Plant vitality enhancers containing amino acids or their salts and oligosaccharides, and their use

Abstract

The purpose of this invention is to control and strengthen the physiological process of plants, and improve the vitality, harvest, quality and post-harvest storage life of agricultural produce.　This plant activator characterized by containing an amino acid or a salt thereof, and an oligosaccharide, is applied to plants.

Description

Technical Field

[0001] The present invention relates to a plant vitalizer containing an amino acid or a salt thereof and an oligosaccharide, and a method for cultivating and producing a plant using the plant vitalizer.

Background Art

[0002] The yield of plants is reduced by abiotic stresses such as sunshine duration, temperature and rainfall, as well as biotic stresses such as pests and diseases. In order to increase the yield of agricultural crops in particular, various fertilizers and pesticides have been used so far. Fertilizers are nutrient sources required for plant growth, but do not have the function of alleviating stress. Pesticides directly eliminate pests and diseases that parasitize plants and eliminate biotic stress. However, when pesticides are used, although their safety has been sufficiently confirmed, there are concerns about the impact on the human body and the environment caused by excessive intake. In particular, there is a concern that agents such as pesticides produced by chemical synthesis methods will remain in soil and the like for a long period of time once sprayed, and it has been desired to impart resistance to biological stress by other methods if possible. For this reason, in recent years, the use of biostimulants as substances that are safe for both the human body and the environment has attracted attention in addition to the above.

[0003] "Biostimulants," also known as "biostimulants" or "plant stimulants," are substances containing various groups of materials and microorganisms that, when applied to plants or their root systems, stimulate a series of processes that occur naturally within the plant, thereby improving nutrient absorption, increasing fertilization efficiency, conferring stress tolerance, and enhancing quality. They do not have a direct effect on pests or diseases and are therefore not classified as any insecticide or fungicide. In other words, they are naturally occurring components (including microorganisms) that are not plant hormones or nutrients, but even in very small amounts, stimulate plant vitality and promote growth. It is believed that applying biostimulants to plants increases nutrient absorption and utilization, promotes growth, and improves crop yield and quality. Agricultural biostimulants include a diverse range of formulations of compounds, materials, and other products applied to plants or soil to control and enhance the physiological processes of crops. Biostimulants act on plant physiology through pathways different from those of nutrients to improve crop vitality, yield, quality, and post-harvest shelf life. In this way, biostimulants can stimulate the inherent abilities of plants and promote their growth without causing the problems associated with conventional pesticides and fertilizers.

[0004] To date, the following have been reported in relation to such biostimulants: a plant vitality agent combining chitin oligosaccharides and chitosan having antibacterial activity (Patent Document 1); a plant vitality agent containing oligosaccharides and plant extracts in vinegar (Patent Document 2); a plant growth promoter containing cellulose (Patent Document 3); a plant growth regulator containing hexofuranose derivatives (Patent Document 4); a method for improving plant disease resistance using low-molecular-weight chitin or chitosan (Patent Document 5); a fertilizer containing chitin and / or chitosan (Patent Document 6); and a plant growth promoter containing glutamic acid and proline as constituent amino acids (Patent Document 7). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 9-143013 [Patent Document 2] Japanese Patent Publication No. 2001-64112 [Patent Document 3] Japanese Patent Publication No. 2002-114610 [Patent Document 4] Japanese Patent Publication No. 2013-151438 [Patent Document 5] Japanese Patent Publication No. 2015-48436 [Patent Document 6] Japanese Patent Publication No. 2017-95352 [Patent Document 7] Japanese Patent Publication No. 2019-006774 [Patent Document 8] International Publication No. 2017 / 104687 [Overview of the project] [Problems that the invention aims to solve]

[0006] The problem that this invention aims to solve is to control and enhance the physiological processes of plants, thereby improving the vitality, yield, quality, and post-harvest shelf life of crops. [Means for solving the problem]

[0007] The inventors of this invention diligently studied and conducted numerous experiments to solve the above-mentioned problems. As a result, they discovered the remarkable finding that a combination of amino acids or their salts and oligosaccharides promotes plant growth and increases the elicitor activity of plants, thus completing the present invention.

