Use of a legume seed-derived powder in an inoculum

Abstract

The current invention relates to a use of legume seed-derived powder for increasing the survivability of microorganisms in an inoculum. The invention also relates to a seed coating, to a method for coating a seed, and a coated seed. The invention further relates to a method for enhancing the survivability of microorganisms coated on a seed surface.

Description

[0001] USE OF A LEGUME SEED-DERIVED POWDER IN AN INOCULUM

[0002] FIELD OF TH E INVENTION

[0003] The present invention relates to the field of agricultural technology, specifically to seed coatings. It addresses improving the survival and efficacy of microorganisms applied to seed surfaces.

[0004] BACKGROUN D

[0005] Soybean Glycine max') and Yellow Pea Pisum sativum) are leguminous plants, or legumes, indigenous to East Asia, historically rooted 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; however, the effective establishment and survival of these symbiotic microbes on seeds poses challenges, as well as the survival of such microbes in an inoculum, such as a seed coating to be used to coat such seeds. Gram-negative rhizobia, a group of bacteria forming symbiotic relationships with the roots of legume plants, are sensitive to environmental conditions. Seed coating liquids containing such rhizobia generally need refrigeration to maintain viability. As a result, seeds are typically coated with the symbiotic strain(s) immediately before sowing to ensure sufficient viable rhizobia upon planting.

[0008] Some existing solutions claim a shelf life of seed coatings comprising rhizobia of 90- 120 days through overdosing the microorganisms. The results however fall short compared to the efficacy of a fresh coating, as the microorganisms tend to lose their effectiveness / survivability over time in the inoculum, coating, and / or when coated on a seed surface. In addition, inocula, seed coatings and / or methods for improving the shelf life of said inocula and coatings are for instance known from W02017143130, CN100566574, Chun Hwi Cho et al. (2011), Yi Qui et al. (2020), Masoume Amirkhani et al. (2016), and W02021081018. Coating legume seeds with a microbial strain is thus a time-consuming and expensive task, generally performed right before sowing when farmers are already busy with various other agricultural activities.

[0009] Furthermore, although the above focuses on nitrogen-fixation and legumes, it is essential to note that the potential applications of improved inoculums and seed coating methods extend beyond the specific context of improving nitrogen-fixation in legumes. Current agricultural research explores the utilization of beneficial microbes as alternatives to traditional pesticides and stimulants, for a wide range of plants. In instances where application of such microbes is required or desired, whether nitrogen-fixing strains on legumes or other beneficial microbes on diverse crops, similar challenges emerge in maintaining their viability in inoculums, seed coatings, on plant growth media, and on plant surfaces including seed surfaces. Therefore, a versatile inoculum addressing these challenges holds significant relevance in a broader spectrum of agricultural practices, contributing to sustainable farming methods and optimizing seed performance across various crops.

[0010] Recognizing the pivotal role of effective inoculums and / or seed coatings in modern agriculture, efforts to address challenges are paramount. Inoculum liquids and seed coatings act as a protective shield for microorganisms, contributing to sustainable farming practices and enhancing seed performance. However, maintaining the viability of microorganisms in an inoculum or seed coating, or when applied to a plant growth medium or coated on a seed surface, especially under variable environmental conditions, remains a persistent challenge.

[0011] To address these challenges, there is a growing demand for improved inoculums, seed coating methods and products that enhance microbial survival while ensuring a longer shelf life of the inoculum, coating products, and coated seeds, preferably alleviating the farmers of the need to perform the inoculation or seed coating just before sowing.

[0012] SUMMARY OF TH E INVENTION

[0013] The present invention and embodiments thereof serve to provide a solution to one or more of above-mentioned disadvantages. To this end, the present invention relates to a use of a legume seed-derived powder according to claim 1. In particular, said legume seed-derived powder is used for increasing the survivability of microorganisms in an inoculum.

[0014] In a second aspect, the present invention also relates to a seed coating according to claim 17, wherein the coating comprises a legume seed-derived powder and optionally one or more microorganisms. In addition, an aspect of the invention relates to a method for coating a seed according to claim 21, to a coated seed according to claim 23, and to a method for enhancing the survivability of microorganisms coated on a seed surface according to claim 24.

[0015] Preferred embodiments of the use are shown in any of the claims 2 to 16, of the seed coating in any of the claims 18 to 20, in the method of coating a seed in claim 22, and in the method for enhancing the survivability of microorganisms coated on a seed surface in claim 25.

[0016] The use of legume seed-derived powder was shown to ensure prolonged survival of microorganisms in an inoculum and on a seed surface, thereby extending the shelf life of inoculums and of seeds coated with such inoculum or seed coating 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.

[0017] DEPOSIT OF BIOLOGICAL MATERIAL

[0018] The Bradyrhizobium japonicum strain described in present application was deposited by VIB vzw (Rijvisschestraat 120, 9052 Gent, Belgium) at the BOOM (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. DESCRIPTION OF FIGURES

[0019] 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.

[0020] Figure 1 highlights the positive influence on adding soy powder to a seed coating, resulting in an enhanced survival rate compared to conditions without this addition, as discussed in Example 1.

[0021] Figure 2 provides a comparative analysis on the survival rate of a bacterial strain on soy seed surfaces, coated with a seed coating comprising either soy seed-derived powder or vermiculite. The figure shows an enhanced survival rate when the coating comprises soy seed-derived powder, as discussed in Example 1.

[0022] Figure 3 highlights the importance of autoclaving the soy powder when used in a seed coating, resulting in an enhanced survival rate compared to conditions without autoclaving the powder, as discussed in Example 1.

