Plant growth promoting agent

Abstract

To provide a method and a material for promoting the growth of leguminous plants included in beans.SOLUTION: Provided is an agent, which is a growth promoting agent for leguminous plants included in beans. The agent comprises a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent. The (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms and dimethyl sulfoxide, and the proportion (mass ratio) of the (A) ascorbic acid or a salt thereof is 10 to 3,000,000 when the component (B) is set as 1.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to methods and materials for promoting the growth of legumes, including pulses. [Background technology]

[0002] Approximately one-third of the Earth's land area is arid, and future global warming is expected to further increase this aridity. To address the serious food shortages caused by population growth, there is an urgent need to develop technologies to improve, maintain, and increase yields of soybeans, rice, wheat, corn, and other grains in arid, saline, high-temperature, and low-temperature regions—regions where growth is difficult or where growth is impaired and yields are reduced. Soybeans, in particular, are an important grain and are widely consumed around the world, including in Japan. Furthermore, unlike other grains, soybeans have high protein and lipid content and are highly nutritious. Therefore, they are also important as feed and a source of oils and fats, and technologies to increase yields are being developed.

[0003] Oxygen is an essential substance for the survival of many plants and animals, but at the same time, oxygen generates highly reactive oxygen species within the cells of plants and animals, which cause serious damage to living organisms by damaging genes and deactivating enzymes. For this reason, plants store many antioxidants, including L-ascorbic acid, and a complex enzyme system works in various parts of the cell to use these antioxidants to eliminate reactive oxygen species.

[0004] From this perspective, antioxidants containing ascorbic acid are often incorporated into materials applied to plants to promote their growth. For example, Non-Patent Document 1 discloses the growth-promoting and yield-increasing effects of ascorbic acid application to olives, sugarcane, wheat, and other crops. Non-Patent Document 2 also discloses that a maximum yield increase of 30% can be achieved by foliar spraying a fixed concentration of ascorbic acid dissolved in distilled water onto legume crops three times during the vegetative or reproductive growth phase. Patent Document 1 also discloses the promotion of plant growth using an aqueous composition containing an iron (II) compound and L-ascorbic acid, while Patent Document 2 discloses the promotion of plant growth using an aqueous solution containing glycyrrhizin and L-ascorbic acid. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 60-202805 [Patent Document 2] Japanese Patent Application Publication No. 08-143406

[0006] [Non-Patent Document 1] Akram et al. (2017) Ascorbic Acid-A Potential Oxidant Scavenger and Its Role in Plant Development and Abiotic Stress Tolerance. Frontiers in Plant Science,8:613 [Non-patent document 2] Zarghamnejad et al.,(2014)Chickpea response to ascorbic acid foliar application at vegetable and reproductive stages. International Journal of Biosciences, 5:166-170 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to providing methods and materials for promoting the growth of legumes, including pulses. [Means for solving the problem]

[0008] As a result of investigating the use of ascorbic acid to promote plant growth, the present inventors discovered that when an ascorbic acid solution dissolved in water used in agricultural fields, such as tap water or well water, is sprayed on legumes, including pulses, the growth-promoting effect of ascorbic acid is hardly obtained. They also found that the use of a specific antioxidant in which ascorbic acid is dissolved in an organic solvent in combination with the plant exhibits the effects of promoting root nodule activity and growth.

[0009] That is, the present invention relates to the following 1) to 9). 1) A growth promoter for legumes, including pulses, comprising a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms, and dimethyl sulfoxide, and the ratio (mass ratio) of (A) ascorbic acid or a salt thereof is 10 to 3,000,000 relative to the ratio of component (B) being 1. 2) A growth promoter for legumes, which comprises a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms and dimethyl sulfoxide, and the proportion (mass ratio) of the (C) organic solvent is 4 to 100,000 relative to the proportion of component (B) as 1. 3) A root nodule activity promoter comprising a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or fewer carbon atoms and dimethyl sulfoxide, and the ratio (mass ratio) of (A) ascorbic acid or a salt thereof is 10 to 3,000,000 when the component (B) is taken as 1. 4) A root nodule activity promoter comprising a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms and dimethyl sulfoxide, and the ratio (mass ratio) of the (C) organic solvent is 4 to 100,000 when the component (B) is taken as 1. 5) A yield-enhancing agent for legumes, including pulses, comprising a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms and dimethyl sulfoxide, and the ratio (mass ratio) of (A) ascorbic acid or a salt thereof is 10 to 3,000,000 relative to the ratio of component (B) being 1. 6) A yield-enhancing agent for legumes, including pulses, comprising a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms and dimethyl sulfoxide, and the proportion (mass ratio) of the (C) organic solvent is 4 to 100,000 relative to the proportion of component (B) as 1. 7) A method for promoting the growth of legumes, including pulses, comprising the step of combining (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent with water and applying the resulting mixture to soil or plants, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms and dimethyl sulfoxide, and the ratio (mass ratio) of (A) ascorbic acid or a salt thereof is 10 to 3,000,000 relative to the ratio of component (B) being 1. 8) A method for promoting root nodule activity, comprising the step of combining (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent with water and applying the resulting mixture to soil or a plant, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms and dimethyl sulfoxide, and the ratio (mass ratio) of (A) ascorbic acid or a salt thereof is 10 to 3,000,000 relative to the ratio of component (B) being 1. 9) A method for increasing the yield of legumes, including pulses, comprising a step of combining (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent with water and applying the resulting mixture to soil or plants, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms and dimethyl sulfoxide, and the ratio (mass ratio) of (A) ascorbic acid or a salt thereof is 10 to 3,000,000 relative to the ratio of component (B) being 1. [Effects of the Invention]

[0010] According to the method of the present invention, the growth of legumes included in pulses can be increased. Furthermore, nodule activity is promoted in legumes included in pulses that have formed nodules, improving the ureide-producing ability of the plant. That is, according to the present invention, the yield of fruits, seeds, or grains of legumes included in pulses can be increased. [Brief explanation of the drawings]

[0011] [Figure 1] Seed weight per soybean plant. [Figure 2] Solubility when the mixing ratio of BHT and isobutyl alcohol is changed. [Figure 3] Solubility of BHT in organic solvent mixtures (1:4). [Figure 4] Soybean nodule weight one week after foliar application. [Figure 5] The amount of allantoic acid contained in soybean exudate one week after foliar spray. [Figure 6] Amount of allantoin contained in soybean exudate one week after foliar spray. [Figure 7] The amount of allantoic acid contained in soybean exudate one week after foliar spray. [Figure 8] Soybean plants in test area 7 one week after foliar spray. [Figure 9] Seed weight per soybean plant. [Figure 10] The amount of allantoic acid contained in soybean exudate one week after foliar spray. [Figure 11] Dry weight of aboveground parts of adzuki beans one week after foliar spray. [Figure 12] Dry weight of the underground parts of adzuki beans one week after foliar spray. [Figure 13] Dry weight of chickpea above ground one week after foliar application. [Figure 14] Number of pods per Lotus japonicus plant. [Figure 15] Seed weight per Lotus japonicus plant. [Figure 16] Number of beans per broad bean plant. [Figure 17] Seed weight per broad bean plant. [Figure 18] Appearance of one- or two-dose compositions after one week of storage. [Figure 19] The amount of allantoic acid contained in soybean exudate one week after foliar spray. [Figure 20]Dry weight of soybean aboveground parts one week after foliar spray. [Figure 21] Dry weight of soybean underground one week after foliar spray. DETAILED DESCRIPTION OF THE INVENTION

[0012] In the growth promoter for legumes included in pulses of the present invention, "growth promotion" means increasing the growth amount (fresh weight, elongation, etc.) of legumes included in pulses and increasing the yield of fruits, seeds, or grains. When the growth promoter for legumes included in pulses of the present invention is intended solely to increase grain yield, it is referred to as a "yield-increasing agent for legumes included in pulses." In the present invention, legume plants included in pulses are so-called "pulses," the seeds of which are harvested and used, such as soybean (including edamame), adzuki bean, chickpea, lotus flower, kidney bean, pea, broad bean, runner bean, lima bean, mung bean, cowpea, black eye bean, hyacinth bean, jack bean, lentil, winged bean, etc. Preferred legume plants included in pulses are soybean, adzuki bean, kidney bean, pea, broad bean, chickpea, and lotus flower, with soybean being more preferred.

[0013] In the nodule activity promoter of the present invention, "promotion of nodule activity" means promoting nodule activity in nodule-forming plants, that is, the nitrogen fixation function in a host plant exerted by nodules. Here, "nodules" refer to nodules that form on the roots of plants through symbiosis with bacteria (rhizobia). Within the nodules, rhizobia reduce atmospheric nitrogen and convert it into ammonia nitrogen, which is then supplied to the host, a process known as symbiotic nitrogen fixation. The term "nodule-forming plant" refers to a host plant on which nodules are formed, and in the present invention, preferred examples include legumes included in pulses.

[0014] The legume growth promoter or nodule activity promoter included in the pulses of the present invention comprises a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent. The combination of (A) ascorbic acid or a salt thereof, (B) antioxidant, and (C) organic solvent is carried out by mixing components (A), (B), and (C) in an appropriate mass ratio in advance or at the time of use. In one embodiment, components (A), (B), and (C) are combined to form a single preparation (single-dose preparation (composition)). In another embodiment, for example, a preparation containing component (A) and a preparation containing components (B) and (C) are prepared separately and then combined at the time of use to form a two-dose preparation (kit).

[0015] In the present invention, the IUPAC systematic name of ascorbic acid, which is component (A), is (R)-3,4-dihydroxy-5-((S)-1,2-dihydroxyethyl)furan-2(5H)-one. Ascorbic acid may be in any of the D-, L-, and DL-forms, but the L-form (so-called L-ascorbic acid) is preferred. Commercially available ascorbic acid of various grades can be used. Examples of salts of ascorbic acid include sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, ammonium salts, and salts with nitrogen-containing organic bases such as pyridine, trimethylamine, triethylamine, tributylamine, and diethylamine.

[0016] When applying the legume growth promoter or root nodule activity promoter of the present invention to a plant, the concentration of (A) ascorbic acid or a salt thereof in the composition can be adjusted appropriately depending on the application method, for example, within a range of 100 ppm by mass or more and 300,000 ppm by mass or less. For example, when spraying using a sprayer (e.g., a boom sprayer), the concentration in the spray solution is preferably 100 ppm by mass or more, more preferably 300 ppm by mass or more, and even more preferably 500 ppm by mass or more, and preferably 20,000 ppm by mass or less, more preferably 10,000 ppm by mass or less, and even more preferably 4,500 ppm by mass or less. Furthermore, the concentration is preferably 100 to 20,000 ppm by mass, more preferably 300 to 10,000 ppm by mass, and even more preferably 500 to 4,500 ppm by mass. In the case of aerial spraying, the concentration in the spray solution is preferably 20,000 mass ppm or more, more preferably 80,000 mass ppm or more, and even more preferably 150,000 mass ppm or more, and is preferably 300,000 mass ppm or less, more preferably 250,000 mass ppm or less, and even more preferably 200,000 mass ppm or less. Also, the concentration is preferably 20,000 to 300,000 mass ppm, more preferably 80,000 to 250,000 mass ppm, and even more preferably 150,000 to 200,000 mass ppm.

