JP2020029406A - Plant growth regulator - Google Patents

Abstract

To identify a plant growth-regulating active substance of Phlebia tremellosa and use the same as a plant growth regulator.SOLUTION: Provided is a sesquiterpene compound represented by formula (I) or (II). These compounds especially have an activity to enhance extension of a main root of a plant and are useful as a plant growth regulator.SELECTED DRAWING: None

Description

  The present invention relates to a sesquiterpene compound produced by Shiitake mushroom, a plant growth regulator containing the compound, and a method for regulating plant growth, which comprises using the compound.

A mushroom refers to a fungus belonging to the Basidiomycota or Ascomycota and forms a fruiting body having a size that can be observed with the naked eye. There are 4,000 to 5,000 wild species of mushrooms in Japan alone, of which about 2,000 have been identified. Mushrooms often interact with other organisms, and many have a variety of physiologically active substances, but research on their functional components is extremely limited.
Shiitake mushroom is a kind of mushroom called white rot fungus and a microorganism that causes white rot of wood. Although not suitable for edible use, its culture filtrate extract is known to contain various physiologically active compounds. (Non-Patent Documents 1 to 3). In addition, it was also found to have aflatoxin biosynthesis inhibitory activity (Non-Patent Document 4). However, no plant growth promoting and / or inhibiting action as in the present application is known.
Merlactone has been reported as a sesquiterpene isolated from Shiitake mushroom (Non-Patent Document 5), but the effect of merlactone on plant growth is extremely weak.

On the other hand, in the agricultural field, promoting or controlling plant growth is an important technique for improving productivity. At present, various types of plant growth regulators for the purpose of promoting and / or suppressing plant growth have been put to practical use, and the plant growth regulators have contributed to the improvement of crop yield and product quality.
Specifically, fruiting accelerators utilizing auxin's fruiting-enhancing action, fruit-enlargement action, and fruit-enlargement promoting agents are known. As a coloring accelerator, 4-chlorophenoxyacetic acid, dichlorprop, ethiclozate and the like have been put to practical use. However, such plant growth regulators are also small in number, have insufficient effects, and often have more undesirable effects.
Recently, plant growth regulators derived from natural products have been demanded from the viewpoint of safety orientation.

H. Anke, O. Sterner, W. Steglich. Structure-activity relatonships of unsaturated dialdehydes 3. Mutagenic, antimicrobial, cytotoxic, and phytotoxic activities of merulidial derivarives. J. Antibiotic. 42, 738-744 (1989) M. G. Bruno, B. Steffan, W. Steglih W: Antibiotics from basidiomycetes. Part 23. Merulidial, an isolactarane derivative from Merulius tremellosus. Terahedron, 42, 3579-3586 (1986) W. Quack, T. Anke, O. Oberwinkler, M. G. Giannetti, W. Steglich: Antibiotics from basidiomycetes. Part 5. Merulidial, an isolactarane derivative from Merulius tremellosus. Antibiotics, 31.737-741 (1978) Mizuki Inoue, Naoki Endo, Kosuke Saotome, Nitaro Maekawa, Akira Nakagiri, Kotomi Ueno, Toru Ishihara, Search for Aflatoxin Biosynthesis Inhibitors from Basidiomycetes, Japan Society for Agricultural Chemistry Osaka Conference, Abstracts p.101 Sterner O, Anke T, Sheldrick W, Steglich W (1990) New sterpurane and isolactarane sesquiterpenes from the fungus Merulius tremellosus.Tetrahedron, 46 (7) 2389-2400

  The problem to be solved by the present invention was to isolate and identify a plant growth regulating, particularly a growth promoting compound from Shiitake mushroom and use it as an agricultural chemical.

The present inventors have conducted intensive studies, and as a result, have found that the compound promoting growth of Shiitake mushroom is represented by the following formula (I) or (II):
The present invention was completed by further confirming the plant growth promoting activity of these compounds.

