JP7801816B2 - Plant growth promoting composition - Google Patents

Description

The present invention relates to a plant growth-promoting composition and a plant growth-promoting method.

With the current explosive population growth, producing sufficient supplies of plants for food has become a global challenge. Other issues include desertification due to the reduction in green space, and rising earth temperatures due to global warming, which are creating increasing environmental problems for plant growth. In other words, excessive stress on plants caused by global environmental factors often causes problems for plant growth.

There is therefore a demand for technologies that promise more efficient harvesting than conventional technologies, and biostimulants are attracting attention as a new technology that reduces damage to plants caused by climate and soil conditions by controlling environmental stress on plants, thereby providing healthy plants.

Patent Document 1 discloses a plant stomatal opening promoter containing stevia, and reports that by artificially promoting the opening of plant stomatal pores with stevia, heat stress resistance can be improved.
Patent Document 2 discloses a composition for activating genes that activate the ethylene and/or jasmonic acid signaling system, which comprises a yeast cell wall decomposition product, and reports that applying the yeast cell wall decomposition product to plants activates the expression of plant defensin genes that are involved in the antibacterial properties of plants and disease prevention.
Patent Document 3 discloses a method for increasing the yield of grains by inoculating a cell wall-containing substance of a methanol-utilizing bacterium into a plant for cultivating grains.

Patent No. 6741262 Patent No. 4931388 JP 2019-180361 A

As mentioned above, research has been conducted to date on improving plant environmental stress tolerance and increasing yields using various materials, but no examples have been reported using persimmon extract or persimmon powder.

After extensive research, the inventors used a highly accurate screening method they developed themselves to discover that, among various materials, persimmon extract or persimmon powder is an effective ingredient for promoting plant growth, leading to the completion of this invention.

That is, the present invention is as follows.
[1]
A plant growth promoting composition comprising, as an active ingredient, one or more selected from the group consisting of persimmon extract and persimmon powder.
[2]
The plant growth-promoting composition described in [1] above has one or more plant growth-promoting effects selected from the group consisting of elongation of stems, leaves or roots, increased leaf number, promotion of flowering or fruiting, increased number of flowers or fruits, increased plant weight or crop yield, greening, and promotion of tillering.
[3]
The plant growth-promoting composition according to [1] or [2] above, which has an effect of improving environmental stress tolerance.
[4]
The plant growth-promoting composition described in [3] above, which has the effect of improving environmental stress tolerance against one or more stresses selected from the group consisting of temperature stress, nutritional stress, chemical stress, light stress, drought stress, pH stress, salt stress, hypoxic stress, herbicide stress, physical stress, and disease stress.
[5]
The plant growth-promoting composition according to [3] above, which activates genes in the stress response system.
[6]
A method for promoting plant growth, comprising applying a plant growth-promoting composition containing one or more active ingredients selected from the group consisting of persimmon extract and persimmon powder to a plant or to soil or culture solution in which the plant grows.
[7]
The plant growth promoting method according to [6] above, wherein the amount of the plant growth promoting composition used is 0.001 to 50% by mass or 0.001 to 50% by volume relative to the culture solution or dilution solvent.

By applying a composition containing one or more selected from the group consisting of persimmon extract and persimmon powder of the present invention to plants, plant growth can be promoted more safely and inexpensively than with conventional materials.

These images show the improvement in root growth of rice plants when no persimmon extract or persimmon powder was added (left) and when 4.5 mL of a plant growth-promoting composition containing persimmon extract (Example 1) was added (right). This is a graph showing the root weight of rice plants when no persimmon extract or persimmon powder was added and when 4.5 mL of a plant growth-promoting composition containing persimmon extract was added. This is an image showing a heat map of the expression levels of high temperature stress resistance genes in rice when no persimmon extract or persimmon powder was added and when 4.5 mL of a plant growth-promoting composition containing persimmon extract was added. This is an image showing the expression level of the rice high temperature stress tolerance gene Os05g0110100 when no persimmon extract or persimmon powder was added and when 4.5 mL of a plant growth promoting composition containing persimmon extract was added. This is an image showing a heat map of the expression levels of drought stress tolerance genes in rice when no persimmon extract or persimmon powder was added and when 4.5 mL of a plant growth-promoting composition containing persimmon extract was added. This is an image showing the expression level of the rice drought stress tolerance gene Os03g0225100 when no persimmon extract or persimmon powder was added and when 4.5 mL of a plant growth-promoting composition containing persimmon extract was added. This is an image showing a heat map of the expression levels of salt stress tolerance genes in rice when no persimmon extract or persimmon powder was added and when 4.5 mL of a plant growth-promoting composition containing persimmon extract was added. This is an image showing the expression level of the rice salt stress tolerance gene Os01g0200700 when no persimmon extract or persimmon powder was added and when 4.5 mL of a plant growth-promoting composition containing persimmon extract was added.

