Method for coating seeds with microorganisms

Abstract

The present invention provides a method for coating seeds using a seed coating composition containing microorganisms useful for the healthy growth of plants, thereby increasing the survival rate of microorganisms inoculated onto the seeds and improving the efficiency of microorganism establishment on the plant body grown from the seeds. [Solution] The present invention provides a method of coating a seed surface by spraying a coating solution containing encapsulated microorganisms in a coating solution containing a microbial protective agent such as glycerol, using an electrospinning device.

Description

[Technical Field]

[0001] The present invention relates to a method for coating plant seeds. More specifically, the present invention relates to a method for coating plant seeds with a plant seed coating agent containing microorganisms. [Background technology]

[0002] To effectively cultivate agricultural plants, research and development is being conducted on agricultural microbial materials that utilize specific microorganisms that can influence plants through some form of interaction, such as symbiotic relationships. Currently, at least 140 types of agricultural microbial materials are distributed domestically, mainly as soil conditioners and seed coating agents.

[0003] In agricultural microbial materials, in addition to mycorrhizal fungi and rhizobia, whose mechanisms of action have been well studied, there is a growing expectation for the use of various microorganisms, such as Plant Growth Promoting Rhizobacteria (PGPR), Plant Growth Promoting Fungi (PGPF), or antagonistic microorganisms, which are expected to have loose symbiotic relationships with plants or specific interactions with other microorganisms.

[0004] Seed coating agents are agricultural materials that are expected to contribute to seed storage and, after sowing seeds in soil, promote germination, plant growth, and increase yield by coating seeds with a coating agent containing components useful for seed protection and plant growth. Seed coating agents that contain microorganisms as one of their useful components are expected to be useful as agricultural microbial materials. Many seed coating agents consist of a base material for coating seeds, such as polymers like cellulose, chitosan, gum arabic, alginic acid, starch, polyethylene glycol, polyvinyl alcohol, or polyvinyl acetate; fillers (bulkers) such as bentonite clay, calcium carbonate, talc, or diatomaceous earth; microbial protective agents; nutritional components; microorganisms; and other additives (Non-patent Literature 1: Pedrini et al., Trends Plant Sci. 2017 Feb;22(2):106-116). Examples of microorganisms used in seed coatings include spores of antibiotic-producing fungi (Patent Document 1: Japanese Patent Publication No. 2022-046489), bacteria with plant disease control properties (Patent Document 2: Japanese Patent Publication No. 2015-093595), and rhizobia (Patent Document 3: Japanese Patent Publication No. 11-004606).

[0005] The main methods for coating seed surfaces with seed coating agents containing these known microorganisms involve either spraying a solution containing suspended microbial cells directly onto the seed surface or immersing the seeds in the solution. However, recently, a case using a method called electrospinning has been reported (see below).

[0006] Electrospinning is a technology developed in the 1930s that generates fine fibers or capsules by applying a high voltage while spraying a polymer solution through a nozzle. While electrospinning has been used in textile applications such as filters and nonwoven fabrics, and in industrial products such as batteries and separators, recent research and development is progressing in biomedical fields such as cell sheets and artificial blood vessels, as well as in cosmetics, food, and pharmaceutical formulations with enhanced sustained-release effects, stability, and solubility. An example of electrospinning application using live microorganisms in the food industry is the technology for encapsulating lactic acid bacteria (Non-patent document 2: Gomez-Mascaraque et al., LWT-Food Sci. Technol. 2016;69:438-446). Furthermore, in the field of microbial materials for agriculture, a technique has already been reported in which rhizobia and plant growth-promoting bacteria are incorporated into polyvinyl alcohol microfibers using the electrospinning method to coat seeds (Non-patent Literature 3: De Gregorio et al., PLoS One. 2017 May 4;12(5):e0176930; Non-patent Literature 4: Damasceno et al., Can J Microbiol. 2013 Nov;59(11):716-9). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2022-046489 [Patent Document 2] Restatement No. 2015-093595 [Patent Document 3] Japanese Patent Application Publication No. 11-004606 [Non-patent literature]

[0008] [Non-Patent Document 1] Pedrini et al., Trends Plant Sci. 2017 Feb;22(2):106-116 [Non-Patent Document 2] Gomez-Mascaraque et al., LWT-Food Sci. Technol. 2016;69:438-446 [Non-Patent Document 3] :De Gregorio et al., PLoS One. 2017 May 4;12(5):e0176930 [Non-Patent Document 4] Damasceno et al., Can J Microbiol. 2013 Nov;59(11):716-9 [Overview of the project] [Problems that the invention aims to solve]

[0009] However, conventional seed coating agents containing microorganisms did not adequately achieve sufficient survival rates for the microorganisms inoculated onto the seeds, nor did they ensure efficient establishment of the microorganisms on the plants that grew from the seeds. The present invention provides a method for coating seeds using a seed coating composition containing microorganisms useful for the healthy growth of plants, with the aim of increasing the survival rate of microorganisms inoculated onto the seeds and improving the efficiency of microorganism establishment on the plant body grown from the seeds. [Means for solving the problem]

[0010] As a result of diligent research to solve the above problems, the inventors of the present invention have found that by using an electrospinning device to spray a coating solution containing a microbial protective agent such as glycerol onto the surface of a seed, thereby coating the seed, the survival rate of the microorganisms inoculated onto the seed and the efficiency of their establishment on the plant body grown from the seed can be significantly increased. Furthermore, they have found a coating solution composition that can further increase the survival rate and establishment efficiency of the microorganisms, thus completing the present invention.

