data. Statistical significance will be determined at p ≤ 0.05, while means were compared using least significant difference (LSD) test. Treatment combinations on each unit of the design

Treatment factors are listed in the order: Treat1.

Meteorological Data The meteorological data during the trial periods were recorded. Results Assessment Schedule Assessment was done on growth, yield and quality parameters as shown in Table 5 during the trial periods in all the four sites due to treatment application. Table 5. Activity, treatment application and assessment schedule

NB: The dates are for the Kiambu site which was set first followed by Kirinyaga, Machakos and lastly Murang’a at an interval of 3 days apart Effect of treatments on the growth parameters of maize There were significant differences between the growth and quality parameters of maize due to treatment application in Kiambu (Table 6). In the treatments where Biolevel NP was applied there was significant increase on the maize height, root zone diameter and the stand count which was comparable to the reference product. Also, the quality parameters of maize viz number of cobs per plant and the ear length were significantly increased on the Biolevel NP treatments except where it was used solely. Table 6: Influence of treatments on the plant height, root diameter, stand count, number of cobs per plant and the ear length of maize in Kiambu

In Kirinyaga, it was observed that Biolevel NP application in combination with the conventional fertilizer practice, there were significant increases on the plant height, root zone dimeter and the establishment of the crop stand. The seed emergence rate stood at 100% on the 250g application of Biolevel NP and the 500g stood at 99.7% compared to the recommended nutrition program alone which had 92% while the untreated control had 76%. An average of 2 cobs was recorded on the Biolevel NP treatments as well as an increase on the ear length by 1-2cm (Table 7). Table 7: Influence of treatments on the stand count, plant height, number of cobs per plant, ear length and root diameter of maize at Kirinyaga

Treatments with the same letter along the columns are not significantly different according to DMRT at P≤0.05. RFP-Recommended Fertilizer Program Significant differences (P≤0.05) were observed in Machakos on the Biolevel NP trial where the treatments of the combined test product with the conventional fertilizer program was comparable to the reference product in increasing the growth parameters i.e. the plant height and the root zone diameter and improving the stand establishment with over 98% (Table 8). The application of Biolevel NP Alone had lower effect compared to the integrated treatment probably because of the nutritional composition compared to what the full program will add to the soil. However, the positive influence compared to the untreated control shows the efficacy of Biolevel NP and if used judiciously with the other nutritional soil supplements there would be greater fertility and nutritional improvement. Table 8: Effect of test treatments on the plant height, root zone diameter, stand count, number of cobs per plant and the ear length of maize in Machakos Treatments with the same letter along the columns are not significantly different according to DMRT at P≤0.05. RFP-Recommended Fertilizer Program In Murang’a, there were great and significant increases on the growth and quality of maize due to application of Biolevel NP compared to the recommended fertilizer program for maize alone and the untreated control (Table 9). A 10cm increase was recorded on the height which was majorly facilitated by a wider root zone which also led to a double cob formation and a longer ear of an average of 12cm where the 500g rate was used which was however not significantly different from the half rate of 250g. Table 9: Influence of treatments on the stand count, number of cobs per plant, ear length, plant height and root diameter of maize in Murang’a

Treatments with the same letter along the columns are not significantly different according to DMRT at P≤0.05. RFP-Recommended Fertilizer Program Effect of treatments on yield parameters of maize Treatments differed significantly (P≤0.05) on the number of complete cobs, ear length, fodder yield, grain yield and green cob yield after applications of treatments in Kiambu (Table 10). Application of Biolevel NP plus the standard fertilization program were as effective as the reference product on most of the tested parameters while application of Biolevel NP alone was significantly lower than when combined with a fertilizer program. This is so because Biolevel NP only enhances the effectiveness and efficiency of the applied nutritional supplements but does not provide the essential nutrients by itself. Table 10: Mean number of cobs per plant, ear length, green cob yield, fodder yield, cob weight and grain yield as influenced by treatments in Kiambu

Treatments with the same letter along the columns are not significantly different according to DMRT at P≤0.05. RFP-Recommended Fertilizer Program Application of Biolevel NP increased the yield and yield parameters of maize in Kirinyaga significantly due to the improvement of uptake of nutrients and proper mobilization (Table 11). The grain yield increased by over 2 tonnes per hectare where Biolevel NP was added and was comparable to the reference product. Table 11: Treatment effect on the yield parameters of maize in Kirinyaga in the Biolevel NP efficacy trial

Treatments with the same letter along the columns are not significantly different according to DMRT at P≤0.05. RFP-Recommended Fertilizer Program The yield components in Machakos showed a decrease compared to the other sites but there were significant differences between the treatments with the same trend as other sites recorded with addition of Biolevel NP doubling the grain yield of maize compared to the conventional fertilizer program used in the study site (Table 12). Table 12: Mean number of weight of cobs, grains per cob, green fodder yield, total green cob yield and grain yield as influenced by treatments in Machakos

Treatments with the same letter along the columns are not significantly different according to DMRT at P≤0.05. RFP-Recommended Fertilizer Program Significant differences were observed in Murang’a after application of treatments on the number of number of grains per cob, yield of green cobs, fodder yield, cob weight and grain yield (Table 13). The yield almost doubled compared to the conventional practice while sole application of Biolevel NP had lower effect than all the other treatments except the untreated control in most of the yield parameters. The number of grains per cob in the combined treatment of Biolevel NP and the conventional fertilizer practice of maize treatment which directly explains the increased green cob yield as well as the weight of the individual edible cobs. Table 13: Mean number of cobs per plant, ear length, weight of cobs, total green cob yield and grain yield as influenced by treatments in Murang’a

Treatments with the same letter along the columns are not significantly different according to DMRT at P≤0.05. RFP-Recommended Fertilizer Program Discussion Application of Biolevel NP at the rate of 250g and 500g in addition to 75% of the recommended fertilizer program significantly increased the growth, quality and yield components of maize across the four sites in Kenya.