[0023] Figure 4 shows the effect of autoclaving the seed-derived powder on bacterial survival on the coated seeds as discussed in Example 2. (A) The legume seed-derived powder is either not autoclaved (control, 0 minutes), or autoclaved at 121 °C either for 5 minutes, 10 minutes, 15 minutes, or 21 minutes. (B) The legume seed-derived powder is either not autoclaved (control, 0 minutes), or autoclaved for 21 minutes either at 115 °C, 121 °C, or 132 °C.

[0024] Figure 5 shows the effect of different legume seed-derived powders on bacterial survival on the coated seeds as discussed in Example 3. The seed coating comprised trehalose and CMC, and a carrier which was either vermiculite (control) or autoclaved legume powder (autoclaved at 121°C for 21 min), chosen from Soy Glycine max'), cowpea (Vigna unguiculata), chickpea (Cicer arietinum), common vetch (Vicia sativa), lupin (Lupinus spp.), lima bean (Phaseolus lunatus), adzuki bean Vigna angularis), grass pea (Lathyrus sativus), alfalfa (Medicago sativa), yam bean (Pachyrhizus spp.), bean prelude (Phaseolus vulgaris), mung bean (Vigna radiata), fava bean (Vicia faba).

[0025] DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention concerns a use of legume seed-derived powder for increasing the survivability of microorganisms in an inoculum. The invention also concerns a method for coating a seed, and a coated seed. The method further concerns a method for enhancing the survivability of microorganisms coated on a seed surface. Definitions

[0027] 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.

[0028] As used herein, the following terms have the following meanings:

[0029] "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.

[0030] "About" as used herein referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / - 20% or less, preferably + / -10% or less, more preferably + / -5% or less, even more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier "about" refers is itself also specifically disclosed.

[0031] "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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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. 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).

[0038] 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.

[0039] 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.

[0040] 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.

[0041] In the present context, an "inoculum" refers to any substance or material comprising microorganisms or a plurality or mixture of said substances or materials, such as a liquid substance or solid substance comprising microorganisms, preferably a liquid substance.

[0042] In the context of the present application, "increasing the survivability of microorganisms in an inoculum" refers to a substantial improvement in the proportion of microorganisms that maintain viability over time when incorporated into the inoculum. This enhancement results in a significantly reduced rate of microbial decline, leading to a higher overall survival rate compared to an inoculum without the use of legume seed-derived powder. The baseline survival level is defined as the microbial titers observed in an inoculum without the legume seed-derived powder. The term also implies a notable increase in the proportion of microorganisms within the inoculum that remains viable. The preferred enhancement results in a survival rate of at least 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000%, 4000%, 5000%, 6000%, 7000%, 8000%, 9000%, 10000%, 11000%, 12000%, 13000%, 14000%, or 15000% of the microbial population of an inoculum without the addition of legume seed-derived powder after a specified duration, such as storage for 30 or 60 days. The storage conditions for the inoculum involve exposure to relatively high room temperature (25°C), and the inoculum is stored protected from light, preferably in airtight sealed containers.

[0043] In the context of the present application "increasing the survivability of microorganisms coated onto a seed surface" refers to a notable reduction in the proportion of microorganisms that lose viability over time, resulting in a higher percentage of microorganisms remaining viable compared to uncoated seeds or seeds coated only with the microorganisms without the use of legume seed-derived powder. This improvement is characterized by a significant decrease in the rate of microbial decline, leading to a higher overall survival rate. The baseline survival level is defined as the microbial titers observed on uncoated seeds or seeds coated using conventional methods without addition of legume seed-derived powder. The term also implies a noteworthy increase in the proportion of microorganisms within the coating population that maintains viability. The preferred enhancement results in a survival rate of at least 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000%, 4000%, 5000%, 6000%, 7000%, 8000%, 9000%, 10000%, 11000%, 12000%, 13000%, 14000%, or 15000% of the microbial population of uncoated seeds or seeds coated using conventional methods without addition of legume seed-derived powder after a specified duration, such as storage for 30 or 60 days, compared to the initial population at the onset of coating. The storage conditions for the seeds involve exposure to relatively high room temperature (25°C), and the seeds are stored protected from light, preferably in airtight sealed plastic bags.

[0044] 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.

[0045] "Cellulose-derivative" is a compound derived from cellulose, a natural polymer found in plant cell walls.

[0046] 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.

[0047] 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.

[0048] "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.

[0049] "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.

[0050] "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.

[0051] 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.

[0052] Description

[0053] In a first aspect, the invention provides / relates to a use of a legume seed-derived powder for increasing the survivability of microorganisms in an inoculum.

[0054] Said "legume seed-derived powder", also referred to as "legume-derived powder" or "legume powder", or optionally a derivative thereof, provides a surface for the microorganisms to adsorb to or to embed into. It can thus act as a protective layer around the microbial cells in the inoculum and / or coated on a seed surface, shielding them from environmental stressors like temperature fluctuations and moisture changes that could potentially compromise their viability. It also provides support to the microorganisms, thereby acting as carrier. In addition, the powder assists in moisture retention, creating a favorable microenvironment for the microorganisms. When the inoculum is used as (part of) a seed coating product, and is coated on a seed surface, the favorable microenvironment may also have a positive effect on seed germination. The legume seed-derived powder also provides nutrients such as amino acids, vitamins, and minerals, and further proteins, fats, oils, and more which serve as a nutritional source for beneficial microorganisms. In the occasion the inoculum is used as (part of) a seed coating, said nutrients, proteins and more are also beneficial to seeds on which said legume-derived powder is applied to. Also, irrespective of whether the powder is provided prior to, simultaneously and / or after said microorganisms, it helps adhere the microorganisms, optionally part of a seed coating product, to the seed surface. Said legume-derived powder helps sustain the microorganisms' metabolic activity and viability, ensuring that these microorganisms remain active and capable of, for instance, promoting plant health if the inoculum is to be used as (part of) a seed coating product. Preferably, when used in a seed coating, 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. In an embodiment, the legume seed-derived powder, or derivative thereof, is obtained by a method comprising the steps of milling legume seeds, thereby forming said legume seed-derived powder, and subsequently autoclaving said powder.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] Autoclaving of legume seed-derived powder or derivative thereof was shown to be an important step when the powder is intended to increase the survivability of microorganisms.

