WO2025169910A1 - Agricultural composition, seed composition, method for promoting plant growth, and method for soil improvement - Google Patents

Abstract

The purpose of the present disclosure is to provide an agricultural composition, a seed composition, a method for promoting plant growth, and a method for soil improvement that are capable of improving plant growth and that use a combination of a biosurfactant and a microbial inoculant.　One embodiment of the present invention is an agricultural composition containing a biosurfactant and a microbial inoculant.

Description

Agricultural composition, seed composition, plant growth promotion method, and soil improvement method

This disclosure relates to agricultural compositions, seed compositions, plant growth promotion methods, and soil improvement methods.

Various studies have been conducted to date to efficiently grow plants (vegetables, fruit trees, etc.). For example, Patent Document 1 discloses the use of at least one lipopeptide as a plant stimulant for plant growth.

Special Publication No. 2020-504768

Patent Document 1 did not consider the combined use of lipopeptides and microbial materials.

The purpose of this disclosure is to provide agricultural compositions, seed compositions, plant growth promotion methods, and soil improvement methods that use a combination of a biosurfactant and a microbial material, which can promote plant growth.

An example aspect of the first embodiment is described as follows:

[1] An agricultural composition comprising a biosurfactant and a microbial material.
[2] The agricultural composition according to [1], wherein the mass ratio of the biosurfactant to the microbial material is 1:50 to 1:20,000.
[3] The agricultural composition according to [1] or [2], wherein the biosurfactant is at least one biosurfactant selected from surfactin, rhamnolipid, sophorolipid, and salts thereof.
[4] The agricultural composition according to any one of [1] to [3], wherein the microbial material is a mycorrhizal fungus material or a Bacillus subtilis material.
[5] A seed composition comprising seeds, a biosurfactant, and a microbial material.
[6] The seed composition according to [5], wherein the mass ratio of the biosurfactant to the microbial material is 1:50 to 1:20,000.
[7] The seed composition according to [5] or [6], wherein the biosurfactant is at least one biosurfactant selected from surfactin, rhamnolipid, sophorolipid, and salts thereof.
[8] The seed composition according to any one of [5] to [7], wherein the microbial material is a mycorrhizal fungus material or a Bacillus subtilis material.
[9] A method for promoting plant growth, comprising applying a biosurfactant and a microbial material to a plant.
[10] The plant growth promotion method according to [9], wherein the mass ratio of the biosurfactant to the microbial material is 1:50 to 1:20,000.
[11] The plant growth-promoting method according to [9] or [10], wherein the biosurfactant is at least one biosurfactant selected from surfactin, rhamnolipid, sophorolipid, and salts thereof.
[12] The plant growth-promoting method according to any one of [9] to [11], wherein the microbial material is a mycorrhizal fungus material or a Bacillus subtilis material.
[13] A soil improvement method comprising applying a biosurfactant and a microbial material to soil.
[14] The soil improvement method according to [13], wherein the mass ratio of the biosurfactant to the microbial material is 1:50 to 1:20,000.
[15] The soil improvement method according to [13] or [14], wherein the biosurfactant is at least one biosurfactant selected from surfactin, rhamnolipid, sophorolipid, and salts thereof.
[16] The soil improvement method according to any one of [13] to [15], wherein the microbial material is a mycorrhizal fungus material or a Bacillus subtilis material.

The agricultural composition, seed composition, plant growth promotion method, and soil improvement method disclosed herein are capable of promoting plant growth.

FIG. 1 is a photograph of the plants produced in Experimental Example 11 and Experimental Example 13 (Comparative Example). FIG. 2 is a photograph of the plants produced in Reference Examples 1 and 3. FIG. 3 is a photograph showing the rhizosphere soil. FIG. 4 is a diagram showing the occupancy rate of each microorganism analyzed in Experimental Examples 17, 18, and 19. FIG. 5 is a diagram showing the occupancy rate of the microorganisms in the upper part of FIG.

The present disclosure will be described in detail below.
First embodiment (disclosure of Japanese Patent Application No. 2024-043018)
The agricultural composition according to the first embodiment includes a biosurfactant and a microbial material. The seed composition according to the first embodiment includes a seed, a biosurfactant, and a microbial material. The plant growth promotion method according to the first embodiment applies a biosurfactant and a microbial material to a plant. The soil improvement method according to the first embodiment applies a biosurfactant and a microbial material to soil. The agricultural composition according to the present disclosure can promote plant growth by applying it to a plant. The plant growth promotion composition can be applied directly to the seeds, leaves, stems, roots, etc. of a plant, or to the soil, or can be applied by adding it to water given to the plant. The plant growth promotion method according to the present disclosure can promote plant growth by applying a biosurfactant and a microbial material to a plant. The plant growth promotion method may involve applying a biosurfactant and a microbial material simultaneously, or applying a biosurfactant first and then applying a microbial material, or applying a microbial material first and then applying a biosurfactant. The soil improvement method of the first embodiment can improve the quality of the soil by applying a biosurfactant and a microbial material to the soil, and plants can be favorably grown in the soil with improved quality. The soil improvement method may involve applying a biosurfactant and a microbial material simultaneously, or applying a biosurfactant first and then applying a microbial material, or applying a microbial material first and then applying a biosurfactant.

In the present disclosure, promoting plant growth may mean promoting the initial growth of the plant, or may mean increasing the final yield of the plant.

A state in which the initial growth of a plant is promoted typically means a state in which the growth of at least one of the above-ground parts and underground parts, and preferably both, is promoted. According to the inventors' studies, many conventional compositions for promoting plant growth have been confirmed to promote the growth of either the above-ground parts or the underground parts, and there has been a desire to promote the growth of both the above-ground parts and the underground parts. The plant growth-promoting composition of the first embodiment is preferable because it is capable of promoting the growth of both the above-ground parts and the underground parts.

A state in which the final yield of a plant is increased refers to a state in which the weight of any part of the plant that is harvested as an agricultural crop, or the weight of the plant itself, has increased. When the final yield of a plant is increased, for example, in the case of dent corn, any part can refer to the weight of the entire above-ground part, or just the weight of the pistil. For example, in the case of soybeans and wheat, it refers to just the grains. When the final yield of a plant is increased, it is preferable that the weight of the fruit, seeds, leaves, or stems of the plant that is harvested as an agricultural crop has increased.

<Plants>
In the present disclosure, the plant is not particularly limited, but is preferably a crop plant, more preferably an edible plant. Examples of crop plants include corn (maize), wheat, barley, rye, oat, rice, soybean, canola (rapeseed), cotton, sunflower, sugar beet, potato, tobacco, broccoli, lettuce, cabbage, spinach, komatsuna (Japanese mustard spinach), cauliflower, coconut, tomato, cucumber, eggplant, melon, pumpkin, okra, bell pepper, watermelon, carrot, radish, onion, leek, fruit trees, ornamental plants, turf, and pasture grass. In the present disclosure, the seed is not particularly limited, but includes seeds of the above plants. The seed composition contains seeds.

<Biosurfactants>
The agricultural composition and seed composition contain a biosurfactant. The plant growth promotion method and the soil improvement method use a biosurfactant. Examples of the biosurfactant include at least one biosurfactant selected from peptide biosurfactants and sugar biosurfactants. The composition may contain one biosurfactant alone or two or more biosurfactants. The method may use one biosurfactant alone or two or more biosurfactants. Because the composition contains a biosurfactant and the method uses a biosurfactant, plant growth can be promoted. The reason for this is unclear, but this effect was not observed when synthetic surfactants were used; it was an effect specific to biosurfactants.

In addition, in the plant growth-promoting composition, it is preferable to use a biosurfactant that is not derived from Wickerhamomyces anomalus yeast, from the standpoint of odor and food hygiene.

Peptide biosurfactants include lipopeptide biosurfactants. Lipopeptide biosurfactants have peptides containing a hydrophobic group and a hydrophilic portion, exhibit surface-active properties, and are produced by microorganisms. Examples of lipopeptide biosurfactants include surfactin, arthrofactin, iturin, fengycin, serawettin, lykesin, viscosin, and salts thereof.

The peptide biosurfactant is preferably at least one peptide biosurfactant selected from surfactin and salts thereof. Surfactin and surfactin salts can be represented by the following general formula (1). One or more types of surfactin and surfactin salts may be used.

[In formula (1), X represents a residue of an amino acid selected from leucine, isoleucine, and valine, R represents an alkyl group having 9 to 18 carbon atoms, and each M ⁺ independently represents a hydrogen ion (H ⁺ ), an alkali metal ion, an ammonium ion, or a pyridinium ion.]

When M ⁺ is a hydrogen ion, it means that CO ₂ ⁻ (M ⁺ ) is a hydroxyl group (COOH group). When two M ⁺ are hydrogen ions, it is surfactin, and when at least one M ⁺ is an alkali metal ion, ammonium ion or pyridinium ion, it means a salt of surfactin. The general formula of surfactin is shown in the following general formula (1′).

[In formula (1′), X and R have the same meanings as in formula (1)]

X is an amino acid residue selected from leucine, isoleucine, and valine, but may be an L-amino acid residue or a D-amino acid residue, with an L-amino acid residue being preferred.

