JPH04342576A - New vegetable physiologically active substance mbh-001, microorganism capable of producing the same, its production and herbicide - Google Patents

Abstract

PURPOSE:To obtain a new vegetable physiologically active substance MBH-001, isolated from a culture of a specific insect-symbiotic microorganism, capable of powerfully inhibiting growth of various plants and utilizable as a herbicide. CONSTITUTION:A vegetable physiologically active substance MBH-001 expressed by the formula is obtained by culturing N1-Ishi-YC-50807-1-7 strain (FERM P-12098) which is a microorganism separated from eggs of brown planthopper in a culture medium containing various nutrient substances under aerobic conditions by spinner culture with aeration (pH6.0-8.0 of the culture medium; 18-32 deg.C culture temperature) and collecting the aforementioned substance according to a method for separating and collecting a normal antibiotic substance, etc., from the above-mentioned culture solution after completing the culture. The aforementioned substance has the following physico- chemical properties. Specific rotatory power [alpha]<25>D=0 (C=0.01, methanol). Color of the substance ; colorless. Ultraviolet absorption spectrum; lambda (ethanol, max.) 210nm. Solubility; readily soluble in methanol, butanol, dimethyl sulfoxide and ethyl acetate and soluble in chloroform and hexane. Color reaction; positive to the ninhydrin reaction, Dragendorff reaction, etc.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to a novel plant physiologically active substance MBH-001, a microorganism that produces it, a method for producing it, and a herbicide containing it as an active ingredient. More specifically, the novel plant physiologically active substance MBH-001 of the present invention is
Nl-Ishi-YC50807, an insect symbiotic microorganism
-1-7 strain can be cultured and obtained from the culture solution, and can be used as a herbicide.

0002

[Prior Art] Glutamine synthetase inhibitors are known as herbicidal active substances produced by microorganisms belonging to the genus Streptomyces. It is a new substance different from herbicidal active substances.

0003

[Means for Solving the Problems] The plant physiologically active substance MBH-001 of the present invention is derived from Nl-Ishi-YC50, an insect symbiotic microorganism newly isolated by the present inventors.
It is collected from a culture of strain 807-1-7. The mycological properties of this strain are as follows.

[0004]A. Form (1) Shape and size: short bacterium 0.4 x 0.6 x 0.
7-1.0μ (2) Pleomorphism: None (3) Motility: None (4) Presence of spores: None (5) Gram staining (3% KOH): Positive

[0005]
B. Growth form (1) Broth agar plate culture: Forms circular yellow colonies. The size is 1 to 1.5 mm, hill-like, with entire edges, smooth, and moistly shiny. (2) Meat juice agar slant culture: Grows thinly on the surface and is translucent and yellow in color. (3) Meat juice liquid culture: Slight growth. The bacterial body is milky white~
It has a milky yellow color. (4) Meat juice gelatin puncture culture: Grows at the puncture site,
Does not liquefy.

C. Physiological properties (1) Nitrate reduction: None (2) Denitrification reaction: None (3) MR test: Positive (4) VP test: Positive (5) Indole production: None (6) Hydrogen sulfide production: None (7) Production of pigment: produces an insoluble yellow pigment on the broth agar medium. (8) Urease: Negative (9) Oxidase: Negative (10) Catalase: Positive (11) Attitude towards oxygen: Microaerobic to aerobic (12)
Fermentative acid production from glucose: None

[0007]
D. Presence or absence of acid production from carbon source (API 50CH
Results after 96 hours) L-arabinose, D-xylose, D-glucose, D-mannose, D-fructose, D-galactose, maltose, sucrose, lactose, trehalose, D-mannite and glycerin Sorbitol, inositol and starch do not produce acids. Basal medium composition: NH4 H2 PO4 1g, K
Cl 0.2g, MgSO4 ・7H2 O 0
．． 2g, Bromthymol Blue 0.03g, distilled water 1,000ml, pH 6.8.