[0059] "Autoclaving" is a sterilization method that utilizes high-pressure steam to eliminate a wide range of microorganisms, including bacteria, fungi, viruses, and spores, from materials. This process involves placing the materials to be sterilized inside a sealed chamber known as an autoclave, where steam is generated and introduced at a controlled temperature and pressure, typically at 121°C (250°F) and 15 psi (about 1 bar) for a duration of 15 to 30 minutes. The purpose of autoclaving is generally to ensure that materials are free from viable microorganisms prior to their use in laboratory, medical, or industrial applications. This sterilization method is relevant for preventing contamination, ensuring the integrity of biological experiments, and mitigating health risks associated with the use of potentially contaminated materials. Legume seed-derived powder, being a natural product, may harbor various microorganisms, including various plant pathogens, bacteria, fungi, viruses, or spores. Autoclaving helps eliminate or reduce these contaminants, preventing unwanted microbial interactions that could negatively affect the intended microorganisms of the invention in surviving in an inoculum, which is optionally applied to a seeds surface. In addition, the intended microorganisms in the inoculum or on the seed surface 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 microorganisms. 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 or toxic impact on microbe survival.

[0060] In the present context, autoclaving of the legume seed-derived powder or derivative thereof may be performed at least 3 minutes or at least 5 minutes at a temperature of between 100 °C and 145 °C. Preferably, the autoclaving is conducted at a temperature of between 110 °C and 140 °C for a duration of at least 3 minutes or at least 5 minutes. More preferably, the autoclaving is carried out at a temperature of between 115 °C and 132 °C for a duration of at least 3 minutes or at least 5 minutes. Autoclaving of the legume seed-derived powder may also be performed at a temperature of between 100 °C and 145 °C for a duration of between 3 and 25 minutes, preferably at a temperature of between 110 °C and 140 °C for a duration of between 3 and 25 minutes, more preferably at a temperature of between 115 °C and 132 °C for a duration of between 5 and 25 minutes or between 5 and 21 minutes.

[0061] In an embodiment, the legume seeds that are milled to legume seed-derived powder are whole legume seeds.

[0062] 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 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.

[0063] 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.

[0064] In an embodiment, the legume seed-derived powder is obtained from non-genetically modified legume seeds. In another embodiment, the legume seed-derived powder is obtained from genetically modified legume seeds.

[0065] In an embodiment, the legume seed-derived powder or derivative thereof has a particle size of 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 and / or minimally 0.1pm, preferably minimally 0.5 pm or even 1 or 5 pm.

[0066] 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 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. Preferably, the particle size is verified or obtained by sieving the particles after the milling step, thereby removing larger particles.

[0067] 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 has a particle size of minimally 0.1 pm, preferably minimally 0.5 pm, or even 1 or 5 pm.

[0068] This particle size was optimized for easy administration of an inoculum comprising the powder, for instance to a seed surface, potentially as part of a coating, and for increasing survivability of microorganisms in said inoculum and / or on said seed surface. The controlled particle size plays a dual role: it facilitates adherence of the inoculum to a surface, such as a seed surface and influences nutrient release from the ground legume seeds. Smaller particles improve nutrient availability, and may contribute to effective adhesion. When the particles are too small, they will be less likely act as carriers for microorganisms. However, excessively large particles could compromise adherence, could lead 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 inoculums and seed coatings, ensuring both enhanced adhesion and nutrient release for optimal seed performance.

[0069] In an embodiment, said legume seed-derived powder or derivative thereof comprises a protein content of at least 30 wt%, thereby possibly providing a nutritional source for beneficial microorganisms and promoting their survival in an inoculum and / or 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.

[0070] 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%.

[0071] 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. 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.

[0072] As already briefly mentioned above, in an embodiment, the inoculum comprising the legume seed-derived powder is coated directly onto a seed surface or used to prepare a seed coating to be coated on a seed surface.

[0073] Incorporating said powder in an inoculum to be used in seed coatings ensures even distribution and adhesion of the microorganisms across the powder and onto the seed surface. This promotes stability, prevents uneven application, and contributes to improved adherence to the seed surface. Moreover, incorporating legume seed- derived powder into the inoculum to be used in seed coatings creates a protective environment for the microorganisms. This encapsulation shields the microorganisms from external factors even before application to the seed surface. It provides the microorganisms with the opportunity to acclimate to the coating environment before being applied to the seed surface, thereby enhancing their survivability and effectiveness. In an embodiment, the microorganisms are embedded in and / or are adsorbed to the surface of the legume-seed derived powder.

[0074] In an embodiment, at least 105, 106, 107, 108, 109, or even at least IO10microorganisms or colony forming units (CFUs) for each gram of legume seed-derived powder is provided on a seed surface. This amount was shown to be sufficient to ensure effective colonization of the seed surface upon providing said powder and said microorganisms, optionally as part of a seed coating, to said seed surface, 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.

[0075] In an embodiment, said legume seed-derived powder or derivative thereof is incorporated in the inoculum in an amount ranging from 10 m / v% to 35 m / v%.

[0076] In an preferred embodiment, the legume seed-derived powder or derivative thereof is incorporated in the inoculum in an amount of about 10 m / v%, 12.5 m / v%, 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%.