[0011] In other words, the present invention relates to, but is not limited to, the following. [1] A method for covering plant reproductive bodies, (1) Providing a first coating solution comprising a microbial cell, a polymer, a nonionic surfactant, and a microbial protectant; and (2) Spraying the first coating solution filled in the first syringe part of an electrospinning device onto a propagule through a first nozzle connected to the first syringe part to form microcapsules in which microbial cells are embedded, thereby coating the surface of the plant propagule The method comprising the above. [2] The polymer is whey protein or whey protein and xanthan, the nonionic surfactant is one or more nonionic surfactants selected from the group consisting of polyoxyethylene (20) sorbitan monolaurate, 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, octylphenoxypoly(oxyethylene)ethanol, octylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, and nonylphenyl polyethylene glycol, and the microbial protectant is one or more microbial protectants selected from the group consisting of glycerol, trehalose, dimethyl sulfoxide, and sucrose. The method according to [1]. [3] The method according to [1] or [2], comprising (3) Spraying a second coating solution containing zein and a nonionic surfactant filled in the second syringe part of the electrospinning device onto the propagule through a second nozzle connected to the second syringe part to form fine fibers The method further comprising the above. [4] The method according to [3], wherein steps (2) and (3) are each repeated alternately two or more times. [5] The method according to any one of [1] to [4], wherein the first coating solution further contains a nutrient component. [6] The method according to [5], wherein the first coating solution contains microbial cells, whey protein concentrate, xanthan, polyoxyethylene (20) sorbitan monolaurate, glycerol, and ammonium molybdate. [7] The method according to any one of [3] to [6], wherein the second coating solution contains an ethanol solution of zein and polyoxyethylene (20) sorbitan monolaurate. [8] The method according to any one of [1] to [7], wherein the propagule is 0.1 cm or more in diameter. [9] The method according to [8], wherein the propagule is a seed, a bulb, or a tuber.

[10] The method according to [9], wherein the seeds are selected from soybean, rice, wheat, and corn seeds, the bulbs are selected from garlic, onion, lily, tulip, gladiolus, and crocus bulbs, and the tubers are selected from potato, taro, ginger, and konjac tubers.

[11] The method according to any one of [1] to

[10] , wherein the microorganism is selected from rhizobia and other Gram-negative bacteria, Gram-positive bacteria, archaea, mycorrhizal fungi and other fungi, and combinations thereof.

[12] A method for producing a coated propagule of a plant, comprising the method according to any one of [1] to

[11] .

[13] A composition for coating a propagule of a plant, the composition comprising microbial cells, a polymer, a nonionic surfactant, and a microbial protectant.

[14] The composition according to

[13] , comprising microbial cells, whey protein, xanthan, polyoxyethylene (20) sorbitan monolaurate, glycerol, and ammonium molybdate.

[15] The composition according to

[13] or

[14] , wherein the microorganism is selected from rhizobia and other gram-negative bacteria, gram-positive bacteria, archaea, mycorrhizal fungi and other fungi, and combinations thereof.

[16] A plant reproductive body coated with any one of the compositions described in

[13] to

[15] .

[17] The reproductive organism according to claim

[16] , which is a seed, bulb, or seed potato. [Effects of the Invention]

[0012] By using the seed coating method and coating composition of the present invention, the survival rate of microorganisms useful for the healthy growth of plants can be increased on coated seeds, and the efficiency of microbial colonization in plants grown from coated seeds can be increased. The present invention enhances the stability of microorganisms against environmental stress on plant reproductive bodies, improves storage period and microbial inoculation efficiency, and thereby enhances the effectiveness of microbial materials. [Brief explanation of the drawing]

[0013] [Figure 1] Figure 1A shows a scanning electron microscope image of the surface of a microcapsule produced by the method of the present invention. Figure 1B shows a transmission electron microscope image showing the inoculated microorganisms contained within the microcapsule produced by the method of the present invention. Figure 1C shows a scanning electron microscope image of the surface of a microcapsule and a microfiber produced by multiple spraying of a first coating solution and a second coating solution by the method of the present invention. [Figure 2]Figure 2A shows the changes in survival rate over the seed storage period for each coating method on soybean seeds coated with a coating solution containing the rhizobia SG09-DsRed strain. Figure 2B shows the microbial survival rate after 60 days of seed storage for each coating method, with the number of viable microorganisms inoculated on the day of production set to 100%. C1: Microbial solution coating method (microbial solution is applied while stirring the seeds); C2: Microcapsules (17% WPC + 5% Tween20 + 10% Fibersol (composition described in Non-Patent Literature 2)); C3: Microfibers (13% PVA + 10% Glycerol (composition described in Non-Patent Literature 3 and 4)); C4: Microcapsules (17% WPC + 5% Tween20); P1: Microcapsules (17% WPC + 5% Tween20 + 0.1% Ammonium Molybdate + 10% Glycerol + 0.1% Xanthan Tane); P2: Multiple coating of microcapsules (same as P1) and microfibers (20% Zein + 5% Tween20). [Figure 3] Figure 3 shows the survival rate of microorganisms on soybean seeds coated by the control (microbial solution coating method and microcapsules according to the method of Reference 2) and the method of the present invention, with the number of viable microorganisms inoculated on the coated seeds on the day of production set to 100%, and the difference in the composition of the coating solution, for 30 days of seed storage. C1: Micromicrobial solution coating method (microorganism solution applied while stirring the seeds); C4: Microcapsules (17% WPC + 5% Tween20); C5: Microcapsules (17% WPC + 5% Tween20 + 0.1% ammonium molybdate); C6: Microcapsules (17% WPC + 5% Tween20 + 10% glycerol); C7: Microcapsules (17% WPC + 5% Tween20 + 0.1% xanthan gum). [Figure 4] Figure 4A shows scanning electron microscope images of the surface of soybean seeds treated with the control method (conventional method, bacterial solution coating method), soybean seeds coated with microcapsules according to the method of the present invention, and soybean seeds coated with microcapsules and microfibers according to the method of the present invention. Figure 4B shows the evaluation of plant growth and microbial colonization in soybean plants after 3 weeks of cultivation. The legend is the same as in Figure 1. [Figure 5] Figure 5 shows the germination rate (Figure 5A) 4 days after sowing, the fresh weight of the above-ground parts (Figure 5B) 3 weeks after cultivation, the number of root nodules attached to the roots of a single plant (Figure 5C), and the weight of the root nodules (Figure 5D) of soybean seeds coated with the rhizobia SG09-DsRed strain using various methods. The legend is the same as in Figure 1. [Figure 6] Figure 6 shows the initial microbial inoculation per seed (Figure 6A, B) and the proportion of root nodules formed by the inoculated rhizobia rather than native rhizobia present in the soil, out of the total number of root nodules attached to the roots of a single plant (Figure 6C, D). [Figure 7] Figure 7 shows scanning electron microscope images of the colonization of inoculated microorganisms on the surface layer of the soybean root base after 7 days of cultivation, following sowing on sterile agar 35 days after seed storage, and comparing untreated seeds with soybean seeds coated with the rhizobia SG09-DsRed strain using various methods. [Figure 8] Figures 8A and 8B show scanning electron microscope images illustrating the effect of adding the thickening polysaccharide xanthan gum to the coating solution to increase the size of the microcapsules (Figure 8A: no xanthan gum added, Figure 8B: 0.1% xanthan gum added). Figure 8C shows a scanning electron microscope image of microcapsule C6 (WPC + Tween20 + glycerol) from Figure 3. [Figure 9] Figures 9A-D are scanning electron microscope images showing the effect of different types and concentrations of thickening polysaccharides on the manufacturing stability of microcapsules. [Figure 10] Figure 10A shows the difference in the survival rate of the rhizobia SG09-DsRed strain under seed storage conditions, depending on the presence or absence of xanthan gum in the microcapsule components. Figure 10B shows the difference in the nodule occupancy rate, an indicator of inoculated microbial colonization in soybeans 30 days after cultivation, depending on the presence or absence of xanthan gum in the microcapsule components. [Figure 11]Figure 11 shows scanning electron microscope images (Figures 11A and 11B) of the surface of rice seeds sprayed with microcapsules or microfibers containing the plant growth-promoting bacterium PsJN strain, and the initial microbial inoculation amount per seed (Figure 11C). [Figure 12] Figure 12 shows scanning electron microscope images of the surface of rice seeds sprayed with microcapsules containing the bacterium UWC1-166 strain (Figure 12A) and the initial microbial inoculation amount per seed (Figure 12B). [Modes for carrying out the invention]

