Plant growth promotion agent composition housed in container and housing container thereof

Abstract

To provide a composition for promoting growth of leguminous plant contained in beans housed in a container and a container housing the same.SOLUTION: A growth promoting composition of legumes contained in legumes consisting of a combination of (A) a first agent containing ascorbic acid or a salt thereof, (B) an antioxidant and (C) a second agent containing an organic solvent, where (B) the antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene and butylhydroxyanisole, and (C) the organic solvent is one or more selected from alcohols and dimethyl sulfoxides having 5 or fewer carbon atoms, and (A) a ratio (mass ratio) of ascorbic acid or its salts is 10-3,000,000 when component (B) is 1, or (C) the ratio (mass ratio) of the organic solvent is 4-100,000 when component (B) is 1, and the first and second agents are housed in one of the two-headed bottle bodies connected by connecting members, respectively.SELECTED DRAWING: Figure 22

Description

[Technical field]

[0001] The present invention relates to a composition for promoting the growth of legumes, including beans contained in a container, and a container for containing the composition. [Background technology]

[0002] Approximately one-third of the land on Earth is arid, and future global warming is expected to lead to an even greater increase in arid areas. In addition, to address the serious food shortage caused by population growth, there is an urgent need to develop technology to improve, maintain, and increase yields for soybeans, rice, wheat, corn, and other grains in arid regions, salt-accumulated regions, and regions with high or low temperatures, i.e., regions where growth is difficult or where growth is impaired and yields are reduced. Soybeans are particularly important grains, and are widely eaten around the world, including in Japan. Also, unlike other grains, soybeans have a high protein and lipid ratio and are rich in nutritional value. Therefore, they are also important as feed and a raw material for oils and fats, and technology to increase yields is being developed.

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

[0004] From this viewpoint, antioxidants containing ascorbic acid are often blended into materials that are applied to plants to promote their growth. For example, Non-Patent Document 1 discloses the growth promotion and yield increase effects of ascorbic acid application to olives, sugarcane, wheat, etc. In addition, Non-Patent Document 2 discloses that a maximum yield increase effect of 30% can be achieved when a certain concentration of ascorbic acid dissolved in distilled water is sprayed on the leaves of legume crops three times in total during the vegetative growth or reproductive growth stage. In addition, Patent Document 1 discloses the promotion of plant growth using an aqueous composition containing an iron (II) compound and L-ascorbic acid, and 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 a composition for promoting the growth of legumes, including beans contained in a container, and a container for containing the composition. [Means for solving the problem]

[0008] As a result of studying 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 beans, the growth promoting effect of ascorbic acid is hardly obtained.Then, they discovered that the root nodule activity promoting effect and growth promoting effect can be achieved by combining ascorbic acid with a specific antioxidant dissolved in an organic solvent, specifically, by storing them in separate containers and mixing them when used.

[0009] That is, the present invention relates to the following 1) and 2). 1) A growth-promoting composition for legumes contained in beans, comprising a combination of (A) a first agent containing ascorbic acid or a salt thereof and (B) a second agent containing an antioxidant and (C) an organic solvent, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, and the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms and dimethyl sulfoxide, 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, or the ratio (mass ratio) of (C) the organic solvent is 4 to 100,000 when the component (B) is taken as 1, and the first agent and the second agent are each contained in one of two-headed bottle bodies in which a first bottle body and a second bottle body are connected by a connecting member. 2) A double-headed bottle body containing the composition described in 1), in which a first bottle body having an external threaded portion at the bottom, a narrow-mouthed bottle head, and a cap body is connected to the lower part of a second bottle body having a bottle body and a wide-mouthed bottle head via a connecting member. Effect of the Invention

[0010] According to the present invention, it is possible to increase the growth of legumes, including beans, promote root nodule activity, and increase the yield of the fruits, seeds, or grains of the plants. Also, according to the present invention, the first and second agents, which have different functions, are contained in independent bottles with good sealing properties, and are sold as a single unit. When used, the consumer can separate the two connected bottles and easily prepare a mixture of the first and second agents in a predetermined ratio. [Brief description of the drawings]

[0011] [Figure 1] Seed weight per soybean plant. [Diagram 2] Solubility when the mixture ratio of BHT and isobutyl alcohol is changed. [Diagram 3] Solubility of BHT in organic solvent mixtures (1:4). [Figure 4] Soybean nodule weight one week after foliar application. [Diagram 5] 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] Amount of allantoic acid contained in soybean exudate one week after foliar spray. [Figure 8] Soybean plants one week after foliar spray in test area 7. [Figure 9] Seed weight per soybean plant. [Figure 10] Amount of allantoic acid contained in soybean exudate one week after foliar spray. [Figure 11] Dry weight of above-ground adzuki bean one week after foliar spray. [Figure 12]Dry weight of adzuki bean underground parts 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 individual. [Figure 16] Number of beans per broad bean plant. [Figure 17] Seed weight per broad bean plant. [Figure 18] Appearance of one- or two-formulation compositions after one week of storage. [Figure 19] Amount of allantoic acid contained in soybean exudate one week after foliar spray. [Figure 20] Dry weight of soybean above ground one week after foliar spray. [Figure 21] Dry weight of soybean underground one week after foliar spray. [Figure 22] 1A and 1B are a front view and a cross-sectional view showing a double-headed bottle body (in which a first bottle body and a second bottle body are connected) according to a first embodiment. [Diagram 23] FIG. 23 is an exploded perspective view of the double-headed bottle body shown in FIG. 22. [Figure 24] 24A and 24B are a perspective view and a cross-sectional view of the connecting member shown in FIG. 23. [Diagram 25] 13A and 13B are a front view and a cross-sectional view showing a double-headed bottle body (in a state in which a first bottle body and a second bottle body are connected) according to a second embodiment. [Figure 26] FIG. 26 is an exploded perspective view of the double-headed bottle body shown in FIG. 25. [Figure 27] 27A and 27B are a perspective view and a cross-sectional view of the connecting member shown in FIG. 26. [Figure 28] 1 is a perspective view of a double-headed bottle body according to a first embodiment, the left being a state in which the first bottle body and the second bottle body are connected, and the right being a state in which the first bottle body is separated. [Figure 29] 13 is a perspective view of a double-headed bottle body according to a second embodiment, Left: a state in which a first bottle body and a second bottle body are connected, Right: a state in which the first bottle body is separated. [Diagram 30] Soybean nodule activity one week after foliar application. [Diagram 31]Dry weight of soybean above ground two days after foliar spray. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] <Growth-promoting composition for legumes included in beans> In the present invention, the "growth promotion" of legumes in the legume plant growth promoter composition (hereinafter also referred to as "the composition of the present invention") means increasing the growth (wet weight, elongation, etc.) of legumes in the legume plant and increasing the yield of fruits, seeds, or grains. In addition, when the plant is a nodule-forming plant, the growth promotion includes the promotion of nodule activity. The promotion of nodule activity means promoting nodule activity, i.e., the nitrogen fixation function in the host plant exerted by nodules. Here, "nodule" means a nodule that occurs on the root of a plant through symbiosis with bacteria (rhizobia). Rhizobia reduce atmospheric nitrogen in the nodules, convert it into ammonia nitrogen, and supply it to the host, so-called symbiotic nitrogen fixation.

[0013] In the present invention, the legumes include so-called "beans" whose seeds are harvested and used, such as soybean (including edamame), adzuki bean, chickpea, lotus grass, kidney bean, pea, broad bean, pea, scarlet bean, lima bean, mung bean, cowpea, black eye bean, hyacinth bean, sword bean, lentil, winged bean, etc. As the legumes included in the legumes, soybean, adzuki bean, kidney bean, pea, broad bean, chickpea, lotus grass, soybean is more preferable.

[0014] The composition of the present invention is a growth promoter composition for legumes that is included in beans, which is a combination of a first agent containing (A) ascorbic acid or a salt thereof and a second agent containing (B) an antioxidant and (C) an organic solvent, in which 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, 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, or the ratio (mass ratio) of (C) the organic solvent is 4 to 100,000 when the component (B) is taken as 1, and the first agent and the second agent are each contained in one of the two-headed bottle bodies in which the first bottle body and the second bottle body are connected by a connecting member. The composition is used by mixing the two when used.