[0077] In an embodiment, said inoculum further comprises a clay.

[0078] The "clay" or "clay mineral" of may also act as a carrier for the microorganisms and / or provides structure to the inoculum or coating to help adhere it to the seed. Said clay can be any clay usable in inoculums or 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 waterabsorbing 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.

[0079] Known inoculums to be used in 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 inoculum, these traditional materials lack certain desirable properties. These were shown to be less advantageous increasing the survivability of microorganisms in the inoculum or on a seed surface.

[0080] In an embodiment, 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, and / or minimally 0.1pm, preferably minimally 0.5 pm or even 1 or 5 pm.

[0081] 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 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.

[0082] Preferably, the particle size is obtained or verified by sieving the particles, thereby removing larger particles.

[0083] 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 clay has a particle size of minimally 0.1 pm, preferably minimally 0.5 pm, or even 1 or 5 pm.

[0084] This particle size was optimized for easy administration of an inoculum comprising the powder, for instance toa seed surface, and for increasing survivability of microorganisms in said inoculum and / or on said seed surface. The controlled particle size plays a dual role: it facilitates adherence of the inoculum to a surface, such as to a seed surface and influences nutrient release from the ground legume seeds. Smaller particles improve in moisture retention, and may contribute to effective adhesion. When the particles are too small, they will be less likely act as carriers for microorganisms. However, excessively large particles could compromise adherence, 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 inoculums and seed coatings, ensuring both enhanced adhesion and moisture retention for optimal seed performance.

[0085] In an embodiment, wherein the inoculum comprising the legume seed-derived powder is coated directly onto a seed surface or used to prepare a seed coating to be coated on a seed surface, one or more sugars or sugar alcohols are added to the seed surface, optionally as part of said seed coating.

[0086] In an embodiment, one or more sugars or sugar alcohols or a mixture thereof are provided to the seed surface prior to, simultaneously with and / or after said legume seed-derived powder and microorganisms are applied to said seed surface.

[0087] "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.

[0088] Providing sugars and / or sugar alcohols to a seed surface offers a range of advantages to microorganisms and their survival on a seed surface. 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 seed coatings, 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.

[0089] Where the sugars or sugar alcohols are provided simultaneously with said legume - seed derived powder and microorganisms to said seed surface, they may be part of the inoculum, however they may also be provided as such, or as part of a further seed coating composition.

[0090] In a further embodiment, said one or more sugars or sugar alcohols are chosen from trehalose, maltose and sorbitol.

[0091] In an embodiment, wherein the inoculum comprising the legume seed-derived powder is coated directly onto a seed surface or used to prepare a seed coating to be coated on a seed surface, one or more cellulose derivatives are added to the seed surface, optionally as part of said seed coating.

[0092] In an embodiment, one or more cellulose derivatives are provided to the seed surface prior to, simultaneously with and / or after said legume seed-derived powder and microorganisms are applied to said seed surface.

[0093] The cellulose derivatives, such as carboxymethylcellulose (CMC), methylcellulose, and ethyl cellulose, provide a protective barrier around the seed and around the microorganisms, shielding them from external factors such as moisture, drought, and pathogens. In addition, they provide adhesion to the seed surface, ensuring the legume seed-derived powder and the microorganisms, optionally as the microbial seed coating, and optionally the further seed coating composition adhere well to the seeds. CMC, in particular, but other cellulose derivatives as well, also aids controlled release of compounds and retains water, collectively increasing the survivability of microorganisms coated on said seed surface, and supporting successful plant germination and growth.

[0094] Where the cellulose derivatives are provided simultaneously with said legume seed- derived powder and microorganisms to said seed surface, they may be part of the inoculum, however they may also be provided as such, or as part of a further seed coating composition which may also comprises said sugars or sugar alcohols or a combination thereof.

[0095] In an embodiment, the microorganisms in the inoculum can be any microbial strain(s) able to survive as part of the inoculum, further comprising said legume seed-derived powder, and optionally on a seed surface. The strain can for instance be a bacterial or fungal strain. Non-limiting examples of such microorganisms, are 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 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.

[0096] "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.

[0097] In an embodiment, the microorganisms are rhizosphere microorganisms. A "rhizosphere microorganisms" pertains to a microorganism, including bacterial strains, "rhizobacterial microorganisms" and fungal strains "rhizosphere fungal microorganisms", 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 abovelisted microorganisms can all be associated with the rhizosphere.

[0098] In an embodiment, the microorganism are symbiotic strains, able to engage in a symbiotic relationship with a plant.

[0099] Non-limiting examples include microorganisms 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 growth-promoting symbiosis), Xanthomonas (plant growth-promoting symbiosis), Bacillus (symbiotic relationships with plants), Pseudomonas (symbiotic relationships with plants), and Acinetobacter (plant growth-promoting symbiosis).

[0100] Non-limiting examples include microorganisms 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).

[0101] "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.

[0102] Thus, in an embodiment, the microorganisms coated on the seed surface are selected from the group consisting of bacterial strains, fungal strains, or a combination thereof, wherein said bacterial strains are chosen from strains of genera Rhizobium, Sinorhizobium, Bacillus, Pseudomonas, Azospirillum, Azotobacter, Clostridium, Klebsiella, Stenotrophomonas, Halomonas, Pianococcus, Agrobacterium, Enterobacter, Serratia, Paenibacillus, Streptomyces, Lactobacillus, Corynebacterium, Micrococcus, Achromobacter, Burkholderia, Ralstonia, Xanthomonas, Acinetobacter, Bradyrhizobium, Frankia, Mezorhizobium, Ensifer, Herbaspirillum, and Brevibacillus ; and wherein said fungal strains are chosen from genera Trichoderma, Beauveria, , Rhizophagus, Piriformospora, Laccaria, Tuber, Pisolithus, Russula, Lepista, Hebeloma, Cenococcum, Amanita, Suillus, Rhizopogon, Penicillium, Fusarium, Cladosporium, Aspergillus, Verticillium, Metarhizium, Gliocladium, Paecilomyces,

[0103] Acremonium, and Talaromyces, and / or are Mycorrhizae.