[0014] <Plant reproductive organs> The methods and compositions of the present invention can, in principle, be applied not only to seeds but to the reproductive bodies of all plants by using an electrospinning apparatus. Considering the properties of the electrospinning method, the size of the plant reproductive bodies used for the purposes of the present invention is preferably 0.1 cm or larger in diameter, more preferably 0.3 cm or larger, and even more preferably 0.5 cm or larger.

[0015] In this specification, examples of plant reproductive bodies include, but are not limited to, seeds, bulbs, cuttings, and seed potatoes, and also include other vegetative reproductive bodies such as clonal reproductive bodies. Seeds are organs formed by seed-reproducing plants (gymnosperms and angiosperms) as a result of sexual reproduction. Bulbs are organs formed by the enlargement of mainly underground parts (roots, stems, and leaves) of perennial plants to store nutrients, and in this specification, refer to anything other than seed potatoes. Cuttings are parts of a plant body such as leaves, stems, and roots of a plant intended for propagation, used to germinate adventitious roots and buds and to propagate an independent individual vegetatively. In this specification, this includes rootstock and scion in grafting, but refers to anything other than seed potatoes. Seed potatoes are tubers or rhizomes used for the vegetative propagation of tubers. Specifically, plant propagates that can be used for the purposes of the present invention include crops including legumes, vegetables, grains, and potatoes; trees including ornamental trees, garden trees, and trees for hedges; and seeds, bulbs, and seed tubers of flowering plants. More specifically, plant propagates that can be used for the purposes of the present invention include, but are not limited to, seeds selected from soybean, rice, wheat, and maize seeds; bulbs selected from garlic, onion, lily, tulip, gladiolus, and crocus bulbs; and seed tubers selected from potato, taro, Japanese yam, and konjac seed tubers. <Microorganisms, microbial cells, microbial cells useful for plants> The method and composition of the present invention can coat the surface of a plant's reproductive body with a seed coating solution containing microorganisms that have beneficial effects on the healthy growth of plants.

[0016] Generally, microorganisms refer to organisms too small to be observed with the naked eye, and include bacteria, fungi, microalgae, and protozoa. In this specification, when referring to microorganisms, it means microorganisms that are useful to plants. In this specification, microorganisms useful to plants refer to microorganisms that exhibit agriculturally beneficial effects on plants, and include, for example, microorganisms that have the function of maintaining or promoting plant growth, the function of controlling harmful organisms that inhibit plant growth, and / or the function of improving the composition of the medium in which plants grow, such as soil. Typically, microorganisms useful to plants that can be used in the method or composition of the present invention have one or more of the above functions and are generally called symbiotic microorganisms, plant growth promoting microorganisms (PGPM), or plant disease control microorganisms. Since the size of the capsules produced by the method or composition of the present invention is less than 20 μm in diameter at most, the microbial cells used in the present invention are preferably 10 μm in diameter or less, and more preferably 5 μm in diameter or less.

[0017] Examples of microorganisms that have the function of maintaining or promoting plant growth include microorganisms that have the ability to fix nitrogen, produce plant growth-promoting hormones, inhibit the synthesis of plant growth inhibitory factors, and / or promote plant phosphate absorption, and include, but are not limited to, rhizobia, including bacteria of the genera Rhizobium, Bradyrhizobium, and Azorhizobium; bacteria of the genera Pseudomonas; bacteria of the genera Bacillus; and arbuscular mycorrhizal fungi.

[0018] Examples of microorganisms that have the function of controlling harmful organisms that inhibit plant growth include, but are not limited to, microorganisms that exhibit antagonistic effects against harmful organisms such as bacteria, fungi, nematodes, or insects that damage plants; such as actinomycetes, including bacteria of the genera Penicillium, Trichoderma, Pseudomonas, Bacillus, and Streptomyces.

[0019] Examples of microorganisms that have the function of improving the composition of the medium in which plants grow, such as soil, include, but are not limited to, microorganisms that have the ability to improve the nutritional balance of soil, the ability to prevent continuous cropping problems, and / or the ability to improve salinity, such as bacteria of the genera Pseudomonas, Bacillus, Lactobacillus, and yeast.

[0020] In this specification, a microbial cell refers to the cell of a single-celled organism such as a bacterium, yeast, or microalgae, or to its spore, hyphae, or fragments of hyphae in the case of a multicellular organism such as a fungus, which is capable of reproduction. <Method for covering plant reproductive structures> One aspect of the present invention relates to a method for covering plant reproductive bodies. Specifically, a method for covering plant reproductive bodies, (1) A step of providing a first coating solution comprising microbial cells, a polymer, a nonionic surfactant, and a microbial protective agent; and (2) A step of coating the surface of a plant reproductive organ by spraying the first coating solution, which is filled in the first syringe of the electrospinning apparatus, onto the reproductive organ through the first nozzle connected to the first syringe, thereby forming microcapsules in which microbial cells are embedded. The method includes the foregoing.

