WO2012120604A1 - Growth regulation agent for plants, growth regulation method for plants and use of same - Google Patents

Abstract

A growth regulation agent for plants which contains, as an active ingredient, at least one kind of compound selected from the group consisting of 3-methyl-2-pentanone, 2, 5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone.

Description

Plant growth regulator, plant growth control method and use thereof

The present invention relates to a plant growth regulator, a plant growth control method, and use thereof.

Plant hormones such as auxin, cytokinin, and ethylene are known as chemical substances that control plant growth. Details of the biosynthetic pathway of these plant hormones have already been clarified, and at the same time, it has long been thought that plant hormones are exclusively biosynthesized by plants themselves. However, fungi that have been found in recent years and exhibiting the activity of directly or indirectly promoting the growth of plants, and bacteria collectively called plant growth promoting rhizobacteria (PGPR) (eg, non-patent literature) Some of them are known to control plant growth by producing plant hormones such as ethylene (see Non-Patent Document 2, for example).

On the other hand, chemical substances derived from soil microorganisms that are not derived from plants themselves and are not plant hormones themselves but regulate plant growth are also known. For example, Patent Document 1 discloses a plant growth regulator containing 2-azahypoxanthine.

However, most plant growth control activities of PGPR are active, such as plant growth control activity factor derived from Bacillus subtilis (for example, GB03 strain, see Non-patent Document 3), which is a typical PGPR, only includes candidate factors. No compound necessary and sufficient for the reconstitution of the product has yet been found.

Furthermore, as a method of using PGPR, mixing with soil is common, but this method has a problem that it is difficult to artificially control the number of PGPR grown, and the effect of PGPR cannot be stably maintained. In addition, research on plant growth control by artificial manipulation of plant hormone-related genes using so-called genetic recombination technology has been conducted, but safety to humans and other organisms has not been established, and there is concern about the potential impact on the environment. Opinions that are deeply rooted in general consumers. For this reason, a plant growth control agent and a growth control method that are more safe for living organisms such as humans and that can stably exhibit excellent growth control action are desired.

JP 2009-001558 A

In view of the present situation, the present invention is highly effective for ingredients derived from natural products that are highly safe for humans and the environment, can stably exert plant growth control action, and have uniform quality and high stability. It aims at providing the plant growth control agent used as a component, the agricultural material containing this control agent, a plant growth control method, etc.

The inventors of the present invention have made extensive studies in view of the above-mentioned problems, and produce Gram-positive bacteria Bacillus subtilis (Bacillus subtilis) when cultured in a medium containing sugar and a carbon source other than sugar. It has been found that volatile metabolites exhibit both inhibitory and promoting activities on plant growth. The growth control action of the volatile metabolite of Bacillus subtilis depends on its concentration. When the concentration of the volatile metabolite is high, the growth of the plant is suppressed. On the other hand, plant growth was promoted at low concentrations of volatile metabolites. Such a plant growth control action by the volatile metabolite of Bacillus subtilis was not observed when cultured in a medium containing only sugar using Bacillus subtilis as a carbon source. Although Bacillus subtilis is sometimes used to promote plant growth, it has been a completely new finding that Bacillus subtilis inhibits plant growth depending on conditions.
In the present specification, “medium containing sugar and a carbon source other than sugar” is also simply referred to as “organic medium”. The “medium containing only sugar as a carbon source” is also simply referred to as “inorganic medium”.

The inventors of the present invention also have a gram-negative bacterium, Agrobacterium tumefacines, and Escherichia coli cultured in an organic medium in addition to Bacillus subtilis. Similarly, it has been found that it has a plant growth-controlling effect, and has come up with the idea that a volatile metabolite generated by culturing microorganisms such as Bacillus subtilis in an organic medium has a plant growth-controlling effect.

The present inventors further isolated components contained in volatile metabolites generated by culturing Bacillus subtilis or the like in an organic medium, and examined the growth control activity of plants for each component. It was found that 3-methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone (acetoin), and 2-pentadecanone contained therein have plant growth control activity. So far, there has been no report on volatile components other than plant hormones such as ethylene having a plant growth control action. When the active ingredient having the plant growth control action is volatile, compared to the case where it is not volatile, for example, even if the agricultural material containing the active ingredient is not in contact with the plant, the active ingredient is uniformly distributed throughout the plant body. It is considered to be an advantage that it can act on plants. For this reason, plant growth control can be performed simply and efficiently. Such a substance having a growth control action is useful in agriculture and forestry, and is also a component contained in the metabolite of microorganisms originally growing in the soil, so it is highly safe for humans and the environment. is there. In addition, since the active ingredient is a component contained in the metabolite of the microorganism, the production of the active ingredient can be realized by culturing the microorganism, or the growth of the plant can be controlled by applying the microorganism itself. .
The present inventors have further studied based on the above findings and have completed the present invention.

That is, the present invention relates to the following (1) to (15).
(1) containing at least one compound selected from the group consisting of 3-methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone as an active ingredient. Characteristic plant growth regulator.
(2) 3-methyl- produced by culturing at least one microorganism selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, fungi and molds in a medium containing sugar and a carbon source other than sugar. (1) containing a volatile metabolite containing at least one compound selected from the group consisting of 2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone. The plant growth regulator as described.
(3) Volatile metabolites generated by culturing at least one microorganism selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, fungi and molds in a medium containing sugar and a carbon source other than sugar. A plant growth regulator characterized by comprising.
(4) The plant growth regulator according to (2) or (3), wherein the microorganism is a gram positive bacterium, a gram negative bacterium, or a filamentous fungus.
(5) The plant growth regulator according to any one of (2) to (4), wherein the microorganism is Bacillus subtilis, Agrobacterium, or mycorrhizal fungus.
(6) The plant growth control agent according to any one of (1) to (5), wherein the plant growth control is plant growth suppression or growth promotion.
(7) The plant growth regulator according to (1) or (2), wherein the plant growth control is plant growth inhibition.
(8) The plant body, plant cultivation area or plant seed is selected from the group consisting of 3-methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone. A method for controlling plant growth, comprising applying at least one compound.
(9) 3-methyl- produced by culturing at least one microorganism selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, fungi and fungi in a medium containing sugar and a carbon source other than sugar. The volatile metabolite comprising at least one compound selected from the group consisting of 2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone is applied as described in (8) above Plant growth control method.
(10) At least one microorganism selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, fungi, and fungi is included in the plant body, plant cultivation area, or plant seed, including sugar and a carbon source other than sugar. A plant growth control method comprising applying a volatile metabolite generated by culturing in a medium.
(11) The plant growth control method according to (9) or (10), wherein the microorganism is a gram positive bacterium, a gram negative bacterium, or a filamentous fungus.
(12) The plant growth control method according to any one of (9) to (11), wherein the microorganism is Bacillus subtilis, Agrobacterium, or mycorrhizal fungus.
(13) The plant growth control method according to any one of (8) to (12), wherein the plant growth control is plant growth suppression or growth promotion.
(14) The method for controlling plant growth according to (8) or (9), wherein the plant growth control is plant growth suppression.
(15) controlling plant growth of at least one compound selected from the group consisting of 3-methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone Use for.

According to the present invention, using a substance that is safe to humans and the environment that does not cause soil degradation, environmental pollution, and the like, it is possible to stably promote plant growth in order to improve plant yield, work efficiency, etc. Can be suppressed.

FIG. 1 is a graph showing plant growth inhibitory activity by volatile metabolites of Bacillus subtilis. FIG. 2 is a graph showing changes over time in Arabidopsis root length caused by volatile metabolites of Bacillus subtilis. FIG. 3 is a diagram showing the influence of volatile metabolites of Bacillus subtilis on plant wet weight. FIG. 4 is a diagram showing the analysis results of volatile metabolites of Bacillus subtilis by GC-MS. FIG. 5 is a diagram showing the analysis results of volatile metabolites of Bacillus subtilis by GC-MS. FIG. 6 is a graph showing the growth inhibitory activity of Arabidopsis thaliana by 3-methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone. FIG. 7 is a diagram showing the influence of 3-methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone on the elongation of the main root of Arabidopsis thaliana. FIG. 8 is a graph showing the rice growth-inhibiting activity by volatile metabolites of Bacillus subtilis. FIG. 9 is a diagram showing the length of the main root of Arabidopsis thaliana when each of Bacillus subtilis (ATCC9372), Escherichia coli (DH5a) and Agrobacterium (EHA101) is cultured in an organic medium or an inorganic medium in an Arabidopsis culture system. . FIG. 10 is a diagram showing the growth inhibitory activity of sweet basil by a volatile metabolite of Bacillus subtilis. FIG. 11 is a graph showing the growth inhibitory activity of periwinkle by a volatile metabolite of Bacillus subtilis. FIG. 12 is a diagram showing rice growth-inhibiting activity by volatile metabolites of Bacillus subtilis and Agrobacterium.

