JP2024044044A - Biostimulant composition, foliar spray agent, and plant cultivation method - Google Patents

Abstract

[Problem] To provide a highly active biostimulant composition that induces disease resistance in plants. [Solution] The biostimulant composition contains xylotetraose and is used to induce disease resistance in plants, and the content of xylotetraose is 30% by mass or more based on the total mass of xylose and xylooligosaccharides having a degree of polymerization of 2 to 10. [Selected Figure] None

Description

The present invention relates to biostimulant compositions, foliar sprays, and plant cultivation methods.

Plant yields are reduced by abiotic stresses such as sunlight hours, temperature, and rainfall, as well as by biotic stresses such as pests and diseases. To date, various fertilizers and pesticides have been used to increase crop yields. Fertilizers are a source of nutrients necessary for plant growth, but do not have the function of alleviating stress. Pesticides directly eliminate pests that parasitize plants and eliminate biotic stress. However, when using pesticides, although their safety has been fully confirmed, there are concerns about the effects on the human body or the environment from overdosing. In particular, there is a concern that pesticides and other chemicals manufactured by chemical synthesis may remain in the soil for a long time once sprayed, so there has been a desire to deal with biotic stress by other methods. For this reason, in addition to these, the use of biostimulants has been attracting attention in recent years as substances that are safe for both the human body and the environment.

"Biostimulant" is also referred to as "biological stimulant" or "plant stimulant", etc., and contains a wide variety of substance groups or microorganisms, and when applied to the plant body or its root system, it restores its natural state. By stimulating a series of processes that occur within the plant, it is possible to improve crop quality by improving nutrient absorption, increasing fertilization efficiency, and imparting stress tolerance. However, it does not have a direct effect on pests and is therefore not classified as any type of insecticide or fungicide. That is, a biostimulant is a component (including microorganisms) that exists in nature, and although it is not a plant hormone or a nutrient, it refers to a substance that stimulates the vitality of plants and promotes their growth even in very small amounts. It is said that applying biostimulants to plants increases the plant's nutrient absorption and utilization rate, promotes growth, and improves the yield and quality of agricultural crops. Agricultural biostimulants include a wide variety of formulations such as compounds, substances and other products that are applied to plants or soil to control and enhance physiological processes in crops. Biostimulants act on plant physiology through different pathways than nutrients to improve crop vigor, yield, quality and post-harvest storage.

In this way, biostimulants can stimulate the natural abilities of plants to promote their growth without causing the problems associated with traditional pesticides or fertilizers.

Patent Document 1 (JP Patent Publication 63-215606) describes a method for cultivating plants using oligosaccharides, and Example 1 describes the results of investigating the plant growth-promoting effect of xylooligosaccharides with a degree of polymerization of 2 to 10 using radish sprouts.

Japanese Unexamined Patent Publication No. 63-215606

The xylo-oligosaccharide having a degree of polymerization of 2 to 10 described in Cited Document 1 is a mixture containing a plurality of xylo-oligosaccharides having different degrees of polymerization. In Cited Document 1, the amount of xylooligosaccharides added is discussed, but the active ingredient with the plant growth promoting effect has not been specifically elucidated, and the mechanism of action other than the plant growth promoting effect has not been disclosed.

The objective of the present invention is to provide a highly active biostimulant composition that induces disease resistance in plants.

The present inventors have discovered that xylooligosaccharide with a degree of polymerization of 4, that is, xylotetraose, is a compound that has activity as a biostimulant that induces disease resistance in plants, and has completed the present invention. .

That is, the present invention includes the following [1] to [4].

[1]
A biostimulant composition containing xylotetraose, which is used to induce disease resistance in plants, wherein the content of the xylotetraose is 30% by mass or more based on the total mass of xylose and xylooligosaccharides having a degree of polymerization of 2 to 10.
[2]
A foliar spray agent containing the biostimulant composition described in [1].
[3]
A plant cultivation method comprising foliar spraying of the biostimulant composition according to [1].
[4]
Use of xylotetraose as a biostimulant to induce disease resistance in plants.

The present invention provides a highly active biostimulant composition that induces disease resistance in plants.