[0008] The present invention is as follows: [1] A plant vitality enhancer characterized by containing an amino acid or a salt thereof and an oligosaccharide. [2] The plant vitality agent according to claim 1, wherein the oligosaccharide is at least one selected from the group consisting of chitin oligosaccharide, cellooligosaccharide and xylooligosaccharide. [3] The plant vitality agent according to claim 1 or 2, comprising all three types of oligosaccharides: chitin oligosaccharide, cellooligosaccharide, and xylooligosaccharide. [4] The plant vitality agent according to any one of 1 to 3, wherein the amino acid is at least one selected from the group consisting of glycine, glutamic acid, and histidine. [5] The plant vitality agent according to any one of 1 to 4, wherein the amino acid is glycine. [6] The plant stimulant according to any one of 1 to 5, wherein the total content of the oligosaccharides in the plant stimulant is 0.05 to 10% by mass. [7] The plant vitality agent according to any one of 1 to 6, wherein the total content of the amino acids or salts thereof in the plant vitality agent is 2.5 to 30% by mass. [8] A plant stimulant according to any one of 1 to 7, further comprising a spreading agent. [9] A method of growing plants, comprising applying a plant stimulant described in any of items 1 to 8 to a plant.

[10] The method according to 9, comprising applying the plant stimulant to a plant at a concentration such that the total content of the oligosaccharides is 0.1 to 500 ppm by mass.

[11] The method according to 9 or 10, wherein the plant stimulant is applied to the plant by foliar spraying.

[12] A method for producing a plant or a part thereof having increased elicitor activity compared to when no plant stimulant described in any of 1 to 8 is applied, comprising cultivating the plant by the method described in any of 9 to 11.

[13] The method according to 12, wherein the elicitor activity is determined by measuring the amount of glucanase produced in the plant.

[14] A fertilizer composition containing a plant vitality enhancer as described in any of 1 to 8. [Effects of the Invention]

[0009] According to the present invention, it is possible to control and enhance the physiological processes of plants without causing problems such as the effects on human health and the environment caused by conventional pesticides and fertilizers, thereby improving the vitality, yield, quality, and post-harvest shelf life of crops. [Modes for carrying out the invention]

[0010] In a first aspect of the present invention, a plant vitality agent is provided that is characterized by containing an amino acid or a salt thereof and an oligosaccharide. The "plant vitality agent" according to the present invention includes not only those that have an alleviating effect on abiotic stresses such as temperature, light, water, and salt that affect plant growth, but also those that have an alleviating effect on biological stresses such as diseases and pests.

[0011] The oligosaccharide used in the present invention is preferably at least one selected from the group consisting of chitin oligosaccharide, cellooligosaccharide, and xylooligosaccharide. More preferably, the plant vitality agent according to the present invention contains two or more oligosaccharides selected from the group consisting of chitin oligosaccharide, cellooligosaccharide, and xylooligosaccharide, and even more preferably contains all three types of chitin oligosaccharide, cellooligosaccharide, and xylooligosaccharide. In the present invention, chitin oligosaccharides are used as elicitors (i.e., "exogenous elicitors") of biological substances other than plants, such as fungi, insects, and crustaceans, and cellooligosaccharides and xylooligosaccharides are used as elicitors (i.e., "endogenous elicitors") of plant-derived substances.

[0012] Elicitors are a general term for substances that induce biological defense responses in the tissues or cultured cells of higher plants, inducing disease resistance in the plant's immune system. Plants sense elicitors with receptors present on their leaves and other surfaces, triggering a pathogen resistance response. This results in a biological defense mechanism (immunity) in which various compounds are secreted against various pathogens. When elicitors act on plants, defense responses such as the synthesis and accumulation of phytoalexins and infection-specific proteins, reactive oxygen species generation, reactive nitrogen generation, hypersensitive reactive cell death, and gene expression changes are induced. It is believed that these reactions protect plants from pathogens and enhance their disease resistance. Phytoalexins are antibacterial compounds that are synthesized and accumulated in plants under the action of elicitors, and the antibacterial compounds produced differ for each plant species. Representative phytoalexins include flavonoids, terpenoids, fatty acid derivatives and the like. Reactive oxygen species have the effect of killing pathogenic microorganisms, and further, reactive oxygen species and reactive nitrogen species function alone or in concert as signals that trigger various defense responses. Such disease resistance due to the elicitor effect is expected for agricultural applications, for example, because it enhances resistance to a wide range of diseases.