The plant growth in the present invention is not particularly limited as long as it is a phenomenon accompanied by normal differentiation or proliferation of plant cells, and includes not only elongation and expansion of organs constituting the plant body but also germination from seeds. .

The plant growth regulator of the first aspect of the present invention (hereinafter also referred to as the plant growth regulator of the present invention 1) is 3-methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2. -Containing at least one compound selected from the group consisting of butanone and 2-pentadecanone as an active ingredient. Among these, it is preferable to contain one or two of 3-methyl-2-pentanone and 2,5-dimethylpyrazine as essential. More preferably, it contains at least 3-methyl-2-pentanone. Also, a plant growth regulator containing 3-methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone is one of the preferred embodiments of the present invention 1.
In the present specification, suppression of growth includes suppression of germination. Growth promotion includes germination promotion, biomass promotion, and harvest improvement.

The 3-methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone, which are the active ingredients of the growth regulator of the present invention 1, are all commercially available. Can be used. Further, for example, when a microorganism such as Gram-positive bacteria, Gram-negative bacteria, fungi or mold is cultured in a medium containing sugar and a carbon source other than sugar under conditions according to the type of the microorganism, usually 3- A volatile metabolite of a microorganism containing at least one compound selected from the group consisting of methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone is added to the culture system. Generate.
In the present invention, such volatile metabolites of microorganisms can be collected by a known technique and used as an active ingredient in the present invention 1. Moreover, the said compound can be obtained by isolate | separating and refine | purifying the collected volatile metabolite by a well-known method.

In the present invention, the active ingredient is generated by culturing at least one microorganism selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, fungi and molds in a medium containing sugar and a carbon source other than sugar. A volatile metabolite containing at least one compound selected from the group consisting of 3-methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone is preferable. Can be used. The fact that the volatile metabolite contains the compound can be confirmed by collecting the volatile metabolite by a known method and analyzing the collected component by GC-MS or the like.

The microorganisms that produce the volatile metabolites are preferably soil microorganisms, among which bacteria such as Gram-positive bacteria, Gram-negative bacteria, and filamentous fungi are preferred. As Gram-positive bacteria, Bacillus subtilis (for example, ATCC9372 strain, A001 strain, GB03 strain, etc.), Firmicutes typified by Bifidobacterium (Lactobacillus bifidus), etc .; typified by Corynebacterium Examples include Actinobacteria. Examples of gram-negative bacteria include Agrobacterium (for example, EHA101 strain), E. coli (for example, DH5α strain), and the like. Examples of filamentous fungi include mycorrhizal fungi and the like. These microorganisms are available from ATCC and the like. Among them, the microorganism that generates a volatile metabolite is preferably Bacillus subtilis, Agrobacterium, or mycorrhizal fungus. Among these, Bacillus subtilis, which has been proven to be purely cultured and has been traditionally used in foods such as natto, is more preferable because it has a lower influence on the human body. As long as the effect of the present invention is exhibited, the microorganism may be a wild type, a naturally occurring mutant, or an artificial mutant by genetic manipulation. The microorganism used for obtaining the volatile metabolite may be one kind or two or more kinds.

A medium containing a sugar and a carbon source other than sugar used for culturing a microorganism may be appropriately selected according to the type of microorganism. The medium may be a solid medium or a liquid medium. The sugar may be any sugar that is normally used for culturing microorganisms. Examples thereof include glucose, fructose, sucrose, mannose, maltose, mannitol, xylose, galactose, and the like. it can. The concentration of sugar in the medium may be appropriately set according to the type of microorganism, but is usually about 0.1 to 30% by mass, preferably about 0.5 to 20% by mass, It is more preferably about 1 to 20% by mass, further preferably about 1 to 10% by mass, and particularly preferably about 2 to 8% by mass.

Examples of carbon sources other than sugar include amino acid, protein hydrolyzate, peptone, polypeptone, yeast extract, dry yeast, corn steep liquor, defatted soybean hydrochloric acid hydrolyzate and the like, and one or more are used. can do. The concentration of the carbon source other than sugar in the medium may be appropriately set according to the type of microorganism, for example, usually about 0.1 to 30% by mass and about 0.5 to 20% by mass. It is preferably about 1 to 20% by mass, more preferably about 1 to 10% by mass, and particularly preferably about 2 to 8% by mass.

The culture conditions using a medium containing sugar and a carbon source other than sugar may be those normally used according to the type of microorganism. For example, the culture temperature is usually about 5 to 50 ° C., preferably about 27 to 37 ° C. for Bacillus subtilis and the like. In the case of Escherichia coli, Agrobacterium, etc., it is usually about 16 to 37 ° C. The culture temperature of E. coli is preferably about 20 to 37 ° C, more preferably about 25 to 37 ° C, still more preferably about 30 to 37 ° C, and most preferably about 37 ° C. The culture temperature of Agrobacterium is preferably about 20 to 35 ° C, more preferably about 25 to 35 ° C, still more preferably about 28 to 32 ° C, and most preferably about 30 ° C. What is necessary is just to let pH of a culture medium be pH normally used according to microorganisms. For example, when a liquid medium is used for culture, it is preferable that the medium is appropriately stirred or shaken during the culture. The culture time is not particularly limited, but usually, the amount of volatile metabolites generated increases after the logarithmic growth phase after the start of culture. Moreover, the light conditions at the time of microorganism culture are not particularly limited, and may be dark conditions or normal growth conditions for plants. For example, the light intensity is preferably about 150 to 100,000 lux, more preferably about 300 to 30000 lux, further preferably about 400 to 10,000 lux, and particularly preferably about 500 to 10,000 lux. The light condition may be a dark condition or a bright condition for 24 hours, or may be a day length condition in which a plant usually grows. For example, it may be about 4 hours light condition 20 hours dark condition, or about 8 hours light condition 16 hours dark condition. Furthermore, about 12 hours light condition 12 hours dark condition, about 16 hours light condition 8 hours dark condition, about 20 hours light condition 4 hours dark condition, etc. may be sufficient.

The growth regulator of the present invention 1 comprises 3-methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2, which is generated by culturing the microorganism in a medium containing sugar and a carbon source other than sugar. It preferably contains a volatile metabolite containing at least one compound selected from the group consisting of butanone and 2-pentadecanone. Such a volatile metabolite is more preferably a volatile metabolite containing at least one of 3-methyl-2-pentanone and 2,5-dimethylpyrazine, more preferably at least 3-methyl- It is a volatile metabolite containing 2-pentanone. Preferably, a volatile metabolite containing 3-methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone is used. Such a volatile metabolite is generated, for example, by culturing the aforementioned Bacillus subtilis, Agrobacterium, Escherichia coli, filamentous fungus or the like in a medium containing sugar and a carbon source other than sugar.

The collection of the volatile metabolites can be performed by a known method, for example, a known volatile metabolite collector, a cold trap method using liquid nitrogen, or the like. The collected volatile metabolite can be used as an active ingredient of the growth regulator of the first invention. In addition, as long as the effects of the present invention are exhibited, a processed product such as a diluted product or a diluted product obtained by diluting or concentrating the collected volatile metabolite can be used as an active ingredient. Further, the volatile metabolite or a processed product thereof may be subjected to a treatment such as concentration to dryness, spray drying, freeze drying, etc. and used as a dried product. Processing such as concentration can be performed by a known method.
The collected volatile metabolites are selected from the group consisting of 3-methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone by a known purification method. At least one compound can be isolated and purified. The isolated and purified compound can be used as the active ingredient of the present invention 1.