1 shows the results of HPLC analysis of the xylooligosaccharide powder produced in Example 1. 1 shows the results of HPLC analysis of the xylotetraose-containing aqueous solution of Example 1. 1 is an HPLC analysis result of a xylobiose-containing aqueous solution of Comparative Example 1. 3 shows the HPLC analysis results of the xylotriose-containing aqueous solution of Comparative Example 2. 3 shows the HPLC analysis results of the xylopentaose-containing aqueous solution of Comparative Example 3. 1 shows the results of measuring the elicitor activity of aqueous xylooligosaccharide solutions of Example 1 and Comparative Examples 1 to 3, together with the results of the control (water). 1 is a graph showing the elicitor activity of the xylo-oligosaccharide aqueous solutions of Example 1 and Comparative Examples 1 to 3, and also shows the results of a control.

Embodiments of the present invention will be described below. Note that the embodiments described below show typical examples of the present invention, and the present invention is not limited thereto.

[Use of xylotetraose as a biostimulant]
One embodiment is the use of xylotetraose as a biostimulant to induce disease resistance in plants.

Biostimulants are materials with elicitor activity, and have the effect of enhancing immune activity in plants. By applying biostimulants to plants, the defenses against biological stresses such as pests, bacteria, mold, and viruses are activated. As an effect of activating the plant's defenses, they can also be used to promote plant growth. Biostimulants can also have an effect of mitigating abiotic stresses such as temperature, light, water, and salt that are related to plant growth.

An elicitor is a general term for a substance that induces a biological defense response in the tissues or cultured cells of higher plants, and induces disease resistance in the plant's immune system. Plants sense elicitors with receptors present on the leaf surface, etc., and initiate a pathogen resistance response. This results in a biological defense action (immunity) in which various compounds are secreted against pathogens, etc. When an elicitor acts on a plant, defense responses such as synthesis and accumulation of phytoalexins or infection-specific proteins, production of reactive oxygen species (ROS), production of reactive nitrogen, callose deposition, MAPK phosphorylation, hypersensitive reactive cell death, and changes in gene expression are induced, and it is believed that these defense responses enable plants to protect themselves from biological stress caused by pathogens and the like, and increase disease resistance.

Phytoalexins are antibacterial compounds that are synthesized and accumulated within plants by the action of elicitors, and different antibacterial compounds are produced depending on the plant species. Representative phytoalexins include, for example, flavonoids, terpenoids, and fatty acid derivatives. Active oxygen has the effect of killing pathogenic microorganisms. Active oxygen and nitrogen function individually or in concert as signals that trigger various defense reactions.

Oligosaccharides derived from plant cell walls are a source of damage-associated molecular patterns (DAMPs) that are recognized by pattern recognition receptors leading to the induction of biological defense responses. The present inventors evaluated the elicitor activity of xylo-oligosaccharides with different degrees of polymerization by using an Arabidopsis transformant containing a luciferase marker gene under the control of the promoter of the transcriptional regulator AtWRKY33 gene as a fluorescent biosensor. As a result, they discovered that xylotetraose is a compound that exhibits elicitor activity. The WRKY gene is a regulator of systemic acquired resistance in plants. WRKY genes are a family of transcription factors unique to plants, and are thought to have regulatory functions in response to biological stresses such as pests, pathogens, and other stresses. It is known that most of Arabidopsis' disease defense systems are shared with other plants. Therefore, elicitor activity evaluation using a transformant containing the Arabidopsis WRKY33 gene promoter can be applied to disease resistance in a wide variety of plants. Therefore, based on the above findings of the present inventors, xylotetraose can be used as a biostimulant to induce disease resistance in various plants.

[Biostimulant composition]
In one embodiment, a biostimulant composition is used for inducing disease resistance in plants and includes xylotetraose. The biostimulant composition contains xylotetraose in an amount of 30% by mass or more based on the total mass of xylose and xylooligosaccharide having a degree of polymerization of 2 to 10. The biostimulant composition preferably contains xylotetraose in an amount of 40% by mass or more, more preferably 50% by mass, based on the total mass of xylose and xylooligosaccharide having a degree of polymerization of 2 to 10. In one embodiment, the biostimulant composition contains 30% to 100% by weight, preferably 40% to 100% by weight of xylotetraose, based on the total weight of xylose and xylooligosaccharides with a degree of polymerization of 2 to 10. , more preferably 50% to 100% by mass.

Xylotetraose is a xylooligosaccharide with a degree of polymerization of 4, that is, an oligosaccharide in which four xyloses are polymerized through β-glycosidic bonds. Xylotetraose preferably has a β-1,4-glycosidic bond, and more preferably has the following chemical structure.

Xylotetraose can be obtained by isolating xylotetraose from a mixture of xylooligosaccharides. The isolation means is not particularly limited, but HPLC fractionation is preferred.