[0013] Chitin oligosaccharides include partially deacetylated chitosan oligosaccharides, are oligosaccharides in which several N-acetylglucosamine units are linked in series, and are generally obtained by hydrolyzing chitin derived from crustaceans or the like. They are also referred to as oligo-N-acetylglucosamine. That is, chitin oligosaccharides can be obtained by chemically or enzymatically partially hydrolyzing chitin, which is prepared by a conventional method from the shells of crustaceans such as crabs and shrimps. As the chitin oligosaccharide, one or a mixture of a plurality selected from N-acetylchitobiose, N-acetylchitotriose, N-acetylchitotetraose, N-acetylchitopentaose, N-acetylchitohexaose, N-acetylchitoheptaose, N-acetylchitooctaose and the like is preferably used. Among these, N-acetylchitopentaose, N-acetylchitohexaose, and N-acetylchitoheptaose in particular have high elicitor effects.

[0014] The chitin oligosaccharide used in the present invention particularly preferably has the following chemical structure.

Chemical Formula

[0015] Cello-oligosaccharides are short sugars formed by the polymerization of multiple glucose molecules via β-glycosidic bonds. Recently, functionalities such as moisturizing properties, anti-stickiness, flavor enhancement, starch retrogradation reduction, and protein denaturation inhibition have been discovered, leading to expectations for their use in pharmaceuticals, cosmetics, food, and animal feed. In particular, cello-oligosaccharides with a degree of polymerization of 3 or higher are attracting greater attention due to their enhanced functionality and the potential for the provision of new functionalities. Currently, industrially used cello-oligosaccharides are produced by enzymatic reactions, but their main components are glucose and the dimer cellobiose, with little to no oligomers beyond the trimer cellotriose. However, in recent years, the applicants have reported a method for producing cello-oligosaccharides containing oligomers with a degree of polymerization of 3-6 by controlling the heating rate, cooling rate, reaction temperature, and reaction time during a hydrolysis reaction of plant biomass using a carbon catalyst to induce a hydrothermal reaction (Patent Document 8). When obtaining cellooligosaccharides by hydrolysis of cellulose, it is preferable to use crystalline fine cellulose such as Avicel (manufactured by Merck) or cotton linter pulp as the cellulose raw material.

[0016] The cellooligosaccharide used in the present invention is particularly preferred to have the following chemical structure. [ka]

[0017] Xylooligosaccharides are short sugars in which several xylose molecules are polymerized by β-glycosidic bonds. They are generally obtained by hydrolysis of xylan, the main component of hemicellulose, and are mainly sold for food applications.

[0018] In the present invention, xylooligosaccharides having the following chemical structure are particularly preferred. [ka]

[0019] When a plant vitality enhancer contains only two types of oligosaccharides, chitin oligosaccharide and xylooligosaccharide, the mass ratio of chitin oligosaccharide to xylooligosaccharide is preferably 1:1 to 1:5, and more preferably 1:1.5 to 1:4. When a plant vitality enhancer contains only two types of oligosaccharides, chitin oligosaccharide and cello-oligosaccharide, the mass ratio of chitin oligosaccharide to cello-oligosaccharide is preferably 1:1 to 1:5, and more preferably 1:1.5 to 1:4.

[0020] When a plant vitality enhancer contains three types of oligosaccharides—chitin oligosaccharide, cellooligosaccharide, and xylooligosaccharide—it is preferable that the proportion of each oligosaccharide to the total content of chitin oligosaccharide, cellooligosaccharide, and xylooligosaccharide be 10-50% by mass for chitin oligosaccharide, 10-50% by mass for cellooligosaccharide, and 10-60% by mass for xylooligosaccharide. More preferably, the proportions of each oligosaccharide are 20-40% by mass for chitin oligosaccharide, 20-40% by mass for cellooligosaccharide, and 20-55% by mass for xylooligosaccharide.

[0021] The amino acid used in the present invention is preferably at least one selected from the group consisting of glycine, glutamic acid, and histidine, and more preferably glycine. The amino acid salts used in this invention are not particularly limited, but examples include sodium salts and potassium salts of amino acids. A specific example of an amino acid salt is monosodium glutamate.

[0022] The plant vitality agent according to the present invention may be manufactured in any form, such as powder, granules, or liquid, but it is generally preferable to use a liquid form that is easy to spray. The plant vitality agent according to the present invention can be supplied as a stock solution obtained by dissolving amino acids or their salts and oligosaccharides at a high concentration in a solvent such as water. In one embodiment, the total content of the oligosaccharides in the plant vitality agent stock solution is preferably 0.05 to 10% by mass, more preferably 0.1 to 8% by mass, and even more preferably 0.5 to 6% by mass. In another embodiment, the total content of the oligosaccharides in the plant vitality agent stock solution is preferably 1 to 15% by mass, more preferably 3 to 12% by mass, and even more preferably 5 to 10% by mass.