[0015] In the present invention, the IUPAC systematic name of ascorbic acid, which is the component (A), is (R)-3,4-dihydroxy-5-((S)-1,2-dihydroxyethyl)furan-2(5H)-one. Ascorbic acid may be any of D-, L-, and DL-isomers, but is preferably L-isomer (so-called L-ascorbic acid). Ascorbic acid, commercially available products 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] The concentration of (A) ascorbic acid or a salt thereof in the composition when the composition of the present invention is applied to a plant can be appropriately adjusted depending on the supply method, for example, within the range of 100 mass ppm or more and 300,000 mass ppm or less. For example, when spraying using a sprayer (e.g., a boom sprayer), the concentration in the spray solution is preferably 100 mass ppm or more, more preferably 300 mass ppm or more, and more preferably 500 mass ppm or more, and preferably 20,000 mass ppm or less, more preferably 10,000 mass ppm or less, and more preferably 4,500 mass ppm or less. In addition, it is preferably 100 to 20,000 mass ppm, more preferably 300 to 10,000 mass ppm, and more preferably 500 to 4,500 mass ppm. 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 further 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 further 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 further preferably 150,000 to 200,000 mass ppm.

[0017] In the composition of the present invention, the antioxidant (B) is specifically at least one selected from the group consisting of tocopherol (vitamin E), dibutylhydroxytoluene (BHT) and butylhydroxyanisole (BHA). Among these, preferred are phenol-based antioxidants BHT and BHA, and more preferred is BHT. 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 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 body, they react with lipids present in the cell membrane, causing continuous lipid peroxidation reactions, generating lipid radicals and lipid peroxy radicals. By using antioxidants, it becomes possible to capture such radicals. In the present invention, (B) the antioxidant is used in the form of a solution dissolved in (C) an organic solvent, whereby the plant growth promoting effect or the root nodule activity promoting effect of ascorbic acid is effectively exhibited.

[0019] In addition, according to a two-component composition of the present invention in which a formulation (first agent) containing component (A) and a formulation (second agent) containing components (B) and (C) are separately prepared and then combined, discoloration that occurs when a formulation containing all of components (A), (B), and (C) is stored can be suppressed. The first agent containing component (A) may contain an antioxidant other than tocopherol, BHT, and BHA, such as one or more selected from sodium sulfite, potassium sulfite, potassium pyrosulfite, chlorogenic acid, catechin, glutathione, and uric acid.

[0020] The concentration of the antioxidant (B) in the composition (mixture of the first and second agents) when the composition of the present invention is applied to plants is preferably 0.001 mass ppm or more, more preferably 0.01 mass ppm or more, and further preferably 0.1 mass ppm or more, and is preferably 100 mass ppm or less, more preferably 20 mass ppm or less, and further preferably 5 mass ppm or less. Also, it is preferably 0.001 to 100 mass ppm, more preferably 0.01 to 20 mass ppm, and further preferably 0.1 to 5 mass ppm.

[0021] The organic solvent, which is 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] The concentration of the (C) organic solvent in the composition (mixture of the first and second agents) when the composition of the present invention is applied to plants can be appropriately adjusted 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 content in the spray liquid is preferably 1 ppm by mass or more, more preferably 10 ppm by mass or more, and further 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 further preferably 1,000 ppm by mass or less. Also, the content is preferably 1 to 10,000 ppm by mass, more preferably 10 to 5,000 ppm by mass, and further 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. Also, the concentration 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, of component (A) 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, in the case of spraying by a sprayer, 500 to 45,000 is preferable, in the case of spraying by aerial spraying, 150,000 to 2,000,000 is preferable, further, in the case of spraying by a sprayer, 500 to 4,500 is more preferable, and in the case of spraying by aerial spraying, 150,000 to 200,000 is preferable. In addition, when component (B) is 1, component (C) is preferably 4 or more, more preferably 10 or more, more preferably 20 or more, more preferably 50 or more, and preferably 100,000 or less, more preferably 50,000 or less, more preferably 20,000 or less, and more preferably 10,000 or less. Also, it is preferably 4 to 100,000, more preferably 10 to 50,000, more preferably 20 to 20,000, and more preferably 50 to 10,000. Among these, in the case of spraying by a sprayer, 20 to 10,000 is preferable, and in the case of spraying by aerial spraying, 2,000 to 100,000 is preferable. Furthermore, in the case of spraying by a sprayer, 50 to 1,000 is more preferable, and in the case of spraying by aerial spraying, 5,000 to 10,000 is more preferable.

[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 permeability of (A) ascorbic acid or a salt thereof onto a plant surface, thereby enhancing the effect of (A) ascorbic acid or a salt thereof, or enabling the effect to be exerted more efficiently. The (D) surfactant is preferably contained in the second agent containing components (B) and (C). The surfactant may include a non-ionic surfactant and / or an anionic surfactant. Examples of the nonionic surfactant 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 alkyl aryl 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 phosphoric acid, 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, 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 the composition of the present invention is applied to plants, the concentration of the (D) surfactant in the composition (mixture of the first and second agents) can be appropriately adjusted depending on the supply method within the range of 10 ppm by mass or more and 30,000 ppm by mass or less. For example, when spraying using a sprayer (e.g., a boom sprayer), the content in the spray liquid is preferably 10 ppm by mass or more, more preferably 100 ppm by mass or more, and further 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 further preferably 500 ppm by mass or less. Also, the content is preferably 10 to 5,000 ppm by mass, more preferably 10 to 1,000 ppm by mass, and further 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. Also, the concentration 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. In addition, when the surfactant (D) is combined, the ratio (mass ratio) of the component (D) is preferably 10 or more, more preferably 50 or more, and even more preferably 100 or more, and is preferably 300,000 or less, more preferably 100,000 or less, and even more preferably 5,000 or less, when the component (B) is taken as 1. Also, the ratio is preferably 10 to 300,000, more preferably 50 to 100,000, and even more preferably 100 to 5,000. Among these, in the case of spraying with a sprayer, 100 to 5,000 is preferred, and in the case of spraying by aerial spraying, 1,000 to 50,000 is preferred. Furthermore, in the case of spraying with a sprayer, 100 to 500 is more preferred, and in the case of spraying by aerial spraying, 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. The chelating agent (E) is preferably contained in the first agent containing component (A). Examples of the chelating agent include aminocarboxylic acid chelating agents, phosphonic acid chelating agents, hydroxycarboxylic acid chelating agents, polyvalent carboxylic acid chelating agents, etc. Examples of the aminocarboxylic acid chelating agent 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 the phosphonic acid chelating agent 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 the polyvalent carboxylic acid chelating agent include succinic acid, oxalic acid, glutaric acid, adipic acid, fumaric acid, malonic acid, and salts thereof.

[0028] The concentration of the chelating agent (E) in the composition (mixture of the first and second agents) when the composition of the present invention is applied to plants is preferably 0.01 ppm by mass or more, more preferably 0.1 ppm by mass or more, and further 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 further 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 further 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 the component (B) is taken as 1. The ratio is 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) an antioxidant, (C) an organic solvent, and (D) a 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, it has been shown that adding a combination of an antioxidant such as BHT and an organic solvent (second agent) to ascorbic acid (first agent) increases nodule weight and nodule activity, promotes the production of ureides in the nodules, and promotes the growth of the host plant. Therefore, the combination of ascorbic acid (first agent), a specific antioxidant and an organic solvent (second agent) is a composition for promoting the growth of legumes, including beans, and for promoting nodule activity. It has been reported that the amount of ureide derived from nodules correlates with grain yield (Total amount of basal nitrogen diagnostic method for rotational field soybeans, Ibaraki Prefectural Agricultural Research Center, Agricultural Research Institute, H18 Main Results; http: / / www.pref.ibaraki.jp / nourinsuisan / noken / seika / h18pdf / documents / 27.pdf), so the composition of the present invention is also useful for increasing grain yield. Since the nitrogen fixation function in the host plant can be evaluated as the ureide production ability or amide production ability, the promoting effect of the composition of the present invention on the nodule activity can be specifically evaluated as the amount of ureide or amide produced per wet weight of nodule. The amount of ureide or amide produced can be calculated by measuring the amount of ureide or amide in the exudate collected when the plant body is cut (for example, the above-ground part is cut just below the cotyledon). Here, the ureide includes allantoin, allantoic acid, and citrulline, and the amide includes asparagine and glutamine, but it is preferable to measure the amount of allantoic acid or asparagine.

[0031] A composition comprising the above-mentioned first and second agents in combination is a composition for promoting the growth or nodule activity of legumes, including beans, and for increasing the yield of fruits, seeds or grains, and can be, for example, a formulation for adding or blending to various agricultural or horticultural materials, or cultivation substrates for cultivating plants, such as soil, culture medium, and nutrient solution for hydroponics. The composition may be in the form of a liquid or gel, or may be in the form of a powder or granule. The first agent and the second agent are each contained in one of the bottle bodies of a double-headed bottle body formed by connecting a first bottle body and a second bottle body with a connecting member.

[0032] The composition may contain any other components in addition to the above components (A) to (C) and further (D) and (E), such as solvents (e.g., water, buffer solutions, culture media, solutions for hydroponics, etc.), 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, alumina, etc.), pH adjusters for promoting dissolution of the compounds, spreading agents for increasing the spreading ability of the compounds to plants or soil, fertilizer components for increasing the fertilizing effect, agricultural chemical components, binders, and bulking agents. Examples of 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.