[0104] In an embodiment, the microorganism 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.

[0105] 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.

[0106] In an embodiment, the microorganism are mycorrhizae.

[0107] In an embodiment, the microorganism 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. 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 non-limiting 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.

[0108] In an embodiment, the legume seed-derived powder is provided simultaneously with said microorganisms to said seed surface, as part of the inoculum, preferably as part of a seed coating as described in any one of the embodiments below.

[0109] In an embodiment, the legume seed-derived powder in combination with the microorganisms promotes, and preferably increases, nitrogen fixation in legumes, when applied to said legume seed surface.

[0110] In an aspect, the present invention relates to an inoculum comprising one or more microorganisms and a legume seed-derived powder or derivative thereof.

[0111] A person of ordinary skill will appreciate that features of the first aspect relating to the use as described above return in the further aspects 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.

[0112] Said legume seed-derived powder or derivative thereof is preferably as described above in any one of the embodiments, and said microorganisms may be any type of microorganism, such as any of the ones described above, preferably said microorganisms are nitrogen-fixing microorganisms. Preferably, said seed-derived powder is autoclaved, preferably as described above.

[0113] Such inoculum comprising said microorganisms and legume seed-derived powder provides a prolonged survivability of said microorganisms in said inoculum.

[0114] Said inoculum can be used as a seed coating coated directly on a seed surface, or can be used to prepare a seed coating to be coated on a seed surface.

[0115] Advantages of the legume seed-derived powder in the inoculum are its effect on increasing the survivability of the microorganisms in the inoculum. The inoculum can therefore be prepared longer in advance of its use, for example as a seed coating or as part of a seed coating. Further advantages of the powder in the inoculum are as provided above.

[0116] In an embodiment, said inoculum comprises at least 1 m / v%, preferably at least 2 m / v%, preferably at least 3 m / v%, preferably at least 4 m / v%, preferably at least 5 m / v%, preferably at least 6 m / v%, preferably at least 7 m / v%, preferably at least 8 m / v%, preferably at least 9 m / v%, preferably at least 10 m / v%, preferably at least 12 m / v%, preferably at least 14 m / v%, preferably at least 16 m / v%, preferably at least 18 m / v%, preferably at least 20 m / v% of said legume seed-derived powder.

[0117] In an embodiment, said inoculum comprises said legume seed-derived powder in an amount ranging from 10 m / v% to 35 m / v%.

[0118] In an preferred embodiment, said inoculum comprises said legume seed-derived powder in an amount of about 10 m / v%, 12.5 m / v%, 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%.

[0119] In an aspect, the present invention relates to a seed coating, comprising a legume seed-derived powder and optionally one or more microorganisms.

[0120] In a preferred embodiment, the seed coating comprises one or more microorganisms.

[0121] Said legume seed-derived powder is preferably as described above in any one of the embodiments, and said microorganisms may be any type of microorganism, such as any of the ones described above, preferably said microorganisms are nitrogen-fixing microorganisms. Such seed coating comprising said microorganisms and legume seed-derived powder provides a prolonged effectivity of the coating by increasing the survivability of said microorganisms when coated on a seed surface.

[0122] The seed coating thereby extends the shelf life of seeds coated with such seed coating 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 is generally done. As a result, valuable time is saved for farmers, contributing to more efficient planting processes.

[0123] In an embodiment, said seed coating comprises at least 1 m / v%, preferably at least

[0124] 2 m / v%, preferably at least 3 m / v%, preferably at least 4 m / v%, preferably at least

[0125] 5 m / v%, preferably at least 6 m / v%, preferably at least 7 m / v%, preferably at least

[0126] 8 m / v%, preferably at least 9 m / v%, preferably at least 10 m / v%, preferably at least

[0127] 12 m / v%, preferably at least 14 m / v%, preferably at least 16 m / v%, preferably at least 18 m / v%, preferably at least 20 m / v% of said legume seed-derived powder.

[0128] In an embodiment, said seed coating comprises said legume seed-derived powder in an amount ranging from 10 m / v% to 35 m / v%.

[0129] In an preferred embodiment, the seed coating comprises said legume seed-derived powder in an amount of about 10 m / v%, 12.5 m / v%, 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%.

[0130] In an embodiment, said seed coating further comprises a clay.

[0131] In an embodiment, at least 105, 106, 107, 108, 109, or even at least 1010microorganisms or colony forming units (CFUs) for each gram of legume seed-derived powder is provided in said seed coating. This amount was shown to be sufficient to ensure effective colonization of the seed surface upon providing said seed coating to said seed surface, 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. In an embodiment, the seed coating further comprises at least one component chosen from the list of one or more sugars or sugar alcohols or a mixture thereof, one or more cellulose derivatives, and a clay.

[0132] The one or more sugars or sugar alcohols are preferably chosen from maltose, trehalose, and sorbitol, or a combination thereof. The cellulose derivatives are preferably chosen from CMC, methylcellulose, or ethyl cellulose, or a combination thereof. The clay is preferably vermiculite.

[0133] Advantages are as described above in any one of the above embodiments.

[0134] In an embodiment, the seed coating 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 comprises between 5 m / v% and 7.5 m / v% of said sugars or sugar alcohols.