[0023] In one embodiment, the total content of the amino acids or their salts in the plant vitality agent stock solution according to the present invention is preferably 2.5 to 30% by mass, more preferably 5 to 20% by mass, and even more preferably 9 to 16% by mass. In another embodiment, the total content of the amino acids or their salts is preferably 10 to 70% by mass, more preferably 15 to 60% by mass, and even more preferably 20 to 50% by mass.

[0024] The plant vitality agent according to the present invention may further contain other components besides the active ingredient amino acid or its salt and oligosaccharide, such as preservatives, spreading agents, anti-precipitation agents, thickeners, and excipients. Examples of preservatives include potassium sorbate, parahydroxybenzoic acid esters, benzoic acid, sodium dehydroacetate, hinokitiol, phenoxyethanol, polyaminopropyl biguanide, and polylysine. The spreading agent is a viscous liquid mainly composed of a surfactant and is not particularly limited as long as it can be used as a spreading agent for the plant vitality agent, but examples include polyoxyethylene nonylphenyl ether, sorbitan fatty acid ester, and polyoxyethylene hexitane fatty acid ester. Examples of anti-precipitation agents include polyphosphate or salts of polyphosphate, or polycarboxylic acid-type polymer surfactants. Examples of thickeners include water-soluble polymers such as carboxymethylcellulose (CMC), polyacrylamide, and starch, or molasses, alcohol fermentation concentrate wastewater, amino acid fermentation concentrate wastewater, etc. Excipients include lactose and starch.

[0025] In a second embodiment of the present invention, a method for cultivating plants is provided, which includes applying the plant vitality agent according to the present invention to plants.

[0026] The plants to which the plant vitality agent according to the present invention is applied are not particularly limited, but typically include agricultural crops, such as plants of the Asteraceae, Solanaceae, Brassicaceae, Poaceae, Fabaceae, Rosaceae, Cucurbitaceae, Convolvulaceae, Chenopodiaceae, Liliaceae, Apiaceae, Malvaceae, Zingiberaceae, and Nelumbonaceae families. Specifically, this includes Brassicaceae plants such as Chinese cabbage, cabbage, broccoli, flowering vegetables, komatsuna, mizuna, radish, and turnip; Solanaceae plants such as potatoes, tomatoes, eggplants, bell peppers, chili peppers, shishito peppers, and tobacco; Asteraceae plants such as garland chrysanthemum, lettuce, leaf lettuce, burdock, and butterbur; Cucurbitaceae plants such as watermelon, melon, pumpkin, cucumber, bitter melon, loofah, and gourd; Chenopodiaceae plants such as spinach, Swiss chard, sea beans, and beets; carrots, and celery. Examples include plants of the Apiaceae family such as parsley and Japanese parsley; plants of the Fabaceae family such as soybeans (edamame), adzuki beans, kidney beans, broad beans, peas, winged beans, and peanuts; plants of the Convolvulaceae family such as sweet potatoes and water spinach; plants of the Liliaceae family such as chives, leeks, onions, garlic, and asparagus; plants of the Rosaceae family such as strawberries, apples, pears, and loquats; plants of the Malvaceae family such as okra and cotton; plants of the Zingiberaceae family such as ginger; plants of the Nelumbonaceae family such as lotus; and plants of the Poaceae family such as corn, rice, barley, wheat, and sugarcane. Among these, Brassicaceae plants such as cabbage and komatsuna, Solanaceae plants such as tomatoes and eggplants, Asteraceae plants such as lettuce and leaf lettuce, and Rosaceae plants such as strawberries and apples are preferred, with komatsuna and tomatoes being more preferred among them.

[0027] The plant vitality agent according to the present invention is generally used by adding water or the like to the stock solution and diluting it to a desired concentration (for example, by diluting it 1000 times), and is applied to plants at a concentration in which the total content of the oligosaccharides in the plant vitality agent is preferably 0.1 to 500 ppm by mass. More preferably, the total content of the oligosaccharides in the plant vitality agent is 0.5 to 200 ppm by mass, and even more preferably, it is applied to plants at a concentration in which it is preferably 1 to 100 ppm by mass.