[0021] The method of the present invention uses an electrospinning apparatus. The electrospinning apparatus used in the present invention comprises a syringe section for filling with the coating solution or composition of the present invention, a nozzle section connected to the syringe section for spraying the coating solution or composition of the present invention, a collector section for placing a plant reproductive body and coating the surface of the plant reproductive body with the sprayed coating solution or composition of the present invention, and an application section for applying a high voltage when spraying the coating solution or composition of the present invention. The size and material of each section may be as described, and additional components may be included. The electrospinning apparatus used in the present invention comprises one or more syringe sections and nozzle sections. For example, if the electrospinning apparatus used in the present invention comprises two syringe sections and two nozzle sections, these may be referred to as the first syringe section and the first nozzle section, and the second syringe section and the second nozzle section, respectively. The application section in the electrospinning apparatus used in the present invention is configured to apply a high voltage to the nozzle section and / or the collector section. Any electrospinning apparatus can be used in this invention as long as it has the above configuration and can achieve the effects of the present invention. For example, a commercially available apparatus such as the Fluidnatek LE-50 manufactured by Bioinicia may be used.

[0022] The method of the present invention includes the steps of providing a first coating solution and spraying the first coating solution, which is filled in a first syringe of an electrospinning apparatus, onto a plant reproductive body through a first nozzle connected to the first syringe, thereby coating the surface of the plant reproductive body by forming microcapsules in which microbial cells are embedded.

[0023] The first coating solution comprises (1) microbial cells, (2) polymer, (3) nonionic surfactant, and (4) microbial protective agent, and optionally (5) nutrients and / or (6) other additives. (1) Microbial cells The number of microbial cells contained in the first coating solution can be appropriately set depending on the purpose. For example, the number of cells in 1 mL of the first coating solution is 10 8 Less than a cell, 10 7 Less than a cell, 10 6 Less than a cell, 10 5 Less than a cell, or 10 4 The number of cells can be set to less than one cell. The number of microbial cells per colony can be appropriately adjusted by those skilled in the art depending on the size, shape, and coverage of the colony. (2) Polymer The polymer contained in the first coating solution may be any polymer compound that can function as a substrate capable of forming microcapsules and / or microfibers when the composition is sprayed onto the surface of a plant reproductive body using an electrospinning method. Examples of such polymers include, but are not limited to, one or more polymers selected from the group consisting of cellulose, chitosan, gum arabic, alginic acid, starch, polyethylene glycol, polyvinyl alcohol, polyvinyl acetate, whey protein, and xanthan gum. Whey protein, or whey protein and xanthan gum, are preferred polymers for use in the composition of the present invention.

[0024] When whey protein is used as the polymer, it may be included in the composition in the form of a whey protein concentrate. The whey protein concentrate is an aqueous solution containing whey-derived protein and contains 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more by dry weight of protein. The whey-derived protein content in the whey protein concentrate can be measured using known methods such as the Kjeldahl method, the Dumas method, and modified or improved methods thereof. The whey protein concentrate used in the composition of the present invention may be produced from milk using known methods, or a commercially available whey protein concentrate may be used. The content of the whey protein concentrate contained in the first coating solution is preferably 5% to 30% by weight, more preferably 10% to 20% by weight, and even more preferably 17% by weight, when the whey protein concentrate contains 70% to 80% by weight of whey protein.

[0025] In this specification, xanthanate refers to a polymer consisting of repeating units of monomers comprising two molecules of glucose, two molecules of mannose, and glucuronic acid, and a salt thereof. When xanthanate is used in the first coating solution, the content of xanthanate in the first coating solution is preferably 0.01% to 1% by weight, more preferably 0.05% to 0.5% by weight, and even more preferably 0.1% by weight. (3) Nonionic surfactants The nonionic surfactant used in the first coating solution may be any surfactant that has the function of helping to form microcapsules and / or microfibers based on the polymer when the composition is sprayed onto the surface of a plant reproductive body using the electrospinning method. Examples of preferred nonionic surfactants include, but are not limited to, polyoxyethylene (20) sorbitan monolaurate (Tween20), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (TritonX-100), octylphenoxy(polyoxyethylene)ethanol, octylphenoxypolyethoxyethanol (Nonidet), and nonylphenyl polyethylene glycol. When polyoxyethylene (20) sorbitan monolaurate is used as the nonionic surfactant, the content of polyoxyethylene (20) sorbitan monolaurate in the first coating solution is preferably 1% to 10% by weight, more preferably 3% to 8% by weight, and even more preferably 5% by weight. (4) Microbial protective agents The first coating solution can increase the survival rate of microorganisms inoculated onto the surface of plant reproductive bodies by containing a microbial protective agent. Examples of microbial protective agents include, but are not limited to, one or more microbial protective agents selected from the group consisting of glycerol, trehalose, dimethyl sulfoxide (DMSO), and sucrose. A preferred microbial protective agent is glycerol. When the first coating solution contains glycerol, the glycerol content in the first coating solution is preferably 1% to 20% by weight, more preferably 5% to 15% by weight, and even more preferably 10% by weight. (5) Nutritional components The first coating solution may optionally contain nutrients in addition to the components (1) to (4) described above. Nutrients refer to components that can serve as nutrients for microorganisms after the first coating solution has been sprayed onto the surface of the plant's reproductive body. Examples of nutrients that may be included in the first coating solution include, but are not limited to, ammonium molybdate. When ammonium molybdate is used as a nutrient, the content of ammonium molybdate in the first coating solution is preferably 0.01% to 1% by weight, more preferably 0.05% to 0.5% by weight, and even more preferably 0.1% by weight. (6) Other additives The first coating solution may optionally contain other additives in addition to the components (1) to (4) or (1) to (5) described above. Other additives include, but are not limited to, fillers such as bentonite clay, calcium carbonate, talc, or diatomaceous earth.

[0026] Preferably, the first coating solution comprises microorganisms, whey protein concentrate, xanthan gum, glycerol, polyoxyethylene (20) sorbitan monolaurate, and ammonium molybdate.

[0027] In the method of the present invention, conditions such as the spraying time of the first coating solution onto the plant reproductive body and the voltage applied during spraying can be appropriately set considering the size of the plant reproductive body and other factors.