[0017] In the present invention, the antioxidant, component (B), is specifically at least one selected from the group consisting of tocopherol (vitamin E), dibutylhydroxytoluene (BHT), and butylhydroxyanisole (BHA). Among these, the phenolic antioxidants BHT and BHA are preferred, and BHT is more preferred. In the present invention, antioxidants other than tocopherol, BHT, and BHA, such as sodium erythorbate, propyl gallate, sodium sulfite, potassium sulfite, potassium pyrosulfite, chlorogenic acid, catechin, glutathione, uric acid, etc., preferably one or more selected from sodium sulfite, potassium sulfite, potassium pyrosulfite, chlorogenic acid, catechin, glutathione, and uric acid, can also be used in combination as the antioxidant. Therefore, a preferred embodiment is to use one or more phenolic antioxidants selected from BHT and BHA as the antioxidant, which is component (B), in combination with one or more antioxidants selected from sodium sulfite, potassium sulfite, glutathione, and uric acid.

[0018] When ascorbic acid is dissolved in water containing metal ions, such as tap water, the metal ions react with ascorbic acid to generate hydrogen peroxide, which then undergoes a Fenton reaction with the metal ions to generate hydroxyl radicals. When the hydroxyl radicals enter the plant, they react with lipids present in the cell membrane, triggering a series of lipid peroxidation reactions, generating lipid radicals and lipid peroxy radicals. The use of antioxidants can sequester these radicals.

[0019] In the present invention, (B) the antioxidant is used in the form of a solution dissolved in (C) the organic solvent, whereby the plant growth-promoting or nodule activity-promoting effect of ascorbic acid is effectively exhibited. The legume growth promoter or nodule activity promoter included in the pulses of the present invention may be either a one- or two-component formulation as described above, but is used in a two-component formulation (kit) in which a formulation containing component (A) (first formulation) and a formulation containing components (B) and (C) (second formulation) are separately prepared and combined, and the two are mixed at the time of use. By using a two-component formulation, discoloration that occurs when a formulation containing all of components (A), (B), and (C) is stored can be suppressed. In this case, the first agent containing component (A) may contain one or more antioxidants other than tocopherol, BHT, and BHA, such as sodium sulfite, potassium sulfite, potassium pyrosulfite, chlorogenic acid, catechin, glutathione, and uric acid.

[0020] When the legume growth promoter or root nodule activity promoter of the present invention is applied to a plant, the concentration of the antioxidant (B) in the composition is preferably 0.001 mass ppm or more, more preferably 0.01 mass ppm or more, and even more preferably 0.1 mass ppm or more, and is preferably 100 mass ppm or less, more preferably 20 mass ppm or less, and even more preferably 5 mass ppm or less, and is preferably 0.001 to 100 mass ppm, more preferably 0.01 to 20 mass ppm, and even more preferably 0.1 to 5 mass ppm.

[0021] The organic solvent, component (C), is a solvent used to dissolve the antioxidant (B), and is specifically one or more solvents selected from alcohols having 5 or less carbon atoms and dimethyl sulfoxide (DMSO). Here, examples of alcohols having 5 or less carbon atoms include preferably ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-2-propanol, isobutyl alcohol, 1-pentanol, 3-methyl-1-butanol, 2,2-dimethyl-1-propanol, ethylene glycol, diethylene glycol, propylene glycol, and glycerol, and more preferably isobutyl alcohol and DMSO.

[0022] When one or more selected from BHT and BHA are used as the (B) antioxidant, it is preferable to use DMSO, isobutyl alcohol, or a mixture thereof as the (C) organic solvent.

[0023] When applying the legume growth promoter or nodule activity promoter included in the pulses of the present invention to plants, the concentration of the (C) organic solvent in the composition can be adjusted appropriately depending on the supply method within the range of 1 ppm by mass or more and 100,000 ppm by mass or less. For example, when spraying using a sprayer (e.g., a boom sprayer), the concentration in the spray solution is preferably 1 ppm by mass or more, more preferably 10 ppm by mass or more, and even more preferably 50 ppm by mass or more, and is preferably 10,000 ppm by mass or less, more preferably 5,000 ppm by mass or less, and even more preferably 1,000 ppm by mass or less. Also, the concentration is preferably 1 to 10,000 ppm by mass, more preferably 10 to 5,000 ppm by mass, and even more preferably 50 to 1,000 ppm by mass. In the case of aerial spraying, the concentration in the spray solution is preferably 100 ppm by mass or more, more preferably 1,000 ppm by mass or more, and even more preferably 5,000 ppm by mass or more, and is preferably 100,000 ppm by mass or less, more preferably 50,000 ppm by mass or less, and even more preferably 10,000 ppm by mass or less, and is preferably 100 to 100,000 ppm by mass, more preferably 1,000 to 50,000 ppm by mass, and even more preferably 5,000 to 10,000 ppm by mass.

[0024] The ratio (mass ratio) of the combination of (A) ascorbic acid or a salt thereof, (B) antioxidant, and (C) organic solvent, when component (B) is taken as 1, is preferably 10 or more, more preferably 100 or more, even more preferably 500 or more, and is preferably 3,000,000 or less, more preferably 2,000,000 or less, even more preferably 200,000 or less. Also, it is preferably 10 to 3,000,000, more preferably 100 to 2,000,000, even more preferably 500 to 200,000. Among these, when spraying by a sprayer, 500 to 45,000 is preferable, when spraying by aerial spraying, 150,000 to 2,000,000 is preferable, and further, when spraying by a sprayer, 500 to 4,500 is more preferable, and when spraying by aerial spraying, 150,000 to 200,000 is preferable. Furthermore, when component (B) is taken as 1, component (C) preferably has a molecular weight of 4 or more, more preferably 10 or more, even more preferably 20 or more, and even more preferably 50 or more, and is preferably 100,000 or less, more preferably 50,000 or less, even more preferably 20,000 or less, and even more preferably 10,000 or less. It is also preferably 4 to 100,000, more preferably 10 to 50,000, even more preferably 20 to 20,000, and even more preferably 50 to 10,000. Of these, 20 to 10,000 is preferred for application by a sprayer, and 2,000 to 100,000 is preferred for application by aerial spraying. Furthermore, 50 to 1,000 is more preferred for application by a sprayer, and 5,000 to 10,000 is more preferred for application by aerial spraying.

[0025] In the present invention, (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent can be used in combination with (D) a surfactant. The use of a surfactant can improve the wettability, adhesion, and penetration of (A) ascorbic acid or a salt thereof onto the plant surface, thereby enhancing the effect of (A) ascorbic acid or a salt thereof or enabling the effect to be exerted more efficiently. As described above, when the legume growth promoter or nodule activity promoter included in the pulses of the present invention is made into a two-component formulation (kit) combining a formulation (first formulation) containing component (A) with a formulation (second formulation) containing components (B) and (C), it is preferable that surfactant (D) be contained in the second formulation containing components (B) and (C). The surfactant may include a nonionic surfactant and / or an anionic surfactant. Examples of nonionic surfactants include one or more selected from sorbitan fatty acid esters, glycerin fatty acid esters, polyoxyalkylene sorbitan fatty acid esters, polyoxyethylene fatty acid esters, polyglycerin fatty acid esters, polyalkylene glycol fatty acid esters, polyoxyethylene resin acid esters, polyoxyethylene alkyl ethers, polyoxyethylene alkylaryl ethers, polyoxyalkylene aryl ethers, polyoxyethylene alkenyl ethers, alkyl polyglycosides, polyoxyalkylene alkyl polyglycosides, and sucrose fatty acid esters. Examples of the anionic surfactant include one or more selected from alkyl sulfates, alkylbenzene sulfonates, polyoxyalkylene alkyl ether sulfates, polyoxyalkylene alkenyl ether sulfates, polyoxyalkylene alkylaryl ether sulfates, fatty acid salts, pyrophosphates, lauryl phosphate, polycarboxylic acid type polymers, polyoxyethylene alkylene alkyl acetates, aromatic sulfonate formalin condensates, polyoxyethylene distyrenated ether sulfates, alkyl diphenyl ether disulfonates, dialkyl sulfosuccinates, alkyl naphthalene sulfonates, and the like. Of these, from the viewpoint of preventing phytotoxicity to plants when excessively applied, the nonionic surfactant is preferably one or more selected from sorbitan fatty acid esters, glycerin fatty acid esters, polyoxyalkylene sorbitan fatty acid esters, polyalkylene glycol fatty acid esters, polyoxyethylene resin acid esters, polyoxyethylene alkyl ethers, alkyl polyglycosides, and sucrose fatty acid esters, and more preferably one or more selected from sorbitan fatty acid esters, glycerin fatty acid esters, polyalkylene glycol fatty acid esters, polyoxyethylene resin acid esters, and polyoxyethylene alkyl ethers, and the anionic surfactant is preferably one or more selected from alkyl sulfates (e.g., sodium lauryl sulfate, ammonium lauryl sulfate, triethanolamine lauryl sulfate, etc.) and fatty acid salts.

[0026] When applying a growth promoter or nodule activity promoter for legumes included in the pulses of the present invention to plants, the concentration of surfactant (D) in the composition can be adjusted appropriately depending on the supply method within the range of 10 mass ppm or more and 30,000 mass ppm or less. For example, when spraying using a sprayer (e.g., a boom sprayer), the concentration in the spray solution is preferably 10 ppm by mass or more, more preferably 100 ppm by mass or more, and even more preferably 200 ppm by mass or more, and is preferably 5,000 ppm by mass or less, more preferably 1,000 ppm by mass or less, and even more preferably 500 ppm by mass or less. Also, the concentration is preferably 10 to 5,000 ppm by mass, more preferably 10 to 1,000 ppm by mass, and even more preferably 100 to 500 ppm by mass. In the case of aerial spraying, the concentration in the spray solution is preferably 100 ppm by mass or more, more preferably 500 ppm by mass or more, and even more preferably 1,000 ppm by mass or more, and is preferably 10,000 ppm by mass or less, more preferably 8,000 ppm by mass or less, and even more preferably 5,000 ppm by mass or less, and is preferably 100 to 10,000 ppm by mass, more preferably 500 to 8,000 ppm by mass, and even more preferably 1,000 to 5,000 ppm by mass. Furthermore, when a surfactant (D) is combined, the ratio (mass ratio) of component (D) is preferably 10 or more, more preferably 50 or more, and even more preferably 100 or more, relative to component (B) being 1, and is preferably 300,000 or less, more preferably 100,000 or less, and even more preferably 5,000 or less. Also, the ratio is preferably 10 to 300,000, more preferably 50 to 100,000, and even more preferably 100 to 5,000. In the case of spraying with a sprayer, a ratio of 100 to 5,000 is preferred, and in the case of spraying by aerial spraying, a ratio of 1,000 to 50,000 is preferred. In the case of spraying with a sprayer, a ratio of 100 to 500 is more preferred, and in the case of spraying by aerial spraying, a ratio of 1,000 to 5,000 is more preferred.

[0027] In the present invention, (E) a chelating agent can be used in combination with (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent. Use of a chelating agent can improve the stability of (A) ascorbic acid or a salt thereof, and as a result, the effect of ascorbic acid or a salt thereof can be stabilized. As described above, when the legume growth promoter or nodule activity promoter included in the pulses of the present invention is made into a two-component formulation (kit) combining a formulation (first formulation) containing component (A) with a formulation (second formulation) containing components (B) and (C), it is preferable that the chelating agent (E) is contained in the first formulation containing component (A). Examples of chelating agents include aminocarboxylic acid chelating agents, phosphonic acid chelating agents, hydroxycarboxylic acid chelating agents, and polycarboxylic acid chelating agents. Examples of aminocarboxylic acid chelating agents include ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), diethylenetriaminepentaacetic acid (DTPA), methylglycine diacetic acid (MGDA), triethylenetetraminehexaacetic acid (TTHA), glutamic acid diacetic acid (GLDA), hydroxyethyliminodiacetic acid (HIDA), dihydroxyethylglycine (DHEG), aspartic acid diacetic acid (ASDA), ethylenediaminesuccinic acid (EDDS), and salts thereof. Examples of phosphonic acid chelating agents include hydroxyethylidene diphosphonic acid (HEDP), nitrilotrismethylenephosphonic acid (NTMP), phosphonobutanetricarboxylic acid (PBTC), ethylenediaminetetramethylenephosphonic acid (EDTMP), and salts thereof. Examples of the hydroxycarboxylic acid chelating agent include citric acid, malic acid, tartaric acid, gluconic acid, lactic acid, and salts thereof. Examples of polycarboxylic acid chelating agents include succinic acid, oxalic acid, glutaric acid, adipic acid, fumaric acid, malonic acid, and salts thereof.