The following describes in detail the mode for carrying out the present invention (hereinafter also referred to as the "present embodiment"). Note that the present invention is not limited to this embodiment, and various modifications can be made within the scope of its gist.

<Plant growth promoting composition>
The plant growth-promoting composition of this embodiment contains one or more active ingredients selected from the group consisting of persimmon extract and persimmon powder. Persimmon (Diospyros kaki) is a plant of the genus Diospyros in the family Ebenaceae. Specific examples include Diospyros lotus, and more specifically Diospyros melanoxylon. The variety and origin of persimmon are not particularly limited, and examples include Ichida persimmon, Fuyu persimmon, and Gosho persimmon.

In this embodiment, persimmon extract refers to specific components extracted from various parts of persimmons, and is not particularly limited. Persimmon extract can be obtained, for example, by the method described below in "Method for preparing a plant growth-promoting composition." Furthermore, persimmon powder refers to various parts of persimmons or persimmon extract that have been dried and pulverized, and is not particularly limited.

The plant growth-promoting composition in this embodiment is a composition that has a plant growth-promoting effect. Here, the plant growth-promoting effect includes, for example, promoting growth such as elongation of stems, leaves, or roots, increasing the number of leaves, promoting flowering or fruiting, increasing the number of flowers or fruits, increasing plant weight or crop yield, greening, or promoting tillering.

More specifically, a case where a plant growth-promoting effect is observed refers to, for example, an increase in the actual weight increase (%) or dry weight increase (%) when the plant growth-promoting composition of this embodiment is used compared to when it is not used. The actual weight or dry weight may be the actual weight or dry weight of the entire plant, or the actual weight or dry weight of each part of the plant (preferably the roots). When the plant growth-promoting composition is used, the actual weight increase (%) or dry weight increase (%) is preferably 10% or more, more preferably 20%, and even more preferably 30% or more, compared to when the plant growth-promoting composition of this embodiment is not used. A plant growth-promoting effect includes a case where no growth is observed without the use of the plant growth-promoting composition, but growth is observed after use of the plant growth-promoting composition. Note that the above values are average values for the entire plant to which the plant growth-promoting composition is applied.

In addition, the plant growth promoting effect also includes the effect of improving the activity of plants as follows.
1) Root elongation and improved root survival rate, 2) Increased nutrient absorption efficiency, 3) Induction of flower buds, promotion of flowering, and increased fruit production, 4) Accumulation of sugars and other components, 5) Increased water-saving effect, 6) Callus induction effect, 7) Increased plant size, particularly elongation or thickening of above-ground and below-ground parts, 8) Wound repair. In other words, the plant growth-promoting composition of this embodiment may have the effects 1) to 8) above in addition to growth-promoting effects such as elongation of stems, leaves, or roots, increased leaf number, promotion of flowering or fruiting, increased number of flowers or fruits, increased plant weight or crop yield, greening, or promotion of tillering. By exerting the effect 1) above, nutrient absorption by the roots may be promoted, and the growth of the entire plant may be promoted.

The plant growth-promoting composition of this embodiment may confer tolerance to a variety of environmental stresses to which plants are exposed. The plant growth-promoting composition of this embodiment may have the effect of improving environmental stress tolerance. Here, examples of environmental stress include biotic stress and abiotic stress, and specific examples include herbicide stress, disease stress, temperature stress such as high temperature stress and low temperature stress, nutritional stress, chemical stress, light stress, drought stress, pH stress such as oxidative stress, salt stress such as osmotic stress, hypoxic stress, and physical stress. Nutritional stress includes, for example, nutrient deficiency stress and nutrient excess stress.

