JPS5926269B2 - New antibiotic SF-1836 substance, its manufacturing method and its uses - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a new antibiotic substance SF-1836, its production method, and its uses.

More specifically, SF belonging to the genus Streptomyces
This invention relates to a new antibiotic SF-1836 substance collected from the culture obtained by culturing -1836 substance-producing bacteria in a medium, its production method, and its use as a water surface application as a rice leaf blight control agent. The present inventors have demonstrated that a culture of a certain strain belonging to the genus Streptomyces shows antibacterial activity against bacteria, filamentous fungi, and mycoplasma in conventional antibacterial tests such as the agar plate method and liquid dilution method. However, the rice leaf blight fungus Xanthomonas oryzae (Xanthomo-nasory
We have discovered that a substance that suppresses the onset of rice leaf blight is produced only when applied to the water surface of rice inoculated with 2ae), and we have isolated the effective substance purely from the culture material.
As a result of investigating its properties, it was confirmed that it was a new antibiotic that was different from known substances.
The present invention was completed by naming the substance as a substance.

Streptomyces SF- used in the method of the present invention
An example of a 1836 substance-producing bacterium is Strebmyces.
StreptOmycecus SF-1836 strain (StreptOmy
cesza Omyceticus SF-1836).

This strain was used as the 32nd contracted microorganism strain by the Microbial Technology Research Institute.
It has been deposited as No. 54. The mycological properties of Streptomyces zaomyceticus SF-1836 strain are as follows. Form I Aerial mycelium grows well on starch agar, oatmeal agar, yeast/malt agar, tyrosine agar, etc., and forms abundant spores.

The branches are simple and no axle branches are seen. The tips of aerial hyphae are straight or wavy, and do not form spiral threads. No sclerotia formation is observed.

The surface structure of the spores observed by electron microscopy is smooth. Spores are oval to cylindrical and 0.8-1.1 x 0.6-0.8 microns. Spores usually form chains of 10 or more spores. Growth status on various media Physiological properties (1) Growth temperature range: Good growth on starch yeast agar media in the temperature range of 15 to 40°C.

(2) Liquefaction of gelatin: Gradually liquefy by culturing at 20°C for 14 days.

(3) Hydrolysis of starch: Positive (4) Coagulation of skim milk: Positive (28°C) Negative (37°C) Peptonization of skim milk: Positive (28°C, 37(C) (5) Production of melanin-like pigments : Utilization of positive carbon sources (Pridham-Gottlieb agar medium) (1) Utilized: D-glucose, D-xylose, L-arabinose (2) Not utilized: D-Flagdose, D-mannitol, I-inositol, rhamnose , Sucrose, Raffinose To summarize the mycological characteristics of strain SF-1836 from the above,
Aerial hyphae are straight or wavy, and the spore surface structure is smooth.

The color tone of development is white - cream - yellowish brown - blackish brown.P
No change at H. Aerial mycelium is abundant on starch agar, oatmeal agar, and tyrosine agar, and the color is white to gray. Melanin-like pigments are produced in organic media, but weakly in tyrosine agar, and other soluble pigments are not observed.

These characteristics of the SF-1836 strain are related to Streptomyces zaomyceticus (Streptomyces spp.), a member of the genus Streptomyces.
The properties most closely match those of Omycesza Omycetieus).

That is, the aerial hyphae are straight or wavy, the spore surface is smooth, white to gray aerial hyphae are attached, and both are well matched in terms of producing melanin pigment and availability of carbon sources. We are doing so. ISP (International Streptomyces Project) description (Inter-NatiO
nalJOurnalOfSystemat-1cBa
cter1010gy vol.22, p.374-375, 19
Streptomyces zaomyceticus and S.
When strain F21836 is compared, differences are observed in the ability of aerial mycelia to settle on glycerin/asparagine agar and starch agar, but they are also similar in growth color tone. From the above, the SF-1836 strain is different from the ISP-listed strain.
Although they differ in their ability to attach to aerial mycelia on asparagine agar and starch agar, their basic properties are very similar, so it is reasonable to attribute this to the species Streptomyces zaomyceticus. Therefore, the present inventors SF-1836 strain was converted to Streptomyces zaomyceticus SF-1836 (StreptOmyceszaO
It was named my-Ceticus SF-1836).