[0028] The application of plant stimulants to plants can be carried out by methods customary in this industry, and the application method is not particularly limited. For example, it may be applied directly to the leaves and stems of plants, applied to the culture medium or soil in which the plants are grown, or mixed with fertilizers and applied to the culture medium or soil. When mixed with fertilizers, the type of fertilizer is not limited and can be any chemical fertilizer containing nitrogen, phosphorus, and potassium, or organic fertilizers such as oil cake, fish meal, bone meal, seaweed powder, sugars, and vitamins. In particular, foliar application is preferred for effectively expressing the elicitor activity. Foliar application can be carried out by methods customary in this industry, such as power sprayers, shoulder sprayers, broadcasters, sprayers, manned or unmanned helicopters, fogging devices, and hand sprayers.

[0029] When a plant vitality enhancer is mixed with fertilizer and sprayed, the content of the oligosaccharide is preferably 10 to 50% by mass, and more preferably 15 to 40% by mass, based on 100% by mass of solids in the fertilizer composition. The content of the amino acid or its salt is preferably 10 to 50% by mass, and more preferably 15 to 40% by mass, based on 100% by mass of solids in the fertilizer composition. In addition to the oligosaccharide and the amino acid or its salt, the fertilizer composition preferably contains at least one nutrient selected from nitrogen, phosphorus, and potassium, and more preferably contains all three nutrients: nitrogen, phosphorus, and potassium. In the case of a liquid fertilizer, the fertilizer composition preferably contains 70 to 99% by mass of water, more preferably 75 to 99% by mass, and it is preferable to dilute this stock solution 100 to 1000 times before spraying.

[0030] By cultivating plants in this manner, it becomes possible to produce plants or parts thereof (e.g., roots, stems, leaves, flowers, fruits, seeds, tissues, cells, etc.) that possess elicitor activity, compared to cases where plant stimulants are not applied. This, in turn, can improve the vitality, yield, quality, and post-harvest shelf life of crops.

[0031] As mentioned above, the elicitor effect is an important indicator of disease resistance. The inventors have now discovered that elicitor activity can be evaluated by measuring the enzyme activity of glucanase, which is one of the signals of the elicitor effect. By taking a portion of the leaves of a cultivated plant and analyzing its glucanase activity, it becomes possible to evaluate the same individual plant over time.

[0032] The following is an overview of the procedure for evaluating elicitor activity: (i) Sample and pre-process the plants; (ii) Create a calibration curve using BSA as a protein standard (using absorbance at a wavelength of 600 nm by the dye-binding method); (iii) Measure the protein concentration of the sample prepared in (i); (iv) Measure the glucanase activity of the sample prepared in (i). Specifically, the activity is evaluated as an absorbance value at a wavelength of 590 nm using a B-HS reagent that develops color when soluble low-molecular-weight degradation products are released by glucanase; (v) Calculate the glucanase activity per protein unit. The specific procedure for evaluating this elicitor activity is described in detail in the following example.

[0033] The present invention will be further illustrated by the following examples, but the present invention is not limited thereto. [Examples]

[0034] [1. Prepare the oligosaccharides] (1) Chitin oligosaccharide 10 g of chitin (purified chitin, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dispersed in 30 mL of water containing 1.2 g of phosphoric acid. The resulting powder, dried under reduced pressure, was placed in a 250 mL alumina pot along with 100 g of 5 mm diameter alumina balls. This pot was then set in a planetary ball mill (PULVERISETTE 6, manufactured by Fritsch) and processed continuously at 500 rpm for 6 hours to obtain the reaction product. The reaction was started at room temperature, and the temperature rise due to shear heating was left to occur naturally. Next, the reaction mixture was suspended in water and neutralized with calcium hydroxide to obtain a slurry. This slurry was then filtered using 5B filter paper in a Nutsche filter, and the recovered filtrate was freeze-dried to obtain chitin oligosaccharide powder.