In this embodiment, "environmental stress tolerance" refers to a trait that allows a plant to grow normally or near-normally, even under growth conditions that cause environmental stress, without substantially suffering from undesirable effects such as inability to grow (withering), poor growth (e.g., bleaching or yellowing of the plant body, reduced root length or reduced leaf number), reduced growth rate, or reduced plant weight or crop yield. By improving the plant's environmental stress tolerance with the plant growth-promoting composition of this embodiment, the plant can grow normally even in external environments that have undesirable effects on the growth of ordinary plants.

The temperature stress tolerance-imparting effect of the plant growth-promoting composition of this embodiment includes, for example, the effect of promoting normal growth under high-temperature and low-temperature conditions. High-temperature conditions may be appropriately determined by a person skilled in the art based on common general technical knowledge in the technical field, but are preferably 35°C or higher, for example, 35°C to 50°C. Low-temperature conditions may be appropriately determined by a person skilled in the art based on common general technical knowledge in the technical field, but are preferably 10°C or lower, for example, 0°C to 10°C. Furthermore, the salt stress-imparting effect allows normal growth in the presence of 0.1 to 3.0% by mass of sodium chloride. The drought stress-imparting effect allows normal growth even at a relative humidity of 10 to 40%.
The effect of the plant growth-promoting composition of this embodiment in imparting nutritional stress tolerance includes, for example, promoting normal growth in plants that are deficient in chemical substances that serve as nutrient sources for the plant (nutrient-deficient state). Here, a nutrient-deficient state may be, for example, a state in which the amount of each nutrient is less than the concentration indicated for the test period in Table 1 of the Examples. Specifically, the amount of each nutrient may be approximately one-half to one-third, one-third to one-quarter, one-quarter to one-fifth, one-fifth to one-sixth, one-sixth to one-seventh, or one-seventh to one-eighth of the amount indicated for the test period concentration in Table 1. The test period concentrations in Table 1 are concentrations commonly used in the cultivation of crops such as rice.

In this embodiment, the environmental stress tolerance of a plant can be evaluated, for example, by the following means. Salt stress tolerance can be confirmed by growing the target plant in a medium or soil containing an appropriate concentration of salt at a growth temperature and growth period suitable for the plant, and evaluating the phenotype. Drought stress tolerance can be confirmed by growing the target plant at a growth temperature, growth humidity, and growth period suitable for the plant, and evaluating the phenotype. High temperature stress tolerance can be confirmed by acclimating the target plant to a high temperature, such as 35°C to 50°C, and then growing it at a growth temperature and growth period suitable for the plant, and evaluating the phenotype. Low temperature stress tolerance can be confirmed by acclimating the target plant to a low temperature that does not cause freezing, and then growing it at a growth temperature and growth period suitable for the plant, and evaluating the phenotype.

When the plant growth-promoting composition of the present embodiment has an effect of improving environmental stress tolerance, it may activate a gene in a stress response system. Examples of the gene in the stress response system include, but are not limited to, genes generally known as indicators of response to environmental stresses such as temperature stress, nutritional stress, chemical stress, light stress, drought stress, pH stress, salt stress, hypoxic stress, herbicide stress, physical stress, and disease stress.
Genes generally known as indicators of response to environmental stress are genes disclosed in known databases such as the National Center for Biotechnology Information (NCBI), Ensemble Plants, The Arabidopsis Information Resource (TAIR), and The Rice Annotation Project (RAP). Specifically, genes generally known as indicators of response to high temperature stress include Os01g0135900, Os01g0273500, Os01g0875700, Os02g0753800, Os03g0161900, Os03g0266300, Os03g0426900, Os05g0110100, and Os05g03645. 00, Os05g0460000, Os05g0530400, Os06g0682900, Os06g0716700, Os06g0727200, Os08g0127600, Os08g0191100, Os08g0500700, Os09g0133600, Os11g0216100, Os11g0661200, etc. For example, by applying the plant growth-promoting composition of this embodiment to a plant, the following genes can be increased in the plants acclimated to high temperatures: Os01g0135900, Os01g0273500, Os01g0875700, Os02g0753800, Os03g0161900, Os03g0266300, Os03g0426900, Os05g0110100, Os05g03645 ... The expression levels of genes such as s05g0460000, Os05g0530400, Os06g0682900, Os06g0716700, Os06g0727200, Os08g0127600, Os08g0191100, Os08g0500700, Os09g0133600, Os11g0216100, and Os11g0661200 are increased compared to the control.