[0028] When the legume growth promoter or root nodule activity promoter of the present invention is applied to a plant, the concentration of the chelating agent (E) in the composition is preferably 0.01 ppm by mass or more, more preferably 0.1 ppm by mass or more, and even more preferably 1 ppm by mass or more, and is preferably 100 ppm by mass or less, more preferably 50 ppm by mass or less, and even more preferably 10 ppm by mass or less, and is preferably 0.01 to 100 ppm by mass, more preferably 0.1 to 50 ppm by mass, and even more preferably 1 to 10 ppm by mass. When a chelating agent (E) is combined, the ratio (mass ratio) of component (E) is preferably 0.01 or more, more preferably 0.1 or more, even more preferably 1 or more, and is preferably 100 or less, more preferably 50 or less, even more preferably 10 or less, when component (B) is taken as 1. The ratio is also preferably 0.01 to 100, more preferably 0.1 to 100, more preferably 1 to 100, even more preferably 1 to 10.

[0029] Suitable combinations of (B) antioxidant, (C) organic solvent, and (D) surfactant used together with (A) ascorbic acid include, for example, the following. A combination in which component (B) is an antioxidant containing one or more selected from BHT and BHA, component (C) is an organic solvent containing isobutyl alcohol or DMSO, and component (D) is a surfactant containing one or more selected from sorbitan fatty acid esters, glycerin fatty acid esters, polyalkylene glycol fatty acid esters, polyoxyethylene resin acid esters, and polyoxyethylene alkyl ethers, and optionally further containing an alkyl sulfate. A combination in which component (B) is an antioxidant containing BHT, sodium sulfite, glutathione, and uric acid, component (C) is an organic solvent containing isobutyl alcohol or DMSO, and component (D) is one or more surfactants selected from sorbitan fatty acid esters, glycerin fatty acid esters, polyalkylene glycol fatty acid esters, polyoxyethylene resin acid esters, and polyoxyethylene alkyl ethers, and optionally further containing an alkyl sulfate.

[0030] As shown in the examples below, when soybean seeds, which are nodule-forming plants, are sown and then inoculated with rhizobia to cultivate the soybeans, adding a combination of an antioxidant such as BHT and an organic solvent to ascorbic acid increased nodule weight and nodule activity, promoted the production of ureides in the nodules, and promoted the growth of the host plant. Therefore, a combination of ascorbic acid, a specific antioxidant, and an organic solvent can be a growth promoter or a nodule activity promoter for legumes included in beans, and can be used to promote the growth or nodule activity of legumes included in beans, or can be used to produce a growth promoter or a nodule activity promoter for legumes included in beans. It has been reported that the amount of ureide derived from nodules correlates with grain yield (Diagnosing method for total basal nitrogen application in rotational field soybeans, Ibaraki Prefectural Agricultural Center, Agricultural Research Institute, H18 Main Results; http: / / www.pref.ibaraki.jp / nourinsuisan / noken / seika / h18pdf / documents / 27.pdf). Sex enhancers are believed to be useful in increasing grain yield.

[0031] Since the nitrogen fixation function in a host plant can be evaluated as the ability to produce ureide or amide, the nodule activity of the present invention can be evaluated specifically as the amount of ureide or amide produced per wet nodule weight. The amount of ureide or amide produced can be calculated by measuring the amount of ureide or amide in exudate collected after cutting the plant (e.g., cutting the above-ground part just below the cotyledons). Examples of ureides include allantoin, allantoic acid, and citrulline, and examples of amides include asparagine and glutamine. It is preferable to measure the amount of allantoic acid or asparagine.

[0032] The growth promoters or nodule activity promoters for legumes included in the above-mentioned pulses can be used as compositions (e.g., various agricultural or horticultural materials) for promoting the growth or nodule activity of legumes included in pulses, or as materials (single substances) or formulations to be added or incorporated into cultivation substrates for cultivating plants, such as soil, culture medium, or nutrient solution for hydroponics. The composition may be in the form of a liquid or gel, or may be in the form of a solid (block, powder, granules, etc.). Here, the composition may be a composition in which components (A), (B), and (C) are mixed in advance, or may be a composition in which a formulation containing component (A) and a formulation containing components (B) and (C) are separately prepared and then mixed together at the time of use.

[0033] The composition may contain any other components in addition to the components (A) to (C) and further (D) and (E) used in the present invention, such as solvents (e.g., water, buffer solutions, culture media, and solutions for hydroponics), carriers (e.g., zeolite, silica, bentonite, Glauber's salt, diatomaceous earth, vermiculite, perlite, peat moss, activated carbon, humus, talc, clay, carbon black, pulp, straw, soybean meal, kaolin, montmorillonite, and alumina), pH adjusters for promoting dissolution of the compounds, wetting agents for increasing the adhesiveness to plants or soil, fertilizer components for increasing fertilizer efficacy, agricultural chemical components, binders, and bulking agents. Examples of suitable compounds include plant growth-promoting microorganisms such as rhizobia and mycorrhizal fungi, essential nutrients for plants, flavonoids, organic acids, amino acids, peptides, nucleosides, nucleotides, nucleic acid bases, sugars, monohydric alcohols, food additives, microbial extracts, plant hormones, NOD factors, i.e., lipo-chitooligosaccharides, synthetic lipo-chitooligosaccharides, chitooligosaccharides, chitinous compounds, linoleic acid or its derivatives, linolenic acid or its derivatives, karrikins, acyl-homoserine lactone derivatives, betaine compounds, and phenolic compounds.

[0034] When the legume growth promoter or root nodule activity promoter of the present invention is used as a one-component formulation, it is preferable to incorporate one or more carriers selected from zeolite, silica, bentonite, and sodium sulfate in order to improve the storage stability or inhibit browning of the formulation, and it is more preferable to incorporate silica in order to improve storage stability and inhibit browning. In this case, the concentration of the carrier (component (F)) in the composition is preferably 50 ppm by mass or more, more preferably 100 ppm by mass or more, and even more preferably 250 ppm by mass or more, and preferably 1,000 ppm by mass or less, more preferably 750 ppm by mass or less, and even more preferably 650 ppm by mass or less. It is also preferably 50 to 1,000 ppm by mass, more preferably 100 to 750 ppm by mass, and even more preferably 250 to 650 ppm by mass. When the (F) carrier is combined, the mass ratio of component (F) is preferably 50 or more, more preferably 100 or more, even more preferably 250 or more, and is preferably 1,000 or less, more preferably 750 or less, even more preferably 650 or less, assuming that component (B) is 1. The mass ratio is also preferably 50 to 1,000, more preferably 100 to 750, even more preferably 250 to 650.

[0035] Compositions for aerial application may also contain emulsifiers such as polyoxyethylene fatty acid esters and oils such as decyl alcohol.

[0036] Examples of the composition include, but are not limited to, cultivation substrates (e.g., agricultural or horticultural soil, culture medium, culture solution for hydroponics, water, etc.) containing at least components (A) to (C) of the present invention, fertilizers, water for watering, microbial materials such as rhizobia materials, soil conditioners, pesticides, sowing materials, and plant supplements (e.g., activators, nutrients, etc.).

[0037] The fertilizers, microbial materials, soil conditioners, sowing materials, and plant supplements are preferred because they contribute to improving the soil in which plants are grown. The fertilizers, microbial materials, soil conditioners, sowing materials, and plant supplements may be solid or liquid, and if solid, may be in the form of blocks, powders, granules, etc., but are preferably powders or granules. In addition to containing components (A) to (C) as active ingredients, the fertilizers, microbial materials, soil conditioners, sowing materials, and plant supplements may also contain components of fertilizers, microbial materials, soil conditioners, sowing materials, and plant supplements that are typically used in plant cultivation.

[0038] The cultivation substrate, fertilizer, microbial materials such as rhizobia materials, soil conditioners, pesticides, sowing materials, and plant supplements may be prepared by adding components (A) to (C) of the present invention to ordinary cultivation substrates (e.g., agricultural or horticultural soil, culture medium, culture medium, hydroponic solution, water, etc.), fertilizer, microbial materials such as rhizobia materials, soil conditioners, pesticides, sowing materials, and plant supplements (e.g., activators, nutrients, etc.).

[0039] The composition may be prepared by separately preparing a formulation containing component (A) and a formulation containing components (B) and (C) and mixing them together at the time of use. When a two-dose formulation is prepared by mixing a formulation containing component (A) (first agent) and a formulation containing components (B) and (C) (second agent) at the time of use, the weight ratio of the first agent to the second agent is 1 to 100, preferably 1 to 50, and more preferably 1 to 25. When preparing the first agent and the second agent by dissolving each in water, the concentration of the first agent in the aqueous solution is preferably 100 ppm by mass or more and 300,000 ppm by mass or less, and the concentration of the second agent in the aqueous solution is preferably 10 ppm by mass or more and 100,000 ppm by mass or less.

[0040] The method of supplying the growth promoter or nodule activity promoter for legumes included in the pulses of the present invention is not particularly limited, as long as it is applied to plants so as to exert the effects of the present invention. That is, there are no particular limitations as long as the composition comes into contact with or is delivered to the plant body of a legume plant included in pulses or to the soil in the rhizosphere of the plant, and examples thereof include surface spraying on the soil, irrigation, plowing in, foliar spraying on plants, application by mixing with fertilizer, addition to a hydroponic solution, or application or smearing on seeds before sowing (for example, seed dressing), but it is preferable for the components of the present invention to be applied in the form of a spray solution diluted with water, and foliar spraying is particularly preferred. The spray solution may be prepared at the time of application, and the dilution water used in this case may be any of agricultural water, well water, groundwater, river water, lake water, tap water, etc.

[0041] The method of application is not particularly limited, but examples thereof include a spray method, i.e., a method of spraying the application liquid in the form of a mist by atomization. By using such a method, the legume growth promoter or root nodule activity promoter included in the pulses of the present invention adheres to the plant and then exhibits good spreadability on the plant. Specific examples of spraying methods include manual spraying using a sprayer, atomizer, or sprayer (e.g., a boom sprayer), and aerial spraying using an airplane, helicopter, drone, etc.

[0042] The application amount of the legume growth promoter or root nodule activity promoter included in the pulses of the present invention depends on the concentrations of components (A) to (C) contained in the composition at the time of application. For example, when the concentration of component (A) contained in the spray solution is 100 to 300,000 mass ppm, the amount of component (A) used per plant in the composition is preferably 1 mg or more, more preferably 5 mg or more, more preferably 10 mg or more, and preferably 150 mg or less, more preferably 100 mg or less, more preferably 50 mg or less. Also, the amount is preferably 1 to 150 mg, more preferably 5 to 100 mg, more preferably 10 to 50 mg. The legume growth promoter or root nodule activity promoter included in the pulses of the present invention may be applied in an amount within the above range at once or in multiple applications.