[0035] (2) Cellooligosaccharides Examples 1 and 2 and Comparative Examples 1 and 4 below used cellooligosaccharides produced by "Manufacturing Method 1," while Examples 6, 8, and 10 used cellooligosaccharides produced by "Manufacturing Method 2." (Manufacturing method 1: Obtained from crystalline fine cellulose powder) 10g of Avicel (crystalline fine cellulose manufactured by Merck) and 1.5g of activated carbon BA50 (manufactured by Ajinomoto Fine Techno Co., Ltd.) were placed together with 2000g of 1.5cm diameter alumina balls in a 3600mL ceramic pot mill. This was then set on a benchtop pot mill turntable (manufactured by Nittokagaku Co., Ltd., benchtop pot mill model ANZ-51S) and processed at 60rpm for 48 hours to obtain the reaction raw materials. The process was started at room temperature, and the temperature rise due to shear heating was left to occur naturally. Next, 0.374 g of the reaction raw materials and 40 mL of water were placed in a high-pressure reactor (internal volume 100 mL, autoclave manufactured by OM Labtec Co., Ltd., made of Hastelloy C22). The mixture was then heated to 230 °C at a rate of 10-30 °C / min (average heating rate 11.3 °C / min) while stirring at 600 rpm. Immediately after heating, the reactor was cooled by air cooling at a rate of 10-30 °C / min (average cooling rate 16.7 °C / min) to prepare the reaction solution. Next, the reaction mixture was collected using a centrifuge, and the supernatant was freeze-dried to obtain cellooligosaccharide powder.

[0036] (Manufacturing method 2: Obtained from cotton linter pulp) 271g (1.8% moisture content, 266g dry weight) of cotton linter pulp (Tohoku Kosen Co., Ltd., 97% cellulose content) was mixed with 38g of 85% by mass phosphoric acid (special grade reagent, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) using a food blender (model: HBF500S, Hamilton Beach Corporation) to obtain 309g of reaction raw material (3.4% moisture content, 10.4% phosphoric acid content). Next, 309g of the reaction material was placed in a vibrating mill (device name: MB-1 type, manufactured by Chuo Kakoki Co., Ltd., pot size 5L) together with 13kg of φ3 / 4 inch carbon steel balls. A hydrolysis reaction was carried out by dry grinding for 24 hours under conditions of a total amplitude of 8mm, a vibration frequency of 16.2Hz, and a jacket flow water temperature of 75℃, after which the reaction powder was recovered. 10 g of this reaction powder and 90 g of deionized water were placed in a 200 L beaker and stirred at 25°C for 1 hour using a magnetic stirrer to obtain an extract of cellulose hydrolysate. Next, 1.3 g of a 40% by mass aqueous calcium hydroxide solution was added to the extract, and the mixture was stirred using a magnetic stirrer at 25°C for 1 hour to prepare a neutralized solution. The supernatant was collected using a centrifuge, and then freeze-dried to obtain cellooligosaccharide powder.

[0037] (3) Xylooligosaccharides Examples 1 and 2 and Comparative Examples 1 and 4 below used xylooligosaccharides from the "Commercial Products" section, while Examples 6 to 9 and 11 used xylooligosaccharides produced by the "Manufacturing Method" section.

[0038] (Manufacturing method: Obtained from corn cob powder) Acremonium Cellulolyticus TN strain (FERM P-18508) was cultured in a 500 mL flask containing 100 mL of liquid medium (Avicel 50 g / L, KH2O4 24 g / L, ammonium sulfate 5 g / L, potassium tartrate 1 / 2H2O 4.7 g / L, urea 4 g / L, Tween80 1 g / L, MgSO4·7H2O 1.2 g / L, ZnSO4·7H2O 10 mg / L, MnSO4·5H2O 10 mg / L, CuSO4·5H2O 10 mg / L) at 30°C for 6 days with shaking. 5 g of corn cob powder was suspended in 50 mL of the supernatant obtained by centrifugation of the culture solution, and the reaction was carried out with stirring at 50°C for 72 hours. The supernatant obtained by centrifugation of the reaction solution was freeze-dried to obtain xylooligosaccharide raw material. (Commercially available product) Xylooligosaccharide 95P, manufactured by Bussan Food Science Co., Ltd., was used. [2. Preparation of Amino Acids] (1) Glycine: Manufactured by Showa Denko Corporation (2) Lysine: Manufactured by Fujifilm Wako Pure Chemical Corporation (3) Sodium glutamate (MSG): Manufactured by Fujifilm Wako Pure Chemical Corporation (4) Histidine: Manufactured by Fujifilm Wako Pure Chemical Corporation (5) Alanine: Manufactured by Fujifilm Wako Pure Chemical Corporation (6) Arginine: Manufactured by Fujifilm Wako Pure Chemical Corporation

[0039] [3. Measuring the dry weight of frilly lettuce roots] (1) Preparation of plant vitality enhancers The oligosaccharides prepared in [1. Preparation of Oligosaccharides] and the amino acids prepared in [2. Preparation of Amino Acids] were dissolved in water by stirring with a stirrer in their respective compositional ratios so that the plant vitality agent concentration (mass ppm) was 1000 times that of Comparative Examples 1-2 and Examples 1, 6-7 in the table below. After that, the mixture was sterilized using a 0.45 μm filter to obtain the plant vitality agent stock solution.