[0033] Compositions for aerial application may contain emulsifiers such as polyoxyethylene fatty acid esters and oils such as decyl alcohol.

[0034] Examples of the composition of the present invention include, but are not limited to, a cultivation substrate containing the composition of the present invention (e.g., a solution for nutrient culture for agricultural or horticultural use), fertilizer, water for watering, microbial materials such as root nodule bacteria materials, soil conditioners, pesticides, seeding materials, and plant supplements (e.g., activators, nutrients, etc.). Among these, fertilizer, microbial materials, soil conditioners, seeding materials, and plant supplements are preferred because they contribute to the improvement of the soil in which plants are grown. The fertilizer, microbial materials, soil conditioners, seeding materials, and plant supplements may be solid or liquid, and if solid, are preferably powder or granules. The fertilizer, microbial materials, soil conditioners, seeding materials, and plant supplements may contain components of fertilizer, microbial materials, soil conditioners, seeding materials, and plant supplements that are usually used in plant cultivation, in addition to containing components (A) to (C) as active ingredients.

[0035] The cultivation substrate, fertilizer, microbial materials such as root nodule bacteria materials, soil conditioners, pesticides, seeding materials, and plant supplements may be prepared by adding the composition of the present invention to ordinary cultivation substrates (e.g., agricultural or horticultural soil, culture medium, culture medium, nutrient solution, water, etc.), fertilizer, microbial materials such as root nodule bacteria materials, soil conditioners, pesticides, seeding materials, plant supplements (e.g., activators, nutrients, etc.), and the like.

[0036] When using the composition of the present invention, the two connected bottles are separated and the first and second agents are mixed in a predetermined ratio to use the composition. The mass ratio of the first agent is 1 to 100, preferably 1 to 50, and more preferably 1 to 25, relative to the mass ratio of the second agent taken as 1. 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.

[0037] The method for supplying the composition of the present invention is not particularly limited, so long as it is applied to a plant so as to exert the effects of the present invention. That is, there are no particular limitations as long as the composition (a mixture of the first and second agents) is brought into contact with or delivered to the plant body of a legume plant included in pulses or to the soil in the rhizosphere of the plant, and examples of the method 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 (e.g., 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.

[0038] The method of application is not particularly limited, and examples thereof include a spray method, i.e., a method of spraying the application liquid in the form of a mist by spraying. By using such a method, the legume plant growth promoter or root nodule activity promoter included in the beans of the present invention adheres to the plant and then exhibits good spreading properties on the plant. Specific examples of spraying methods include manual spraying using a mister, sprayer, or sprayer (e.g., a boom sprayer), and aerial spraying using an airplane, helicopter, drone, etc.

[0039] The application amount of the composition of the present invention depends on the concentration of the components (A) to (C) contained in the composition when applied. For example, when the concentration of the component (A) contained in the spray solution is 100 to 300,000 mass ppm, the amount of the 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, it is preferably 1 to 150 mg, more preferably 5 to 100 mg, more preferably 10 to 50 mg. The composition may be applied in an amount within the above range at once, or may be applied in several divided doses.

[0040] 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 filling period after the reproductive growth period.

[0041] <Double-headed bottle for containing the composition of the present invention> In the composition of the present invention, the first and second agents are each contained in one of the bottle bodies of a double-headed bottle in which a first bottle body and a second bottle body are connected by a connecting member. The double-headed bottle of the present invention is a double-headed bottle having two bottle heads connected via a connecting member: a first bottle body having a bottle body with an external thread at the bottom, a narrow-mouthed bottle head, and a cap body, and a second bottle body having a bottle body and a wide-mouthed bottle head. Due to the difference in the structure of the connecting member, the double-headed bottle body shown in Figures 22 to 24 as a first embodiment and the double-headed bottle body shown in Figures 25 to 27 as a second embodiment can be mentioned. In either case, the first bottle body comprises a bottle body 3 having an external thread at the bottom, a narrow-mouth bottle head 4, and a lid body 5, and the second bottle body comprises a bottle body 6 and a wide-mouth bottle head 7 (Figs. 23 and 26). The narrow-mouth bottle head 4 of the first bottle body and the wide-mouth bottle head 7 of the second bottle body are provided on their outer walls with external threads for screwing onto the lid body 5 or the connecting member 8 or 18, respectively.

[0042] <First embodiment> The double-headed bottle body equipped with a connecting member 8 will be described with reference to Figs. The connecting member 8 includes a cover plate 9, a first ring wall 10 extending upward along the outer circumferential edge of the cover plate 9, and a second ring wall 11 formed by folding the first ring wall 10 downward (FIG. 24). An internal thread portion 15 is provided on the inner wall of the first ring wall 10. Also, an internal thread portion 17 is provided on the inner wall of the second ring wall 11 (FIG. 24).

[0043] An external threaded portion provided at the bottom of the bottle trunk 3 of the first bottle body 1 is connected to the first ring wall 10 of the connecting member 8 via a screw-type structure 13, and the connecting member 8 is connected to the wide-mouth bottle head 7 of the second bottle body 2 via a screw-type structure 14 (Figure 23).

[0044] The screw structure 13 is composed of an external thread portion 12 of the bottle body 3 having an external thread portion at the bottom of the first bottle body 1, and an internal thread portion 15 provided on the inner wall of the first ring wall 10. The screw structure 14 is composed of an external thread portion 16 provided on the outer wall of the wide-mouth bottle head 7 of the second bottle body 2, and an internal thread portion 17 provided on the inner wall of the second ring wall 11 (FIG. 23). That is, the internal thread portion 15 of the first ring wall 10 screws into the external thread portion 12 of the bottle body 3 which has an external thread portion at the bottom of the first bottle body 1, and the internal thread portion 17 of the second ring wall 11 screws into the external thread portion 16 provided on the outer wall of the wide-mouth bottle head 7 of the second bottle body 2 (Figure 23). The screwing direction of the screwing structure 13 between the first bottle body 1 and the connecting member 8 may be the same as or different from the screwing direction of the screwing structure 14 between the second bottle body 2 and the connecting member 8.

[0045] <Second embodiment> The double-headed bottle body equipped with the connecting member 18 will be described with reference to Figs. The connecting member 18 includes a cover plate 19, and a first ring wall 20 and a second ring wall 21 that protrude vertically from the outer periphery of the cover plate 19 (FIG. 27). An internal thread portion 22 is provided on the inner wall of the first ring wall 20. An internal thread portion 24 is provided on the inner wall of the second ring wall 21 (FIG. 27).

[0046] An external threaded portion provided at the bottom of the bottle barrel 3 of the first bottle body 1 is connected to the first ring wall 20 of the connecting member 18 via a screw-type structure 23, and the connecting member 18 is connected to the wide-mouth bottle head 7 of the second bottle body 2 via a screw-type structure 25 (Figure 26).

[0047] The screw structure 23 is composed of an external thread portion 12 of the bottle body 3 having an external thread portion at the bottom of the first bottle body 1, and an internal thread portion 22 provided on the inner wall of the first ring wall 20. The screw structure 25 is composed of an external thread portion 16 provided on the outer wall of the wide-mouth bottle head 7 of the second bottle body 2, and an internal thread portion 24 provided on the inner wall of the second ring wall 21 (FIG. 26). That is, the internal thread portion 22 of the first ring wall 20 screws into the external thread portion 12 of the bottle body 3 which has an external thread portion at the bottom of the first bottle body 1, and the internal thread portion 24 of the second ring wall 21 screws into the external thread portion 16 provided on the outer wall of the wide-mouth bottle head 7 of the second bottle body 2 (Figure 26). The screwing direction between the first bottle body 1 and the connecting member 18 may be the same as or different from the screwing direction between the second bottle body 2 and the connecting member 18.

[0048] The first bottle body and the second bottle body can be made of synthetic resin or metal. Examples of synthetic resin include polyethylene terephthalate, polypropylene, polyethylene such as high density polyethylene, and vinyl chloride. Examples of metal include aluminum and tinplate. The synthetic resin may be either transparent or opaque. The first and second bottle bodies made of synthetic resin or metal can be manufactured by injection molding or press blow molding using a mold. It is preferable that the bottle body having an external thread at the bottom of the first bottle body and the narrow-mouth bottle head, and the bottle body and wide-mouth bottle head of the second bottle body are each an integral part made of synthetic resin.

[0049] The material of the connecting member may be any of the various materials listed as the materials of the first bottle body and the second bottle body. The material of the connecting member may be the same as that of the bottle body. From the viewpoint of formability and handling of the thread, the material of the connecting member is preferably a synthetic resin. When the material of the connecting member is a synthetic resin, it can be manufactured by injection molding using a mold. The connecting member is preferably a one-piece product made of a synthetic resin.

