JP3256265B2 - Novel antibiotic RS-28A, method for producing the same and fungicide for agricultural and horticultural use - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel antibiotic, a method for producing the same, and a fungicide for agricultural and horticultural use containing the antibiotic as an active ingredient.

[0002]

2. Description of the Related Art Conventionally, heavy metal compounds such as copper agents, mercury agents, arsenic agents, and organic chlorine-based agents have been used as agricultural and horticultural fungicides. However, all of them are harmful to animals and human bodies, cause soil contamination, and remain in nature to act on plants for a long time. Therefore, environmental pollution by these chemicals has become a serious social problem, and their use has been banned or restricted. However, various plant diseases including the main disease of rice tend to increase with the decrease of the target drug and the emergence of resistant bacteria. Therefore, as a countermeasure, there is a strong demand for the development of a new pesticide which shows a remarkable effect on the target disease and is highly safe.

[0003]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a novel antibiotic effective against various plant diseases and a method for producing the same. It is a further object of the present invention to provide a fungicide for agricultural and horticultural use containing the above antibiotic as an active ingredient.

[0004]

SUMMARY OF THE INVENTION Antibiotic RS-28A
Is a substance having the following physicochemical properties. Melting point: 126-128 ° C Elemental analysis: carbon 59.01%, hydrogen: 8.67% Specific rotation: [α] _D ²⁰ = + 35.4 ° (C = 0.5
% Methanol) UV absorption spectrum: λ ^MeOH _max = 212 nm (E
^1% _{1 cm} = 98) Infrared absorption spectrum: 3375, 2920, 173
0, 1630, 1595, 1455, 1368, 128
0, 1180, 1065, 967, 900, 875, 5
75 cm ^-1 Solubility: Easily soluble in methanol, ethanol and butanol, poorly soluble in water, insoluble in acetone, aceto = tolyl, chloroform, ethyl acetate and benzene. Molecular weight: 1554 (by HRFABMS) (molecular formula: C ₇₈ H ₁₃₈ 0 ₃₀₎ Color reaction: Lemieux, KM _n O _4, iodine vapor, anisaldehyde ─ positive in sulfuric acid reaction, ninhydrin, radon Smith, negative for Dragendorff reaction. Material color: white powder

[0005] The antibiotic RS-28A was isolated from a culture obtained by culturing RS-28, a microorganism belonging to the genus Streptomyces, isolated from soil collected from Fujian Province, China. RS-28 bacteria are microorganisms having the following mycological properties.

(1) Morphological characteristics The present strain, RS-28 strain, is 1% yeast extract,
The cells were cultured in a liquid medium containing 1% glucose.
Although N, hydrochloric acid was hydrolyzed at 110 ° C. for 18 hours, thin layer chromatography detected L, L-diaminopimelic acid, but did not detect meth-diaminopimelic acid. Electron microscopic observation of the growth on the agar plate,
The aerial system is screw-shaped, with a smooth, egg-shaped spore surface.
A large number of spore chains are formed at the tip of the aerial system.

2 Growth on various media (27
C., cultivation for 20 days, color tone was determined by Color Harmony Manual 4th edition (Container).

1) Starch yeast agar medium Growth: Normal Aerial system: partially formed Aerial system color: white (a) Back color: bright yellow (2na) Soluble dye: none

2) Yeast extract / malt extract agar medium Growth: Good Aerobic: Normal Aerobic color: Pearl (2ba) Back color: mustard gold (2pe) Soluble dye: None

3) Oatmeal agar medium Growth: good Aerobacteria: Many Aerobacteria Color: Shell pink (5ba) Back color: Maize (2ga) Soluble dye: None

4) Starch / inorganic salt agar medium Growth: good Aerobacteria: Many Aerobacteria Color: Pink tint (7ba) Back color: Light melon yellow (3ea) Soluble pigment: None

5) Tyrosine agar medium Growth: good Aerobic system: Normal Aerobic system Color: Pearl (2ba) Back color: Amber (3pe) Soluble pigment: Dark brown (3pn)

6) Sucrose nitrate agar medium Growth: Poor Aerobic system: None Back color: Pearl (2ba) Soluble pigment: None

7) Glucose / asparagine agar medium Growth: good Aerial: Normal Aerial Color: Shell Pink (5ba) Back Color: Maize (2ga) Soluble Pigment: None

8) Glycerol-asparagine agar medium Growth: Normal Aerobic: Low Aerobic color: White (a) Back color: dark brown (3 pn) Soluble pigment: Covert brown (3 nl)

9) Nutrient agar medium Growth: Ordinary aerial system: None Back color: Light wheat (2ea) Soluble pigment: None

