JPH0276594A - Novel antibiotic y-9 and y-10, production thereof and plant disease controlling agent containing the same - Google Patents

Abstract

PURPOSE:To obtain antibiotics Y-9 and Y-10 and a plant disease controlling agent containing the above-mentioned antibiotics as active ingredients by culturing a bacterium belonging to the genus Bacillus and capable of producing antibiotics Y-9 and Y-10 under aerobic conditions in a culture medium containing various kind of nutrious foods. CONSTITUTION:A bacterium, e. g., Bacillus subtillis TM-105 bacterium (FRM P-10013) belonging to the genus Bacillus and having ability capable of producing antibiotics Y-9 and Y-10 is subjected to aerobic culture in a culture medium containing various kind of nutrious foods under aerobic conditions. The culture medium is preferably liquid medium and preferably used at pH6.0-8.0 and 20-35 deg.C. After culturing, the antibiotics Y-9 and Y-10 are collected from the cultured product in combination with extraction by various kind of organic solvent, chromatography by various kind of active absorbent, etc. The obtained antibiotics Y-9 and Y-10 have excellent action to various kind of plant pathogenic mold fungus.

Description

[Detailed Description of the Invention] [Object of the Invention] (Industrial Application Field) The present invention relates to novel antibiotics Y-9 and Y-10, a method for producing the same, and a plant disease control agent containing the same as an active ingredient. It is.

More specifically, the novel antibiotics Y-9 and Y-10 of the present invention
is obtained by culturing antibiotic Y-9 and Y-10-producing bacteria belonging to the genus Bacillus and separating and collecting the culture solution.

(Prior art and problems to be solved by the invention) Conventionally,
Many peptide antibiotics are known as antibiotics produced by microorganisms belonging to the genus Bacillus. Unlike substances, there are no known documents on it.

[Structure of the Invention] (Means and Effects for Solving the Problems) The novel antibiotics Y-9 and Y-10 of the present invention and their manufacturing method will be described in detail below.

The microorganisms used in the present invention are antibiotics Y-9 and Y-1.
,0 production capacity, and Bacillus (Bacillus).
It is a bacterial species belonging to the genus S. llus). One example is Bacillus subtilis TM-105 (Bacillus subtilis TM-105).
lus subtillisTM-105) has the property of advantageously producing the antibiotics Y-9 and y-io of the present invention, and can be effectively used in the method of the present invention.

The Bacillus subtilis TM-105 bacterium (rTM
-105 bacterium) was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on May 2, 1986, under the microbial accession number, Kikoken Bacteria No. 10013.

TM-105 bacteria has the following mycological properties.

a) Morphology (1) Bacterial shape: rod-shaped (2) Cell size 0.5-0.8 μ x 1.5-3.0 μ (3) Pleomorphism: None 4) Motility: Yes Lateral flagella (5) Spores: Yes, ellipsoidal or cylindrical spores, neutral spores.

(6) Durham staining: positive b) Growth status The cells were cultured at 30°C and observed for 7 days.

+1) Meat juice agar plate culture: After 3 days at 30°C, it is roughly 4+n++ irregularly circular.

It is hemispherical, white, and has an opaque consistency.

(2) Meat juice agar slant culture: It grows on the surface and is opaque, milky white with a slightly yellowish tinge.

(3) Meat juice liquid culture: It grows in large quantities, becomes cloudy, and then precipitates.

(4) Ridmus Milk: It is peptonized and the pH is slightly alkaline.

C) Physiological properties (1) Nitrate reduction: Positive (21 VP test: Positive (3) Indole formation: Positive (4) Starch hydrolysis: Positive (5) Utilization of citric acid (Koser medium) Medium properties (6
) Utilization of propionic acid: Negative (7) 7% NaCl2 medium: Growth (8) Pigment production (1% glucose and tyrosine were added to meat juice agar medium to examine pigment production): Negative (9) Casein degradation: Disassemble.

(10) Catalase: Positive (11) Anaerobic culture: Positive (12) Egg yolk reaction: Negative (13) Tyrosine decomposition: Negative (14) Hippurate decomposition: Negative (15) Oxidase: Positive (16) From carbon source Acid production Glycerol: positive L-arabinose: positive D-xylose: positive galactose: negative D-glucose: positive D-fructose: positive D-mannose: positive inositol, positive mannitol: positive sorbitol, positive salicin: positive maltose, positive lactose : Positive sucrose: Positive trehalose, Positive starch: Positive (17) Growth range: pH: 5.0-10. O1 optimum pH +7.2~7
．． 5 Temperature: 15-55°C (does not grow at 60°C) Optimum temperature = 20-25°C d) Sabaloud dextrose medium (manufactured by Difco),
Bergy's Manual Determinative Batteriology (Bergy's M
annual of Deternative Ba
Compared to the description in cteriol-ogyl 8th edition, 1974, this strain is an aerobic Gram-positive bacillus and has motility using lateral flagella, so it is similar to Bacillus.
s) is determined to belong to the genus. Among the Bacillus genus, it was identified as a strain of Bacillus subtilis based on characteristics such as hippurate decomposition, growth temperature, and availability of carbon sources.