[0050] The above-mentioned first bottle and second bottle separately contain (A) a first agent containing ascorbic acid or a salt thereof, and (B) a second agent containing an antioxidant and (C) an organic solvent, but the first agent and the second agent may be contained in either the first bottle or the second bottle. (A) The first agent containing ascorbic acid or a salt thereof may be contained in a powder or granular form. In that case, a liquid agent can be prepared by adding tap water, agricultural water, well water, etc. to the bottle.

[0051] The first bottle body and the second bottle body are separated at the time of use (Figures 28 and 29), and the first agent and the second agent are mixed. For example, the composition in the first bottle body is placed in the second bottle body, or the composition in the first bottle body and the composition in the second bottle body are placed in separate containers and mixed.

[0052] For example, in one embodiment, the powdered first agent is contained in the second bottle, and the liquid second agent is contained in the first bottle. Next, the liquid second agent contained in the first bottle is dissolved in tap water, agricultural water, well water, or the like that has been added to another container, and then the powdered first agent contained in the second bottle is dissolved. Alternatively, the liquid agent can be prepared by dissolving the first agent and the second agent in the reverse order.

[0053] In relation to the above-described embodiment, the present invention further discloses the following aspects. <1> A growth-promoting composition for legumes contained in beans, comprising a combination of a first agent containing (A) ascorbic acid or a salt thereof and a second agent containing (B) an antioxidant and (C) an organic solvent, wherein the (B) antioxidant is at least one selected from tocopherol, dibutylhydroxytoluene, and butylhydroxyanisole, and the (C) organic solvent is at least one selected from alcohols having 5 or less carbon atoms and dimethyl sulfoxide, 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, or the ratio (mass ratio) of (C) the organic solvent is 4 to 100,000 when the component (B) is taken as 1, and the first agent and the second agent are each contained in one of two-headed bottle bodies in which a first bottle body and a second bottle body are connected by a connecting member. <2> The first bottle body has a bottle body having an external thread at the bottom, a narrow-mouth bottle head, and a lid body, and the second bottle body has a bottle body and a wide-mouth bottle head. <1> The composition described in <3> The first agent is contained in a second bottle body, and the second agent is contained in a first bottle body. <1> or <2> The composition described in <4> The ratio (mass ratio) of (A) ascorbic acid or a salt thereof and (C) organic solvent is, when the ratio of component (B) is taken as 1, that of component (A) is 10 to 3,000,000, preferably 100 to 2,000,000, more preferably 500 to 200,000, and that 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> ~ <3> 2. The composition according to claim 1 . <5> (B) The antioxidant further comprises one or more selected from sodium sulfite, potassium sulfite, glutathione, and uric acid; <1> ~ <3> 2. The composition according to claim 1 . <6> (C) the organic solvent is at least one selected from isobutyl alcohol and dimethyl sulfoxide; <1> ~ <3> 2. The composition according to claim 1 .

[0054] <7> Furthermore, the second agent is combined with a surfactant (D), <1> ~ <6> The agent according to any one of the preceding claims. <8> The ratio (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, relative to the ratio of the component (B) taken as 1. <7> The composition described in <9> (D) the surfactant comprises a nonionic surfactant and / or an anionic surfactant; <7> or <8> The composition described in <10> (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 salt. <9> The composition described in <11> (D) the surfactant includes a nonionic surfactant and an anionic surfactant; <9> The agent described in. <12> (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 salts and fatty acid salts. <11> The composition described in <13> Furthermore, the first agent is combined with a chelating agent (E), <1> ~ <12> 2. The composition according to claim 1 . <14> The ratios (mass ratios) of (A) ascorbic acid or a salt thereof, (C) organic solvent, (D) surfactant and (E) chelating agent, when the ratio of component (B) is taken as 1, are: component (A) is 10 to 3,000,000, preferably 100 to 2,000,000, more preferably 500 to 200,000; component (C) is 4 to 100,000, preferably 10 to 50,000, more preferably 20 to 20,000, even more preferably 50 to 10,000; component (D) is preferably 10 to 300,000, more preferably 50 to 100,000, 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, even more preferably 1 to 10. <13> The composition described in <15> (E) the chelating agent is one or more selected from ethylenediaminetetraacetic acid and ethylenediamine-N,N'-disuccinic acid; <13> or <14> The composition described in

[0055] <16> <1> ~ <15> 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, hyacinth bean and jack bean, more preferably beans selected from soybean, adzuki bean, kidney bean, pea, broad bean, chickpea and lotus grass, more preferably soybean.

[0056] <17> <1> A bottle body containing the composition described in claim 1, wherein the bottle body is a double-headed bottle body in which a second bottle body having a bottle body and a wide-mouth bottle head is connected to the lower part of a first bottle body having an external threaded portion at the bottom, a narrow-mouth bottle head, and a cap via a connecting member. <18> The connecting member comprises a cover plate, a first ring wall extending upward along the outer periphery of the cover plate, and a second ring wall formed by folding the first ring wall downward, and the first bottle body and the second bottle body are connected by an external thread provided on the bottom of the bottle barrel of the first bottle body screwing into an internal thread provided on the inner wall of the first ring wall of the connecting member, and a wide-mouth bottle head of the second bottle body screwing into an internal thread provided on the inner wall of the second ring wall of the connecting member. <17> The bottle body described in. <19> The connecting member comprises a cover plate and a first ring wall and a second ring wall each protruding perpendicularly from the outer periphery of the cover plate, and the first bottle body and the second bottle body are connected by an external thread provided on the bottom of the bottle barrel of the first bottle body screwing into an internal thread provided on the inner wall of the first ring wall of the connecting member, and a wide-mouth bottle head of the second bottle body screwing into an internal thread provided on the inner wall of the second ring wall of the connecting member. <17> The bottle body described in. EXAMPLES

[0057] Test Example 1: Effect of application of ascorbic acid solution Ascorbic acid was dissolved in water to prepare an aqueous solution of ascorbic acid, and ascorbic acid was dissolved in deionized water to prepare an aqueous solution of ascorbic acid. The effects of application of the aqueous solution of ascorbic acid to 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) Section 2: foliar spray of 500 ppm ascorbic acid (dissolved in tap water) Section 3: foliar spray of 500 ppm ascorbic acid (dissolved in deionized water)

[0058] (1)Cultivation conditions Soybean cultivation was carried out in a field in Tochigi Prefecture, and the soybean cultivar "Sato no Hohoemi" was used. In the field, foliar spray treatment was carried out in plots of 2m width, excluding the surrounding areas. For 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 cultivated within the 2m area set as one plot.

[0059] (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. An aqueous solution of 500 ppm by mass of ascorbic acid was prepared by dissolving it in tap water or deionized water. Using a battery-powered sprayer (GT-5HS, Koshin Co., Ltd.), 20 mL of the aqueous solution of ascorbic acid per plant was sprayed so that it was applied to the entire soybean plant body. Spraying was performed on the 60th day after sowing, and the growth stage was the time corresponding to the seed filling stage. Harvesting was performed on the 108th day after sowing. After harvesting, all the seeds were collected from each individual and dried at 100 °C for 48 hours. As yield data, the dry seed mass was measured.

[0060] (3) Results After calculating the average seed weight of each individual in one plot, the average and standard deviation of the three plots were calculated (Figure 1). The graph in the figure shows the average value ± standard deviation. The same concentration of ascorbic acid was sprayed on the leaves. When the water was used in agricultural fields, 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 in the water used in agricultural fields (tap water).

[0061] Test Example 2: Solubility of dibutylhydroxytoluene in various solvents The amount of BHT dissolved in 100μL of isobutyl alcohol was varied. It was confirmed that BHT was uniformly dissolved up to 25mg, 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 examined, BHT was uniformly dissolved in ethanol, methanol, 2-propanol, 1-butanol and dimethyl sulfoxide (DMSO), except for glycerin (Figure 3). BHT is known to be poorly soluble in water. Each reagent used was manufactured by Fujifilm Wako Pure Chemical Corporation.

[0062] 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 components. One or more selected from BHT, BHA and tocopherol, which is component (B), was dissolved in component (C) in advance, and then component (D) was mixed to prepare the second part. Next, one or more selected from component (A) and component (E) were mixed to prepare the first part. Each composition example shown in Table 2 was prepared as a two-part composition in which components (A), (B), (C), (D) and (E) were blended in the amounts shown in the table.

[0063] (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.

[0064] (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 used from Fujifilm Wako Pure Chemical Industries, Ltd. EDTA was used from Dojindo Laboratories, Inc. The surfactants used were sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, polyoxyethylene lauryl ether, polyoxyethylene alkyl ether, and polyethylene glycol monolaurate.