R is an alkyl group having 9 to 18 carbon atoms, and is a linear or branched monovalent saturated hydrocarbon group having at least 9 but no more than 18 carbon atoms. Examples of alkyl groups having 9 to 18 carbon atoms include n-nonyl, 6-methyloctyl, 7-methyloctyl, n-decyl, 8-methylnonyl, n-undecyl, 9-methyldecyl, n-dodecyl, 10-methylundecyl, n-tridecyl, 11-methyldodecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, and n-octadecyl, with 10-methylundecyl being preferred.

Each M ⁺ is independently a hydrogen ion (H ⁺ ), an alkali metal ion, an ammonium ion, or a pyridinium ion. The alkali metal ion is not particularly limited, but may represent a lithium ion, a sodium ion, a potassium ion, or the like. The ammonium ion is not particularly limited, but may, for example, be an ammonium ion represented by N(R ¹ ) ₄ ⁺ . Each R ¹ independently represents hydrogen or an organic group. A preferred embodiment of the ammonium ion is a quaternary ammonium ion in which all R ^1s are organic groups. Examples of the organic group include an alkyl group, an aralkyl group, and an aryl group. Specifically, examples of the alkyl group include alkyl groups having 1 to 10 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, and t-butyl. Examples of the aralkyl group include aralkyl groups having 7 to 12 carbon atoms, such as benzyl, methylbenzyl, and phenylethyl. Examples of the aryl group include aryl groups having 6 to 15 carbon atoms, such as phenyl, toluyl, and xylyl. Examples of the ammonium ion include tetramethylammonium ion and tetraethylammonium ion. The pyridinium ion is not particularly limited. In the pyridinium ion, a hydrogen atom bonded to a carbon atom constituting a pyridine ring may be substituted with an organic group. In addition, in the pyridinium ion, the bond to N ⁺ constituting the pyridine ring may be, for example, hydrogen or an organic group. Note that, as the organic group possessed by the pyridinium ion, the organic groups listed in the description of ^R1 can be appropriately used.

The two M ⁺ groups present in general formula (1) may be the same or different. In one preferred embodiment, some of the M ⁺ groups present in general formula (1) are hydrogen ions and some of the M ^{+ groups} are alkali metal ions. The alkali metal ions are not particularly limited, but include lithium ions, sodium ions, and potassium ions. When the two M ⁺ groups present in general formula (1) are two or more types of ions, the two M ⁺ groups may be the same type of ions when focusing on a single molecule (salt). When the M ⁺ groups are two types of ions, the ratio (molar ratio) of a certain ion A to a certain ion B is, for example, 1:10 to 10:1, preferably 1:5 to 5:1, and more preferably 1:3 to 3:1. In one preferred embodiment, some of the two M ^{+ groups} present in general formula (1) are hydrogen ions and some are sodium ions (Na ⁺ ).

Peptide biosurfactants such as surfactin or surfactin salts can be obtained by culturing a microorganism, for example a strain of Bacillus subtilis, and isolating it from the culture medium according to known methods. Purified products can be used, or they can be used unpurified, for example in the culture medium itself. Furthermore, those obtained by chemical synthesis methods can also be used in the same way, as long as they have the same molecular structure. Commercially available products can also be used.

Glycosurfactants include rhamnolipids, sophorolipids, mannosylerythritol lipids, cellobiose lipids, trehalose lipids, succinoyltrehalose lipids, glucose lipids, polyol lipids, oligosaccharide fatty acid esters, and salts thereof.

As a glycobiosaturant, at least one glycobiosaturant selected from rhamnolipids, sophorolipids, and salts thereof is preferred.

Glycosurfactants can be obtained according to known methods. Commercially available products can also be used.

A preferred embodiment of the biosurfactant is at least one biosurfactant selected from surfactin, rhamnolipid, sophorolipid, and salts thereof. Furthermore, a particularly preferred embodiment of the biosurfactant is at least one biosurfactant selected from surfactin and salts thereof.

<Microbial materials>
The agricultural composition and seed composition contain a microbial material. The plant growth promotion method and the soil improvement method use a microbial material. The microbial material is not particularly limited, and examples include a microbial material containing at least one species selected from Trichoderma fungi, mycorrhizal fungi, nitrogen-fixing bacteria, yeast, Bacillus subtilis, and rhizobia. The microbial material may be used alone or in combination with two or more species. In one preferred embodiment, the microbial material is a mycorrhizal fungal material. The amount of microorganisms in the microbial material is not limited. The amount of microorganisms in the microbial material may be, for example, 1.0 x 10 ⁸ CFU/g to 1.0 x 10 ¹⁰ CFU/g. In one preferred embodiment, the microbial material is substantially free of a rhizobia material. Therefore, the microbial material is preferably a microbial material containing at least one species selected from Trichoderma fungi, mycorrhizal fungi, nitrogen-fixing bacteria, yeast, and Bacillus subtilis. In the present disclosure, "substantially free of" a rhizobia material means that the rhizobia material is 0.1 parts by mass or less, and preferably 0.01 parts by mass or less, when the entire microbial material is taken as 100 parts by mass. In one more preferred embodiment, the microbial material does not contain a rhizobia material, i.e., the rhizobia material is 0 parts by mass.

<Additives>
The agricultural composition may contain, as necessary, components other than the biosurfactant and soybean meal hydrolysate as additives. The seed composition may contain, as necessary, components other than the seed, biosurfactant, and soybean meal hydrolysate as additives. The plant growth-promoting method and the soil improving method may use, as necessary, components other than the biosurfactant and soybean meal hydrolysate as additives.

The composition may contain one or more additives. The method may use one or more additives. Additives include, but are not limited to, thickeners, dispersants, humectants, colorants, antifoaming agents, UV protectants, antifreeze agents, preservatives, biological control agents or biocides, emulsifiers, sequestrants, plasticizers, phospholipids, flow agents, coalescing agents, waxes, preservatives, fillers (e.g., clay, talc, glass fiber, cellulose, micronized wood, etc.) and/or elements necessary for plant growth (e.g., one or more selected from the group consisting of Mo, Co, B, Fe, Cu, Zn, Mn, S, Mg, Ca, N, P, and K).

<Agricultural composition>
The agricultural composition comprises a biosurfactant and a microbial agent as described above.

The agricultural composition preferably has a mass ratio of biosurfactant to microbial material of 1:50 to 1:20,000, more preferably 1:100 to 1:15,000, and even more preferably 1:200 to 1:10,000.

The agricultural composition contains a biosurfactant and a microbial material, and may also contain additives. A seed composition can be obtained by coating plant seeds with the agricultural composition.

The method for producing the agricultural composition is not particularly limited, but examples include a method of preparing a liquid agricultural composition by dissolving or dispersing the components that make up the agricultural composition, i.e., the biosurfactant, microbial material, and any additives used, in water or the like. Examples of liquids, such as water, that can be used to prepare a liquid agricultural composition include water, organic solvents, and mixed solvents of water and organic solvents, with water being a preferred embodiment.

When water is used to prepare an agricultural composition for coating plant seeds, the amount of water can be adjusted depending on the water absorption of the target seeds. For example, in the case of seeds with low water absorption (e.g., corn, soybean, wheat, etc.), an agricultural composition for coating seeds with low water absorption can be prepared by adjusting the amount of water so that 8 to 10 L of agricultural composition is used per ton of seeds. For example, in the case of seeds with medium water absorption (e.g., barley, rice, oilseed rape, oats, etc.), an agricultural composition for coating seeds with medium water absorption can be prepared by adjusting the amount of water so that 10 to 12 L of agricultural composition is used per ton of seeds. For example, in the case of seeds with high water absorption (e.g., sugar beet, spinach, etc.), an agricultural composition for coating seeds with high water absorption can be prepared by adjusting the amount of water so that 12 to 150 L of agricultural composition is used per ton of seeds. The agricultural composition prepared in this manner can be used to coat the target seeds. The seeds coated with the agricultural composition can then be used as a seed composition by drying the water as needed.

<Seed composition>
The seed composition includes seeds, a biosurfactant, and a microbial material as described above. The seed composition includes the biosurfactant and the microbial material, and may also include additives. In the seed composition, these components may be present on the surface of the seeds, or may be present inside the seeds after being impregnated into the seeds, or may be present partially on the surface of the seeds and partially inside the seeds.

The mass ratio of the biosurfactant to the microbial material in the seed composition is preferably 1:50 to 1:20,000, more preferably 1:100 to 1:15,000, and even more preferably 1:200 to 1:10,000.

The seed composition may contain the biosurfactant in an amount of 0.25×10 ⁻⁴ to 2.0×10 ⁻² mass%, 0.3×10 ⁻⁴ to 1.5×10 ⁻² mass%, 0.35×10 ⁻⁴ to 1.3×10 ⁻² mass%, or 0.4×10 ⁻⁴ to 1.1×10 ⁻² mass%, based on 100 mass% of the seed.

The method for producing the seed composition is not particularly limited, but examples include a method in which the aforementioned agricultural composition is prepared as a liquid agricultural composition (coating solution) and the seed surface is coated with the coating solution to prepare the seed composition. Alternatively, the seed composition may be prepared by separately coating the seeds with the aforementioned biosurfactant and microbial material.

<Plant growth promotion method>
The plant growth-promoting method involves applying a biosurfactant and a microbial material to plants as described above. There are no particular limitations on the method for applying the biosurfactant and the microbial material to plants, and the method may involve preparing the agricultural composition described above and applying the composition to plants. Alternatively, the method may involve applying the biosurfactant and the microbial material separately to plants. When applying the biosurfactant and the microbial material to plants, they may be applied directly to the seeds, leaves, stems, roots, etc. of the plants, to the soil, or by adding them to water given to the plants.