[0040] (2) Cultivation experiment In the test plots, 150g of concentrated plant stimulant solution for each condition was added to 150L of nutrient solution prepared for hydroponics and dispersed (diluted 1000 times). Cultivation of 120 plants was started at a temperature of 20-22°C. Cultivation was carried out for a total of 60 days, and 150g of concentrated plant stimulant solution was added every week for a total of 5 times after the start of cultivation, and the dry weight of the frilly lettuce roots was measured. The dry weight of the roots was measured by cutting the root portion, drying the cut root in a constant-temperature dryer at 50°C for 12 hours, and then measuring its weight. Note that the root dry weight in Table 1 represents the percentage (%) of the root dry weight in the untreated group (where no plant stimulant was added), with the untreated group being set at 100%.

[0041] [Table 1]

[0042] [4. Evaluation of elicitor activity in Komatsuna] (1) Preparation of plant vitality enhancers The oligosaccharides prepared in [1. Preparation of Oligosaccharides] and the amino acids prepared in [2. Preparation of Amino Acids] were stirred in water in their respective composition ratios to achieve a concentration (mass ppm) of the plant stimulant 1000 times that of Comparative Examples 4-10 and Examples 2 and 8-9 in the table below. After stirring, the mixture was sterilized using a 0.45 μm filter to obtain the plant stimulant stock solution. This stock solution was diluted 1000 times with water and used in the following cultivation tests.

[0043] (2) Preparation of MS medium Komatsuna was grown on Murashigeskoug (MS) agar medium. The prepared plant stimulant was added to the MS medium to the final concentrations shown in each comparative example and example, and then autoclaved at 121°C for 20 minutes.

[0044] (3) Sowing and growth methods On a clean bench, the MS medium, sterilized by autoclaving, was transferred to a plant box. After it had cooled completely, 10 seeds of komatsuna (wakami, Sakata Seed Co.) were sown in each box. The plants were then grown in a bright place at 22°C for 24 hours under long-day conditions for 6 days.

[0045] (4) Protein extraction A protein extraction buffer with the following composition was prepared.

[0046] [Table 2]

[0047] 300 μl of the protein extraction buffer prepared above was added to a 1.5 ml tube provided with the biomassher (Nippi Corporation), and the sampled leaves (plant material), cut to approximately 4 x 4 mm with scissors, were placed in it. This procedure was repeated five times for each sample. Next, the plant material was crushed by rotating the stirring rod by hand until almost no visible solid material remained. The mixture was centrifuged at 15,000 × g for 10 minutes at 4°C, and the aqueous layer was collected in a new 1.5 ml tube to prepare the extract.

[0048] (5) Adjustment of protein concentration Standards were prepared by serially diluting 2 mg / ml of bovine serum albumin (BSA) of known purity (1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64 dilutions). Using the prepared standards, the average absorbance at 600 nm (Abs600) was taken to create a calibration curve (n=3). 300 μl of Coomassie Brilliant Blue (CBB) solution was dispensed into a 96-well plate, and 6 μl of the extract prepared above was added. Abs600 was measured. MilliQ was used as the blank. The absorbance of the extract was fitted to the calibration curve created using serially diluted 2 mg / ml BSA to determine the protein concentration. If the Abs600 measurement of the extract fell outside the calibration curve, the protein concentration was determined by diluting it appropriately with ultrapure water (MilliQ) and re-measuring. The obtained values ​​were used to dilute the extract to a constant concentration, and then used for the following glucanase activity measurements.