Genes generally known to be indicators of response to drought stress include Os01g0615050, Os01g0864500, Os02g0115700, Os02g0178800, Os02g0682300, Os02g0753800, Os02g0766700, Os03g0225100, and Os03g059440. 0, Os05g0127200, Os05g0247100, Os05g0382900, Os05g0542500, Os07g0684000, Os08g0492500, Os09g0271100, Os09g0421700, Os10g0562900, Os11g0167800, Os11g0704500, etc. For example, by applying the plant growth-promoting composition of this embodiment to a plant, the following can be achieved in plants grown under dry conditions: Os01g0615050, Os01g0864500, Os02g0115700, Os02g0178800, Os02g0682300, Os02g0753800, Os02g0766700, Os03g0225100, Os03g0594400 The expression levels of genes such as Os05g0127200, Os05g0247100, Os05g0382900, Os05g0542500, Os07g0684000, Os08g0492500, Os09g0271100, Os09g0421700, Os10g0562900, Os11g0167800, and Os11g0704500 are increased compared to the control.

Genes generally known as indicators of response to salt stress include Os01g0141000, Os01g0200700, Os01g0615100, Os01g0624500, Os01g0864500, Os02g0132300, Os02g0327000, Os02g0594700, and Os03g0285700. , Os03g0364400, Os03g0594400, Os03g0678800, Os04g0493000, Os04g0511200, Os04g0530900, Os05g0127200, Os05g0542500, Os07g0684000, Os08g0492500, Os10g0370500, etc. For example, by applying the plant growth-promoting composition of this embodiment to a plant, the following growth factors can be increased in the plants grown in a medium or soil containing salt at an appropriate concentration: Os01g0141000, Os01g0200700, Os01g0615100, Os01g0624500, Os01g0864500, Os02g0132300, Os02g0327000, Os02g0594700, Os03g The expression levels of genes such as Os0285700, Os03g0364400, Os03g0594400, Os03g0678800, Os04g0493000, Os04g0511200, Os04g0530900, Os05g0127200, Os05g0542500, Os07g0684000, Os08g0492500, and Os10g0370500 are increased compared to the control.

The plant growth-promoting composition of this embodiment may contain one or more components selected from the group consisting of persimmon extract and persimmon powder, either alone or in combination with one or more agriculturally acceptable ingredients. Agriculturally acceptable ingredients are not particularly limited, but include, for example, solvents, carriers, excipients, binders, solubilizers, stabilizers, thickeners, leavening agents, lubricants, surfactants, oily liquids, buffers, disinfectants, antifreeze agents, antifoaming agents, colorants, antioxidants, and additional active ingredients. Agriculturally acceptable carriers are preferably agriculturally acceptable liquid carriers such as water, mineral oil fractions such as kerosene or diesel oil, vegetable or animal oils, cyclic or aromatic hydrocarbons (e.g., paraffin, tetrahydronaphthalene, alkylated naphthalenes or their derivatives, or alkylated benzenes or their derivatives), alcohols (e.g., methanol, ethanol, propanol, butanol, or cyclohexanol), ketones (e.g., cyclohexanone), amines (e.g., N-methylpyrrolidone), or mixtures thereof.

When the plant growth-promoting composition of this embodiment contains one or more additional active ingredients, the additional active ingredients can be various compounds known in the art that have the activity of improving environmental stress tolerance. By including one or more such additional active ingredients in the plant growth-promoting composition, the plant's environmental stress tolerance tends to be further improved.

The plant growth-promoting composition of this embodiment can be used in combination with conventional agricultural materials such as pesticides and fertilizers. Examples of agricultural materials include pesticides, fertilizers, soil conditioners, and potting soil (culture soil). Specifically, pesticides may be fungicides, insecticides, insecticides and fungicides, acaricides, nematicides, rodenticides, insect repellents, attractants, spreaders, hormones (growth regulators), herbicides, and the like. Fertilizers may be organic fertilizers, chemical fertilizers, liquid fertilizers, solid fertilizers, compound fertilizers, compost, growth promoters, stimulants, calcium supplements, and the like. Soil conditioners may be those intended for physical, chemical, or biological soil improvement, and may be oxygen suppliers, etc. Furthermore, pesticides, fertilizers, soil conditioners, and potting soil may contain components derived from plants, animals, lime, ores, or microorganisms.

