JPH0577395B2 - - Google Patents

Description

[Detailed description of the invention]

[Technical Field] The present invention relates to a novel antibiotic RS-44, a method for producing the same, and an agricultural and horticultural fungicide containing RS-44 as an active ingredient. [Background of the Invention] Conventionally, heavy metal compounds such as copper agents, mercury agents, and arsenic agents, as well as organochlorine agents and organic phosphate agents, have been used as disinfectants for agriculture and horticulture. These drugs are harmful to the human body, contaminate the soil, and remain in nature and act on animals and plants for long periods of time.Environmental pollution caused by these drugs has become a serious social problem, and their use is currently prohibited or restricted. . However, various plant diseases, including major diseases of rice, are showing an increasing trend due to the decrease in target drugs and the appearance of resistant bacteria. There is a strong desire for the development of new high-performance agricultural chemicals. [Object of the Invention] An object of the present invention is to provide a method for producing a novel antibiotic effective against various plant diseases. A further object of the present invention is to provide an agricultural and horticultural fungicide containing the above-mentioned antibiotic as an active ingredient. [Structure of the Invention] The antibiotic RS-44 of the present invention is a novel antibiotic that has not been described in any literature and has the physicochemical properties described below.
It has excellent control effects against various plant diseases such as major rice diseases such as rice sheath blight and rice blast, as well as cucumber gray mold, cucumber anthracnose, pear black spot, and grape late rot. It is an excellent agricultural and horticultural fungicide that does not cause any phytotoxicity to plants and has no effect on the human body when applied. The present invention will be explained in detail below. <Production of antibiotic RS-44> (Microorganism used) The microorganism that produces antibiotic RS-44 of the present invention is a bacterial species that belongs to the genus Streptomyces and has the ability to produce antibiotic RS-44. As an example, Streptomyces sp. RS-44 (hereinafter referred to as "RS-44 strain") can be mentioned,
The microorganism has the above-mentioned characteristics, advantageously produces the antibiotic RS-44 of the present invention, and can be effectively used in the production method of the present invention. In addition, not only natural and artificial mutant strains of the above-mentioned RS-44 strain, but also all microorganisms belonging to the genus Streptomyces that have the ability to produce the antibiotic RS-44 described below can be used in the method of the present invention. . The above strain RS-44 is a soil actinomycete isolated from soil collected in Jiangsu Province, China, and was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on March 5, 1985, and its microorganism accession number is , Microtechnology Research Institute
No. 8684 (FERM P-8684). The RS-44 strain has the following mycological properties. 1 Morphological characteristics This strain was obtained by culturing the RS-44 strain in a cell analysis medium containing glucose yeast extract, separating and drying the cells, hydrolyzing them with 6NHCl at 110°C for 20 hours, and using paper chromatography to determine the cell wall amino acids. When this was detected, L,L-diaminopimelic acid was detected. In addition, the aerial hyphae that grew on the agar plate formed a spiral hyphae, on which dozens of spores were formed, but no sporangia or motile spores were observed. Based on the above, this bacterium belongs to Streptomyces. The spores have a cylindrical shape and a smooth surface. 2 Growth conditions on various media (27℃, 3 weeks culture, color tone is based on Descriptive Color Names)
(1) Sucrose nitrate agar medium Growth: No growth (2) Glucose-aspartate agar medium Growth: Normal Aerial hyphae: Trace amount b (Oyster white) Reverse side: b (Oyster white) Pigment: None (3) Glycerin/asparagine agar medium Growth: Normal Aerial mycelium: Trace amount b (Oyster white) Back side: b (Oyster white) Pigment: None (4) Starch/Inorganic salt agar medium Growth: Good Aerial mycelium: Large amount 2ge ( Reverse side: 2ie (light mustard) Pigment: None (5) Tyrosine agar medium Growth: Good Aerial mycelia: Lots of 3DC (natural) Reverse side: 2le (mustard) Pigment: 2ea (light maize) (6) Nutrient agar medium Growth : Good Aerial mycelium: None Back side: 11/2Ca (cream) Pigment: None (7) Yeast malt agar growth: Good Aerial mycelium: Lots of 5fe (Atsushi) Back side: 2lg (Mustard tan) Pigment: None (8) Oatmeal agar Medium Growth: Poor Aerial mycelium: None Back side: Unknown Pigment: None (9) Peptone/yeast/iron agar medium Growth: Good Aerial mycelium: None Back side: 1 1/2ea (light yellow) Pigment: None 3 Assimilation of carbon sources (Pridham-Gottlieb agar medium) L-arabinose - D-xylose + D-glucose + D-fructose - sucrose - inositol - L-rhamnose - raffinose - D-mannitol - +... grows. −……Does not develop. 4 Other physiological properties 1 Optimum temperature 25-35℃ 2 Liquefaction of gelatin (glucose, peptone,
On gelatin medium) Liquefy 3 Hydrolysis of starch (on starch inorganic salt agar medium) Hydrolyze 4 Coagulation of skim milk Coagulation 5 Liquefaction of skim milk Liquefy 6 Formation of melanin-like pigment (on tyrosine agar medium) None ( peptone-yeast iron agar) None Streptomyces with the above properties were added to Bergey's Manual of Determinative Bacteriology, 8th edition.