Examples of microorganisms used in the present invention include TM-105 bacteria, as well as its natural and artificial mutant strains, as well as bacterial species belonging to the genus Bacillus, including antibiotic Y-9, which will be described later.
Any microorganism capable of producing Y-10 can be used in the present invention.

In carrying out the present invention, antibiotic Y-9 and Y-10 producing bacteria belonging to the genus Bacillus can be cultured by a conventional method for producing antibiotics. For industrially advantageous production, bacteria producing antibiotics Y-9 and Y-10 may be cultured with aeration under aerobic conditions in a medium containing various nutrients.

Culture conditions and medium composition may be selected based on those used in the production of general antibiotics.

That is, the medium basically contains a carbon source, a nitrogen source, and an inorganic salt, and if necessary, vitamins, precursors, and the like may be added. Examples of carbon sources include glucose, arabinose, xylose, starch, dextrin, glycerin,
Mannitol, organic acids, molasses, potato, etc. are used alone or as a mixture, and nitrogen sources include, for example, peptone, soy flour, corn stew liquor, malt extract, amino sugar, rice sugar, malt, urea, ammonium salts. etc. or a mixture thereof may be used. Further, if necessary, antifoaming agents such as silicone oil, soybean oil, and surfactants may be added.

The medium is preferably a liquid medium, the pH of the medium is preferably about 6.0 to about 8.0, and the culture temperature is preferably adjusted to about 20 to about 35°C.

After the completion of the culture, the antibiotics ¥-9 and Y-10 can be separated and collected from the culture in accordance with the usual method for separating and collecting fermentation products from the culture. That is, by appropriately combining extraction methods using various organic solvents, chromatography using various active adsorbents, etc., antibiotics Y-9 and Y-10 were extracted.
Collect.

(Examples of the Invention) Hereinafter, the present invention will be explained in more detail with reference to Examples, but these Examples are not intended to limit the scope of the present invention in any way.

Example I TM-1,05 bacteria were grown in a seed medium (glucose 1%, peptone 0.5%, potassium phosphate 0.01%, magnesium sulfate 0.005%, pH 6,8) at 30°C for 24 hours. The resulting culture solution was inoculated into a fermentation medium (composition is the same as the seed medium), and cultured at 30°C for 5 days.

After removing the bacterial cells from the resulting culture solution at 5° C. by centrifugation, the pH was adjusted to 2.0, and the resulting precipitate was collected by centrifugation. This was extracted with methanol to obtain a methanol-extracted fraction. The methanol extraction section was subjected to silica gel TLC and solvent system: 1-butanol:acetic acid:water = 65:10
:25 (V/V/V)), the active fraction was scraped off, eluted with methanol, and concentrated to dryness under reduced pressure to obtain a powder.

The obtained powder was dissolved in methanol and 5henshuP
AK (manufactured by Senshu Kagaku) ODS-1 (-4251) was packed in a high-pressure column chromatography column, methanol, water: 10% diethylamine monoacid buffer (pH
7,5)/8:1:1 (V/V/V) antibiotics Y-9 and Y-10 were eluted in this order. Y-9 and Y-10 were each further applied to a Sephadex L H-20 column and eluted with 100% methanol. Y-921mg and Y-1012m
g was obtained.

Antibiotics Y-9 and Y-10 thus obtained are novel antibiotics having physicochemical and biological properties as described below.

(1) Molecular weight and molecular formula % formula %) (2) Ultraviolet absorption spectrum λ::g” 224.nm (IIL+ 105.4
)】2 276r+a+ (EjL 8.8)￥-10 λm::' 224nm (Eir-99,3) 276
nm (Ejcn, 8. 7) (see Figure 1) (3) Infrared absorption spectrum
(See Figure 2) (4) Solubility in solvents Soluble in methanol and dimethylformamide, insoluble in methylene chloride and ether (5) Color reaction Lydon-Smith, ninhydrin color reaction positive, Mauritschko reaction negative.

