Seed coating product and method

Abstract

The current invention relates to a seed coating product comprising: a cellulose derivative; one or more sugars, sugar alcohols, or a mixture thereof; one or more clays, a milk-derived powder, a legume seed-derived powder, or a combination thereof; and one or more microbial strains. The current invention also relates to a method for coating seeds, and to a coated seed.

Description

[0001] SEED COATING PRODUCT AND METHOD

[0002] FIELD OF THE INVENTION

[0003] This invention relates to a seed coating product and methods for enhancing the survival and effectiveness of microbes on seeds, particularly legume seeds.

[0004] BACKGROUND

[0005] Soybean (Glycine max) and Yellow Pea (Pisum sativum) are leguminous plants, or legumes, indigenous native to East Asia, with historical origins in regions including China, Japan, and Korea. Legume plants have gained popularity as a valuable crop for both human consumption and animal feed due to their high protein content and nutritional benefits.

[0006] These legumes form a symbiotic relationship with nitrogen fixing microbes such as Rhizobium and Bradyrhizobium bacteria, facilitating nitrogen fixation and enhancing plant growth. This unique interaction allows soybean and pea plants to convert atmospheric nitrogen into a form that is readily available for their growth.

[0007] Legume seeds can be inoculated with symbiotic microbial strains, such as for instance in the form of a seed coating. Seed coatings or seed dressings are for instance known from Teresa Berninger et al. (2017), CN100566574, WO2022217038, W02017143130, CN116548472, W02008002371,

[0008] W02021081018, WO2017044473, Yi Qiu et al. (2020) and Masoume Amirkhani et al. (2016), however the effective establishment and survival of these symbiotic microbes on seeds poses challenges. Gram-negative rhizobia, for example, a group of bacteria that can form symbiotic relationships with the roots of legume plants, are sensitive to environmental conditions, and seed coating liquid containing such rhizobia generally needs to be refrigerated to maintain their viability. As a result, such seeds are generally only inoculated or coated with the symbiotic strain(s) immediately prior to sowing to ensure that sufficient viable rhizobia are present on the seeds upon sowing.

[0009] Coating legume seeds with a strain is therefore a time consuming and expensive task, that needs to be performed right before sowing, a time when farmers are already busy with many other tasks.

[0010] To address these challenges, there is a growing demand for improved seed coating methods and products that enhance microbial survival while ensuring a longer shelf life for coated seeds, preferably alleviating the farmers of the need to perform the seed coating just before sowing.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention and embodiments thereof serve to provide a solution to one or more of above-mentioned disadvantages. To this end, in a first aspect, the present invention relates to a seed coating product according to claim 1. In particular, said seed coating product comprises:

[0013] - a cellulose derivative; one or more sugars, sugar alcohols, or a mixture thereof; one or more clays, a milk-derived powder, a legume seed-derived powder, or a combination thereof; and one or more microbial strains.

[0014] Preferred embodiments of the seed coating product are shown in any of the claims 2 to 18.

[0015] In a second aspect, the present invention relates to a method for coating seeds according to claim 19. More particular, the method comprises applying a seed coating product according to any one of the claims 1 to 18 to a seed surface. A preferred embodiment of the seed coating method is shown in claim 21 or 21.

[0016] In a third aspect the present invention relates to a coated seed according to claim 22, wherein the seed is coated with a seed coating product according to any one of the claims 1 to 18.

[0017] The seed coating product, method and coated seed of the invention, ensure prolonged survival of the microbial strain on the seed, thereby extending the shelf life of seeds coated with such seed coating product considerable. This, in turn, presents the advantage of offering pre-coated seeds to farmers, alleviating the need for them to perform seed coating just before sowing. As a result, valuable time is saved for farmers, contributing to more efficient planting processes.

[0018] DEPOSIT OF BIOLOGICAL MATERIAL

[0019] The Bradyrhizobium japonicum strain described in present application was deposited by VIB vzw (Rijvisschestraat 120, 9052 Gent, Belgium) at the BCCM (Belgian Coordinated Collections of Microorganisms) consortium (BCCM represented by Laboratorium voor Microbiologie - Bacterienverzameling (LMG), Universiteit Gent, K.L. Ledeganckstraat 35, 9000 Gent, Belgium), recognized as an International Depositary Authority by the World Intellectual Property organization since March 1, 1992 and in accordance with the Budapest Treaty as specified in Rule 31(1) EPC2000 for the purpose of patent procedure and the regulations thereunder. The Bradyrhizobium japonicum strain of current application has been deposited as Bradyrhizobium japonicum RHG_Soy_223 with deposit number LMG P-32018. The original deposit has been done on 05.10.2020. Throughout the text, RHG_Soy_223 is used interchangeably with Soy_223, or Soy223.

[0020] DESCRIPTION OF FIGURES

[0021] The following description of the figures of specific embodiments of the invention is merely exemplary in nature and is not intended to limit the present teachings, their application or uses.

[0022] Figures 1 to 5 illustrate the prolonged survival of the bacterial strain over a 60-day period under different coating conditions, as shown in Table 1 and Example 1. The conditions represent seeds coated with a bacterial strain, and further various combination of sugar (alcohol), cellulose derivatives, and / or carrier substances, and in various concentrations.

[0023] Figure 6 depicts the nodulation outcomes on soy plants grown from seeds either inoculated with the bacterial strain or coated with a seed coating product comprising said strain, 25 m / v % Trehalose, 5 m / v % CMC, and 5 m / v % vermiculite according to an embodiment of the present invention. Additionally, it illustrates the disparity in nodulation between plants grown from seeds coated at the time of sowing and those coated after one month of storage at 25 °C in airtight sealed plastic bags, shielded from sunlight.

[0024] Figure 7: Boxplot of the number of nodules per plant in function of the treatment 34 DAS according to Example 2.

[0025] Figure 8: Boxplots showing the number of nodules per plant in function of various treatments as shown in Example 3. Figure 8A is showing variety Gml704-454 and Figure 8B is showing variety Gml704-533.

[0026] Figure 9: Boxplots of the average SPAD value in function of various treatments as shown in Example 3. Figure 9A is showing variety Gml704-454 and Figure 9B is showing variety Gml704-533.

[0027] Figure 10: Boxplots of the EXGreen index of plants in function of various treatments as shown in Example 3. Figure 10A is showing variety Gml704-454 and Figure 10B is showing variety Gml704-533.

[0028] Figure 11: Boxplots of the seed yield (ton / ha) adjusted to a moisture content of 14%, as shown in Example 3. Figure 11A is showing variety Gml704-454 and Figure 11B is showing variety Gml704-533. Figure 12: Boxplots of the average protein content (%) in seeds, as shown in Example 3. Figure 12A is showing variety Gml704-454 and Figure 12B is showing variety Gml704-533.

[0029] Figure 13 presents the survival rates of the bacterial strain in a seed coating over a 94-day period under different conditions, as shown in Table 2 and Example 4. The conditions represent seeds coated with a bacterial strain, trehalose, carboxymethylcellulose, and further vermiculite; vermiculite + milk powder; or vermiculite + milk powder + soy powder.

[0030] Figure 14 shows the survival rates of a Rhizobium sophorae pea6 strain in a 'seed coating' comprising trehalose, CMC, and autoclaved vermiculite, as compared to the strain in a 'liquid inoculation' without said trehalose, CMC, and vermiculite over a 90- day period as described in Example 5.

[0031] Figure 15 presents the survival rates of the B. japonicum SOY223 strain in a seed coating comprising CMC, autoclaved vermiculite and varying sugar(alcohol) concentrations, in particular varying trehalose concentrations, as described in Example 6.

[0032] Figure 16 presents the survival rates of B. japonicum SOY223 strain in a seed coating comprising trehalose, autoclaved vermiculite, and varying cellulose derivatives, in particular cellulose derivatives chosen from hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), a combination of 2.5 m / v% HPMC and 2.5 m / v% ethyl cellulose (EC), methylcellulose (MC), hydroxyethyl cellulose (HEC), and cellulose monoacetate (CA), as described in Example 7.

[0033] Figure 17 shows (A) the number of nodules per plant at flowering time in function of the log(CFU / seed) at sowing time, and (B) the average yield (ton / ha) in function of the log(CFU / seed) at sowing time, of soy seeds inoculated with B. japonicum SOY223 and a seed coating formulation comprising trehalose, CMC, and vermiculite, as descried in Example 8.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention concerns a seed coating product comprising:

[0036] - a cellulose derivative; one or more sugars, sugar alcohols, or a mixture thereof; one or more clays, a milk-derived powder, a legume seed-derived powder, or a combination thereof; and one or more microbial strains.

[0037] The current invention also relates to a method for coating seeds, and to a coated seed. The seed coating product, method for coating and the coated seed of the invention, ensure prolonged survival of the microbial strain(s) on the seed, thereby extending the shelf life of seeds coated with such seed coating product considerable. This, in turn, presents the advantage of offering pre-coated seeds to farmers, alleviating the need for them to perform seed coating just before sowing. As a result, valuable time is saved for farmers, contributing to more efficient planting processes.

[0038] Definitions

[0039] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

[0040] As used herein, the following terms have the following meanings:

[0041] "A", "an", and "the" as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more than one compartment.

[0042] "Comprise", "comprising", and "comprises" and "comprised of" as used herein are synonymous with "include", "including", "includes" or "contain", "containing", "contains" and are inclusive or open-ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.

[0043] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0044] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints. The expression "% by weight", "weight percent", "%wt" or "wt%", here and throughout the description unless otherwise defined, refers to the relative weight of the respective component based on the overall weight of the formulation.

[0045] The expression "% by mass / volume", "mass / volume percent", "m / v%" or "%m / v", here and throughout the description unless otherwise defined, refers to the relative mass of the respective component based on its concentration in a solution, with the mass measured in grams and the volume measured in milliliters.

[0046] Whereas the terms "one or more" or "at least one", such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.

[0047] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, definitions for the terms used in the description are included to better appreciate the teaching of the present invention. The terms or definitions used herein are provided solely to aid in the understanding of the invention.

[0048] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0049] As used herein, the term "microorganism" or "microbe" or "microbial strain" refers to any species or taxon of microorganism, including, but not limited to, archaea, bacteria, microalgae, fungi (including mold and yeast species), mycoplasmas, microspores, nanobacteria, oomycetes, and protozoa. In some embodiments, a microbe or microorganism encompasses individual cells (e.g., unicellular microorganisms) or more than one cell (e.g., multi-cellular microorganism).

[0050] As used herein, the term "bacterium", "bacteria", or "bacterial" refers in general to any prokaryotic organism, and may reference an organism from either Kingdom Eubacteria (Bacteria), Kingdom Archaebacteria (Archaea), or both. In some cases, bacterial genera have been reassigned due to various reasons (such as, but not limited to, the evolving field of whole genome sequencing), and it is understood that such nomenclature reassignments are within the scope of any claimed genus.

[0051] As used herein, the term "fungus", "fungi", or "fungal" refers to a diverse group of eukaryotic microorganisms that typically possess chitin in their cell walls and lack chlorophyll. Fungi encompass a wide array of organisms, including yeasts, molds, and mushrooms.

[0052] The "rhizosphere" is a narrow soil zone that surrounds and is influenced by the root systems of plants. It is a dynamic interface where complex interactions occur between plant roots, microorganisms, and soil particles. The rhizosphere is characterized by unique chemical, physical, and biological properties that arise due to the release of root exudates (compounds released by plant roots), microbial activity, and nutrient exchange. This zone serves as a critical site for nutrient uptake, symbiotic associations, and various ecological processes that impact plant growth and soil health.

[0053] A "legume" or "leguminous plant" is a type of plant belonging to the Fabaceae family, commonly known as the pea or bean family. Legumes are characterized by their unique fruit, known as a legume, which is a pod that typically contains multiple seeds. These plants are highly valued for their agricultural and nutritional significance. Legumes come in various forms, including bushes, vines, and trees. They can be annual or perennial and are found in a wide range of climates and ecosystems around the world. Some well-known examples of legume crops include: soybeans (Glycine max), cowpeas (Vigna unguiculata), chickpeas (Cicer arietinum), common vetch (Vicia sativa), peas (Pisum sativum), lupins (such as Lupinus angustifolius and Lupinus albus), lima beans (Phaseolus lunatus), adzuki beans (Vigna angularis), grass peas (Lathyrus sativus), alfalfa (Medicago sativa), yam beans (Pachyrhizus spp.), mung beans (Vigna radiata), fava beans (Vicia faba), and lentils (Lens culinaris).. Legumes have a unique ability to form symbiotic relationships with nitrogen-fixing bacteria called rhizobia. These bacteria convert atmospheric nitrogen into a form that plants can utilize, thus enriching the soil with nitrogen, an essential nutrient for plant growth. This makes legumes not only valuable food sources but also beneficial for crop rotation practices that help improve soil fertility. Because of their nutritional value, versatility, and role in sustainable agriculture, legumes are considered an important component of human diets and agricultural systems around the world.

