Plant growth stimulants containing exogenous and endothelial elicitors and their use

Abstract

The present invention controls or strengthens plant physiological processes and improves the vitality, yield, quality, and post-harvest shelf life of crops.　A plant vitalizing agent characterized by containing an exogenous elicitor and an endogenous elicitor is applied to plants.

Description

Technical Field

[0001] The present invention relates to a plant vitality agent containing an exogenous elicitor and an endogenous elicitor, and a method for cultivating and producing plants using the plant vitality agent.

Background Art

[0002] The yield of plants decreases due to abiotic stresses such as sunlight hours, temperature, and rainfall, as well as biotic stresses such as pests and diseases. In particular, in order to increase the yield of agricultural crops, 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 control pests parasitic on plants and eliminate biotic stress. However, when using pesticides, although safety has been sufficiently confirmed, there are concerns about the effects on the human body and the environment due to excessive intake. In particular, drugs such as pesticides produced by chemical synthesis methods may remain in the soil and the like for a long time once sprayed, and it has been desired to confer resistance to biotic stress by other methods if possible. For this reason, in recent years, in addition to these, the use of biostimulants as substances safe for both the human body and the environment has attracted attention.

[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 improving quality. They do not have a direct effect on pests or diseases, and therefore are 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 variety of formulations such as 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] Related to such biostimulants, the following have been reported: 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), and a fertilizer containing chitin and / or chitosan (Patent Document 6). [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] 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 exogenous and endogenous elicitors 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 exogenous elicitors and endogenous elicitors. [2] The plant stimulant according to claim 1, wherein the exogenous elicitor is a chitin oligosaccharide and the endogenous elicitor is at least one oligosaccharide selected from cellooligosaccharides and xylooligosaccharides. [3] The plant stimulant according to claim 1 or 2, wherein the total content of the exogenous elicitor and the endogenous elicitor in the plant stimulant is 0.05 to 10% by mass. [4] The plant stimulant according to any one of 1 to 3, wherein the mass ratio of the exogenous elicitor to the endogenous elicitor in the plant stimulant is 0.2 to 5. [5] The plant vitality agent according to any one of 1 to 4, comprising xylooligosaccharide as the endogenous elicitor. [6] The plant stimulant according to claim 5, comprising both cellooligosaccharide and xylooligosaccharide as the endogenous elicitor. [7] The plant stimulant according to 6, wherein the mass ratio of cellooligosaccharide to xylooligosaccharide in the plant stimulant is 0.2 to 5. [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 vitality agent to a plant at a concentration such that the total content of the exogenous elicitor and the endogenous elicitor 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 embodiment of the present invention, a plant vitality agent is provided that comprises an exogenous elicitor and an endogenous elicitor. 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] 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 antimicrobial compounds synthesized and accumulated in plants through the action of elicitors, and the antimicrobial compounds produced vary depending on the plant species. Representative phytoalexins include flavonoids, terpenoids, and fatty acid derivatives. Reactive oxygen species have the effect of killing pathogenic microorganisms, and furthermore, reactive oxygen species and reactive nitrogen function as signals that activate various defense reactions, either individually or in combination. Disease resistance due to such elicitor effects is expected to have agricultural applications, such as enhancing resistance to a wide range of diseases.

[0012] In this specification, "exogenous elicitor" means an elicitor derived from organisms other than plants, such as fungi, insects, or crustaceans, and is not particularly limited as long as it has an elicitor effect, but typically includes chitin, chitosan, and their oligosaccharides, as well as various biomolecules derived from insects. The plant vitality enhancer according to the present invention preferably contains chitin oligosaccharide as an exogenous elicitor.

[0013] Chitin oligosaccharide includes partially deacetylated chitosan oligosaccharide, and is an oligosaccharide in which several N-acetylglucosamines are linked together. Generally, it is obtained by hydrolyzing chitin derived from crustaceans, etc., and is also referred to as oligo-N-acetylglucosamine. That is, chitin oligosaccharide is obtained by chemically or enzymatically partially hydrolyzing chitin 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-acetylchitoctaose, etc. is preferably used. Among these, N-acetylchitopentaose, N-acetylchitohexaose, and N-acetylchitoheptaose particularly have a high elicitor effect.