The growth control agent of the present invention 1 is applied to a plant so that a volatile active ingredient acts on the plant to suppress or promote the growth of the plant. For this reason, it is used suitably for plant growth suppression or growth promotion. Preferably, it is used for plant growth inhibition. Plant growth inhibitor containing as an active ingredient at least one compound selected from the group consisting of 3-methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone Is one of the preferred embodiments of Invention 1.

As the growth control agent of the first aspect of the present invention, at least one microorganism selected from the group consisting of the gram-positive bacteria, gram-negative bacteria, fungi, and molds, or a processed product of the microorganisms can also be used. In this case, the microorganism is preferably a living bacterium. The use of viable microorganisms as the growth control agent of the first aspect of the present invention is preferable because the effect lasts for a long time by one application. The microorganism produces the volatile metabolite containing the active ingredient in the presence of the sugar and the carbon source other than sugar as described above. Therefore, when applied to a plant as described later, the effect of the present invention is exhibited. . When the plant growth regulator of the present invention 1 contains the microorganism, it is preferable to culture the microorganism in advance in the presence of sugar and a carbon source other than sugar.

In the first aspect of the present invention, when the microorganism or a processed product thereof is used, for example, a microbial solution obtained by culturing the microorganism in the presence of the aforementioned sugar and a carbon source other than sugar, a culture solution, etc. For example, in the case of using a cell fluid (culture solution) that can be suitably used as a control agent, it is preferably about 10 ³ to 10 ¹⁰ (viable cell count) / mL, more preferably 10 ⁵ to 10 ^9. Plant growth control by sterilizing (viable cell count) / mL of the cell fluid (culture solution), more preferably about 10 ⁶ to 10 ⁸ (viable cell count) / mL, if desired It can be used as an agent. Such a plant growth regulator (bacterial fluid) can be used after appropriately diluted.

Examples of processed microorganisms include dry cells obtained by drying cells cultured in the presence of sugar and a carbon source other than sugar. In addition, an extract of a microbial cell cultured in the presence of sugar and a carbon source other than saccharide, a disrupted solution by ultrasonic waves, a solid liquid such as filtration or centrifugation of the culture supernatant, culture solution or culture supernatant of the microbial cell A solid residue or the like separated by the separation means is also exemplified as the processed product. In addition, a treated liquid obtained by removing a cell wall of a microorganism cultured in the presence of sugar and a carbon source other than sugar by an enzyme or mechanical means is also exemplified. Furthermore, these concentrates, these dilutions, or these dried products are also included in the treated product. In addition, the treated product in the present invention may be obtained by further adding, for example, various chromatographic separations to the bacterial cell extract, ultrasonic disruption solution, the cell culture solution or culture supernatant. include.

The application amount of the plant growth control agent of the present invention 1 is, for example, an application form (for example, application, spraying, foliar application, soil application, etc.) to be described later, and the purpose of application (for example, growth inhibiting action, growth promoting action, etc.) What is necessary is just to set suitably by.

For example, when the plant growth regulator of the present invention 1 is used for suppressing plant growth, the application amount of the active ingredient to the plant may be appropriately set depending on the kind and part of the plant to be described later. Further, for example, the plant can be killed by continuously applying the plant growth regulator of the first aspect of the present invention. For example, when used in the form of an aqueous solution such as an aqueous solvent, the concentration of the active ingredient is, for example, preferably about 1 to 10,000 μM, more preferably about 2 to 1000 μM. In addition, when the plant growth regulator of the present invention is used in soil for plant growth inhibition, the active ingredient is also preferably about 1 to 10000 μmol / kg, more preferably about 2 to 1000 μm. It is mixed with soil so as to be mol / kg. In addition to the case where the solid is mixed with the soil, it can be applied in such an amount when it is sprayed on the foliage or the aqueous solution is irrigated on the soil.

When the volatile metabolite of the microorganism described above is contained as an active ingredient of the plant growth regulator of the present invention 1, the concentration of the compound contained in the volatile metabolite is set within the above range. preferable.
When the application rate is within the above range, the growth of plant roots, bulbs, cultured roots and the like can be effectively suppressed by applying the plant growth regulator of the first aspect of the present invention.

When at least one microorganism selected from the group consisting of the gram-positive bacteria, gram-negative bacteria, fungi, and molds is used in the first aspect of the present invention, the usage amount of the plant growth regulator containing the microorganism is also described later. It can be set as appropriate according to the plant type, site, application method, application purpose, and the like. For example, when the above-mentioned microorganisms are used to suppress the growth of plants, the application rate is preferably about 10 ³ to 10 per one cell in terms of the number of living bacteria when used in a closed system mixed with room temperature fumes. About 10 to 100 times (preferably about 20 times) of about 50 to 500 mL (preferably about 300 mL) of a cell solution (culture solution) of about ¹⁰ (viable bacteria) / mL is diluted, more preferably about 10 ⁵ to It is preferable to dilute about 50 to 500 mL (preferably about 300 mL) of about 10 ⁹ (viable cell count) / mL of the bacterial cell solution (culture solution) and to spray about 10 to 100 times (preferably about 20 times). In the case of foliar spraying on the foliage, etc., about 10 ³ to 10 ¹⁰ (viable cell count) / mL of about 20 to 500 mL (preferably about 200 mL) of bacterial cell solution (culture solution) about 50 to 1000 times Dilute (preferably about 500 times), more preferably about 10 ⁵ to 10 ⁹ (viable cell count) / mL about 20 to 500 mL (preferably about 200 mL) of about 20 to 500 mL (preferably about 200 mL) It is preferable to use after diluting to the extent (preferably about 500 times). In the case of irrigation, a cell solution (culture solution) of about 10 ³ to 10 ¹⁰ (viable cell count) / mL is diluted about 10 to 500 times (preferably about 100 times), and more preferably about A bacterial cell solution (culture solution) of about 10 ⁵ to 10 ⁹ (viable cells) / mL is diluted about 10 to 500 times (preferably about 100 times), and 1 to 10 L per one can be used. In addition to the above, when solid matter such as dried microbial cells is mixed with soil, it can be appropriately diluted as well.

The plant growth regulator of the second aspect of the present invention (hereinafter also referred to as the plant growth regulator of the present invention 2) is at least one selected from the group consisting of gram-positive bacteria, gram-negative bacteria, fungi and molds. It contains volatile metabolites generated by culturing seed microorganisms in a medium containing sugar and a carbon source other than sugar.

The microorganism that produces the volatile metabolite is preferably a soil microorganism, and examples thereof include gram-positive bacteria and gram-negative bacteria. As Gram-positive bacteria, Bacillus subtilis (for example, ATCC9372 strain, A001 strain, GB03 strain), Firmicutes typified by Bifidobacterium (Lactobacillus bifidus), etc .; radiation represented by Corynebacterium Examples include Actinobacteria. Examples of gram-negative bacteria include Agrobacterium and E. coli. Among these, Bacillus subtilis, which has been proven to be purely cultured and has been traditionally used in foods such as natto, is more preferable because it has a lower influence on the human body. As long as the effect of the present invention is exhibited, the microorganism may be a wild type, a naturally occurring mutant, or an artificial mutant by genetic manipulation. The microorganism used for obtaining the volatile metabolite may be one kind or two or more kinds.

For example, a plant growth regulator containing a volatile metabolite generated by culturing Bacillus subtilis in a medium containing sugar and a carbon source other than sugar is a preferred embodiment of the plant growth regulator of the present invention 2. One.

The medium used for culturing microorganisms and a medium containing a carbon source other than sugar, the culture conditions, and the like are the same as the medium, the culture conditions, and the like in the plant growth regulator of the first invention described above.
The method for collecting the generated volatile metabolite is the same as that in the plant growth regulator of the first aspect of the present invention.

The collected volatile metabolite can be used as an active ingredient of the growth regulator of the present invention 2. In addition, as long as the effects of the present invention are exhibited, a treated product such as a diluted product or a concentrated product obtained by subjecting the collected volatile metabolite to a treatment such as dilution or concentration can be used as an active ingredient. Further, the volatile metabolite or a processed product thereof may be subjected to a treatment such as concentration to dryness, spray drying, freeze drying, etc. and used as a dried product.