[0054] "Cellulose-derivative" is a compound derived from cellulose, a natural polymer found in plant cell walls. Examples include carboxymethylcellulose (CMC), ethyl cellulose (EC), methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), and cellulose monoacetate (CA). CMC is widely used in various industries for its unique properties, including its ability to act as a thickener, binder, stabilizer, and emulsifier. Ethyl cellulose is a cellulose derivative obtained through the ethylation of cellulose, resulting in improved solubility in organic solvents. Ethyl cellulose finds applications in the pharmaceutical and food industries for controlled-release drug formulations and as a coating for food products. Methylcellulose is a cellulose derivative produced by substituting hydroxyl groups of cellulose with methyl groups. It is commonly used in the food industry as a thickener, emulsifier, and gelling agent due to its ability to form a gel-like structure when mixed with water. Hydroxypropyl methylcellulose is another derivative, known for its role in pharmaceutical coatings and as a controlled-release agent. Hydroxypropyl cellulose is used in tablets and other pharmaceutical formulations for its film-forming and thickening properties, while hydroxyethyl cellulose finds applications in personal care products as a thickener and stabilizer. Cellulose monoacetate is used in fiber production, film coatings, and even as a biodegradable plastic.

[0055] In the context of the present invention, cellulose derivatives exhibit adhesive or binding properties, enabling them to function as 'glue' to hold components of the seed coating together and / or allow easy coating to the seeds. These derivatives help ensure cohesive interactions between the components and maintain uniform coverage on the seed surface. The above described derivatives are particularly suited for this role, as their ability to form gel-like or adhesive structures provides stability and adhesion to the coating, thereby enhancing the durability and effectiveness of the seed coating. "Trehalose" is a naturally occurring carbohydrate composed of two glucose molecules and is known for its remarkable ability to stabilize biological structures under challenging conditions, including dehydration and extreme temperatures. It is commonly utilized as a protectant and stabilizer in various industries, such as food and pharmaceuticals, to maintain the integrity of sensitive products.

[0056] "Sorbitol" is a sugar alcohol derived from fruits and is recognized for its ability to retain moisture. It is widely used as a sugar substitute due to its lower calorie content and mild sweetness. Sorbitol's humectant properties, which enable it to hold onto water, make it a sought-after ingredient in cosmetics, foods, and medications.

[0057] "Maltose", a disaccharide composed of two glucose molecules, is a natural product of starch digestion. This sugar is often utilized in the food industry for its sweetening and browning properties, while also serving as an energy source for microorganisms.

[0058] The term "Colony Forming Units" (CFU) refers to a quantifiable measure of viable microorganisms, encompassing bacteria and other microbes, capable of forming colonies under specific cultivation conditions. In the present disclosure, CFU is used interchangeably with "microbial titers" to denote the concentration or quantity of viable microbes in a given sample. The assessment of CFU involves plating a known volume of a diluted sample onto a solid growth medium and counting the resulting colonies, providing a standardized and widely accepted method for microbial quantification. The term "microbial titers" is used interchangeably with "Colony Forming Units" (CFU) and refers to the concentration or quantity of viable microbes in a given sample. Throughout this disclosure, both terms are used synonymously to convey the measurement of viable microorganisms, including bacteria, fungi, and other microbes. The quantification may involve various methods, with the specific approach depending on the experimental context. The use of these terms interchangeably is intended to encompass any suitable method for determining the concentration of viable microbes.

[0059] Description

[0060] In a first aspect, the invention relates to a seed coating product comprising:

[0061] - a cellulose derivative; one or more sugars, sugar alcohols, or a mixture thereof; one or more clays, a milk-derived powder, a legume seed-derived powder, or a combination thereof; and one or more microbial strains. This seed coating product provides a unique combination of ingredients, all tailored to be used in seed coatings thereby enhancing the survival and effectiveness of microbes on seeds such as legume seeds.

[0062] The cellulose derivatives, such as carboxymethylcellulose (CMC), ethyl cellulose (EC), methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), and cellulose monoacetate (CA), or a combination thereof, provide a protective barrier around the seed, shielding it from external factors such as moisture, drought, and pathogens. In addition, they provide adhesion to the seed surface, ensuring further ingredients of the coating adhere well. CMC, in particular, but other cellulose derivatives as well, also aids controlled release of compounds and retains water. Cellulose derivatives exhibit adhesive or binding properties, enabling them to function as 'glue' to hold components of the seed coating together and / or allow easy coating to the seeds. These derivatives help ensure cohesive interactions between the components and maintain uniform coverage on the seed surface. The above-described derivatives are particularly suited for this role, as their ability to form gel-like or adhesive structures provides stability and adhesion to the coating, thereby enhancing the durability and effectiveness of the seed coating, and supporting successful plant germination and growth.

[0063] "Sugars" are simple carbohydrates that provide a readily available source of energy for various organisms, including microbes. "Sugar alcohols" or "polyols" are compounds derived from sugars by replacing one or more hydrogen and / or oxygen atoms with hydroxyl groups.

[0064] Sugars and sugar alcohols play pivotal roles in seed coatings, offering a range of advantages. Serving as osmoprotectants, they enable seeds to withstand environmental stresses like drought. Simultaneously, these compounds act as readily available energy reservoirs, promoting microbial growth and metabolism during seed colonization. This dual functionality enhances seed germination and early seedling growth. Their presence not only contributes to the stability of the coating, ensuring uniform application but also facilitates the initial adhesion of microbial strains to seed surfaces. This establishes a robust foundation for long-term symbiotic interactions, improving microbial survival under stress conditions. Overall, sugars and sugar alcohols contribute to the resilience of the coating, aiding seeds in navigating environmental challenges and fostering enhanced plant growth and health.

[0065] Clays, milk-derived powder such as milk powder, legume seed-derived powder such as pea-derived powder or soy seed-derived powder or a derivative thereof, may provide structure to the coating, helping it adhere to the seed. In addition, they may provide a surface for the strains to adsorb to or to embed into. Further, they may assist in moisture retention, creating a favorable microenvironment for seed germination. They may also provide nutrients and proteins, fats, oils, and more which are beneficial to the microbial strains and / or to the coated seed.

[0066] The "clay" or "clay mineral" may acts as a carrier for the microbial strain(s) and / or provides structure to the coating to help adhere it to the seed. Said clay can be any clay usable in seed coatings. Non-limiting examples are vermiculite, kaolin, bentonite, montmorillonite, halloysite, attapulgite and sepiolite. "Vermiculite" is a naturally occurring lightweight mineral with a porous structure. Its unique properties include excellent water retention and aeration capabilities, making it valuable for improving soil structure and moisture availability. "Kaolin" or "kaolinite" is a common clay mineral that is often used in seed coatings. It offers good particle adherence, which helps the coating stick to the seeds. Kaolin also has reflective properties that can provide protection against sunlight and heat stress. "Bentonite" is a versatile clay used in various industries, including seed coatings. It swells when wet, forming a gel-like substance that can help retain moisture around the seed. This can promote germination and provide a protective barrier. "Montmorillonite" is a type of smectite clay with excellent water retention properties. It can form a gel-like structure when hydrated, aiding seed germination and establishment. "Halloysite" is a tubular clay mineral that can be used to enhance seed coatings. Its porous structure allows for good water retention and gradual release of nutrients to the seed. "Attapulgite" is another clay used in seed coatings for its water-absorbing and water-retaining properties. It can improve moisture availability to seeds during germination. "Sepiolite" is a clay mineral known for its absorbent and adsorbent properties. It can help retain moisture around seeds and provide a stable environment for germination.

[0067] The one or more microbial strains play a crucial role in promoting plant health. They can facilitate nutrient uptake by forming symbiotic relationships with plant roots, enhance plant growth by producing growth-promoting substances, contribute to disease resistance and overall soil health, and / or improve seedling establishment and root development. Known seed coatings often utilize perlite, peat, and / or lignite as components instead of clays and / or the above-mentioned powders. However, in comparison to the components used in the present seed coating product, these traditional materials lack certain desirable properties. These were shown to be less advantageous increasing the survivability of microbial strains on a seed surface.

[0068] The microbial strain can be any microbial strain able to survive in the present seed coating product, and preferably on a seed coated with such seed coating product. The strain can for instance be a bacterial or fungal strain.

[0069] Non-limiting examples of such microbial strains, are strains of the following bacterial genera: Rhizobium, Sinorhizobium, Bacillus, Pseudomonas, Azospirillum, Azotobacter, Clostridium, Klebsiella, Stenotrophomonas, Halomonas, Pianococcus, Agrobacterium, Enterobacter, Clostridium, Serratia, Paenibacillus, Streptomyces, Lactobacillus, Corynebacterium, Micrococcus, Achromobacter, Burkholderia, Ralstonia, Xanthomonas, Acinetobacter, Bradyrhizobium, Frankia, Mezorhizobium , Ensifer, Herbaspirillum, and Brevibacillus; and strains of the following fungal genera : Trichoderma, Beauveria, Rhizophagus, Piriformospora, Tuber, Suillus, Rhizopogon, Penicillium, Fusarium, Cladosporium, Aspergillus, Verticillium, Metarhizium, Gliocladium, Paecilomyces, Acremonium, Talaromyces, Amanita, Laccaria, Pisolithus, Russula, Lepista, Hebeloma, and Cenococcum, and further Mycorrhizae.

[0070] "Mycorrhizae" refers to a symbiotic association between plant roots and certain fungi, enhancing nutrient absorption and overall plant health. The fungi form a network of hyphae around or within plant roots, facilitating a mutual exchange of nutrients and carbohydrates.

[0071] In an embodiment, the microbial strain is a rhizosphere microbial strain. A "rhizosphere microbial strain" pertains to a microbial strain, including bacterial strains, "rhizobacterial strains" and fungal strains "rhizosphere fungal strains", thriving within the rhizosphere, a narrow soil zone surrounding and influenced by the root system of plants. These microorganisms contribute to plant health, nutrient cycling, and ecosystem dynamics in the rhizosphere. The above-listed microorganisms can all be associated with the rhizosphere.

[0072] In an embodiment, the microbial strain is a symbiotic strain, able to engage in a symbiotic relationship with a plant. Non-limiting examples include strains of the following bacterial genera: Rhizobium (nitrogen-fixing symbiosis), Bradyrhizobium (nitrogen-fixing symbiosis), Frankia (nitrogen-fixing symbiosis), Mesorhizobium (nitrogen-fixing symbiosis), Sinorhizobium (nitrogen-fixing symbiosis), Azospirillum (plant growth-promoting symbiosis), Ensifer (formerly known as Sinorhizobium, nitrogen-fixing symbiosis), Agrobacterium (symbiotic relationships with plants), Herbaspirillum (plant growthpromoting symbiosis), Klebsiella (nitrogen-fixing symbiosis), Stenotrophomonas (plant growth-promoting symbiosis), Halomonas (plant growth-promoting symbiosis), Pianococcus (symbiotic relationships with plants), Enterobacter (plant growth-promoting symbiosis), Serratia (plant growth-promoting symbiosis), Paenibacillus (plant growth-promoting symbiosis), Streptomyces (plant growthpromoting symbiosis), Lactobacillus (symbiotic relationships with plants), Corynebacterium (plant growth-promoting symbiosis), Micrococcus (symbiotic relationships with plants), Achromobacter (plant growth-promoting symbiosis), Burkholderia (plant growth-promoting symbiosis), Ralstonia (plant growthpromoting symbiosis), Xanthomonas (plant growth-promoting symbiosis), Bacillus (symbiotic relationships with plants), Pseudomonas (symbiotic relationships with plants), and Acinetobacter (plant growth-promoting symbiosis).

[0073] Non-limiting examples include strains of the following fungi or fungal genera: Mycorrhizae (includes various fungal genera forming mutualistic symbiotic associations with plant roots), Rhizophagus (formerly Glomus, mycorrhizal fungi), Trichoderma (symbiotic interactions with plants), Piriformospora (plant growthpromoting symbiosis), Laccaria (ectomycorrhizal symbiosis), Tuber (ectomycorrhizal symbiosis), Pisolithus (ectomycorrhizal symbiosis), Russula (ectomycorrhizal symbiosis), Lepista (ectomycorrhizal symbiosis), Hebeloma (ectomycorrhizal symbiosis), Cenococcum (ectomycorrhizal symbiosis), Amanita (ectomycorrhizal symbiosis), Suillus (ectomycorrhizal symbiosis), and Rhizopogon (ectomycorrhizal symbiosis).