The invention provided by the present application is as follows.
(1) A compound represented by the formula (I) or (II).
(2) A plant growth regulator containing the compound as an active ingredient and containing necessary additives and carriers.
(3) The plant growth regulator of (2), which is a plant growth promoter.
(4) A method for regulating plant growth, comprising using the above compound.
(5) The method for regulating plant growth according to (4), which is a method for promoting plant growth.

  According to the present invention, a plant growth regulating active compound produced by Shiitake mushroom was identified. Since these compounds (I) and (II) are derived from natural products, they are considered to be highly safe for humans and animals and have little effect on the environment.

FIG. 1 shows the effects of the mushroom extract on the extension of the main root, the extension of the lateral root and the number of lateral roots. Bars indicate standard deviation, and asterisks indicate statistically significant differences (* P <0.05, ** P <0.01, Kruskal-Wallis test). FIG. 2 shows the effects of fractions obtained by silica gel dry column chromatography on elongation of main roots, elongation of lateral roots and the number of lateral roots. The bars and asterisks in the figure are the same as in FIG. FIG. 3 shows a purification scheme of a Shiitake mushroom culture filtrate extract by a column. FIG. 4 shows the effects of the fraction obtained by silica gel wet column chromatography on the elongation of the main root, the elongation of the lateral root, and the number of lateral roots. The bars and asterisks in the figure are the same as in FIG. FIG. 5 shows a purification scheme of Compound 1. FIG. 6 shows a purification scheme of Compound 2. FIG. 7 shows the effects of Compound 1 and Compound 2 on the extension of the main root, the extension of the lateral root and the number of lateral roots. The bars and asterisks in the figure are the same as in FIG.

The plant growth regulator of the present invention has the formula (I) and / or (II):
Is contained as an active ingredient.
The compounds of the formulas (I) and / or (II) may be cultured from Shiitake mushrooms according to the examples described below and extracted from the culture filtrate, or may be chemically synthesized.
The term “plant growth regulation” as used in the present invention includes both promotion and inhibition of plant growth, such as plant dwarfing (suppression of elongation), regulation of flowering time, induction of upright leaves (and accompanying photosynthesis). Efficiency improvement, biomass increasing effect), pollen growth suppression, flower freshness retention, plant anti-stress agents (heat, dryness, cold etc.), weed control by reproduction control, plant aging suppression, root enlargement, fruit set It should be interpreted in the broadest sense, including promotion, promotion of fruit enlargement, prevention of fruit drop, and promotion of peel coloration. For example, rooting promoters, plant growth promoters, root system development promoters and the like are typical examples of the plant growth regulator of the present invention, but the plant growth regulator of the present invention is not limited to these. Absent.

  The plant growth regulator of the present invention, an aqueous solution of the above compound can be used as a plant growth regulator as it is, but wettable powders, emulsions, granules, powders, surfactants, and the like are used in ordinary plant growth regulators. It may be formulated using the carrier obtained. For example, solid carriers include mineral powders (kaolin, bentonite, clay, montmorillonite, talc, diatomaceous earth, mica, vermiculite, gypsum, calcium carbonate, phosphorus lime, etc.), and vegetable powders (soy flour, flour, wood flour, tobacco) Powders, starch, crystalline cellulose, and the like, polymer compounds (petroleum resin, polyvinyl alcohol resin, polyvinyl acetate resin, polyvinyl chloride, ketone resin, and the like), and alumina, waxes, and the like can be used. As the liquid carrier, for example, alcohols (methanol, ethanol, propanol, butanol, ethylene glycol, benzyl alcohol, etc.), aromatic hydrocarbons (toluene, benzene, xylene, etc.), chlorinated hydrocarbons (chloroform, Carbon tetrachloride, monochlorobenzene, etc., ethers (dioxane, tetrahydrofuran, etc.), ketones (acetone, methyl ethyl ketone, etc.), esters (ethyl acetate, butyl acetate, etc.), acid amides (N, N-dimethylacetamide, etc.) , Ether alcohols (such as ethylene glycol ethyl ether), or water.

