Root nodule activity promoting agent

Abstract

To provide a method and a material for promoting growth of soybean.SOLUTION: A root nodule activity promoting agent for soybean includes ascorbic acid or its salt as an active ingredient, and ascorbic acid in a concentration of 5,000-20,000 mass ppm is applied to the soybean.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a nodule activity promoter that promotes nodule activity in soybeans. [Background technology]

[0002] Various legumes are used as important crops around the world. Among them, soybeans, which have a high protein content per unit area, are extremely important to the world's food supply as a high-protein, high-calorie crop. Soybeans are edible as they are, but they are also processed into soybean oil and high-protein soybean meal by pressing. They are also used as a raw material for lecithin, a natural emulsifier.

[0003] Soybean oil is used as a raw material for cosmetics, soap, and other daily necessities, and is also used as biofuel. Soybean meal is also important as livestock feed. With the recent increase in animal protein consumption in developing and emerging countries and the growing food demand due to population growth, there is a growing need to increase the production of legumes, especially soybeans, for livestock feed.

[0004] Rhizobium is a soil microorganism that forms nodules on the roots of legumes and performs symbiotic nitrogen fixation. Rhizobium reduces atmospheric nitrogen within the nodules, converting it into ammonia nitrogen, which is then converted into ureides such as allantoin and allantoic acid, or amides such as asparagine, and supplies the resulting nitrogen to the host. Soybeans absorb a large amount of nitrogen, most of which is believed to be derived from rhizobium. Therefore, enhancing nodules is considered important for ensuring soybean productivity, and techniques for increasing and activating rhizobium by adding specific compounds have been reported. For example, Patent Document 1 reports that N-acyllactam compounds have the effect of increasing rhizobium proliferation (Patent Document 1), and that inoculating soybeans with an inoculant containing glycine betaine increases the number of nodules, nodule weight, and aboveground dry weight (Patent Document 2).

[0005] Meanwhile, antioxidants containing ascorbic acid are often incorporated into materials applied to plants to promote growth. For example, Non-Patent Document 1 discloses that the number of leaves increased when olive trees were sprayed twice with ascorbic acid at concentrations of 500, 1,000, and 2,000 ppm. Non-Patent Document 2 also discloses that chickpea, a legume, can achieve a yield increase of up to 30% when foliar sprays of 100, 200, and 300 ppm aqueous ascorbic acid solutions were applied three times. However, even in Non-Patent Document 2, it is estimated that the maximum yield-increasing effect can be achieved at 226 ppm, and application of ascorbic acid at high concentrations has not been carried out to date. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 63-304977 [Patent Document 2] Japanese Patent Application Laid-Open No. 2003-40720 [Patent Document 3] Japanese Patent Application Laid-Open No. 2009-73772 [Non-patent literature]

[0007] [Non-Patent Document 1] Mayi et al., (2014) Effect of Foliar Spray of Humic acid, Ascorbic acid, Cultivars and their Interactions on Growth of Olive (Olea European L.) Transplants cvs. Khithairy and Sorany. IOSR Journal of Agriculture and Veterinary Science, 7:18-30). [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]

[0008] The present invention relates to providing methods and materials for promoting soybean growth. [Means for solving the problem]

[0009] The present inventors have found that high concentrations of ascorbic acid have the effect of increasing the amount of ureide produced in nodulated soybeans, and are useful for improving soybean seed yield.

[0010] That is, the present invention relates to the following 1) to 4). 1) A nodule activity promoter for soybeans, which contains ascorbic acid or a salt thereof as an active ingredient, and which is applied to soybeans at a concentration of 5,000 to 20,000 ppm by mass as ascorbic acid. 2) A soybean growth promoter containing ascorbic acid or a salt thereof as an active ingredient, which is applied to soybeans at a concentration of 5,000 to 20,000 ppm by mass as ascorbic acid. 3) A method for promoting nodule activity in soybeans, comprising the step of applying a treatment solution containing ascorbic acid or a salt thereof to soybeans at 5,000 to 20,000 ppm by mass. 4) A method for promoting soybean growth, comprising the step of applying a treatment solution containing ascorbic acid or a salt thereof to soybeans at 5,000 to 20,000 ppm by mass. [Brief explanation of the drawings]

