JPS6120273B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is a novel physiologically active substance, herbimycin.
B, its production method, and agricultural and horticultural chemicals containing it as an active ingredient. First, the present inventors cultured a microorganism belonging to the genus Streptomyces in a medium, and thereby incorporated herbimycin (J. Antibiotics32, 255-261,
(1979)), and named this substance ``AM-3672 substance'', and filed a patent application in 1983-
A patent application was filed as 086080. The present inventors discovered that the herbimycin-producing bacterium, Streptomyces hygroscopicus strain AM-3672, produces another unknown compound in addition to herbimycin (AM-3672 substance). As a result of separating and purifying this unknown compound and analyzing its physicochemical and biological properties, this compound was found to be a new compound with a molecular formula of C ₂₈ H ₃₈ N ₂ O ₈ and was named "herbimycin B." It has also been found that this herbimycin B has germination inhibitory and herbicidal activity on higher plants. Herbimycin B is useful as a herbicide because it can kill broad-leaved weeds by pre-emergent soil treatment. It has also been found that this herbimycin B is extremely effective in controlling plant virus infections and does not cause any phytotoxicity, and is therefore useful as a plant virus disease control agent. As a result of further research based on these new findings, we have completed the present invention. (1) Physical and chemical properties of herbimycin B The physical and chemical properties of herbimycin B, the compound of the present invention, are as shown in Table 1.