[0074] "Ectomycorrhizal symbiosis" is a mutually beneficial partnership between certain fungi and plant roots, where the fungal hyphae envelop the root tips without penetrating the plant cells. The fungi enhance nutrient uptake for the plant, while the plant provides sugars to the fungi.

[0075] In an embodiment, the microbial strain is a nitrogen-fixing strain. Such "nitrogenfixing strain" has the ability to convert atmospheric nitrogen gas (N2) into biologically useful forms of nitrogen, such as ammonia (NH3) or nitrate (NO3-). This process is known as nitrogen fixation and is crucial for making nitrogen available to plants and other organisms in ecosystems. These microorganisms form symbiotic relationships with certain plants, like legumes (e.g., peas, beans, clover), and provide them with a source of nitrogen.

[0076] In addition to or as an alternative to nitrogen-fixing strains, the coating may also incorporate Plant Growth-Promoting Rhizobacteria (PGPR) to enhance the overall plant health and vigor. PGPR, encompassing a diverse array of beneficial bacterial genera such as Pseudomonas, Bacillus, and Stenotrophomonas, contribute to plant growth by facilitating nutrient uptake, producing growth-promoting substances, and conferring resistance to stressors. Including PGPR in the seed coating broadens the spectrum of benefits, promoting not only nitrogen availability but also fostering a favorable rhizosphere environment. This dual approach harnesses the synergistic potential of nitrogen-fixing strains and PGPR, offering a comprehensive solution for optimizing plant performance and crop yield.

[0077] In an embodiment, the microbial strain is of the genus Bradyrhizobium, Ensifer, or Rhizobium. "Bradyrhizobium" and "Ensifer" strains are nitrogen-fixing bacteria that form a symbiotic relationship with legume plants, including soybeans. "Rhizobium" strains are nitrogen-fixing bacteria that form a symbiotic relationship with yellow pea, a further legume plant. Bradyrhizobium, Ensifer, and Rhizobium strains all have the ability to convert atmospheric nitrogen into a form that can be absorbed and utilized by the plants, thereby enriching the soil with essential nutrients and promoting plant growth. Bradyrhizobium strains are commonly used in agricultural practices to improve crop yield and reduce the need for synthetic fertilizers. By establishing a symbiotic relationship with the host plants, these bacteria contribute to increased plant health, nodulation, and overall productivity.

[0078] Each ingredient in the seed coating product, the cellulose derivative, the sugars or sugar alcohols, the clays, milk-derived powder, legume seed-derived powder or derivative thereof and the one or more microbial strains thus serves a specific purpose, and their combination aims to create an optimal environment for seed germination, early seedling growth, and overall plant health. The coating acts as a protective layer while providing essential nutrients and support for the seed and emerging seedling. The combination of seed coating product ingredients ensures prolonged microbial survival, thereby extending considerable the shelf life of seeds coated with such seed coating product. This, in turn, presents the advantage of offering pre-coated seeds to farmers, alleviating the need for them to perform seed coating just before sowing. As a result, valuable time is saved for farmers, contributing to more efficient planting processes.

[0079] In an embodiment, the seed coating product comprises at least 5 m / v% of said sugars or sugar alcohols, preferably at least 5.25 m / v%, preferably at least 5.5 m / v%, preferably at least 5.75 m / v%, preferably at least 6 m / v%, or preferably at least 6.25 m / v% of said sugars or sugar alcohols. In an embodiment, the seed coating product comprises between 5 m / v% and 7.5 m / v% of said sugars or sugar alcohols.

[0080] The inclusion of at least 5 m / v% of said sugars or sugar alcohols in the seed coating product provides improved moisture retention, microbial protection, increased available energy for the microbial strain(s), and improved initial adhesion of the strain(s) to the seed surface, in addition to improved microbial survival under (environmental) stress conditions, thereby maintaining microbial-plant interactions that contribute to improved plant growth and health.

[0081] In another or further embodiment, the seed coating product comprises at least 6.25 m / v% of said sugars or sugar alcohols, preferably at least 6.5 m / v%, preferably at least 6.75 m / v%, preferably at least 7 m / v%, preferably at least 7.25 m / v%, preferably at least 7.5 m / v%, preferably at least 7.75 m / v%, preferably at least 8 m / v%, preferably at least 8.25 m / v%, preferably at least 8.5 m / v%, preferably at least 8.75 m / v%, preferably at least 9 m / v%, preferably at least 9.25 m / v%, preferably at least 9.5 m / v%, preferably at least 9.75 m / v%, preferably at least 10 m / v%, preferably at least 10.25 m / v%, preferably at least 10.5 m / v%, preferably at least 10.75 m / v%, preferably at least 11 m / v%, preferably at least 11.25 m / v%, preferably at least 11.5 m / v%, preferably at least 11.75 m / v%, preferably at least 12 m / v%, preferably at least 12.25 m / v%, preferably at least 12.5 m / v%, preferably at least 12.75 m / v%, preferably at least 13 m / v%, preferably at least 13.25 m / v%, preferably at least 13.5 m / v%, preferably at least 13.75 m / v%, preferably at least 14 m / v%, preferably at least 14.25 m / v%, preferably at least 14.5 m / v%, preferably at least 14.75 m / v%, preferably at least 15 m / v% of said sugars or sugar alcohols. In an embodiment, the seed coating product comprises between 10 m / v% and 14 m / v% of said sugars or sugar alcohols. Further, increasing the concentration to at least 10 m / v% or even 12.5 m / v% of said sugars or sugar alcohols enhances the protective capabilities of the seed coating product, supporting the symbiotic microbes' viability during storage and planting.

[0082] In a preferred embodiment, the seed coating product comprises at least 6.25 m / v% of said sugars or sugar alcohols, such as 6.25 m / v%, or 7.5 m / v%, or 8.75 m / v%, or 10 m / v%, or 11.25 m / v%, or 12.5 m / v%, or 13.75 m / v%, or 14 m / v%. In a preferred embodiment, the seed coating product comprises between 5 m / v% and 14 m / v% of said sugars or sugar alcohols.

[0083] In an embodiment, the seed coating product comprises between 0.5 m / v% and 2.5 m / v% of cellulose derivatives. Preferably, the seed coating product includes 0.5 m / v%, 0.625 m / v%, 0.75 m / v%, 0.875 m / v%, 1.0 m / v%, 1.125 m / v%, 1.25 m / v%, 1.375 m / v%, 1.5 m / v%, 1.625 m / v%, 1.75 m / v%, 1.875 m / v%, 2.0 m / v%, 2.125 m / v%, 2.25 m / v%, 2.375 m / v%, 2.5 m / v% of cellulose derivatives, and more preferably, the seed coating product comprises 1.25 m / v% of cellulose derivatives.

[0084] Controlling the cellulose derivatives content between 0.5 m / v% and 2.5 m / v% offers an ideal balance between adhesion and protection, optimizing the overall seed coating process.

[0085] In a preferred embodiment, where the cellulose derivative is CMC, the seed coating product comprises between 0.5 m / v% and 2.5 m / v% of CMC. Preferably, the seed coating product includes 0.5 m / v%, 0.625 m / v%, 0.75 m / v%, 0.875 m / v%, 1.0 m / v%, 1.125 m / v%, 1.25 m / v%, 1.375 m / v%, 1.5 m / v%, 1.625 m / v%, 1.75 m / v%, 1.875 m / v%, 2.0 m / v%, 2.125 m / v%, 2.25 m / v%, 2.375 m / v%, 2.5 m / v% of CMC, and more preferably, the seed coating product comprises 0.5 to 1.25 m / v% of CMC, most preferably about 1.25 m / v% of CMC.

[0086] Increasing the concentration of cellulose derivative, and in particular CMC, leads to better adherence of the coating to seeds and a higher microbial load over time. Although higher concentrations lead to better adherence, the concentration of the cellulose derivative is preferably not higher than 2.5 m / v%, not higher than 1.875 m / v%, and preferably about 1.25 m / v%.

[0087] A CMC or methylcellulose concentration of about 1.25 m / v% was shown to offer the most optimal concentration. In a specific embodiment, the cellulose-derivative incorporated into the seed coating product undergoes a sterilization process involving heating without boiling. While numerous constituents of the seed coating product are subject to optional autoclaving for sterilization, cellulose-derivatives are deliberately exempted from this method to prevent polymer breakdown. Instead, the preferred approach involves subjecting cellulose-derivatives to a heating process prior to their incorporation into the seed coating product. This meticulous procedure ensures the preservation of polymer integrity, aligning with the optimal conditions for effective seed coating.

[0088] In an embodiment, said sugars or sugar alcohols are chosen from trehalose, sorbitol, maltose or a combination thereof. It was shown that those listed sugar (alcohols) are particularly effective in increasing the viability of one or more strains coated on the seeds.

[0089] "Trehalose" is a naturally occurring carbohydrate commonly utilized as a protectant and stabilizer in various industries to maintain the integrity of sensitive products. In the realm of seed coatings, trehalose's protective properties come into play, ensuring the viability of microbial strains on coated seeds even in unfavorable environmental situations.

[0090] "Sorbitol" is a sugar alcohol derived from fruits and is recognized for its ability to retain moisture. In seed coatings, sorbitol's moisture-retaining capability proves advantageous by preserving optimal moisture levels around the seeds, thus fostering effective germination and early-stage plant growth, in addition to ensuring viability of the strain(s) coated on said seeds.

[0091] "Maltose", a disaccharide composed of two glucose molecules, is a natural product of starch digestion. In seed coatings, maltose can serve a dual role: as a source of nourishment for beneficial microorganisms, and potentially as a factor that aids in seed germination by providing a readily accessible energy source. Its incorporation enhances the microbial activity and overall effectiveness of seed coatings comprising one or more microbial strain(s) designed to promote successful plant-microbe interactions.

[0092] In the most favored embodiment, trehalose is the preferred sugar for use. Extensive studies have demonstrated that trehalose significantly enhances microbial survival on coated seeds compared to alternative sugars such as sorbitol. Remarkably, microbial survival rates with trehalose are found to be nearly equivalent to those observed when using maltose. It is noteworthy, however, that maltose exhibits a lower heat stability, rendering it unsuitable for autoclaving-based sterilization processes. Consequently, trehalose emerges as the optimal choice, offering both superior microbial survival benefits and robust stability under heat conditions, making it a prime candidate for seed coating compositions. As sugar (alcohols) are carbon sources, they facilitate non preferential microbial growth as well. To avoid this, the sugar(alcohol) is preferably autoclaved before use.

[0093] In an embodiment, the seed coating product comprises between 10 m / v% and 25 m / v% of clay, milk-derived powder, legume seed-derived powder or derivative thereof, or a combination thereof.

[0094] In an preferred embodiment, the seed coating product comprises 10 m / v%, 12.5 m / v%, 15 m / v%, 17.5 m / v%, 20 m / v%, 22.5 m / v% or 25 m / v% of clay, milk- derived powder, legume seed-derived powder or derivative thereof, or a combination thereof.

[0095] In an embodiment, where the seed coating product comprises a clay, and said clay preferably is vermiculite.

[0096] "Vermiculite" known for its expanded and porous structure upon heating, stands out as a particularly effective clay choice when combined with a beneficial microbial strain. This naturally occurring mineral possesses exceptional water retention and aeration capabilities, making it invaluable for soil improvement and moisture management. In seed coatings, vermiculite excels as a carrier, facilitating optimal interaction between seeds and coating constituents. Its porous nature not only retains moisture essential for germination but also ensures proper aeration, creating an environment conducive to microbial growth. Vermiculite further enhances the uniform distribution of the microbes on the seed surface, promoting their colonization and interaction with the legume roots upon germination. The synergistic effect of vermiculite and the microbial strain fosters successful seed coating outcomes, where seeds receive both the necessary moisture and the environment conducive to establishing symbiotic relationships and promoting robust plant growth.

[0097] Vermiculite is chosen over above-described alternative clays kaolin, bentonite, montmorillonite, halloysite, attapulgite and sepiolite as compared to these clays, vermiculite holds a balanced advantage due to its dual role as a water retainer and an aerator. Its expanded structure accommodates water retention while allowing oxygen exchange, fostering a favorable environment for seed germination and microbial activity. This unique combination positions vermiculite as an ideal choice for enhancing seed coating success by optimizing both moisture availability and aeration.