[0049] (6) Measurement of glucanase activity Samples for each comparative example and example were prepared by mixing 100 μl of B-HS substrate solution (1 tablet of B-HS reagent (Megazyme) suspended in 10 ml of MilliQ), 50 μl of 0.2 M phosphate buffer (pH 6.0), and 50 μl of the dilution prepared above or ultrapure water (blank) in a 1.5 ml tube. The enzyme reaction was carried out in a 30°C water bath for 1 hour and 30 minutes, shaking the sample well every 15 minutes. The reaction was stopped by adding 100 μl of 0.2 N NaOH stop solution (300 μl total). The mixture was centrifuged at 15,000 rpm for 5 minutes, and 200 μl of the supernatant was dispensed into a 96-well plate. To evaluate glucanase activity, the absorbance at 590 nm (Abs590) was measured and compared with the case without plant stimulant application (Comparative Example 3).

[0050] [Table 3-1]

[0051] [Table 3-2]

[0052] [5. Measuring the raw weight of komatsuna] (1) Preparation of plant vitality enhancers The oligosaccharides prepared in [1. Preparation of Oligosaccharides] and the amino acids prepared in [2. Preparation of Amino Acids] were stirred with a stirrer in their respective composition ratios to achieve a plant stimulant concentration (mass ppm) 1000 times that of Comparative Examples 12-14 and Examples 3-5 and 10-11 in the table below. Dissolved in water, and then sterilized through a 0.45 μm filter, to obtain the plant stimulant stock solution. This stock solution was diluted 1000 times with water and used in the following cultivation tests.

[0053] (2) Measurement of raw body weight We prepared komatsuna (wakami, Sakata Seed Co.) as the crop, organic vegetable soil (Hirota Shoten) as the growing medium, and Shin-Taki Organic Liquid Fertilizer No. 3 (Taki Chemical Co., Ltd.) as the fertilizer. After sprouting the crop on filter paper, the seedlings were transplanted into 25-cell connected pots (280 x 280 mm overall, 50 x 50 x 50 mm per cell) filled with potting soil and fertilizer. After transplanting, the komatsuna was grown in a culture room for 34 days, with each plant growth stimulant applied as a foliar spray at one-week intervals. The fresh weight was then measured and compared with that of the plants without plant growth stimulants (Comparative Example 11).

[0054] [Table 4]

Claims

1. A plant vitality enhancer characterized by containing an amino acid or a salt thereof and an oligosaccharide, The oligosaccharide is at least one selected from the group consisting of chitin oligosaccharide, cellooligosaccharide, and xylooligosaccharide. When the oligosaccharide is a chitin oligosaccharide, the amino acid is at least one selected from the group consisting of glycine, glutamic acid, and histidine. When the oligosaccharide is a xylooligosaccharide, the amino acid is glycine. If the oligosaccharide is a cellooligosaccharide, the amino acid is glycine. When the oligosaccharides are chitin oligosaccharides and xylooligosaccharides, the amino acid is glycine. When the oligosaccharides are chitin oligosaccharides and cellooligosaccharides, the amino acid is glycine. When the oligosaccharides are chitin oligosaccharides, xylooligosaccharides, and cellooligosaccharides, the amino acid is glycine, and A plant stimulant wherein the total content of the amino acids or their salts in the plant stimulant is 2.5 to 30% by mass.

2. The plant vitality agent according to claim 1, wherein the oligosaccharides include all three types: chitin oligosaccharide, cellooligosaccharide, and xylooligosaccharide.

3. The plant vitality agent according to claim 1 or 2, wherein the rate of deacetylation of the chitin oligosaccharide is 30% or less.

4. The plant stimulant according to any one of claims 1 to 3, wherein the total content of the oligosaccharides in the plant stimulant is 0.05 to 10% by mass.

5. A plant vitality agent according to any one of claims 1 to 4, further comprising a spreading agent.

6. A method for cultivating plants, comprising applying a plant vitality agent according to any one of claims 1 to 5 to a plant.

7. The method according to claim 6, comprising applying the plant vitality agent to a plant at a concentration such that the total content of the oligosaccharides is 0.1 to 500 ppm by mass.

8. The method according to claim 6 or 7, wherein the plant vitality agent is applied to the plant by foliar spraying.

9. A method for producing plants or a portion thereof having increased elicitor activity compared to when no plant stimulant described in any one of claims 1 to 5 is applied, the method comprising cultivating the plants by the method described in any one of claims 6 to 8.

10. The method according to claim 9, wherein the elicitor activity is determined by measuring the amount of glucanase produced in the plant.

11. A fertilizer composition containing a plant vitality enhancer according to any one of claims 1 to 5.

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