[0011] [Figure 1]Amount of allantoin in soybean exudate one week after foliar spray of ascorbic acid. [Figure 2] Amount of allantoic acid in soybean exudate one week after foliar spray of ascorbic acid. [Figure 3] Soybean plants one week after foliar spray of ascorbic acid. [Figure 4] Chickpea nodule weight one week after foliar application of ascorbic acid. [Figure 5] Amount of allantoic acid in chickpea exudate one week after foliar application of ascorbic acid. [Figure 6] Asparagine content in chickpea exudate one week after foliar application of ascorbic acid. [Figure 7] Seed weight per Lotus japonicus plant one week after foliar spray of ascorbic acid. [Figure 8] Number of pods per Lotus japonicus plant one week after foliar spray of ascorbic acid. [Figure 9] Seed weight per pod of Lotus japonicus one week after foliar spray of ascorbic acid. Detailed Description of the Invention

[0012] In the present invention, the term "nodule" refers to a nodule that forms on the root of a plant through symbiosis with bacteria (rhizobia). Within the nodule, rhizobia reduce atmospheric nitrogen to ammonia nitrogen, which is then supplied to the host, thereby performing so-called symbiotic nitrogen fixation. Rhizobium is known to be specific to the plants with which it can coexist symbiotically. Examples of rhizobium that are known to host soybeans include Bradyrhizobium diazoefficiens, Bradyrhizobium japonicum, Bradyrhizobium elkanii, and Ensifer fredii.

[0013] In the present invention, "soybean" refers to soybean (scientific name: Glycine max), an annual plant of the legume family. There are a wide variety of soybean varieties, including Fukuyutaka, Enrei, Sato no Hohoemi, Yuagari Musume, Ryuho, and Suzuyutaka, but the present invention is not limited to these.

[0014] In the present invention, ascorbic acid may be any of D-, L-, and DL-isomers, but is preferably L-isomer (so-called L-ascorbic acid). Commercially available ascorbic acid of various grades can be used. Examples of salts of ascorbic acid include sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, ammonium salts, and salts with nitrogen-containing organic bases such as pyridine, trimethylamine, triethylamine, tributylamine, and diethylamine.

[0015] As shown in the Examples below, when soybean seeds are sown and then inoculated with rhizobia to cultivate soybeans (nodule-forming plants), application of ascorbic acid at a certain concentration or higher to the soybean plants has been shown to increase the amount of ureido compounds such as allantoic acid produced, thereby promoting ureido production in the nodules. Therefore, the use of ascorbic acid or a salt thereof at a certain concentration or higher can serve as a soybean nodule activity promoter or soybean growth promoter, and can be used to promote soybean nodule activity or growth, and can also be used to manufacture a soybean nodule activity promoter or soybean growth promoter. It has been reported that the amount of ureide derived from nodules correlates with grain yield (Diagnosing method for total basal nitrogen fertilizer application in soybeans in rotational upland fields, Ibaraki Prefectural Agricultural Center, Agricultural Research Institute, H18 Main Results; http: / / www.pref.ibaraki.jp / nourinsuisan / noken / seika / h18pdf / documents / 27.pdf). Therefore, such a nodule activity promoter is useful for increasing grain yield. It is thought that this is the case.

[0016] In the present invention, "nodule activity" refers to the nitrogen fixation function in a host plant exerted by nodules, and "promotion of nodule activity" refers to promoting the nitrogen fixation function exerted by nodules. Here, the nitrogen fixation function in a host plant can be evaluated as the ability to produce ureide or amide. Therefore, the nodule activity of the present invention can be evaluated more specifically as the amount of ureide or amide produced per wet nodule weight. The amount of ureide or amide produced can be calculated by measuring the amount of ureide or amide in exudate collected after cutting the plant (e.g., cutting the above-ground part just below the cotyledons). Examples of ureides include allantoin, allantoic acid, and citrulline, and examples of amides include asparagine and glutamine. It is preferable to measure the amount of allantoic acid or asparagine.