The properties of the SF-1836 strain tend to change artificially or naturally as in the case of other strains of the genus Streptomyces, but the Streptomyces zaomyceticus strain SF-1836 referred to in the present invention is This includes all of these mutant bacteria. For example, the SF-1836 strain can be mutated by bacterial strain mutation methods such as ultraviolet rays, X-rays, radiation, chemicals, transduction, transformation, and genetic combination; All Strebtomyces bacteria capable of producing the -1836 substance can be used in the method of the present invention. In the method of the present invention, the strain is cultured in a medium containing nutrients that can be used by common microorganisms.

As the nutrient source, any known nutrient source that has been conventionally used for culturing Streptomyces bacteria can be used. For example, glucose, starch, glycerin, starch syrup, molasses, soybean oil, etc. can be used as carbon sources. Also, soybean flour, wheat germ, meat scratches, peptone, dried yeast, cornstarch, ammonium sulfate, sodium nitrate, etc. can be used as the nitrogen source. In addition, calcium carbonate, salt,
In addition to adding inorganic salts such as potassium chloride and phosphates, organic and inorganic substances that aid the growth of bacteria and promote production of the SF-1836 substance may be appropriately added.
The most suitable culture method is the liquid culture method, especially the deep culture method, as is the case with general antibiotic production methods.

Cultivation is carried out under aerobic conditions, and the appropriate temperature for cultivation is 2.
The temperature is 5 to 38°C, but in most cases it is cultured at around 28 to 30°C. The production of SF-1836 substance reaches its maximum in 3 to 6 days in both shaking culture and aeration agitation culture. The culture conditions described above can be applied by selecting the optimum conditions for each depending on the characteristics of the production strain used.

The SF-1836 substance produced in the culture solution is collected and purified using a conventional method for producing antibiotics.

That is, the SF-1836 substance mainly exists in the liquid part of the culture medium, but as described later, since it is a water-soluble amphoteric substance, its depiction and purification was carried out using Amberlite RA-400, . IR-120, Dowex 1X2
This can be adsorbed using an ion exchange resin such as . For example, after decolorizing the culture oral fluid with activated carbon, the active ingredient is added to Amberlite IRA-400 (0H-type).
The adsorbed material was eluted with hydrochloric acid solution, and the
The method of adsorption to 0W×2 (H+ type) and elution with aqueous ammonia is an effective means for purifying the SF-1836 substance.

The crude purified product of SF-1836 substance obtained by this method is added to Dowex 50W x 2 (Na+ type,) and then chromatographed using citric acid buffer, paper chromatography, etc. By doing so, a pure product can be isolated. SF-1836 substance (free type)
The physical and chemical properties of are described below.

(1) Properties: Amphoteric substance with white needle-like crystals (2) Melting point: Does not show a clear melting point up to 300°C.

It starts to change color at 173°C.

(3) Specific rotation: [α] R-140° (C21.0, water) (4) Ultraviolet absorption: As shown in FIG. 1, this is terminal absorption.

(Water) (5) Infrared absorption spectrum: The infrared absorption spectrum obtained by the potash bromide tablet method is shown in FIG.

(6) Hydrogen nuclear magnetic resonance spectrum: The hydrogen nuclear magnetic resonance spectrum in heavy water is shown in FIG.

(100z.TM? section standard) (7) Elemental analysis values: Carbon 53.18, Hydrogen 6.21, Nitrogen 12.21, Oxygen 28.40% Calculated value as C5H7NO2, Carbon 53.10, Hydrogen 6.19, Nitrogen 12.3
9. Oxygen 28.32% (8) Molecular formula: C5H7NO2 (
9) Molecular weight: 113 (self) Stability: Stable between PH2 and 9 a1) Color reaction: Ninhydrin, Greig-Leaback, silver mirror, SOrensen, potassium permanganate reaction positive ( Solubility: Soluble in water, slightly soluble in methanol, ethanol, n-butanol, ethyl acetate, chloroform, benzene, ethyl ether (auto) Rf value in silica gel thin layer chromatography: developing solvent
Rf value n-butanol:acetic acid:water (3:1:1) 0.25phenol:water (75:25) 0.41a4
) Rf value in paper chromatography: developing solution Rf value n-butanol:acetic acid:water (3:1:1) 0.26n-butanol:water (4:1:2) 0. 30SF-1836
The following methods are used to test substances.