[0043] The timing and frequency of application may vary depending on the type of legume included in the beans, but in the case of soybeans, when applying by surface spraying, irrigation, plowing into the cultivation substrate such as soil, or seed dressing, it is usually preferable to apply once or 1 to 3 times before or at the same time as sowing, and when applying after sowing, it is preferable to apply from the early vegetative growth period before the reproductive growth period to the grain enlargement period after the reproductive growth period.

[0044] In relation to the above-described embodiment, the present invention further discloses the following aspects. <1> A growth promoter for legumes, including beans, comprising a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms, and dimethyl sulfoxide, and the ratio (mass ratio) of (A) ascorbic acid or a salt thereof is 10 to 3,000,000 when the component (B) is taken as 1. <2> A growth promoter for legumes, including beans, comprising a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms and dimethyl sulfoxide, and the proportion (mass ratio) of the (C) organic solvent is 4 to 100,000 relative to the proportion of component (B) taken as 1. <3> A root nodule activity promoter comprising a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms, and dimethyl sulfoxide, and the ratio (mass ratio) of (A) ascorbic acid or a salt thereof is 10 to 3,000,000 when the component (B) is taken as 1. <4> A root nodule activity promoter comprising a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms and dimethyl sulfoxide, and the proportion (mass ratio) of the (C) organic solvent is 4 to 100,000 when the proportion of component (B) is 1. <5> A yield-enhancing agent for leguminous plants, including pulses, comprising a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms, and dimethyl sulfoxide, and the ratio (mass ratio) of (A) ascorbic acid or a salt thereof is 10 to 3,000,000 relative to the ratio of component (B) being 1. <6> A yield-enhancing agent for leguminous plants, including pulses, comprising a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms and dimethyl sulfoxide, and the proportion (mass ratio) of the (C) organic solvent is 4 to 100,000 relative to the proportion of component (B) taken as 1. <7> The ratio (mass ratio) of (A) ascorbic acid or a salt thereof to (C) the organic solvent is, when the ratio of component (B) is 1, 10 to 3,000,000, preferably 100 to 2,000,000, more preferably 500 to 200,000, and the ratio of component (C) is 4 to 100,000, preferably 10 to 50,000, more preferably 20 to 20,000, and even more preferably 50 to 10,000. <1> ~ <6> The agent according to any one of the above. <8> (B) The antioxidant further includes one or more selected from sodium sulfite, potassium sulfite, glutathione, and uric acid. <1> ~ <7> The agent according to any one of the above. <9> (C) the organic solvent is at least one selected from isobutyl alcohol and dimethyl sulfoxide; <1> ~ <8> The agent according to any one of the above.

[0045] <10> Furthermore, (D) a surfactant is combined, <1> ~ <9> The agent according to any one of the above. <11> The proportion (mass ratio) of the surfactant (D) is preferably 10 to 300,000, more preferably 50 to 100,000, and even more preferably 100 to 5,000, when the proportion of the surfactant (B) is 1. <10> The agent described in <12> (D) the surfactant comprises a nonionic surfactant and / or an anionic surfactant; <10> or <11> The agent described in <13> (D) The nonionic surfactant is at least one selected from sorbitan fatty acid esters, glycerin fatty acid esters, polyalkylene glycol fatty acid esters, and polyoxyethylene alkyl ethers, and the anionic surfactant is an alkyl sulfate ester salt. <12> The agent described in <14> (D) The surfactant includes a nonionic surfactant and an anionic surfactant. <12> The agent described in <15> (D) The nonionic surfactant is at least one selected from sorbitan fatty acid esters, glycerin fatty acid esters, polyalkylene glycol fatty acid esters, polyoxyethylene resin acid esters, and polyoxyethylene alkyl ethers, and the anionic surfactant is at least one selected from alkyl sulfate ester salts and fatty acid salts. <14> The agent described in <16> Furthermore, (E) a chelating agent is combined, <1> ~ <15> The agent according to any one of the above. <17> The ratios (mass ratios) of (A) ascorbic acid or a salt thereof, (C) organic solvent, (D) surfactant, and (E) chelating agent, when component (B) is taken as 1, are such that component (A) is 10 to 3,000,000, preferably 100 to 2,000,000, and more preferably 500 to 200,000; component (C) is 4 to 100,000, preferably 10 to 50,000, more preferably 20 to 20,000, and even more preferably 50 to 10,000; component (D) is preferably 10 to 300,000, more preferably 50 to 100,000, and even more preferably 100 to 5,000; and component (E) is preferably 0.01 to 100, more preferably 0.1 to 100, more preferably 1 to 100, and even more preferably 1 to 10. <16> The agent described in <18> (E) The chelating agent is at least one selected from ethylenediaminetetraacetic acid and ethylenediamine-N,N'-disuccinic acid; <16> or <17> The agent described in <19> Furthermore, (F) a carrier is combined, <1> ~ <18> The agent according to any one of the above. <20> The ratio (mass ratio) of the (F) carrier is 10 to 10,000, preferably 50 to 1,000, more preferably 100 to 750, and even more preferably 250 to 650, when the ratio of component (B) is 1. <19> The agent described in <21> (F) The carrier is one or more selected from zeolite, silica, bentonite, and sodium sulfate; <19> or <20> The agent described in

[0046] <22> The composition comprises a first agent containing the component (A) and a second agent containing the components (B) and (C), and the two agents are combined when used. <1> ~ <9> The agent according to any one of the above. <23> A method for promoting the growth of legumes, including pulses, comprising the step of combining (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent with water and applying the resulting mixture to soil or plants, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms and dimethyl sulfoxide, and the ratio (mass ratio) of (A) ascorbic acid or a salt thereof is 10 to 3,000,000 relative to the ratio of component (B) being 1. <24> A method for promoting root nodule activity, comprising the step of applying to soil or a plant a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent with water, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms and dimethyl sulfoxide, and the ratio (mass ratio) of (A) ascorbic acid or a salt thereof is 10 to 3,000,000 relative to the ratio of component (B) being 1. <25> A method for increasing yield of legumes, including pulses, comprising a step of combining (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent with water and applying the resulting mixture to soil or plants, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms and dimethyl sulfoxide, and the ratio (mass ratio) of (A) ascorbic acid or a salt thereof is 10 to 3,000,000 when the component (B) is taken as 1. <26> A first agent containing the component (A) and a second agent containing the components (B) and (C) are prepared, and the two are combined and applied to soil or plants when in use. <23> ~ <25> A method according to any one of the preceding claims. <27> The weight ratio of the first agent is 1 to 100 when the weight ratio of the second agent is 1. <22> The agent described in <28> Dissolve the first and second agents in water before use. <26> The method described below. <29> The concentration of the first agent in the aqueous solution is 100 mass ppm or more and 300,000 mass ppm or less, and the concentration of the second agent is 10 mass ppm or more and 100,000 mass ppm or less. <28> The method described below.

[0047] <30> Use of a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent for producing a growth promoter for legumes, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms, and dimethyl sulfoxide, and the ratio (mass ratio) of (A) ascorbic acid or a salt thereof is 10 to 3,000,000 relative to the ratio of component (B) being 1. <31> Use of a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent for producing a growth promoter for legumes, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms and dimethyl sulfoxide, and the ratio (mass ratio) of the (C) organic solvent is 4 to 100,000 relative to the ratio of component (B) to component (B) as 1. <32> Use of a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent for producing a nodule activity promoter, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or fewer carbon atoms and dimethyl sulfoxide, and the ratio (mass ratio) of (A) ascorbic acid or a salt thereof is 10 to 3,000,000 when the component (B) is taken as 1. <33> Use of a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent for producing a nodule activity promoter, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms and dimethyl sulfoxide, and the ratio (mass ratio) of the (C) organic solvent is 4 to 100,000 when the component (B) is taken as 1. <34> A use of a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent for producing a yield-enhancing agent for legumes, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms and dimethyl sulfoxide, and the ratio (mass ratio) of (A) ascorbic acid or a salt thereof is 10 to 3,000,000 relative to the ratio of component (B) to be 1. <35> A use of a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent for producing a yield-enhancing agent for legumes, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms and dimethyl sulfoxide, and the ratio (mass ratio) of the (C) organic solvent is 4 to 100,000 relative to the ratio of component (B) to component (B) as 1. <36> The ratio (mass ratio) of (A) ascorbic acid or a salt thereof to (C) the organic solvent is, when the ratio of component (B) is 1, 10 to 3,000,000, preferably 100 to 2,000,000, more preferably 500 to 200,000, and the ratio of component (C) is 4 to 100,000, preferably 10 to 50,000, more preferably 20 to 20,000, and even more preferably 50 to 10,000. <30> ~ <35> The use according to any one of the preceding claims. <37> (B) The antioxidant further includes one or more selected from sodium sulfite, potassium sulfite, glutathione, and uric acid. <30> ~ <36> The use according to any one of the preceding claims. <38> (C) the organic solvent is at least one selected from isobutyl alcohol and dimethyl sulfoxide; <30> ~ <37> The use according to any one of the preceding claims. <39> Furthermore, (D) a surfactant is combined, <30> ~ <38> The use according to any one of the preceding claims. <40> The proportion (mass ratio) of the surfactant (D) is preferably 10 to 300,000, more preferably 50 to 100,000, and even more preferably 100 to 5,000, when the proportion of the surfactant (B) is 1. <39> Use as described in. <41> (D) the surfactant includes at least a nonionic surfactant or an anionic surfactant; <39> or <40> Use as described in. <42> (D) The surfactant further contains an anionic surfactant in addition to the nonionic surfactant. <41> Use as described in. <43> (D) The nonionic surfactant is at least one selected from sorbitan fatty acid esters, glycerin fatty acid esters, polyalkylene glycol fatty acid esters, polyoxyethylene resin acid esters, and polyoxyethylene alkyl ethers, and the anionic surfactant is at least one selected from alkyl sulfate ester salts and fatty acid salts. <42> Use as described in. <44> Furthermore, (E) a chelating agent is combined, <30> ~ <43> The use according to any one of the preceding claims. <45> The ratios (mass ratios) of (A) ascorbic acid or a salt thereof, (C) organic solvent, (D) surfactant, and (E) chelating agent, when component (B) is taken as 1, are such that component (A) is 10 to 3,000,000, preferably 100 to 2,000,000, and more preferably 500 to 200,000; component (C) is 4 to 100,000, preferably 10 to 50,000, more preferably 20 to 20,000, and even more preferably 50 to 10,000; component (D) is preferably 10 to 300,000, more preferably 50 to 100,000, and even more preferably 100 to 5,000; and component (E) is preferably 0.01 to 100, more preferably 0.1 to 100, more preferably 1 to 100, and even more preferably 1 to 10. <44> Use as described in. <46> (E) The chelating agent is at least one selected from ethylenediaminetetraacetic acid and ethylenediamine-N,N'-disuccinic acid; <44> or <45> Use as described in. <47> The composition comprises a first agent containing the component (A) and a second agent containing the components (B) and (C), and the two agents are combined when used. <30> ~ <38> The use according to any one of the preceding claims. <48> <1> , <2> , <5> , <6> , <23> , <25> , <30> , <31> , <34> and <35> In the above, the legumes included in the beans are preferably beans selected from soybean, adzuki bean, chickpea, lotus grass, kidney bean, peanut, broad bean, pea, scarlet runner bean, lima bean, mung bean, cowpea, lamb bean and jack bean, more preferably beans selected from soybean, adzuki bean, kidney bean, pea, broad bean, chickpea and lotus grass, more preferably soybean. [Example]

[0048] Test Example 1: Effect of application of ascorbic acid solution Ascorbic acid was dissolved in water and deionized water to prepare aqueous solutions of ascorbic acid. The effects of application of the aqueous solutions of ascorbic acid on soybeans (Glycine max) were examined. The effect of a single foliar spray of 500 ppm ascorbic acid on soybean yield was evaluated. The test plots 1 to 3 evaluated are as follows: 1 plot: control (no application) 2nd section: foliar spray of ascorbic acid 500 ppm (dissolved in tap water) Section 3: foliar spray of 500 ppm ascorbic acid (dissolved in deionized water)

[0049] (1)Cultivation conditions Soybean cultivation was carried out in a field in Tochigi Prefecture, and the soybean cultivar "Sato no Hohoemi" was used. Within the field, foliar spray treatments were carried out in plots of 2m width, excluding the surrounding areas. Within each test plot, 12 plots were used for the control (test plot 1), and 3 plots were used for the ascorbic acid application plots (test plots 2 and 3). An average of 19 plants were grown within the 2m area set as one plot.