[0098] In an embodiment, the milk-derived powder is obtained through a drying process to reduce it to a powdered form. Preferably, this powder is derived from milk and contains essential nutrients, providing a rich and balanced source of proteins, fats, carbohydrates, vitamins, and minerals. Preferably, the milk-derived powder is whole milk powder. Preferably, the milk-derived powder serves as a multifunctional component in the seed coating, functioning as both a carrier for microbial strains and a nutrient source for enhanced seed germination and early plant development. The powder may be derived from various types of milk, including but not limited to cow's milk, goat's milk, or sheep's milk, and can be further processed to achieve desired characteristics for optimal seed coating performance. Further, milk-derived powder may help preserving the microbial strains.

[0099] In an embodiment, where the seed coating product comprises a legume seed-derived powder or a derivative thereof, said powder is obtained by a method comprising the steps of milling legume seeds, thereby forming said legume seed-derived powder, and subsequently autoclaving said powder.

[0100] In the context of the present invention, "grinding" or "milling" are used interchangeably to refer to the mechanical processes of reducing legume seeds into smaller particles or powder. Milling involves the use of specialized equipment, such as hammer mills, ball mills, or air-classifying mills, to break down the seeds, ensuring a consistent and fine texture suitable for various applications. This transformative process enhances the accessibility of nutrients, improves uniformity, and contributes to the desired characteristics of the resulting legume seed-derived powder.

[0101] Preferably, the legume seeds are dried before milling, facilitating easy grinding and ensuring the stability and quality of the resulting powder. This drying process also prevents powder clumping, and allows for easy storage. Preferably, the seeds are dried to a moisture content of between 5 % and 15 %, preferably about 10 % moisture content. Moisture content can be measured by various methods as described in the art. Examples of these methods include the use of moisture analyzers, oven-drying techniques, near-infrared spectroscopy, or capacitance-based moisture meters.

[0102] Autoclaving of legume seed-derived powder was shown to be an important step. Legume seed-derived powder, being a natural product, may harbor various microorganisms, including plant pathogens, bacteria, fungi, viruses, or spores. Autoclaving helps eliminate or reduce these contaminants, preventing unwanted microbial interactions that could negatively affect the intended microbial strains in the seed coating. In addition, the microbial strains used in seed coating product may be sensitive to competition or antagonism from other microorganisms present in the legume seed-derived powder, potentially negatively impacting overall plant health. Autoclaving helps create a more controlled environment, enhancing the viability and effectiveness of the intended microbial strains. Furthermore and in addition to the above, without wishing to be bound by theory, the inventors hypothesized that the heating step of the autoclaving process may denature or destroy contaminant proteins that have a negative impact on microbe survival.

[0103] In an embodiment, said legume seeds that are milled, are whole legume seeds.

[0104] Reference to "whole legume seeds” indicates that legume seeds have not undergone any purification or separation of components. These seeds are intact and haven't had the outer hull, germ, or endosperm removed. In other words, they are in their whole form as they existed in the legume plant. This implies that the entire seed, including all its components, is subjected to the milling process to create the legume seed-derived powder. While the composition of the seeds remains the same, and there is no purification or separation step, the seeds or the resulting powder may have undergone some processing, such as autoclaving, steam sterilization, heat treatment, microwave processing, roasting, enzyme treatment, or ultrasonic processing. It is important to clarify that the term "legume seed-derived powder", when it specifically refers to powder obtained from "whole legume seeds", excludes products such as legume meal, seed extracts, protein isolates, oils, lecithin, and similar items derived from legume seeds, that have undergone significant processing and separation of components.

[0105] As mentioned, said legume seed-derived powder acts as a nutritional source as it contains essential nutrients such as proteins, amino acids, vitamins, and minerals. When incorporated into the coating, it can provide a nutritional source for the beneficial microbes. This can help sustain the microbes' metabolic activity and viability during storage, ensuring that they remain active and capable of establishing a symbiotic relationship with the plants when the seeds are planted. Said seed- derived powder also forms a protective layer around the microbial cells in the coating, shielding them from environmental stressors like temperature fluctuations and moisture changes that could potentially compromise their viability.

[0106] In an embodiment, a derivative of legume seed-derived powder may be used, preferably a derivative of a soy seed-derived powder, such protein isolates, protein concentrates, enriched protein, polysaccharide extracts, starches, fibers, or oils. Enriched protein, protein isolates and concentrates provide a concentrated source of nutrients, promoting microbial viability, while polysaccharide extracts improve the adhesion and moisture retention of the coating. Starches act as thickening agents, ensuring uniform coverage, and fibers contribute to the structural integrity of the coating, offering gradual nutrient release and protection against environmental stressors. Additionally, legume seed oils can add hydrophobicity, improving water resistance and shelf life, while serving as carriers for active ingredients. These derivatives enhance the seed coating's performance, ensuring optimal microbial survival and plant health.

[0107] In an embodiment, said legume seed-derived powder and / or said clay has a particle size of maximally 1.0 mm, preferably maximally 0.9 mm, or 0.8 mm, or 0.7 mm, or 0.6 mm, or preferably maximally 0.5 mm.

[0108] In an embodiment, at least 90 %, preferably at least 91 %, more preferably at least 92 %, more preferably at least 93 %, more preferably at least 94%, or at least 95 % of the legume seed-derived powder and / or clay has a particle size of maximally 1.0 mm, preferably maximally 0.9 mm, or 0.8 mm, or 0.7 mm, or 0.6 mm, or preferably maximally 0.5 mm.

[0109] Preferably, the particle size is obtained or verified by sieving the particles after the milling step, thereby removing larger particles.

[0110] In an embodiment, said legume seed from which said powder is derived, can be any legume seed, but preferably this is a soy seed or pea. Said powder is preferably derived from non-genetically modified legume seeds. This particle size was optimized for easy administration onto a seed surface, potentially as part of a coating, and for increasing survivability of microorganisms on said seed surface. The controlled particle size plays a dual role: it facilitates adherence to the seed surface and influences nutrient release from the ground soybeans. Smaller particles, while aiding in nutrient availability and / or moisture retention, may contribute to effective adhesion. However, excessively large particles could compromise adherence, leading to suboptimal nutrient release, and could potentially block pipelines of administration equipment, such as for instance a seed coater. The particle size is therefore carefully chosen for achieving maximum efficacy in seed coatings, ensuring both enhanced adhesion, nutrient release and / or moisture retention for optimal seed performance.

[0111] In an embodiment, said legume seed-derived powder comprises a protein content of at least 30 wt%, thereby possibly providing a nutritional source for beneficial microorganisms and promoting their survival on a plant surface. Preferably said powder is obtained from legume seeds having a protein content of at least 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt% based on dry weight or more. Preferably, protein content based on dry weight is not more than 55 wt%. Examples of soy cultivars or varieties known for their high protein content are Lenka, Artemis, Aurelina, Primas, and RGT Sphinxa.

[0112] In another or further embodiment, the legume seed-derived powder is derived from legume cultivars or varieties having an average protein content, such as for instance between 30 and 40 wt%.

[0113] Protein content can be measured by any method known in the art. Examples are Kjeldahl Method, Dumas Method, Near-Infrared Spectroscopy (NIRS), Bradford Assay, Biuret Method, Lowry Method, and Elemental Analysis.

[0114] The Kjeldahl method is a classic and widely used technique for determining total nitrogen content, from which protein content is calculated. The nitrogen content is measured by digesting the sample with concentrated sulfuric acid and then distilling the released ammonia, which is subsequently absorbed in an acidic solution. The Dumas method, also known as combustion analysis, involves burning the sample in the presence of excess oxygen and measuring the nitrogen content. Like the Kjeldahl method, protein content is then calculated based on the nitrogen content. NIRS is a rapid and non-destructive technique that analyzes the absorption of near-infrared light by the sample. It is calibrated using reference samples with known protein content to establish a prediction model. The Bradford assay is a colorimetric method that uses the binding of Coomassie Brilliant Blue dye to proteins. The intensity of the color is proportional to the protein concentration and can be measured spectrophotometrically. The biuret method involves the reaction of proteins with copper ions in an alkaline solution. The resulting purple color is proportional to the protein concentration and can be measured spectrophotometrically. The Lowry method is another colorimetric assay that involves the reduction of Folin-Ciocalteu reagent by proteins in an alkaline solution. The intensity of the blue color formed is proportional to the protein concentration. Elemental analysis involves measuring the elemental composition of a sample, including the nitrogen content. Protein content is then calculated based on the nitrogen-to-protein conversion factor.

[0115] In an embodiment, the seed coating product may comprises each of said clay, milk- derived powder, or legume seed-derived powder alone, or in any combination thereof.

[0116] As an example, a combination of a legume seed-derived powder with vermiculite in the seed coating product was shown to be particularly effective in providing a surface to microbial strains which adsorb to or embed into said powder and vermiculite, which further provides an exceptional water retention and aeration capabilities to the coating product, and also provide nutrients and proteins, fats, oils, and more which are beneficial to the microbial strains and / or to the coated seed.

[0117] In an embodiment, the seed coating product comprises at least 105, 106, 107, 108, 109, or even at least IO10microbial cells or colony forming units (CFUs) for each gram of clay, milk-derived powder, legume seed-derived powder, or combination thereof. This amount was shown to be sufficient to ensure effective colonization of the seed surface upon coating seeds with said seed coating product and optionally to ensure effective colonization of the roots or further plant parts, and optionally the growth medium such as soil, in which the seed is subsequently grown.

[0118] As described above, the seed coating product comprises at least one microbial strain. In an embodiment, said microbial strain is a Bradyrhizobium strain, an Ensifer strain, or a Rhizobium strain. In a further embodiment, the Bradyrhizobium strain is a Bradyrhizobium japonicum strain. B. japonicum has shown well-working symbiotic relationships with various legume plants, in particular soybean plants. In another further embodiment, the Rhizobium strain is a Rhizobium spp, shown to establish symbiotic relationships with legume plants, including yellow pea. Preferably the Bradyrhizobium strain is a Bradyrhizobium japonicum strain deposited at BCCM-LMG, having received the deposit accession number LMG P-32018. Using this strain, also referred to in the present context as Bradyrhizobium japonicum strain RHG_Soy_223, offers multiple benefits. This strain has demonstrated an ability to thrive in colder climates, making them well-suited for coating seeds of legume crops in regions with suboptimal temperatures. The incorporation of strain RHG_Soy_223, with its specific deposit accession number LMG P-32018, ensures a consistent and reliable source of beneficial bacteria, enabling dependable and sustainable crop growth enhancements even in colder environments. As a nonlimiting example, said strain is particularly suitable to be used in combination with soybeans when grown in for example Northern Europe and Canada, while this plant generally thrives in warm and temperate climates, such as China, the United States, Brazil, Argentina, and India.

[0119] In an embodiment, the seed coating product further comprises one or more additives suitable for enhancing coated seed shelf-life. In the present context, said additives may be referred to as "additives" or as "shelf-life enhancing additives". Said additives preferably further preserve the coating's stability and microbial viability, contributing to the prolonged viability of the coated seeds, facilitating ease of transportation and ensuring optimal microbial performance during planting.

[0120] A non-limiting examples of said additives is Arabic gum.

[0121] "Arabic gum" imparts additional protection to the coating, by improving the overall stability and durability of the coating, protecting the seeds and any added materials from environmental factors. Additionally, Arabic gum is soluble in water, which allows it to form a gel-like substance when mixed with water, aiding in the adhesion of other components during the coating process.

[0122] In an embodiment, the seed coating product further comprises one or more of a bacterial inoculant, a fungal inoculant, or a combination thereof.

[0123] The inclusion of bacterial and / or fungal inoculants in the seed coating product introduces novel symbiotic microorganisms to further promote plant growth. Bacterial inoculants from diverse genera as discussed below can establish beneficial interactions with plants, fixing nitrogen, enhancing nutrient availability, and conferring resistance to stress. Fungal inoculants, spanning various families and phyla, contribute to improved soil structure, nutrient absorption, and disease resistance.

[0124] The fungal inoculant can comprise a fungal inoculant of the family Glomeraceae,

[0125] Claroidoglomeraceae, Gigasporaceae, Acaulosporaceae, Sacculosporaceae

[0126] Entrophosporaceae, Pacidsporaceae, Diversisporaceae, Paraglomeraceae

[0127] Archaeosporaceae, Geosiphonaceae, Ambisporaceae, Scutellosporaceae

[0128] Dentiscultataceae, Racocetraceae, Trichoderma, or a fungal inoculant of the phylum Basidiomycota, Ascomycota, Zygomycota, or any of the genera as described above, or a combination thereof.