(6) Water-soluble amino acid composition (analysis using an amino acid analyzer of hydrolyzate) Y-912111, [11111 Y-101211011111 Hydrolysis was performed under the conditions of 6N-HCI 2.110″C for 18 hours.

(7) Fatty acid Y-94-hydroxyhexadecanoic acid molecular weight 272 (EI analysis after TMS conversion) Y-104-hydroxyhexadecanoic acid molecular weight 272
(E1 analysis after TMS conversion) Analysis of chloroform extract of hydrolyzed product (3N-HC, e, 110°C for 18 hours) (8) Nuclear magnetic resonance spectrum (see Figure 3) Using FX-90 manufactured by JEOL It was measured. Solvent double methanol.

Antibiotics Y-9 and Y-10 with the above physicochemical properties
differs from similar known peptide antibiotics in the structure of its constituent amino acids and fatty acids. That is, when antibiotics Y-9 and Y-10 are compared with Fengycin, the water-soluble amino acid composition of Fengycin is 3.
G1u, 0G1n, whereas Y-
9, ¥-10 is different in that it is 2GLu, 1G1n,
In the fatty acid structure, Fengycin has a structure from C15 to C
], up to 8 saturated or unsaturated fatty acids, whereas Y
-9 and Y-10 differ in that 4-hydroxyhexadecanoic acid is a constituent fatty acid. Details of the differences from known antibiotics are shown in Table 1 below.

Next, the biological activities of antibiotics Y-9 and Y-10 will be explained. Antibiotics Y-9 and Y-10 are for Durham positive bacteria,
Although it does not have antibacterial activity against negative bacteria, yeast, and green algae, it does show growth-inhibiting action against filamentous fungi. The antibacterial activity against typical bacterial strains is shown in Table 2. The antibacterial activity test shown in Table 2 was conducted by a diffusion method using filter paper soaked with Y-9250 ppm:Y-10115 ppm.

As mentioned above, antibiotics Y-9 and Y-10 can be used as agricultural fungicides because they exhibit excellent effects against various plant-pathogenic fungi. When this compound is used as an agricultural fungicide, it can be prepared into granules, powders, emulsions, and water using suitable solid carriers, liquid carriers, emulsifying dispersants, etc., in the same way as pesticide formulations known in the art. Japanese medicine,
It can be formulated into any dosage form such as tablets, oils, sprays, atomizers, etc. and applied. These carriers include clay, kaolin, bentonite, acid clay, diatomaceous earth, calcium carbonate, solid carriers such as nitrocellulose, starch, gum arabic, and liquid carriers such as water, methanol, ethanol, acetone, dimethylformamide, and ethylene. Examples include glycol and the like. In addition, in terms of formulation,
Commonly used adjuvants, such as sulfuric esters of higher alcohols, polyoxyethylene alkyl allyl ethers, alkyl allyl sorbitan monolaurates, alkyl allyl sulfonates, quaternary ammonium salts, polyalkylenes Oxide etc. can be appropriately blended.

The blending ratio of active ingredients is 10 to 9 for emulsions and hydrating agents.
Approximately 0% is appropriate, and for powders, oils, etc., 0.1
Approximately 10% to 10% is appropriate, but these concentrations may be increased or decreased as appropriate depending on the purpose of use.

In addition, the drug of the present invention may be used with other fungicides, herbicides, insecticides,
It can be used by appropriately mixing it with fertilizer substances and soil conditioners. The present invention will be specifically explained below using formulation examples.
The present invention is not limited to this in any way.

In addition, "parts" in the formulation examples represent parts by weight.

Formulation Example 1 (Wettable powder) 10 parts of antibiotic Y-10, 5 parts of sodium lauryl sulfate, 2 parts of dinaphthylmethane disulfonic acid soda formalin condensate, and 83 parts of clay were mixed and ground to obtain 100 parts of a wettable powder.

Formulation Example 2 (Emulsion) 108 parts of antibiotic Y-1, 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.

Next, the effect of controlling various plant diseases by the agricultural and horticultural fungicide of the present invention will be specifically explained using test examples.

Test Example 1 (Control test against rice blast) Rice paddy (variety: Aichi Asahi) was sown directly into cultivation pots, 10 pots/
At the four-leaf stage, a hydrating powder prepared according to Formulation Example 1 was diluted to a predetermined concentration and 50 ml per pot was sprayed onto the complement using a spray gun. After the spray solution had dried, rice blast fungus (Pyricularia oryzae) was cultured at 28°C for 14 days in an oatmeal medium (1 g of oatmeal, 0.4 g of sucrose, 0.3 g of agar, 20 ml of water).
) was made into a suspension and sprayed onto the complement in an inoculation box.