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

Chemical formula

[0015] In the present specification, "endogenous elicitor" means an elicitor of plant-derived substances, and is not particularly limited as long as it has an elicitor effect. Typically, it is cellulose, xylan, and their oligosaccharides produced from plants, etc. The plant vigor agent according to the present invention preferably contains at least one oligosaccharide selected from cellooligosaccharide and xylooligosaccharide as an endogenous elicitor.

[0016] Cello-oligosaccharides are short sugars in which multiple glucose molecules are polymerized by β-glycosidic bonds. In recent years, functionalities such as moisturizing properties, anti-stickiness, flavor enhancement, starch retrogradation reduction, and protein denaturation inhibition have been discovered, and their use in the pharmaceutical, cosmetic, food, and animal feed fields is expected. In particular, cello-oligosaccharides with a degree of polymerization of glucose of 3 or higher are attracting even greater attention in terms of increased functionality and the conferral of new functionalities. Cello-oligosaccharides currently used industrially are produced by enzymatic reactions, but their main components are glucose and the dimer cellobiose, and they contain almost no oligomers of the trimer cellotriose or higher. However, in recent years, the applicants have reported a method for producing cello-oligosaccharides containing oligomers with a degree of polymerization of glucose of 3 to 6 by controlling the heating rate, cooling rate, reaction temperature, and reaction time to induce a hydrothermal reaction in a hydrolysis reaction of plant biomass using a carbon catalyst (Patent Document 7). 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.

[0017] The cellooligosaccharide used in the present invention is particularly preferred to have the following chemical structure. [ka]

[0018] 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.

[0019] In the present invention, xylooligosaccharides having the following chemical structure are particularly preferred. [ka]

[0020] 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 that is easy to spray. The plant vitality agent according to the present invention can be supplied as a stock solution in which exogenous elicitors and endogenous elicitors are dissolved at a high concentration in a solvent such as water. In one embodiment, the total content of the exogenous elicitors and endogenous elicitors 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 exogenous elicitors and endogenous elicitors 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.

[0021] In one embodiment, the mass ratio of the exogenous elicitor to the endogenous elicitor in the plant vitality agent according to the present invention (i.e., exogenous elicitor content / endogenous elicitor content) is preferably 0.2 to 5, more preferably 0.3 to 3, and even more preferably 0.5 to 1.5. In another embodiment, the mass ratio of the exogenous elicitor to the endogenous elicitor is preferably 0.1 to 4, more preferably 0.2 to 2, and even more preferably 0.3 to 1.

[0022] The plant vitality agent according to the present invention more preferably contains xylooligosaccharide as an endogenous elicitor, and optimally contains both cellooligosaccharide and xylooligosaccharide. In one embodiment, the mass ratio of cellooligosaccharide to xylooligosaccharide in the plant vitality agent according to the present invention (i.e., cellooligosaccharide content / xylooligosaccharide content) is preferably 0.2 to 5, more preferably 0.3 to 3, and even more preferably 0.5 to 1.5. In another embodiment, the mass ratio of cellooligosaccharide to xylooligosaccharide is preferably 0.1 to 4, more preferably 0.2 to 2, and even more preferably 0.3 to 1.

[0023] When a plant vitality enhancer contains chitin oligosaccharide as an exogenous elicitor and both cellooligosaccharide and xylooligosaccharide as an endogenous elicitor, it is preferable that the proportion of each oligosaccharide to the total content of chitin oligosaccharide, cellooligosaccharide, and xylooligosaccharide is 10-50% by mass for chitin oligosaccharide, 10-50% by mass for cellooligosaccharide, and 10-60% by mass for xylooligosaccharide. More preferably, the proportion of each oligosaccharide is 20-40% by mass for chitin oligosaccharide, 20-40% by mass for cellooligosaccharide, and 20-55% by mass for xylooligosaccharide.

[0024] The plant vitality agent according to the present invention may further contain other components besides the exogenous and endogenous elicitors, which are the active ingredients, 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 to the desired concentration (for example, by diluting it 1000 times), and is applied to plants at a concentration in which the total content of exogenous elicitors and endogenous elicitors in the plant vitality agent is preferably 0.1 to 500 ppm by mass. The total content of exogenous elicitors and endogenous elicitors in the plant vitality agent is more preferably 0.5 to 200 ppm by mass, and even more preferably 1 to 100 ppm by mass, when applied to plants.