[0028] The method of the present invention may further include, in addition to steps (1) and (2) above, a step (3) in which a second coating solution containing zein and a nonionic surfactant, which is filled in the second syringe of an electrospinning apparatus, is sprayed onto the plant growth through a second nozzle connected to the second syringe to form fine fibers. Step (3) allows for the formation and coating of additional fine fibers on the surface of the plant growth. If necessary, the method of the present invention may involve repeating steps (2) and (3) alternately two or more times.

[0029] The second coating solution contains zein. Zein is one of the major proteins in corn, making up about 5% of the corn, and can be extracted with alcohol. Zein is a type of prolamin, a group of proteins that are insoluble in water but soluble in 50-90% ethanol. It is rich in proline and glutamic acid, but lacks the essential amino acids tryptophan and lysine. The zein used in this invention may be extracted from corn using known methods, or it may be commercially available as a biochemical reagent, for example from Fujifilm Wako Pure Chemical Industries, Ltd. Compared to the fine fibers of polyvinyl alcohol (PVA) or polycaprolactone (PCL), the fine fibers of zein are poorly soluble in water. Therefore, by layer-coating plant reproductive bodies with these and easily soluble microcapsules, it is expected that the microbial capsules will be retained near the reproductive bodies for a long period of time, even in high-humidity environments such as plant cultivation environments.

[0030] The zein contained in the second coating solution is preferably 5% to 30% by weight, more preferably 10% to 25% by weight, and even more preferably 20% by weight, as a final concentration of zein dissolved in 82% ethanol.

[0031] The nonionic surfactant contained in the second coating solution is not particularly limited as long as the objectives of the present invention can be achieved, but is preferably polyoxyethylene (20) sorbitan monolaurate. The content of the nonionic surfactant contained in the second coating solution is preferably 1% to 10% by weight, more preferably 3% to 8% by weight, and even more preferably 5% by weight, when polyoxyethylene (20) sorbitan monolaurate is used.

[0032] In the method of the present invention, conditions such as the spraying time of the second coating solution onto the plant reproductive body and the voltage applied during spraying can be appropriately set considering the size of the plant reproductive body, etc. <Method for Producing Plant Propagules> The present invention also relates, in one aspect, to a method for producing coated plant propagules, which includes a method for coating the above-mentioned plant propagules. <Composition for Coating Plant Propagules> The present invention relates, in one aspect, to a composition for coating plant propagules, which comprises (1) microbial cells, (2) a polymer, (3) a nonionic surfactant, and (4) a microbial protectant, and optionally, (5) nutrient components and / or (6) other additives. The composition of the present invention can be suitably used for coating plant propagules using the electrospinning method as a coating solution. Specifically, the composition of the present invention can be used to form microcapsules and / or fine fibers containing microorganisms on the surface of plant propagules by spraying on the surface of the plant propagules using the above electrospinning apparatus, so as to coat the surface of the propagules. (1) Microbial cells The number of microbial cells contained in the composition of the present invention can be appropriately set according to the purpose. For example, the number of cells contained in 1 mL of the composition in solution form is less than 10 8 cells, less than 10 7 cells, less than 10 6 cells, less than 10 5 cells, or less than 10 4 cells, and can be set accordingly. The number of microbial cells per propagule can be appropriately adjusted by those skilled in the art according to the size and shape of the propagule and the coating amount. (2) Polymer The polymer contained in the composition of the present invention may be any polymer compound that can function as a substrate capable of forming microcapsules and / or microfibers when the composition is sprayed onto the surface of a plant reproductive body using an electrospinning method. Examples of such polymers include, but are not limited to, one or more polymers selected from the group consisting of cellulose, chitosan, gum arabic, alginic acid, starch, polyethylene glycol, polyvinyl alcohol, polyvinyl acetate, whey protein, and xanthan gum. Whey protein, or whey protein and xanthan gum, are preferred polymers for use in the composition of the present invention.

[0033] When whey protein is used as the polymer, it may be included in the composition in the form of a whey protein concentrate. The whey protein concentrate is an aqueous solution containing whey-derived protein and contains 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more by dry weight of protein. The whey-derived protein content in the whey protein concentrate can be measured using known methods such as the Kjeldahl method, the Dumas method, and modified or improved methods thereof. The whey protein concentrate used in the composition of the present invention may be produced from milk using known methods, or a commercially available whey protein concentrate may be used. The content of the whey protein concentrate included in the composition of the present invention is preferably 5% to 30% by weight, more preferably 10% to 20% by weight, and even more preferably 17% by weight, when the whey protein concentrate contains 70% to 80% by weight of whey protein.

[0034] In this specification, xanthan gum refers to a polymer consisting of repeating units of monomers comprising two molecules of glucose, two molecules of mannose, and glucuronic acid, and a salt thereof. When xanthan gum is used in the composition of the present invention, the content of xanthan gum in the composition is preferably 0.01% to 1% by weight, more preferably 0.05% to 0.5% by weight, and even more preferably 0.1% by weight. (3) Nonionic surfactants The nonionic surfactant used in the composition of the present invention may be any surfactant that has the function of assisting in the formation of microcapsules and / or microfibers based on the polymer when the composition is sprayed onto the surface of a plant reproductive body using the electrospinning method. Examples of preferred nonionic surfactants include, but are not limited to, polyoxyethylene (20) sorbitan monolaurate (Tween20), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (Triton X-100), octylphenoxy(polyoxyethylene)ethanol, octylphenoxypolyethoxyethanol (Nonidet), and nonylphenyl polyethylene glycol. When polyoxyethylene (20) sorbitan monolaurate is used as the surfactant, the content of polyoxyethylene (20) sorbitan monolaurate in the composition is preferably 1% to 10% by weight, more preferably 3% to 8% by weight, and even more preferably 5% by weight. (4) Microbial protective agents The compositions of the present invention, by containing a microbial protective agent, can increase the survival rate of microorganisms inoculated onto the surface of plant reproductive bodies. Examples of microbial protective agents include, but are not limited to, one or more microbial protective agents selected from the group consisting of glycerol, trehalose, dimethyl sulfoxide (DMSO), and sucrose. A preferred microbial protective agent is glycerol. When the composition of the present invention contains glycerol, the glycerol content in the composition is preferably 1% to 20% by weight, more preferably 5% to 15% by weight, and even more preferably 10% by weight. (5) Nutritional components In addition to the components (1) to (4) above, the composition of the present invention may optionally contain nutritional components. Nutritional components refer to components that can serve as nutrients for microorganisms after the composition has been sprayed onto the surface of a plant's reproductive body. Examples of nutritional components that may be included in the composition of the present invention include, but are not limited to, ammonium molybdate. When ammonium molybdate is used as a nutritional component, the content of ammonium molybdate in the composition is preferably 0.01% to 1% by weight, more preferably 0.05% to 0.5% by weight, and even more preferably 0.1% by weight. (6) Other additives The compositions of the present invention may optionally contain other additives in addition to the components (1) to (4) or (1) to (5) described above. Other additives include, but are not limited to, fillers such as bentonite clay, calcium carbonate, talc, or diatomaceous earth.