In the plant growth promotion method, the mass ratio of the biosurfactant to the microbial material applied to the plant is preferably 1:50 to 1:20,000, more preferably 1:100 to 1:15,000, and even more preferably 1:200 to 1:10,000.

For example, when the agricultural composition is sprayed on leaves in the plant growth-promoting method, the agricultural composition for coating plant seeds described above can be diluted 10 to 4,000 times with water to prepare an agricultural composition for foliar spray, and the agricultural composition can be used for foliar spraying.

<Soil improvement method>
The soil improvement method involves applying a biosurfactant and a microbial material to soil as described above. The method for applying the biosurfactant and the microbial material to soil is not particularly limited, and may involve preparing the agricultural composition described above and applying the composition to the soil. Alternatively, the soil improvement method may involve applying the biosurfactant and the microbial material separately to the soil.

In the soil improvement method, the mass ratio of biosurfactant to microbial material applied to the soil is preferably 1:50 to 1:20,000, more preferably 1:100 to 1:15,000, and even more preferably 1:200 to 1:10,000.

For example, when carrying out the soil improvement, the agricultural composition for coating plant seeds described above can be diluted 10 to 4,000 times with water to prepare an agricultural composition for soil improvement, and the soil can be improved by applying the agricultural composition to the soil.

Second embodiment (disclosure of Japanese Patent Application No. 2024-017099)
A second embodiment of the present disclosure relates to a composition for activating microorganisms, a composition for promoting plant growth, a method for activating microorganisms, a method for diversifying microorganisms, a method for producing plants, a method for producing seeds, and seeds.

Rhizosphere microorganisms, which live in the rhizosphere, a soil space influenced by plant root secretions and soil microorganisms, are used as microbial materials in the agricultural field. Furthermore, it is believed that a sustainable and/or stable supply of food will be increasingly required in the future. Microorganisms, such as rhizosphere microorganisms, that live in plant growth environments can be efficiently utilized to contribute to a sustainable and/or stable supply of food, and thus microorganisms are attracting attention. Among rhizosphere microorganisms, plant growth-promoting rhizobacteria (PGPR) (C. K. Jha1 and M. Saraf, E3 J. Agric. Res. Develop., Vol. 5 (2), pp. 108-119, Ap ril, 2015 and M. Vocciante, et al., Appl. Sci. 2022, 12, 1231), and Plant Growth Promoting Fungi (A. A. Adedayo and O. O. Babalola, J. Fungi 2023, 9, 239) are known.

JP 2022-509204 A describes a soil treatment composition for improving plant immune health, growth, and/or yield, which contains Wickerhamomyces anomalus yeast and/or its growth by-products. The soil treatment composition is described as a microbial-based soil treatment composition for improving the health, growth, and overall yield of crop plants by improving the health and/or growth of the plant's root system and by stimulating the plant's innate immune and other metabolic systems that contribute to plant health and productivity.

However, Wickerhamomyces anomalus is a yeast-like fungus that produces ethyl acetate, and is known to cause a thinner odor when attached to food products. Therefore, there is a strong demand for a microbial activation composition that can efficiently activate microorganisms, such as rhizosphere microorganisms.

The second embodiment of the present disclosure aims to solve the above-mentioned problems of the prior art and achieve the following objectives. That is, the present invention aims to provide a composition for activating microorganisms, a composition for promoting plant growth, a method for activating microorganisms, a method for diversifying microorganisms, a method for producing plants, a method for producing seeds, and seeds that can efficiently activate microorganisms.

As a result of extensive research conducted by the inventors to achieve the above-mentioned objective, they have discovered that it is possible to provide a composition for activating microorganisms comprising a biosurfactant, a composition for promoting plant growth comprising a biosurfactant, a method for activating microorganisms comprising the step of treating seeds with a composition containing a biosurfactant, a method for diversifying microorganisms comprising the step of treating seeds with a composition containing a biosurfactant, a method for producing plants comprising the step of treating seeds with a composition containing a biosurfactant, a method for producing seeds comprising the step of treating seeds with a composition containing a biosurfactant, or a composition for activating microorganisms, a composition for promoting plant growth, a method for activating microorganisms, a method for diversifying microorganisms, a method for producing plants, a method for producing seeds, or seeds that can efficiently activate microorganisms using seeds that have a biosurfactant on their surface.

The second embodiment of the present disclosure is based on the above-mentioned findings of the inventors, and the means for solving the problems of the second embodiment are as follows.

[1] A composition for activating microorganisms, characterized by containing a biosurfactant.
[2] A plant growth-promoting composition characterized by containing a biosurfactant.
[3] A method for activating microorganisms, comprising a step of treating seeds with a composition containing a biosurfactant.
[4] A method for diversifying microorganisms, comprising a step of treating seeds with a composition containing a biosurfactant.
[5] A method for producing a plant, comprising a step of treating seeds with a composition containing a biosurfactant.
[6] A method for producing seeds, comprising a step of treating seeds with a composition containing a biosurfactant.
[7] Seeds characterized by having a biosurfactant on the surface.

Further example aspects of the second embodiment are described as follows.
(1) A composition for activating microorganisms, characterized by containing a biosurfactant.
(2) The composition for activating microorganisms according to (1), wherein the biosurfactant is surfactin or a salt thereof.
(3) The composition for activating microorganisms described in (1), which is a microbial activator that increases microorganisms with an occupancy rate of 1% or less.
(4) The composition for activating microorganisms described in (1), which is a microbial diversification agent.
(5) The composition for activating microorganisms according to (1), which is used for seed treatment.
(6) A plant growth-promoting composition comprising a biosurfactant.
(7) A method for activating microorganisms, comprising a step of treating seeds with a composition containing a biosurfactant.
(8) The method for activating microorganisms according to (7), comprising the step of sowing seeds treated with the composition in soil and allowing microorganisms to grow at an occupancy rate of 1% or less.
(9) A method for diversifying rhizosphere microorganisms, comprising a step of treating seeds with a composition containing a biosurfactant.
(10) The method for diversifying microorganisms according to (9), comprising the step of sowing seeds treated with the composition in soil and allowing microorganisms with an occupancy rate of 1% or less to grow.
(11) A method for producing a plant, comprising a step of treating seeds with a composition containing a biosurfactant.
(12) A method for producing a plant according to (11), comprising the step of sowing seeds treated with the composition in soil and allowing microorganisms to grow at an occupancy rate of 1% or less.
(13) A method for producing seeds, comprising a step of treating seeds with a composition containing a biosurfactant.
(14) A seed characterized by having a biosurfactant on its surface.

The second embodiment of the present disclosure can solve the above-mentioned problems of the prior art and achieve the above-mentioned objective, and can provide a microbial activation composition containing a biosurfactant, a plant growth promotion composition containing a biosurfactant, a method for activating microorganisms comprising the step of treating seeds with a composition containing a biosurfactant, a method for diversifying microorganisms comprising the step of treating seeds with a composition containing a biosurfactant, a method for producing plants comprising the step of treating seeds with a composition containing a biosurfactant, a method for producing seeds comprising the step of treating seeds with a composition containing a biosurfactant, or a composition for activating microorganisms, a composition for promoting plant growth, a method for activating microorganisms, a method for diversifying microorganisms, a method for producing plants, a method for producing seeds, or seeds that can efficiently activate microorganisms using seeds that have a biosurfactant on their surface.

<Composition for microbial activation>
The composition for activating microorganisms includes a biosurfactant and may further include other ingredients.

<Biosurfactants>
The biosurfactants (biological surface-active substances) are substances having surface-active properties that are produced inside or outside of cells by living organisms. Among these, surfactants produced outside the cells of microorganisms are preferred, and biosurfactants not derived from Wickerhamomyces anomalus yeast are more preferred because they are widely accepted by users.

The biosurfactant is not particularly limited and can be selected appropriately depending on the purpose. Examples include peptide-type biosurfactants and sugar-type biosurfactants. Among these, peptide-type biosurfactants are preferred because they can efficiently activate microorganisms. The biosurfactants may be used alone or in combination of two or more types.

The peptide biosurfactant is not particularly limited and can be selected appropriately depending on the purpose. Examples include lipopeptide biosurfactants. The lipopeptide biosurfactant has a peptide containing a hydrophobic group and a hydrophilic portion.

The lipopeptide biosurfactant is not particularly limited and can be selected appropriately depending on the purpose. Examples include surfactin, arthrofactin, iturin, fengycin, serawettin, lykesin, viscosin, and salts thereof. Among these, surfactin or a salt thereof is preferred because it can efficiently activate microorganisms.

The surfactin or salt thereof is represented by the following general formula (1):

[In formula (1), X represents an amino acid residue selected from leucine, isoleucine, and valine, R represents an alkyl group having 9 to 18 carbon atoms, and each M ⁺ independently represents a hydrogen ion (H ⁺ ), an alkali metal ion, an ammonium ion, or a pyridinium ion.]

There are no particular limitations on X, so long as it is an amino acid residue selected from leucine, isoleucine, and valine, and it can be selected appropriately depending on the purpose. However, leucine is preferred, and L-leucine is preferred, as it can efficiently activate microorganisms.