[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 chemical fertilizers containing nitrogen, phosphorus, and potassium, or organic fertilizers such as oil cake, fish meal, bone meal, seaweed powder, amino acids, sugars, and vitamins. As for the application method, foliar application is particularly preferable 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 exogenous elicitor is preferably 5 to 30% by mass, and more preferably 8 to 20% by mass, based on 100% by mass of solids in the fertilizer composition. The content of the endogenous elicitor is preferably 15 to 60% by mass, and more preferably 25 to 50% by mass, based on 100% by mass of solids in the fertilizer composition. In addition to the exogenous and endogenous elicitors, 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 1-7 and Comparative Examples 9, 10, and 15 below used cellooligosaccharides produced by "Manufacturing Method 1," while Examples 9, 10, 12, 14, 16, and 17 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, 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 1-8 and Comparative Examples 11, 12, and 16 below used xylooligosaccharides from the "Commercial Products" section below, while Examples 9-15, 17, and 18 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.

[0039] [2. Measurement of edible portion yield and root dry weight of frilly lettuce] (1) Preparation of plant vitality enhancers Each oligosaccharide prepared in [1. Preparation of Oligosaccharides] was stirred with a stirrer and dissolved in water at its respective composition ratio so that the concentration of the plant vitality agent (mass ppm) was 1000 times that of Examples 1 and 9-11 in the table below. After sterilization using a 0.45 μm filter, the resulting solution was used as the plant vitality agent stock.

[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 hydroponic cultivation 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, with additional additions of 150g of concentrated plant stimulant solution made every week for a total of 5 times after the start of cultivation. The yield of edible portion and dry root weight of frilly lettuce were measured and compared with the case where no plant stimulant was applied (Comparative Example 1). The edible portion yield was measured by cutting off the root portion and measuring the upper part as the edible portion. The dry weight of the roots was measured by drying the cut roots in a constant temperature dryer at 50°C for 12 hours and then measuring their weight.

[0041] [Table 1]

[0042] [3. Measurement of edible portion yield and sugar content of tomatoes] (1) Preparation of plant vitality enhancers The oligosaccharides prepared in [1. Preparation of Oligosaccharides] were dissolved in water by stirring with a stirrer at their respective composition ratios so that the plant vitality agent concentration (mass ppm) was 1000 times that of Comparative Examples 3-6 and Examples 2-3 and 12-13 in the table below. After sterilization using a 0.45 μm filter, this was used as the plant vitality agent stock solution. This stock solution was diluted 1000 times with water and used in the following cultivation tests.

[0043] (2) Cultivation experiment The day before planting in the field, I thoroughly sprayed a plant stimulant solution onto the leaves of the medium-sized tomato seedlings that were being grown in pots, as well as onto the soil in the pots. Tomato field cultivation takes place in greenhouses, covering a total area of ​​334m². 2 Using the field, 40 medium-sized tomato seedlings were planted in each plot at 50cm intervals, and conventional farming methods using chemical fertilizers were employed. A 1.5 kg / plot aqueous solution of the plant stimulant, prepared to the plant stimulant concentration for each condition, was prepared each time. Foliar spraying using a watering can and irrigation of the soil near the base of the plant were performed once every two weeks for two months, starting two weeks after planting, for a total of five times. After that, the yield of edible parts and sugar content were measured and compared with the case where no plant stimulant was applied (Comparative Example 2). The edible portion yield was measured by cutting off only the edible tomato fruit and weighing it, while the sugar content was measured by extracting the juice from the tomato fruit and measuring it using a refractometer.

[0044] [Table 2]

[0045] [4. Measuring the dry weight of tomatoes] (1) Preparation of plant vitality enhancers The oligosaccharides prepared in [1. Preparation of Oligosaccharides] were dissolved in water by stirring with a stirrer at their respective composition ratios so that the plant vitality agent concentration (mass ppm) was 1000 times that of Comparative Examples 8-12 and Examples 4-5 and 14-15 in the table below. After sterilization using a 0.45 μm filter, this was used as the plant vitality agent stock solution. This stock solution was diluted 1000 times with water and used in the following cultivation tests.

[0046] (2) Cultivation experiment Tomato seeds were soaked in distilled water for 6 hours, then the stratum corneum was removed, and they were dried in an aerated area for 30 minutes. Next, 10 seeds were placed in each of several culture dishes lined with absorbent paper, and each culture dish was filled with a plant stimulant solution for each condition and soaked for 6 hours. After that, three seeds of uniform size were selected from each culture dish and planted in pots for 11 days according to the conditions. The dry weight of the germinated seeds was measured and compared with that of the case where no plant stimulant was applied (Comparative Example 7). The dry weight of the plants was measured after the root portion was cut off and the remaining upper part was dried in a constant temperature dryer at 50°C for 12 hours.