Xylooligosaccharides can be obtained by oligomerizing xylan by enzymatic or chemical methods. There are no particular limitations on the xylan raw material. For example, when xylooligosaccharides are obtained by a saccharification enzymatic reaction of xylan using Acremonium cellulolyticus as an enzyme-producing fungus, corncob powder can be used. Other plant biomass can also be used as the xylan raw material.

The biostimulant composition may contain, as xylooligosaccharides other than xylotetraose (degree of polymerization: 4), xylobiose (degree of polymerization: 2), xylotriose (degree of polymerization: 3), xylopentaose (degree of polymerization: 5), xylohexaose (degree of polymerization: 6), xyloheptaose (degree of polymerization: 7), xylooctaose (degree of polymerization: 8), xylononaose (degree of polymerization: 9), xylodecaose (degree of polymerization: 10), or combinations thereof.

The biostimulant composition may further include additives, such as other plant growth promoters, pesticides, spreading agents, preservatives, suspending agents, thickeners, excipients, solvents, and combinations thereof.

A spreading agent is a viscous liquid whose main component is a surfactant. Examples of the spreading agent include polyoxyethylene nonylphenyl ether, sorbitan fatty acid ester, and polyoxyethylene hexitane fatty acid ester.

Examples of preservatives include potassium sorbate, paraoxybenzoic acid ester, sodium dehydroacetate, hinokitiol, phenoxyethanol, polyaminopropyl biguanide, and polylysine.

Solvents can be used to facilitate application of the biostimulant composition to plants or to render the biostimulant composition into a liquid composition containing the elicitor active ingredient at a suitable concentration. The solvent is preferably water.

The form of the biostimulant composition is not particularly limited, and examples of the form include liquid, powder, and granule. The biostimulant composition is preferably in liquid form, since it can be easily sprayed as is or diluted to prepare a spray solution.

A biostimulant composition can be produced by mixing xylotetraose and optionally additives. The xylotetraose used to produce the biostimulant composition may be purified or in the form of a mixture with other xylooligosaccharides.

The biostimulant composition may be supplied as a highly concentrated stock solution that is diluted at the time of use, or it may be supplied as a composition pre-adjusted to the concentration for use. At the time of use, the stock solution is diluted, for example, 1000 times with water and applied to plants. The content of xylotetraose in the stock solution is preferably 0.05% to 10% by weight, more preferably 0.1% to 8% by weight, even more preferably 0.5% to 6% by weight.

[How to use biostimulant composition]
The biostimulant composition can be applied to induce disease resistance in various plants. The target plants to which the biostimulant composition is applied are not particularly limited, but are typically agricultural crops, such as Brassicaceae, Solanaceae, Asteraceae, Cucurbitaceae, Chenopodiaceae, Apiaceae, Fabaceae, and Convolvulaceae. Examples include plants of the family Liliaceae, Rosaceae, Malvaceae, Zingiberaceae, Lotus family, and Poaceae.

Specific examples include Brassica plants such as Chinese cabbage, cabbage, broccoli, cauliflower, komatsuna, mizuna, radish, and turnip; Solanaceae plants such as potato, tomato, eggplant, bell pepper, chili pepper, shishito pepper, and tobacco; Asteraceae plants such as chrysanthemum, lettuce, leaf lettuce, burdock, and butterbur; Cucurbit plants such as watermelon, melon, pumpkin, cucumber, bitter melon, loofah, and gourd; Chenopodiaceae plants such as spinach, Japanese mustard grass, Swiss chard, hijiki seaweed, and beet; carrots and celery , parsley, mitsuba, and other Apiaceae plants, legumes such as soybeans (edamame), red beans, kidney beans, broad beans, peas, winged beans, and peanuts, convolvulaceae plants such as sweet potatoes and water spinach, lilies such as chives, leeks, onions, garlic, and asparagus, roses such as strawberries, apples, pears, and loquats, mallows such as okra and cotton, ginger, lotus, and other lotus plants, and corn, rice, barley, wheat, and sugarcane, and other grasses. Among these, cruciferous plants such as cabbage and komatsuna, solanaceous plants such as tomato and eggplant, Asteraceae plants such as lettuce and leaf lettuce, and roses such as strawberries and apples are preferred, and komatsuna and tomato are more preferred.

Generally, a biostimulant composition is prepared by adding water or the like to its stock solution and diluting it to a desired concentration (for example, 1000-fold dilution) so that the content of xylotetraose in the biostimulant composition is adjusted to a suitable level. It is applied to plants at a concentration of 0.1 ppm to 500 ppm by weight. The concentration of xylotetraose in the biostimulant composition is more preferably 0.5 ppm to 200 ppm by weight, and even more preferably 1 ppm to 100 ppm by weight.