The R group is not particularly limited as long as it is an alkyl group having 9 to 18 carbon atoms (a linear or branched monovalent saturated hydrocarbon group having from 9 to 18 carbon atoms), and can be selected appropriately depending on the purpose. However, from the viewpoint of efficient activation of microorganisms, an alkyl group having 9 to 17 carbon atoms is preferred, an alkyl group having 9 to 16 carbon atoms is more preferred, an alkyl group having 9 to 15 carbon atoms is even more preferred, an alkyl group having 10 to 14 carbon atoms is particularly preferred, and an alkyl group having 11 to 13 carbon atoms is most preferred.

Specific examples of R are not particularly limited and can be selected appropriately depending on the purpose. Examples include n-nonyl, 6-methyloctyl, 7-methyloctyl, n-decyl, 8-methylnonyl, n-undecyl, 9-methyldecyl, n-dodecyl, 10-methylundecyl, n-tridecyl, 11-methyldodecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, and n-octadecyl. Of these, the 10-methylundecyl group is preferred due to its ability to efficiently activate microorganisms.

Each M ⁺ is independently a hydrogen ion (H ⁺ ), an alkali metal ion, an ammonium ion, or a pyridinium ion.

The alkali metal ions are not particularly limited and can be selected appropriately depending on the purpose. Examples include lithium ions, sodium ions, and potassium ions.

The ammonium ion is not particularly limited and can be appropriately selected depending on the purpose. Examples include ammonium ions represented by N(R ¹ ) ₄ ⁺ . Each R ¹ independently represents hydrogen or an organic group. Among these, a quaternary ammonium ion in which all R ^1s are organic groups is preferred. Examples of the organic group include alkyl groups, aralkyl groups, and aryl groups. Examples of the alkyl group include alkyl groups having 1 to 10 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, and t-butyl. Examples of the aralkyl group include aralkyl groups having 7 to 12 carbon atoms, such as benzyl, methylbenzyl, and phenylethyl. Examples of the aryl group include aryl groups having 6 to 15 carbon atoms, such as phenyl, toluyl, and xylyl. Specific examples of the ammonium ion include tetramethylammonium ion and tetraethylammonium ion. The pyridinium ion is not particularly limited and can be appropriately selected depending on the purpose, and a hydrogen atom bonded to a carbon atom constituting a pyridine ring may be substituted with an organic group, or hydrogen or an organic group may be bonded to N ⁺ constituting a pyridine ring. The organic group contained in the pyridinium ion is as described for the organic group in the ammonium ion.

The two M ⁺ groups present in general formula (1) may be the same or different. The two M ⁺ groups present in general formula (1) are not particularly limited and can be selected appropriately depending on the purpose. However, it is preferable that some of the M ⁺ groups are hydrogen ions and some of the M ⁺ groups are alkali metal ions. The alkali metal ions are not particularly limited and can be selected appropriately depending on the purpose. Examples include lithium ions, sodium ions, and potassium ions. Among these, sodium ions are preferred. When the two M ⁺ groups present in general formula (1) are two or more types of ions, in the case of a single molecule (salt), the two M ⁺ groups may be the same type of ion. When the M ⁺ groups are two types of ions, the ratio (molar ratio) of a certain ion A to a certain ion B is, for example, 1:10 to 10:1, preferably 1:5 to 5:1, and more preferably 1:3 to 3:1. When M ⁺ is a hydrogen ion, it means that CO ₂ ⁻ (M ⁺ ) is a hydroxyl group (COOH group). When two M ⁺ are hydrogen ions, it means surfactin, and when at least one M ⁺ is an alkali metal ion, ammonium ion or pyridinium ion, it means a salt of surfactin.

The general formula of surfactin is shown below as general formula (1').

[In formula (1′), X and R have the same meanings as in formula (1)]

The peptide biosurfactants can be obtained by culturing a microorganism, such as a strain of Bacillus subtilis, and isolating them from the culture medium according to known methods. A purified product can be used, or they can be used unpurified, for example, in the culture medium. Furthermore, those obtained by chemical synthesis can also be used as long as they have the same molecular structure. Commercially available products can also be used.

The sugar-type biosurfactant is not particularly limited and can be selected appropriately depending on the purpose, and examples thereof include sodium ion sugar-type biosurfactants. The sodium ion sugar-type biosurfactant is not particularly limited and can be selected appropriately depending on the purpose, and examples thereof include rhamnolipid, sophorolipid, mannosylerythritol lipid, cellobiose lipid, trehalose lipid, succinoyltrehalose lipid, glucose lipid, polyol lipid, oligosaccharide fatty acid ester, and salts thereof. Of these, at least one selected from rhamnolipid, sophorolipid, and salts thereof is preferred.

The glycosurfactant can be obtained according to known methods. Commercially available products can also be used.

The biosurfactant is not particularly limited and can be selected appropriately depending on the purpose, but at least one selected from surfactin, rhamnolipid, sophorolipid, and salts thereof is particularly preferred because it can efficiently activate microorganisms.

The lower limit of the content of the biosurfactant in 100% by mass of the microbial activation composition is not particularly limited and can be selected appropriately depending on the purpose; however, from the viewpoint of efficient activation of microorganisms, it is preferably 0.0001% by mass or more, more preferably 0.001% by mass or more, even more preferably 0.002% by mass or more, particularly preferably 0.005% by mass or more, and most preferably 0.01% by mass or more. The upper limit of the content of the biosurfactant in 100% by mass of the microbial activation composition is not particularly limited and can be selected appropriately depending on the purpose; however, from the viewpoint of efficient activation of microorganisms, it is preferably 10% by mass or less, more preferably 1% by mass or less, even more preferably 0.5% by mass or less, particularly preferably 0.2% by mass or less, and most preferably 0.1% by mass or less. Among these, from the viewpoint of efficient activation of microorganisms, a concentration of 0.0001% by mass to 10% by mass is preferred, 0.001% by mass to 1% by mass is more preferred, 0.002% by mass to 0.5% by mass is even more preferred, 0.005% by mass to 0.2% by mass is particularly preferred, and 0.01% by mass to 0.1% by mass is most preferred.

<Other ingredients>
The other components are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include carriers, additives, etc. The other components may be used alone or in combination of two or more.

The carrier is not particularly limited and can be selected appropriately depending on the purpose. Examples include liquid carriers and solid carriers. The carriers may be used alone or in combination of two or more types.

The liquid carrier is not particularly limited and can be selected appropriately depending on the purpose. Examples include water and organic solvents. Examples of organic solvents include methyl ether, ethyl ether, propyl ether, and butyl ether. The water used as the carrier is not limited to pure water, but may be an aqueous solution, aqueous suspension, aqueous gel, or aqueous slurry, and may have viscosity. The organic solvent used as the carrier is not limited to pure organic solvents, but may be an organic solvent-based solution, suspension, gel, or slurry, and may have viscosity.

The solid carrier is not particularly limited and can be selected appropriately depending on the purpose, and examples thereof include hydratable substances. The solid carrier can be in the form of a powder or granules. The hydratable substance is not particularly limited and can be selected appropriately depending on the purpose, and examples thereof include polyvinylpyrrolidone, random and block copolymers of alkylene oxides, vinyl acetate/vinylpyrrolidone copolymers, alkylated vinylpyrrolidone copolymers, polyalkylene glycols including polypropylene glycol and polyethylene glycol, polyvinyl acetate, polyvinyl alcohol, gelatin, agar, gum arabic, karaya gum, tragacanth gum, guar gum, locust bean gum, xanthan gum, ghatti gum, carrageenan, alginate, casein, dextran, pectin, chitin, 2-hydroxyethyl starch, 2-aminoethyl starch, 2-hydroxyethyl cellulose, methylcellulose, carboxymethyl cellulose salts, cellulose sulfate, polyacrylamide, alkali metal salts of maleic anhydride copolymers, and alkali metal salts of poly(meth)acrylates. Among these, polyvinyl alcohol is preferred because it can efficiently activate microorganisms.

The additives are not particularly limited and can be appropriately selected depending on the purpose. Examples of the additives include moisturizers, colorants, antifoaming agents, UV protectants, antifreeze agents, preservatives, biological control agents, biocides, emulsifiers, extenders, sequestrants, plasticizers, phospholipids, flow agents, coalescing agents, waxes, and/or fillers (e.g., clay, talc, glass fiber, cellulose, finely divided wood, etc.).
The carriers may be used alone or in combination of two or more.

The microbial activation composition is capable of activating the microorganisms.

The microorganisms refer to microorganisms that inhabit the plant's growing environment, and examples of the plant's growing environment include the rhizosphere and phyllosphere. The rhizosphere is a soil space influenced by plant root secretions and soil microorganisms, and includes the inner rhizosphere, which is the internal environment of the root, such as the intercellular spaces of the root epidermis and cortex, the root surface, which is the root's surface, and the outer rhizosphere, which is the soil area surrounding the root. The phyllosphere is defined as the surface of the above-ground part of the plant, and phyllosphere microorganisms include epiphytes (ectophytic fungi) that exist on the leaf surface and endophytes (endophytic fungi) that exist inside the leaf tissue. The microorganisms are not particularly limited and can be selected appropriately depending on the purpose, and examples include bacteria and fungi. Among these, plant growth-promoting rhizobacteria (PGPR) and plant growth-promoting fungi (PGPF) are preferred. Specific examples of the microorganisms are not particularly limited and can be selected appropriately depending on the purpose, including bacteria such as those of the Bradyrhizobium, Bacillus, and Clostridium genera, and fungi such as those of the Penicillium genus.