[0050] (2) Foliar spray treatment and measurement of seed weight Ascorbic acid was used as food additive grade vitamin C (L-ascorbic acid) fine mesh type SSS manufactured by Fuso Chemical Co., Ltd. A 500 ppm by mass aqueous solution of ascorbic acid was prepared by dissolving it in tap water or deionized water. Using a battery-powered sprayer (GT-5HS, Koshin Corporation), 20 mL of aqueous ascorbic acid solution per plant was sprayed so that it covered the entire soybean plant. Spraying was performed 60 days after sowing, at a growth stage corresponding to the seed filling stage. Harvesting was performed 108 days after sowing. After harvesting, all seeds were collected from each plant and dried at 100°C for 48 hours. Dry seed mass was measured as yield data.

[0051] (3) Results After calculating the average seed weight of each individual in one plot, the average value and standard deviation of the three plots were calculated (Fig. 1). The graph in the figure shows the average value ± standard deviation. When the water was used in agriculture, it was found that tap water and deionized water had different effects on seed weight. In other words, the effect of ascorbic acid was lost when using tap water, which is used in agriculture.

[0052] Test Example 2: Solubility of dibutylhydroxytoluene in various solvents The amount of BHT dissolved in 100 μL of isobutyl alcohol was varied. It was confirmed that BHT dissolved uniformly up to 25 mg, i.e., a BHT:isobutyl alcohol ratio of 1:4 (Figure 2). Solubility was also confirmed for other solvents at a BHT:solvent ratio of 1:4. Of the solvents tested, BHT dissolved uniformly in ethanol, methanol, 2-propanol, 1-butanol, and dimethyl sulfoxide (DMSO), with the exception of glycerin (Figure 3). BHT is known to be poorly soluble in water. All reagents used were manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

[0053] Example 1 Preparation of a spray solution having plant growth promoting, nodule activity promoting and yield increasing effects (1) Preparation of two-dose composition The two-part composition shown in Table 1 was prepared using the following blended ingredients. Component (B), one or more selected from BHT, BHA, and tocopherol, was dissolved in component (C) beforehand, and then component (D) was mixed in to prepare a second part. Next, one or more selected from component (A) and component (E) were mixed to prepare a first part. Each composition example shown in Table 2 was prepared as a two-part composition in which component (A), component (B), component (C), component (D), and component (E) were blended in the amounts shown in the table.

[0054] (2) Preparation of spray solution and solubility evaluation The solubility of the spray solution was evaluated by dissolving the prepared two-part composition in 100 mL of tap water heated to 30°C. That is, the second part was dissolved in heated tap water, and then the first part was dissolved therein, and the solubility was evaluated.

[0055] (3) Reagents used Ascorbic acid was used as "Food Additive Grade Vitamin C (L-Ascorbic Acid) Fine Mesh Type SSS" manufactured by Fuso Chemical Co., Ltd. Sodium ascorbate, tocopherol, BHT, BHA, ethanol, methanol, 2-propanol, isobutyl alcohol, 1-butanol, DMSO, and citric acid were manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. EDTA was manufactured by Dojindo Laboratories, Inc. Surfactants used included sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, polyoxyethylene lauryl ether, polyoxyethylene alkyl ether, and polyethylene glycol monolaurate.

[0056] (4) Solubility evaluation results The aqueous solubility of the spray solution prepared using each composition was evaluated by its appearance according to the following criteria. The results are shown in Table 2. ◯: No residue remains and the solution is homogeneous. △: Some undissolved material is observed, but the mixture becomes uniform immediately after stirring. ×: Some remains undissolved, and the mixture does not become uniform even immediately after stirring.

[0057] The results in Table 2 show that in Composition Examples 1 to 17, a uniform spray solution suitable for spraying as a plant growth promoter can be obtained.

[0058] [Table 1]

[0059] [Table 2]

[0060] Example 2 Evaluation of nodule weight and nodule activity (1) Soil preparation and sowing A medium-term fertilizer release soil (Takii Hydrated Cell Fertilizer Medium-Term Release, Takii Seed Co., Ltd.) and vermiculite granules (Akagi Engei Co., Ltd.) were mixed at a volume ratio of 1:1, and the soil was filled into polypots (10.5 cm diameter, 9 cm height). Soybean seeds "Enrei" (purchased from Nikko Seed Co., Ltd.) were used. After adding 250 mL of tap water per pot, two seeds were sown per pot, one at a depth of approximately 1-2 cm from the soil surface. Each test plot had six replicates (n = 6).

[0061] (2) Inoculation of rhizobia Yeast-Mannitol (YM) medium (K2HPO4 0.5g, MgSO4 7 H2O 0.2g, NaCl 0.1g, Yeast Extract 0.4g, Ma A solid medium was prepared by adding 1.5% agar (Wako Pure Chemical Industries, Ltd.) to 10 g of nitritol and 1 L of distilled water (pH 6.8). The soybean rhizobia (Bradyrhizobium japonicum) strain NBRC14783T was grown on the solid medium. A platinum loop of the grown rhizobia was inoculated into 50 mL of YM liquid medium in a 500 mL Sakaguchi flask and cultured with shaking at 30°C for approximately 36 hours. A rhizobia culture solution with a bacterial turbidity (OD600) of approximately 0.3 was prepared. After sowing, 1 mL of the rhizobia culture solution was dropwise inoculated onto the seeds using a micropipette.

[0062] (3)Cultivation conditions Cultivation from sowing to thinning was carried out in an artificial climate chamber (LPH-411SP, Nippon Medical and Chemical Instruments Manufacturing Co., Ltd.) under photoperiod light conditions (light intensity 440–480 μmol / m 2 / s)16 The light period was 30°C / dark period was 25°C, and the humidity was 50%. Afterwards, the plants were thinned out so that there was one plant per pot. After thinning, the plants were grown outdoors. Watering was carried out by adding tap water to the tray placed under the pot after the water had run out, so that the bottom 5 cm of the pot was covered.

[0063] (4) Foliar spray treatment The spray solution shown in Table 3 was prepared and sprayed at a rate of 6.7 mL per plant using a spray bottle on the 14th day after sowing. Foliar spray was performed only once, and seven test plots were examined. To prepare the spray solution, BHT from component (B) was first dissolved in DMSO from component (C), and then components (C) and (D) were mixed to prepare the second agent. Next, component (A), uric acid (antioxidant), and component (E) were mixed to prepare the first agent. After dissolving the second agent in the water used, the first agent was further dissolved to prepare the spray solution.

[0064] Here, ascorbic acid was used "Food Additive Grade Vitamin C (L-Ascorbic Acid) Fine Mesh Type SSS" manufactured by Fuso Chemical Co., Ltd. BHT, uric acid, DMSO, and EDTA were manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., and EDDS was used "Chelest EDDS-35" manufactured by Chelest Co., Ltd. DMSO and isobutanol were used as component (C). Of component (D), sorbitan monolaurate, sorbitan fatty acid ester, or polyoxyethylene resin acid ester was used as the nonionic surfactant. Furthermore, of component (D), fatty acid soap potassium salt was used as the anionic surfactant.

[0065] [Table 3]

[0066] (5) A method for collecting exudate fluid to measure the amount of ureido nitrogen, which is an index of nodule activity After 21 days of cultivation, soybean stems were cut at the cotyledonary node using pruning shears, and a 1.5 mL microtube (Eppendorf) filled with #10 cotton balls (Kawamoto Sangyo Co., Ltd.) was placed over the cut stem to collect exudation fluid from the stem for 2 hours. After exudation fluid collection, the cotton balls were stored in a -80°C freezer. The volume of exudation fluid was calculated by comparing the weight of the cotton balls before and after collection.

[0067] (6) Extraction of exudate components collected on a cotton ball The exudate components contained in the cotton ball were eluted with ultrapure water and filtered using a Micro Bio-Spin Chromatography column (Bio-Rad Laboratories, Inc.). The filtrate remaining in the column was collected by centrifugation using a centrifuge (CR15RN, Hitachi, Ltd.) at 15,000 rpm for 1 minute. The filtrate was diluted 1,000 times and quantified.

[0068] (7) Apparatus and method for determining the amount of ureido nitrogen The HPLC and mass spectrometers used were an Agilent 1260 Infinity LC system (Agilent Technologies) and an AB SCIEX TripleQuad 4500 system (AB SCIEX Corporation), respectively. The column used was a Scherzo SS-C18 (100 mm x 2 mm, 3 μm) (Intact Corporation), with an oven temperature of 40°C. Five μL of appropriately diluted sample was injected at a flow rate of 0.5 mL / min. The eluents were 0.1% formic acid (eluent A) and 50 mM ammonium acetate / methanol (eluent B). The system was equilibrated at a ratio of 95:5 for eluent A:eluent B before application. After 5 minutes, ureido nitrogen was eluted using a linear gradient of 80:20 for eluent A:eluent B.

[0069] (8) Standards used and quantification The amount of ureido nitrogen in the exudate was analyzed by LC-MS. Allantoin (Tokyo Chemical Industry Co., Ltd.) and allantoic acid (Toronto Research Chemicals Inc.) were used as standards. The standards were analyzed by LC-MS, and a calibration curve was prepared in the range of 10-1000 ppb. Allantoin and allantoic acid were identified in each sample based on the retention time, accurate mass, and MS / MS spectrum match with each reagent. Allantoin and allantoic acid in the samples were quantified using the calibration curve, and the total amount per plant was calculated.

[0070] (9) Results The results of measuring nodule weight and nodule activity are shown in Figures 4 and 5. Of the six replicates in each test plot, the highest and lowest values ​​were excluded, and the average and standard deviation were calculated from the data for four replicates. The graphs in the figures show the average ± standard deviation. Test Example 1 demonstrated that differences in the water quality used to dissolve ascorbic acid affect soybean yield. Test plots 1 to 3 in this example confirmed that even at the early soybean growth stage, nodule weight and nodule activity increased when ascorbic acid was dissolved in deionized water, whereas the effect of ascorbic acid application tended to decrease when ascorbic acid was dissolved in tap water. Comparison of test plots 4 and 5 demonstrated that the addition of an organic solvent was necessary to restore the nodule activity reduced by dissolving ascorbic acid in tap water. Furthermore, comparison of test plots 6 and 7 suggested that EDTA was a more preferable chelating agent for restoring nodule activity.

[0071] Example 3: Examination of the mixing ratio of ascorbic acid and antioxidant The soil preparation and sowing, rhizobia inoculation, exudate collection method for measuring the amount of ureido nitrogen, which is an indicator of nodule activity, experimental procedures before quantifying the exudate, the equipment and measurement method used to quantify the amount of ureido nitrogen, and the standards and quantification items used were the same as in Example 2, and soybeans were cultivated for 21 days.