[0135] The inclusion of at least 5 m / v% of said sugars or sugar alcohols in the seed coating provides improved moisture retention, microbial protection, increased available energy for the microorganisms, 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.

[0136] In another or further embodiment, the seed coating 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 comprises between 10 m / v% and 14 m / v% of said sugars or sugar alcohols.

[0137] 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, supporting the symbiotic microbes' viability during storage and planting.

[0138] In a preferred embodiment, the seed coating 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 comprises between 5 m / v% and 14 m / v% of said sugars or sugar alcohols.

[0139] In an embodiment, the seed coating comprises between 0.5 m / v% and 2.5 m / v% of cellulose derivatives. Preferably, the seed coating 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 comprises 1.25 m / v% of cellulose derivatives.

[0140] 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.

[0141] In a preferred embodiment, where the cellulose derivative is CMC, the seed coating comprises between 0.5 m / v% and 2.5 m / v% of CMC. Preferably, the seed coating 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 comprises 0.5 to 1.25 m / v% of CMC, most preferably about 1.25 m / v% of CMC.

[0142] 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%.

[0143] A CMC or methylcellulose concentration of about 1.25 m / v% was shown to offer the most optimal concentration.

[0144] In a specific embodiment, the cellulose-derivative incorporated into the seed coating undergoes a sterilization process involving heating without boiling. While numerous constituents of the seed coating 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.

[0145] As mentioned said sugars or sugar alcohols are preferably 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 microorganisms coated on the seeds.

[0146] 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.

[0147] This seed coating provides a unique combination of ingredients, all tailored to enhance the survival and effectiveness of microorganisms on seeds.

[0148] In an embodiment, the seed coating further comprises one or more of a bacterial inoculant, a fungal inoculant, or a combination thereof. The inclusion of bacterial and / or fungal inoculants in the seed coating 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.

[0149] The fungal inoculant can comprise a fungal inoculant of the family Glomeraceae,

[0150] Claroidoglomeraceae, Gigasporaceae, Acaulosporaceae, Sacculosporaceae

[0151] Entrophosporaceae, Pacidsporaceae, Diversisporaceae, Paraglomeraceae

[0152] Archaeosporaceae, Geosiphonaceae, Ambisporaceae, Scutellosporaceae

[0153] 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.

[0154] 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.

[0155] 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.

[0156] In an embodiment, the seed surface on which the microorganisms are coated and survive, can be any plant seed, preferably a legume seed, such as but not limited to a soybean or a yellow pea. In an aspect, the present invention relates to a method for coating a seed, comprising applying one or more coating layers to said seed, wherein said seed is coated with one or more microorganisms and a legume seed-derived powder.

[0157] In an embodiment, the seed coating is a seed coating as described above in any one of the embodiments, or an inoculum as described above in any one of the embodiments.

[0158] Preferably, the seed coating further comprises one or more sugars or sugar alcohols or a mixture thereof, one or more cellulose derivatives, and / or a clay. The one or more sugars or sugar alcohols are preferably chosen from maltose, trehalose, and sorbitol, or a combination thereof. The cellulose derivatives are preferably chosen from CMC, methylcellulose, or ethyl cellulose, or a combination thereof. The clay is preferably vermiculite.

[0159] In an embodiment, the seed coating comprises two compositions, one applied to the seed after the other as separate layers.

[0160] Preferably a first layer directly applied to the seed comprises a cellulose derivative as described above, while a second layer applied on top of said first layer comprises the microorganisms and the legume seed-derived powder, and optionally the clay.

[0161] It is thus clear that the second layer may be the inoculum as described above in any one of the embodiments.

[0162] Further optionally, the one or more sugars, sugar alcohols, or mixtures thereof are present in the composition either applied as first or as second layer, thus either in combination with the cellulose derivative or with the microorganisms and powder respectively.

[0163] In an embodiment, the coating and / or one or more of its layers are applied by spraying on said seed surface. In another embodiment, the coating and / or one or more of its layers are applied by dipping said seeds into a solution containing said first coating composition. In another embodiment, the coating and / or one or more of its layers are applied by roll coating said first coating composition onto said seeds. In another embodiment, the coating and / or one or more of its layers are applied by vacuum coating said first coating composition onto said seeds. In another embodiment, the coating and / or one or more of its layers are applied using a rotary seed coater, where the seeds undergo a specialized coating process facilitated by rotary motion for efficient and uniform coverage.

[0164] In an embodiment, wherein multiple layers are applied, the method further comprises the step of drying the seeds after applying a first layer and before applying a second layer. In another embodiment, the method does not comprise this active drying step.

[0165] In an aspect, the present invention relates to a coated seed, coated with a seed coating comprising a legume seed-derived powder and one or more microorganisms.

[0166] Said legume seed-derived powder is preferably as described above in any one of the embodiments, and said microorganisms may be any type of microorganism, such as any of the ones described above, preferably said microorganisms are nitrogen-fixing microorganisms.

[0167] Seed coated with such seed coating have an extended shelf life compared to uncoated seeds and compared to seeds coated without said legume seed-derived powder, as the microorganisms have an increased survivability on the seed surface. The alleviates the requirement of coating seeds with microorganisms at the time of sowing the seeds, thereby saving valuable time for farmers.

[0168] In an embodiment, the seed coating further comprises at least one component chosen from the list of one or more sugars or sugar alcohols or a mixture thereof, one or more cellulose derivatives, and a clay. The one or more sugars or sugar alcohols are preferably chosen from maltose, trehalose, and sorbitol, or a combination thereof. The cellulose derivatives are preferably chosen from CMC, methylcellulose, or ethyl cellulose, or a combination thereof. The clay is preferably vermiculite.

[0169] In an embodiment, the seeds are coated with the seed coating as described above in any one of the embodiments.