According to Determinative Bacteriology (8 th ed.), it belongs to the WiSiC-iSM group and is most closely related to Streptomyces and Cacaoi in terms of color tone and spore morphology; It is estimated that this bacterium is very closely related to this. (Culture method and purification method) In obtaining the antibiotic RS-44 of the present invention,
The antibiotic-producing bacteria belonging to the genus Streptomyces can be cultured by a conventional method for producing antibiotics. The form of culture may be liquid culture or solid culture, and for industrially advantageous culture,
A spore suspension or culture solution of the producing bacteria may be inoculated into a medium and cultured with aeration and stirring. The nutrient source of the medium is not particularly limited, and the medium may contain carbon sources, nitrogen sources, and other sources commonly used for culturing microorganisms. Carbon sources include starch, dextrin, glycerin, glucose, sucrose, galactose, inositol, mannitol, etc. Nitrogen sources include peptone, soybean flour, meat extract, rice bran, koji, urea, cornstap liquor, and ammonium salts. , nitrates and other organic or inorganic nitrogen compounds are used. In addition, inorganic salts such as common salt, phosphates, potassium, calcium,
Metal salts such as zinc, manganese, iron, etc. may be added as appropriate.
Vegetables, mineral oil, etc. may be added. Culture conditions such as culture temperature and culture time are suitable for the growth of the bacteria used, and
Conditions are chosen to maximize RS-44 production. For example, the pH of the medium should preferably be 4 to 9, especially around neutrality, and the optimum culture temperature should be about 25° to 35°C. However, these culture conditions such as culture composition, hydrogen ion concentration of the medium, culture temperature, and stirring conditions should be adjusted as appropriate to obtain favorable results depending on the type of bacterial strain used and external conditions. Needless to say, it is. To obtain RS-44 from the culture thus obtained, it can be collected using appropriate means commonly used for collecting metabolites. For example, any of the methods that utilize the solubility difference between RS-44 and impurities, the ionic binding force difference, the adsorption affinity difference, and the molecular weight difference may be used alone or , used in appropriate combinations or repeatedly. Specifically, RS-44 is mostly present in the culture filtrate. The pH of the culture filtrate is adjusted to an acidic side, for example PH4.0, extracted with an organic solvent such as ethyl acetate, and the resulting oil is subjected to adsorption chromatography, silica gel chromatography, or liquid chromatography. When purified by a combination of methods such as chromatography, ion exchange chromatography, and gel filtration chromatography, a fraction containing RS-44 and other active ingredients can be obtained. For example, apply the oil to silica gel laminate and chloroform-
Develop with methanol system to obtain active fraction. This was further subjected to high-performance liquid chromatography (for example, Nucleosil ₅ C ₁₈ , methanol/water (8:2)).
to obtain a coarse powder. This is then further processed using high-performance liquid chromatography (for example, High-performance liquid chromatography).
carbon CDS) to obtain a purified white powder of RS-44. The physicochemical and biological properties of RS-44 thus obtained are as follows. <Physicochemical and biological properties of antibiotic RS-44> Decomposition point: Gradual decomposition at 160° or higher. Elemental analysis: Carbon 63.36% Hydrogen 8.69%, Oxygen 26.72
% Specific rotation: [α] ²⁰ _D +3.4° (C = 1%, chloroform) Ultraviolet absorption spectrum: λ methanol Max 210,
250nm (sh) (as shown in Figure 1) Infrared absorption spectrum: (KBr method) (as shown in Figure 2) 3400, 2900, 1755, 1695, 1620, 1445,
1375, 1240, 1155, 1085, 970, 900, 715 cm ^-1 Solubility: Easily soluble in methanol, acetone, ethyl acetate, chloroform, and benzene, slightly soluble in water, insoluble in hexane and pentane. Molecular weight: (by FD mass spectrum) 766 (Molecular formula: C ₄₂ H ₇₀ O ₁₂ ) Color reaction: Anisaldehyde-sulfuric acid, positive for 2,4-dinitrophenylhydrazine FeCl ₃ , negative for ninhydrin, Lydon-Smith Color: White powder thin layer chromatography (Merck, USA)
0.25mm silica gel (solvent: chloroform: methanol =
2:1) Rf value 0.42 (solvent ethyl acetate:benzene = 4:1) Rf
Value: 0.16 Antibacterial spectrum: Tested using the standard agar plate dilution method and expressed as minimum inhibitory concentration (MIC).
Bacteria were measured using bouillon agar medium, and molds were measured using potato sucrose agar medium. MIC (mcg/ml) Botrytis cinerea 125 Colletotrichum lagenarium
lagenarium) 125 Pyricularia oryzae
125 Rhizoctonia solani
125 Aspergillus niger
125 Alternaria mali 500 Glomerella singillulata
cingurata) 500 Xanthomonas oryzae
oryzae) 32 Xanthomonas citri
32 Stoohylococcus aureus >500 Escherichia coli BE1186
>500 Bacillus subtilis
＞500 <Comparison with other substances> In the UV spectrum, antibiotics that absorb at 240 nm (shoulder) include Bandamycin, Chalcomycin, and Neutramycin; The molecular weights are different: 500, 600, and 513, and the molecular formulas are also different. Therefore, the RS-44 substance is clearly different from these substances, and it was concluded that this substance is a new substance. (Agricultural and Horticultural Fungicide Containing RS-44 as an Active Ingredient) When using the active ingredient of the present invention as an agricultural and horticultural fungicide, it is generally necessary to use an appropriate pesticide formulation as well as pesticide formulations known in the art. Granules, powders, emulsions, etc. using solid carriers, liquid carriers, emulsifying dispersants, etc.