[0008]Nl-Ishi-YC50807-1-7
Although the mycological properties of the strain were compared with the description in Virgie's Manual of Systematic Bacteriology (1984 edition), it was difficult to identify the genus, and the strain was isolated from the eggs of an insect (the brown planthopper). Since it is a fungus, there is a possibility that it is a new species, and it has not yet been identified. This strain was designated as Nl-Ishi-YC50807-1-7 at the National Institute of Microbiological Technology, Agency of Industrial Science and Technology.
It has been deposited as No. 098.

[0009] The microorganism of the present invention also includes Nl-Ish.
The i-YC50807-1-7 strain is, of course, its natural and artificial mutant strains, and is an insect symbiotic microorganism, MBH-0.
All microorganisms having the production ability of 01 are included in the microorganisms of the present invention.

[0010] To produce MBH-001, MBH-
001-producing bacteria are cultured in a conventional manner. For industrially advantageous production, the producing bacteria are cultured with aeration under aerobic conditions in a medium containing various nutrients.

[0011] The culture conditions and composition of the medium are selected from those used in the production of general antibiotics. That is,
The medium contains a carbon source, a nitrogen source, and an inorganic salt, and vitamins, precursors, etc. may be added as necessary. Examples of carbon sources include glucose, sucrose, starch, molasses,
Potato etc. are used alone or as a mixture, and as nitrogen sources, for example, ammonium salts, nitrates, peptone, soybean flour, corn steep liquor, malt extract, yeast extract, etc. are used alone or as a mixture thereof. It will be done. Further, if necessary, antifoaming agents such as silicone oil, soybean oil, and surfactants may be added.

[0012] The medium is preferably a liquid medium, the pH of the medium is preferably 6.0 to 8.0, and the culture temperature is adjusted to 18 to 32°C. The culture period is usually 2 to 7 days. Note that the optimum culture conditions may be selected depending on the characteristics of the producing bacteria used.

[0013] After completion of the culture, the plant physiologically active component MBH-001 can be separated and collected from the culture solution in accordance with the method for separating and collecting ordinary antibiotics and the like from the culture. That is, the sample is collected by appropriately combining extraction methods using various organic solvents, chromatography using various adsorbents, and the like.

The plant physiologically active substance MBH-001 is a new compound having the following physical and chemical properties. (a) Molecular weight: 199 (b) Compositional formula: Cl0, Hl7, N1, O3 (c) Specific rotation: [α]25D = 0 (C = 0.01, methanol) (d) Color of substance: Colorless ( e) Ultraviolet absorption spectrum: λ (ethanol, max
) 210 nm, shown in Figure 1. (f) Infrared absorption spectrum: ν(CHCl3, max
) cm-13388, 2960, 2874, 1779
, 1650, 1376, 1166, 920, 908, as shown in FIG. (g) Solubility in solvents: methanol, butanol,
Easily soluble in dimethyl sulfoxide and ethyl acetate, soluble in chloroform and hexane. (h) Color reaction: ninhydrin reaction positive, Dragendorff reaction positive (i) Acidic, neutral, basic: Neutral (j) Rf value: Silica gel thin layer chromatography (Merck HPTLC, Kieselgel 60) );Hexane/
Ethyl acetate/acetic acid = 60:38:2, Rf value = 0.4 (
k): Proton nuclear magnetic resonance spectrum: shown in FIG. 3. (l) C-13 nuclear magnetic resonance spectrum: shown in FIG. 4.

Furthermore, from the above-mentioned physicochemical properties and spectral analysis results, the chemical structure of the novel plant physiologically active substance MBH-001 was estimated as follows.