[0017] In the present invention, "promoting soybean growth" means increasing the growth amount (fresh weight, elongation, etc.) of soybeans and increasing the yield of fruits, seeds, or grains, preferably increasing the yield of grains.

[0018] The above-mentioned soybean nodule activity promoter or soybean growth promoter can be a composition (e.g., various agricultural or horticultural materials) for promoting nodule activity in soybeans or promoting soybean growth, or a material (single substance) or formulation to be added to or incorporated into various agricultural or horticultural materials. The composition may be in the form of a liquid or gel composition, or may be in a solid state (block, powder, granules, etc.). That is, it may be in the form of a liquid that can be applied directly to soybean plants, or a composition that is diluted or dissolved before use.

[0019] The composition may contain any component in addition to the ascorbic acid or salt thereof of the present invention, such as a solvent (e.g., water, buffer solution, culture medium, solution for hydroponics, etc.), a carrier (e.g., diatomaceous earth, vermiculite, perlite, peat moss, activated carbon, humus, talc, zeolite, clay, carbon black, pulp, straw, soybean meal, bentonite, kaolin, montmorillonite, alumina, etc.), a pH adjuster, a spreading agent for increasing the spreading ability of the fertilizer to plants, a fertilizer component for increasing the fertilizer effect, an agricultural chemical component, a binder, a bulking agent, a plant growth-promoting microorganism such as rhizobia or mycorrhizal fungi, These include essential plant nutrients, flavonoids, organic acids, amino acids, peptides, nucleosides, nucleotides, nucleic acid bases, sugars, monohydric alcohols, nonionic surfactants, 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.

[0020] Examples of the composition include, but are not limited to, agricultural or horticultural materials such as fertilizers, water for watering, soil conditioners, pesticides, and plant supplements (e.g., activators, nutrients, etc.). The compositions may be prepared by adding ascorbic acid or a salt thereof to conventional agricultural or horticultural materials.

[0021] The content of ascorbic acid or a salt thereof in the root nodule activity promoter or soybean growth promoter of the present invention can be appropriately set to suit the application amount, and is, for example, 50% by mass or more, preferably 60% by mass or more, more preferably 70% by mass or more, and 100% by mass or less, preferably 98% by mass or less, more preferably 95% by mass or less, based on the total mass of the composition. Alternatively, it is 50 to 100% by mass, preferably 60 to 98% by mass, more preferably 70 to 95% by mass.

[0022] The application of the nodule activity promoter or soybean growth promoter of the present invention may be carried out by supplying the composition to soybeans so that the effects of the present invention can be exerted, and is preferably by spraying the composition so that it comes into contact with the soybean plant body, preferably by foliar spraying. In this case, if the composition is in liquid form, it is used as is or diluted with water, preferably ion-exchanged water, to prepare a spray solution, and if the composition is in solid form, it is dissolved in water, preferably ion-exchanged water, to prepare a spray solution, which is then supplied to soybean plants. The method of application is not particularly limited, but examples thereof include a spray method, i.e., a method of spraying the application solution in the form of a mist by atomization. By using such a method, the root nodule activity promoter or soybean growth promoter of the present invention adheres to plants and then exhibits good spreadability on the plants. Specific examples of spraying methods include manual spraying using a sprayer, atomizer, or sprayer (e.g., a boom sprayer), and aerial spraying using an airplane, helicopter, drone, etc.

[0023] The application amount of the root nodule activity promoter or soybean growth promoter may vary depending on the application method, application time, cultivation density, growth stage, etc., but for example, the application amount of ascorbic acid is, as a concentration in the spray solution when sprayed on soybeans foliage, 5,000 mass ppm or more, preferably 8,000 mass ppm or more, more preferably 10,000 mass ppm or more, and 20,000 mass ppm or less, preferably 18,000 mass ppm or less, more preferably 15,000 mass ppm or less, or 5,000 to 20,000 mass ppm, preferably 8,000 to 18,000 mass ppm, more preferably 10,000 to 15,000 mass ppm. The amount of ascorbic acid applied per soybean plant is 33 mg or more, preferably 53 mg or more, more preferably 66 mg or more, and 133 mg or less, preferably 120 mg or less, more preferably 100 mg or less, or 33 to 133 mg, preferably 53 to 120 mg, more preferably 66 to 100 mg.