[0129] The bacterial inoculant, for purposes of the present invention, can include a bacterial inoculant of the genus Rhizobium, Bradyrhizobium, Mesorhizobium, Azorhizobium, Allorhizobium, Sinorhizobium, Kluyvera, Azotobacter, Pseudomonas, Azospirillium, Bacillus, Streptomyces, Paenibacillus, Paracoccus, Enterobacter, Alcaligenes, Mycobacterium, Gliocladium, Glomus, Klebsiella, Stenotrophomonas, Halomonas, Pianococcus, any of the genera described above, or a combination thereof.

[0130] Within the domain of seed coating formulations, strategic focus is placed on specific microbial strains crucial for regulatory compliance. Notably, Azotobacter spp., Mycorrhizal fungi, Rhizobium spp., and Azospirillum spp. hold paramount significance as they are part of the positive list outlined by the EU Fertilising Product Regulation under PFC6 microbial category. These strains, acknowledged for their biostimulant effects, emerge as pivotal contributors, ensuring alignment with current and future regulatory standards in the dynamic landscape of microbial seed treatments.

[0131] In an embodiment, the seed coating product comprises a first coating composition and a second coating composition, suited to coat a seed with a first coating layer of said first coating composition and a second coating layer of said second coating composition.

[0132] In a further embodiment, a) said first coating composition comprises a cellulose derivative; and wherein b) said second coating composition comprises one or more microbial strains, and one or more clays, a milk-derived powder, or legume seed-derived powder, or a combination thereof. As mentioned above, said cellulose derivative such as carboxymethylcellulose (CMC), methylcellulose, or ethyl cellulose, provides adhesion to the seed surface, ensuring further ingredients of the coating adhere well. Said derivatives further retain water, collectively enhancing seed coating effectiveness and supporting successful plant germination and growth.

[0133] By providing said cellulose derivative in a first coating composition suited to coat a seed with a first layer of said first coating composition, it ensures efficient adherence of the second layer comprising the second coating composition, i.e. the strains and clays and / or powders.

[0134] As also mentioned above, said clays, milk-derived powder such as milk powder, legume seed-derived powder provide a surface for the microbial strains to adsorb to or to embed into. Further, they may assist in moisture retention, creating a favorable microenvironment for the stains and for seed germination. Further, they also provide nutrients and proteins, fats, oils, and more which are beneficial to the microbial strains and / or to the coated seed. By combining said clays and / or said powders in a second coating composition suited to be coated as a second layer on a seed surface, an optimal microenvironment is created which perfectly adheres to the seed because of said first layer comprising cellulose derivatives.

[0135] In a further embodiment, said one or more sugars, sugar alcohols, or mixtures thereof are either present in the first coating composition or in the second coating composition.

[0136] Said sugars or sugar alcohols, such as trehalose, sorbitol, and maltose, serving as osmo-protectants and readily available energy reservoirs, promote microbial growth and metabolism during seed colonization. This establishes a robust foundation for long-term symbiotic interactions, improving microbial survival under stress conditions. As they also facilitate the initial adhesion of microbial strains to seed surfaces, the sugar (alcohols) may perfectly well be provided in a first or second layer of the coating, and thus be present in either in the first coating composition or the second coating composition.

[0137] This seed coating product provides a unique combination of a first coating composition and a second coating composition, tailored to be using in seed coating, thereby enhancing the survival and effectiveness of microbes on seeds such as legume seeds. The two coating compositions were shown to be particularly effective when used as first and second layers of a seed coating.

[0138] In an embodiment, the ratio of the first seed coating composition to second coating composition is about 4: 1, 3: 1, 2: 1, 1: 1, 1 :2, 1 :3, or 1:4. Most preferably, the ratio is 1:3.

[0139] In an embodiment, the first coating composition comprises between 2.5 m / v% and 10 m / v% of cellulose-derivatives, preferably the seed coating product comprises 2.5 m / v%, 3.0 m / v%, 3.5 m / v%, 4.0 m / v%, 4.5 m / v%, 5.0 m / v%, 5.5 m / v%, 6.0 m / v%, 6.5 m / v%, 7.0 m / v%, 7.5 m / v%, 8.0 m / v%, 8.5 m / v%, 9.0 m / v%, 9.5 m / v%, or 10 m / v% of cellulose-derivatives, and more preferably, the first coating composition comprises 5 m / v% of cellulose-derivatives.

[0140] Controlling the cellulose-derivatives content between 2.5 m / v% and 10 m / v% offers an ideal balance between adhesion and protection, optimizing the overall seed coating process.

[0141] In a preferred embodiment, where the cellulose-derivative is CMC, the first coating composition comprises between 2.5 m / v% and 10 m / v% of CMC, preferably the seed coating product comprises 2.5 m / v%, 3.0 m / v%, 3.5 m / v%, 4.0 m / v%, 4.5 m / v%, 5.0 m / v%, 5.5 m / v%, 6.0 m / v%, 6.5 m / v%, 7.0 m / v%, 7.5 m / v%, 8.0 m / v%, 8.5 m / v%, 9.0 m / v%, 9.5 m / v%, or 10 m / v% of cellulose-derivatives, and more preferably, the first coating composition comprises 2.5 to 5 m / v% of CMC, most preferably about 5 m / v% of CMC.

[0142] Increasing the concentration of cellulose-derivative, and in particular CMC, led to a better adherence of the coating to seeds, and to a higher microbial load over time. Although higher concentrations lead to a better adherence, the concentration of the cellulose-derivative is preferably not higher than 10 m / v%, not higher than 7,5 m / v% and preferably about 5 m / v%, as higher concentrations increase solubility issues of the cellulose-derivative.

[0143] A CMC or methylcellulose concentration of about 5 m / v% was shown to offer a most optimal balance between adherence and solubility.

[0144] In an embodiment, the first or second coating composition comprises at least 20 m / v% of said sugars or sugar alcohols, preferably at least 21 m / v%, preferably at least 22 m / v%, preferably at least 23 m / v%, preferably at least 24 m / v%, or preferably at least 25 m / v% of said sugars or sugar alcohols. In an embodiment, the first or second coating composition comprises between 20 m / v% and 30 m / v% of said sugars or sugar alcohols.

[0145] The inclusion of at least 20 m / v% of said sugars or sugar alcohols in said first or second coating composition provides improved moisture retention, microbial protection, increased available energy for the microbial strain(s), and improved initial adhesion of the strain(s) to the seed surface, in addition to an improved microbial survival under (environmental) stress conditions, thereby maintaining microbial-plant interactions that contributes to improved plant growth and health.

[0146] In another or further embodiment, the first or second coating composition comprises at least 26 m / v% of said sugars or sugar alcohols, preferably at least 27 m / v%, preferably at least 28 m / v%, preferably at least 29 m / v%, preferably at least 30 m / v%, preferably at least 31 m / v%, preferably at least 32 m / v%, preferably at least 33 m / v%, preferably at least 34 m / v%, preferably at least 35 m / v%, preferably at least 36 m / v%, preferably at least 37 m / v%, preferably at least 38 m / v%, preferably at least 39 m / v%, preferably at least 40 m / v%, preferably at least 41 m / v%, preferably at least 42 m / v%, preferably at least 43 m / v%, preferably at least 44 m / v%, preferably at least 45 m / v%, preferably at least 46 m / v%, preferably at least 47 m / v%, preferably at least 48 m / v%, preferably at least 49 m / v%, preferably at least 50 m / v%, preferably at least 51 m / v%, preferably at least 52 m / v%, preferably at least 53 m / v%, preferably at least 54 m / v%, or preferably at least 55 m / v% of said sugars or sugar alcohols. In an embodiment, the first or second coating composition comprises between 40 m / v% and 55 m / v% of said sugars or sugar alcohols.

[0147] Further, increasing the concentration to at least 40 m / v% or even 50 m / v% of said sugars or sugar alcohols enhances the protective capabilities of the seed coating product, supporting the symbiotic microbes' viability during storage and planting.

[0148] In a preferred embodiment, the first or second coating composition comprises at least 25 m / v% of said sugars or sugar alcohols, such as 25 m / v%, or 30 m / v%, or 35 m / v%, or 40 m / v%, or 45 m / v%, or 50 m / v%, or 55 m / v%. In a preferred embodiment, the first or second coating composition comprises between 20 m / v% and 55 m / v% of said sugars or sugar alcohols. Said sugar (alcohol) concentrations above preferably not higher than 55 m / v%, as higher concentrations increase solubility issues of the sugar(alcohol)s.

[0149] In an embodiment, the second coating composition comprises between 15 m / v% and 35m / v% of clay, milk-derived powder, legume seed-derived powder, or a combination thereof.

[0150] In an preferred embodiment, the second coating composition comprises 15 m / v%, 17.5 m / v%, 20 m / v%, 22.5 m / v%, 25 m / v%, 27.5 m / v%, 30 m / v%, 32.5 m / v%, or 35 m / v% of clay, milk-derived powder, legume seed-derived powder, or a combination thereof.

[0151] In a particular embodiment, the seed coating product comprises a first coating composition and a second coating composition, suited to coat a seed with a first coating layer of said first coating composition and a second coating layer of said second coating composition, wherein: a) said first coating composition comprises carboxymethylcellulose (CMC) and one or more sugars or sugar alcohols or a mixture thereof; and wherein b) said second coating composition comprises one or more microbial strains and a clay, a milk-derived powder, and / or a legume seed-derived powder.

[0152] Preferably, said second coating composition comprises one or more microbial strains, and vermiculite and / or soy seed-derived powder.

[0153] The first coating composition, comprising CMC and one or more sugars or sugar alcohols or a mixture thereof, provides adhesion, acts as a protective matrix, ensuring optimal microbial viability and protection against environmental stressors.

[0154] The second coating composition comprising one or more microbial strains and clay milk-derived powder, and / or a legume seed-derived powder, facilitates the establishment and growth of the microorganisms, preferably symbiotic microorganisms, on the seed surface.

[0155] In a second aspect, the present invention relates to a method for coating seeds, the method comprises applying a seed coating product to a seed surface, said seed coating product comprises a cellulose derivative; one or more sugars, sugar alcohols, or a mixture thereof; one or more clays, a milk-derived powder, a legume seed- derived powder, or a combination thereof; and one or more microbial strains. A person of ordinary skill will appreciate that features of the first aspect relating to the seed coating product as described above return in the aspects of the method, the coated seed, and uses thereof, of the invention as described throughout the present text. Consequently, all aspects of the present invention are related. All features and related advantages as described in one of the aspects can relate to any of these aspects, even if they are described in conjunction with a specific aspect.

[0156] Preferably, the method comprises applying a seed coating product as described above in any one of the embodiments to a seed surface.

[0157] In an embodiment of the method, the seed coating product is applied in at least two steps, comprising:

[0158] - applying to said seed surface a first coating composition comprising a cellulose derivative; and

[0159] - applying on top of said first coating composition a second coating composition comprising one or more microbial strains and one or more clays, a milk- derived powder, a legume seed-derived powder, or a combination thereof.

[0160] In a further embodiment, said one or more sugars, sugar alcohols, or mixtures thereof are either present in the first coating composition or in the second coating composition.

[0161] In particular, the first coating composition described in relation with the method, may be the first coating composition as described above in any one of the embodiments related to the coating product, and its components may be present in any percentage, for example in the percentages as described above. The second coating composition comprising a microbial strain and a clay, may for example comprise a Bradyrhizobium strain such as a B. japonicum strain having the deposit accession number LMG P-32018. Where the second coating composition comprises a clay, it may be any type of clay, and can for instance be vermiculite. Where the second coating composition comprises a milk-derived powder or a legume seed- derived powder, said powder may have any characteristics and / or advantages as already described above.

[0162] The method comprising providing two coating compositions as two coating, and thus two layers, on seeds, was shown to be particularly effective. In particular, also the order of administering first the first coating composition, followed by the second coating composition, showed to be advantageous. So was the present method shown to improve strain survival on the seed over a similar method in which both first and second compositions were combined into one mixture, that was applied as one coating layer to the seed. In addition, the present method was shown to improve strain survival on the seed over a similar method in which first the second composition was applied, after which the first composition was applied to the seed.

[0163] In a particular embodiment of the method, the first coating composition applied as a first coating comprises carboxymethylcellulose (CMC) and one or more sugars or sugar alcohols or a mixture thereof; and the second coating composition applied as a second coating comprises the one or more microbial strains and a clay, milk- derived power or legume seed-derived powder or a combination thereof.