As the growth control agent of the present invention 2, at least one kind of microorganism selected from the group consisting of the gram-positive bacteria, gram-negative bacteria, fungi and molds, or a processed product thereof can also be used. In this case, the microorganism is preferably a living bacterium. The use of viable microorganisms as the growth control agent of the second aspect of the present invention is preferable because the effect lasts for a long time by one application. Since the microorganism produces a volatile metabolite containing the active ingredient in the presence of sugar and a carbon source other than sugar, the effect of the present invention is exhibited when applied to a plant as described later. When the growth control agent of the present invention 2 contains the microorganism, it is preferable to culture the microorganism in advance in the presence of sugar and a carbon source other than sugar. Examples of the processed product of the microorganism include the same processed product of the microorganism in the above-described growth control agent of the first invention.

In the second aspect of the present invention, when the microorganism or a processed product thereof is used, for example, a culture solution obtained by culturing the microorganism in the presence of the aforementioned sugar and a carbon source other than sugar is suitable as a plant growth regulator. Can be used. Preferably, about 10 ³ to 10 ¹⁰ (viable cell count) / mL, more preferably about 10 ⁵ to 10 ⁹ (viable cell count) / mL, or more preferably 10 ⁶ to 10 A bacterial cell solution (culture solution) of about ⁸ (viable cell count) / mL can be sterilized if desired and used as a plant growth regulator. Such a plant growth regulator (bacterial fluid) can be used after appropriately diluted. Such plant growth regulators can be used after appropriately diluted. The use of viable microorganisms as the growth control agent of the second aspect of the present invention is preferable because the effect lasts for a long time by one application. Moreover, it is preferable to sterilize and use the microorganism because the application rate can be easily controlled.

When the plant growth regulator of the present invention 2 is applied to a plant, a volatile active ingredient acts on the plant to suppress or promote the growth of the plant. For this reason, it is used suitably for plant growth suppression or growth promotion. Preferably, it is used for promoting the growth of plants. A plant containing a volatile metabolite generated by culturing at least one microorganism selected from the group consisting of gram-positive bacteria, gram-negative bacteria, fungi and molds in a medium containing sugar and a carbon source other than sugar This growth promoter is one of the preferred embodiments of the present invention 2. When the plant growth regulator of the present invention 2 is used to promote plant growth, the application amount of the active ingredient to the plant may be appropriately set depending on the type of plant, site, application method, application purpose, etc. described later. There is no particular limitation. For example, when a plant growth control agent containing the microorganism is used to promote plant growth, the amount used is preferably one in terms of the number of viable bacteria when used in a closed system mixed with room temperature fumes. About 10 ³ to 10 ¹⁰ (viable cell count) / mL of about 50 to 500 mL (preferably about 300 mL) of a bacterial cell solution (culture solution) is diluted about 10 to 100 times (preferably about 20 times) Preferably, about 50 to 500 mL (preferably about 300 mL) of about 10 ⁵ to 10 ⁹ (viable cell count) / mL or about 50 to 500 mL (preferably about 300 mL) is diluted about 10 to 100 times (preferably about 20 times), Spraying is preferred. In the case of foliar spraying on the foliage, etc., about 10 ³ to 10 ¹⁰ (viable cell count) / mL of about 20 to 500 mL (preferably about 200 mL) of bacterial cell solution (culture solution) about 50 to 1000 times Dilute (preferably about 500 times), more preferably about 10 ⁵ to 10 ⁹ (viable cell count) / mL about 20 to 500 mL (preferably about 200 mL) of about 20 to 500 mL (preferably about 200 mL) The degree (preferably about 500 times) is preferably diluted about 500 times. In the case of irrigation, a cell solution (culture solution) of about 10 ³ to 10 ¹⁰ (viable cells) / mL is diluted about 10 to 500 times (preferably about 100 times), and more preferably about 10 times. A bacterial cell solution (culture solution) of about ⁵ to 10 ⁹ (viable cell count) / mL is diluted about 10 to 500 times (preferably about 100 times), and 1 to 10 L per one can be used. In addition to the above, when solid matter such as dried microbial cells is mixed with soil, it can be appropriately diluted as well.

The plant to which the plant growth regulator of the present inventions 1 and 2 (hereinafter referred to as the plant growth regulator of the present invention) is applied may be a gymnosperm or an angiosperm. In the case of angiosperms, either monocotyledonous plants or dicotyledonous plants may be used, and there is no particular limitation. For example, grains such as rice, barley, corn, and wheat; vegetables such as tomato, lettuce, basil, radish, Japanese mustard spinach, spinach, cabbage, turnip, pumpkin, bell pepper; green manure plants such as alfalfa, clover, and lotus; cosmos, torenia , Chrysanthemum, gerbera, pansies, orchids, peonies, tulips and other florets; beans such as azuki bean, green beans, soybeans, peanuts, broad bean, peas; lawn grasses such as pebbles, bentgrass, pears; Etc.

The plant in the present invention may be, for example, the whole plant body or a part of a plant body such as a plant tissue or organ, and also includes plant cells such as protoplasts and callus. Examples of plant tissues or organs include seeds, germinated seeds, roots, stems, leaves, petals, fruits, bulbs and the like. Plant cells include suspension cultures, embryos, division regions, callus tissues, shoots, gametes, spores, pollen, and microspores.

The plant growth regulator of the present invention can contain known pharmaceutical additives. Known additives for pharmaceutical preparations include excipients, emulsifiers, wetting agents, disintegrants and the like. Further, the plant growth regulator of the present invention can be used by mixing with known agricultural chemicals, herbicides, nutrients and the like. When using the above-mentioned living microorganism of the microorganism for the plant growth regulator of the present invention, it is preferable not to use a chemical pesticide harmful to the microorganism.

The form of the plant growth regulator of the present invention may be any of liquid, oil, emulsion, aqueous solvent, paste, wettable powder, flowable, granule, fine granule, powder, tablet, aerosol, film, net, sheet, etc. There is no particular limitation.

The plant growth regulator of the present invention is suitably used for agricultural materials and the like. Agricultural materials containing the plant growth regulator of the present invention are also included in the present invention. Agricultural materials include solid fertilizers, liquid fertilizers, seed coats, haze agents, herbicidal sheets, greenhouse exteriors, chills, shading films, and curing sheets. Agricultural materials may contain known additives in addition to the plant growth regulator of the present invention. The method for incorporating the plant growth regulator of the present invention into an agricultural material is not particularly limited. For example, a plant growth control agent is contained in a herbicidal sheet, an outer wall of a greenhouse, a cold chill, a light-shielding film, a curing sheet, etc. by kneading or applying the plant growth control agent of the present invention to the surface by a known method. Can be made.

The plant growth can be controlled by applying the plant growth regulator of the present invention to a plant body, a plant cultivation site or a plant seed. For example, the growth regulator of the present invention 1 is suitably used for plant growth inhibition. The growth regulator of the present invention 2 is suitably used for promoting plant growth. In addition to applying directly to soil, culture medium or hydroponic liquid in hydroponics, etc., it can be applied to the plant cultivation space by spraying, haze, etc. Is also included.

At least one selected from the group consisting of 3-methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone for the plant body, plant cultivation site or plant seed A plant growth control method to which the above compound is applied is also one aspect of the present invention. Such a plant growth control method is also referred to as the plant growth control method of the third aspect of the present invention. In the method of the present invention 3, for example, the above-described plant growth regulator of the present invention 1 may be applied to a plant body, a plant cultivation site or a plant seed.

Culturing at least one microorganism selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, fungi, and molds in a medium containing a carbon source other than sugar on a plant body, plant cultivation site or plant seed A plant growth control method applying a volatile metabolite generated by the above is also encompassed by the present invention. Such a plant growth control method is also referred to as the plant growth control method of the fourth invention. In the method of the present invention 4, for example, the above-described plant growth regulator of the present invention 2 may be applied to a plant body, a plant cultivation site or a plant seed.