[0035] The composition of the present invention may be used in combination with another composition for coating plant reproductive bodies, which comprises zein and a nonionic surfactant. <Plant reproductive bodies coated with the composition> In one aspect, the present invention relates to a plant reproductive body coated with the above composition. The plant reproductive body is as described above. The plant reproductive body of the present invention can be produced using the above method of coating the plant reproductive body or the method of producing a coated plant reproductive body.

[0036] Plant reproductive bodies coated with the composition of the present invention exhibit high stability against environmental stress of microorganisms on the surface of the reproductive body, and have improved storage period and microbial inoculation efficiency, thus providing a high application effect as a microbial material.

[0037] The number of viable microbial cells on the surface of plant reproductive bodies can be measured using known methods such as colony counting. The plant reproductions of the present invention maintain their effect of improving the survival rate of inoculated microorganisms even after a storage period of 10 to 60 days, or 10 to 90 days.

[0038] The present invention will be described below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0039] Unless otherwise specified, the percentage of each component is given in weight percentage. Also, unless otherwise specified, A and B indicate values ​​greater than or equal to A and less than or equal to B, respectively (A and B are numerical values). (Example 1) Method for culturing bacterial strains and preparing concentrated bacterial cell solution Verification tests of the present invention were conducted using two strains of genetically modified rhizobia: Bradyrhizobium ottawaensis SG09-DsRed strain (hereinafter referred to as SG09-DsRed strain) and Bradyrhizobium diazoefficiens USDA110-DsRed strain (hereinafter referred to as USDA110-DsRed strain), both of which constitutively express the red fluorescent protein DsRed. These strains used in the tests were selectively and aerobically cultured with shaking at 30°C in HM liquid medium containing 100 ng / μl of the antibiotic spectinomycin. After 3 days of culture, the culture solution was centrifuged, and the supernatant was removed and washed twice with an equal volume of sterile water to prepare an aqueous solution (bacterial cell concentrate) containing the microbial cells. The number of viable cells in the bacterial cell concentrate was calculated by colony counting, and the bacterial cell concentration was approximately 10 10 The culture was diluted to a concentration of CFU / mL (CFU: colony-forming units). The prepared mycelial fluid was to be used for seed inoculation on the same day and was stored on ice until then. (Example 2) Method for coating plant reproductive bodies using an electrospinning device As the first coating solution, a coating solution containing 10% (vol.) of the bacterial suspension of the rhizobia SG09-DsRed strain prepared in Example 1 was prepared. In addition to the microbial cells, this solution consists of 17% final concentration whey protein concentrate (WPC, containing 70% to 80% whey protein), 5% Tween20, 0.1% ammonium molybdate ((NH4)6Mo(III)7O24·4H2O), 10% glycerol, and 0.1% xanthan gum. This was ejected from the first nozzle of an electrospinning device (Fluidnatek LE-50, Bioinicia) at a flow rate of 400 μl / h and a voltage difference of 29.3 kV (nozzle side +24.3 kV, base side -5 kV) to produce microcapsules containing microbial cells with a particle size of approximately 3-5 μm (minimum 1 μm or more, maximum less than 20 μm) (Figure 1A, Figure 8B). No microbial cells were observed outside the capsules; they were all contained within the capsules (Figure 1B). During the total spraying time of 37.5 minutes, the seeds on the base were randomly moved approximately every 6 minutes to uniformly coat the entire surface of the seeds with microcapsules.

[0040] The second coating solution, used for producing the microfibers, consisted of a final concentration of 20% zein (dissolved in 82% ethanol) and 5% Tween20. This solution was ejected from a second nozzle, separate from the first nozzle of the electrospinning apparatus, at a flow rate of 920 μl / h and a voltage difference of 18.6 kV (nozzle side +8.6 kV, base side -10 kV), generating microfibers less than 1 μm in diameter. By alternating between spraying microcapsules for approximately 6 minutes and spraying microfibers for approximately 5 seconds, a higher-order structure in which capsules and fibers were stacked was created (Figure 1C). The total spraying time of the microfibers onto the seeds was 25 seconds. Since zein microfibers are poorly water-soluble, they are expected to have the effect of retaining microcapsules near the seeds under high humidity conditions. (Example 3) Effect of microcapsules, or microcapsules and microfibers, on improving microbial survival under seed storage conditions Soybean seeds (variety: Hatayutaka) were used in the microbial coating test without sterilization treatment. First, two types of seeds were produced using the manufacturing method described in Example 2. Specifically, two types of seeds were produced by either a method of coating seeds with microcapsules containing microorganisms using an electrospinning method with the coated first coating solution of the present invention (P1), or a method of coating seeds with microcapsules containing microorganisms and fine fibers containing zein by combining an electrospinning method using the first coating solution of the present invention with an electrospinning method using the second coating solution of the present invention (P2). Furthermore, for comparison, four types of comparative seeds were produced using the following methods: a conventional general method of applying a microbial solution while stirring the seeds (hereinafter referred to as the bacterial solution coating method: C1); a method of coating seeds with microcapsules containing microorganisms produced by the method described in Non-Patent Literature 2 (C2); a method of coating seeds with microfibers containing microorganisms produced by the methods described in Non-Patent Literature 3 and Non-Patent Literature 4 (C3); or a method of coating seeds with microcapsules containing microorganisms by electrospinning using a coating solution composed only of whey protein concentrate and a nonionic surfactant, with xanthan gum, microbial protective agent (glycerol), and nutritional components (ammonium molybdate) removed from the first coating solution of P1 (C4). The types of seeds produced are shown in Table 1.