[0024] The nodule activity promoter or soybean growth promoter may be applied in an amount within the above range at once or in divided doses.

[0025] The timing and frequency of application is usually such that application is carried out once or 1 to 3 times at R4 (pod elongation stage), R5 (early grain filling stage), and R6 (grain filling stage) in the growth stage indication of the reproductive growth period of soybeans according to Fehr et al. (Fehr, WR, Caviness, CE, 1977. Stages of soybean development. Cooperative Extension Service, Agriculture and Home Economics Experiment Station, Iowa State University, Ames, Iowa), i.e., approximately 5 to 13 weeks, preferably 6 to 12 weeks, after sowing.

[0026] In relation to the above-described embodiment, the present invention further discloses the following aspects. <1> A nodule activity promoter for soybeans, which contains ascorbic acid or a salt thereof as an active ingredient, and is applied to soybeans at a concentration of 5,000 to 20,000 ppm by mass as ascorbic acid. <2> A soybean growth promoter containing ascorbic acid or a salt thereof as an active ingredient, which is applied to soybeans at a concentration of 5,000 to 20,000 ppm by mass in terms of ascorbic acid. <3> Application to soybeans is by foliar spray; <1> or <2> The agent described in <4> A method for promoting nodule activity in soybeans, comprising the step of applying a treatment solution containing ascorbic acid or a salt thereof to soybeans at 5,000 to 20,000 ppm by mass. <5> A method for promoting soybean growth, comprising the step of applying a treatment solution containing ascorbic acid or a salt thereof to soybeans at a concentration of 5,000 to 20,000 ppm by mass. <6> Application to soybeans is by foliar spray; <4> or <5> The method described below. <7> Apply to soybeans once or 1 to 3 times during the seed filling stage or 6 to 12 weeks after sowing. <6> The method described below. <8> Use of ascorbic acid or a salt thereof for producing a soybean nodule activity promoter to be applied to soybeans at a concentration of 5,000 to 20,000 ppm by mass as ascorbic acid. <9> Use of ascorbic acid or a salt thereof for producing a soybean growth promoter to be applied to soybeans at a concentration of 5,000 to 20,000 ppm by mass as ascorbic acid. <10> <1> ~ <9> In the above, the application amount of ascorbic acid is, as a concentration in the spray solution when sprayed on the leaves of soybeans, preferably 8,000 ppm by mass or more, more preferably 10,000 ppm by mass or more, and preferably 18,000 ppm by mass or less, more preferably 15,000 ppm by mass or less, or preferably 8,000 to 18,000 ppm by mass, more preferably 10,000 to 15,000 ppm by mass. <11> <1> ~ <9> In this study, the amount of ascorbic acid applied per soybean plant was 33 to 133 mg. <12> <1> or <2> In the agent described above, the content of ascorbic acid or a salt thereof in the composition is 50% by mass or more, preferably 60% by mass or more, more preferably 70% by mass or more, and 100% by mass or less, preferably 98% by mass or less, more preferably 95% by mass or less, based on the total mass of the composition, or 50 to 100% by mass, preferably 60 to 98% by mass, more preferably 70 to 95% by mass. [Example]

[0027] Example 1 Growth-promoting effect of ascorbic acid on soybean The effect of a single foliar spray of high-concentration ascorbic acid on soybean nodule activity was evaluated. The experimental plots evaluated were as follows: Test area 1: Control (no application) Test area 2: Foliar spray of ascorbic acid 1,500 ppm Test area 3: Ascorbic acid 3,000 ppm foliar spray Test area 4: Ascorbic acid 5,000 ppm foliar spray Test area 5: Ascorbic acid 10,000 ppm foliar spray Test area 6: Ascorbic acid 20,000 ppm foliar spray

[0028] (1) Soil preparation and sowing A medium-term fertilizer release soil (Takii Hydrated Cell Fertilizer Medium-Term Release, Takii Seed Co., Ltd.) and vermiculite granules (Akagi Engei Co., Ltd.) were mixed at a volume ratio of 1:1, and the soil was filled into polypots (10.5 cm diameter, 9 cm height). Soybean seeds "Enrei" (purchased from Nikko Seed Co., Ltd.) were used. After adding 250 mL of tap water per pot, two seeds were sown per pot, one at a depth of approximately 1-2 cm from the soil surface. Each test plot had six replicates (n = 6).