The method for applying the plant growth regulator of the present invention to plants is not particularly limited as long as the plant growth regulator can be applied substantially. For example, you may apply so that the plant growth regulator of this invention may contact a plant directly. In addition, since the active ingredient in the present invention is volatile, the growth control agent can be applied to a plant by allowing the growth control agent to coexist in a plant cultivation system in a non-contact manner with the plant. . As application forms, for example, treatment to plant bodies such as foliage spraying, treatment to plant cultivation areas such as soil treatment, treatment to seeds such as seed soaking, application to hydroponic plants by mixing in hydroponic liquid Etc. Specifically, for example, a method of spraying powders, granules, liquids, etc. on soil; a method of spraying diluted aqueous solutions, aerosols, etc. directly on plants such as leaves, stems, fruits, etc .; plants by spraying, fumes, etc. Method of spraying in the cultivation system space; Method of pouring into the soil; Dilution mixing with hydroponics or feed water in contact with the roots such as hydroponics, rock wool, or semi-permeable membrane film bags And a method of applying or mixing to a plant culture medium; and a method of non-contact co-culture of the microorganisms in a plant cultivation system as in Examples described later. When the microorganism is used as a growth control agent, for example, the active ingredient can be applied to the plant by co-culturing the microorganism and the plant in a non-contact manner or in contact with each other. In addition, for example, when diluting and supplying to hydroponic liquid or supply water, the active ingredients of various concentrations (for example, about 1 μM to 0.5 M) are dissolved in liquid fertilizer and processed, and the seedlings are grown by hydroponic. The optimal concentration can be determined. The supply of the active ingredient to the plant can be appropriately determined with reference to the above concentration range even when the application method is changed.

In order to exhibit the effects of the present invention more efficiently, it is preferable to apply the plant growth regulator of the present invention to plants in a closed or semi-closed system. More preferably, it is applied to the plants in a closed system (eg, greenhouse, greenhouse, plant factory, space station, etc.). In the present invention, the application time of the plant growth regulator may be appropriately selected according to the purpose, and may be any of, for example, before sowing, sowing, seedling, growth period, flowering period, maturation period, etc., and is particularly limited. It is not something.

The method for inhibiting plant growth using at least one microorganism selected from the group consisting of the Gram-positive bacteria, Gram-negative bacteria, fungi and molds as a plant growth regulator of the first aspect of the present invention. An example will be described.
(A) Method of irrigating the soil Preferably, a bacterial cell solution (culture solution) of about 10 ³ to 10 ¹⁰ (viable bacteria) / mL is diluted to about 10 to 1000 times (preferably about 100 times), and more Preferably, about 10 ⁵ to 10 ⁹ (viable cell count) / mL of a cell solution (culture solution) can be used even after being diluted about 10 to 1000 times (preferably about 100 times). The microorganism is cultured in a medium containing sugar and a carbon source other than sugar. The culture solution can be used as it is without being sterilized or after being sterilized by a sterilizing filter or the like. Or the extract of the said microorganisms or the collected volatile substance can be used. The timing of irrigation may be appropriately determined depending on the plant. For example, in leaf vegetables and the like, the soil volume is 700 to 800 g in a 15 cm pot at about 20 ° C. ± 5 ° C. through growth, and about 50 mL of a 500 to 1000 times solution may be injected into one pot.
(B) Leaf surface spraying method Apply about 50 mL of 50-1000 times solution to the main leaf once using the solution of (a).
Although depending on the plant, either of the above methods (a) and (b) may be usually performed once, but may be appropriately processed a plurality of times.

(C) Method of spraying the granules In the process of manufacturing existing granules containing minerals such as nitrogen, phosphoric acid, potassium, and three major plant nutrients, iron (a) is mixed into the soil. Mix or spread on the ground.
(D) Method of mixing with hydroponic liquid The liquid of the above (a) is mixed with the hydroponic liquid, diluted as appropriate, and applied to a plant that is hydroponically cultivated using a support or the like.
(E) Method of spraying in the air The liquid of the above (a) is fogged in the space by mixing with existing agricultural chemicals or the like by moving spray or an ultrasonic smoker. It is suitably used for plant cultivation in a closed plant factory, an artificial weather device, or a greenhouse.

In the case of using a living microorganism of the microorganism in the methods (a), (b), (c), (d), (e) and the like, a bactericidal agent harmful to the living microorganism, such as an organic phosphorus type or a heavy metal type It is preferable not to use such disinfectants at the same time. However, when the microbial extract or the collected volatile substance is used, since the expected physiological activity does not depend on the living bacteria, it can be used at the same time as the fungicide, for example.

When the plant growth regulator and the plant growth control method of the present invention are used, plant growth can be promoted or suppressed without causing soil degradation, environmental pollution, and the like. Further, for example, by using a strain that is the same as or similar to a soil microorganism naturally present in normal farmland, the plant growth control agent and the plant growth control method of the present invention are safe and secure for consumers and producers. Technology can be provided. For example, it is suitably used as a herbicide or the like by utilizing the action of suppressing plant growth. The plant growth regulator of the present invention can be used in combination with a known herbicide. Moreover, for example, when the growth of cultivated plants is promoted, the cultivation efficiency can be increased. Furthermore, by promoting the growth, the morbidity rate can be reduced by shortening the seedling period that is generally susceptible to diseases. It is also preferable to mix the plant growth regulator of the present invention with known agricultural chemicals or nutrients and use them in crops. The present invention is suitably applied to, for example, a plant factory.

The present invention controls plant growth of at least one compound selected from the group consisting of 3-methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone. The use for doing is also included.
The present invention relates to a volatile metabolite generated by culturing at least one microorganism selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, fungi and molds in a medium containing sugar and a carbon source other than sugar. Of the use of the plant for controlling plant growth.
Preferred embodiments of the present invention are the same as the plant growth regulator and the plant growth control method described above.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereby. In this example, “%” means “% by mass” unless otherwise specified.

<Example 1>
1. Experimental Method 1-1 Seed Sterilization Seeds of Arabidopsis thaliana (Ecotype, Colombia) were purchased from Implanta Innovations. Rice (Oryza sativa, variety: Nihonbare) seeds were distributed by Professor Shige Prefectural University Professor Hiroshi Hasegawa. The seeds of this rice (variety name: Nipponbare) were used in Example 2, including Catharanthus. Roseus, variety name: Little Delicata, and sweet basil (Ocimum basilicum) are commercially available, and are generally available after soaking the seeds in a 1% sodium hypochlorite solution for 15 minutes before using distilled water. The rice seed was sterilized after removing the seed coat.

1-2 Seeding Sterilized seeds were sown on plant agar medium. In the case of Arabidopsis seeds with small seeds, 0.1% agarose and seeds were mixed and seeded on the surface of the agar medium with a pipette in order to facilitate the operation.

1-3 Plant cultivation In the case of Arabidopsis thaliana, low temperature treatment at 4 ° C. for 5 days promoted germination synchronization. Rice and Arabidopsis were cultivated under conditions of 23 ° C. and long days (16 hours of illumination and 8 hours of illumination (light period 16 hours and dark period 8 hours)). The composition of the plant agar medium is as follows.

Arabidopsis MS (Murashige-Skoog) Medium: 1 liter NH ₄ NO ₃ 1,650 mg, ZnSO ₄ 7H ₂ 0 8.6 mg, KNO ₃ 1,900 mg, KI 0.83 mg, CaCl ₂ 2H ₂ O 440 mg, Na ₂ MoO ₄ 2H ₂ O 0.25 mg, MgSO ₄ 7H ₂ O 370 mg, CuSO ₄ 5H ₂ O 0.025 mg, KH ₂ PO ₄ 170 mg, CoCl ₂ 6H ₂ O 0.025 mg, H ₃ BO ₃ 6.2 mg, MnSO ₄ 4H ₂ O 22.3 mg, FeCl ₃ 16.2 mg, sucrose 30 g, Phytoblend (trade name, manufactured by Caisson Laboratories) 7 g

Rice Hoagland No.2 Medium: 1 NH ( ₄ ) ₃ PO ₄ 115.03 mg, H ₃ BO ₃ 2.86 mg, Ca (NO ₃ ) ₂ 656.4 mg, CuSO ₄ 5H ₂ O 0.08 mg, FeCl ₃ 16.2 mg, MgSO ₄ 240.76 mg, MnCl ₂ 4H ₂ O 1.81 mg, MoO ₃ 0.016 mg, KNO ₃ 606.6 mg, ZnSO ₄ 7H ₂ 0 0.22 mg, Phytoblend (trade name, manufactured by Caisson Laboratories) 7 g