[0041] [Table 1]

[0042] These seeds were air-dried at 20°C for 60 minutes, then sealed in resealable bags with a desiccant (OZO-S, manufactured by OZO Chemical Technology Co., Ltd.) in batches of five seeds each, and stored at 4°C. Five seeds were collected on the day of storage, and on the 10th, 30th, 60th, and 90th day of storage. Microbial cells were collected from the seed surface by stirring in a 0.85% NaCl solution, and the number of viable cells was calculated by colony counting. The microbial survival rate and the change in viable cell count were also investigated, with the number of viable cells of the inoculated microorganisms on the seed on the day of storage set to 100%. As a result, compared to methods in other literature such as bacterial solution coating and electrospinning devices, and when microcapsules were made using only WPC and Tween20 as constituent materials, the coated seeds of the present invention did not show a rapid decrease in survival rate at the 10th day of storage, the survival rate was almost 100% at the 30th day of storage, the survival rate was almost 50% at the 60th day of storage, and the survival rate remained above 20% at the 90th day of storage (Figure 2A). The microbial survival rate on the seeds at the 60th day of storage was more than 20 times higher than that of the general-purpose bacterial solution coating method (Figure 2B).

[0043] Since no improvement in microbial survival was observed in microcapsules made with WPC and Tween20 alone, we compared the differences in microbial survival in microcapsules made with WPC and Tween20, respectively, in order to investigate which of the newly added constituent materials in this invention—ammonium molybdate, glycerol, or xanthanum—is the main cause of the effect. Specifically, coated seeds were manufactured and storage tests were conducted using the same method as in Example 2 and the storage test described above, except that ammonium molybdate was added (C5), glycerol was added (C6), and xanthanum was added (C7) in addition to WPC and Tween20. The types of seeds manufactured are shown in Table 2.

[0044] [Table 2]

[0045] As a result, particularly high survival rates were observed under conditions where glycerol was added (Figure 3). While not theoretically bound, it is possible that glycerol acted as a cytoprotective agent for microorganisms. Surprisingly, the survival-enhancing effect of glycerol was far greater than an additive effect. In the case of microcapsules produced using the present invention's method and coating solution, which further contained ammonium molybdate and xanthan in addition to glycerol, the 30-day survival rate of microorganisms was further increased (Figure 2A), suggesting that a combined effect including materials other than glycerol may also be provided. (Example 4) Evaluation of plant establishment of inoculated microorganisms under competition with indigenous microorganisms in an indoor soybean cultivation test using non-sterilized soil For each type of seed produced by the method of Example 3, the surface layer of the coated soybean seeds was examined using a scanning electron microscope.

[0046] When comparing the surface layers of soybean seeds coated by the conventionally used bacterial solution coating method (C1), the generation of microcapsules using an electrospinning device with the first coating solution of the present invention (P1), and the generation of microcapsules and microfibers using an electrospinning device with the first and second coating solutions of the present invention (P2), electron microscope images confirmed that a microstructure was formed on the surface of seeds coated using the method of the present invention (Figure 4A). Indoor cultivation tests were conducted using seeds produced by these three methods. Four seeds were sown in each of the non-sterilized Kuroboku soil in the upper section of a Leonardo jar (a system consisting of two 125 ml poly containers connected together, with a cotton string attached in the center to supply tap water from the lower section to the soil in the upper section), moisture-absorbing vermiculite was placed over the seeds, and after 7 days, the seedlings were thinned to two individuals per Leonardo jar and cultivated for 3 weeks in a growth chamber (26°C, photoperiod 16h-light / 8h-dark) (Figure 4B).

[0047] Next, plant colonization was evaluated by calculating the percentage of rhizobia inoculated from an external source within the total number of root nodules. Specifically, the fresh weight and total number of root nodules (>1 mm in diameter) attached to soybean roots three weeks after cultivation were measured. These were then surface-washed with sterile water, arranged one by one on a multi-well plate, and treated with an enzyme complex solution (cellulase / pectinase / hemicellulase) to prepare a suspension in which rhizobia were eluted from the outside of the root nodules. Fluorescent root nodules derived from genetically modified rhizobia strains inoculated from an external source and non-fluorescent root nodules from indigenous bacteria were distinguished by analysis using a fluorescence spectrophotometer and fluorescence microscope. Seeds C1, P1, and P2 were prepared using the same method as described above.

[0048] The germination rate of soybean seeds coated by the method of the present invention four days after sowing (Figure 5A), the fresh weight of the above-ground parts three weeks after cultivation (Figure 5B), the number of root nodules (Figure 5C), and the weight of root nodules (Figure 5D) were all equivalent to or better than those obtained by the fungal solution coating method. From this, it is clear that the seeds produced by the present invention do not inhibit plant growth.

[0049] The initial microbial inoculation amount per seed on the day of microbial treatment using the present invention was significantly less than 1 / 20th of that using the bacterial solution coating method (Figure 6A). This is because there are limitations to the flow rate conditions under which microcapsules can be produced using an electrospinning device and the total ejection time that can be processed in one day. On the other hand, the occupancy rate of rhizobia inoculated from an external source was equivalent to that of the bacterial solution coating method (Figure 6C). Similar results were obtained in indoor cultivation tests using other rhizobia strains, such as USDA110-DsRed (Figures 6B, D). This suggests that even with a smaller initial microbial inoculation amount compared to conventional methods, the present invention can achieve a comparable occupancy rate in a competitive environment with indigenous rhizobia and establish itself in plants after seed germination. (Example 5) Establishment of inoculum microorganisms on the surface of roots that have sprouted from stored seeds Untreated sterilized seeds, seeds treated with the bacterial cell coating method (C1 above), and seeds treated with the method of the present invention (P1 above) were each stored for 35 days under the conditions described in Example 3. Each stored seed was sown on sterile agar and cultivated in a growth chamber for 7 days. Microbial localization of microorganisms on the root surface was confirmed by electron microscopy at the base of roots (approximately 3 cm away from the seed) from seeds treated with the method of the present invention (Figure 7). This suggests that roots from seeds treated with the method of the present invention contained more microbial cells compared to roots from seeds stored using the general bacterial solution coating method. This indicates that microorganisms inoculated on the seed surface can colonize the roots after germination, even after seed storage for more than one month. (Example 6) Functional improvement by increasing the size of microcapsules using the thickening polysaccharide xanthan gum. To investigate the effect of adding xanthan gum on microcapsules, three types of first coating solutions were prepared with the compositions shown in Table 3 below, and soybean seeds were coated using the electrospinning method described in Example 2.