[0164] In another or further preferred embodiment of the method, the first coating composition applied as a first coating comprises carboxymethylcellulose (CMC) and one or more sugars or sugar alcohols or a mixture thereof; and the second coating composition applied as a second coating comprises the one or more microbial strains and a soy seed-derived powder and / or vermiculite.

[0165] In an embodiment, the first or second coating composition, preferably the second coating composition, further comprises one or more additives suitable for enhancing the shelf life of the coated seeds. In a preferred embodiment, said additives may comprise Arabic gum.

[0166] In an embodiment, the first coating composition is applied by spraying on said seed surface. In another embodiment, the first coating composition is applied by dipping said seeds into a solution containing said first coating composition. In another embodiment, the first coating composition is applied by roll coating said first coating composition onto said seeds. In another embodiment, the first coating composition is applied by vacuum coating said first coating composition onto said seeds. In another embodiment, the first coating composition is applied using a rotary seed coater, where the seeds undergo a specialized coating process facilitated by rotary motion for efficient and uniform coverage.

[0167] In an embodiment, the second coating composition is applied by spraying on said seed surface. In another embodiment, the second coating composition is applied by dipping said seeds into a solution containing said second coating composition. In another embodiment, the second coating composition is applied by roll coating said second coating composition onto said seeds. In another embodiment, the second coating composition is applied by vacuum coating said second coating composition onto said seeds. In another embodiment, the second coating composition is applied using a rotary seed coater, where the seeds undergo a specialized coating process facilitated by rotary motion for efficient and uniform coverage.

[0168] In an embodiment, the method further comprises the step of drying the seeds after applying the first coating composition and before applying the second coating composition. In another embodiment, the method does not comprise this active drying step.

[0169] In a preferred embodiment, said seeds are legume seeds. Preferably the legume seeds are of the genus Glycine such as Glycine max or soybean, of the genus Pisum such as Pisum sativum or yellow pea, of the genus Phaseolus such as Phaseolus vulgaris or bean, of the genus Lens such as Lens culinaris or lentil, of the genus Lupinus such as Lupinus angustifolius or Lupinus albus, the genus Vicia such as Vicia faba, or of the genus Cicer such as Cicer arietinum.

[0170] The method's applicability to legume seeds, including soybean (Glycine max), yellow peas (Pisum sativum), beans (such as Phaseolus vulgaris and Vicia faba), lentils (Lens culinaris), lupins (Lupinus angustifolius or Lupinus albus), and chickpeas (Cicer arietinum), brings significant advantages to agricultural practices in various climates. Legume seeds, spanning across the genera Glycine, Pisum, Phaseolus, Vicia, Lens, Lupinus, and Cicer, are particularly receptive to symbiotic interactions, making them ideal candidates for this method. Specifically, legume seeds of the genus Glycine (e.g., Glycine max), the genus Pisum (e.g., Pisum sativum), the genus Phaseolus (e.g., Phaseolus vulgaris), the genus Lens (e.g., Lens culinaris), the genus Lupinus (e.g., Lupinus angustifolius or Lupinus albus), the genus Vicia (e.g. Vicia faba), and the genus Cicer (e.g., Cicer arietinum) benefit from the invention's tailored approach, ensuring optimal symbiotic relationships even in colder climates.

[0171] In another aspect, the present invention also relates to a seed coated with the seed coating product as described above in any of the embodiments. The survival of microbial strains was shown to be particularly effective on seeds coated with said seed coating product.

[0172] In a preferred embodiment, wherein seed coating product comprises said first and second coating composition, the first coating composition of the seed coating product is applied as a first coating layer directly on the seed, and the second coating composition of the seed coating product is applied as a second coating layer on top of said first coating composition.

[0173] The survival of microbes on the seed surface of seeds coated with two coating compositions as two layers on the seed surface as described above, was shown to be enhanced over such similar seeds coated with a mixture of both first and second compositions combined applied as one coating layer. In addition, the survival of microbes on the seed surface of seeds coated first with the second composition, on top of which subsequently the first coating composition was applied, was shown to be lower compared to the survival on coated seeds of the invention in which first the first coating composition was applied, on top of which the second coating layer was applied.

[0174] In a preferred embodiment, at least 104, 105106, 107, 108, 109, or even at least IO10microbial cells or colony forming units (CFUs) are delivered to each seed. Preferably, after two months, said colony forming units (CFUs) on said seeds coated with the seed coating product as described above has decreased maximally one or two order of magnitude, reaching at least 103, 104, 105, 106, 107, 108, or even at least 109CFUs. For this preferably the seeds are stored over these two months at rather harsh conditions to simulate adverse scenarios, i.e. a room temperature of 25 °C and the seeds are stored in airtight sealed plastic bags protected from light.

[0175] In another aspect, the present invention also relates to a seed coated using a method as described above in any of the embodiments.

[0176] In a preferred embodiment, the first coating composition of the seed coating product is applied as a first coating layer directly on the seed, and the second coating composition of the seed coating product is applied as a second coating layer on top of said first coating composition.

[0177] In embodiments, the seed is coated with the seed coating product as described above in any of the embodiments following the method as described above in any of the embodiments.

[0178] In embodiments, the seed is a legume seed, preferably of the genus Glycine such as Glycine max or soybean, of the genus Pisum such as Pisum sativum or yellow pea, of the genus Phaseolus such as Phaseolus vulgaris or bean, of the genus Lens such as Lens culinaris or lentil, of the genus Lupinus such as Lupinus angustifolius or Lupinus albus, the genus Vicia such as Vicia faba, or of the genus Cicer such as Cicer arietinum.

[0179] In embodiments, the coated seeds comprise at least 104, 105106, 107, 108, 109, or even at least IO10microbial cells or colony forming units (CFUs) per seed, as described above.

[0180] The present invention also relates to a plant grown from a seed coated with the seed coating product as described in any of the embodiments, and / or grown from a seed coated using the method as described in any of the embodiments.

[0181] The invention's coated seeds offer numerous benefits for agricultural productivity. The seed coating product imparts a powerful advantage to the seeds by enhancing their symbiotic interactions with microbes and improving survival rates. The coated seeds exhibit improved microbial viability, nodulation, and plant growth compared to conventional coatings, and has a longer shelf life compared to seed coated with such conventional coatings. As described above, the extended shelf life of said seeds presents the advantage of offering pre-coated seeds to farmers, alleviating the need for them to perform seed coating just before sowing. As a result, valuable time is saved for farmers, contributing to more efficient planting processes.

[0182] In an aspect, the present invention also relates to various uses of the seed coating product as described above in any one of the embodiments.

[0183] In particular, the invention also relates to the use of the seed coating product as described above in any one of the embodiments for increasing the survivability of microbial strains on a seed surface.

[0184] In the context of the present invention, the term "increasing the survivability of microbial strains on a seed surface" refers to a substantial increase in microbial titers within the same time lapse on the coated seed compared to uncoated seeds or seeds which only comprise the microbial strain. This enhancement is characterized by a remarkable elevation in microbial titers ranging from 10 to 150-fold beyond the baseline survival level. The baseline survival level is defined as the microbial titers on uncoated seeds or seeds coated with conventional methods. Additionally, "increasing the survivability of microbial strains on a seed surface" implies a significant boost in the proportion of microbial strains within the coating population that maintains viability, wherein the preferred enhancement leads to at least 1000%, 2000%, 3000%, 4000%, 5000%, 6000%, 7000%, 8000%, 9000%, 10000%, 11000%, 12000%, 13000%, 14000%, or 15000% survival of the initial microbial population after 60 days, compared to the initial population at the onset of coating. During storage, the seeds are subjected to relatively high room temperature: 25°C, and they are stored protected from light, preferably in airtight plastic bags.

[0185] The invention also relates to the use of the seed coating product as described above in any one of the embodiments for promoting cold stress resistance of a seed coated with said seed coating product.

[0186] "Cold stress resistance" as referred to in the present context, encompasses the capacity of a plant to endure non-freezing low temperatures without or with limited injury, damage, or yield reduction. Plants without said cold stress resistance are highly sensitive to cold or chilling stress and are injured or killed by non-freezing low temperatures or have a reduced nitrogen fixing capacity. They exhibit various symptoms of chilling injury such as chlorosis, necrosis, or growth retardation. This cold stress resistance shows when subjected to temperatures between 5 and 20°C, 8 and 18°C, or 10 and 15°C, particularly as measured in the plant's growth medium or soil, for durations of at least 2h, 4h, 6h, or 8h per day, or during specific time frames, such as during the night. Notably, the seed coating product can significantly elevate cold stress resistance, as evidenced by a substantial reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in injury, damage, or yield reduction, preferably seed yield reduction, due to low temperatures in plants grown from said seeds, preferably leguminous species such as soybean or yellow pea. This cold stress resistance also extends to increased tolerance of nitrogen fixation under such conditions, showcasing the potent role of the seed coating product in increasing plant endurance against non-freezing low temperatures. Importantly, nitrogen fixation is a temperature-sensitive phenomenon, and the advantage of having a cold-adapted strain also lies in its ability to enhance nitrogen fixation, thereby contributing to increased plant vigor and yield.

[0187] The present invention will be now described in more details, referring to examples that are not limitative.

[0188] EXAMPLES AND DESCRIPTION OF FIGURES

[0189] The data provided below, pertaining to soy seeds and plants, yields comparable results when employing pea seeds and plants. Anticipated is the likelihood that analogous outcomes would be observed in other plant varieties, particularly among legume seeds and plants as described above. Furthermore, it is reasonable to expect similar trends in the survivability of various microorganisms, including other bacteria and fungi.

[0190] Example 1: laboratory seed coating process

[0191] Introduction:

[0192] A novel seed coating product comprising a first coating composition and a second coating composition, suited to coat a seed with a first coating layer of said first coating composition and a second coating layer of said second coating composition has been developed as well as a method for coating seeds to efficiently deliver a microbial strain to seeds, thereby enhancing the survival of the strain on the seeds.

[0193] Methodology (general) - specifics / variations see below per experiment:

[0194] To initiate the process, a Bradyrhizobium japonicum strain, for example a B. japonicum strain with deposit accession number LMG P-32018, (Soy223), was grown for 6 days in Yeast Mannitol Broth (YMB) in a shaking incubator at 30 °C, after which it was centrifuged. Out of 50 ml, 5 ml concentrate was obtained.

[0195] A first coating composition comprising trehalose, sorbitol, and / or maltose and / or carboxymethylcellulose (CMC) was prepared. For concentrations, see further.

[0196] A second coating composition comprising bacterial culture (5ml concentrate) and clay and / or soy powder (ground and sieved - 400 microns; the powder subseguently autoclaved for 15 to 21 minutes at 121 °C) was prepared. For concentrations, see further.

[0197] For each treatment, 100g of soy seeds were coated as follows:

[0198] First the first coating composition was applied using a rotary seed coating for 10 seconds after which the second coating composition was applied, again using the rotary seed coater for 10 seconds. The volume ratio first to second composition was about 1:3.

[0199] Right after coating, the seeds were sampled (10 seeds per treatment). The rest was stored in airtight bags at 25 °C protected from sunlight. During set timepoints 10 seeds were sampled, to determine the amount of CFU per seed. For this, the seed coatings of the 10 seeds were dissolved in 10ml lOmM MgSO4. This solution was further diluted and plated on YMA in duplicate.

[0200] For the present experiment, the following combinations were tested: see Table 1. Concentrations of sugar(alcohols) and CMC are expressed based on the volume of the first coating composition.

[0201] Table 1

[0202] Results of bacterial titers and survival rate over time

[0203] Results are shown in Figures 1-5.

[0204] Figure 1 illustrates the prolonged survival of the bacterial strain over a 60-day period under different coating conditions.

[0205] Conditions 1 represents seeds coated without the addition of sugar (alcohol), cellulose derivative, or carrier substances (Control - 5 ml concentrated bacterial culture).

[0206] Condition 2 involves seeds coated with (per 100 g of seeds):

[0207] 2 ml of a first composition comprising 25 m / v % Trehalose and 5 m / v % CMC, and a second composition comprising 5 ml concentrated bacterial culture and 1.5g vermiculite. The amount of CFU per seed was determined on day 0, 1, 8, 15, 30 and 63, and the survival rate compared to day 0 was calculated.

[0208] The results highlight a remarkable difference in bacterial strain survival between the two conditions. After 30 days, Condition 1 shows a survival rate of only 0.53%, while Condition 2 demonstrates a significantly higher survival rate of 3.8%. As the evaluation period extends to 63 days, the bacterial strain's survival rate further declines in Condition 1 to a mere 0.01%, emphasizing the vulnerability of the strain under these conditions. In contrast, Condition 2 maintains a robust survival rate of 0.51% after 60 days.