[0170] In another or further embodiment, the seeds are coated according to the method as described above in any one of the embodiments. 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.

[0171] 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 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.

[0172] 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.

[0173] The present invention also relates to a plant grown from a seed coated with the seed coating as described in any of the embodiments, and / or grown from a seed coated using the method as described in any of the embodiments.

[0174] It is further clear that, in an aspect, the present method thus also relates to a method for enhancing the survivability of microorganisms in an inoculum. Said method comprises providing a legume seed-derived powder to said inoculum.

[0175] It is further clear that, in an aspect, the present method thus also relates to a method for enhancing the survivability of microorganisms coated on a seed surface. Said method comprises providing a legume seed-derived powder prior to, simultaneously with and / or after applying said microorganisms to said seed surface.

[0176] Said legume seed-derived powder is preferably as described above in any one of the embodiments, and said microorganisms may be any type of microorganism, such as any of the ones described above, preferably said microorganisms are nitrogen-fixing microorganisms. Said legume seed-derived powder and said microorganisms may be delivered to the seed surface as part of a seed coating as described above in any one of the embodiments, and / or by a method as described above in any one of the embodiments.

[0177] 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.

[0178] The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended to, nor should they be interpreted to, limit the scope of the invention.

[0179] EXAM PLES AN D DESCRIPTION OF FIGURES

[0180] 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.

[0181] Example 1: laboratory seed coating process

[0182] Introduction:

[0183] A novel seed coating comprising soy seed-derived powder 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.

[0184] Methodology (general) - specifics / variations see below per experiment:

[0185] 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. A first coating composition comprising trehalose and carboxymethylcellulose (CMC) was prepared. For concentrations, see further.

[0186] A second coating composition comprising bacterial culture (5ml concentrate) and clay and / or soy powder (ground and sieved - 400 microns; the powder subsequently autoclaved for 15 to 21 minutes at 121 °C) was prepared. For concentrations, see further.

[0187] For each treatment, 100g of soy seeds were coated as follows:

[0188] 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.

[0189] 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.

[0190] For the present experiment, the following combinations were tested: see Table 1. Concentration of trehalose and CMC are expressed based on the volume of the first coating composition.

[0191] Table 1 Results of bacterial titers and survival rate over time

[0192] Results are shown in Figures 1 and 2, and present the survival rate of the bacterial strain on soy seeds coated with said strain over about 60 days under different coating conditions.

[0193] Figure 1 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.

[0194] Contrary to the general methodology described above, in this experiment the bacterial strain was only grown for 5 days instead of 6 days.

[0195] Both coatings (Condition 1 and 2) involves seeds coated with (per 100 g of seeds):

[0196] 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.

[0197] In addition, the second coating composition of Condition 2 further comprises 0.5 g of autoclaved ground soy seed powder per 100 g of seeds.

[0198] 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.

[0199] The results indicate an overall increased survival with soy powder at all time points, as shown in Figure 1.

[0200] Figure 2 illustrates the comparative survival rates of bacterial strains in a seed coating comprising either soy powder (Condition 4) or vermiculite (Condition 3) until 60 days.

[0201] Condition 3 involves seeds coated with (per 100 g of seeds):

[0202] 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.

[0203] Condition 4 involves seeds coated with (per 100 g of seeds):

[0204] 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. 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.

[0205] The data clearly demonstrates that soy powder outperforms vermiculite at all evaluated time points.

[0206] Figure 3 presents the survival rates of the bacterial strain in a seed coating over 60 days under different conditions, in which soy powder is added which is either autoclaved or not.

[0207] Conditions 5, 6 and 7 involve seeds coated with (per 100 g of seeds):

[0208] 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.

[0209] In addition, the second coating composition of Condition 6 and 7 further comprises 0.5 g of ground soy seed powder per 100 g of seeds. The powder used in Condition 7 was not autoclaved, contrary to the general method described above.

[0210] 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.

[0211] The results show a decrease in survival rate of the bacterial strain when the soy powder was not autoclaved, as shown in Figure 3, indicating non-autoclaved powder comprises a contaminant affecting the survival rate of the strain that is eliminated or reduced when the powder is autoclaved.

[0212] These findings show how autoclaved soy seed-derived powder plays an important role in enhancing the survival of Bradyrhizobium japonicum on soy seeds. This not only expands our understanding of effective seed coating strategies but also presents opportunities for further optimization, ultimately contributing to advancements in agricultural practices and outcomes.

[0213] Example 2: autoclaving the seed-derived powder

[0214] Method:

[0215] 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. A first coating composition comprising 25 w / v% trehalose and 5 w / v% CMC was prepared.

[0216] A second coating composition comprising bacterial culture (5ml concentrate) and autoclaved soy powder.

[0217] For a first experiment the soy powder was autoclaved at 121°C for different time periods: either 5 minutes, 10 minutes, 15 minutes, or 21 minutes. The control soy powder was not autoclaved (0 minutes).

[0218] Example 2B

[0219] For a second experiment the soy powder was autoclaved for 21 minutes at different temperatures: either 115 °C, 121 °C, or 132 °C. The control soy powder was not autoclaved (0 minutes).

[0220] 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.

[0221] 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.

[0222] Results:

[0223] As shown in Figure 4A, autoclaving soy powder at a temperature of 121 °C for a period ranging from 5 to 21 mins significantly increased bacterial survival on the seeds coated with the seed coating formulation.

[0224] As shown in Figure 4B, autoclaving soy powder for 21 min at temperatures between 115 and 132 °C increased the bacterial survival on the seeds coated with the seed coating formulation.

[0225] Example 3: the use of different legume seed powders

[0226] 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. A first coating composition comprising 25 w / v% trehalose and 5 w / v% CMC was prepared.