10) Peptone / Yeast extract / Iron agar medium Growth: Normal Aerobic system: None Back color: Light wheat (2ea) Soluble pigment: None

Use of 3 sugars D-glucose good growth ++ D-fructose good growth ++ L-rhamnose does not grow ─ raffinose does not grow ─ D-xylose has good growth ++ L-arabinose has good growth ++ sucrose does not grow ─ inositol growth Good ++ D-mannitol good growth ++ D-galactose good growth ++

From the above properties, it is clear that the RS-28 bacterium belongs to the genus Streptomyces. The RS-28 strain has been deposited with the Research Institute of Microbial Industry of the National Institute of Advanced Industrial Science and Technology under the deposit number No. 12737 (FERM P-12737) on February 3, 1992.

In addition, naturally occurring and artificial mutants of the above RS-28 strain, as well as microorganisms belonging to the genus Streptomyces and capable of producing the antibiotic RS-28A, are all used in the method of the present invention. Can be.

From the above, it is clear that the present bacterium belongs to Streptomyces, and the present strain is referred to as Streptomyces sp. Called RS-28.

[Culture method and purification method] The antibiotic R of the present invention
In obtaining S-28A, the antibiotic-producing bacterium belonging to the genus Streptomyces can be cultured by an ordinary method for producing an antibiotic. The form of the culture may be liquid culture or solid culture. For industrially advantageous culture, a spore suspension or culture solution of the above-mentioned production bacterium may be inoculated into a medium, and aerated and stirred culture may be performed.

The nutrient source of the medium is not particularly limited, and a carbon source, a nitrogen source and the like usually used for culturing microorganisms can be contained in the medium. As a carbon source, starch, dextrin, glycerin, glucose,
Galactose, inositol, mannitol, etc., and nitrogen sources include peptone, soy flour, meat extract, rice bran, koji, urea, corn steep liquor, ammonium salts, nitrates, and other organic or inorganic nitrogen compounds. In addition, inorganic salts, for example, salt, phosphates, potassium, calcium, zinc, manganese, metal salts such as iron and the like may be appropriately added. Etc. may be added.

Culture conditions such as culture temperature and culture time are selected so as to be suitable for the growth of the bacterium to be used and to maximize the production of RS-28A. For example, the pH of the medium is 4-9,
In particular, it is preferably around neutral, and the optimal temperature for culture is preferably about 25 to 35 ° C. However, these culture compositions, the culture conditions such as the hydrogen ion concentration of the culture medium, the culture temperature, and the stirring conditions should be appropriately adjusted so as to obtain favorable results depending on the type of the strain used and the external conditions. Needless to say,

From the culture thus obtained, RS
In order to obtain -28A, the metabolite can be collected by appropriately using a commonly used means. For example, RS
Means utilizing the difference in solubility between -28A and impurities, means utilizing the difference in ionic binding force, means utilizing the difference in adsorption affinity, and means utilizing the difference in molecular weight are each used alone or in combination as appropriate. Or used repeatedly. Specifically, RS-28A is mostly present in the culture solution. When the culture solution is purified by a combination of various ion exchange chromatography, gel filtration chromatography, liquid chromatography, cellulose partition chromatography and the like, a fraction containing RS-28A and other active ingredients is obtained.

The powder obtained by freeze-drying this fraction is further subjected to high performance liquid chromatography (for example, High-
Carbon ODS, acetonitrile {methanol}
Purified with water (0.15% formic acid)
A purified white powder of 28A is obtained.

Thin layer chromatography: Thin layer plate manufactured by Merck, USA 0.25 mm silica gel Solvent Chloroform: Methanol = 2: 1 Rf value 0.23 Solvent Chloroform: Methanol = 1: 1 Rf value 0.36 Solvent Butanol: Methanol: Water = 4 : 1: 2 Rf value 0.61

Minimum growth inhibitory concentration of antibiotic RS-28A for various microorganisms Test strain MIC (μg / ml) ────────────────────────────────── Botrytis cinerea 30 (Botrytis cinerea) Colletotricum lagenarium 2 (Colletotrichum lagenarium) Pyricularia oryzae 30 (Pyricularia oryzae) Chlorella vulgaris 1 (Chlorera vulgaris) Albicans 2 (Candida albicans) ──────────────────────────────────

[Comparison with other substances] A known antibiotic having a terminal absorption in the UV spectrum and having a molecular weight of 1500 or more and 1600 or less was compared and searched with a computer using a computer. There was no one. Also, no antibiotic has the molecular formula C ₇₈ H ₁₃₈ O ₃₀ . Therefore, it was concluded that RS-28A was a novel antibiotic. [Agricultural and horticultural fungicide containing RS-28A as an active ingredient]