  As the surfactant used for the purpose of emulsification, dispersion, diffusion and the like, any of nonionic, anionic, cationic and amphoteric surfactants can be used. Examples of surfactants that can be used in the present invention include polyoxyethylene alkyl ether, polyoxyethylene alkyl aryl ether, polyoxyethylene fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, and oxyethylene polymer. , Oxypropylene polymer, polyoxyethylene alkyl phosphate, fatty acid salt, alkyl sulfate, alkyl sulfonate, alkyl aryl sulfonate, alkyl phosphate, alkyl phosphate, polyoxyethylene alkyl sulfate, Quaternary ammonium salts, oxyalkylamines, lecithin, saponins and the like. If necessary, gelatin, casein, sodium alginate, starch, agar, polyvinyl alcohol and the like can be used as auxiliary agents.

The shape of the plant growth regulator of the present invention is not limited, and it can be formed into any formulation such as powder, granule, granule, wettable powder, flowable, emulsion and paste. It can be produced by mixing, stirring, spray-drying, etc. the other components according to a conventional method.
When applied to plants, they can be used as soil treatment agents, foliage treatment agents, seed treatment agents before sowing, treatment agents for plants before transplantation, treatment agents for plants at the time of transplantation, and the like. In hydroponics, it may be used by mixing with a hydroponic solution, and in tissue culture, it may be used by suspending or dissolving it in a medium.

In the case of compound 1, the concentration of the plant growth regulator of the present invention is preferably in the range of 10 to 2000 ppm, more preferably 50 to 2000 ppm, and particularly preferably 100 to 1000 ppm. In the case of compound 2, the content is preferably in the range of 1 to 500 ppm, more preferably 10 to 300 ppm.
Hereinafter, the present invention will be described in more detail and specifically with reference to Examples, but these Examples do not limit the present invention.

1. Plant growth regulation activity of Shiitake mushroom extract (1) Liquid culture of Shiitake mushroom A large-scale culture was carried out using a mushroom (Phlebia tremellosa TUFC11737) which was inoculated from a stored strain into a PDA medium (potato dextrose agar medium) and grown. 5 L of malt liquid medium was dispensed into a 500 mL Erlenmeyer flask in 200 mL portions and sterilized in an autoclave. Mushroom mushrooms were inoculated from the PDA medium to the malt liquid medium and cultured at 25 ° C. for 52 days.
After separation into a culture filtrate and a mycelium by filtration, the respective plant growth regulating activities were measured according to the following method.
(2) Plant growth regulating activity test A filter paper was spread on a 24-well plate, and the sample was dissolved in methanol and added to the filter paper. After completely removing the methanol by air drying, 300 µL of distilled water was added. Four lettuce seeds were sown per hole and incubated in a cycle artificial weather machine at 28 ° C., a light period of 16 hours, and a dark period of 8 hours. Six days later, the image of the plant was captured by a scanner, and the extension of the main root, the extension of the lateral root, and the number of lateral roots were analyzed using the image processing software ImageJ.
FIG. 1 shows the measurement results of the extract of the culture filtrate and the mycelium of the Shiitake mushroom extract. The experiment was performed on eight individuals, and the average value was shown.
(3) Fractionation of extract of cultured filtrate of Shiitake mushroom and activity for regulating plant growth The culture of Shiitake mushroom was filtered by suction, and the filtrate was partitioned and extracted three times with about one third of ethyl acetate. The obtained ethyl acetate layer was dried over anhydrous sodium sulfate overnight, and concentrated under reduced pressure using an evaporator. Then, it was completely dried in a desiccator to obtain an extract (946.4 mg).
Subsequently, fractionation was performed by silica gel dry column chromatography (Daisogel IR-60-63 / 210, Daiso Corporation, Osaka, 85.0 g). Using an acetone-hexane mixed solvent for elution, the acetone concentration was increased stepwise from 0 %% to 60% in 10% steps, and then eluted with 100% acetone and then methanol. 650 mL of solvent was used per fraction. As a result, the nine fractions, namely, 0% fraction (36.6 mg), 10% fraction (32.3 mg), 20% fraction (325.0 mg), 30% fraction (136.1 mg), 40% % Fraction (68.3 mg), 50% fraction (23.9 mg), 60% fraction (32.9 mg), 100% fraction (28.8 mg) and methanol fraction (27.5 mg) were obtained. These nine fractions were subjected to the above-mentioned plant growth regulating activity test. The results are shown in FIG.
As a result of the plant test, a 20% fraction (325.0 mg) showed a high activity. This fraction (288.5 mg) was further fractionated by silica gel column chromatography (Daisogel, 28.5 g). A mixed solvent of ethyl acetate-hexane was used for elution, and the ethyl acetate concentration was increased stepwise by 10% from 20% to 60%, followed by elution with 100% ethyl acetate and then methanol. 285 mL of solvent was used per fraction. As a result, the seven fractions, ie, 20% fraction (3.6 mg), 30% fraction (7.3 mg), 40% fraction (22.0 mg), 50% fraction (17.8 mg), with respect to the ethyl acetate concentration, A 60% fraction (60.8 mg), a 100% fraction (72.6 mg) and a methanol fraction (37.8 mg) were obtained. FIG. 3 shows the purification scheme so far, and FIG. 4 shows the results of the plant growth regulating activity test on the obtained seven fractions.