[0029] (2) Inoculation of rhizobia Yeast-Mannitol (YM) medium (K2HPO4 0.5g, MgSO4 7 H2O 0.2g, NaCl 0.1g, Yeast Extract 0.4g, Ma A solid medium was prepared by adding 1.5% agar (Fujifilm Wako Pure Chemical Industries, Ltd.) to 10 g of nitritol and 1 L of distilled water (pH 6.8). The soybean rhizobia (Bradyrhizobium japonicum) strain NBRC14783T was grown on the solid medium. A platinum loop of the grown rhizobia was inoculated into 50 mL of YM liquid medium in a 500 mL Sakaguchi flask and cultured with shaking at 30°C for approximately 36 hours. A rhizobia culture solution with a bacterial turbidity (OD600) of approximately 0.3 was prepared. After sowing, 1 mL of the rhizobia culture solution was dropwise inoculated onto the seeds using a micropipette.

[0030] (3)Cultivation conditions Cultivation was carried out indoors under the following conditions: 16 hours of light, 25°C, LED light source, and light intensity of 400-440 μmol / m 2 7 days after sowing, the plants were grown in a pot with one plant per pot. Watering was carried out by adding tap water to the tray placed under the pots after the water had run out, so that the bottom 5 cm of the pots were covered.

[0031] (4) Foliar spray treatment On the 21st day after sowing, 6.7 mL of ascorbic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) dissolved in tap water was sprayed per plant using a spray bottle. Ascorbic acid was applied only once, at concentrations ranging from 1,500 to 20,000 ppm.

[0032] (5) Measurement of nodule activity 1) A method for collecting exudate fluid to measure the amount of ureido nitrogen, which is an index of nodule activity After 28 days of cultivation, soybean stems were cut at the cotyledonary node using pruning shears, and a 1.5 mL microtube (Eppendorf) filled with #10 cotton balls (Kawamoto Sangyo Co., Ltd.) was placed over the cut stem to collect exudation fluid from the stem for 2 hours. After exudation fluid collection, the cotton balls were stored in a -80°C freezer. The volume of exudation fluid was calculated by comparing the weight of the cotton balls before and after collection.

[0033] 2) Experimental procedures before quantifying exudate The exudate contained in the cotton ball was eluted with ultrapure water and filtered using a Micro Bio-Spin Chromatography column (Bio-Rad Laboratories, Inc.). The filtrate was collected by centrifugation using a centrifuge (CR15RN, Hitachi, Ltd.) at 15,000 rpm for 1 minute. The filtrate was diluted 1,000 times and quantified.

[0034] 3) Apparatus and measurement method used to quantify the amount of ureido nitrogen The HPLC and mass spectrometers used were an Agilent 1260 Infinity LC system (Agilent Technologies, Inc.) and an AB SCIEX TripleQuad 4500 system (AB SCIEX Corporation), respectively. The column was a Scherzo SS-C18 (100 mm × 2 mm, 3 μm) (intact). The oven temperature was set to 40°C. 5 μL of the appropriately diluted sample was injected. The flow rate was 0.5 mL / min, and the eluents were 0.1% formic acid aqueous solution (eluent A) and 50 mM ammonium acetate / methanol solution (eluent B). After equilibration at eluent A:eluent B = 95:5, the sample was applied, and ureido nitrogen was eluted using a linear gradient such that after 5 minutes the eluent A:eluent B = 80:20.

