JPS6320519B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention has an effect of inhibiting spore germination and germ tube elongation of plant pathogenic bacteria, and is thereby useful for preventing and treating undesirable plant diseases. ,
Spore germination inhibitor lturin-A
The present invention relates to a new method for efficiently manufacturing.

Conventional method Aitiurin-A is a general formula isolated from a culture of a certain type of soil fungus belonging to the genus Bacillus. (R in the formula is an alkyl group) It is a known substance having the chemical structure shown below, and is known to have antibacterial activity. {Literature,
(Akira lsogai, Seiji Takayama, Shigeo
Murakoshi and Akinori Suzuki.
Tetrahedron Letters, Vol23, No.30. (1982)
pp3065−3068): (Franqise Peypoux,
Micheline Guinand, Geogres Michel, Lucien
Delcambe, Bhupesh C.Dad and Edgar
Lederer, Biochemistry.Vol17, No.19 (1978)
p3992-3996)} It has been known that Aitiurin-A is produced in the culture solution of soil bacteria belonging to the genus Bacillus, but this method has a low yield and is not suitable for industrial use. It was appropriate.

Problems to be Solved by the Invention The present invention uses a bacterium that has an even better ability to produce aityulin-A than the soil bacteria belonging to Bacillus, and that produces aityulin-A in a short period of time and with high yield. The object of the present invention is to provide a method suitable for industrially producing Aitiurin-A.

Means for Solving the Problems In order to search for spore germination inhibitors of plant pathogenic bacteria, the present inventors used spore germination as an indicator, and conducted extensive searches to find microorganisms with excellent productivity of spore germination inhibitors. As a result, it was unexpectedly discovered that a significant amount of aitiurin-A was produced in the culture solution of bacteria isolated from rice leaves, and based on this finding, the present invention was completed. That is, according to the present invention, Bacillus
subtilis No.NA-apb-1 strain (Feikoken Bacterium 7661
No.) was cultured aerobically in a nutrient medium, and the culture solution contained the phytopathogen spore germination inhibitor Aitiurin-A.
Aitiurin-A can be efficiently produced by producing and accumulating it and then collecting it.

The plant pathogenic bacteria spore germination inhibitor Aitiurin-A-producing bacterium belonging to the species Bacillus subtilis used in the method of the present invention was isolated from rice leaves cultivated in Sendai City, Miyagi Prefecture, and is described below. It has the mycological properties shown in

(a) Form (1) Bacteria shape: rod-shaped (2) Bacteria size: 0.8-3.5μm (3) Polyplasia: none (4) Motility: Yes, with lateral flagella (5) Spores: Yes, (6) Gram staining: positive (7) Anti-acidity: None (b) Growth status The cells were cultured at 28°C and observed for 7 days.

(1) Broth agar plate culture: Massive growth, 2 days later,
1.0-2.0 μm, irregularly circular, cream-colored, and does not produce soluble pigments.

(2) Meat juice agar slant culture: Grows on the surface, opaque, milky white and slightly yellowish.

(3) Meat juice liquid culture: Grows in large quantities, becomes cloudy and then precipitates, giving an orange color.

(4) Litmus milk: It is peptonized and has a slightly alkaline pH.

(5) Growth range: PH6-8 (c) Physiological properties (1) Nitrate reduction: Positive (2) VP test: Positive (3) Indole production: Positive (4) Starch hydrolysis: Positive (5 ) Utilization of citric acid: Positive (6) Utilization of propionic acid: Negative (7) 7% NaCl medium: Growth (8) Azide medium: No growth (9) Production of pigment (1% glucose and Tyrosine was added to check pigment production): Negative (10) Casein decomposition: Decomposed (11) Catalase: Positive (12) Anaerobic culture: Negative (13) Egg yolk reaction: Negative (14) Tyrosine decomposition: Not decomposed (15) Liquefaction of gelatin: Dissolution (16) Lysozyme resistance test: Positive (17) Decomposition of hippurate: Negative (18) Oxidase test: Negative (19) OF test: Oxidation (20) MR test: Negative (21 ) Generation of acids from carbonate sources Glucose: Positive Arabinose: Positive Xylose: Positive Mannitol: Positive Mannose: Positive Sorbitol: Positive Initiate: Positive Trehalose: Positive Fructose: Positive Glycerin: Positive Soluble starch: Positive Satucalose: Positive Maltose: Positive Galactose: Positive Lactose: Positive (c) Sabaroud. Dextrose medium (manufactured by Difco): Growth The above properties are described in Bergeys Manual of Determinative Bacteriology.
Bacteriology) 8th edition, 1974, this strain is an aerobic Gram-positive bacillus with motility using lateral flagella, and is therefore judged to belong to the genus Bacillus. 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, and Bacillus subtilis No.
NA-apb-1 (Bacillus subtilis No.NA-apb-
1) and was given the microorganism accession number 7661 to the Institute of Microbial Technology, Agency of Industrial Science and Technology (June 1982).
11).