After inoculation, the mice were left in an inoculation box at 25° C. and 90% relative humidity for 24 hours, and then taken out of the inoculation box and left to stand, resulting in disease onset 5 to 7 days after inoculation.

In addition, the control value was calculated according to the following calculation method.

The results are shown in Table 3.

Mouse 11 Sasei Kaori] Belly drum 1 Marosukedo L Sakamasho] L No antibiotic Y-920085 Y-1020087 // 100 81 // Test example 2 [Control test against rice sesame leaf blight] Rice paddy (variety: Aichi Asahi) directly sown in cultivation pots 10 plants/
At the 5-leaf stage, a hydrating powder prepared according to Formulation Example 1 was diluted to a predetermined concentration and 50 ml per pot was sprayed onto the complement using a spray gun. After the spray liquid has dried, the PSA
Medium (20 ml of potato broth, 0.4 g of cane sugar, 0.0 g of agar)
Rice sesame leaf blight fungus (Cochliobolus mlyabeanus) cultured at 28°C for 114 days at 3g)
The spores were suspended and spray inoculated onto the complement in an inoculation box.

After inoculation, the mice were left in an inoculation box at 25° C. and 90% relative humidity for 24 hours, then taken out of the inoculation box and left to stand, and the disease developed 3 to 4 days after inoculation.

The control value was calculated according to the following calculation method.

The results are shown in Table 4.

×100 Summer A-s Antibiotic Y-920086 None 100 3 Q // Y-10200901/ 100 82 // Triazine 500 99 // From the above results, antibiotics Y-9, Y-10 of the present invention or a mixture thereof It has been revealed that a drug containing as an active ingredient is effective as a fungicide for agricultural chemicals, showing an extremely high control value against various plant diseases and causing no phytotoxicity.

[Effects of the Invention] According to the present invention, it is possible to provide novel antibiotics Y-9 and Y-10 that exhibit excellent effects against various plant pathogenic filamentous fungi.

[Brief explanation of the drawing]

Figure 1 (1) and (2) are antibiotics Y-9 and Y-, respectively.
Figure 2 (1), (2) shows the ultraviolet absorption spectra of 10
) show the infrared absorption spectra of antibiotics Y-9 and ¥-10, respectively, and FIGS. 3(1) and (2) show the 13c nuclear 6R gas resonance spectra of antibiotics Y-9 and Y-10, respectively. Figure 1 (1) Figure 1 (2) District's Σ---- District's

Claims (1)

[Claims] 1. New antibiotic Y-9 and Y-10 substances characterized by having the following physicochemical and biological properties (1) Molecular weight and molecular formula Y-9 1462 (FAB-MS) C_7_2H_1_1_0N_1_2O_2_0Y-1
0 1490 (FAB-MS) C_7_4H_1_1_4N_1_2O_2_0 (2) Ultraviolet absorption spectrum λ^M^e^O^H_m_a_x224nm (E^1^
%_1_c_m105.4)276nm(E^1^%_
1_c_m8.8)λ^M^e^O^H_m_a_x2
24nm (E^1^%_1_c_m99.3) 276n
m (E^1^%_1_c_m8.7) (3) Infrared absorption spectrum Y-9 λ^K^B^r_m_a_x1750.1658.15
40cm^-^Y-10 λ^K^B^r_m_a_x1760, 1660, 15
50cm^-^1 (4) Solubility in solvents Soluble in methanol, dimethylformamide, methylene chloride, insoluble in ether (5) Color reaction Lydon-Smith, ninhydrin color reaction positive, Molish reaction negative. (6) Water-soluble amino acid composition [there is an amino acid sequence] (7) Fatty acid Y-94-hydroxyhexadecanoic acid molecular weight 272 (EI analysis after TMS conversion) Y-104-hydroxyhexadecanoic acid molecular weight 272 (EI analysis after TMS conversion) 2 , bacteria producing antibiotics Y-9 and Y-10 belonging to the genus Bacillus were cultured, and new antibiotics Y-9 and Y-10 were obtained from the culture.
Novel antibiotic Y characterized by separating and collecting 10
-9, manufacturing method of Y-10. 3. Bacillus subtilis TM-105 (Bacillus subtilis TM-105, Microtechnical Research Institute No. 10013) is cultivated under aerobic conditions in a nutrient medium containing assimilable carbon and nitrogen sources.
Method described. 4. A plant disease control agent containing the novel antibiotic Y-9 or Y-10 as an active ingredient.