[Table] (2) Characteristics and culture of producing bacteria Streptomyces hygroscopicus, a Streptomyces actinomycete, is used for the production of herbimycin B.
hygroscopicus) can be used. For culturing this bacterium, a commonly used method for culturing actinomycetes can be used. As a nutrient source for culture, glucose, starch, mannitol, fructose, inositol, etc. are used as a carbon source alone or in combination. Nitrogen sources can be inorganic and organic, such as ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, etc., as well as natural nitrogen sources such as peptone, meat extract, yeast extract, dried yeast, corn steep liquor, soybean. Flour, soybean flour, casamino acids, soluble vegetable protein, etc. can be used alone or in combination. In addition to adding other inorganic salts such as common salt, potassium chloride, calcium carbonate, and phosphates, do not hinder the addition of organic or inorganic substances that promote the growth of this fungus and the production of herbimycin B. The most suitable culture method is a liquid culture method, especially a deep stirring culture method. Culture temperature is 27-32
It is desirable to culture at around neutral °C and pH. When culture is carried out in liquid culture for usually 2 to 4 days, herbimycin B, the substance of the present invention, is produced in the culture solution. The activity of herbimycin B was confirmed by its germination inhibitory effect on radish seeds, and the culture solution was extracted with ethyl acetate, concentrated to dryness, dissolved in methanol, made alkaline, and measured by absorbance at 530 nm. (3) Purification and isolation of herbimycin B For the purification and isolation of the substance of the present invention from the culture solution, commonly used separation and purification methods for microbial metabolic products can be used. Since the substance of the present invention is highly soluble in common organic solvents, ethyl acetate, butyl acetate, isoamyl acetate, chloroform, etc. can be used alone or in combination for extraction. It can then be purified by methods such as solvent fractionation, activated carbon adsorption, and silica gel column chromatography. An example of purification and isolation is shown below. First, the culture solution is extracted with ethyl acetate, and immediately washed with a 5% sodium bicarbonate solution and then a saturated solution of sodium carbonate and sodium bicarbonate to remove impurities, and the resulting neutral fraction is concentrated and dissolved in methanol. After that, activated carbon adsorption is performed. Next, elute with ethyl acetate, concentrate until the ethyl acetate layer becomes slightly cloudy, and heat at 5°C.
Coarse crystals can be obtained by leaving the mixture at . A pure product can be obtained by repeating recrystallization with ethyl acetate. (4) Herbicide containing herbimycin B as an active ingredient To prepare a herbicide containing the compound of the present invention, herbimycin B, as an active ingredient, herbimycin B is prepared according to a known formulation. In combination with suitable active carriers, emulsions, wettable powders, powders,
It is preferable to formulate it into a form convenient for use, such as granules. The carrier used in this case is a solid carrier such as clay, talc, bentonite,
Examples include diatomaceous earth. Examples of liquid carriers include water, alcohols, acetone, and dimethylformamide. Compensation agents in formulations include emulsifiers, dispersants,
Surfactants such as penetrants can be used as appropriate. Next, a herbicide formulation containing herbimycin B as an active ingredient will be specifically explained. However, the composition is not limited to these formulation examples. Note that "parts" below indicate parts by weight. Formulation example 1 Emulsion Herbimycin B 50 parts Dimethylformamide 35 parts Toximal (trade name of Sanyo Kasei) 15 parts Mix and dissolve the above completely to make an emulsion. When using, dilute the above emulsion 50 to 1000 times with water, and add 0.01 to 100% of herbimycin B per 10 ares.
Spread to 1 kg. Formulation example 2 Wettable powder Herbimycin B 25 parts Zeekrite A (trade name) 45 parts Solpol 5039 (surfactant, manufactured by Toho Chemical) 20 parts Carplex (anti-caking agent) 10 parts Mix and grind the above ingredients uniformly. and use it as a hydrating agent. When using, dilute the above hydrating agent 50 to 1000 times with water so that herbimycin B is 0.01 to 10 times per 10 ares.
Spread to 1 kg. (5) Plant virus disease control agent containing herbimycin B as an active ingredient To prepare a plant virus disease control agent containing the compound of the present invention, herbimycin B, as an active ingredient, herbimycin B can be mixed with a known formulation. Accordingly, in combination with a suitable inert carrier, an emulsion,
It is preferable to formulate it into a form convenient for use, such as a wettable powder or a powder. The carrier used in this case is a solid carrier such as clay, talc, bentonite,
Examples include diatomaceous earth. Examples of liquid carriers include water, alcohols, acetone, and dimethylformamide. As formulation aids, emulsifiers, dispersants,
Surfactants such as penetrants can be used as appropriate. Next, a specific formulation example of a plant virus disease control agent containing herbimycin B as an active ingredient will be explained. However, the composition is not limited to these examples. Formulation example 3 Wettable powder Herbimycin B 1 part Siegrite A (trade name) 95 parts Solpol 5039 (surfactant: manufactured by Toho Chemical Co., Ltd.) 2 parts Carplex (anti-caking agent: manufactured by Shionogi & Co., Ltd.)
Mix and grind 2 or more parts uniformly to make a wettable powder. When used, the above-mentioned hydrating agent is diluted with water and sprayed at a predetermined concentration. Hereinafter, the present invention will be explained in detail with reference to Examples. Example 1 Streptomyces hygroscopicus AM- precultured on Waxmann agar medium containing glucose as the main component.
Glucose
2.5%, soybean flour 1%, potassium chloride 0.4%, yeast extract 0.25%, meat extract 0.1%, ammonium sulfate
0.5%, dipotassium phosphate 0.02%, calcium carbonate
15 ml of medium containing 0.3% (PH7.2 before sterilization) (caliber 24
m/m test tube) and cultured with shaking at 30°C for 3 days to prepare a seed culture. This seed culture solution 1
ml was inoculated into a 500 ml Erlenmeyer flask containing 100 ml of a medium with the same composition as above, and cultured at 30°C in a shaking culture machine with a rotation speed of 240 rpm. Culture was stopped at 72 hours and purification was performed. This culture solution 1 was centrifuged at 7000 rpm, and the resulting supernatant (PH6.6) was extracted twice with 500 ml and 300 ml of ethyl acetate. Wash this extract with 500ml of 5% sodium bicarbonate, then add 500ml
and twice with 300 ml of a saturated solution of sodium bicarbonate and sodium carbonate. The ethyl acetate layer was concentrated to dryness to obtain 450 mg of crude powder. After dissolving this concentrate in 100 ml of methanol, 1 g of activated carbon "Refined Shirasagi" was added and stirred for 1 hour. 50ml of activated carbon powder obtained through this process
The mixture was extracted with ethyl acetate, and the ethyl acetate layer was concentrated until it became slightly cloudy and left at 5°C to obtain crude crystals. By repeating this process and reconsolidating, herbimycin B becomes a crystal.
4.5 mg was obtained. When we measured the physical and chemical properties of this herbimycin B crystal, we found that the first
Obtained the results in the table. Example 2 A liquid medium with the same composition as shown in Example 1 was
Dispense 20 of each into three 30-volume jar fermenters, and after sterilizing them, add a medium of the same composition in advance.
300ml of seed culture solution cultivated for 72 hours for each plant
The cells were inoculated and cultured at 30°C for 43 hours (aeration rate
20/min, rotation speed 350rpm, internal pressure 0.5Kg/cm ² ). Transfer this culture solution to a shear press type centrifuge.
The bacterial cells were removed at 10,000 rpm, and the resulting supernatant was concentrated to about 1/4 volume, and then extracted with 10 portions of ethyl acetate. This ethyl acetate layer was washed with a 5% sodium hydrogen carbonate solution in Steps 5 and 2 and a saturated sodium hydrogen carbonate solution in Step 3, and further washed with distilled water in Step 7 to remove impurities. When the ethyl acetate layer was concentrated, a gummy substance appeared. was gotten. Dissolve this gummy substance in 2 methanol,
60g of activated carbon "Refined Shirasagi" was added and stirred for 1 hour. After washing the activated carbon obtained through this process with methanol, it was extracted with 2.5 ml of ethyl acetate, and the extract was concentrated to about 150 ml and left at 5°C. The obtained crude crystals are repeatedly re-crystallized to obtain a pure product, and then
Herbimycin B, which has the physicochemical properties shown in the table,
600 mg was obtained. Example 3 Sterilized diluvial soil was placed in a plastic box measuring 15 cm long, 22 cm wide, and 6 cm deep, and rice, Japanese wild grass, crabgrass, cyperus, core grass, purslane, dog mustard, leafminer chrysanthemum, corn, and soybean were sown in a plastic box of approximately 1.5 cm in width and 6 cm in depth. After covering the soil with cm, the active ingredient of the compound herbimycin B of the present invention obtained in Example 2 and the control drug were sprayed on the soil surface at a predetermined ratio. The chemical solution used for spraying was the emulsion of Formulation Example 1 diluted with a predetermined amount of water, and was uniformly sprayed over the entire surface of the soil using a small sprayer. Three weeks after spraying the chemical solution, the herbicidal effect on rice and various weeds was investigated according to the following criteria. The results are shown in Table 2. Judgment criteria Weed killing rate 90% or more………………………………5 〃 70-90%………………………………4 〃 40-70%………………………………3 〃 20-40%………………………………2 〃 5-20………………………………1 〃 5% or less (almost no effect) 0 However, the above weed killing rate is The weight of above-ground plants in the chemically treated area and the weight of above-ground plants in the untreated area were measured and calculated using the following formula. Weed killing rate (%) = (1 - above-ground grass weight in treated area / above-ground grass weight in non-treated area) × 100