The content of one or more ingredients selected from the group consisting of persimmon extract and persimmon powder in the plant growth-promoting composition relative to the entire plant growth-promoting composition may be any value as long as the one or more ingredients selected from the group consisting of persimmon extract and persimmon powder function as active ingredients for promoting plant growth. For example, the content may be 5, 10, 20, 30, 40, 50, 60, 70, 80, or 90% by mass (volume %) or more, and may be 99, 95, 90, 80, 70, 60, 50, 40, 30, 20, or 10% by mass (volume %) or less.

The plant growth-promoting composition of this embodiment can be used as an agricultural chemical formulation or pesticide to promote plant growth. The plant growth-promoting composition may be in any form, such as a solid (e.g., powder or granules) or a liquid (e.g., a solution or suspension), and is preferably used in a liquid form such as a solution or suspension. When used as a solution, it may be in liquid form, or may be used as a liquid prepared just before use.

The formulation of the plant growth-promoting composition of this embodiment is not particularly limited, and can be formulated into formulations commonly used in the technical field, such as emulsions, wettable powders, liquids, water-soluble formulations, dusts, powders, pastes, or granules.

In this embodiment, the plants to which the plant growth-promoting composition is applied are not particularly limited, and examples include angiosperms and gymnosperms. Examples of target plants include Asteraceae plants such as chrysanthemums and gerberas, Solanaceae plants such as potatoes, tomatoes, and eggplants, Brassicaceae plants such as rapeseed and rapeseed, Poaceae plants such as rice, corn, wheat, sugarcane, and barley, Legumes such as soybeans, Apiaceae plants such as carrots, Lamiaceae plants such as basil, mint, and rosemary, Convolvulaceae plants such as morning glory, Salicaceae plants such as poplars, and Euphorbiaceae plants such as castor beans, cassava, and jatropha. , Convolvulaceae plants such as sweet potatoes, Rutaceae plants such as oranges and lemons, Rosaceae plants such as cherry blossoms and roses, Orchidaceae plants such as Phalaenopsis, Gentianaceae plants such as bellflowers, Primulaces such as cyclamen, Violets such as pansies, Liliaceae plants such as lilies, Amaranthaceae plants such as sugar beets, Vitaceae plants such as grapes, Cupressaceae plants such as cedars and cypresses, Oleaceae plants such as olives and Osmanthus, and Pinaceae plants such as red pine.

Target plants may include not only the entire plant (i.e., the complete plant body), but also plant tissues or organs (e.g., cut flowers, or vegetative propagation organs such as rhizomes, tuberous roots, corms, or runners), cultured cells, and/or callus, among other plant parts.

The plant growth-promoting composition of this embodiment can be applied to the whole or part of a plant at any stage of growth, including before or after plant emergence (e.g., the whole or part of a seed, seedling, or mature plant).

<Method for preparing plant growth-promoting composition>
The plant growth-promoting composition can be prepared, for example, by obtaining one or more selected from the group consisting of persimmon extract and persimmon powder, and then mixing it with the agriculturally acceptable ingredients described above as necessary.
The method for obtaining persimmon extract is not particularly limited, and general methods for extracting plant extracts can be used. Specifically, persimmon extract can be obtained by a method comprising the steps of heating or crushing persimmon fruit, pulp, skin, stem, branch, or root, and then removing impurities to obtain a persimmon extract raw material; concentrating the persimmon extract raw material obtained in the above step to obtain a persimmon concentrate; and fractionating the persimmon concentrate obtained in the above step to obtain a persimmon extract containing specific components. Persimmon extract can also be obtained from one or more persimmon fruit, pulp, skin, stem, branch, or root that have undergone each of the steps for obtaining persimmon extract. For example, persimmon extract raw material or persimmon concentrate can be obtained from persimmon residue, such as impurities removed in the persimmon extract raw material obtaining step, using a method similar to that described above, and then persimmon extract can be obtained from the persimmon extract raw material or persimmon concentrate using a method similar to that described above. Alternatively, persimmon extract can be obtained from persimmon residue, such as impurities obtained during the production of any persimmon processed product. The persimmon extract raw material or persimmon concentrate can be used directly as persimmon extract. In each step or after each step, the persimmon extract raw material, persimmon concentrate, or persimmon extract may be heated or pressurized. When heating, the heating conditions such as heating temperature and heating time can be appropriately determined depending on the variety, part, condition, etc. of the persimmon used.
There are no particular limitations on the method for obtaining persimmon powder, and it can be obtained by drying persimmon residue such as impurities that arise when obtaining persimmon extract raw material, persimmon concentrate, or persimmon extract, fresh or frozen persimmon extract raw material, persimmon concentrate, persimmon extract, or persimmon (one or more selected from the group consisting of persimmon fruit, pulp, skin, stem, branch, or root) using methods such as hot air drying, air drying, dry heat drying, or vacuum drying, and then crushing it into powder.