When the active ingredient of the present invention is used as a fungicide for agricultural and horticultural use, an appropriate solid carrier, liquid carrier, emulsifying dispersant and the like are usually used in the same manner as in agricultural chemical formulations known in the art. Granules, powders, emulsions, wettable powders, tablets,
It can be formulated into any dosage form such as oils, sprays and aerosols and applied. As these carriers, clay, kaolin, bentonite, acid clay, silicon earth, calcium carbonate, as a solid carrier, nitrocellulose, starch,
Gum arabic and the like, and the liquid carrier include water, methanol, ethanol, acetone, dimethylformamide, ethylene glycol and the like. In the preparation, commonly used adjuvants, for example, higher alcohol sulfates, polyoxyethylene alkyl allyl ether, alkyl allyl polyethylene glycol ether, alkyl allyl sorbitan monolaurate, alkyl Allyl sulfonate, quaternary ammonium salt, polyalkylene oxide and the like can be appropriately blended.

The mixing ratio of the active ingredient is suitably about 10 to 90% for emulsions and wettable powders, and about 0.1 to 10% for powders and oils. These concentrations may be increased or decreased as appropriate.

The agent of the present invention can be used by being appropriately mixed with other fungicides, herbicides, insecticides, fertilizer substances, and soil conditioners.

[Examples] The present invention will be specifically described below with reference to production examples, examples and test examples. In addition,
“%” And “parts” represent 1% by weight and parts by weight, respectively.

Example 1 Glucose 2%, soluble starch 1%, meat extract 0.1
%, Dried yeast 0.4%, soy flour 2.5%, salt 0.2
% And a composition of 0.005% dipotassium phosphate 7
Wherein the culture medium 0ml inoculation with RS-28 strain was 48 hours with shaking cultured at 27 ° C. in 100 of K ₁ flask.

This culture was centrifuged to separate the cells and the culture filtrate. Each was extracted with butanol, and the butanol layers were combined and concentrated. This concentrated solution was used as Diaion HP-1.
0 column (3 × 30 cm) was eluted in the order of 50% methanol, 70% methanol, and 100% methanol. Since the 100% methanol eluted fraction had activity, it was concentrated and dried to obtain 519 mg. This was added to the top of an MCl gel column (2.1 × 81 cm) and eluted in the order of 80% methanol, 90% methanol. The eluate was fractionated, and the active fraction was collected, concentrated and dried to obtain 108 mg. This was further purified by high performance liquid chromatography. The column is Nucleosil High-Carbon ODS (20 mm in diameter,
(Length: 25 cm) and the solvent is 78% (acetonitrile: methanol = 5: 3) in a system of 22% water and 0.15% formic acid at a flow rate of 8.5 ml / min.
The RS-28A material appeared in minutes. The active fraction is repeatedly collected, concentrated and lyophilized to give the substance (R
10 mg of pure white powder of S-28A) were obtained.

Example 2 [Wettable powder] 10 parts of RS-28A, 5 parts of sodium lauryl sulfate, 2 parts of a condensate of sodium dinaphthylmethanedisulfonate and 83 parts of clay were mixed and pulverized to obtain 100 parts of a wettable powder. Obtained.

Example 3 [Emulsion] 8 parts of RS-28A, ethylene glycol, 1 part
0 parts, 20 parts of dimethylformamide, 10 parts of alkyl dimethylbenzyl ammonium chloride and 52 parts of methanol were mixed and dissolved to obtain 100 parts of an emulsion.

Example 4 [Powder] 0.2 part of RS-28A, 0.5 part of calcium stearate, 50 parts of talc and 9.3 parts of clay were mixed and pulverized to obtain 100 parts of powder.

Example 5 [Granules] 10 parts of RS-28A, 15 parts of starch, 72 parts of bentonite and 3 parts of sodium salt of lauryl alcohol sulfate were mixed and pulverized to obtain 100 parts of granules.

Test Example 1 [Control Test for Cucumber Anthracnose] Cucumber (cultivar:
After sowing Sagami Hanjiro), the wettable powder prepared according to Example 2 was diluted to a predetermined concentration and sprayed on seedlings grown for 15 days, and then air-dried. Cucumber anthracnose (Colletot)
Ricum legenarium) was cultured on a potato glucose agar plate medium, and the obtained spores were suspended in distilled water. After the drug solution was dried, the cucumber was transferred to an inoculation box, and the suspension was spray-inoculated with 4 ml per four cucumber seedlings using a spray gun. Cucumber seedlings after inoculation
After 24 hours, the cells were placed under artificial lighting under constant temperature and high humidity conditions, and after 4 days, the degree of onset was examined. Table 1 shows the results.