[0050] [Table 3]

[0051] By adding 0.1% xanthan gum to the microcapsule preparation solution, microcapsules with a particle size of 1 μm or larger and a maximum of less than 20 μm were stably produced, significantly larger than those without xanthan gum (Figure 8A, B). This particle size was also significantly larger than that of the microcapsules made from WPC, Tween20, and glycerol produced in Example 3 (Figure 8B, C). After testing with other thickening polysaccharides and other addition concentrations (0.2% xanthan gum, 0.2% alginic acid, 0.5% pectin, 1.0% pectin), the addition of 0.1% xanthan gum showed the best stability in producing large-sized capsules (Figure 9A, B, C, D).

[0052] Soybean seeds produced by conventionally used bacterial solution coating methods, microcapsules without xanthanthu, and microcapsules containing xanthanthu, were subjected to seed storage tests (Example 3) and indoor cultivation tests (Example 4). As a result, the microcapsules with added xanthanthu showed improved survival rates under seed storage conditions (Figure 10A) and root nodule occupancy rates after 30 days of cultivation (Figure 10B). The superior microbial survival and plant establishment in seeds produced by the method of the present invention are not achieved solely by microbial encapsulation with WPC, but are also achieved by the microbial protective effect of glycerol, and it is believed that this effect is further enhanced by adding xanthanthu and modifying the capsule particle size. (Example 7) Application of the present invention to plant seeds other than soybeans and microorganisms other than rhizobia. Regarding the microorganisms, a solution for producing microcapsules containing 10% (vol.%) bacterial suspension was prepared in the same manner as in Example 1, except that Paraburkholderia phytofirmans strain PsJN or Pseudomonas putida strain UWC1-166 were used (P1 and P2 in Figure 11: PsJN strain; P1 in Figure 12: UWC1-166 strain). Rice seeds (variety: Nipponbare) were sterilized by immersion in 70% ethanol for 1 minute and shaking in a 2% sodium hypochlorite solution for 30 minutes, followed by three washes with sterile water, and then thoroughly air-dried. The dried rice seeds were used for the coating test. Seeds produced using the electrospinning apparatus of Example 2 were air-dried at 20°C for 60 minutes. The formation of the microstructure on the seed surface was confirmed by electron microscopy, and the number of viable cells on the seed surface was calculated by colony counting by stirring four seeds at a time in a 0.85% NaCl solution.

[0053] It was confirmed that microcapsules could also coat rice seeds. Compared to soybean seeds, the microcapsules on rice seeds tended to be localized rather than uniformly sprayed, which is presumed to be due to the uneven surface structure and water repellency of the rice seed (Figures 11A and 12A). By using fine fibers and multiple spraying, the microcapsules could be uniformly layered over the entire seed (Figure 11B), and even for seeds with the same microbial cell spraying time, an improvement in the initial microbial inoculation was confirmed in rice seeds by using fine fibers and multiple spraying (Figure 11C). Although rice seeds tend to have a lower initial microbial inoculation per seed due to their smaller size compared to soybean seeds, it was confirmed that both the PsJN strain and UWC1-166 strain of bacteria other than rhizobia survived within the microcapsules encapsulated by the electrospinning device (Figures 11C and 12B). From these results, it is considered that the present invention is applicable to a wide range of plant seeds and microorganisms and is expected to be used in the supply of various coated seeds.

Claims

1. A method for covering plant reproductive bodies, (1) A step of providing a first coating solution comprising microbial cells, a polymer, a nonionic surfactant, and a microbial protective agent; and (2) A step of coating the surface of a plant reproductive organ by spraying the first coating solution, which is filled in the first syringe of the electrospinning apparatus, onto the reproductive organ through the first nozzle connected to the first syringe, thereby forming microcapsules in which microbial cells are embedded. The method, including the method described above.

2. The method according to claim 1, wherein the polymer is whey protein, or whey protein and xanthan gum, the nonionic surfactant is one or more nonionic surfactants selected from the group consisting of polyoxyethylene (20) sorbitan monolaurate, 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, octylphenoxy(polyoxyethylene)ethanol, octylphenoxypolyethoxyethanol, and nonylphenyl polyethylene glycol, and the microbial protective agent is one or more microbial protective agents selected from the group consisting of glycerol, trehalose, dimethyl sulfoxide, and sucrose.

3. A method according to claim 1 or 2, (3) A step of forming fine fibers by spraying a second coating solution containing zein and a nonionic surfactant, which is filled in the second syringe of the electrospinning apparatus, onto the organism through a second nozzle connected to the second syringe. The method further includes the method described above.

4. The method according to claim 3, wherein step (2) and step (3) are each repeated alternately two or more times.

5. The method according to claim 1, wherein the first coating solution further comprises nutritional components.

6. The method according to claim 5, wherein the first coating solution comprises microbial cells, whey protein concentrate, xanthan gum, polyoxyethylene (20) sorbitan monolaurate, glycerol, and ammonium molybdate.

7. The method according to claim 3, wherein the second coating solution comprises an ethanol solution of zein and polyoxyethylene (20) sorbitan monolaurate.

8. The method according to claim 1, wherein the reproductive body has a diameter of 0.1 cm or more.

9. The method according to claim 8, wherein the reproductive organism is a seed, a bulb, or a seed potato.

10. The method according to claim 9, wherein the seeds are selected from soybean, rice, wheat, and corn seeds, the bulbs are selected from garlic, onion, lily, tulip, gladiolus, and crocus bulbs, and the seed tubers are selected from potato, taro, yam, and konjac seed tubers.

11. The method according to claim 1, wherein the microorganism is selected from rhizobia and other gram-negative bacteria, gram-positive bacteria, archaea, mycorrhizal fungi and other fungi, and combinations thereof.

12. A method for producing a covered plant propagule, comprising the method described in claim 1.

13. A composition for coating plant reproductive bodies, comprising microbial cells, a polymer, a nonionic surfactant, and a microbial protective agent.

14. The composition according to claim 13, comprising microbial cells, whey protein, xanthan gum, polyoxyethylene (20) sorbitan monolaurate, glycerol, and ammonium molybdate.

15. The composition according to claim 13 or 14, wherein the microorganisms are selected from rhizobia and other gram-negative bacteria, gram-positive bacteria, archaea, mycorrhizal fungi and other fungi, and combinations thereof.

16. A plant reproductive body coated with the composition according to claim 13 or 14.

17. The reproductive organism according to claim 16, which is a seed, bulb, or seed potato.

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