1-4 Bacterial culture Bacillus subtilis standard strains (Bacillus subtilis, A001 and ATCC9372), other Bacillus subtilis strains (DB9011 and QST-713), Agrobacterium (EHA101), and Escherichia coli (DH5a) include the following media: In culture. These bacteria are available from public institutions or are generally commercially available.
TS (Tryptic Soy) Agar medium (organic medium): Tryptic Soy Agar (trade name, manufactured by Sigma, lot number 22091-500G)
TS liquid medium (organic medium): Bacto Tryptic Soy Broth (trade name, manufactured by Becton Dickinson, lot number 211824)
Synthetic agar medium (inorganic medium): per liter KH ₂ PO ₄ 2.72 g, Na ₂ HPO ₄ 10.75 g, MgSO ₄ 7H ₂ O 0.10 g, NH ₄ Cl 2.00 g, CaCl ₂ 0.01 g, vitamin B1 0.01 g, FeSO ₄ 7H ₂ O 0.30 mg, MnSO ₄ H ₂ O 0.01 mg, biotin 3.00 mg, sucrose 15.5 g, Phytoblend (trade name, manufactured by Caisson Laboratories) 7 g

One platinum loop was taken from each bacterial cryopreserved stock (50% glycerol, 50% TS medium), and cultured with shaking in TS medium at 23 ° C. and 100 rpm for 2 days to prepare a saturated culture solution. 30 μl of this saturated culture broth was inoculated into 3 μml of TS medium and cultured overnight at 23 ° C. and 100 rpm for use in the assay. The cryopreservation stock refers to a saturated culture solution that is mixed at the above saturated culture solution with an equal amount of glycerol and stored at −80 ° C.

1-5 Assay Sprouts that had been previously cultivated in the absence of bacterial co-culture were used for the assay. In the case of Arabidopsis thaliana, preculture was performed for 5 days, and in the case of rice, 3 days. A plant medium for assay was prepared in which a 35 mm dish was placed at one corner of the plant medium (see ad in FIG. 1).

1-5-1 Assay using Bacillus subtilis A 35 mm dish was filled with a bacterial TS agar medium (organic medium) or inorganic medium, and this was placed in one corner of the plant medium to prepare a plant medium for assay (FIG. 1). A to d). The medium in the 35 mm dish and the plant medium are separated by the 35 mm dish and are not in contact with each other. The gas phase is continuous between the 35 mm dish and the plant medium. In FIG. 1a and FIG. 1b, the 35 mm dish is filled with a bacterial TS agar medium (organic medium), and in FIG. 1 c and FIG. 1 d, the 35 mm dish is filled with an inorganic medium.

Bacillus subtilis cultivated overnight (turbidity is usually measured at a wavelength of 600 nm and around 0.8) is inoculated into the medium in a 35 mm dish along with the seedling transplant, and the plant medium plate is sealed with breathable surgical tape (A in FIG. 1 and c in FIG. 1). Therefore, co-culture was performed in a closed system. In FIG. 1 b and FIG. 1 d, Arabidopsis and rice seedlings were transplanted in the same manner except that Bacillus subtilis was not inoculated. That is, the Bacillus subtilis standard strain (ATCC9372) was cultured in a 35 mm dish 1 using a bacterial TS agar medium in the plant medium plate of FIG. In the plant culture medium plate of FIG. 1b, the 35 mm dish 2 is filled with a bacterial TS agar medium not inoculated with the Bacillus subtilis standard strain. In the plant medium plate of FIG. 1c, a Bacillus subtilis standard strain was cultured in a 35 mm dish 3 using an inorganic medium. In the plant culture medium plate of FIG. 1 d, the 35 mm dish 4 is filled with an inorganic culture medium in which the Bacillus subtilis standard strain is not inoculated.
The plates a to d in Fig. 1 are placed on a cultivation shelf at an angle of 60-90 degrees, and are grown under long-day conditions (lighting for 16 hours, turning off lighting for 8 hours). Observed over time. The standard co-culture was 5 days for both Arabidopsis and rice. Co-culture was performed at 23 ° C.

1-5-2 Assay using compounds 2-pentadecanone, 2,5-dimethylpyrazine, 3-methyl-2-pentanone (3-methyl-2) -pentanone) and 3-hydroxy-2-butanone (acetoin) (2-pentadecanone is Kanto Chemical Co., Inc., all manufactured by Sigma), chloroform was used for 2-pentadecanone. Except for the above, methanol was used as a solvent, and an appropriately diluted solution was used in place of the aforementioned Bacillus subtilis 1-5-1. The diluted solution was applied directly to the plant medium, or immersed in a filter paper placed in a 35 mm dish of the plant medium for assay and used for the assay. The diluted solution was applied to the plant medium or dropped on filter paper only once at the start of the assay. Plant culture was performed according to the above.

1-6 Collection of volatile components Small scale: Three Monotrap (registered trademark) DCC18 (manufactured by GL Science) were placed at a distance of 1.5 cm from the 35 mm dish, and volatile components were collected over 3 days.
Three Monotrap (registered trademark) disks were transferred to a Spitz tube, 2 mL of methylene chloride was added, and sonication was performed for 10 minutes. Put methylene chloride extract after ultrasonic treatment through a column packed with absorbent cotton in a Pasteur pipette and washed in advance with methylene chloride. After removing fine debris, evaporate methylene chloride in a 65 ° C water bath. The extract was concentrated. After concentration to 20 μL, 1 μL was used for GC-MS analysis. Monotrap (registered trademark) and a method for collecting volatile components using the same are described in detail at the following URL. http://www.glsciences.com/products/monotrap/catalogue.pdf

1-7 GC-MS Analysis GC-MS was measured under the following conditions.
GC-MS (apparatus): 6890N GC (model number) with 5975 Inert Mass Selective Detector (model number) (quadrupole mass spectrometer manufactured by Agilent Technologies)
Column: HP5ms capillary column (trade name), 30 m × 0.25 mm id, 0.25 μm df (Agilent Technologies)
Carrier gas: helium methylene chloride with a head pressure of 50 KPa (constant pressure) was used as a solvent, and 1 μL of the sample was split-injected at a 1:10 ratio. The injection port was 230 ° C. and the oven temperature was 40 ° C. After the sample injection, the temperature was increased at 5 ° C./minute after 10 minutes and maintained at 220 ° C. for 30 minutes.
In quadrupole mass spectrometry, the EM voltage (EM voltage) was set to 1,176 V, and the ionization voltage (ionization voltage) was set to 70 eV.

2. Results 2-1 Effect of Bacillus subtilis volatile metabolites on the growth of Arabidopsis seedlings B. subtilis was inoculated in 35 mm dish 1 of the medium of FIG. 1 and 35 mm dish 3 of the medium of FIG. Five Arabidopsis seedlings that had been sterilized in advance were transplanted at different distances from Bacillus subtilis and co-cultured for 5 days. Co-culture was performed in a closed system. FIG. 1b and FIG. 1d are plates obtained by cultivating Arabidopsis seedlings in the same manner as in FIG. 1a and FIG. 1c except that Bacillus subtilis was not inoculated on a 35mm plate. The black bars in the plates of FIGS. 1a to 1d represent the position of the base at the time of implantation, and the dots represent the position of the root tip.

As a result, it was revealed that the elongation of the main roots of Arabidopsis plants transplanted at a close distance from Bacillus subtilis cultured in an organic medium (a medium containing sugar and a carbon source other than sugar) was significantly inhibited (FIG. 1). A). The root position is indicated by a white line. P, M, and D indicate the distance from the 35 mm plate (P: 1-3 cm, M: 4-6 cm, D: 7-9 cm). Furthermore, it was also found that when co-culturing was continued as it was, it died in order from Arabidopsis thaliana transplanted at a short distance from Bacillus subtilis within a week.
In FIG. 1c in which Bacillus subtilis was cultured in an inorganic medium, the growth of Arabidopsis thaliana plants was not inhibited, and grew to the same extent as b in FIG. 1 and d in FIG. 1 cultured in the absence of Bacillus subtilis.