[0065] (4) Solubility evaluation results The aqueous solubility of the spray solution prepared using each composition was evaluated based on the appearance according to the following criteria. The results are shown in Table 2. ◯: There is no residue left and the solution is homogeneous. △: Some undissolved material is observed, but the mixture becomes uniform immediately after stirring. ×: Some remains undissolved, and the mixture is not uniform even immediately after stirring.

[0066] 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.

[0067] [Table 1]

[0068] [Table 2]

[0069] Example 2 Evaluation of nodule weight and nodule activity (1) Soil preparation and sowing Medium-term fertilizer-effect soil (Takii Hydrated Cell Soil Medium-term Fertilizer-effect, 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 a polypot (diameter 10.5 cm, height 9 cm). "Enrei" soybean seeds (purchased from Nikko 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 about 1 to 2 cm from the soil surface. Each test plot was repeated 6 times (n=6).

[0070] (2) Inoculation of rhizobia Yeast-Mannitol (YM) medium (K 2 HPO 4 0.5g MgSO 4 7 H 2 O 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 nnitol and 1 L of distilled water (pH 6.8), and the soybean root nodule bacteria (Bradyrhizobium japonicum) NBRC14783T strain was grown on the solid medium. A loopful of the grown root nodule bacteria was taken and 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 root nodule culture solution with a bacterial turbidity OD600 value of approximately 0.3 was prepared. After sowing, 1 mL of the root nodule culture solution was dropwise inoculated onto the seeds using a micropipette.

[0071] (3)Cultivation conditions The cultivation from sowing to thinning was carried out in an artificial climate chamber (LPH-411SP, Nippon Medical and Chemical Equipment Manufacturing Co., Ltd.) under light conditions of 440-480 μmol / m 2 / s)16 The light period was 30°C / dark period 25°C, and the humidity was 50%. 7 days after sowing Afterwards, the plants were thinned out so that there was one plant per pot. After thinning, the plants were grown outdoors. Watering was done 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.

[0072] (4) Foliar spray treatment The spray solution shown in Table 3 was prepared and sprayed at 6.7 mL per plant using a spray bottle on the 14th day after sowing. Foliar spray was performed only once, and seven types of test plots were examined. The spray solution was prepared by dissolving BHT in DMSO (component (B)) in advance, then mixing component (C) with component (D) to prepare the second agent. Next, component (A), uric acid (antioxidant) and component (E) were mixed to prepare the first agent. The second agent was dissolved in the water used, and the first agent was further dissolved to prepare the spray solution.

[0073] Here, ascorbic acid was used as "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 as "Chilest EDDS-35" manufactured by Chelest Co., Ltd. DMSO and isobutanol were used as component (C). Among component (D), sorbitan monolaurate or sorbitan fatty acid ester, polyoxyethylene resin acid ester were used as nonionic surfactants. Among component (D), fatty acid soap potassium salt was used as anionic surfactants.

[0074] [Table 3]

[0075] (5) A method for collecting exudate 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 then covered with 1.5 mL microtubes (Eppendorf, Inc.) filled with cotton balls #10 (Kawamoto Sangyo Co., Ltd.), and the exudation fluid exuding from the cut surface of the stem was collected for 2 hours. The cotton balls after collection 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.

[0076] (6) Extraction of exudate components collected on a cotton ball The exudate components 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.) operated at 15,000 rpm for 1 minute. The filtrate was diluted 1,000 times and quantified.

[0077] (7) Apparatus and method for determining the amount of ureido nitrogen The HPLC and mass spectrometers used were Agilent 1260 Infinity LC system (Agilent Technologies, Inc.) and AB SCIEX TripleQuad 4500 system (AB SCIEX, Inc.), respectively. The column used was Scherzo SS-C18 (100 mm x 2 mm, 3 μm) (Intact Corporation), and the oven temperature was 40°C. 5 μL of appropriately diluted sample was injected, and the flow rate was 0.5 mL / min. The eluents were 0.1% formic acid aqueous solution (eluent A) and 50 mM ammonium acetate / methanol solution (eluent B). The sample was supplied after equilibration with eluent A:eluent B = 95:5, and ureido nitrogen was eluted with a linear gradient of eluent A:eluent B = 80:20 after 5 minutes.

[0078] (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 created in the range of 10-1000 ppb. Allantoin and allantoic acid were identified for 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 based on the calibration curve, and the total amount per plant was calculated.

[0079] (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 showed that the difference in water quality in which ascorbic acid is dissolved affects soybean yield. Test plots 1 to 3 in this example confirmed that even at the early growth stage of soybeans, when ascorbic acid was dissolved in deionized water, the nodule weight and nodule activity tended to increase, and when ascorbic acid was dissolved in tap water, the effect of application of ascorbic acid tended to decrease. A comparison of test plots 4 and 5 showed that the addition of an organic solvent was necessary to restore the nodule activity that was reduced by dissolving ascorbic acid in tap water. In addition, a comparison of test plots 6 and 7 suggested that EDTA was a more preferable chelating agent for restoring nodule activity.

[0080] Example 3: Examination of the mixing ratio of ascorbic acid and antioxidant The soil preparation and sowing, inoculation of rhizobia, method of collecting exudate to measure the amount of ureido nitrogen, which is an index of nodule activity, experimental procedures before quantifying the exudate, equipment and measurement method used to quantify the amount of ureido nitrogen, and standards and quantitative items were the same as in Example 2, and soybeans were cultivated for 21 days.

[0081] (1)Cultivation conditions The plants were grown in an artificial climate chamber (LPH-411SP, Nippon Medical and Chemical Equipment Manufacturing Co., Ltd.) under light conditions of 440–480 μmol / m 2 The light / dark period was 16 hours / s, the temperature was 30°C light / 25°C dark, and the humidity was 50%. Seven days after sowing, the plants were thinned out so that there was one plant per pot. Watering was done 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.

[0082] (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 types of 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.

[0083] [Table 4]

[0084] (3) Results The measurement results of nodule activity are shown in Figures 6 and 7. The graphs in the figures show the average value ± standard deviation. In Figures 6 and 7, when ascorbic acid dissolved in tap water was sprayed on the leaves, the nodule activity did not change or rather decreased compared to when it was not applied. In test area 3, which was a composition that did not contain ascorbic acid, the nodule activity decreased, but by adding ascorbic acid to the composition of test area 3, the nodule activity was restored or improved. In a test using a water volume that assumed the case of spraying using a sprayer (e.g., a boom sprayer), the test area of ​​ascorbic acid:BHT=500:1 in test area 4 had the highest nodule activity, and the test area of ​​ascorbic acid:BHT=20,000:1 in test area 7 showed a tendency for the nodule activity to decrease. In addition, as shown in Figure 8, the ascorbic acid:BHT=20,000:1 test caused a problem of leaf color fading, indicating that the mixture ratio of ascorbic acid, component (B), and component (C) is preferably 100-20,000:1:2-100. It was also shown that component (C) had the effect of restoring nodule activity when isobutyl alcohol was used.

[0085] Example 4: Increased yield of soybeans in fields The effect of foliar application of a composition containing 1,500 ppm ascorbic acid on soybean yield was evaluated. Foliar application was performed only once, and eight test plots were examined (Table 5).

[0086] [Table 5]

[0087] (1)Cultivation conditions Soybean cultivation was carried out in a field in Tochigi Prefecture, and the soybean cultivar "Sato no Hohoemi" was used. In the field, foliar spray treatment was carried out in a 2m-wide section of the ridge, excluding the periphery. Three sections were tested per test area. An average of 10 plants were cultivated in the 2m area set as one section.

[0088] (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 the ascorbic acid solution per plant was sprayed so that it covered the entire soybean plant. 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 the seeds were collected from each individual and dried at 100°C for 48 hours. Dry seed mass was measured as yield data.

[0089] (3) Results After calculating the average seed weight of each individual in each plot, the average value and standard deviation of the eight plots were calculated (Figure 9). The graph in the figure shows the average value ± standard deviation. It was confirmed again 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 even when polyoxyethylene lauryl ether was used.

[0090] Example 5 Evaluation of nodule activity by high concentration application of ascorbic acid The soil preparation and sowing, inoculation of rhizobia, cultivation conditions, method for collecting exudation fluid to measure the amount of ureido nitrogen, which is an index of nodule activity, experimental procedures before quantifying the exudation fluid, the equipment and measurement method used to quantify the amount of ureido nitrogen, and the standards and quantitative items used were the same as in Example 3, and soybeans were cultivated for 21 days.

[0091] (1) Foliar spray treatment Foliar spray treatment was carried out on the 14th day 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 reagents were dissolved in tap water or deionized water to prepare spray solutions. Foliar spray was carried out 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 mister at 6.7 mL per plant, and test plots 4 to 8 were sprayed at 0.125 mL per plant.