(Crushing process)
Persimmons can be crushed using fresh or dried persimmons, for example, in a mixer. The crushed persimmons are filtered using filter cloth or filter paper to obtain a persimmon extract raw material from which impurities have been removed. When filtering, an extraction solvent such as an organic solvent may be added beforehand. Alternatively, impurities may be removed by collecting the supernatant using a centrifuge instead of filtration.

(concentration process)
The resulting persimmon extract raw material can be concentrated using methods such as vacuum concentration, freeze concentration, and membrane concentration to obtain a persimmon concentrate. The concentration ratio is preferably 3 to 30 times, and more preferably 4 to 20 times. A concentration ratio of 5 times or more tends to improve handling and reduce transportation costs because the amount of plant growth-promoting composition added to plants is reduced, while a concentration ratio of 30 times or less tends to reduce sediment in the plant growth-promoting composition, making it easier to handle.
In addition, in preparing the plant growth-promoting composition, it is not necessary to concentrate the persimmon extract raw material, and the obtained persimmon extract raw material may be subjected to the following fractionation step as is.

(Fractionation step)
The resulting persimmon concentrate can be fractionated by solvent extraction or other methods to obtain persimmon extract containing specific components. In the fractionation process, for example, hydrophilic and hydrophobic fractions can be obtained by liquid-liquid partitioning. Further, more purified fractions can be obtained by passing the fraction through a column.

If necessary, the persimmon extract obtained in the fractionation process may be further purified or subjected to separation of highly active fractions. Examples of purification processes include filtration and adsorption (ion exchange resin column, activated carbon column, etc.). Examples of separation processes for highly active fractions include gel filtration, adsorption, silica gel column chromatography, and HPLC.

<Plant growth promotion method>
The plant growth promotion method of this embodiment promotes plant growth by applying a composition containing one or more active ingredients selected from the group consisting of persimmon extract and persimmon powder to a plant, or to the soil or culture solution in which the plant grows. In this method, the plant growth promotion composition can be applied to a plant or part thereof (e.g., a seed, a seedling, or a mature plant) at any stage of growth, including before or after germination. Furthermore, the composition can be applied not only to the plant itself, but also to the soil, culture medium, or culture solution in which the plant grows. By applying the plant growth promotion composition to a plant at any stage of growth, or to the soil, culture medium, or culture solution in which the plant grows, plant growth can be promoted.

The plant growth-promoting composition may be applied to a plant once or multiple times. Furthermore, if the application period is multiple, the number of applications at each period may be either once or multiple times.

Methods for applying the plant growth-promoting composition to plants include, for example, addition, spraying, coating, and immersion. These methods may be used alone or in combination of two or more. The plant growth-promoting composition may be applied to plants manually or by machine. When using a machine, there are no particular limitations, but application (spraying) may also be done using a drone, for example.

When the plant growth-promoting composition is applied to a plant in a culture solution in which the plant grows, or when diluted with a diluent and applied to a field in which the plant grows, the amount of plant growth-promoting composition used relative to the culture solution or diluent is preferably 0.001 to 50% by mass or 0.001 to 50% by volume, more preferably 0.01 to 30% by mass or 0.01 to 30% by volume, and even more preferably 0.01 to 10% by mass or 0.01 to 10% by volume. While a sufficient plant growth-promoting effect tends to be achieved when the amount of plant growth-promoting composition used falls within the above ranges, the amount used may be adjusted appropriately depending on the content of one or more selected from the group consisting of persimmon extract and persimmon powder in the plant growth-promoting composition, the type and growth condition of the plant to which the plant growth-promoting composition is applied, and the plant's growth environment, such as temperature, humidity, and light. The effective concentration range may be investigated using a known cultivation test method, and the amount used may be determined. When using a drone to apply a plant growth-promoting composition diluted with a diluting solvent to plants, the amount of solution that can be carried on the drone is limited by factors such as the drone's carrying capacity, so the amount of plant growth-promoting composition used relative to the diluting solvent is preferably 1 to 50% by mass or 1 to 50% by volume.