FIG. 2 shows the results of measuring the main root lengths of Arabidopsis cultured on the plate of FIG. 1a and Arabidopsis cultured on the plate of FIG. The vertical axis | shaft of FIG. 2 shows the length (mm) of a root. As a result of measuring the main root length of Arabidopsis thaliana from the start of the assay, when the Bacillus subtilis is not cocultured (represented as CTL in FIG. 1 b and FIG. 2), the number of days of culture is independent of the distance from the 35 mm plate for bacteria. The roots grew with the progress of. On the other hand, when co-cultured with Bacillus subtilis, culturing Bacillus subtilis in an organic medium (referred to as ATCC9372 in FIG. 1a and FIG. 2), when the distance from Bacillus subtilis is short, 1-2 days after the start of co-culture It was already found that elongation was completely inhibited at that time (FIG. 2). It was also revealed that this root elongation inhibition was gradually reduced as the distance from the bacterial 35 mm plate was increased. In the results of FIG. 2, 5 individuals were observed per experimental group, and the standard deviation was represented by a bar.

Fig. 3 shows the effect of Bacillus subtilis volatile components on the wet plant weight. In FIG. 3, “(A) without Bacillus subtilis” is Arabidopsis thaliana cultured on the plate of FIG. 1 b, and “(B) with Bacillus subtilis” is Arabidopsis thaliana cultured on the plate of FIG. As described above, P, M, and D indicate the distance from the 35 mm plate in FIG. 1 (P: 1-3 cm, M: 4-6 cm, D: 7-9 cm). As can be seen from FIG. 3, about the wet weight (mg) of Arabidopsis thaliana, about an individual close to Bacillus subtilis (P in a of FIG. 1), compared to the case without Bacillus subtilis (P in b of FIG. 1). On the other hand, in the individual far from Bacillus subtilis (D in a of FIG. 1), the increase was about 1.7 times as opposed to the case without Bacillus subtilis (D in b of FIG. 1). From the above results, it becomes clear that Bacillus subtilis can control Arabidopsis thaliana growth 1) in a distance-dependent manner and 2) in a Bacillus subtilis medium-dependent manner when co-cultured with Bacillus subtilis and Arabidopsis thaliana. It was. Since Arabidopsis thaliana and Bacillus subtilis are non-contact, the active factor was expected to be volatile. The result shown in FIG. 3 is an average of the results of observing 5 individuals per experimental group, and the standard deviation is represented by a bar.

Table 1 summarizes the effects of Bacillus subtilis volatile components on Arabidopsis thaliana growth when Bacillus subtilis and Arabidopsis were co-cultured for 5 days. At a long distance from Bacillus subtilis, root elongation was slightly suppressed as compared to the case without Bacillus subtilis, but the wet weight increased. Since the action of the volatile component of Bacillus subtilis differs depending on the plant organ, it was considered that the response mechanism itself or the sensitivity on which the volatile component of Bacillus subtilis acts differs depending on the plant organ.

Subsequently, in order to investigate the presence or absence of volatile components preferentially released under the culture conditions in which Bacillus subtilis exhibits plant growth inhibitory activity, the volatile components of Bacillus subtilis cultured under different culture conditions are collected with Monotrap (registered trademark). And analyzed by GC-MS. The results are shown in FIGS. 4a is a GC-MS chromatogram of volatile components generated when Bacillus subtilis (ATCC9372) is cultured in an organic medium (FIG. 1a). The volatile component a in FIG. 4 showed plant growth inhibitory activity. That is, the collected concentrated solution of Bacillus subtilis volatile components exhibited root elongation inhibitory activity. This indicates that the active ingredient can be concentrated and is stable for at least several days after extraction. FIG. 4b is a GC-MS chromatogram of volatile components of an organic medium not containing Bacillus subtilis (FIG. 1b). FIG. 4c is a GC-MS chromatogram of volatile components generated from Bacillus subtilis when Bacillus subtilis is cultured in an inorganic medium (FIG. 1c). The volatile component of FIG. 4b and the volatile component of FIG. 4c did not show plant growth inhibitory activity.
As a result of analysis by GC-MS, at least five compound peaks indicated by arrows in FIG. 4a were preferentially detected when Bacillus subtilis was cultured on an organic agar medium.

When these five compound fragmentation patterns were collated with the compound library by MS / MS, as shown in FIG. 5, 3-methylpentanone (3-methyl-2-pentanone) and 2,5-dimethylpyrazine ( 2,5-dimethylpyrazine), 2-heptanone, 2-tridecanone, and 2-pentadecanone. FIGS. 5a to 5c correspond to FIGS. 4a to 4c, respectively. That is, a in FIG. 5 is a GC-MS chromatogram of volatile components generated when Bacillus subtilis is cultured in an organic medium (a in FIG. 1). FIG. 5b is a GC-MS chromatogram of the volatile components of the organic medium without Bacillus subtilis (FIG. 1b). FIG. 5c is a GC-MS chromatogram of volatile components generated from Bacillus subtilis when Bacillus subtilis is cultured in an inorganic medium (FIG. 1c).

2-2 Assay using compounds Prepare 10 mM solutions (solvent is methanol) for 3-methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone (acetoin), and 2-pentadecanone. did. After transplanting Arabidopsis thaliana to the plant medium, 5 μL or 50 μL of a 10 mM solution of each compound was dropped onto a filter paper in a 35 mm dish and cultured at 23 ° C. for 5 days. A photograph of each plate after culture is shown in FIG. In FIG. 6, the black bar indicates the position of the base at the time of transplantation. The position of the root end is indicated by a white line.

FIG. 7 shows the results of measuring the length of the main roots of Arabidopsis thaliana after transplanting (after culturing at 23 ° C. for 5 days). The vertical axis of the graph in FIG. 7 represents the length (mm) of the main root that has been elongated after transplantation (after culturing at 23 ° C. for 5 days). CTL is a control.
From the results of FIG. 6 and FIG. 7, 3-methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone all suppress the elongation of the main root through the gas phase. It was found that the plant growth inhibitory activity was exhibited.
Moreover, although data are not shown, in the said experiment, instead of dripping the solution of each compound in a 35 mm dish, 100 microliters of 250 micromol or 50 micromol solution of each compound was directly apply | coated to the plant culture medium, and it experimented similarly. . Also in this case, the elongation of the main root was suppressed by each compound.

2-3 Effects of Bacillus subtilis volatile metabolites on the growth of rice seedlings The seed coat of rice (Oryza sativa variety: Nihonbare) was removed, sterilized as described above, and sown after 7 days. ATCC9372) and co-cultured. In order to adapt to the growth conditions of rice, the same operation as in the Arabidopsis experiment was performed except that 0.5 × Hoagland No. 2 medium was used as the plant medium and the growth temperature was 27 ° C.

As a result, when Bacillus subtilis was cultured in an organic medium, the growth of rice was inhibited by the volatile components. The results are shown in FIG. The plant growth inhibitory activity of Bacillus subtilis was effective not only on Arabidopsis but also on rice. FIGS. 8a to 8d show the results of examining the influence of volatile metabolites of Bacillus subtilis on the growth of rice. In the plant medium plate of FIG. 8a, a Bacillus subtilis standard strain was cultured in a 35 mm dish using a bacterial TS agar medium (organic medium). In the plant medium plate of FIG. 8b, the 35 mm dish is filled with a bacterial TS agar medium not inoculated with the Bacillus subtilis standard strain. In the plant medium plate of FIG. 8c, the Bacillus subtilis standard strain was cultured in a 35 mm dish using an inorganic medium. In the plant culture medium plate of FIG. 8 d, the 35 mm dish is filled with an inorganic culture medium not inoculated with the Bacillus subtilis standard strain. The black bars a to d in FIG. 8 represent the base of rice.

2-4 Effect of Escherichia coli and Agrobacterium volatile metabolites on the growth of Arabidopsis seedlings Along with Arabidopsis, E. coli (DH5a) or Agrobacterium (EHA101) is transformed into (A) organic medium or (B) inorganic medium. FIG. 9 shows the length (mm) of the main root of Arabidopsis thaliana cultured in The experiment using Escherichia coli or Agrobacterium was the same as the experiment using Bacillus subtilis standard strain and Arabidopsis thaliana except that Escherichia coli (DH5a) or Agrobacterium (EHA101) was used instead of the Bacillus subtilis standard strain. The operation was performed. In FIG. 9, ATCC9372 represents Bacillus subtilis (standard strain, ATCC9372), DH5a represents E. coli (DH5a), and EHA101 represents Agrobacterium (EHA101). In FIG. 9, “No bacteria” means that the microorganisms were not co-cultured.