The method of applying the biostimulant composition is not particularly limited, and examples thereof include foliar spraying, soil spraying, culture medium spraying, irrigation, and seed application. For soil application, medium application and irrigation, biostimulant compositions may be incorporated into fertilizers. Examples of fertilizers include chemical fertilizers containing nitrogen, phosphoric acid, and potassium; and organic fertilizers such as oil cake, fish cake, bone meal, seaweed powder, amino acids, sugars, and vitamins.

By spraying the biostimulant composition on the leaves, the elicitor activity can be effectively expressed while increasing application efficiency. The method of foliar spraying is not particularly limited, and can be performed using, for example, a power sprayer, a shoulder sprayer, a broadcaster, a sprayer, a manned or unmanned helicopter, a drone, a smoker, a hand sprayer, or the like.

In one embodiment, a method of growing plants is provided that includes foliar spraying a biostimulant composition.

Foliar spraying is preferably performed so that the amount of xylotetraose sprayed per ^{1 cm2} of leaf surface is 0.1 ng to 100 ng, and more preferably so that the amount of xylotetraose sprayed per ^{1 cm2} of leaf surface is 1 ng to 20 ng. In an actual field, it is difficult to selectively spray only the leaf surface and to have all of the sprayed material adhere to the leaf surface, so it is preferable to adjust the concentration of xylotetraose in the biostimulant composition to 1 ppm by mass to 500 ppm by mass, preferably 10 ppm by mass to 100 ppm by mass, by dilution, etc., and spray 0.01 g to 20 g, preferably 0.1 g to 10 g, of xylotetraose per ¹⁰⁰ m2 of cultivated area evenly over the plant body. For example, a typical foliar application involves diluting the stock solution of the biostimulant composition 1000 times and applying 100 L (or in some cases 30 L to 200 L) of the resulting diluted solution per 10 ares (1000 ^m2 ) of cultivated area. In the case of drone application, the stock solution of the biostimulant composition may be diluted 10 times and 10 L of the resulting diluted solution may be applied per 10 ares (1000 ^m2 ) of cultivated area.

In one embodiment, the biostimulant composition is provided in the form of a foliar spray containing the composition.

The timing and frequency of application of the biostimulant composition are not particularly limited. For example, the biostimulant composition can be applied before the onset of plant disease, or it can be applied to a plant that has already developed a disease.

The present invention will be described more specifically with reference to the following examples, but the present invention is not limited thereto.

[Example 1]
1. Production of xylooligosaccharides (obtained from corn cob powder)
Acremonium cellulolyticus TN strain (FERM P-18508) was cultured in a liquid medium (Avicel 50 g/L, KH ₂ PO ₄ 24 g/L, ammonium sulfate 5 g/L, potassium tartrate 1/2 H ₂ O 4.7 g/L, urea 4 g/L, Tween (registered trademark) 80 1 g/L, MgSO _{4.7H 2} O 1.2 g/L, ZnSO 4.7H ₂ O 10 mg/L, MnSO _{4.5H 2} O 10 mg/L, CuSO _{4.5H 2} O 10 mg/L, and 10 mg/L of _{10 ...} A 500-mL flask was charged with 100 mL of 100 mL of 10 mg/L xylooligosaccharides (100 g/mL) and cultured at 30°C for 6 days with shaking. 50 mL of the centrifuged supernatant of the culture was then suspended in 50 mL of corncob powder, and the resulting reaction solution was stirred at 50°C for 72 hours. The centrifuged supernatant was then freeze-dried to obtain xylooligosaccharide powder.

The obtained xylooligosaccharide powder was dissolved in pure water to a concentration of 50 mass ppm, and analyzed by high performance liquid chromatography (HPLC). The HPLC analysis results are shown in FIG. FIG. 1 also shows the peaks of xylobiose and xylotriose as standard samples.