[0072] (1)Cultivation conditions Cultivation was carried out in an artificial climate chamber (LPH-411SP, Nippon Medical and Chemical Instruments Manufacturing Co., Ltd.) under photoperiod light conditions (light intensity 440–480 μmol / m 2 The temperature was 30°C light / 25°C dark, and the humidity was 50%. Seven days after sowing, the plants were thinned out to one per pot. After the water in the tray placed under the pot had run out, fresh tap water was added to the tray, enough to cover the bottom 5 cm of the pot.

[0073] (2) Foliar spray treatment As in Example 2, the spray solution shown in Table 4 below was prepared, and 6.7 mL per plant was sprayed using a spray bottle on the 14th day after sowing. Foliar spray was performed only once, and seven test plots were examined (Table 4). The amount of water sprayed was kept constant, and the concentration of ascorbic acid in the spray solution was adjusted by changing the amount of ascorbic acid per plant. The reagents used were the same as those in Example 2.

[0074] [Table 4]

[0075] (3) Results The results of measuring nodule activity are shown in Figures 6 and 7. The graphs in the figures show the mean value ± standard deviation. As shown in Figures 6 and 7, when ascorbic acid dissolved in tap water was applied to the leaves, nodule activity remained unchanged or even decreased compared to when no application was performed. Nodule activity decreased in Test Plot 3, which contained no ascorbic acid. However, adding ascorbic acid to Test Plot 3 restored or improved nodule activity. In tests using a water volume simulating application using a sprayer (e.g., a boom sprayer), Test Plot 4, with an ascorbic acid:BHT ratio of 500:1, showed the highest nodule activity, while Test Plot 7, with an ascorbic acid:BHT ratio of 20,000:1, showed a tendency for nodule activity to decrease. Furthermore, as shown in Figure 8, the ascorbic acid:BHT ratio of 20,000:1 caused leaf discoloration, indicating that a mixture ratio of ascorbic acid, component (B), and component (C) of 100-20,000:1:2-100 is preferable. It was also shown that component (C) had the effect of restoring nodule activity even when isobutyl alcohol was used.

[0076] Example 4: Increased soybean yield in the field The effect of foliar application of a composition containing 1,500 ppm ascorbic acid on soybean yield was evaluated. Only one foliar application was performed, and eight test plots were examined (Table 5).

[0077] [Table 5]

[0078] (1)Cultivation conditions Soybean cultivation was carried out in a field in Tochigi Prefecture, and the soybean cultivar "Sato no Hohoemi" was used. Foliar spray treatments were carried out in plots of 2m wide ridges, excluding the surrounding areas. Three plots were tested per test area. An average of 10 plants were grown in the 2m area set as one plot.

[0079] (2) Foliar spray treatment and measurement of seed weight In addition to the reagents described in Example 2, polyoxyethylene lauryl ether was used as a nonionic surfactant. Urea, potassium dihydrogen phosphate, sodium sulfite, glutathione, and EDTA were all manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. The spray solution was prepared by dissolving the reagent in tap water or deionized water. Using a battery-powered sprayer (GT-5HS, Koshin Corporation), 6.7 mL of ascorbic acid solution per plant was sprayed so that the entire soybean plant was covered. Spraying was performed 75 days after sowing, at a growth stage corresponding to the seed filling stage. Harvesting was performed 173 days after sowing. After harvesting, all seeds were collected from each plant and dried at 100°C for 48 hours. Dry seed mass was measured as yield data.

[0080] (3) Results After calculating the average seed weight of each individual plant in each plot, the average value and standard deviation for the eight plots were calculated (Figure 9). The graph in the figure shows the average value ± standard deviation. It was again confirmed that when the same concentration of ascorbic acid was sprayed on leaves, tap water and deionized water had different effects on seed weight. It was shown that adding components (B), (C), and (D) to ascorbic acid could increase yield even when tap water was used. It was also shown that component (D) had a yield-increasing effect when polyoxyethylene lauryl ether was used.

[0081] Example 5 Evaluation of nodule activity by application of high concentrations of ascorbic acid The soil preparation and sowing, rhizobia inoculation, cultivation conditions, exudate collection method for measuring the amount of ureido nitrogen, which serves as an indicator of nodule activity, experimental procedures before quantifying the exudate, the equipment and measurement method used to quantify the amount of ureido nitrogen, and the standards and quantification items used were the same as in Example 3, and soybeans were cultivated for 21 days.

[0082] (1) Foliar spray treatment Foliar spray treatment was performed 14 days after sowing. In addition to the reagents described in Examples 2, 3, and 4, polyethylene glycol monolaurate and glycerin fatty acid ester were used as nonionic surfactants. The spray solution was prepared by dissolving the reagents in tap water or deionized water. Foliar spray was performed only once, and eight test plots containing high concentrations of ascorbic acid were examined, assuming aerial spray (Table 6). Test plots 2 and 3 were sprayed with a spray bottle at 6.7 mL per plant, and test plots 4 to 8 were sprayed at 0.125 mL per plant.

[0083] [Table 6]

[0084] (2) Results The results of measuring nodule activity are shown in Figure 10. Of the six replicates in each test plot, the highest and lowest values ​​were excluded, and the average and standard deviation were calculated from the data for four replicates. The graph in the figure shows the average ± standard deviation. As shown in Figure 10, soybean nodule activity tended to increase in plots 4 and 5, where ascorbic acid was applied with a small water volume, compared with plots 2 and 3. Regardless of the amount of water used for application, using deionized water to dissolve ascorbic acid improved nodule activity compared with tap water. A comparison of plots 5 and 6 revealed that the highest nodule activity was achieved by adding BHT as compound (B), DMSO as compound (C), and polyethylene glycol monolaurate as compound (D) to ascorbic acid. On the other hand, in plot 7, when the ascorbic acid concentration was increased to 300,000 ppm, nodule activity decreased compared with plot 6, indicating that an ascorbic acid concentration of 300,000 ppm or less is preferable when applying with a small water volume. Furthermore, a comparison of plots 8 and 6 indicated that DMSO was preferable to isobutyl alcohol as compound (C). It was also shown that compound (D) had the effect of restoring nodule activity when polyethylene glycol monolaurate was used.

[0085] Example 6 Evaluation of early growth index in adzuki bean (1) Soil preparation and sowing A medium-term fertilizer-release soil (Takii Hydrated Cell Culture Medium-Term Fertilizer, Takii Seed Co., Ltd.) and vermiculite granules (Akagi Engei Co., Ltd.) were mixed at a volume ratio of 1:1, and the soil was filled into polypots (10.5 cm diameter, 9 cm height). Adzuki bean seeds, "Tamba Dainagon Azuki" (purchased from Takii Seed Co., Ltd.), were used. After adding 250 mL of tap water per pot, two seeds were sown in each pot, one at a depth of approximately 1–2 cm from the soil surface. Each test plot had six replicates (n = 6).

[0086] (2) Inoculation of rhizobia A solid medium was prepared by adding 1.5% agar (Wako Pure Chemical Industries, Ltd.) to yeast-mannitol (YM) medium (0.5 g K2HPO4, 0.2 g MgSO4·7H2O, 0.1 g NaCl, 0.4 g yeast extract, 10 g mannitol, and 1 L distilled water (pH 6.8)). Bradyrhizobium japonicum strain NBRC14783T was grown on the solid medium. A loopful of the grown rhizobia was inoculated into 50 mL of YM liquid medium in a 500 mL Sakaguchi flask and cultured with shaking at 30°C for approximately 36 hours. A rhizobia culture solution with a bacterial turbidity (OD600) of approximately 0.3 was prepared. After sowing, 1 mL of the rhizobia culture solution was dropwise inoculated onto the seeds using a micropipette.

[0087] (3)Cultivation conditions Cultivation was carried out in an artificial climate chamber (LPH-411SP, Nippon Medical and Chemical Instruments Manufacturing Co., Ltd.) under photoperiod light conditions (light intensity 440–480 μmol / m 2 The temperature was 25°C light / 20°C dark, and the humidity was 50%. Seven days after sowing, the plants were thinned out to one per pot. After the water in the tray placed under the pot had run out, fresh tap water was added to the tray, enough to cover the bottom 5 cm of the pot.

[0088] (4) Foliar spray treatment A spray solution was prepared, and 21 days after sowing, 6.7 mL per plant was sprayed once using a spray bottle. The spray solution was prepared by dissolving BHT in isobutyl alcohol, mixing the isobutyl alcohol and sorbitan monolaurate, and then dissolving the mixture in water. The ascorbic acid was then dissolved in water to prepare the solution. The reagents used were the same as in Example 2. Test plots 1 to 4 evaluated are as follows: On the 28th day after sowing, the plants were dried at 90°C for 24 hours, and then the dry weight of the above-ground and underground parts of the plants was measured.

[0089] 1 plot: control (no application) 2nd section: foliar spray of ascorbic acid 1,500 ppm (dissolved in tap water) 3rd section: foliar spray of ascorbic acid 1,500 ppm by mass (dissolved in deionized water) 4 sections: 1,500 ppm ascorbic acid + 1 ppm BHT + 50 ppm isobutyl alcohol + 350 ppm sorbitan monolaurate (dissolved in tap water) applied to leaves

[0090] (5) Results The measurement results of the dry weight of the aboveground part and the dry weight of the underground part are shown in Figures 11 and 12. The graphs in the figures show the mean value ± standard deviation. Test Example 1 and Example 2 show that when the same concentration of ascorbic acid is sprayed on the leaves of soybeans, tap water and deionized water have different effects on seed weight and early growth indices. As with soybeans, adzuki beans also show that the effect on early growth indices varies depending on the water quality in which ascorbic acid is dissolved. A comparison of Test Areas 2 and 4 showed that adding components (B), (C), and (D) to ascorbic acid can achieve growth-promoting effects even when tap water is used.

[0091] Example 7 Evaluation of early growth indicators in chickpea (1) Soil preparation and sowing A medium-term fertilizer release soil (Takii Hydrated Cell Fertilizer Medium-Term Release, Takii Seed Co., Ltd.) and vermiculite granules (Akagi Engei Co., Ltd.) were mixed at a volume ratio of 1:1, and the soil was filled into polypots (10.5 cm diameter, 9 cm height). Chickpea seeds were Kaburi variety (purchased from Nikko Seed Co., Ltd.). After adding 250 mL of tap water per pot, two seeds were sown in each pot, one seed at a depth of approximately 1-2 cm from the soil surface. Each test plot had six replicates (n = 6).

[0092] (2) Inoculation of rhizobia A solid medium was prepared by adding 1.5% agar (Wako Pure Chemical Industries, Ltd.) to yeast-mannitol (YM) medium (0.5 g K2HPO4, 0.2 g MgSO4·7H2O, 0.1 g NaCl, 0.4 g yeast extract, 10 g mannitol, and 1 L of distilled water (pH 6.8)). Chickpea rhizobia (Mesorhizobium ciceri) strain NBRC100389T was grown on the solid medium. A loopful of the grown rhizobia was inoculated into 50 mL of YM liquid medium in a 500 mL Sakaguchi flask and cultured with shaking at 30°C for approximately 36 hours. A rhizobia culture solution with a bacterial turbidity (OD600) of approximately 0.3 was prepared. After sowing, 1 mL of the rhizobia culture solution was dropwise inoculated onto the seeds using a micropipette.

[0093] (3) Foliar spray treatment The cultivation conditions and the method of foliar spray treatment were the same as those in Example 6 (Example of adzuki beans). Test plots 1 to 4 evaluated are as follows. On the 28th day after sowing, the plants were dried at 90°C for 24 hours, and then the dry weight of the above-ground parts of the plants was measured.