[0209] This compelling data underscores the efficacy of the first coating composition comprising 25 m / v % Trehalose, 5 m / v % CMC; and the use of 1.5g vermiculite per 100g of seeds in enhancing the bacterial strain's survival on soy seeds.

[0210] Figure 2 portrays the impact of different concentrations of carboxymethylcellulose (CMC) on bacterial strain survival over time, up to 60days.

[0211] Contrary to the general methodology described above, in this experiment the bacterial strain was only grown for 4 days instead of 6 days.

[0212] 100 of seeds were coated with 2 ml of CMC (1 m / v% - Condition 3; 5 m / v% - Condition 4), and 5 ml concentrated bacterial culture with 1.5g vermiculite. No sugar (alcohol) was added.

[0213] The amount of CFU per seed was determined on day 0, 3, 7, 13, 31 and 60, and the survival rate compared to day 0 was calculated.

[0214] The results reveal that 5 m / v % CMC (Condition 4) leads to a notably better survival rate compared to 1 m / v % CMC (Condition 3) from 10 to 50 days, with the difference disappearing after 60 days. Further, it was seen that a higher CMC concentration resulted in composition with a higher viscosity and less dust (particles of the coating) coming off the seeds, indicating a better adherence of the coating.

[0215] Figure 3 showcases the survival rates of the bacterial strain over 60 days under different sugar (alcohol) conditions.

[0216] Condition 5 serves as a control (no sugar (alcohol)) and comprises per 100 g of seeds 2 ml of first coating composition with only a 5 m / v% CMC solution, an a second coating composition with 5 ml concentrated bacterial culture, and 1.5 g of vermiculite. For conditions 6 to 9 the 2ml of the first coating composition comprises 5 % CMC and further either 25 m / v % Trehalose, 50 m / v % Trehalose, 50 m / v % Sorbitol or 50 m / v% Maltose, respectively. The second coating composition remains the same as for the control.

[0217] The amount of CFU per seed were determined on day 0, 1, 5, 15, 30, 46, 60 and the survival rate compared to day 0 was calculated.

[0218] After 30 days, the survival rate for each of the conditions comprising sugar (alcohols) is markedly higher compared to control Condition 5. After 60 days, the highest survival rates are observed with 25 m / v % Trehalose (Condition 6) and 50 m / v % Maltose (Condition 9), which was slightly lower for 50 m / v % Trehalose (Condition 7), and again lower for Sorbitol (Condition 8). Condition 8 with Sorbitol, however, still results in a higher survival rate compared to control Condition 5.

[0219] The addition of sugar (alcohols) thus leads to increased survivability of the strain on seeds.

[0220] Figure 4 presents the survival rates of the bacterial strain in a seed coating over 59 days under different conditions, one in which soy powder is added.

[0221] Contrary to the general methodology described above, in this experiment the bacterial strain was only grown for 5 days instead of 6 days.

[0222] Both coatings (Condition 10 and 11) involves seeds coated with (per 100 g of seeds): 2 ml of a first composition comprising 25 m / v % Trehalose and 5 m / v % CMC, and a second composition comprising 5 ml concentrated bacterial culture and 1.5g vermiculite.

[0223] In addition, the second coating composition of Condition 11 further comprises 0.5 g of autoclaved ground soy seed powder per 100 g of seeds.

[0224] The amount of CFU per seed were determined on day 0, 1, 3, 7, 15, 29, 45, and 59 and the survival rate compared to day 0 was calculated.

[0225] The results indicate an overall increased survival with soy powder at all time points, as shown in Figure 4.

[0226] Figure 5 illustrates the comparative survival rates of bacterial strains in a seed coating comprising either soy powder (Condition 13) or vermiculite (Condition 12) until 60 days.

[0227] Condition 12 involves seeds coated with (per 100 g of seeds): 2 ml of a first composition comprising 25 m / v % Trehalose and 5 m / v % CMC, and a second composition comprising 5 ml concentrated bacterial culture and 1.5g vermiculite.

[0228] Condition 13 involves seeds coated with (per 100 g of seeds):

[0229] 2 ml of a first composition comprising 25 m / v % Trehalose and 5 m / v % CMC, and a second composition comprising 5 ml concentrated bacterial culture and 1.5g autoclaved ground soy seed powder.

[0230] The amount of CFU per seed were determined on day 0, 1, 3, 8, 15, 31, 40, and 60 and the survival rate compared to day 0 was calculated.

[0231] The data clearly demonstrates that soy powder outperforms vermiculite at all evaluated time points.

[0232] Conclusion:

[0233] This exemplary seed coating formulation and protocol exemplifies an effective and innovative means of delivering Bradyrhizobium japonicum to soy seeds. The results underscore the importance of specific components, such as sugar (alcohols), cellulose derivatives, clays and legume seed-derived powders, in significantly enhancing the bacterial strain's survival on soy seeds. This advancement holds promise for optimizing seed coating strategies for improved agricultural outcomes.

[0234] Example 2: SPAD and amount of nodules of plants grown from coated or uncoated seeds, sown at coating or after one month of storage

[0235] Objective: Sov plant inoculation trial to test the effectiveness of a seed coating according to the present invention compared to liquid inoculation.

[0236] Treatments:

[0237] Negative control: Seeds were not treated before or during planting.

[0238] Positive control: During planting 4 seeds / pot were inoculated with 1 ml of fresh Soy223 broth (0.25 ml per seed).

[0239] Freshly coated: Per 100g of seeds: 2 ml of first coating composition (5%CMC, 25% trehalose), 5 ml of concentrated Soy223 broth to which 1.5g of fine vermiculite and 0.5g of autoclaved soy powder was added. The coating was performed a half day before sowing.

[0240] Fresh liquid: Per 100g of seeds: 5 ml of concentrated Soy223 broth was added. The coating was performed half a day before sowing. Old coating: Per 100g of seeds: 2 ml of first coating composition (5%CMC, 25% trehalose), 5 ml of concentrated Soy223 broth to which 1.5g of fine vermiculite and 0.5g of autoclaved soy powder was added. The coating was performed one month before sowing. The seeds were stored at 25 °C.

[0241] Old liquid: 5 ml of concentrated Soy223 was added to 100g of seeds and stored in an airtight bag for 1 month at 25°C before sowing.

[0242] Protocol for seed coating: Soy223 was grown for 6 days in YMB in a shaking incubator at 30 °C, after which it was centrifuged. Out of 50 ml, 5 ml concentrate was obtained. For each treatment, 100g of seeds were coated as described in the treatment. First the first coating composition was applied, and seeds were coated for 10 seconds after which the carrier (vermiculite and soy powder) plus inoculum was applied and the coater was spun for 10 seconds. Right after coating, the seeds were sampled (10 seeds per treatment). The rest was stored in airtight bags at 25 °C.

[0243] Protocol for planting: 4 seeds were planted per pot (P13). Each pot contained 500 g of non-autoclaved 1 / 4OPM (a potting mix with low amount of nutrients). Each pot has an individual saucer for water gift. The plants were positioned in a raster according to the experimental design. The plants were grown in a growth room at 26°C with a 12 / 12 day / night cycle. The plants were watered from above. After 7 days one seedling was selected per pot. The experiment was stopped after 34 days.

[0244] At day 34 the SPAD was measured. SPAD stands for Soil Plant Analysis Development, and it refers to a method used to measure the chlorophyll content in plant leaves. The SPAD meter is a handheld device that provides a quick and non-destructive way to estimate chlorophyll levels, which is an indicator of the plant's photosynthetic activity. SPAD was measured as follows: per plant 6 measurements were taken of the upper most fully developed leaf, one on each of the six leaf lobes.

[0245] The roots were washed and the number of nodules was determined. After this, the leaves and roots were dried for 4 days at 50°C. The dried foliar and root mass was weighted.

[0246] Results:

[0247] When looking at the SPAD values at 34 days past sowing and inoculation, the inoculated treatments can be clearly distinguished from the negative control. The SPAD value of the old liquid treatment is the lowest although not significantly different from the positive control (Figure 6).

[0248] When looking at the number of nodules per plant in function of the treatment (Figure 7), a clear reduction can be noticed when comparing the liquid inoculations to the coatings. Although a lower number of nodules not always results in a lower yield, as plants can regulate the activity of nodules depending on their nitrogen status, a coating resulting in lower number of nodules could be more vulnerable in adverse conditions. Furthermore, it can be noted that decrease in number of nodules per plant after one month of storage is less drastic for the coated seeds than for the liquid inoculations.

[0249] Hence, inoculating through a seed coating is an advantageous method that ensures higher bacteria viability during storage.

[0250] Example 3: effectiveness of seed coating in field trials

[0251] Objective: A field trial was set up to determine the effectiveness of our coating stored in warehouse conditions in time and to compare it to commercial seed coatings.

[0252] Treatments (for more details, see Example 2):

[0253] 1. Non-inoculated

[0254] 2. Fresh SOY223, inoculated on sowing day (no coating)

[0255] 3. Fresh coat (SOY223), stored for 1 day at RT (Room Temperature)

[0256] 4. 1-month-old coat (SOY223), stored at warehouse temperature

[0257] 5. Turbosoy fresh, stored for 1 day at RT

[0258] 6. Turbosoy 21 days before, stored at warehouse temperature.

[0259] 7. Rhizoliq Top fresh, stored for 1 day at RT

[0260] 8. Vitalianz R Soja, inoculated on sowing day

[0261] Tubosoy, Rhizoliq Top en Vitalianz R Soja are commercial seed inoculation products, and were applied according to manufacturer's instructions.

[0262] Protocol:

[0263] Each treatment was applied to two soy varieties (Gml704-454 - short: 454 ; and Gml704-533 - short: 533). For each variety 4 replicates were foreseen. Each plot had a size of 7.5m2 sown at average sowing density. The plants were sown on 03 / 05 / 2023.

[0264] The field was visited multiple times during the growing season to take drone images. The data of the following days in 2023 was analyzed: 6 / 07, 7 / 08, 25 / 08, 6 / 09. The drone flights gave data for the following spatial indexes: CIVE, ExG.Norm, ExG, VARI. All are parameters for the greenness of the plant which is correlated to the chlorophyll content and in turn correlated to the nitrogen status of the plant.

[0265] On 20 / 06 4 plants per plot were harvested to determine the average number of nodules per plant per plot. On 7 / 08 SPAD values were determined.

[0266] Subsequently, also seed yield (corrected to 14% moisture content) and Seed protein content were determined as detailed below.

[0267] Results:

[0268] Not all graphs and data are shown below, only some are selected.

[0269] 20 / 06: When looking at the number of nodules per plant, It can be seen that variety Gml704-454 does not respond as much compared to variety Gml704-533 to the different inoculation treatments. The graph of variety 533 shows that a coating according to the present invention performs similar to the fresh commercial inoculants up to 1 month (Figure 8).

[0270] 7 / 08: When looking at the average SPAD value, all inoculated treatments result in a clear and significant increase compared to the negative control. The one-month- old coating is performing at the same level of other fresh inoculum (Figure 9).

[0271] Spatial indexes: EXGreen: The ExG index was selected as it is showing the clearest difference between the negative control and the inoculated treatments in both varieties. In this index, darker (healthier) plants score lower values, while lighter ones score higher. EXGreen values (ExG_230825) are shown on the Y-axis. Only in variety 454, there is a difference between inoculated treatments as Vitalianz is underperforming. The coating according to the present invention up to one month is performing as good or better as the control (Figure 10).

[0272] Seed yield corrected to 14% moisture content: In order to determine the seed yield (ton / ha), a plot is harvested and the fresh weight per plot is determined. A representative sample between 190g and 220g is taken and weighted before the sample is completely dried in an oven at 70°C for 3 days. Immediately after drying, the dry weight of the sample is measured. Based on the total fresh weight per plot, the fresh sample weight, the dried sample weight and the size of the plot, the yield

[0273] (ton / ha) at 14% moisture is calculated.

[0274] Results show a clear improvement of seed yield in all inoculated treatments. The one-month-old coating is working as good or better as the fresh commercial coatings (Figure 11).

[0275] Seed protein content:

[0276] The seed protein content (%) was determined via Near-Infrared Reflectance (NIR). For this method, seed samples between 190 and 220g were taken per plot. The samples were analysed by measuring their absorbance in a Polytec NIR spectrometer (5 measurements for each sample). The average absorbance value was calculated per sample and the nitrogen content calculated through a regression using a calibration curve previously prepared based on samples of which protein content had been experimentally determined through the Kjeldahl method.