[0016]

[Case 2]

Next, the biological activity of the plant physiologically active substance MBH-001 will be explained. MBH-001 has a strong growth-inhibiting effect on both legumes and broad-leaved plants, and can non-selectively control various weeds growing in agricultural land from pre-emergence to the growing season. can. For example, it can control paddy field weeds such as field weeds, Japanese knotweed, and azalea, as well as upland weeds such as Japanese knotweed, Japanese knotweed, Japanese knotweed, blueberry, oat, blackberry, and goldenrod. Furthermore, paddy fields,
It can control various weeds that grow not only in fields, but also in orchards, mulberry orchards, lawns, and non-agricultural lands from before they emerge until the growing season.

When MBH-001 is used as a herbicide, it can be prepared into granules, powders, emulsions, or wettable powders using appropriate solid carriers and emulsifying dispersants in the same way as pesticide formulations known in the art. It can be formulated into any dosage form such as tablets, oils, sprays, and aerosols. As the solid carrier, clay, kaolin, bentonite, acid clay, diatomaceous earth, calcium carbonate, nitrocellulose, starch, gum arabic, etc. can be used. Furthermore, water, methanol, ethanol, dimethylformamide, ethylene glycol, etc. can be used as a liquid carrier. In addition, adjuvants commonly used in formulations, such as sulfuric esters of higher alcohols, polyoxyethylene alkylaryl ethers and their sulfonates, alkylaryl sorbitan monolaurates, alkylaryl sulfonates, alkylaryl polyethylene glycol ethers, A condensate of sodium dinaphthylmethane disulfonate and formalin, alkyldimethylbenzyl ammonium chloride, and the like can be appropriately blended.

Furthermore, the herbicide of the present invention can be used by appropriately mixing with other fungicides, herbicides, insecticides, fertilizers, and soil conditioners.

[0020]

EXAMPLES The present invention will be explained in more detail with reference to Examples below, but these Examples are not intended to limit the scope of the present invention in any way.

Example 1 Nl-Ishi-YC50807-1-7 strain was used as a seed medium,
That is, in a medium (pH 7.3) containing 1.7% tryptone, 0.3% soytone, 0.25% glucose, 0.5% sodium chloride and 0.25% dipotassium phosphate,
After culturing at 28°C for 48 hours, the resulting culture solution was supplemented with 1.7% tryptone. Soytone 0.3%, glucose 0.25%,
Sodium chloride 0.5% and dipotassium phosphate 0.25
% and cultured at 28°C for 3 days, and the resulting culture solution was stored frozen at -20°C.

Ethyl acetate was added to 4000 ml of the obtained culture solution (pH 9) and extracted with stirring. Then, the aqueous layer is p
After adjusting to H6.5, ethyl acetate was added again and extraction was carried out with stirring to obtain 1.5 g of ethyl acetate extracted fraction. After drying this ethyl acetate extraction fraction under reduced pressure, it was dissolved in methanol,
Purification by silica gel column chromatography (Kieselgel 60; hexane/ethyl acetate/acetic acid =
98/0/2 to 48/50/2, gradient elution) The active fraction was concentrated under reduced pressure to obtain 27 mg of an oil. The obtained oil was purified by silica gel preparative TLC to obtain 5 mg of pure MBH-001.

[0023] Next, formulation examples of the present invention will be explained, but the present invention is not limited thereto. In addition, in the formulation example “
"Parts" means parts by weight.

Formulation Example 1 (hydrating powder) 10 parts of plant physiologically active substance MBH-001, 10 parts of polyoxyethylene alkylaryl ether sulfonate
1 part, 2 parts of a condensate of sodium dinaphthyl methanesulfonate and formalin, and 78 parts of clay were mixed and ground to obtain 100 parts of a wettable powder.

Formulation Example 2 (Liquid) 10 parts of a physiologically active plant substance MBH-001, 20 parts of dimethylformamide, 10 parts of ethylene glycol, 10 parts of alkyldimethylbenzylammonium chloride and 50 parts of methanol were mixed and dissolved to obtain 100 parts of a liquid. Ta.