[0094] 1 plot: control (no application) 2nd section: foliar spray of ascorbic acid 1,500 ppm (dissolved in tap water) 3rd section: foliar spray of ascorbic acid 1,500 ppm by mass (dissolved in deionized water) 4 sections: 1,500 ppm ascorbic acid + 1 ppm BHT + 50 ppm isobutyl alcohol + 350 ppm sorbitan monolaurate (dissolved in tap water) applied to leaves

[0095] (4) Results The results of measuring the dry weight of the aboveground parts are shown in Figure 13. Of the six replicates in each test plot, the highest and lowest values ​​were excluded, and the average and standard deviation were calculated from the data for four replicates. The graph in the figure shows the average ± standard deviation. Test Example 1 and Example 2 show that when the same concentration of ascorbic acid is sprayed on the leaves of soybeans, tap water and deionized water have different effects on seed weight and early growth indices. As with soybeans, chickpeas also showed that the effect on early growth indices differs depending on the water quality in which ascorbic acid is dissolved. A comparison of Test Areas 2 and 4 showed that adding Components (B), (C), and (D) to ascorbic acid can achieve growth-promoting effects even when tap water is used.

[0096] Example 8: Evaluation of seed weight in Lotus japonicus (1) Soil preparation Medium-term fertilizer-effective soil (Takii Hydrated Cell Soil Medium-term Fertilizer-effective, Takii Seed Co., Ltd.) and vermiculite granules (Akagi Engei Co., Ltd.) were mixed in a volume ratio of 1:1, and the soil was filled into a polypot (diameter 6 cm, height 5.5 cm).

[0097] (2) Germination and sowing Lotus japonicus seeds of the Miyakojima MG-20 strain were used. 1 mL of concentrated sulfuric acid (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a 2 mL microtube (Eppendorf Co., Ltd.) filled approximately one-third with seeds, and the tubes were left to stand for 10 minutes. After rinsing five times with tap water, the tubes were submerged for two hours. After adding 100 mL of tap water per pot, three germinated seeds were sown in each pot, one at a depth of approximately 1-2 cm from the soil surface. Each test plot had eight replicates (n = 8).

[0098] (3) Inoculation of rhizobia A solid medium was prepared by adding 1.5% agar (Wako Pure Chemical Industries, Ltd.) to yeast-mannitol (YM) medium (0.5 g K2HPO4, 0.2 g MgSO4·7H2O, 0.1 g NaCl, 0.4 g yeast extract, 10 g mannitol, and 1 L distilled water (pH 6.8)). The Lotus japonicus rhizobia (Mesorhizobium loti) strain MAFF303099 (ML GUS) was grown on the solid medium. A loopful of the grown rhizobia was inoculated into 50 mL of YM liquid medium in a 500 mL Sakaguchi flask and cultured with shaking at 30°C for approximately 36 hours. A rhizobia culture solution with a bacterial turbidity (OD600) of approximately 0.3 was prepared. After sowing, 1 mL of the rhizobia culture solution was dropwise inoculated onto the seeds using a micropipette.

[0099] (4)Cultivation conditions Cultivation was performed indoors under the following conditions: 16 hours of light, 25°C, LED light source (Ogetsu Corporation; model number: VGL-1200W), and light intensity of 400–440 μmol / m 2 / s. Ten days after sowing, the plants were thinned out so that there was one plant per pot. Watering was carried out by adding tap water to the tray placed under the pot after the water had run out, enough to cover the bottom 5 cm of the pot. Starting three weeks after sowing, a 1,000-fold dilution of High Grade Flowering Promoter (Hyponex Japan Co., Ltd.) was added to the water once a week.

[0100] (5) Foliar spray treatment The spray solution shown in Table 7 was prepared, and six weeks after sowing, 6.7 mL per plant was sprayed once using a spray bottle in test plots 2 to 6, and 0.125 mL per plant in test plots 7 to 9. The spray solution shown in Table 13 was prepared, and 14 days after sowing, 6.7 mL per plant was sprayed using a spray bottle. Foliar spray was performed only once, and seven test plots were examined. To prepare the spray solution, BHT (component (B)) was first dissolved in component (C), and then components (C) and (D) were mixed to prepare the second formulation. Next, component (A) and the other components were mixed to prepare the first formulation. The second formulation was dissolved in the water used, and then the first formulation was further dissolved to prepare the spray solution. Mature seeds were harvested one after another, and cultivation was completed 16 weeks after sowing. The number of pods and seed weight of each plant were measured.

[0101] [Table 7]

[0102] (6) Results The measurement results for the number of pods are shown in Figure 14, and the measurement results for the seed weight are shown in Figure 15. The graphs in the figures show the mean value ± standard deviation. Test Example 1 and Example 2 showed that when the same concentration of ascorbic acid was sprayed on the leaves of soybeans, tap water and deionized water had different effects on seed weight. As with soybeans, it was also shown that the effect on seed weight of lotus grass differed depending on the water quality in which ascorbic acid was dissolved. A comparison of Test Plots 3 and 4 showed that adding Components (B), (C), and (D) to ascorbic acid increased the number of pods and seed weight, even when tap water was used. Test Plot 5 showed that adding fertilizer components to the composition also had a yield-increasing effect. Furthermore, as shown in Test Plot 6, sufficient application effects were obtained even when the concentration of the composition was halved. Example 5 shows that application of a high concentration of the composition to soybeans improves nodule activity in the early stages of growth. It was also shown that the addition of components (B), (C), and (D) to ascorbic acid to lotus grass, as with soybeans, can increase yield even when sprayed in small amounts at high concentrations using tap water.

[0103] Example 9 Evaluation of seed weight in broad beans (1) Soil preparation and cultivation conditions Planters (65.3 cm wide, 24.5 cm deep, 18.5 cm high) were filled with 2 L of gravel at the bottom, and then filled with 9 L of medium-term fertilizer-effect soil (Takii Hydrated Cell Culture Medium-Term Fertilizer-Effect, Takii Seed Co., Ltd.). After adding 2 L of tap water per planter, broad bean seedlings (variety: Nintoku Issun, Takii Seed Co., Ltd.) purchased from a home improvement store were transplanted in groups of three. Each test plot was replicated three times (n = 3). Cultivation was carried out outdoors.

[0104] (2) Foliar spray treatment The spray solution was prepared and sprayed 6.7 mL per plant using a spray bottle on the 51st day after planting. Foliar spraying was performed only once, and four types of test plots were examined. The spray solution was prepared by dissolving BHT from component (B) in component (C) beforehand, and then mixing component (C) and component (D) to prepare the second agent. Next, component (A) and the other components were mixed to prepare the first agent. The second agent was dissolved in the water used, and then the first agent was further dissolved therein to prepare the spray solution. The reagents were the same as in Example 1. Test plots 1 to 4 evaluated are as follows: Mature seeds were harvested one by one, and cultivation was terminated 96 days after planting. The number of beans and seed weight of each plant were measured. 1 plot: control (no application) 2nd section: foliar spray of ascorbic acid 1,500 ppm by mass (dissolved in deionized water) 3rd section: foliar spray of ascorbic acid 1,500 ppm (dissolved in tap water) 4 sections: 1,500 ppm ascorbic acid + 1 ppm BHT + 50 ppm isobutyl alcohol + 350 ppm sorbitan monolaurate (dissolved in tap water) applied to leaves

[0105] (3) Results The results of measuring the number of beans per plant are shown in Figure 16, and the results of measuring seed weight are shown in Figure 17. The graphs in the figures show the average value ± standard deviation. A comparison of test plots 1 and 4 showed that adding components (B), (C), and (D) to ascorbic acid increased the number of beans per plant by 7.5% and seed weight by 32.9%, demonstrating that this also has a yield-increasing effect on broad beans. Furthermore, a comparison of test plots 3 and 4 showed that adding components (B), (C), and (D) to ascorbic acid enhanced the yield-increasing effect.

[0106] Example 10 Storage stability test of a composition having plant growth promoting, root nodule activity promoting and yield increasing effects (1) Preparation of one- or two-dose compositions Using 20 g of ascorbic acid as component (A), 13.3 mg of BHT as component (B), 0.67 mL of isobutyl alcohol as component (C), and 4.7 mL of sorbitan monolaurate as component (D), one- or two-component compositions were prepared by the following method. A one-component composition was prepared by dissolving component (B) in component (C), then mixing with component (D), and then mixing with component (A). A two-component composition was prepared by the following method. A second component was prepared by dissolving component (B) in component (C) in advance, and then mixing with component (D). Next, only component (A) was used as the first component. The reagents were the same as in Example 1.

[0107] (2) Storage stability test Half of the prepared one- or two-dose compositions were dispensed into two transparent glass bottles. They were stored for one week in a refrigerator set at 4°C or in a storage cabinet set at 50°C. After one week, the appearance was evaluated, and the aqueous solubility of the stored formulations was evaluated when they were diluted 500 times with water.

[0108] (3) Evaluation results of appearance and solubility of the formulation The results of evaluating the appearance of the formulations are shown in Figure 18. The left photo shows the first and second agents of the two-dose composition after storage at 50°C, the center photo shows the first and second agents of the two-dose composition after storage at 4°C, and the right photo shows the single-dose composition after storage at 50°C and the single-dose composition after storage at 4°C.

[0109] From the results in Figure 18, the two-component composition was found to be different in the samples stored at 4°C and 50°C. No difference in appearance was observed between the two formulations. On the other hand, the color of the single-formulation composition varied depending on the storage temperature. It was also shown that both the single-formulation and two-formulation formulations could produce a uniform spray solution suitable for application as a plant growth promoter. The results of the study suggested that the single-formulation formulation showed a large degree of discoloration during storage, and that the two-formulation formulation was superior in terms of appearance.

[0110] Example 11: Examination of the mixing ratio of organic solvents The soil preparation and sowing, rhizobia inoculation, exudate collection method for measuring the amount of ureido nitrogen, which is an indicator of nodule activity, experimental procedures before quantifying the exudate, the equipment and measurement method used to quantify the amount of ureido nitrogen, and the standards and quantification items used were the same as in Example 2, and soybeans were cultivated for 21 days.

[0111] (1)Cultivation conditions Cultivation was carried out in an artificial climate chamber (LPH-411SP, Nippon Medical and Chemical Instruments Manufacturing Co., Ltd.) under photoperiod light conditions (light intensity 440–480 μmol / m 2 The temperature was 30°C light / 25°C dark, and the humidity was 50%. Seven days after sowing, the plants were thinned out to one per pot. After the water in the tray placed under the pot had run out, fresh tap water was added to the tray, enough to cover the bottom 5 cm of the pot.

[0112] (2) Foliar spray treatment The spray solution shown in Table 8 was prepared, and on the 14th day after sowing, 6.7 mL per plant was sprayed once using a spray bottle to test plots 1 to 5, and 0.125 mL per plant to test plots 6 to 8. Foliar spray was performed only once, and seven test plots were examined (Table 8). The reagents used were the same as those used in Example 5.

[0113] [Table 8]

[0114] (3) Results The results of measuring nodule activity are shown in Figure 19. The graph in the figure shows the mean value ± standard deviation. A comparison of test plots 2 and 3-8 showed that adding components (B), (C), and (D) to ascorbic acid resulted in a greater improvement in nodule activity than when ascorbic acid was applied alone using tap water for dissolution. It was also shown that the effect was achieved even when the mixing ratio of components (B) and (C) was 1:10-10,000.

[0115] Example 12 Evaluation of early growth indicators at low concentrations of antioxidants Soil preparation, sowing, and inoculation with rhizobia were the same as in Example 2, and soybeans were cultivated for 21 days.