Cultivation in the method for producing the plant disease fungus spore germination inhibitor aitiurin-A of the present invention is carried out in a medium containing nutrients that can be used by the strain. The medium composition includes, for example, Jiyagaimo decoction, starch, glycerin, dextrin, carbon sources such as sucrose, maltose, and glucose, peptone, meat extract, yeast extract, casein hydrolyzate, corn, etc.
Starch liquor, gluten meal, inorganic nitrogen sources, etc. are used. In addition, inorganic salts such as calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium chloride, and sodium chloride are added as necessary. A suitable medium temperature is 20 to 30°C. The seed culture may be a solid culture or a liquid culture. In the case of main culture, stirring culture, shaking culture, and aeration culture are used. When antifoaming is required during culture or culture sterilization, antifoaming agents such as silicone oil and surfactants can be used. The inhibitory effect of aityulin-A obtained in the present invention on spore germination of plant pathogenic bacteria can be assayed by the following method. That is, the test bacterial spores are suspended at a predetermined concentration, the spore suspension is mixed with a culture solution containing a bacterial spore germination inhibitor, and the spore suspension is suspended for 24 hours.
After incubating for 24 hours, the specimen was stained with methylene blue and the germination rate was determined.
The effect of aityulin-A can be determined {Reference, Nobuo Ikegawa et al., Bioassay of Physiologically Active Substances (Kodansha Scientific Publishing) (1984)} Cultivation is continued until aityulin-A is substantially accumulated, and the culture of this substance is For extraction from the liquid, since the produced aityulin-A is mainly present in the culture filtrate, it is purified and collected from the supernatant after removing bacterial cells by centrifugation or filtration. In order to purify and collect the inhibitory factor, it is possible to appropriately utilize the means normally used to collect metabolites of microorganisms, such as vacuum concentration, freeze drying, solvent extraction, treatment with a resin, treatment with an adsorbent, The target substance is separated, purified, and collected from the filtrate by crystallization, recrystallization, and other means alone, in combination in any order, or repeatedly.

In a preferred embodiment for collection, after the cultivation is completed, the culture solution is filtered using a filter aid to remove bacterial cells. The obtained filtrate was concentrated under reduced pressure,
Unintended substances are extracted and removed by adding an organic solvent such as ethyl acetate, hexane, chloroform, acetone, etc. The residue obtained here is extracted with alcohol, the extract is concentrated under reduced pressure and chromatographed using an adsorbent such as silica gel. When silica gel is used as an adsorbent, a mixed solvent of butanol, ethanol, chloroform, aqueous ammonia, water, etc. is used as an elution solvent for development and elution. The eluate is appropriately fractionated, fractions containing the target substance are collected, and the fractions are concentrated to dryness under reduced pressure at a low temperature to obtain a crude substance. In order to further purify the obtained crude substance, the target substance is isolated from the culture solution by using an appropriate combination of the above-mentioned means.

Effects of the Invention According to the present invention, Aitiurin-A is a plant pathogenic fungus spore germination inhibitor that is effective for the prevention and treatment of plant diseases.
can be produced efficiently, the method of the present invention is suitable as an industrial method for producing aitiurin.

Examples Next, the present invention will be explained in more detail with reference to Examples.

Example Meat extract, peptone 1%, yeast extract 0.25%,
Bacillus subtilis No.NA-apb-1 strain was inoculated into a liquid medium containing 0.5% sodium chloride, and 28
The cells were cultured aerobically for 50 hours at 30°C. The obtained culture filtrate (30 liters) was concentrated under reduced pressure at low temperature, washed with ethyl acetate, and extracted with methanol. The extract was concentrated to dryness under reduced pressure at low temperature, and the same material was subjected to silica gel (ODS) column chromatography with developing solvent 60.
~80% methanol elutes and contains inhibitory factor, initial flow liquid
2.5 liters was collected. This eluate was concentrated to dryness, and the same operation was repeated three times to obtain 270 milligrams of aithurin-A as a white powder. This product completely matched the standard specimen obtained separately.

Claims (1)

[Claims] 1. Bacillus subtilis No.NA-apb-1 strain was cultured aerobically in a nutrient medium, and the plant pathogenic bacteria spore germination inhibitor Aitiurin-A was added to the culture. 1. A method for producing aitiurin-A, a plant pathogenic fungus spore germination inhibitor, which comprises producing and accumulating a system substance and collecting the same.