2. Isolation of Active Compound and Structure Determination (1) Isolation of Compound 1 The above ethyl acetate 60% fraction (60.8 mg) was subjected to TLC [SiO ₂ glass plate Silica gel 60 F254, Merck, developing solvent: ethyl acetate- Hexane (4: 1)] was used to divide into six fractions, 60% -1, 60% -2, 60% -3, 60% -4, 60% -5 and 60% -6. Rf values are 0.0-0.07 (60% -1), 0.07-0.16 (60% -2), 0.16-0.29 (60% -3), 0.29-0.37 (60% -4), 0.37-0.46 (60% % -5) and 0.46-1.0 (60% -6).
As a result of the plant growth regulating activity test, major compounds were purified from the 60% -5 fractions having activity using preparative HPLC. The retention time of this compound was 4.01 minutes. Table 5 shows the conditions for preparative HPLC. This active compound was designated as Compound 1. FIG. 5 shows a purification scheme of Compound 1, and mass spectrum data is shown below.
Yield of compound 1: 5.9 mg. Positive ESI MS m / z: 247.2 [M + H] ⁺ , high resolution ESI MS m / z: 247.1324 [M + H] ⁺ (calc. For C ₁₅ H ₁₉ O ₃ : 247.1334), GC-MS m / z : 246.10 (35.16), 189.10 (39.66), 115.05 (41.55), 131.10 (43.88), 105.10 (44.14), 157.10 (45.80), 143.10 (50.53), 91.05 (55.19), 145.10 (57.03), 159.15 (90.51) , 43.05 (100.00).
(2) Isolation of Compound 2 The above 50% ethyl acetate fraction (17.8 mg) was purified by TLC [SiO ₂ glass plate Silica gel 60 F254, Merck, developing solvent: ethyl acetate-hexane (4: 1)]. One fraction, 50% -1, 50% -2, 50% -3, 50% -4, 50% -5, 50% -6 was obtained. The respective Rf values are 0.0-0.11 (50% -1), 0.11-0.18 (50% -2), 0.18-0.33 (50% -3), 0.33-0.40 (50% -4), 0.40-0.51 (50% % -5) and 0.51-1.0 (50% -6).
As a result of the plant growth regulating activity test, the active compound was purified from 50% -5 fractions having activity using preparative HPLC. The retention time of this compound was 4.7 minutes. Table 5 shows the conditions for preparative HPLC. This active compound was designated as Compound 2. The purification scheme of Compound 2 is shown in FIG. 6, and the mass spectrum data is shown below.
Yield of compound 2: 9.7 mg. Positive ESI MS m / z: 247 [M + H] ⁺ , High resolution ESI MS m / z: 246.1326 [M + H] ⁺ (calc. For C ₁₅ H ₁₉ O ₃ : 247.1334 ), GC-MS m / z: 246.10 (48.44), 07.10 (49.67), 203.10 (51.22), 159.10 (54.36), 131.10 (54.46), 79.05 (58.27), 119.10 (60.30), 173.15 (71.56), 41.50 (72.83), 77.05 (73.83), 217.10 (74.84), 105.10 (76.41), 43.05 (89.84), 91.05 (100.00).