As shown in FIG. 9, not only Bacillus subtilis (ATCC9372) but also Escherichia coli (DH5a) and Agrobacterium (EHA101) exhibited an Arabidopsis root elongation inhibitory activity when cultured in an organic medium. Therefore, it was found that the main root elongation inhibitory activity of Arabidopsis thaliana was preserved not only in Bacillus subtilis but also in volatile metabolites when Escherichia coli and Agrobacterium were cultured in an organic medium.

<Example 2>
In the assay using Example 1-5-1 Bacillus subtilis in Example 1, seeds of sweet basil (Ocimum basilicum) and Catharanthus roseus cv. Littile Delicata were used instead of Arabidopsis seeds. The same experiment was conducted using Bacillus subtilis standard strain (A001) as The experimental conditions were based on the conditions in Example 1 using Arabidopsis except that the co-culture period was 10 days.

The results are shown in FIGS. The black bars in FIGS. 10a to 10d and 11a to 11d represent the positions of root tips at the time of transplantation.
FIGS. 10a to 10d show the results of examining the influence of volatile metabolites of Bacillus subtilis on the growth of sweet basil. In the plant medium plate of FIG. 10a, a Bacillus subtilis standard strain (A001) was cultured using a TS agar medium for bacteria in a 35 mm dish. In the plant medium plate of FIG. 10b, the 35 mm dish is filled with a bacterial TS agar medium not inoculated with the Bacillus subtilis standard strain. In the plant medium plate of FIG. 10c, the Bacillus subtilis standard strain was cultured in a 35 mm dish using an inorganic medium. In the plant culture medium plate of FIG. 10d, the 35 mm dish is filled with an inorganic culture medium not inoculated with the Bacillus subtilis standard strain.

11a to 11d show the results of examining the influence of volatile metabolites of Bacillus subtilis on the growth of periwinkle. In the plant medium plate of FIG. 11a, a Bacillus subtilis standard strain (A001) was cultured in a 35 mm dish using a bacterial TS agar medium. In the plant culture medium plate of FIG. 11b, the 35 mm dish is filled with a TS agar medium for bacteria that is not inoculated with a standard strain of Bacillus subtilis. In the plant medium plate of FIG. 11c, the Bacillus subtilis standard strain was cultured in a 35 mm dish using an inorganic medium. In the plant culture medium plate of FIG. 11d, the 35 mm dish is filled with an inorganic culture medium in which the Bacillus subtilis standard strain is not inoculated.

As can be seen from FIGS. 10 to 11, the growth of sweet basil and periwinkle was also inhibited by the volatile components of Bacillus subtilis cultured in an organic medium, similar to Arabidopsis thaliana. Although it is difficult to distinguish with a black and white photograph, the plant in the upper left of the panel (a in FIG. 10 and a in FIG. 11) is completely dead.

<Example 3>
In the same manner as in Example 1, the seed coat of rice (Oryza sativa variety: Nihonbare) was removed, sterilized, and then sown, and then cultivated for 4 days. QST-713)) or Agrobacterium (EHA101). B. subtilis Strain DB9011 and B. subtilis Strain QST-713 were obtained from Idemitsu Kosan and Meiji Seika, respectively. In order to match the growth conditions of rice, 0.5 × Hoagland No. The same operation as in the experiment in Arabidopsis thaliana in Example 1 (assay using 1-5-1 Bacillus subtilis in Example 1) was performed except that 2 medium was used and the growth temperature was 27 ° C. .

The results are shown in FIG. The black bars in a to e in FIG. 12 represent the positions where rice was sown.
FIG. 12 a is a control, and the microorganism was not co-cultured. FIG. 12 b, FIG. 12 d, and FIG. 12 e are cases where co-cultured with Bacillus subtilis. FIG. 12c shows the case of co-culture with Agrobacterium (EHA101). The Bacillus subtilis of FIG. 12b is a Bacillus subtilis standard strain (ATCC9372), and the Bacillus subtilis of FIG. 12d is B. subtilis Strain DB9011. The Bacillus subtilis of e of FIG. 12 is B. subtilis Strain QST-713.
As shown in FIG. 12b, FIG. 12d and FIG. 12e, when Bacillus subtilis is cultured in an organic medium (row (A) in FIG. 12), no matter which Bacillus subtilis is used, The volatile components inhibited rice growth. Even when Bacillus subtilis was cultured in an inorganic medium (row (B) in FIG. 12), growth inhibition of rice did not occur.
From FIG. 12c, when Agrobacterium was cultured in an organic medium, the growth of rice was inhibited by its volatile components. Even when Agrobacterium was cultured in an inorganic medium (row (B) in FIG. 12), growth inhibition of rice did not occur.
In addition, since the used rice seeds were not necessarily completely germinated in comparison with Arabidopsis thaliana, there was some variation in the degree of elongation of the main root.
As described above, it has been clarified that rice also suffers from growth inhibition such as main root elongation not only by Bacillus subtilis but also by volatile components of Agrobacterium.

The present invention is useful in fields such as agriculture and forestry and horticulture.

1 35mm dish inoculated with a standard strain of Bacillus subtilis (TS agar medium for bacteria)
2 35mm dish not inoculated with the standard strain of Bacillus subtilis (TS agar medium for bacteria)
3 35mm dish inoculated with a standard strain of Bacillus subtilis (agar medium containing only sugar as a carbon source)
4 35mm dish inoculated with Bacillus subtilis standard strain (agar medium containing only sugar as carbon source)

Claims (15)

1. It contains at least one compound selected from the group consisting of 3-methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone as an active ingredient. Plant growth regulator.
2. 3-methyl-2-pentanone generated by culturing at least one microorganism selected from the group consisting of gram-positive bacteria, gram-negative bacteria, fungi and molds in a medium containing sugar and a carbon source other than sugar A volatile metabolite containing at least one compound selected from the group consisting of 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone. Growth control agent.
3. It contains volatile metabolites generated by culturing at least one microorganism selected from the group consisting of gram-positive bacteria, gram-negative bacteria, fungi, and molds in a medium containing sugar and a carbon source other than sugar. A plant growth regulator characterized by
4. 4. The plant growth regulator according to claim 2 or 3, wherein the microorganism is a gram positive bacterium, a gram negative bacterium, or a filamentous fungus.
5. The plant growth regulator according to any one of claims 2 to 4, wherein the microorganism is Bacillus subtilis, Agrobacterium, or mycorrhizal fungus.
6. The plant growth regulator according to any one of claims 1 to 5, wherein the plant growth control is plant growth suppression or growth promotion.
7. The plant growth regulator according to claim 1 or 2, wherein the plant growth control is plant growth inhibition.
8. At least one selected from the group consisting of 3-methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone for the plant body, plant cultivation site or plant seed A method for controlling plant growth, which comprises applying the above compound.
9. 3-methyl-2-pentanone generated by culturing at least one microorganism selected from the group consisting of gram-positive bacteria, gram-negative bacteria, fungi and molds in a medium containing sugar and a carbon source other than sugar The plant growth according to claim 8, wherein a volatile metabolite comprising at least one compound selected from the group consisting of 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone is applied. Control method.
10. At least one microorganism selected from the group consisting of gram-positive bacteria, gram-negative bacteria, fungi, and molds is cultured in a medium containing a carbon source other than sugar and sugar on the plant body, plant cultivation site, or plant seed. A method for controlling plant growth, which comprises applying a volatile metabolite generated by the process.
11. The plant growth control method according to claim 9 or 10, wherein the microorganism is a gram-positive bacterium, a gram-negative bacterium, or a filamentous fungus.
12. The plant growth control method according to any one of claims 9 to 11, wherein the microorganism is Bacillus subtilis, Agrobacterium, or mycorrhizal fungus.
13. The plant growth control method according to any one of claims 8 to 12, wherein the plant growth control is plant growth suppression or growth promotion.
14. The plant growth control method according to claim 8 or 9, wherein the plant growth control is plant growth inhibition.
15. Use of at least one compound selected from the group consisting of 3-methyl-2-pentanone, 2,5-dimethylpyrazine, 3-hydroxy-2-butanone, and 2-pentadecanone for controlling plant growth .

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