[0041]

[Table 1]

[Test for Control of Cucumber Bacterial Bursal Disease] After seeding of cucumber (variety: Sagami Hanjiro), the wettable powder prepared according to Example 2 was diluted to a predetermined concentration in seedlings grown for 15 days. After spraying, it was air-dried. Cucumber sclerotium (Sc
(Erotinia scerotiorum) was cultured on a potato-glucose agar plate medium, and the bacterial flora was inoculated in two places per leaf of a cucumber seedling. The cucumber seedlings after inoculation were placed under constant temperature and high humidity conditions, moved under artificial lighting 24 hours later, and examined the extent of disease 4 days later. Table 2 shows the results. The control value was determined by the following equation.

[0043]

(Equation 1)

[0044]

[Table 2]

[0045]

According to the above test results, the fungicide for agricultural and horticultural use of the present invention shows an extremely high control value even at a low concentration against the above-mentioned plant diseases, and shows no phytotoxicity and high safety. It became clear.

[Brief description of the drawings]

FIG. 1 shows an ultraviolet absorption spectrum (in methanol) of the antibiotic RS-28A of the present invention.

FIG. 2 is a view showing an infrared absorption spectrum (potassium bromide tablet) of the antibiotic RS-28A of the present invention.

FIG. 3: Proton NM of the antibiotic RS-28A of the present invention
R (CD ₃ in OD) is a diagram showing the spectrum.

FIG. 4 shows carbon 13 of the antibiotic RS-28A of the present invention.
NMR is a diagram showing a (0~110PPM, CD ₃ in OD) spectrum.

FIG. 5 shows carbon 13 of the antibiotic RS-28A of the present invention.
It is a figure which shows an NMR (110-220 PPM) spectrum.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Hiroyuki Nagata 2-1 Hirosawa, Wako-shi, Saitama Pref. (72) Inventor Isamu Yamaguchi 2-1 Hirosawa, Wako-shi, Saitama Pref. (72) Inventor Riki Arie, 2-1 Hirosawa, Wako-shi, Saitama Pref., RIKEN (72) Inventor Toshiyuki Shimizu 2-1 Hirosawa, Wako-shi, Saitama Pref., RIKEN (72) Inventor, Keimei Guo 2354 Shado, Shanghai, China No. Within the Jianghai Agrochemical Research Institute (72) Inventor Shen Tora's first No. 2354, Shado-ro, Shanghai, People's Republic of China Inside the Jianghai Agrochemical Research Laboratory (72) Inventor Ni Changchun No. 2354, Shado-ro, Shanghai, People's Republic of China In the laboratory (72) Inventor Takaaki Ming No. 2354, Shado-ro, Shanghai, People's Republic of China No. 2354, Jianghai Agrochemical Research Institute (72) Inventor Zhang Yunba, Shanghai, China No. 2354, Shangdo Road, Shanghai City Agrochemical Research Institute (56) References JP-A-59-154992 (JP, A) (58) ) Field surveyed (Int. Cl. ⁷ , DB name) C07G 11/00 A01N 63/02 C12P 1/06 BIOSIS (DIALOG) WPI (DIALOG) JICST file (JOIS)

Claims (4)

(57) [Claims] 1. An antibiotic RS having the following physicochemical properties:
-28A. Melting point: 126-128 ° C Elemental analysis: carbon 59.01%, hydrogen: 8.67% Specific rotation: [α] _D ²⁰ = + 35.4 ° (C = 0.5%)
Methanol) UV absorption spectrum: λ ^MeOH _max = 212 nm (E
^1% _{1 cm} = 98) Infrared absorption spectrum: 3375, 2929, 173
0, 1630, 1595, 1455, 1368, 128
0, 1180, 1065, 967, 900, 875, 5
75 cm ^-1 Solubility: Easily soluble in methanol, ethanol and butanol, poorly soluble in water, insoluble in acetone, acetonitrile, chloroform, ethyl acetate and benzene. Molecular weight: 1554 (according to HRABMS) (molecular formula: C ₇₈ H ₁₃₈ O ₃₀ ) Color reaction: Remu, KMnO ₄ , iodine vapor, positive for anisaldehyde-sulfuric acid reaction, negative for ninhydrin, Radon-Smith, and Dragendorff reactions. Material color: white powder 2. Streptomyces
es) culturing an RS-28 producing bacterium belonging to the genus,
2. The antibiotic RS-28A according to claim 1, wherein the antibiotic RS-28A is separated and collected from the culture.
Manufacturing method. 3. The method according to claim 1, wherein the RS-28 producing bacterium is Streptomyces sp. RS-28.
s sp. RS-28). 4. A fungicide for agricultural and horticultural use, comprising the antibiotic RS-28A according to claim 1 as an active ingredient.