The activation is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include an increase in microorganisms and diversification of microorganisms.
The increase in microorganisms is preferably an increase in microorganisms (rare species) that occupy 1% or less in a specific growth environment.
The term "microorganisms with an occupancy rate of 1% or less" means microorganisms whose number is 1% or less when the total number of microorganisms in a specific growth environment is taken as 100%.
An increase in microorganisms with an occupancy rate of 1% or less means an increase in the number of microorganisms of 1% or less when the total number of microorganisms contained in a specific growth environment before activation or before activation is taken as 100%.
The microbial diversification means an increase in the variety of microorganisms in a particular growing environment.

In another embodiment, the activation refers to the exhibiting of at least one of the following effects i) to iii):
i) an increase in the number of rhizosphere microorganisms, more specifically, an increase in the total number of rhizosphere microorganisms in the target rhizosphere;
ii) an increase in plant growth-promoting microorganisms (PGPM), such as plant growth-promoting rhizobacteria (PGPR) and plant growth-promoting fungi (PGPF), more specifically, an increase in PGPR and/or PGPF;
iii) Diversification of rhizosphere microorganisms, specifically, an increase in the number of microbial species growing in the target rhizosphere, and/or an increase in the number of rhizosphere microorganisms (rare species) that account for less than 1% of the total rhizosphere microorganisms.

The microbial activation composition can diversify the microorganisms, and can therefore be used as a microbial diversification agent.

The microorganism-activating composition can promote plant growth and can therefore be used as a plant growth-promoting composition. There are no particular restrictions on the growth and it can be selected appropriately depending on the purpose, but early growth is preferred. Early growth refers to the period from germination to the middle of the vegetative growth stage, when dry matter production begins to increase rapidly. For example, it refers to growth within 90 days, 60 days, 30 days, 25 days, 21 days, or 14 days after germination.

The microorganism-activating composition is applied to plants, soil, or water given to plants. Among these, it is preferable to apply it to leaves, seeds, seedlings, fruits, etc. of plants, and it is even more preferable to use it for seed treatment.

The seed treatment method is not particularly limited and can be selected appropriately depending on the purpose. Examples include a method of applying the microbial activating composition and a method of soaking the microbial activating composition. Among these, the method of applying the microbial activating composition is preferred because it can efficiently activate microorganisms. A method of uniformly applying the microbial activating composition while rotating seeds is more preferred, and a method of uniformly applying the microbial activating composition while rotating dried seeds is even more preferred.

The microorganism-activating composition can be used for seed treatment to efficiently activate the microorganisms. The microorganism-activating composition can be used as a seed treatment composition or a seed coating composition.

The lower limit of the amount of the biosurfactant per ton of seeds is not particularly limited and can be selected appropriately depending on the purpose. However, from the viewpoint of efficiently activating microorganisms, it is preferably 0.1 g or more, more preferably 0.2 g or more, and even more preferably 0.5 g or more. The upper limit of the amount of the biosurfactant per ton of seeds is not particularly limited and can be selected appropriately depending on the purpose. However, from the viewpoint of efficiently activating microorganisms, it is preferably 500 g or less, more preferably 200 g or less, and even more preferably 100 g or less. Of these, from the viewpoint of efficiently activating microorganisms, it is preferably 0.1 g or more and 500 g or less, more preferably 0.2 g or more and 200 g or less, and even more preferably 0.5 g or more and 100 g or less.

The seeds are not particularly limited and can be selected appropriately depending on the purpose, and examples include gymnosperm seeds and angiosperm seeds. Of these, angiosperm seeds are preferred. The angiosperms are not particularly limited and can be selected appropriately depending on the purpose, and examples include grasses, lilies, Musaceae, Bromeliaceae, Orchidaceae, Cruciferae, Legumes, Solanaceae, Cucurbits, Convolvulaceae, Rosaceae, Mulberry, Malvaceae, Asteraceae, Amaranthaceae, and Polygonaceae. Of these, legumes and grasses are preferred.

The Gramineae plants include rice, wheat, barley, corn, oats, turfgrass, sorghum, rye, foxtail millet, and sugarcane. The Liliaceae plants include leeks and asparagus. The Musaceae plants include bananas. The Bromeliaceae plants include pineapples. The Orchidaceae plants include orchids.

Examples of the Brassicaceae family include Arabidopsis thaliana, Chinese cabbage, rapeseed, cabbage, cauliflower, and radish. Examples of the Leguminaceae family include soybean, adzuki bean, kidney bean, pea, cowpea, and alfalfa. Examples of the Solanaceae family include tomato, eggplant, potato, tobacco, and chili pepper. Examples of the Cucurbitaceae family include oriental melon, cucumber, melon, and watermelon. Examples of the Convolvulaceae family include morning glory, sweet potato (sweet potato), and bindweed. Examples of the Rosaceae family include rose, strawberry, and apple. Examples of the Moraceae family include mulberry, fig, and rubber tree. Examples of the Malvaceae family include cotton and kenaf. Examples of the Asteraceae family include lettuce. Examples of the Amaranthaceae family include sugar beet (sugar beet). Examples of the Polygonaceae family include buckwheat.

The method for producing the microbial activation composition is not particularly limited and can be selected appropriately depending on the purpose. For example, a method in which the biosurfactant and other ingredients are mixed and stirred until homogeneous is included.

<Composition for promoting plant growth>
The plant growth-promoting composition contains a biosurfactant and may further contain other components, as described above in the section (Composition for activating microorganisms).

<Method for activating microorganisms>
The method for activating microorganisms includes a step of treating seeds with a composition containing a biosurfactant, and may further include other steps.

<Step of treating seeds with a composition containing a biosurfactant>
The composition containing the biosurfactant contains the biosurfactant and can further contain other ingredients. The biosurfactant and other ingredients, as well as the seeds and seed treatment, are as described above in (Composition for activating microorganisms).

<Other processes>
The other step is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include a step of sowing seeds treated with the composition in soil. Among these, a step of sowing seeds treated with the composition in soil and growing microorganisms with an occupancy rate of 1% or less is preferred. By sowing seeds treated with the composition in soil, microorganisms with an occupancy rate of 1% or less can be grown.

<Methods for diversifying microorganisms>
The method for diversifying microorganisms includes a step of treating seeds with a composition containing a biosurfactant, and may further include other steps. The step of treating seeds with a composition containing a biosurfactant and other steps are as described in the above section (Method for activating microorganisms).

<Plant manufacturing method>
The method for producing a plant includes a step of treating seeds with a composition containing a biosurfactant, and may further include other steps. The step of treating seeds with a composition containing a biosurfactant and other steps are as described above in (Method for activating microorganisms).

<Method for producing seeds (coated seeds)>
The seed production method includes a step of treating seeds with a composition containing a biosurfactant. The step of treating seeds with a composition containing a biosurfactant is as described above in (Method for activating microorganisms). The seed production method allows for the production of coated seeds coated with the composition containing the biosurfactant.

<Seeds (coated seeds)>
The seeds contain a biosurfactant and may further contain other components. Among these, those having a biosurfactant on the surface of the seeds are preferred. The seeds having a biosurfactant on the surface of the seeds may have the biosurfactant only on the surface of the seeds, or a portion of the biosurfactant may have penetrated into the inside of the seeds. The biosurfactant and other components are as described above in (Composition for activating microorganisms). The seeds are produced by the above-mentioned (Method for producing seeds (coated seeds)).

The first and second embodiments of the present disclosure are independent of each other, but the definitions, regulations, ranges, etc. of terms in the first embodiment of the present disclosure may be replaced with definitions, regulations, ranges, etc. corresponding to those in the second embodiment of the present disclosure, or the definitions, regulations, ranges, etc. in the second embodiment of the present disclosure may be replaced with definitions, regulations, ranges, etc. corresponding to those in the first embodiment of the present disclosure.

Therefore, in one embodiment, the first embodiment of the present disclosure is an invention intended to reinforce the second embodiment of the present disclosure. Furthermore, in one embodiment, the second embodiment of the present disclosure is an invention intended to reinforce the first embodiment of the present disclosure.

This specification includes the disclosure of Japanese Patent Application No. 2024-017099, from which the present application claims priority.
Furthermore, this specification incorporates the disclosure of Japanese Patent Application No. 2024-043018, which is a priority document of the present application.

The present embodiment will be described below using examples, but the present disclosure is not limited to these examples. Note that each experimental example is an independent experiment.

In this example, "SF" refers to surfactin Na (product name: Kaneka Surfactin, manufactured by Kaneka), and "SL" refers to sophorolipid (prepared according to Journal of Oleo Science, 60, (5) pp. 267-273 (2011)).

[Experimental Example 1]
Seed compositions were prepared as follows and corn was grown under normal conditions.

100g of corn seeds (variety: Snowdent Otoha) were coated with 800μL of a coating solution containing SF and a thickener (polyvinyl alcohol (Poval)) in water using a coating device (manufactured by SATEC), and then coated with mycorrhizal fungal material to prepare a seed composition. SF was coated at 0.5g-100g per ton of seeds, and mycorrhizal fungal material at 0.5kg-5kg per ton.

9cm pots were filled with potting soil and one seed composition (coated corn seed) was sown per pot. After sowing, the plants were kept in a closed greenhouse set at 24°C/14 hours during the day and 18°C/10 hours at night, and the growth status was investigated 21 days after sowing. The investigation was carried out by measuring the dry weight of the above-ground and below-ground parts. The test was repeated 12 times and the average value was calculated.