[0092] [Table 6]

[0093] (2) Results The measurement results of 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 value ± standard deviation. In Figure 10, in test plots 4 and 5, where ascorbic acid was sprayed with a small amount of water, the soybean nodule activity tended to increase compared to test plots 2 and 3. Regardless of the amount of water used for spraying, when deionized water was used to dissolve ascorbic acid, the nodule activity was improved compared to when tap water was used. A comparison of test plots 5 and 6 showed 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, when the ascorbic acid concentration was increased to 300,000 ppm in test plot 7, the nodule activity decreased compared to test plot 6, indicating that an ascorbic acid concentration of 300,000 ppm or less is preferable when spraying with a small amount of water. A comparison of test plots 8 and 6 also showed that DMSO is 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.

[0094] Example 6 Evaluation of early growth indices in adzuki beans (1) Soil preparation and sowing Medium-term fertilizer-effect soil (Takii Hydrated Cell-based Medium-term Fertilizer-effect Soil, 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 a polypot (diameter 10.5 cm, height 9 cm). "Tamba Dainagon Azuki" (purchased from Takii Seed Co., Ltd.) was used as azuki bean seeds. After adding 250 mL of tap water per pot, two seeds were sown in each pot, one at a depth of about 1 to 2 cm from the soil surface. The number of replicates for each test plot was 6 (n=6).

[0095] (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, 1 L distilled water (pH 6.8)), and the soybean root nodule bacteria (Bradyrhizobium japonicum) NBRC14783T strain was grown on the solid medium. A loopful of the grown root nodule bacteria was taken and 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 root nodule culture solution with a bacterial turbidity OD600 value of approximately 0.3 was prepared. After sowing, 1 mL of the root nodule culture solution was dropwise inoculated onto the seeds using a micropipette.

[0096] (3)Cultivation conditions The plants were grown in an artificial climate chamber (LPH-411SP, Nippon Medical and Chemical Equipment Manufacturing Co., Ltd.) under light conditions of 440–480 μmol / m 2 The light / dark period was 16 hours / s, the temperature was 25°C light / 20°C dark, and the humidity was 50%. Seven days after sowing, the plants were thinned out so that there was one plant per pot. Watering was done 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.

[0097] (4) Foliar spray treatment The spray solution was prepared, and on the 21st day after sowing, 6.7 mL per plant was sprayed once using a sprayer. The spray solution was prepared by dissolving BHT in isobutyl alcohol, mixing isobutyl alcohol and sorbitan monolaurate, and then dissolving in water. Then, ascorbic acid was dissolved in water to prepare the solution. The reagents used were the same as those 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 weights of the above-ground and below-ground parts of the plants were measured.

[0098] 1 plot: control (no application) 2nd section: foliar spray of ascorbic acid at 1,500 ppm (dissolved in tap water) Section 3: foliar spray of ascorbic acid at 1,500 ppm by mass (dissolved in deionized water) 4th section: Ascorbic acid 1,500 ppm by mass + BHT 1 ppm by mass + isobutyl alcohol 50 ppm by mass + sorbitan monolaurate 350 ppm by mass sprayed on leaves (dissolved in tap water)

[0099] (5) Results The results of measuring 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 average value ± standard deviation. Test Example 1 and Example 2 show that in soybeans, when the same concentration of ascorbic acid is sprayed on the leaves, tap water and deionized water have different effects on seed weight and early growth indexes. In adzuki beans, as in soybeans, it was shown that the effect on early growth indexes differs depending on the water quality in which ascorbic acid is dissolved. A comparison of Test Areas 2 and 4 showed that by adding Components (B), (C) and (D) to ascorbic acid, a growth promotion effect can be obtained even when tap water is used.

[0100] Example 7 Evaluation of early growth indices in chickpea (1) Soil preparation and sowing Medium-term fertilizer-effect soil (Takii Hydrated Cell Soil Medium-term Fertilizer-effect, 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 a polypot (diameter 10.5 cm, height 9 cm). Chickpea seeds of the Kaburi variety (purchased from Nikko 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 about 1 to 2 cm from the soil surface. The number of replicates for each test plot was 6 (n=6).

[0101] (2) Inoculation of rhizobia Yeast-Mannitol (YM) medium (K 2 HPO 4 0.5g MgSO 4 7H 2 A solid medium was prepared by adding 1.5% agar (Wako Pure Chemical Industries, Ltd.) to 0.2 g of O, 0.1 g of NaCl, 0.4 g of yeast extract, 10 g of mannitol, and 1 L of distilled water (pH 6.8), and chickpea root nodule bacteria (Mesorhizobium ciceri) NBRC100389T strain was grown on the solid medium. A platinum loop of the grown root nodule bacteria was taken and inoculated into 50 mL of YM liquid medium in a 500 mL Sakaguchi flask, and cultured with shaking at 30°C for about 36 hours. A root nodule culture solution with a bacterial turbidity OD600 value of about 0.3 was prepared. After sowing, 1 mL of the root nodule culture solution was dropwise inoculated onto the seeds using a micropipette.

[0102] (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). The experimental 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 the dry weight of the above-ground parts of the plants was measured.

[0103] 1 plot: control (no application) 2nd section: foliar spray of ascorbic acid at 1,500 ppm (dissolved in tap water) Section 3: foliar spray of ascorbic acid at 1,500 ppm by mass (dissolved in deionized water) 4th section: Ascorbic acid 1,500 ppm by mass + BHT 1 ppm by mass + isobutyl alcohol 50 ppm by mass + sorbitan monolaurate 350 ppm by mass sprayed on leaves (dissolved in tap water)

[0104] (4) Results The results of measuring the aboveground dry weight 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 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 indexes. As with soybeans, chickpeas also showed that the effect on early growth indexes differs depending on the water quality in which ascorbic acid is dissolved. A comparison of Test Areas 2 and 4 showed that by adding Components (B), (C) and (D) to ascorbic acid, a growth promotion effect can be obtained even when tap water is used.

[0105] Example 8: Evaluation of seed weight in Lotus japonicus (1) Soil preparation Medium-term fertilizer effect soil (Takii hydrous cell soil, medium-term fertilizer effect, 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).

[0106] (2) Germination and sowing The Lotus japonicus seeds used were the Miyakojima MG-20 line. 1 mL of concentrated sulfuric acid (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a 2 mL microtube (Eppendorf Co., Ltd.) containing approximately 1 / 3 of the seeds, and the tubes were left to stand for 10 minutes. The tubes were rinsed five times with tap water, and then soaked for two hours. After supplying 100 mL of tap water per pot, three germinated seeds were planted in each pot, one at a depth of approximately 1-2 cm from the soil surface. Each test plot was repeated eight times (n=8).

[0107] (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, 1 L distilled water (pH 6.8)), and Mesorhizobium loti MAFF303099 (ML GUS) strain was grown on the solid medium. A loopful of the grown rhizobia was taken and 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 value of approximately 0.3 was prepared. After sowing, 1 mL of the rhizobia culture solution was dropwise inoculated onto the seeds using a micropipette.

[0108] (4)Cultivation conditions The plants were grown 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 / m2 / s. Ten days after sowing, the plants were thinned out to one plant per pot. Watering was performed by adding tap water to the pot after the water in the tray placed under the pot had run out, enough to immerse the bottom 5 cm of the pot. Three weeks after sowing, High Grade Flowering Promotion (Hyponex Japan Co., Ltd.) diluted 1,000 times was added to the water once a week.

[0109] (5) Foliar spray treatment The spray solution shown in Table 7 was prepared, and 6 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 types of test plots were examined. The spray solution was prepared by dissolving BHT (component (B)) in component (C) beforehand, then mixing components (C) and (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 to prepare the spray solution. Mature seeds were harvested one by one, and cultivation was terminated 16 weeks after sowing. The number of pods and seed weight of each plant were measured.

[0110] [Table 7]

[0111] (6) Results The measurement results of the pod number are shown in Figure 14, and the measurement results of the seed weight are shown in Figure 15. The graphs in the figures show the average 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. As with soybeans, it was shown that the effect on seed weight of Lotus japonicus varies depending on the water quality in which ascorbic acid is dissolved. Comparison of Test Areas 3 and 4 shows that the number of pods and seed weight increase when tap water is used by adding components (B), (C) and (D) to ascorbic acid. Test Area 5 shows that the yield increase effect can be obtained even when fertilizer components are added to the composition. Also, as shown in Test Area 6, a sufficient application effect was 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 japonicus, as with soybeans, increases yields even when sprayed in small volumes at high concentrations using tap water.