It can be formulated and applied in any dosage form such as wettable powders, tablets, oil solutions, sprays, and aerosols. These carriers include clay, kaolin, bentonite, acid clay, diatomaceous earth, calcium carbonate, solid carriers such as nitrocellulose, starch, gum arabic, etc., and liquid carriers such as water, methanol, ethanol, acetone, dimethylformamide, etc. Examples include ethylene glycol. In addition, adjuvants commonly used in formulations, such as
Higher alcohol sulfate ester, polyoxyethylene, alkyl allyl ether, alkyl allyl polyethylene glycol ether, alkyl allyl sorbitan monolaurate, alkyl allyl sulfonate, alkyl allyl sulfonate, quaternary Grade ammonium salts, polyalkylene oxides, etc. can be appropriately blended. The appropriate blending ratio of the active ingredient is about 10 to 90% for emulsions and wettable powders, and about 0.1 to 10% for powders, oils, etc. However, depending on the purpose of use, these The concentration may be increased or decreased as appropriate. In addition, the drug of the present invention may be used with other fungicides, herbicides,
It can be used by appropriately mixing with pesticides, fertilizer substances, and soil conditioners. The present invention will be specifically explained below using production examples, working examples, and test examples, but the present invention is not limited thereto. In addition, "%" and "part" represent weight % and weight part, respectively. Production example: 2% glucose, 1% soluble starch, meat extract
0.1%, dry yeast 0.4%, soy flour 2.5%, salt 0.2%,
The RS-44 strain was inoculated into a medium containing 0.005% dibasic potassium phosphate, and cultured with shaking at 28°C for 96 hours. This culture solution 36 was extracted with ethyl acetate at pH 4 and then concentrated under reduced pressure to obtain 4.8 g of an oily substance. Thereafter, a silica gel column with a diameter of 55 mm and a length of 700 mm was prepared using a chloroform-methanol (2:1) system as a solvent, and a sample dissolved in methanol was applied and developed. Fraction volume is 10ml and fraction No.101
An activity category was obtained at −140. When the above active fractions are collected and concentrated under reduced pressure, 1.0
g of concentrate was obtained. Thereafter, RS-44 is purified by high performance liquid chromatography. The column used was Nucleosil ₅ C ₁₈ : diameter 20 mm, length 30 cm, and the solvent was methanol water (8:2).
1% diethylamine-formic acid (PH7.5)
When carried out at a flow rate of 8.0 ml/min and a pressure of 170 Kg/ ^cm2 , RS-44 appears in a development time of 20 minutes. this
Collect the RS-44 portion and adjust the pH to 4 with 0.1N hydrochloric acid.
Concentrate under reduced pressure until free of methanol, extract with ethyl acetate, wash with water, concentrate, and the RS
100 mg of -44 was obtained as a powder. After this, purification was performed by high performance liquid chromatography. The column used was High-Carbon ODS: 10 mm in diameter and 25 cm in length, and the solvent was methanol.
Water-1% diethylamine-formic acid (PH7.0) (8:
1:1) system, flow rate 1.4ml/min, pressure 150Kg/cm ²
If you do this, RS-44 will appear at 23 minutes of deployment time. This RS-44 portion was collected, adjusted to pH 4 with 0.1N hydrochloric acid, concentrated under reduced pressure until methanol disappeared,
Extracted with ethyl acetate, washed with 0.1N hydrochloric acid, then water, and concentrated. After this, it was dissolved in methanol and water was added. Concentrate under reduced pressure until free of methanol and lyophilize to give 40% pure white powder.
mg obtained. Example 1 (hydrating powder) RS-44, 10 parts, sodium lauryl sulfate 5 parts,
Mix and grind 2 parts of sinapthylmethane disulfonic acid sodium formalin condensate and 83 parts of clay to make a wettable powder.
Get 100 copies. Example 2 (Emulsion) 8 parts of RS-44, 10 parts of ethylene glycol, 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 3 (Powder) RS-44, 0.2 parts, calcium stearate 0.5
50 parts of talc and 49.3 parts of clay were mixed and ground to obtain 100 parts of powder. Example 4 (Granules) RS-44, 10 parts, starch 15 parts, pentonite
72 parts and 3 parts of sodium salt of lauryl alcohol sulfate are mixed and ground to obtain 100 parts of granules. Test Example 1 (Control test against Botrytis gray mold) A wettable powder prepared according to Example 1 was diluted to a prescribed concentration and sprayed on young seedlings grown for 15 days after sowing of Kiyu (variety: Sagami Hanshiro). After that, it was air-dried. Botrytis cinerea was cultured on a potato-glyose agar plate medium,
Spores induced by irradiation with BLB light were suspended in a 1% solution of 10% glucose and yeast extract. After the chemical solution dries, transfer the cucumber to the inoculation box,
The suspension was sprayed onto young cucumber seedlings using a spray gun.
10 c.c. spray inoculation was carried out per 10 plants. After inoculation, the cucumber seedlings were left under constant temperature and humidity conditions, and the degree of disease onset was examined 4 days later. In addition, the control value was calculated by the following method. (Incidence Index) (Incidence Level) 0 No disease 1 Slight lesions observed. The affected area is less than 210%. The affected area is 310% or more and 20% or less. The affected area is 420% or more and 30% or less. The affected area is 530% or more and 40% or less. The diseased area is more than 640%. Control value (%) = (1
Sum of disease indexes in treated plots/sum of disease indexes in untreated plots) x 10
0 The results are shown in Table 1.