Table 1 shows the measurement results for the dry weight of the aboveground parts of Experimental Example 1, and Table 2 shows the measurement results for the dry weight of the underground parts of Experimental Example 1. In Tables 1 and 2, corn seeds that had not been subjected to the above-mentioned coating treatment were used when the amount of SF used was 0 g/MT-seed and the amount of mycorrhizal fungal material used was 0 kg/MT-seed. Note that in Tables 1 and 2, the dry weight of the aboveground parts and the dry weight of the underground parts are shown as relative values (%), with the dry weight of the aboveground parts and the dry weight of the underground parts of corn seeds that had 0 g/MT-seed of SF used and 0 kg/MT-seed of mycorrhizal fungal material set at 100.

Tables 1 and 2 show that the seed composition of the present disclosure has superior growth potential compared to conventional seeds.

[Experimental Example 2]
Seed compositions were prepared and corn plants were grown under salt stress conditions as described below.

100g of corn seeds (variety: Snowdent Otoha) were coated with 800μL of a coating solution containing SF and a thickener (polyvinyl alcohol (Poval)) in water using a coating device (manufactured by SATEC), and then coated with mycorrhizal fungal material to prepare a seed composition. SF was coated at 0.5g-100g per ton of seeds, and mycorrhizal fungal material at 0.5kg-5kg per ton.

7.5cm pots were filled with potting soil, the bottom was allowed to absorb a sodium chloride solution, and one seed composition (coated corn seed) was sown per pot. After sowing, the plants were kept in a closed greenhouse set at 24°C/14 hours during the day and 18°C/10 hours at night, and the growth status was investigated 21 days after sowing. The investigation was carried out by measuring the dry weight of the above-ground and below-ground parts. The test was repeated 12 times and the average value was calculated.

Table 3 shows the measurement results of the dry weight of the above-ground parts in Experimental Example 2, and Table 4 shows the measurement results of the dry weight of the underground parts in Experimental Example 2. In Tables 3 and 4, corn seeds that had not been subjected to the above-mentioned coating treatment were used when the amount of SF used was 0 g/MT-seed and the amount of mycorrhizal fungal material used was 0 kg/MT-seed. Note that in Tables 3 and 4, the dry weight of the above-ground parts and the dry weight of the underground parts are shown as relative values (%), with the dry weight of the above-ground parts and the dry weight of the underground parts of corn seeds that had 0 g/MT-seed of SF used and 0 kg/MT-seed of mycorrhizal fungal material set at 100.

Tables 3 and 4 show that the seed compositions disclosed herein all showed an increase in aboveground dry weight and/or belowground dry weight, demonstrating superior growth potential compared to conventional seeds even under salt stress conditions.

[Experimental Example 3]
Seed compositions were prepared as follows and corn was grown under normal conditions.

100g of corn seeds (variety: Snowdent Otoha) were coated with 800μL of a coating solution containing biosurfactant (SF or SL) and thickener (polyvinyl alcohol (Poval)) in water using a coating device (manufactured by SATEC), and then coated with mycorrhizal fungal material to prepare a seed composition. The coating was performed so that 10g of biosurfactant was applied per ton of seeds and 5kg of mycorrhizal fungal material was applied per ton of seeds.

9cm pots were filled with potting soil and one seed composition (coated corn seed) was sown per pot. After sowing, the plants were kept in a closed greenhouse set at 24°C/14 hours during the day and 18°C/10 hours at night, and the growth status was investigated 21 days after sowing. The investigation was carried out by measuring the dry weight of the above-ground and below-ground parts. The test was repeated 12 times and the average value was calculated.

Table 5 shows the measurement results for the dry weight of the above-ground and underground parts of Experimental Example 3. In Table 5, corn seeds that had not been subjected to the above-mentioned coating treatment were used as the control. In Table 5, the dry weight of the above-ground and underground parts is shown as a relative value (%), with the dry weight of the above-ground and underground parts of the control corn seeds set at 100.

Table 5 shows that the seed composition of the present disclosure has superior growth potential compared to regular seeds. As a biosurfactant, surfactin Na was superior to sophorolipid.

[Experimental Example 4]
Seed compositions were prepared and corn plants were grown under salt stress conditions as described below.

100g of corn seeds (variety: Snowdent Otoha) were coated with 800μL of a coating solution containing SF and a thickener (polyvinyl alcohol (Poval)) in water using a coating device (manufactured by SATEC), and then coated with mycorrhizal fungal material to prepare a seed composition. SF was coated at 50g per ton of seeds, and mycorrhizal fungal material at 5kg per ton.

7.5cm pots were filled with potting soil, the bottoms were allowed to absorb a sodium chloride solution, and one seed composition (coated corn seed) was sown per pot. After sowing, the plants were kept in a closed greenhouse set at 24°C/14 hours during the day and 18°C/10 hours at night, and the growth status was investigated 21 days after sowing. The investigation was carried out by measuring the dry weight of the above-ground and below-ground parts. The test was repeated 12 times and the average value was calculated.

Table 6 shows the measurement results for the dry weight of the above-ground and underground parts of Experimental Example 4. In Table 6, corn seeds that had not been subjected to the above-mentioned coating treatment were used as the control. In Table 6, the dry weight of the above-ground and underground parts is shown as a relative value (%), with the dry weight of the above-ground and underground parts of the control corn seeds set at 100.

Table 6 shows that the seed composition of the present disclosure has superior growth potential compared to conventional seeds, even under salt stress conditions.

[Experimental Example 5]
(Production of coated dent corn seeds 1)
Coated dent corn seeds were produced by coating 100 g of dent corn seeds (variety: Snow Dent Otoha) with 800 μL of a coating solution containing 0.05 mg of surfactin (Surfactin Na: Kaneka Surfactin, manufactured by Kaneka Corporation), 20 μL of polyvinyl alcohol, and 20 μL of a coloring pigment using a coating device (manufactured by SATEC Co., Ltd.). The amount of surfactin coated per ton of seeds was 0.5 g.

[Experimental Example 6]
(Production of coated dent corn seeds 2)
Coated dent corn seeds were produced in the same manner as in Experimental Example 5, except that the amount of surfactin coated was 100 g per ton of seeds.

[Experimental Example 7 (Comparative Example)]
(Production of coated dent corn seeds 3)
Coated dent corn seeds were produced in the same manner as in Experimental Example 5, except that surfactin was not added to the coating solution.

[Experimental Example 8]
(Production of coated soybean seeds 1)
Coated soybean seeds were produced by coating 100 g of soybean seeds (variety: Fukuyutaka) with 800 μL of a coating solution containing surfactin (Surfactin Na: Kaneka Surfactin, manufactured by Kaneka Corporation), polyvinyl alcohol, and a coloring pigment using a coating device (manufactured by SATEC Co., Ltd.) so that the amount of surfactin coated per ton of seeds was 0.5 g.

[Experimental Example 9]
(Production of coated soybean seeds 2)
Coated soybean seeds were produced in the same manner as in Experimental Example 8, except that the amount of surfactin coated was 100 g per ton of seeds.

[Experimental Example 10 (Comparative Example)]
(Production of coated soybean seeds 3)
Soybean seeds were coated in the same manner as in Experimental Example 8, except that surfactin was not added to the coating solution.

[Experimental Example 11]
(Production of Dent Corn Plants 1)
A 12 cm polypot was filled with soil for field crops (Tsuchitarou (registered trademark), manufactured by Sumitomo Forestry Landscaping Co., Ltd.), and the coated dent corn seeds produced in Experimental Example 5 were sown at four seeds per pot. After germination, one plant with uneven growth was thinned out to three plants per pot. The plants were cultivated in a glass greenhouse, and 21 days after sowing, they were cut 1 cm from the ground and separated into above-ground and underground parts.
The underground parts were washed with water to remove the soil. The above-ground and underground parts were dried at 80°C for two days, and the dry weights were measured. Six replicates were carried out and the average value was calculated. The results are shown in the second row of Table 7.
A photograph of the plants 21 days after sowing is shown on the right side of Figure 1 (SF treatment).

[Experimental Example 12]
(Production of Dent Corn Plants 2)
Dent corn plants were produced in the same manner as in Experimental Example 11, except that the coated dent corn seeds produced in Experimental Example 6 were used instead of the coated dent corn seeds produced in Experimental Example 5, and the dry weights of the above-ground and below-ground parts were measured. The results are shown in the third row of Table 7.

[Experimental Example 13 (Comparative Example)]
(Production of Dent Corn Plants 3)
Dent corn plants were produced in the same manner as in Experimental Example 11, except that the coated dent corn seeds produced in Experimental Example 7 (Comparative Example) were used instead of the coated dent corn seeds produced in Experimental Example 5, and the dry weights of the aboveground and underground parts were measured. The results are shown in the first row of Table 7.
A photograph of the plants 21 days after sowing is shown on the left side of Figure 1 (untreated).

(Reference Example 1: Production of dent corn plants 4)
Dent corn plants were grown in the same manner as in Experimental Example 11, except that sterilized soil prepared by treating the soil for field crops at 120°C for 2 hours was used instead of the soil for field crops, and the dry weights of the above-ground and below-ground parts were measured. The results are shown in the second row of Table 8.
A photograph of the plants 21 days after sowing is shown on the right side of Figure 2 (SF treatment).