The ingredients of the above culture medium and dilution solvent are not particularly limited as long as they do not adversely affect plant growth, and may contain water, pesticides, etc.

The preferred range of the above usage amounts will vary depending on the concentration of the persimmon extract raw material mentioned above, but the above usage amounts are the preferred range when the concentration is 3 to 30 times.

The present invention will be explained in more detail using examples and comparative examples, but the present invention is not limited to these examples in any way.

(Method for measuring root weight)
The roots were identified from the color and shape of the rice plants, and the weight of the identified roots was measured in a bottle using a microbalance.

[Example 1]
After peeling 40 g of persimmons, they were crushed in ultrapure water. The supernatant was removed from the persimmon extract raw material obtained by removing impurities, and then an equivalent amount of ultrapure water was added to the supernatant and resuspended to obtain persimmon extract.
Rice plants were grown in the seedling culture solution shown in Table 1. Then, the rice plants were transferred to 900 mL of the test culture solution shown in Table 1, and 4.5 mL (0.5% by volume) of the obtained persimmon extract was added to investigate the effect on the rice. As a result, the weight of the rice roots increased by 15.5% compared to when the persimmon extract was not added, demonstrating an effect of improving the root establishment of rice.
Furthermore, the concentration of nutrients during the seedling stage was approximately one-sixth to one-seventh of the concentration during the test period, which is sufficient for rice growth, so the rice plants to which persimmon extract had been added were in a state of nutritional deficiency.

[Example 2]
Variation in gene expression in rice was also assessed.

(A) Sample Preparation - Preparation of Rice Plants Rice seedlings grown in the same manner as in Example 1 were transferred to 900 mL of culture solution for the test period shown in Table 1, and 4.5 mL (0.5% by volume) of the persimmon extract used in Example 1 was added to the 900 mL culture solution, and the rice was cultivated.
RNA Extraction RNA samples were obtained using a Maxwell RSC Plant RNA Kit (Promega) and a Maxwell RSC Instrument (Promega) according to the manufacturer's instructions.
RNA quality confirmation: After confirming that there were no problems with the quality of the RNA samples using measuring equipment: Agilent 5400 Bioanalyzer <5312AA> (Agilent Technologies), reagent kit: Agilent RNA kit (15 nt) <DNF-471> (Agilent Technologies), and analysis software: ProSize (Agilent Technologies), the samples were used for library preparation.
Library Preparation A library for the next-generation sequencer DNBSEQ T7 was prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina according to the following steps.
1. Purification of mRNA using oligo(dT) magnetic beads. 2. Fragmentation. 3. Synthesis of single-stranded and double-stranded cDNA using random primers. 4. End repair, polyadenylation, adapter binding, size selection, and amplification are performed.

(B) Analysis using next-generation sequencer. Sequence analysis equipment: DNBSEQ T7 (MGI).
・Sequence (outline)
Sequencing was carried out using DNBSEQ T7 according to the following steps.
1. Addition of sequencing reagents 2. Single-base extension reaction 3. Removal of unreacted bases 4. Incorporation of fluorescent signal 5. Removal of protecting groups and fluorescence 6. Repeat cycles 2...3... up to 300 cycles (C) Data analysis/primary data analysis Analysis computer OS: Ubuntu Desktop 22.04.4 LTS
Analysis software: FastQC, hisat2, samtools, HTSeq, DESeq2
The analysis was performed using the software mentioned above.
1. Checking the quality of the reads FastQC was used to check the quality of the bases contained in the resulting fastq file.
2. Mapping and sorting: Using hisat2, the data was mapped to the Oryza sativa reference genome, Oryza sativa Japonica Group (assembly IRGSP-1.0). Subsequently, the files were sorted and converted to binary data using samtools.
3. Read count The number of mapped reads was counted using htseq-count in HTSeq.
4. Statistical analysis The gene expression levels in the control group and the persimmon extract-added group were compared using the DESeq2 package in the statistical package R.
-Tertiary data analysis: Genes reported to be involved in various environmental stress tolerance in previous studies were searched from the Rice Annotation Project (https://rapdb.dna.affrc.go.jp/). The searched genes were then searched from the gene expression list created in this experiment.
Selection of target genes and plotting of gene expression levels Various environmental stress tolerance genes present in known databases were collected. Various stress tolerance genes were then extracted from the list of genes with elevated expression levels, and their expression levels were plotted as heat maps and bar graphs. In the heat maps in Figures 3, 5, and 7, CTS indicates the control group, and KKS indicates the persimmon extract-added group.