[0277] Results show that the one-month-old coating increases protein content to a similar or slightly higher level than fresh commercial coatings (Figure 12).

[0278] Example 4: laboratory seed coating process - the effect of milk powder

[0279] The setup of Example 1 was used to compare the effectiveness of a coating comprising trehalose, CMC, and further only vermiculite (Condition 14), to a coating further comprising both vermiculite and milk powder (Condition 15), and to a coating further comprising vermiculite, soy powder, and milk powder (Condition 16) as shown in Table 2 below. The amount of CFU per seed were determined on day 0, 1, 3, 8, 15, 31, 40, 60, and 94 and the survival rate compared to day 0 was calculated.

[0280] Table 2

[0281] Figure 13 presents the survival rates of the bacterial strain in a seed coating over 94 days under different conditions, to show the effect of adding milk powder in the seed coating. The results indicate an overall increased survival in conditions where milk powder was added to the coating. Example 5: laboratory seed coating process: seed coating with Rhizobium sophorae pea 6

[0282] Method:

[0283] Rhizobium sophorae pea 6 was grown for 6 days in Yeast Mannitol Broth (YMB) in a shaking incubator at 30 °C, after which it was centrifuged. Out of 50 ml, 5 ml concentrate was obtained.

[0284] A first coating composition comprising trehalose (25 m / v%) and CMC (5 m / v%) was prepared.

[0285] A second coating composition comprising bacterial culture (5ml concentrate) and vermiculite (autoclaved, 21 minutes at 121 °C) was prepared.

[0286] For each treatment, 100g of soy seeds were coated as follows ('seed coating'): First, the first coating composition was applied using a rotary seed coating for 10 seconds after which the second coating composition was applied, again using the rotary seed coater for 10 seconds. The volume ratio first to second composition was about 1 :3. As a control setting ('liquid inoculation'), 5 ml of centrifuged bacterial pellet, without sugar, cellulose derivative and carrier, was added to the seeds.

[0287] Right after coating, the seeds were sampled (10 seeds per treatment). The rest was stored in airtight bags at 25°C protected from sunlight. During set timepoints seeds were sampled, to determine the amount of CFU per seed. For this, the seed coatings of the 10 seeds were dissolved in 10ml lOmM MgSO4. This solution was further diluted and plated on YMA in duplicate.

[0288] Results:

[0289] The results are shown in Figure 14. The seed coating formulation improves the survival of a Rhizobium sophorae strain when applied to soy seeds, compared to the direct application of the inoculum.

[0290] Thus, although the present example section seems to focus on Bradyrhizobium japonicum, similar improvements in survival of other microbial strains such rhizosphere microbial strains, is shown as well.

[0291] Example 6: determining the optimal sugar(alcohol) concentration in the coating

[0292] Method:

[0293] B. japonicum SOY223 was grown for 6 days in Yeast Mannitol Broth (YMB) in a shaking incubator at 30°C, after which it was centrifuged. Out of 50 ml, 5 ml concentrate was obtained.

[0294] A first coating composition was prepared comprising CMC (5 m / v%) and trehalose either at 5 m / v%, 12.5 m / v%, 25 m / v%, 40 m / v% or 55 m / v%. A second coating composition comprising bacterial culture (5ml concentrate) and autoclaved vermiculite (21 minutes at 121 °C) was prepared.

[0295] For each treatment, 100g of soy seeds were coated as follows: First, the first coating composition was applied using a rotary seed coating for 10 seconds after which the second coating composition was applied, again using the rotary seed coater for 10 seconds. The volume ratio first to second composition was about 1 :3.

[0296] Right after coating, the seeds were sampled (10 seeds per treatment). The rest was stored in airtight bags at 25 °C protected from sunlight. During set timepoints seeds were sampled, to determine the amount of CFU per seed. For this, the seed coatings of the 10 seeds were dissolved in 10ml lOmM MgSC . This solution was further diluted and plated on YMA in duplicate.

[0297] Results:

[0298] The optimal trehalose (sugar) concentration is between 25 and 40 m / v% of the first coating composition, as shown in Figure 15, although also 55 m / v% still showed quite good results. As the volume ratio of the first to second composition was about 1 :3, this equals an preferred sugar concentration of between 6.25 m / v% and 10 m / v%, although also 13.75 m / v% showed quite good results. It is therefore deemed that a seed coating having a concentration of between 5 and 15 m / v% of sugar is optimal.

[0299] Example 7: seed coating with various cellulose derivatives

[0300] Method:

[0301] B. japonicum SOY223 was grown for 6 days in Yeast Mannitol Broth (YMB) in a shaking incubator at 30 °C, after which it was centrifuged. Out of 50 ml, 5 ml concentrate was obtained.

[0302] A first coating composition comprising 25 m / v% trehalose and a 5 m / v% cellulose derivative was prepared. Said cellulose derivatives were chosen from hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), a combination of 2.5 m / v% HPMC and 2.5 m / v% ethyl cellulose (EC), methylcellulose (MC), hydroxyethyl cellulose (HEC), and cellulose monoacetate (CA).

[0303] A second coating composition comprising bacterial culture (5ml concentrate) and autoclaved vermiculite (21 minutes at 121 °C) was prepared.

[0304] For each treatment, 100g of soy seeds were coated as follows: First the first coating composition was applied using a rotary seed coating for 10 seconds after which the second coating composition was applied, again using the rotary seed coater for 10 seconds. The volume ratio first to second composition was about 1 :3. As a control setting ('liquid SOY223'), 5 ml of centrifuged bacterial pellet, without sugar, cellulose derivative and carrier, was added to the seeds.

[0305] Right after coating, the seeds were sampled (10 seeds per treatment). The rest was stored in airtight bags at 25 °C protected from sunlight. During set timepoints seeds were sampled, to determine the amount of CFU per seed. For this, the seed coatings of the 10 seeds were dissolved in 10ml lOmM MgSC . This solution was further diluted and plated on YMA in duplicate.

[0306] Results:

[0307] As shown in Figure 16, various cellulose derivatives may be used to replace CMC in the seed coating process and composition, including but not limited to hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), a combination of HPMC and ethyl cellulose (EC), methylcellulose (MC), hydroxyethyl cellulose (HEC), and cellulose monoacetate (CA).

[0308] Example 8: effectiveness of seed coating in field trials for number of nodules and seed yield.

[0309] Method:

[0310] Soy seeds that had previously been inoculated with B. japonicum SOY223 and a seed coating formulation comprising trehalose, CMC, and vermiculite (Condition 2 of Example 1), or with the bacterial strain without coating, were stored at room temperature for different time periods (0, 1, 2 months). These seeds were used to perform a field trial in which various symbiotic parameters were assessed.

[0311] For this trial, 4 plots were sown per treatment, organized in a randomly-generated design. At the moment of sowing, samples of the seeds were taken and plated to determine the CFUs / seed. For this, the seed coatings of the 10 seeds were dissolved in 10ml lOmM MgSC . This solution was further diluted and plated on YMA in duplicate.

[0312] At flowering time, 3 plants were sampled per plot, and the number of symbiotic nodules determined. The average number of nodules for each treatment was determined and plotted in function of the average log(CFU / seed). Subsequently, a linear regression was performed, and an R2values calculated for such.

[0313] Further, at harvest time, plots were harvested and thrashed independently, and seeds stored and dried to 14% moisture content. Seed weight per plot was determined, and based on the plot area, the yield (ton / ha) was calculated for every plot. The average yield was calculated and plotted in function of the average log(CFU / seed) determined for each treatment. A linear regression was performed, and an R2values calculated for such. Results:

[0314] Results of the average number of nodules per plant at flowering time in function of the average log(CFU / seed) at sowing time are shown in Figure 17A. As shown, the number of CFU / seed at sowing time positively (although not linearly) correlates with the average nodule number per plant. Results of the average seed yield in function of the average log(CFU / seed) at sowing time are shown in Figure 17B, the number of CFU / seed at sowing time positively (although not linearly) correlates with the average yield in a soy field trial. The present invention is in no way limited to the embodiments described in the examples and / or shown in the figures. On the contrary, methods according to the present invention may be realized in many different ways without departing from the scope of the invention.

Claims

CLAIMS1. A seed coating product comprising:- a cellulose derivative; one or more sugars, sugar alcohols, or a mixture thereof; one or more clays, a milk-derived powder, a legume seed-derived powder, or a combination thereof; and one or more microbial strains.

2. The seed coating product according to claim 1, comprising at least 5 m / v% of said one or more sugars or sugar alcohols, preferably between 5 m / v% and 15 m / v% of said one or more sugars or sugar alcohols.

3. The seed coating product according to claim 1 or 2, wherein said sugars or sugar alcohols are chosen from trehalose, sorbitol, maltose, or a combination thereof.

4. The seed coating product according to any one of the previous claims, comprising between 0.5 m / v% and 2.5 m / v% of cellulose derivative.

5. The seed coating product according to any one of the previous claims, wherein said cellulose derivative is chosen from carboxymethylcellulose (CMC), ethyl cellulose (EC), methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), and cellulose monoacetate (CA), or a combination thereof.

6. The seed coating product according to any one of the previous claims, comprising between 10 m / v% and 25 m / v% of clay, milk-derived powder, legume seed-derived powder, or a combination thereof.

7. The seed coating product according to any one of the previous claims, wherein the seed coating product comprises a clay, said clay is vermiculite.

8. The seed coating product according to any one of the previous claims, wherein the seed coating product comprises a clay, wherein at least 95 % of the clay has a particle size of maximally 1.0 mm.

9. The seed coating product according to any one of claims, wherein said seed coating product comprises a legume seed-derived powder, said legume is a soy or a pea.

10. The seed coating product according to any one of claims, wherein said seed coating product comprises a legume seed-derived powder, whole legume seed-derived powder.

11. The seed coating product according to any one of the previous claims, wherein said seed coating product comprises a legume seed-derived powder, wherein at least 95 % of the legume seed-derived powder has a particle size of maximally 1.0 mm.

12. The seed coating product according to any one of the previous claims, wherein said milk-derived powder is whole milk powder, preferably derived from cow's milk, goat's milk, or sheep's milk.

13. The seed coating product according to any one of the previous claims, wherein the one or more microbial strains comprise a rhizosphere microbial strain.

14. The seed coating product according to any one of the previous claims, wherein the one or more microbial strains comprise a strain of the genus Bradyrhizobium, Ensifer, Rhizobium, or a combination thereof, preferably comprising a Bradyrhizobium japonicum strain such as a Bradyrhizobium japonicum strain having the deposit accession number LMG P-32018.

15. The seed coating product according to any one of the previous claims, comprising:- between 0.5 m / v% and 2.5 m / v% of cellulose derivative chosen from carboxymethylcellulose (CMC), ethyl cellulose (EC), methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), and cellulose monoacetate (CA), or a combination thereof,- between 5 m / v% and 15 m / v% of one or more sugars or sugar alcohols, chosen from trehalose, sorbitol, maltose, or a combination thereof,- between 10 m / v% and 25 m / v% of clay, milk-derived powder, legume seed-derived powder, or a combination thereof, and- one or more rhizosphere microbial strains, preferably of the genus Bradyrhizobium, Ensifer, or Rhizobium, or a combination thereof.

16. The seed coating product according to any one of the previous claims, comprising a first coating composition and a second coating composition, suited to coat a seed with a first coating layer of said first coating composition and a second coating layer of said second coating composition.

17. The seed coating product according to claim 16, wherein:- said first coating composition comprises the cellulose derivative; and wherein- said second coating composition comprises the one or more microbial strains; and the one or more clays, a milk-derived powder, or legume seed-derived powder; or a combination thereof.

18. The seed coating product according to claim 17, wherein said one or more sugars, sugar alcohols, or mixtures thereof are either present in the first coating composition or in the second coating composition.

19. A method for coating seeds, said method comprises applying a seed coating product according to any one of the claims 1 to 18 to a seed surface.

20. The method according to claim 19, wherein the seed coating product is applied in at least two steps, comprising: - applying to said seed surface a first coating composition comprising a cellulose derivative; and- applying on top of said first coating composition a second coating composition comprising one or more microbial strains; and one or more clays, a milk-derived powder, a legume seed-derived powder, or a combination thereof.

21. The method according to claim 20, one or more sugars, sugar alcohols, or mixtures thereof are either present in the first coating composition or in the second coating composition.

22. A seed, coated with the seed coating product according to any one of the claims 1 to 18.

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