(Reference Example 2: Production of Dent Corn Plant 5)
Dent corn plants were grown in the same manner as in Experimental Example 12, except that sterilized soil prepared by treating the soil for field crops at 120°C for 2 hours was used instead of the soil for field crops, and the dry weights of the above-ground and below-ground parts were measured. The results are shown in the third row of Table 8.

(Reference Example 3: Production of dent corn plants 6)
Dent corn plants were grown in the same manner as in Experimental Example 13 (Comparative Example), except that sterilized soil prepared by treating the soil for field crops at 120°C for 2 hours was used instead of the soil for field crops, and the dry weights of the above-ground and below-ground parts were measured. The results are shown in the first row of Table 8.
A photograph of the plants 21 days after sowing is shown on the left side of Figure 2 (untreated).

The results in Table 7 show that when coated dent corn seeds coated to a biosurfactant amount of 0.5 g per ton of seeds were sown in field crop soil, the dry weight of the aboveground parts increased by a statistically significant 16%, and the dry weight of the belowground parts increased by a statistically significant 16%, compared to when coated dent corn seeds coated to a biosurfactant amount of 0 g per ton of seeds were sown.
Furthermore, when coated dent corn seeds coated with 100 g of biosurfactant per ton of seeds were sown in field crop soil, the dry weight of the aboveground parts increased by a statistically significant 16% and the dry weight of the belowground parts increased by 12% compared to when coated dent corn seeds coated with 0 g of biosurfactant per ton of seeds were sown.

From the results in Table 8, when sterilized bed soil prepared by treating bed soil for field crops at 120°C for 2 hours was used instead of the bed soil for field crops, no effect of the biosurfactant was observed.
Since field crop soil contains microorganisms, it has been shown that biosurfactants significantly promote plant growth through the microorganisms.

[Experimental Example 14]
(Production of soybean plants 1)
A 12 cm polypot was filled with soil for field crops (Tsuchitarou, manufactured by Sumitomo Forestry Landscaping Co., Ltd.), and the coated soybean seeds produced in Experimental Example 8 were sown at four seeds per pot. After germination, one plant that was not growing uniformly was thinned out to three plants per pot. The plants were cultivated in a glass greenhouse, and 21 days after sowing, they were cut 1 cm from the ground and separated into above-ground and underground parts.
The underground parts were washed with water to remove the soil. The above-ground and underground parts were dried at 80°C for 2 days, and the dry weights were measured. Six replicates were carried out and the average value was calculated. The results are shown in the second row of Table 9.

[Experimental Example 15]
(Production of soybean plants 2)
Soybean plants were produced in the same manner as in Experimental Example 14, except that the coated soybean seeds produced in Experimental Example 9 were used instead of the coated soybean seeds produced in Experimental Example 8, and the dry weights of the above-ground and underground parts were measured. The results are shown in the third row of Table 9.

[Experimental Example 16 (Comparative Example)]
(Production of soybean plants 3)
Soybean plants were produced in the same manner as in Experimental Example 14, except that the coated soybean seeds produced in Experimental Example 10 (Comparative Example) were used instead of the coated soybean seeds produced in Experimental Example 8, and the dry weights of the above-ground and underground parts were measured. The results are shown in the first row of Table 9.

(Reference Example 4: Production of soybean plants 4)
Soybean plants were grown in the same manner as in Experimental Example 14, except that sterilized soil for field crops treated at 120°C for 2 hours was used instead of the soil for field crops, and the dry weights of the above-ground and below-ground parts were measured. The results are shown in the second row of Table 10.

(Reference Example 5: Production of soybean plants 5)
Soybean plants were grown in the same manner as in Experimental Example 15, except that sterilized soil prepared by treating the soil for field crops at 120°C for 2 hours was used instead of the soil for field crops, and the dry weights of the above-ground and below-ground parts were measured. The results are shown in the third row of Table 10.

(Reference Example 6: Production of soybean plants 6)
Soybean plants were grown in the same manner as in Experimental Example 16 (Comparative Example), except that sterilized soil prepared by treating the soil for field crops at 120°C for 2 hours was used instead of the soil for field crops, and the dry weights of the above-ground and below-ground parts were measured. The results are shown in the first row of Table 10.

The results in Table 9 show that when coated soybean seeds coated with 0.5 g of biosurfactant per ton of seeds were sown in field crop soil, the dry weight of the aboveground parts increased by 7% and the dry weight of the underground parts increased by a statistically significant 14% compared to when coated soybean seeds coated with 0 g of biosurfactant per ton of seeds were sown.
Furthermore, when coated soybean seeds coated with 100 g of biosurfactant per ton of seeds were sown in soil for field crops, the dry weight of the aboveground parts increased by 7% and the dry weight of the underground parts increased by a statistically significant 19% compared to when coated soybean seeds coated with 0 g of biosurfactant per ton of seeds were sown.

From the results in Table 10, when sterilized bed soil for field crops treated at 120°C for 2 hours was used instead of the bed soil for field crops, no effect of the biosurfactant was observed.
Since field crop soil contains microorganisms, it has been shown that biosurfactants significantly promote plant growth through the microorganisms.

[Experimental Example 17]
(Microbiome analysis 1)
A 9 cm polypot was filled with soil for field crops (Tsuchitarou, manufactured by Sumitomo Forestry Landscaping Co., Ltd.), and the coated soybean seeds produced in Experimental Example 9 were sown. The seeds were cultivated in a glass greenhouse, and on the 19th day after sowing, the rhizosphere soil (the soil adhering to the roots in the area indicated by the circle in Figure 3) was collected with a brush.
The collected rhizosphere soil was subjected to amplicon sequencing analysis by Seibutsu Giken Co., Ltd. to analyze the rhizosphere microorganisms.
The occupancy rate of each microorganism is shown on the right side of Figure 4 (SF treatment (after cultivation)). The occupancy rate of the microorganism in the upper part of Figure 4 (indicated by symbol a in Figure 4) is shown in Figure 5.

[Experimental Example 18]
(Microbiome analysis 2)
Analysis of rhizosphere microorganisms was carried out in the same manner as in Experimental Example 17, except that the coated soybean seeds produced in Experimental Example 10 (Comparative Example) were used instead of the coated soybean seeds produced in Experimental Example 9.
The occupancy rate of each microorganism is shown in the center of Figure 4 (untreated (after cultivation)). The occupancy rate of the microorganisms in the upper part of Figure 4 (indicated by symbol a in Figure 4) is shown in Figure 5.

[Experimental Example 19]
(Microbiome analysis 3)
Amplicon sequencing analysis by Seibutsu Giken Co., Ltd. was performed on field crop soil (Tsuchitarou, manufactured by Sumitomo Forestry Landscaping Co., Ltd.) to analyze the rhizosphere microorganisms in the soil before cultivation.
The occupancy rate of each microorganism is shown on the left side of Figure 4 (soil before cultivation). The occupancy rate of the microorganisms in the upper part of Figure 4 (indicated by the symbol a in Figure 4) is shown in Figure 5.

The results in Figures 4 and 5 show that rare species (e.g., species indicated by symbols b and c in Figure 5) that were present in the field crop soil at an occupancy rate of less than 1% prior to cultivation significantly proliferated when cultivated using seeds with biosurfactants on their surface.

Table 11 summarizes the percentage increase in occupancy of specific microorganisms in Experimental Examples 17 to 19 compared to the untreated control.

The results in Table 11 show that surfactin has the effect of increasing the occupancy rate of plant growth-promoting rhizobacteria (PGPR).
[Experimental Example 20]
Soybeans were drenched under Rhizoctonia disease conditions as described below.

Sterilized barley seeds were inoculated with Rhizoctonia solani and cultured in the dark at 30°C for 10 days to be used as inoculum. Contaminated soil was prepared by mixing the aforementioned inoculum with Tsuchitaro (Sumitomo Forestry Landscaping Co., Ltd.) at a weight ratio of 10%. Soybean seeds (variety: Fukuyutaka) were sown in the contaminated soil at 10 seeds per pot, with three pots prepared for each treatment area. At the time of sowing, the Trichoderma test area was irrigated with a water-suspended solution of Trichoderma material (Trichoderma harzianum strain T22) at 1250 g/ha. Similarly, the surfactin test plot was irrigated with a surfactin aqueous solution at 25 g/ha, while the Trichoderma + surfactin test plot was irrigated with a water-suspended solution of Trichoderma material (Trichoderma harzianum strain T22) at 1,250 g/ha and a surfactin aqueous solution at 25 g/ha. Plants were cultivated in an artificial climate chamber (light period 28°C/12 hours, dark period 25°C/12 hours), and 11 days after sowing, an evaluation index was calculated using the following criteria. Based on this, the disease severity and control value were calculated using the following formula. Table 12 shows the results of the disease severity and control value. Note that a lower disease severity is preferable, and a higher control value is preferable.

(Evaluation index)
0: Healthy 1: Slight water-soaked browning is observed at the base of the plant 2: Water-soaked browning is observed at the base of the plant, and growth is significantly delayed 3: Dead 4: Dampening before emergence (no germination)

(Severity of disease)
Severity = [(0 x number of plants) + (1 x number of plants) + (2 x number of plants) + (3 x number of plants) + (4 x number of plants)] / (4 x total number of plants surveyed) x 100

(Control value)
Control value = (1 - disease incidence in treated area / disease incidence in untreated area) x 100

Table 12 shows that the Trichoderma + surfactin test plot had a lower disease severity and a higher control value compared to both the Trichoderma test plot and the surfactin test plot. Therefore, this experimental example confirmed the synergistic effect of using Trichoderma and surfactin in combination.