Next, the herbicidal effects of the herbicide of the present invention on various weeds will be specifically explained using test examples.

Test Example 1 A PVC pot with an area of 170 cm 2 and a height of 12 cm was filled with paddy soil, and after applying chemical fertilizer in an amount equivalent to 40 kg/ha, water was added and plowed to create a paddy field. Seeds of Japanese millet, Japanese azalea, and azalea were sown here, and two paddy rice seedlings at the two-leaf stage were transplanted into each seedling. 20 pots
The plants were grown in a greenhouse at ~30°C, and two days after transplantation, a hydrating powder prepared according to Formulation Example 1 was diluted with water and applied to a flooded rice field. The herbicidal effect was investigated 30 days after the chemical treatment. The herbicidal effect was evaluated by observing the weed control rate and expressed as a score of 0 to 10 according to the following criteria. The results are shown in Table 1. (Rating) (Weed control rate) 10
100%9
90-99%8
80-89%7
70-79%6 60
~69%5 50-59%
4 40-49%3
30-39%2
20-29%1
10-19%0
0~9%

[0028]

[Table 1]

Test Example 2 A PVC pot with an area of 170 cm 2 and a height of 12 cm was filled with field soil, and chemical fertilizer was applied in an amount equivalent to 30 kg/ha. Seeds of Japanese fir tree, Japanese knotweed, Japanese knotweed, Japanese blueberry, oat, blackberry, and goldenrod were sown here, and two days after sowing, a wettable powder prepared according to Formulation Example 1 was diluted with water and sprayed onto the soil surface. The herbicidal effect was investigated 30 days after the chemical treatment. The herbicidal effect was evaluated by observing the weed suppression rate, and the rating was 0 to 10 based on the same criteria as Test Example 1.
expressed in numbers. The results are shown in Table 2.

[0030]

[Table 2]

From the above results, it was revealed that the preparation containing the plant physiologically active substance MBH-001 of the present invention as an active ingredient exhibits a high herbicidal effect on various weeds and is useful as a herbicide. .

[0032]

Effects of the Invention As is clear from the above test examples, the novel plant physiologically active substance MBH-001 exhibits excellent herbicidal effects against various weeds.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum of MBH-001.

FIG. 2 shows the infrared absorption spectrum of MBH-001.

FIG. 3 shows a proton nuclear magnetic resonance spectrum of MBH-001.

FIG. 4 shows the C-13 nuclear magnetic resonance spectrum of MBH-001.

Claims (1)

[Scope of Claims] [Claim 1] MBH-001 represented by Formula 1. [Claim 1] [Claim 2] MBH- having the following physical and chemical properties
001. (a) Molecular weight: 199 (b) Compositional formula: Cl0, Hl7, N1, O3 (c) Specific rotation: [α]25D = 0 (C = 0.01, methanol) (d) Color of substance: Colorless ( e) Ultraviolet absorption spectrum: λ (ethanol, max
) 210 nm (f) Infrared absorption spectrum: ν(CHCl3, max
) cm-13388, 2960, 2874, 1779
, 1650, 1376, 1166, 920, 908 (g) Solubility in solvents: methanol, butanol,
Easily soluble in dimethyl sulfoxide and ethyl acetate, soluble in chloroform and hexane (h) Color reaction: positive ninhydrin reaction, positive Dragendorff reaction (i) Acidic, neutral, basic: neutral (j) Rf Value: Silica gel thin layer chromatography (Merck HPTLC, Kieselgel 60); hexane/
Ethyl acetate/acetic acid = 60:38:2, Rf value = 0.4 [
Claim 3: Nl-Ishi-YC50807-1-7, which is an insect symbiotic microorganism having MBH-001 productivity.
KK. [Claim 4] Nl-Ishi-YC50807-1
A method for producing MBH-001, which comprises culturing the -7 strain and collecting MBH-001 from the culture. 5. A herbicide containing MBH-001 as an active ingredient.