(3) Structural determination Compound 1 was estimated to be a compound having a molecular weight of 246 from the results of GC-MS and LC-MS analysis. In addition, high-resolution MS revealed that the compound was a compound having a molecular formula of C ₁₅ H ₁₈ O ₃ and an unsaturation degree of 7. This molecular formula is consistent with the loss of two hydrogen atoms from merlactone isolated from Shiitake mushroom (see Non-Patent Document 5). Therefore, it was inferred that Compound 1 had a structure similar to merlactone.
In fact, a comparison of the ¹ H-NMR spectra of merlactone and compound 1 reveals that the chemical properties of H-2, H-5, H-8 to H-10, H-12 to H-15 of the merlactone and the signals corresponding to these are shown. The shift was similar. Also in the ¹³ C-NMR spectrum, the chemical shifts of the signals of C-2 to C-15 of merlactone showed values close to the signals corresponding to these.
On the other hand, protons H-1 is not only one observation, chemical shift was [delta] _H 4.09. Furthermore, it was estimated from the chemical shift of C-1 (δ _C 80.5) that oxygen was bound to C-1. In addition, considering the observation of each spectrum of HH COSY, HH NOESY and HMBC, considering that the oxygen bonded to the 8-position of merlactone is bonded to the carbon at the 1-position to form a cross-link, The structure of 1 was determined as in formula (I). From the same consideration, the structure of Compound 2 was determined as shown in Formula (II).
The ¹ H- and ¹³ C-NMR spectra of Compound 1 and Compound 2 are as shown in the following table.

Plant Growth Regulation Activity of Compounds 1 and 2 A plant growth regulation activity test was performed using the identified compounds 1 and 2. FIG. 7 shows the result.
Compound 1 showed an accelerating activity at 100 ppm and 300 ppm for elongation of the main root. At 3000 ppm, it showed inhibitory activity. The growth and branching of lateral roots were markedly enhanced at 300 ppm, and slightly increased at 1000 ppm.
On the other hand, Compound 2 showed a tendency to promote the elongation of the main root at 10 and 30 ppm, but showed a tendency to inhibit the growth at 300 ppm or more. Concentrations lower than 100 ppm tended to promote lateral root elongation / branching, and remarkable inhibitory activities were observed above 1000 ppm.

  The present invention can be used in the field of agrochemicals. In particular, the compounds of the present invention can be suitably used as plant growth promoters that affect plant physiology and improve crop quality and / or yield.

Claims (7)

The following formula (I) or (II):
A sesquiterpene compound represented by the formula: A plant growth regulator comprising the compound of claim 1 as an active ingredient. A plant growth promoter, wherein the compound of the formula (I) is used at a concentration of 100 to 1000 ppm. A plant growth promoter, wherein the compound of the formula (II) is used at a concentration of 10 to 300 ppm. A method for regulating plant growth, comprising administering the compound of claim 1 to a plant. A method for promoting plant growth, wherein the compound of formula (I) is used at a concentration of 100 to 1000 ppm. A method for promoting plant growth, wherein the compound of formula (II) is used at a concentration of 10 to 300 ppm.