The detailed analysis conditions were as follows.
HPLC device: Shimadzu Corporation Column: Shodex (registered trademark) Asahipak (registered trademark) GF-210 HQ (inner diameter 7.5 mm x 300 mm, manufactured by Showa Denko Co., Ltd.) x 3, connected in series Eluent: H ₂ O
Flow rate: 0.5mL/min
Detector: Shodex (registered trademark) RI (manufactured by Showa Denko K.K.)
Column temperature: 50℃

2. Production of xylotetraose-containing aqueous solution 0.2 g of the obtained xylooligosaccharide powder was dissolved in 10 mL of pure water to obtain a xylooligosaccharide solution. The xylooligosaccharide solution was set in an HPLC fractionator and injected in 50 μL units. Of the eluate from the column, a fraction from 50 minutes to 52 minutes was collected. The fractions obtained after 120 injections were combined and concentrated, and the volume was adjusted to 5 mL with pure water. The concentration of the obtained solution was determined by HPLC analysis under the same analysis conditions as the xylooligosaccharide powder, and the concentration was adjusted to 50 mass ppm with pure water to obtain a xylotetraose-containing aqueous solution. The HPLC analysis results are shown in FIG. 2.

The detailed preparative conditions were as follows.
HPLC device: Manufactured by Shimadzu Corporation Preparation device: Fraction collector FRC-10A (manufactured by Shimadzu Corporation)
Column: Shodex (registered trademark) Asahipak (registered trademark) GF-210 HQ (inner diameter 7.5 mm x 300 mm, manufactured by Showa Denko K.K.) x 3, connected in series Eluent: H ₂ O
Flow rate: 0.5mL/min
Detector: Differential refractive index detector RID-10A (manufactured by Shimadzu Corporation)
Column temperature: 50℃

[Comparative Example 1 to Comparative Example 3]
In the same manner as in Example 1, a xylobiose-containing aqueous solution (Comparative Example 1) was prepared from the 54-minute to 56-minute fraction, a xylotriose-containing aqueous solution (Comparative Example 2) from the 52-minute to 54-minute fraction, and 48 minutes to 50 minutes. A xylopentaose-containing aqueous solution (Comparative Example 3) was obtained from each fraction.

The HPLC analysis results of the xylooligosaccharide-containing aqueous solutions of Comparative Examples 1 to 3 are shown in Figures 3 to 5, respectively.

The solid composition of the xylooligosaccharide-containing aqueous solutions of Example 1 and Comparative Examples 1 to 3 is shown in Table 1.

[Elicitor activity measurement]
The elicitor activity of xylooligosaccharides was measured by the following procedure.

The AtWRKY33 gene (AT2G38470) is a gene encoding a transcriptional regulatory factor that plays a central role in the disease resistance response of Arabidopsis. Seeds of an Arabidopsis transformant containing a luciferase marker gene under the control of the AtWRKY33 gene promoter (H. Kato et al., Recognition of pathogen-derived sphingolipids in Arabidopsis, Science, 376(6595), 857-860 (2022)) were surface sterilized for 1 minute with gentle shaking in a sterilizing solution (3% hydrogen peroxide, 50% ethanol), and then washed with sterile water. Thereafter, the sterilized seeds were added to 150 μL of Murashige-Skoog (MS) liquid medium supplemented with luciferin (1/2 mixed salts for Murashige-Skoog medium, 0.05% [w/v] MES, 0.5% [w/v ] sucrose, adjusted to pH 5.8 with NaOH, 50 μM D-luciferin) and sealed with a transparent plastic cover. This plate was left standing in a growth chamber for about 12 days at 23°C with the light on for 24 hours to grow Arabidopsis. After spraying xylo-oligosaccharides (concentration 25 mass ppm) on grown seedlings, the time course of the chemiluminescence intensity derived from the expressed luciferase was measured eight times using a multiplate reader Mithras LB940 (manufactured by Berthold), and the average I found the value.

FIG. 6 shows the measurement results of the elicitor activity of the xylooligosaccharide aqueous solutions of Example 1 and Comparative Examples 1 to 3. FIG. 7 and Table 2 show the elicitor activities of the xylooligosaccharide aqueous solutions of Example 1 and Comparative Examples 1 to 3 as total counts (average). FIG. 6 also shows the results of a control experiment conducted in the same manner as in Example 1 using pure water instead of the xylooligosaccharide aqueous solution. FIG. 7 and Table 2 also show the results of the control.

Figures 6, 7 and Table 2 show that xylotetraose has specifically high elicitor activity.

The biostimulant composition of the present invention can be advantageously used to induce disease resistance in plants.

Claims (4)

A biostimulant composition containing xylotetraose used to induce disease resistance in plants, the content of said xylotetraose being 30% by mass or more based on the total mass of xylose and xylooligosaccharides having a degree of polymerization of 2 to 10. A foliar spray containing the biostimulant composition according to claim 1. A method for cultivating plants comprising foliar spraying of the biostimulant composition according to claim 1. The use of xylotetraose as a biostimulant to induce disease resistance in plants.

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