[0035] 4) Standards used and quantification The amount of ureido nitrogen in the exudate was analyzed by LC-MS. Allantoin (Tokyo Chemical Industry Co., Ltd.) and allantoic acid (Toronto Research Chemicals Inc.) were used as standards. The standards were analyzed by LC-MS, and a calibration curve was prepared in the range of 10-1000 ppb. Allantoin and allantoic acid were identified 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 using the calibration curve, and the total amount per plant was calculated.

[0036] (6) Results The results of measuring nodule activity are shown in Figures 1 and 2. The graphs in the figures show the mean value ± standard deviation. As shown in Figures 1 and 2, the test plots with higher ascorbic acid concentrations tended to have higher nodule activity. The highest nodule activity was observed in test plot 6, where ascorbic acid was sprayed onto the leaves at a concentration of 20,000 ppm. Furthermore, as shown in Figure 2, higher nodule activity was observed in test plots 4 to 6 compared to test plot 1, indicating that an ascorbic acid concentration of 5,000 ppm or higher is the most preferable concentration.

[0037] Comparative Example 1: Effect of high concentration ascorbic acid on soybeans The effects of a single foliar spray of high-concentration ascorbic acid on soybean leaves were evaluated. Test plots 1 and 2 were as follows: Test area 1: Foliar spray of 20,000 ppm ascorbic acid Test area 2: Ascorbic acid 40,000 ppm foliar spray

[0038] The soil preparation and sowing, rhizobia inoculation, and cultivation conditions were the same as in Example 1, and cultivation was carried out for 28 days. (1) Foliar spray treatment As in Example 1, on the 21st day after sowing, 6.7 mL of ascorbic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) dissolved in tap water was sprayed per plant using a spray bottle. Ascorbic acid was applied only once, at concentrations ranging from 20,000 to 40,000 ppm. Photographs of the plants were taken at the end of cultivation.

[0039] (2) Results Figure 3 shows a photograph of the plant one week after foliar spray. The soybean on the left side of the photograph was sprayed with 40,000 ppm of ascorbic acid, and the soybean on the right side was sprayed with 20,000 ppm of ascorbic acid. After spraying at 40,000 ppm, severe leaf discoloration occurred, indicating that an ascorbic acid concentration of 20,000 ppm or less is preferable.

[0040] Comparative Example 2 Growth-promoting effect of ascorbic acid on chickpea (Cicer arietinum) The effect of a single foliar spray of high concentrations of ascorbic acid on nodule activity in chickpea, a legume similar to soybean, was evaluated. The experimental plots evaluated were as follows: Test area 1: Control (no application) Test area 2: Ascorbic acid 300 ppm foliar spray Test area 3: Ascorbic acid 1,500 ppm foliar spray Test area 4: Ascorbic acid 7,500 ppm foliar spray

[0041] (1) Soil preparation and sowing Medium-term fertilizer release soil (Takii Hydrated Cell Fertilizer Medium-term Release, Takii Seed Co., Ltd.) and vermiculite granules (Akagi Engei Co., Ltd.) were mixed at a volume ratio of 1:1, and the soil was filled into polypots (10.5 cm diameter, 9 cm height). After adding 250 mL of tap water per pot, two chickpea (Kabuli variety) seeds were sown in each pot, one at a depth of approximately 1–2 cm from the soil surface. Each test plot had six replicates (n = 6).

[0042] (2) Inoculation of rhizobia A solid medium was prepared by adding 1.5% agar (Fujifilm Wako Pure Chemical Industries, Ltd.) to yeast-mannitol (YM) medium (0.5 g K2HPO4, 0.2 g MgSO4·7H2O, 0.1 g NaCl, 0.4 g yeast extract, 10 g mannitol, and 1 L distilled water (pH 6.8)). Chickpea rhizobia (Mesorhizobium ciceri) strain NBRC100389T was grown on the solid medium. A loopful of the grown rhizobia was inoculated into 50 mL of YM liquid medium in a 500 mL Sakaguchi flask and cultured at 30°C for approximately 48 hours with shaking. A rhizobia culture solution with a bacterial turbidity (OD600) of approximately 0.3 was prepared. After sowing, 1 mL of the rhizobia culture solution was dropwise inoculated onto the seeds using a micropipette.