[0047] [Table 3]

[0048] [5. Evaluation of elicitor activity in Komatsuna] (1) Preparation of plant vitality enhancers The oligosaccharides prepared in [1. Preparation of Oligosaccharides] were dissolved in water by stirring with a stirrer at their respective composition ratios so that the plant stimulant concentration (mass ppm) was 1000 times that of Comparative Examples 14-16 and Example 6 in the table below. After sterilization using a 0.45 μm filter, the resulting plant stimulant stock solution was prepared. This stock solution was diluted 1000 times with water and used in the following cultivation tests.

[0049] (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.

[0050] (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.

[0051] (4) Protein extraction A protein extraction buffer with the following composition was prepared.

[0052] [Table 4]

[0053] 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.

[0054] (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. A MilliQ was used as the blank. The absorbance of the extract was fitted to a calibration curve created using serially diluted BSA at 2 mg / ml 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.

[0055] (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 13).

[0056] [Table 5]

[0057] [6. Evaluation of cotton elicitor activity] (1) Preparation of plant vitality enhancers The oligosaccharides prepared in [1. Preparation of Oligosaccharides] were dissolved in water by stirring with a stirrer at their respective composition ratios so that the plant stimulant concentration (mass ppm) was 1000 times that of Examples 7-8 and 16-18 in the table below. After sterilization with a 0.45 μm filter, this was used as the plant stimulant stock solution. This stock solution was diluted 1000 times with water and used in the following cultivation tests.

[0058] (2) Sowing and growth methods A cultivation test of Menka was conducted in a planter (640 x 220 x 180 mm) filled with potting soil (organic vegetable soil: Hirota Shoten) and fertilizer (Shin Taki Organic Liquid Fertilizer No. 3: Taki Chemical Co., Ltd.). First, Menka seeds from China were sown, and after germination, 50 ml of the plant vitality agent prepared above was sprayed from above and allowed to grow. The fertilizer was diluted 200 times and applied as a top dressing to approximately 1.5 L of planter the day before spraying the plant vitality agent.

[0059] (3) Measurement of glucanase activity Using the same method as in [5. Elicitor activity test of Komatsuna] above, the glucanase activity of cotton was measured and compared with the case where no plant stimulant was applied (Comparative Example 17).

[0060] [Table 6]

Claims

1. A plant vitality enhancer comprising an exogenous elicitor and an endogenous elicitor, wherein the exogenous elicitor is a chitin oligosaccharide, and the endogenous elicitor is at least one oligosaccharide selected from cellooligosaccharides and xylooligosaccharides.

2. The plant stimulant according to claim 1, wherein the total content of the exogenous elicitor and the endogenous elicitor in the plant stimulant is 0.05 to 10% by mass.

3. The plant vitality agent according to claim 1 or 2, wherein the mass ratio of the exogenous elicitor to the endogenous elicitor in the plant vitality agent is 0.2 to 5.

4. The plant vitality agent according to any one of claims 1 to 3, comprising xylooligosaccharide as the endogenous elicitor.

5. The plant vitality agent according to claim 4, comprising both cellooligosaccharide and xylooligosaccharide as the endogenous elicitor.

6. The plant stimulant according to claim 5, wherein the mass ratio of cellooligosaccharide to xylooligosaccharide in the plant stimulant is 0.2 to 5.

7. A plant vitality agent according to any one of claims 1 to 6, further comprising a spreading agent.

8. A method comprising applying a plant vitality agent according to any one of claims 1 to 7 to a plant.

9. The method according to claim 8, comprising applying the plant vitality agent to a plant at a concentration such that the total content of the exogenous elicitor and the endogenous elicitor is 0.1 to 500 ppm by mass.

10. The method according to claim 8 or 9, wherein the plant vitality agent is applied to the plant by foliar spraying.

11. A method for producing a plant or a part thereof having increased elicitor activity compared to a case where the plant vitality agent described in any one of claims 1 to 7 is not applied, the method comprising cultivating the plant by the method described in any one of claims 8 to 10.

12. The method according to claim 11, wherein the elicitor activity is determined by measuring the amount of glucanase produced in the plant.

13. A fertilizer composition containing a plant vitality enhancer according to any one of claims 1 to 7.

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