[Experimental Example 21]
Potato drench treatments were carried out under normal conditions as follows:

9cm pots were filled with culture soil and one microtuber planted per pot. Before covering with soil, each treatment area was irrigated with a microbial material, a solution containing Bacillus subtilis (Bacillus bacteria) in water, a solution containing Bacillus subtilis and SF, or water. After covering with soil, the plants were kept in a closed greenhouse set at 24°C/14 hours during the day and 18°C/10 hours at night, and growth conditions were investigated 19 days after planting. The investigation was carried out by measuring the dry weight of the above-ground and below-ground parts. The test was repeated 10 times and the average value was calculated.

Table 13 shows the measurement results of the dry weight of the aboveground parts of Experimental Example 21, and Table 14 shows the measurement results of the dry weight of the underground parts of Experimental Example 21. In Tables 13 and 14, the dry weight of the aboveground parts or the dry weight of the underground parts is shown as a relative value (%), with the dry weight of the aboveground parts or the dry weight of the underground parts of microtubers to which SF was applied at an amount of 0 g/ha and Bacillus subtilis material was applied at an amount of 0 kg/ha set at 100.

Tables 13 and 14 show that the composition of the present disclosure increased both the dry weight of above-ground and below-ground parts, demonstrating superior growth potential compared to SF alone and the Bacillus subtilis material alone.

[Experimental Example 22]
Potato drench treatments were carried out under salt stress as shown below.

7.5cm pots were filled with culture soil, the bottom was allowed to absorb a sodium chloride solution, and one microtuber was planted per pot. Before covering with soil, each treatment area was irrigated with a microbial material, a solution containing Bacillus subtilis (Bacillus bacteria) in water, a solution containing Bacillus subtilis and SF, or water. After covering with soil, the plants were kept in a closed greenhouse set at 24°C/14 hours during the day and 18°C/10 hours at night, and growth conditions were investigated 19 days after planting. The investigation was carried out by measuring the dry weight of the above-ground and below-ground parts. The test was repeated 10 times and the average value was calculated.

Table 15 shows the measurement results of the dry weight of the aboveground parts of Experimental Example 22, and Table 16 shows the measurement results of the dry weight of the underground parts of Experimental Example 22. In Tables 15 and 16, the dry weight of the aboveground parts or the dry weight of the underground parts is shown as a relative value (%), with the dry weight of the aboveground parts or the dry weight of the underground parts of microtubers to which SF was applied at an amount of 0 g/ha and Bacillus subtilis material was applied at an amount of 0 kg/ha set at 100.

Tables 15 and 16 show that the composition of the present disclosure increased both the dry weight of above-ground and below-ground parts, demonstrating superior growth potential compared to SF alone and the Bacillus subtilis material alone, even under salt stress.

[Experimental Example 23]
Corn drench treatments were performed under normal conditions as follows:

9cm pots were filled with potting soil and corn seeds (variety: Snowdent Otoha) were sown, one seed per pot. Before covering with soil, each treatment area was irrigated with a microbial material containing Bacillus subtilis (Bacillus bacteria), a solution containing SF in water, a solution containing Bacillus subtilis and SF, or water. After covering with soil, the plants were kept in a closed greenhouse set at 24°C/14 hours during the day and 18°C/10 hours at night, and growth conditions were investigated 21 days after planting. The investigation was carried out by measuring the dry weight of the above-ground and below-ground parts. The test was repeated 12 times and the average value was calculated.

Table 17 shows the measurement results of the above-ground dry weight of Experimental Example 23, and Table 18 shows the measurement results of the below-ground dry weight of Experimental Example 23. In Tables 17 and 18, the above-ground dry weight or below-ground dry weight is shown as a relative value (%), with the above-ground dry weight or below-ground dry weight of corn to which SF was applied at a rate of 0 g/ha and Bacillus subtilis material was applied at a rate of 0 kg/ha set at 100. In Tables 17 and 18, "-" indicates a treatment area where no measurement was performed.

Tables 17 and 18 show that the composition of the present disclosure increased both the dry weight of above-ground and below-ground parts, demonstrating superior growth potential compared to SF alone and the Bacillus subtilis material alone.

[Experimental Example 24]
Corn was irrigated under salt stress conditions as shown below.

7.5cm pots were filled with potting soil, the bottom was allowed to absorb a sodium chloride solution, and one corn seed (variety: Snowdent Otoha) was sown per pot. Before covering with soil, each treatment area was irrigated with a microbial material containing Bacillus subtilis (Bacillus bacteria), a solution containing SF in water, a solution containing Bacillus subtilis and SF, a solution containing Bacillus subtilis and rhamnolipid, or water. After covering with soil, the plants were kept in a closed greenhouse set at 24°C/14 hours during the day and 18°C/10 hours at night, and growth conditions were investigated 21 days after planting. The investigation was carried out by measuring the dry weight of the above-ground and below-ground parts. The test was repeated 12 times and the average value was calculated.

Table 19 shows the results of measuring the dry weight of the aboveground parts of Experimental Example 24, and Table 20 shows the results of measuring the dry weight of the underground parts of Experimental Example 24. In Tables 19 and 20, the dry weight of the aboveground parts or the dry weight of the underground parts is shown as a relative value (%), with the dry weight of the aboveground parts or the dry weight of the underground parts of corn to which SF was applied at an amount of 0 g/ha and Bacillus subtilis material was applied at an amount of 0 kg/ha set at 100. In Tables 19 and 20, "-" indicates a treatment area where no measurement was performed.

Tables 19 and 20 show that the composition of the present disclosure increased both the dry weight of above-ground and below-ground parts, demonstrating superior growth potential compared to SF alone and the Bacillus subtilis material alone, even under salt stress.

[Experimental Example 25]
(Microbiome analysis 4)
A 9 cm polypot was filled with culture soil (Tsuchitarou, manufactured by Sumitomo Forestry Landscaping Co., Ltd.) and corn seeds (variety: Snowdent Otoha) were sown. The surfactin-treated plots were irrigated with a surfactin aqueous solution at 25 g/ha at the time of sowing. Cultivation was carried out in a glass greenhouse, and 19 days after sowing, rhizosphere soil was collected with a brush. The collected rhizosphere soil was subjected to amplicon sequencing analysis by Seibutsu Giken Co., Ltd., and rhizosphere microorganisms were analyzed. Table 21 shows the measurement results of the occupancy rate of each microorganism.

Table 21 shows, as an example, an increase in the occupancy rate of Bacillus. Specifically, the occupancy rate in the untreated area was 0.04%, while in the surfactin-treated area it was 0.1%, an increase of approximately 2.4 times.

The upper and/or lower limit values of the numerical ranges described in this specification can be arbitrarily combined to define a preferred range. For example, the upper and lower limit values of a numerical range can be arbitrarily combined to define a preferred range, the upper limit values of a numerical range can be arbitrarily combined to define a preferred range, and the lower limit values of a numerical range can be arbitrarily combined to define a preferred range. Furthermore, in this application, numerical ranges expressed using the symbol "to" include the numerical values written before and after the symbol "to" as the upper and lower limits, respectively.

Although the present embodiment has been described in detail above, the specific configuration is not limited to this embodiment, and even if there are design changes within the scope that do not deviate from the gist of this disclosure, they are included in this disclosure.
All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims (16)

An agricultural composition comprising a biosurfactant and a microbial material. The agricultural composition according to claim 1, wherein the mass ratio of the biosurfactant to the microbial material is 1:50 to 1:20,000. The agricultural composition according to claim 1, wherein the biosurfactant is at least one biosurfactant selected from surfactin, rhamnolipid, sophorolipid, and salts thereof. The agricultural composition according to claim 1, wherein the microbial material is a mycorrhizal fungus material or a Bacillus subtilis material. A seed composition comprising seeds, a biosurfactant, and a microbial material. The seed composition according to claim 5, wherein the mass ratio of the biosurfactant to the microbial material is 1:50 to 1:20,000. The seed composition according to claim 5, wherein the biosurfactant is at least one biosurfactant selected from surfactin, rhamnolipid, sophorolipid, and salts thereof. The seed composition according to claim 5, wherein the microbial material is a mycorrhizal fungus material or a Bacillus subtilis material. A method for promoting plant growth by applying biosurfactants and microbial materials to plants. The plant growth promotion method according to claim 9, wherein the mass ratio of the biosurfactant to the microbial material is 1:50 to 1:20,000. The plant growth-promoting method according to claim 9, wherein the biosurfactant is at least one biosurfactant selected from surfactin, rhamnolipid, sophorolipid, and salts thereof. The plant growth promotion method according to claim 9, wherein the microbial material is a mycorrhizal fungus material or a Bacillus subtilis material. A soil improvement method that applies biosurfactants and microbial materials to soil. The soil improvement method according to claim 13, wherein the mass ratio of the biosurfactant to the microbial material is 1:50 to 1:20,000. The soil improvement method according to claim 13, wherein the biosurfactant is at least one biosurfactant selected from surfactin, rhamnolipid, sophorolipid, and salts thereof. The soil improvement method according to claim 13, wherein the microbial material is a mycorrhizal fungus material or a Bacillus subtilis material.