[0227] A second coating composition comprising bacterial culture (5ml concentrate) and autoclaved legume powder (autoclaved at 121°C for 21 min), chosen from Soy Glycine max'), cowpea (Vigna unguiculata), chickpea (Cicer arietinum), common vetch (Vicia sativa), lupin (Lupinus spp.), lima bean (Phaseolus lunatus), adzuki bean (Vigna angularis), grass pea (Lathyrus sativus) , alfalfa (Medicago sativa), yam bean (Pachyrhizus spp.), bean prelude (Phaseolus vulgaris , mung bean (Vigna radiata), fava bean (Vicia faba). As a control, vermiculite was added as carrier to the coating formulation instead of legume powder 'coating vermiculite'.

[0228] 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.

[0229] 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.

[0230] Results:

[0231] As shown in Figure 5, aside from soy powder, also other autoclaved legume powders increase the survival of microorganisms on seeds.

[0232] 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. Use of legume seed-derived powder for increasing the survivability of microorganisms in an inoculum.

2. The use according to claim 1, wherein the legume seed-derived 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, wherein said legume seeds are preferably whole legume seeds.

3. The use according to claim 2, wherein said autoclaving is performed at a temperature of between 100 °C and 145 °C for a duration of at least 3 minutes.

4. The use according to any one of the preceding claims, wherein said legume seed-derived powder is whole legume seed-derived powder.

5. The use according to any of the preceding claims, wherein at least 95 % of the legume seed-derived powder has a particle size of maximally 1.0 mm.

6. The use according to any of the preceding claims, wherein the inoculum comprising the legume seed-derived powder is coated directly onto a seed surface or used to prepare a seed coating to be coated on a seed surface.

7. The use according to claim 6, wherein said legume seed-derived powder is incorporated in the inoculum in an amount ranging from 10 m / v% to 35 m / v%.

8. The use according to any of the preceding claims 6 to 7, wherein the inoculum further comprises a clay, such as vermiculite.

9. The use according to any one of the preceding claims 5 to 7, wherein one or more sugars or sugar alcohols or a mixture thereof are added to the seed surface, optionally as part of said seed coating.

10. The use according to claim 9, wherein said one or more sugars or sugar alcohols are chosen from trehalose, maltose and sorbitol.

11. The use according to any one of the preceding claims 6 to 10, wherein cellulose derivatives are added to the seed surface, optionally as part of said seed coating.

12. The use according to claim 11, wherein said one or more cellulose derivatives are 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.

13. The use according to any one of the preceding claims, wherein the microorganisms are embedded in and / or are adsorbed to the surface of the legume seed-derived powder.

14. The use according to any of the preceding claims, wherein the microorganisms in the inoculum are selected from the group consisting of bacterial strains, fungal strains, or a combination thereof.

15. The use according to claim 14, wherein said bacterial strains are chosen from strains of genera Rhizobium, Sinorhizobium, Bacillus, Pseudomonas, Azospirillum, Azotobacter, Clostridium, Klebsiella, Stenotrophomonas, Halomonas, Pianococcus, Agrobacterium, Enterobacter, Serratia, Paenibacillus, Streptomyces, Lactobacillus, Corynebacterium, Micrococcus, Achromobacter, Burkholderia, Ralstonia, Xanthomonas, Acinetobacter, Bradyrhizobium, Frankia, Mezorhizobium, Ensifer, Herbaspirillum, and Brevibacillus, preferably said microorganisms are nitrogen-fixing bacterial strains, preferably of the genus Bradyrhizobium, Ensifer, or Rhizobium.

16. The use according to claim 14, wherein said fungal strains are chosen from the group of Mycorrhizae and the genera Trichoderma, Beauveria, Rhizophagus, Piriformospora, Laccaria, Tuber, Pisolithus, Russula, Lepista, Hebeloma, Cenococcum, Amanita, Suillus, Rhizopogon, Penicillium, Fusarium, Cladosporium, Aspergillus, Verticillium, Metarhizium, Gliocladium,Paecilomyces, Acremonium, and Talaromyces, preferably said microorganisms are Mycorrhizae.

17. A seed coating, comprising a legume seed-derived powder and optionally one or more microorganisms, preferably nitrogen-fixing microorganisms.

18. The seed coating according to claim 17, wherein said legume seed-derived powder has been autoclaved, preferably at a temperature of between 100 °C and 145 °C for a duration of at least 3 minutes.

19. The seed coating according to claim 17 or 18, further comprising at least one component chosen from the list of one or more sugars or sugar alcohols or a mixture thereof, one or more cellulose derivatives, and a clay such as vermiculite.

20. The seed coating according to any one of the preceding claims 17 to 19,- between 10 m / v% and 35 m / v% of legume seed-derived powder,- one or more rhizosphere microbial strains, preferably of the genus Bradyrhizobium, Ensifer, or Rhizobium, or a combination thereof,- 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, and- optionally, one or more clay and / or milk-derived powder.

21. A method for coating a seed, comprising applying one or more coating layers to said seed, wherein said seed is coated with one or more microorganisms and a legume seed-derived powder.

22. The method for coating a seed, wherein said seed is coated with the seed coating of any one of claims 17 to 20.

23. A seed, coated with the seed coating according to any one of claims 17 to 20, and / or coated according to the method of claim 21 or 22.

24. A method for enhancing the survivability of microorganisms coated on a seed surface, said method comprises providing a legume seed-derived powder prior to, simultaneously with and / or after applying said microorganisms to said seed surface.

25. The method according to claim 24, wherein said legume seed-derived powder has been autoclaved, preferably at a temperature of between 100 °C and 145 °C for a duration of at least 3 minutes.

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