[Table] Methyl midazole carbamate (effect of the present invention) From the above test results, the agricultural and horticultural fungicide of the present invention exhibits an extremely high control value against the above plant diseases even at low concentrations, and has no effect on phytotoxicity. It was revealed that this method can provide highly safe agricultural chemicals. In addition, the antibiotic RS-44 of this application is referred to as “Antibiotic SR-44” in the Chinese patent application corresponding to this application.
It is the same as the substance called .

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the ultraviolet absorption spectrum of the antibiotic RS-44 of the present invention, and FIG. 2 is a diagram showing the infrared absorption spectrum of the antibiotic RS-44.

Claims (1)

[Claims] 1. Antibiotic RS- having the following physicochemical properties:
44. Decomposition point: Gradually decomposes above 160°. Elemental analysis: Carbon 63.36% Hydrogen 8.69% Oxygen 26.72
% Specific rotation: [α] ²⁰ _D +3.4° (C = 1%, chloroform) Ultraviolet absorption spectrum: λ methanol Max 210, 250n
m(sh) Infrared absorption spectrum: (KBr method) 3400, 2900, 1755, 1695, 1620, 1445,
1375, 1240, 1155, 1085, 970, 900, 715 cm ^-1 Solubility: Easily soluble in methanol, acetone, ethyl acetate, chloroform, and benzene, slightly soluble in water, insoluble in hexane and pentane. Molecular weight: (by FD mass spectrum) 766 (Molecular formula: C ₄₂ H ₇₀ O ₁₂ ) Color reaction: Positive for anisaldehyde-sulfuric acid, 2,4-dinitrophenylhydrozine FeCl ₃ , negative for ninhydrin, Lydon-Smith Color: White powder 2 Production of antibiotic RS-44, which is characterized by culturing antibiotic RS-44-producing bacteria belonging to the genus Streptomyces and separating and collecting antibiotic RS-44 from the culture. Law. 3 The antibiotic RS-44-producing bacterium is Streptomyces sp.
44) The manufacturing method according to claim 2. 4. An agricultural and horticultural fungicide characterized by containing antibiotic RS-44 as an active ingredient.