[Table] From the results in Table 2, the herbicidal activity of the compound herbimycin B of the present invention is high, and it does not cause any phytotoxic effects on crops such as rice, corn, and soybean, and on crops such as Koakaza, Portulaca, Purslane, and Cucumber. Highly effective against broad-leaved weeds. It also has strong herbicidal activity against Cyperaceae, which is generally difficult to kill. That is, herbimycin B is used for rice,
It has no phytotoxic effects on corn and soybeans, and exhibits strong herbicidal activity against broad-leaved weeds.
It can be used as a herbicide in upland rice cultivation, cotton fields, and soybean fields. Example 4 Tobacco mosaic virus disease infection prevention effect test was conducted using tobacco grown in pots [variety: Nicotiana
Using glutinosa (Nicotiana glutinosa),
A herbimycin B solution adjusted to a predetermined concentration was applied with a brush to one side of one leaf, while only water was applied to the other side to provide an untreated plot. After 24 hours of air drying, apply tobacco mosaic virus solution on both sides.
It was inoculated by rubbing it with carborundum.
After inoculation, the surface of the leaves was washed with water, left to stand in a greenhouse at 25℃, and the number of local lesions that appeared after 3 days was counted.
The inhibition rate was calculated. The results are shown in Table 3. The method for calculating the inhibition rate is as follows. Inhibition rate (%) = (1 - number of local lesions in treated area / number of local lesions in untreated area) × 100

【table】 [Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum (methanol solution) of the compound herbimycin B of the present invention, Figure 2 shows the infrared absorption spectrum (KBr method), and Figure 3 shows the proton nuclear magnetic resonance spectrum (deuterium dimethyl sulfoxide).

Claims (1)

[Claims] 1. Having the following physical and chemical properties and having a molecular formula
Herbimycin B, a new compound represented by C ₂₈ H ₃₈ N ₂ O ₈ . Specific rotation: [α] ²⁵ _D = +109° (C = 1, chloroform) Ultraviolet absorption spectrum: As shown in Figure 1 Infrared absorption spectrum: As shown in Figure 2 Proton nuclear magnetic resonance spectrum: As shown in Figure 3 2 An actinomycete strain belonging to the genus Streptomyces and capable of producing the new compound herbimycin B is aerobically cultured in a medium containing nutrients such as a carbon source, a nitrogen source, inorganic salts, and vitamins. 1. A method for producing a new compound herbimycin B, which comprises producing and accumulating the new compound herbimycin B in a liquid and collecting the same. 3 Streptomyces hygrospicus is an actinomycete of the genus Streptomyces.
2. A method for producing the novel compound herbimycin B according to claim 2, which uses F. hygroscopicus) No. 4335. 4. An agricultural and horticultural herbicide characterized by containing the new compound herbimycin B as an active ingredient. 5. A plant virus disease control agent characterized by containing the novel compound herbimycin B as an active ingredient.