[0112] Example 9. Evaluation of seed weight in broad beans (1) Soil preparation and cultivation conditions After filling a planter (65.3 cm wide, 24.5 cm deep, 18.5 cm high) with 2 L of gravel, 9 L of medium-term fertilizer soil (Takii Hydrated Cell Culture Medium-Term Fertilizer, Takii Seed Co., Ltd.) was added. After adding 2 L of tap water to each 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 area was replicated three times (n=3). Cultivation was carried out outdoors.

[0113] (2) Foliar spray treatment The spray solution was prepared and sprayed with a mist sprayer at 6.7 mL per plant 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 in component (B) in component (C) beforehand, and then mixing components (C) and (D) to prepare the second agent. Next, component (A) and 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 to prepare the spray solution. The reagents were the same as those in Example 1. The 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) Section 2: foliar spray of 1,500 ppm ascorbic acid (dissolved in deionized water) Section 3: foliar spray of 1,500 ppm ascorbic acid (dissolved in tap water) 4th section: Ascorbic acid 1,500 ppm by mass + BHT 1 ppm by mass + isobutyl alcohol 50 ppm by mass + sorbitan monolaurate 350 ppm by mass sprayed on leaves (dissolved in tap water)

[0114] (3) Results The results of measuring the number of beans per plant are shown in Figure 16, and the results of measuring the 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 the seed weight by 32.9%, demonstrating that a yield-increasing effect can also be obtained for broad beans. A comparison of test plots 3 and 4 also showed that adding components (B), (C), and (D) to ascorbic acid increased the yield-increasing effect.

[0115] 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 A one- or two-dose composition was prepared by the following method 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). A one-dose composition was prepared by dissolving component (B) in component (C), mixing with component (D), and then mixing with component (A). A two-dose composition was prepared by the following method. Component (B) was dissolved in component (C) in advance, and then further mixed with component (D) to prepare a second agent. Next, only component (A) was used as the first agent. The reagents were the same as those in Example 1.

[0116] (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 solution solubility of the preparation after storage was evaluated when it was diluted 500 times in water.

[0117] (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-agent composition after storage at 50°C, the center photo shows the first and second agents of the two-agent composition after storage at 4°C, and the right photo shows the one-agent composition after storage at 50°C and the one-agent composition after storage at 4°C.

[0118] From the results in Figure 18, no difference was observed in the appearance of the two-formulation composition samples stored at 4°C and those stored at 50°C. On the other hand, the color of the one-formulation composition's appearance differed depending on the storage temperature. It was also shown that both the one-formulation and two-formulation types were able to produce a uniform spray solution suitable for application as a plant growth promoter. The study results suggested that the one-formulation type showed a large degree of discoloration during storage, and that the two-formulation type was superior in terms of appearance.

[0119] Example 11: Examination of the mixing ratio of organic solvents The soil preparation and sowing, inoculation of rhizobia, method of collecting exudate to measure the amount of ureido nitrogen, which is an index of nodule activity, experimental procedures before quantifying the exudate, equipment and measurement method used to quantify the amount of ureido nitrogen, and standards and quantitative items were the same as in Example 2, and soybeans were cultivated for 21 days.

[0120] (1)Cultivation conditions Cultivation was performed in an artificial weather chamber (LPH-411SP, Nippon Medical and Chemical Equipment Manufacturing Co., Ltd.) with light conditions of 16 hours light (440-480 μmol / m2 / s) / 8 hours dark, temperature of 30°C light / 25°C dark, and humidity of 50%. Seven days after sowing, the plants were thinned out to one per pot. Watering was performed by adding tap water to the pot after the water in the tray placed under the pot had run out, enough to cover the bottom 5 cm of the pot.

[0121] (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 in test plots 1 to 5, and 0.125 mL per plant in test plots 6 to 8. Foliar spray was performed only once, and seven types of test plots were examined (Table 8). The reagents used were the same as those in Example 5.

[0122] [Table 8]

[0123] (3) Results The measurement results of nodule activity are shown in Figure 19. The graph in the figure shows the average 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 higher effect of improving root 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 mixture ratio of components (B) and (C) was 1:10-10,000.

[0124] Example 12 Evaluation of early growth indices at low concentrations of antioxidants Soil preparation, sowing, and inoculation with rhizobia were performed in the same manner as in Example 2, and soybeans were cultivated for 21 days.

[0125] (1)Cultivation conditions Cultivation was performed in an artificial weather chamber (LPH-411SP, Nippon Medical and Chemical Equipment Manufacturing Co., Ltd.) with light conditions of 16 hours light (440-480 μmol / m2 / s) / 8 hours dark, temperature of 30°C light / 25°C dark, and humidity of 50%. Seven days after sowing, the plants were thinned out to one per pot. Watering was performed by adding tap water to the pot after the water in the tray placed under the pot had run out, enough to cover the bottom 5 cm of the pot.

[0126] (2) Foliar spray treatment A spray solution was prepared, and 6.7 mL per plant was sprayed once using a sprayer on the 14th day after sowing. The spray solution was prepared by dissolving BHT in isobutyl alcohol, mixing isobutyl alcohol and sorbitan monolaurate, and then dissolving in water. Then, ascorbic acid was dissolved in water to prepare the solution. The reagents used were the same as those in Example 2. The 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 measured.

[0127] 1 plot: control (no application) 2nd section: foliar spray of ascorbic acid at 1,500 ppm (dissolved in tap water) Section 3: 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 sprayed on leaves (dissolved in tap water)

[0128] (3) Results The results of measuring the aboveground dry weight are shown in Figure 20. The graph in the figure shows the average 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 ppm by mass, the addition of components (B), (C) and (D) to ascorbic acid resulted in a greater growth-promoting effect than the application of ascorbic acid alone, dissolved in tap water.

[0129] Example 13 Evaluation of early growth index when using anionic surfactants Soil preparation, sowing, and inoculation with rhizobia were performed in the same manner as in Example 2, and soybeans were cultivated for 21 days.

[0130] (1)Cultivation conditions Cultivation was performed in an artificial weather chamber (LPH-411SP, Nippon Medical and Chemical Equipment Manufacturing Co., Ltd.) with light conditions of 16 hours light (440-480 μmol / m2 / s) / 8 hours dark, temperature of 30°C light / 25°C dark, and humidity of 50%. Seven days after sowing, the plants were thinned out to one per pot. Watering was performed by adding tap water to the pot after the water in the tray placed under the pot had run out, enough to cover the bottom 5 cm of the pot.

[0131] (2) Foliar spray treatment Foliar spray treatment was carried out on the 14th day 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. Spray solutions were prepared by dissolving the reagents in tap water. Foliar spray was carried out only once, and five types of 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.

[0132] [Table 9]

[0133] (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. Comparing test plots 2 with 3, 4, and 5, it was shown 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 spraying ascorbic acid alone, dissolved in tap water.

[0134] Example 14 Evaluation of soybean nodule activity (1) Soil preparation and sowing Medium-term fertilizer-effect soil (Takii Hydrated Cell Soil Medium-term Fertilizer-effect, 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 a polypot (diameter 10.5 cm, height 9 cm). "Enrei" soybean seeds (purchased from Nikko 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 about 1 to 2 cm from the soil surface. Each test plot was repeated 6 times (n=6).

[0135] (2) Inoculation of rhizobia Yeast-Mannitol (YM) medium (K 2 HPO 4 0.5g MgSO 4 7H 2A solid medium was prepared by adding 1.5% agar (Fujifilm Wako Pure Chemical Industries, Ltd.) to 0.2 g of O, 0.1 g of NaCl, 0.4 g of yeast extract, 10 g of mannitol, and 1 L of distilled water (pH 6.8), and the soybean root nodule bacteria (Bradyrhizobium japonicum) NBRC14783T strain was grown on the solid medium. A platinum loop of the grown root nodule bacteria was taken and inoculated into 50 mL of YM liquid medium in a 500 mL Sakaguchi flask, and cultured with shaking at 30°C for about 36 hours. A root nodule culture solution with a bacterial turbidity OD600 value of about 0.3 was prepared. After sowing, 1 mL of the root nodule culture solution was dropwise inoculated onto the seeds using a micropipette.

[0136] (3)Cultivation conditions The cultivation from sowing to thinning was carried out in an artificial climate chamber (LPH-411SP, Nippon Medical and Chemical Equipment Manufacturing Co., Ltd.) under light conditions of 440-480 μmol / m 2 The light / dark period was 16 hours / s, the temperature was 30°C light / 25°C dark, and the humidity was 50%. Seven days after sowing, the plants were thinned out so that there was one plant per pot. Watering was done 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.