[0043] (3)Cultivation conditions Cultivation was carried out indoors under the following conditions: 16 hours of light, 25°C, LED light source, and light intensity of 400-440 μmol / m 2 Seven days after sowing, the plants were thinned out to one plant per pot. After the water in the tray placed under the pot had run out, fresh tap water was added to the tray so that the bottom 5 cm of the pot was submerged.

[0044] (4) Foliar spray treatment On the 21st day after sowing, 6.7 mL of ascorbic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) dissolved in tap water was sprayed per plant using a spray bottle. Ascorbic acid was applied only once, and concentrations ranging from 300 to 7,500 ppm were investigated.

[0045] (5) Evaluation of nodule weight After 28 days of cultivation, the plants were removed from the pots, the roots were washed with water, and the nodules were removed from the roots and the weight of each nodule was measured.

[0046] (6) Measurement of nodule activity 1) A method for collecting exudate fluid to measure the amount of ureido nitrogen, which is an index of nodule activity Chickpea stems were cut above the third leaf using pruning shears, and a 1.5 mL microtube (Eppendorf) filled with #10 cotton balls (Kawamoto Sangyo Co., Ltd.) was placed over the cut stem to collect exudate from the stem for 2 hours. After exudate collection, the cotton balls were stored in a -80°C freezer. The volume of exudate was calculated by comparing the weight of the cotton balls before and after collection.

[0047] 2) Experimental procedures before quantifying exudate The exudate from the cotton ball was eluted with ultrapure water and filtered using a Micro Bio-Spin Chromatography column (Bio-Rad Laboratories, Inc.). The filtrate remaining in the column was centrifuged in a centrifuge (CR15RN, Hitachi, Ltd.) at 15,000 rpm for 1 minute, and the collected liquid was quantified.

[0048] 3) Apparatus and measurement method used to quantify the amount of ureido nitrogen In the same manner as in Example 1, allantoic acid and asparagine were identified in each sample.

[0049] (7) Results The measurement results are shown in Figures 4 and 5. The graphs in the figures show the mean value ± standard deviation. In Non-Patent Document 2, it is estimated that the maximum yield-increasing effect when ascorbic acid is applied to chickpea is 226 ppm. In this test, an ascorbic acid concentration of 1,500 ppm was found to be more effective in improving nodule activity and increasing nodule weight than an ascorbic acid concentration of 300 ppm. On the other hand, no effect was observed when applied at 7,500 ppm, suggesting that no effect of improving nodule activity is observed at ascorbic acid concentrations above 1,500 ppm. Therefore, the improvement in nodule activity in the concentration range of 5,000 ppm to 20,000 ppm is considered to be specific to soybean.

[0050] Comparative Example 3: Effect of ascorbic acid on increasing seed weight of Lotus japonicus The effect of a single foliar spray of high concentrations of ascorbic acid on seed weight of Lotus japonicus, a legume similar to soybean, was evaluated. The experimental plots 1 to 6 evaluated are as follows: Test area 1: Control (no application) Test area 2: Ascorbic acid 100 ppm foliar spray Test area 3: Ascorbic acid 200 ppm foliar spray Test area 4: Ascorbic acid 500 ppm foliar spray Test area 5: Ascorbic acid 1,000 ppm foliar spray Test area 6: Ascorbic acid 2,000 ppm foliar spray

[0051] (1) Soil preparation and sowing Medium-term fertilizer-effect soil (Takii Hydrated Cell Culture 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 polypots (diameter 6 cm, height 5.5 cm). 100 mL of tap water was added per pot. 1 mL of concentrated sulfuric acid was added to a 2 mL tube filled approximately one-third with Lotus japonicus (system name: Miyakojima MG-20) seeds, and the tube was left to stand for 10 minutes. After rinsing five times with tap water, the seeds were soaked for at least two hours. After soaking, two seeds were sown in each pot, one seed at a depth of approximately 1 cm from the soil surface. Each test plot was replicated six times (n=6).