[0116] (1)Cultivation conditions Cultivation was carried out in an artificial climate chamber (LPH-411SP, Nippon Medical and Chemical Instruments Manufacturing Co., Ltd.) under photoperiod light conditions (light intensity 440–480 μmol / m 2 The temperature was 30°C light / 25°C dark, and the humidity was 50%. Seven days after sowing, the plants were thinned out to one per pot. After the water in the tray placed under the pot had run out, fresh tap water was added to the tray, enough to cover the bottom 5 cm of the pot.

[0117] (2) Foliar spray treatment A spray solution was prepared, and on the 14th day after sowing, 6.7 mL per plant was sprayed once using a spray bottle. The spray solution was prepared by dissolving BHT in isobutyl alcohol, mixing the isobutyl alcohol and sorbitan monolaurate, and then dissolving the mixture in water. The solution was then prepared by dissolving ascorbic acid in water. The reagents used were the same as in Example 2. Test plots 1 to 3 evaluated are as follows. On the 21st day after sowing, the plants were dried at 90°C for 24 hours, and the dry weight of the aboveground parts of the plants was then measured.

[0118] 1 plot: control (no application) 2nd section: foliar spray of ascorbic acid 1,500 ppm (dissolved in tap water) 3 sections: ascorbic acid 1,500 ppm by mass + BHT 0.01 ppm by mass + isobutyl alcohol 50 ppm by mass + sorbitan monolaurate 350 ppm by mass, foliar spray (dissolved in tap water)

[0119] (3) Results The results of measuring the dry weight of the aboveground parts are shown in Figure 20. The graph in the figure shows the mean value ± standard deviation. A comparison of test plots 2 and 3 showed that even when the concentration of BHT in component (B) was set to 0.01 mass ppm, adding components (B), (C), and (D) to ascorbic acid resulted in a greater growth-promoting effect than when ascorbic acid was dissolved in tap water and sprayed alone.

[0120] Example 13 Evaluation of early growth index when anionic surfactants are used Soil preparation, sowing, and inoculation with rhizobia were the same as in Example 2, and soybeans were cultivated for 21 days.

[0121] (1)Cultivation conditions Cultivation was carried out in an artificial climate chamber (LPH-411SP, Nippon Medical and Chemical Instruments Manufacturing Co., Ltd.) under photoperiod light conditions (light intensity 440–480 μmol / m 2 The temperature was 30°C light / 25°C dark, and the humidity was 50%. Seven days after sowing, the plants were thinned out to one per pot. After the water in the tray placed under the pot had run out, fresh tap water was added to the tray, enough to cover the bottom 5 cm of the pot.

[0122] (2) Foliar spray treatment Foliar spray treatment was performed 14 days after sowing. In addition to the reagents described in Example 2, sodium lauryl sulfate, ammonium lauryl sulfate, and triethanolamine lauryl sulfate were used as anionic surfactants. The spray solution was prepared by dissolving the reagents in tap water. Foliar spray was performed only once, and five test plots were examined (Table 9). The spray solution was prepared and sprayed at 6.7 mL per plant using a spray bottle. On the 21st day after sowing, the plants were dried at 90°C for 24 hours, and then the dry weight of the underground parts of the plants was measured.

[0123] [Table 9]

[0124] (3) Results The results of measuring the dry weight of the underground parts are shown in Figure 21. The graph in the figure shows the average value ± standard deviation. A comparison of test plots 2 with 3, 4, and 5 showed that even when component (D) was an anionic surfactant such as sodium lauryl sulfate, ammonium lauryl sulfate, or triethanolamine lauryl sulfate, adding components (B), (C), and (D) to ascorbic acid produced a greater growth-promoting effect than when ascorbic acid was dissolved in tap water and sprayed alone.

[0125] Example 14 Storage stability of single-dose formulation (1) Preparation of a single-dose composition A single-dose composition was prepared using 300 g of ascorbic acid as component (A), 0.2 g of BHT as component (B), 10 mL of isobutyl alcohol as component (C), and 70 mL of sorbitan monolaurate as component (D) according to the following method. After dissolving component (B) in component (C), component (D) was further added, and the resulting mixture was mixed with component (A) to prepare a single-dose composition. The reagents used were the same as those in Example 1.

[0126] (2) Preparation of powder composition The following carriers were used as component (F) and mixed with the single-dose composition to prepare powder compositions. The contents of each component in the resulting powder compositions are shown in Table 10. Zeolite (ZEOLITE (POWDER) manufactured by ZEOBUILDER CO., LTD.), silica (TOKUSIL NP manufactured by Oriental Silicas Corporation), bentonite (ODSOLV K-400 manufactured by Kurosaki Hakudo Kogyo Co., Ltd.), Glauber's salt (a sample of anhydrous Glauber's salt, dry-ground, with an average particle size of 25 μm, manufactured by China-Salt Huaian Hongyun Salt Chemical Co., Ltd.).

[0127] (3) Storage stability test Each powder composition produced was stored at room temperature for one day, and then stacked on a tray using two sieves with 2.0 mm and 9.5 mm openings, starting with the sieve with the smallest opening. 30 g of the composition was added to the top 9.5 mm sieve, the sieve was covered with a lid, and the machine was attached to a mini sieve shaker (manufactured by AS ONE Corporation, shaking speed 3). After shaking for 10 minutes, the mass of the particles remaining on each sieve and on the tray was measured. The sieve passing rate was calculated by dividing the mass that passed through the 2.0 mm sieve by the total mass. Sieve passing rate = (mass passed through sieve / total mass) x 100 The results are shown in Table 10. The results in Table 10 show that each powder composition has a higher sieve permeability than the comparative example, that is, the increase in particle size due to surface adhesion that occurs during storage is suppressed, and therefore the storage stability is excellent.

[0128] (4) Browning prevention test Five grams of each of the prepared powder compositions was dispensed into transparent glass bottles. The bottles were stored in a storage cabinet at a set temperature of 50°C for two weeks. After two weeks, the degree of browning of the appearance was evaluated on a four-point scale (0: no browning, 1: slight browning, 2: browning, 3: significant browning). The results are shown in Table 10. From the results in Table 10, browning after storage was suppressed in the composition to which silica was added. On the other hand, browning of the appearance was observed in the other compositions. The results of the study suggested that the addition of silica is superior from the viewpoint of stability of appearance.

[0129] [Table 10]

Claims

1. An agent for promoting the growth of leguminous plants, comprising (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent, wherein (B) the antioxidant is selected from at least one of tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, (C) the organic solvent is selected from one or more of alcohols having 5 or fewer carbon atoms and dimethyl sulfoxide, and the proportion (mass ratio) of (A) ascorbic acid or a salt thereof is 10 to 3,000,000 when component (B) is set to 1.

2. An agent for promoting the growth of leguminous plants, comprising (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent, wherein (B) the antioxidant is selected from at least one of tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, (C) the organic solvent is selected from one or more of alcohols having 5 or fewer carbon atoms and dimethyl sulfoxide, and the proportion (mass ratio) of (C) the organic solvent is 4 to 100,000 when component (B) is set to 1.

3. A nodule activity enhancer comprising (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent, wherein (B) the antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, (C) the organic solvent is one or more selected from alcohols having 5 or fewer carbon atoms and dimethyl sulfoxide, and the proportion (mass ratio) of (A) ascorbic acid or a salt thereof is 10 to 3,000,000 when component (B) is set to 1.

4. A nodule activity enhancer comprising (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent, wherein (B) the antioxidant is selected from at least one of tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, (C) the organic solvent is selected from one or more of alcohols having 5 or fewer carbon atoms and dimethyl sulfoxide, and the proportion (mass ratio) of (C) the organic solvent is 4 to 100,000 when component (B) is set to 1.

5. A yield-increasing agent for leguminous plants, comprising (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent, wherein (B) the antioxidant is selected from at least one of tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, (C) the organic solvent is selected from one or more of alcohols having 5 or fewer carbon atoms and dimethyl sulfoxides, and the proportion (mass ratio) of (A) ascorbic acid or a salt thereof is 10 to 3,000,000 when component (B) is set to 1.

6. A yield-increasing agent for leguminous plants, comprising (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent, wherein (B) the antioxidant is selected from at least one of tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, (C) the organic solvent is selected from one or more of alcohols having 5 or fewer carbon atoms and dimethyl sulfoxides, and the proportion (mass ratio) of (C) the organic solvent is 4 to 100,000 when component (B) is set to 1.

7. The agent according to any one of claims 1 to 6, wherein the proportion (by mass ratio) of (A) ascorbic acid or a salt thereof and (C) an organic solvent is such that, with component (B) being 1, component (A) is 10 to 3,000,000 and component (C) is 4 to 100,000.

8. (B) The agent according to any one of claims 1 to 6, wherein the antioxidant further comprises one or more selected from sodium sulfite, potassium sulfite, glutathione, and uric acid.

9. (C) The agent according to any one of claims 1 to 6, wherein the organic solvent is one or more selected from isobutyl alcohol and dimethyl sulfoxide.

10. Furthermore, the agent according to any one of claims 1 to 6, comprising (D) a surfactant.

11. (D) The agent according to claim 10, wherein the proportion (mass ratio) of the surfactant is 10 to 300,000 when component (B) is set to 1.

12. (D) The agent according to claim 10, wherein the surfactant comprises a nonionic surfactant and / or an anionic surfactant.

13. (D) The agent according to claim 12, wherein the nonionic surfactant is one or more selected from sorbitan fatty acid ester, glycerin fatty acid ester, polyalkylene glycol fatty acid ester, polyoxyethylene resin acid ester, and polyoxyethylene alkyl ether, and the anionic surfactant is one or more selected from alkyl sulfate salts and fatty acid salts.

14. Furthermore, the agent according to claim 10, comprising (E) a chelating agent.

15. The agent according to claim 14, wherein the proportions (by mass ratio) of (A) ascorbic acid or a salt thereof, (C) an organic solvent, (D) a surfactant, and (E) a chelating agent are such that, with component (B) being 1, component (A) is 10 to 3,000,000, component (C) is 4 to 100,000, component (D) is 10 to 300,000, and component (E) is 0.01 to 100.

16. (E) The agent according to claim 14, wherein the chelating agent is one or more selected from ethylenediaminetetraacetic acid and ethylenediamine-N,N'-disuccinic acid.

17. A method for promoting the growth of leguminous plants, comprising the step of applying a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent with water to soil or plants, wherein (B) the antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, (C) the organic solvent is one or more selected from alcohols having 5 or fewer carbon atoms and dimethyl sulfoxide, and the proportion (mass ratio) of (A) ascorbic acid or a salt thereof is 10 to 3,000,000 when component (B) is set to 1.

18. A method for promoting nodule activity, comprising the step of applying a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent with water to soil or plants, wherein (B) the antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, (C) the organic solvent is one or more selected from alcohols having 5 or fewer carbon atoms and dimethyl sulfoxide, and the proportion (mass ratio) of (A) ascorbic acid or a salt thereof is 10 to 3,000,000 when component (B) is set to 1.

19. A method for increasing the yield of leguminous plants, comprising the step of applying a combination of (A) ascorbic acid or a salt thereof, (B) an antioxidant, and (C) an organic solvent with water to soil or plants, wherein (B) the antioxidant is one or more selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, (C) the organic solvent is one or more selected from alcohols having 5 or fewer carbon atoms and dimethyl sulfoxide, and the proportion (mass ratio) of (A) ascorbic acid or a salt thereof is 10 to 3,000,000 when component (B) is set to 1.

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