Claims

Claims 1. A coating composition for seeds comprising a mixture suspended in a non-polar liquid, the mixture comprising: a. a consortium of bacteria; and b. a bacterial carrier powder; wherein bacteria in the consortium are: iii. saprophytic bacteria; and/or iv. Nitrogen-fixing and Phosphorus-solubilizing bacteria; wherein the non-polar liquid is not bactericidal against the consortium bacteria, and optionally, wherein the seed coating is a liquid at 20 degrees C.

2. A coating composition according to claim 1, wherein the non-polar liquid is inert and so is not reactive with the coating composition components or the intended seeds.

3. A coating composition according to claim 1 or claim 2, wherein non-polar liquid is an oil.

4. A coating composition according to claim 3, wherein the oil is: a. vegetable oil, including but not limited to Rapeseed Oil; Sunflower Oil; Safflower Oil; Soybean Oil; corn Oil; b. is mineral oil; or c. is a mixture of: a) one or more vegetable oils; b) one or more mineral oils; or c) a mixture of one or more vegetable oils and one of more mineral oils.

5. A coating composition according to any one of the preceding claims, wherein the bacterial carrier is a powder, optionally particles with a diameter in the range of 100 micrometres (μm) to 300 micrometres (μm), optionally 200 micrometres (μm).

6. A coating composition according to any one of the preceding claims, wherein the bacterial carrier is a an organic or inorganic powder, optionally: calcified seaweed, talcum, graphite, corn starch, a monosaccharide, disaccharide or polysaccharide powder, optionally dextrose monohydrate.

7. A coating composition according to any one of the preceding claims, wherein the non- polar liquid is sprayable at 20 degrees C.

8. A coating composition according to any one of the preceding claims, wherein the composition has viscosity of between 35 centipoise at 40 degrees centigrade or 40 centipoise at 35 degrees centigrade.

9. A coating composition according to any one of the preceding claims, wherein the consortium comprises: a. at least one Pseudomonas species; b. at least one Bacillus species; and/or c. at least one Paenibacillus species; and, optionally d. at least one Azospirillum species.

10. A coating composition according to any one of the preceding claims, wherein the consortium comprises: a) Pseudomonas putida b) Bacillus subtilis c) Bacillus licheniformis d) Bacillus amyloliquefaciens e) Bacillus megaterium f) Bacillus pumilus; and g) Paenibacillus polymyxa

11. A coating composition according to claim 10, wherein the consortium further comprises i) Azospirillum brasilense

12. A coating composition according to claim 10, wherein the consortium comprises ix. Pseudomonas putida at a minimum concentration of 1x10^8 CFU/g x. Bacillus subtilis at a minimum concentration of 1x10^8 CFU/g xi. Bacillus licheniformis at a minimum concentration of 1x10^8 CFU/g xii. Bacillus amyloliquefaciens at a minimum concentration of 1x10^8 CFU/g xiii. Bacillus megaterium at a minimum concentration of 1x10^8 CFU/g xiv. Bacillus pumilus at a minimum concentration of 1x10^8 CFU/g xv. Paenibacillus polymyxa at a minimum concentration of 1x10^8 CFU/g xvi. Azospirillum brasilense at a minimum concentration of 5x10^7 CFU/g

13. A coating composition according to any one of the preceding claims, further comprising a dye or colourant.

14. A coated seed comprising the coating composition according to any one of the preceding claims.

15. A coated seed according to claim 14, wherein the seed a seed from: a. member of the grass family, Graminacea (a.k.a. Poaceae); or b. member of the Leguminaceae family.

16. A coated seed according to claim 13, wherein the seed is a seed selected from: a. Corn/maize; b. Alfalfa; c. Sorgum; d. Soya; e. Wheat; f. Barley; g. Oats; h. Rice; i. Sugar Cane; or j. a mixture, optionally a forage mixture, of any two or more of the above.

17. A coated granule, wherein the coating is a coating composition according to any one of claims 1-13.

18. A coated granule according to claim 17, wherein the granule is a fertilizer, a liming product, a soil amendment product or shell particles (optionally crushed oyster shell).

19. A coated granule according to claim 18, wherein the granule is an agricultural liming product or agricultural liming material selected from: a. limestone (calcitic or dolomitic); b. burned lime; slaked lime; c. marl; d. shells; and e. industrial by-products fr example sugar beet lime and sludge from water treatment plants.

20. A coated granule according to claim 17 or 18, wherein the granule is a particle in the range of 2 to 4 mm in diameter, for example: poly4 ®

21. A mixture or suspension comprising: a. the coating composition, coated seed or coated granule according to any one of the preceding claims; and b. other seed coatings or applications (such as starter fertilizers , such as ENTEC ).

22. A vessel for storing or transporting the coating composition, coated seed, coated granule or mixture, suspension or emulsion according to any one of the preceding claims.

23. A method of use for the coating composition according to any one of claims 1-13, comprising: a. mixing seeds, granules or providing a mixture, suspension or emulsion with the composition; and b. sowing the coated seed or coated granule or mixture or suspension in a suitable environment such as soil or growth medium that supports a rhizosphere.

24. A method of use for the coated seed, coated granule or mixture or suspension according to any one of claims 14 to 21, comprising sowing the coated seed, coated granule or mixture or suspension in a suitable environment such as soil or growth medium that supports a rhizosphere.