[0052] (2) Inoculation of rhizobia A solid medium was prepared by adding 1.5% agar (Fujifilm Wako Pure Chemical Industries, Ltd.) to yeast-mannitol (YM) medium (0.5 g K2HPO4, 0.2 g MgSO4·7H2O, 0.1 g NaCl, 0.4 g yeast extract, 10 g mannitol, and 1 L distilled water (pH 6.8)). Mesorhizobium loti MAFF303099 was grown on the solid medium. A loopful of the grown rhizobia was inoculated into 50 mL of YM liquid medium in a 500 mL Sakaguchi flask and cultured with shaking at 30°C for approximately 72 hours. A rhizobia culture solution with a bacterial turbidity (OD600) of approximately 0.3 was prepared. After sowing, 1 mL of the rhizobia culture solution was dropwise inoculated onto the seeds using a micropipette.

[0053] (3)Cultivation conditions Cultivation was carried out indoors under the following conditions: 16 hours of light, 25°C, LED light source, and light intensity of 400-440 μmol / m 2 Seven days after sowing, the plants were thinned out to one plant per pot. After the water in the tray placed under the pot had run out, fresh tap water was added to the tray so that the bottom 5 cm of the pot was submerged.

[0054] (4) Foliar spray treatment Seven weeks after sowing (from flowering to pod setting), 6.7 mL of ascorbic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) dissolved in tap water was sprayed per plant using a spray bottle. Ascorbic acid was applied only once, at concentrations ranging from 100 to 2,000 ppm.

[0055] (5) Growth evaluation based on seed weight The plants were cultivated for 14 weeks. Once the pods had turned brown and were confirmed to be mature, they were harvested. The seed weight and number of pods per plant were measured, and the seed weight per pod was calculated.

[0056] (6) Results The measurement results are shown in Figures 7, 8, and 9. The graphs in the figures show the mean ± standard deviation. Compared to the control area, the test area where 200 ppm of ascorbic acid was foliar sprayed showed the greatest increase in seed weight (Figure 7). At 500 and 1000 ppm, seed weight was lower than in the control area. With the foliar spray of 200 ppm ascorbic acid, the number of pods per plant decreased, but the increase in seed weight per pod was thought to be the cause of the increase in seed weight (Figures 8 and 9).

Claims

1. A nodule activity promoter for soybeans, which contains ascorbic acid or a salt thereof as an active ingredient, and which is applied to soybeans at a concentration of 5,000 to 20,000 ppm by mass in terms of ascorbic acid.

2. A soybean growth promoter containing ascorbic acid or a salt thereof as an active ingredient, which is applied to soybeans at a concentration of 5,000 to 20,000 ppm by mass in terms of ascorbic acid.

3. 3. The agent according to claim 1 or 2, which is applied to soybeans by foliar spray.

4. The agent according to claim 3, wherein the application amount of ascorbic acid is 8,000 to 18,000 mass ppm as a concentration in a spray solution when sprayed on soybeans foliage.

5. 3. The agent according to claim 1, wherein the content of ascorbic acid or a salt thereof in the composition is 50 to 100% by mass of the total mass of the composition.

6. The agent according to claim 1 or 2, wherein 33 to 133 mg of ascorbic acid is applied per soybean plant.

7. A method for promoting nodule activity in soybeans, comprising the step of applying a treatment solution containing ascorbic acid or a salt thereof to soybeans at a concentration of 5,000 to 20,000 ppm by mass.

8. A method for promoting soybean growth, comprising the step of applying a treatment solution containing ascorbic acid or a salt thereof to soybeans at a concentration of 5,000 to 20,000 ppm by mass.

9. 9. The method according to claim 7 or 8, wherein application to soybeans is by foliar application.

10. 10. The method according to claim 9, wherein the method is applied to soybeans at the grain filling stage.

11. The method according to claim 9, wherein the application amount of ascorbic acid is 8,000 to 18,000 ppm by mass as a concentration in a spray solution when sprayed on soybeans leaves.

12. The method according to claim 7 or 8, wherein 33 to 133 mg of ascorbic acid is applied per soybean plant.

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