When the rice plants grown in pots in a climate controlled room had grown to the 4-5 leaf stage, a single needle was inoculated with the rice leaf blight bacterium XanthOmOnas Oryzae, and 1 day later, 20 ml of samples at various concentrations were inoculated per pot. Irrigate to the edge of the plant.

When the untreated area is kept in an artificial climate room at 28°C and the disease has sufficiently developed (about 9 to 10 days later), the disease severity is investigated and the control value (%) is calculated when the untreated area is set as 0%. S
Substance F-1836 showed the control value as shown in Table 1 in the assay using this substance, and showed a control value of 5mcg/Wll to 25mcg.
/ml, the relationship between the logarithm of the concentration and the control value shows a linear relationship. Note) The () in "Drug damage" indicates that no drug damage is observed.

At 100 mcg//Tnl, SF-1836 substance has no antibacterial effect on Escherichia coli, Staphylococcus, Bacillus subtilis, Salmonella, acid-fast bacteria, fungi, mycoplasma, and plant pathogenic bacteria and filamentous fungi, including rice leaf blight bacteria. It has no antibacterial activity against Gram-positive and Gram-negative bacteria, and virtually no antibacterial activity is observed.

However, when applied to the water surface of rice inoculated with rice leaf blight bacteria, the onset of rice leaf blight is completely suppressed at 30 mcg/Tnl. Due to the above-mentioned physicochemical properties and its ability to suppress the onset of rice leaf blight despite having no antibacterial effect, SF-1836 substance has no equivalent among known antibiotics. is recognized as a new antibiotic. Rice leaf blight is considered to be an important rice disease in Southeast Asia as well as in Japan, and its appropriate control is strongly desired.

Substance SF-1836 was invented by the present inventors through a systematic study of an agent for controlling rice bacterial leaf blight produced by actinomycetes, and is characterized as a new antibiotic for controlling rice bacterial leaf blight.

Next, examples will be shown, but it goes without saying that many modifications and modification means not exemplified here may be used in the present invention.

Example 1 Bacterial cells containing spores of Streptomyces zaomyceticus SF-1836 strain (Feikoken Bacteria Contract No. 3254) were mixed with 2.0% starch, 1.0% peptone, 0.3% meat scratches, Inoculated into a liquid medium of dipotassium phosphate 0.05% and ρH7O,
Culture with aeration for 24 hours at 28°C and use this as a seed mother.

Glucose 3.0%, starch syrup 10%, wheat germ 25%,
Soluble vegetable protein 0.5%, soybean oil 0.1%, yeast scratches 0.1%, ferrous sulfate 0.00
1%, nickel chloride 0.0001%, cobalt chloride 0.
0001%, PH7. The seed mother was inoculated into O liquid medium 3001 and cultured with aeration at 28° C. for 120 hours.

After finishing the culture solution, the sap was 280. get e. The sap liquid has a pH of 2.0 with sulfuric acid. After adjusting to O, activated carbon 6K9 was added to decolorize. The decolorizing solution (solid content 0.8%) was adjusted to pH 4.0 with caustic soda. Corrected to O, 501 Amberlight IRA
-400 (0H type 1) was adsorbed with SF-1836 substance, washed with water, and eluted with 0.5N hydrochloric acid to obtain a solution (solid content 3.0%) 721 rich in SF-1836 substance. After concentrating the solution to 121, Dowex 50WX2 (H
+ type) 51, washed with water, and eluted with 0.5N aqueous ammonia. SF-183 thus obtained
Fraction 151 rich in 6 substances was concentrated under reduced pressure to remove excess ammonia to a pH of 6.9 and concentrated to 480 ml (solid content 17%). Next, 150ml of this concentrated solution was transferred to a column (11) of Dowex 50W x 2 (Na+ type).
After adding 0.2M taenoic acid buffer (PH3.25
), and the effective fraction (180m0) was developed with Dowex 50
After desalting with W×2 (H+ type), the residue was concentrated under reduced pressure to obtain a brown powder of about 29. Furthermore, dissolve this powder in a small amount of water,
The mixture was added to 6 sheets of paper (Toyo F Paper Washing 525, 40×40 crrL), and paper chromatography was performed using a solvent system of n-butanol:acetic acid:water (3:1:1). S
A portion containing the F-1836 substance was drawn with water and concentrated under reduced pressure to obtain 324 m9 of brown powder. Subsequently, desalting by column chromatography using the aforementioned Dowex 50W x 2 (Na+ type) 02M citrate buffer (PH 3.25) was performed again to obtain a white powder of 107Tf19. Forty-three white needle-shaped crystals were collected from this white powder using aqueous ethanol. As mentioned above, SF-1836
The substance exhibits excellent control effects on rice bacterial leaf blight when applied to the water surface.