Gene expression analysis showed that the expression levels of several high-temperature stress tolerance genes tended to be higher in the shoots of the persimmon extract-treated area than in the control area (Figure 3). In particular, the expression level of the high-temperature stress tolerance gene Os05g0110100 was increased in the shoots (Figure 4). The following findings have been reported regarding these molecules related to high-temperature stress tolerance, and it is thought that they contribute to high-temperature stress tolerance. The expression of molecules homologous to Os05g0110100 is induced by high-temperature stress, suggesting that they play an important role in enabling plants to survive under high-temperature conditions (Reference 1). This gene and similar genes have also been reported to be widely present in Arabidopsis thaliana and rice, as well as in rapeseed, common wheat, soybean, and other plants (Reference 2).

Gene expression analysis showed that the expression levels of several drought stress tolerance genes tended to be higher in the shoots of the persimmon extract-treated area than in the control area (Figure 5). In particular, the expression level of the drought stress tolerance gene Os03g0225100 increased in the shoots (Figure 6). The following has been reported about these molecules related to drought stress tolerance, and therefore they are thought to contribute to drought stress tolerance. The MAP kinase cascade has been suggested to contribute to drought tolerance (Reference 3). This gene and similar genes have been reported to be widely present in plants other than Arabidopsis and rice, such as cotton, soybean, and Physcomitrella patens (Reference 4).

Gene expression analysis showed that the expression levels of several salt stress tolerance genes tended to be higher in the shoots of the persimmon extract-treated area than in the control area (Figure 7). In particular, the expression level of the salt stress tolerance gene Os01g0200700 increased in the shoots (Figure 8). The following has been reported about these molecules related to salt stress tolerance, and therefore they are thought to contribute to salt stress tolerance. It has been reported that when a homologous gene of Os01g0200700 from a salt-tolerant plant is expressed in Arabidopsis, salt stress tolerance is increased (Reference 5). This gene and similar genes have also been reported to be widely present in Arabidopsis and rice, as well as in wild cotton, soybean, common wheat, and corn (Reference 6).

From the above, it is believed that persimmon extract can also improve tolerance to one or more stresses selected from the group consisting of high temperature stress, drought stress, low temperature stress, and salt stress in many plants other than rice by stimulating high temperature stress tolerance genes, drought stress tolerance genes, salt stress tolerance genes, or molecules similar to these.

Reference 1: https://pmc. ncbi. nlm. nih. gov/articles/PMC6862505/#sec4-ijms-20-05321
Reference 2: https://www. arabidopsis. org/locus? key=36137
Reference 3: https://pubmed. ncbi. nlm. nih. gov/28857351/
Reference 4: https://www. arabidopsis. org/locus? key=29640
Reference 5: https://www. nature. com/articles/s41598-017-18263-4
Reference 6: https://www. arabidopsis. org/locus? key=135469

Claims (6)

A composition for improving environmental stress tolerance of a plant, comprising one or more active ingredients selected from the group consisting of persimmon extract and persimmon powder. A composition for improving environmental stress tolerance of a plant as described in claim 1, which has one or more plant growth-promoting effects selected from the group consisting of elongation of stems, leaves or roots, increased leaf number, promotion of flowering or fruiting, increased number of flowers or fruits, increased plant weight or crop yield, greening, and promotion of tillering . 3. A composition for improving environmental stress tolerance of a plant as described in claim 1 or 2, which has the effect of improving environmental stress tolerance against one or more stresses selected from the group consisting of temperature stress, chemical stress, light stress, drought stress, pH stress, salt stress, hypoxic stress, herbicide stress, physical stress, and disease stress. The composition for improving environmental stress tolerance of a plant according to claim 1 or 2, which activates a gene in a stress response system. A method for improving environmental stress tolerance by applying a composition for improving environmental stress tolerance containing one or more active ingredients selected from the group consisting of persimmon extract and persimmon powder to a plant, or to the soil or culture solution in which the plant grows. The method for improving environmental stress tolerance according to claim 5, wherein the amount of the composition for improving environmental stress tolerance used relative to the culture medium or dilution solvent is 0.001 to 50% by mass or 0.001 to 50% by volume.