[0137] (4) Foliar spray treatment The spray solution was prepared and sprayed with a mister at 6.7 mL per plant on the 14th day after sowing. Foliar spray was performed only once, and three types of test plots were examined. Test plots 1 to 3 evaluated are as follows. <Spray liquid> 1 plot: control (no application) 2nd section: foliar spray of ascorbic acid at 1,500 ppm (dissolved in tap water) 3rd section: Ascorbic acid 1,500 ppm by mass + BHT 1 ppm by mass + diethylene glycol 200 ppm by mass + sorbitan monolaurate 350 ppm by mass sprayed on leaves (dissolved in tap water)

[0138] (5) A method for collecting exudate 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, Inc.) filled with cotton balls #10 (Iwatsuki, Inc.) was placed over the cut stem to collect the exudation fluid from the stem for 2 hours. The cotton balls after collection were stored in a -80°C freezer. The volume of the exudation fluid was calculated by comparing the weight of the cotton balls before and after collection.

[0139] (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.) operated at 10,000 rpm for 1 minute. The filtrate was diluted 3,000 times and quantified.

[0140] (7) Apparatus and method for determining the amount of ureido nitrogen The HPLC and mass spectrometers used were Agilent 1260 Infinity LC system (Agilent Technologies, Inc.) and AB SCIEX TripleQuad 4500 system (AB SCIEX, Inc.), respectively. The column used was Scherzo SS-C18 (100 mm x 2 mm, 3 μm) (Intact Corporation), and the oven temperature was 40°C. 5 μL of appropriately diluted sample was injected, and the flow rate was 0.5 mL / min. The eluents were 0.1% formic acid aqueous solution (eluent A) and 50 mM ammonium acetate / methanol solution (eluent B). The sample was supplied after equilibration with eluent A:eluent B = 95:5, and ureido nitrogen was eluted with a linear gradient of eluent A:eluent B = 80:20 after 5 minutes.

[0141] (8) Standards used and quantification The amount of ureido nitrogen in the exudate was analyzed by LC-MS. Allantoic acid (Toronto Research Chemicals Inc.) was used as the standard. The standard was analyzed by LC-MS, and a calibration curve was created in the range of 10-1000 ppb. Allantoic acid was identified for each sample based on the retention time, accurate mass, and MS / MS spectrum match with each reagent. Allantoic acid in the samples was quantified based on the calibration curve, and the total amount per plant was calculated.

[0142] (9) Results The measurement results of nodule activity are shown in Figure 30. The graphs in each figure show the average value ± standard deviation. A comparison of test plots 2 and 3 showed that adding components (B), (C) and (D) to ascorbic acid resulted in a greater effect on improving nodule activity than when ascorbic acid alone was sprayed using tap water for dissolution.

[0143] Example 15 Effect on soybean growth (1) Soil preparation and sowing Medium-term fertilizer-effect soil (Takii Hydrated Cell Soil Medium-term Fertilizer-effect, 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 a polypot (diameter 10.5 cm, height 9 cm). "Enrei" soybean seeds (purchased from Nikko 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 about 1 to 2 cm from the soil surface. Each test plot was repeated 6 times (n=6).

[0144] (2) Inoculation of rhizobia Yeast-Mannitol (YM) medium (K 2 HPO 4 0.5g MgSO 4 7 H 2 O 0.2g, NaCl 0.1g, Yeast Extract 0.4g, Ma A solid medium was prepared by adding 1.5% agar (Fujifilm Wako Pure Chemical Industries, Ltd.) to 10 g of nnitol and 1 L of distilled water (pH 6.8), and the soybean root nodule bacteria (Bradyrhizobium japonicum) NBRC14783T strain was grown on the solid medium. A loopful of the grown root nodule bacteria was taken and 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 root nodule culture solution with a bacterial turbidity OD600 value of approximately 0.3 was prepared. After sowing, 1 mL of the root nodule culture solution was dropwise inoculated onto the seeds using a micropipette.

[0145] (3)Cultivation conditions The plants were grown in an artificial climate chamber (LPH-411SP, Nippon Medical and Chemical Equipment Manufacturing Co., Ltd.) under light conditions of 440–480 μmol / m 2 The light / dark period was 16 hours / s, the temperature was 30°C light / 25°C dark, and the humidity was 50%. After germination, the plants were thinned out so that there was one plant per pot. Watering was done 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.

[0146] (4) Foliar spray treatment The spray solution was prepared and sprayed 6.7 mL per plant using a spray bottle on the 7th day after sowing. Foliar spray was performed only once, and five types of test plots were examined. Test plots 1 to 5 evaluated are as follows. <Spray liquid> 1 plot: control (no application) 2nd section: foliar spray of ascorbic acid at 1,500 ppm (dissolved in tap water) 3rd section: Ascorbic acid 1,500 ppm by mass + BHT 1 ppm by mass + Glycerin 50 ppm by mass (dissolved in tap water) 4th section: Ascorbic acid 1,500 ppm by mass + BHT 1 ppm by mass + propylene glycol 50 ppm by mass (dissolved in tap water) 5th section: Sodium ascorbate 1,500 ppm by mass + tocopherol 10 ppm by mass + glycerin 50 ppm by mass (dissolved in tap water)

[0147] (5) Results On the 9th day after sowing, the plants were dried at 100°C for 7 hours, and the dry weight of the aboveground parts of the plants was measured. The results of measuring the dry weight of the aboveground parts are shown in Figure 31. The graphs in each figure show the average value ± standard deviation. Comparing test plots 1 to 5, it was shown that the growth promotion effect was not confirmed when ascorbic acid was dissolved in tap water and sprayed on the leaves, but by adding components (B) and (C) to ascorbic acid or sodium ascorbate, the growth promotion effect was obtained even when tap water was used as the dissolving water. Compared to the control, the aboveground dry weight increased by an average of about 3.7% in test plot 3, about 9.1% in test plot 4, and about 2.1% in test plot 5. [Explanation of symbols]

[0148] 1. First bottle 2. Second bottle body 3 Bottle body with external thread at bottom 4 Narrow neck bottle head 5 Lid 6 Bottle body 7. Wide-mouth bottle head 8 Connecting members 9 Cover Plate 10 First ring wall of connecting member 8 11 second ring wall of connecting member 8 12 External thread of bottle body 3 13 Screw-fitting structure between first bottle body and connecting member 8 14 Screw-fitting structure between second bottle body and connecting member 8 15 Internal thread portion of inner wall of first ring wall 10 16 External thread of head 7 of wide-mouth bottle 17 Internal thread on the inner wall of the second ring wall 11 18 Connecting members 19 Cover plate 20 first ring wall of connecting member 18 21 second ring wall of connecting member 18 22 Internal thread portion of the inner wall of the first ring wall 20 23 Screw-fitting structure between first bottle body and connecting member 18 24 Internal thread portion of the inner wall of the second ring wall 21 25 Screw-fitting structure between second bottle body and connecting member 18 26a, 26b Two-headed bottle body

Claims

1. A growth-promoting composition for legumes contained in beans, comprising a first agent containing (A) ascorbic acid or a salt thereof and a second agent containing (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, the ratio (mass ratio) of (A) ascorbic acid or a salt thereof being 10 to 3,000,000 when the component (B) is taken as 1, or the ratio (mass ratio) of (C) the organic solvent being 4 to 100,000 when the component (B) is taken as 1, and the first agent and the second agent are each contained in one of the bottles of a double-headed bottle in which a first bottle and a second bottle are connected by a connecting member.

2. 2. The composition of claim 1, wherein the first bottle body comprises a bottle body having an external thread at the bottom, a narrow-mouth bottle head, and a cap, and the second bottle body comprises a bottle body and a wide-mouth bottle head.

3. The composition according to claim 1 or 2, wherein the first agent is contained in a second bottle and the second agent is contained in a first bottle.

4. A bottle body containing the composition described in claim 1, which is a double-headed bottle body in which a second bottle body having a bottle body and a wide-mouth bottle head is connected to the lower part of a first bottle body having a bottle body with an external thread portion at the bottom, a narrow-mouth bottle head, and a lid body via a connecting member.

5. 5. The bottle body according to claim 4, wherein the connecting member comprises a cover plate, a first ring wall extending upward along the outer circumferential edge of the cover plate, and a second ring wall formed by folding the first ring wall downward, and wherein an external thread provided on the bottom of the bottle barrel of the first bottle body screws into an internal thread provided on the inner wall of the first ring wall of the connecting member, and a wide-mouth bottle head of the second bottle body screws into an internal thread provided on the inner wall of the second ring wall of the connecting member, thereby connecting the first bottle body and the second bottle body.

6. The bottle body according to claim 4, wherein the connecting member comprises a cover plate and a first ring wall and a second ring wall each protruding perpendicularly from the outer circumferential edge of the cover plate, and the first bottle body and the second bottle body are connected by an external thread portion provided on the bottom of the bottle barrel of the first bottle body screwing into an internal thread portion provided on the inner wall of the first ring wall of the connecting member, and a wide-mouth bottle head portion of the second bottle body screwing into an internal thread portion provided on the inner wall of the second ring wall of the connecting member.

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