When applying the SF-1836 substance, the substance may be used as is, but in many cases, it may be applied in various forms, such as granules, fine granules, aqueous solutions, and emulsions, which are commonly used as concentrates for water surface application. A dosage form is used, and the preparation is used directly or diluted with water. In this case, the carrier may be either solid or liquid and is not limited to a specific carrier. SF-
Compositions using the 1836 substance can be mixed with other known fungicides, insecticides, acaricides, plant growth regulators, surfactants, fertilizers, etc. to enhance the control effect of rice leaf blight or to save labor. It is possible. When using the SF-1836 substance as a water surface application agent, 1/10 are.
It is preferable to apply the ratio in the range of 00 to 10009. Furthermore, when testing toxicity to fish using medaka and daphnia, no fish toxicity was observed at practical concentrations, and no daphnicidal activity was observed.
Substance 836 can be safely used in rice fields.

Next, some examples of the present invention will be shown, but the content of the main components,
It goes without saying that the types and contents of additives and auxiliary agents are not limited to these examples and can be varied within a wide range.

Example 1 A predetermined amount of a composition obtained by kneading and molding components of granules or more is powdered and used.

Example 2 The above ingredients are mixed and dissolved, and upon use, diluted with water to a predetermined concentration and applied to the root system in a predetermined amount.

Next, the effect of the SF-1836 substance on controlling rice leaf blight will be specifically explained by giving an experimental example.

Experimental Example 1 Test for controlling rice leaf blight by surface application In an artificial climate room (28°C), 8 plants were placed in synthetic resin pots with a diameter of 5 CTfL, and 4 to 5 plants were grown in a paddy field at a water depth of 1 (1-J mo V1). The rice leaf blight bacterium XantOmonas Oryzae was inoculated with a single needle into rice at the leaf stage.

One day later, 50 ml of the sample at a predetermined concentration was injected into the water surface per pot.

The paddy field was kept in an artificial climate room, and when the untreated area had sufficiently developed the disease (about 9 to 10 days later), the lesion length was measured, and the control value (%) was calculated when the untreated area was set as 0%. . As is clear from Table 2, the SF-1836 substance exhibits a high effect of controlling rice leaf blight with a small dose.

[Brief explanation of the drawing]

Figure 1 is an ultraviolet absorption spectral diagram (1,000 PIX [1.H2O) of the SF-1836 substance, and Figure 2 is an infrared absorption spectrum diagram of the SF-1836 substance (KB
Fig. 3 shows the SF-1836 substance 10
It is a proton nuclear magnetic resonance spectrum (in heavy water, TMS external standard) measured at 0M1Iz.

Claims (1)

[Claims] SF-18, a new antibiotic having the following physicochemical constants:
Substance 36: Amphoteric substance exhibiting white needle-like crystals; Specific rotation [α]^2^2_D-140° (C1.0%, water); Average elemental composition in weight ratio: carbon 53.18, hydrogen 6. 21,
Nitrogen 12.21, oxygen 28.40 (%), molecular formula C_5H_7NO_2, molecular weight 113;
It has the ultraviolet absorption spectrum shown in Figure 2, the infrared absorption spectrum (potassium bromide tablet method) shown in Figure 2, and the proton nuclear magnetic resonance absorption spectrum (in heavy water) shown in Figure 3; Soluble in water, sparingly soluble in methanol, ethanol, n-butanol, ethyl acetate, chloroform, benzene, and ethyl ether; positive for potassium permanganate, silver mirror, Seelenzen, ninhydrin, and Gray-Goole back reactions. 2. A method for producing a new antibiotic SF-1836 substance, which comprises culturing SF-1836 substance-producing bacteria belonging to the genus Streptomyces and collecting the SF-1836 substance from the culture. 3. A rice leaf blight control agent for water surface application, characterized by containing the new antibiotic SF-1836 substance as an active ingredient.