JPS6261001B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to the new antibiotic herbicidin E and/or
Or it relates to a bactericidal herbicide containing F as an active ingredient. New actinomycete Streptomyces saganonensis
It is already known that the antibiotics herbicidin A and B produced in the culture solution of strain No. 4075 have antifungal, bactericidal, and herbicidal activities (Japanese Patent Application Laid-open No. 101523/1983). The inventors of the present invention conducted intensive research to further search for antibiotics with bactericidal and herbicidal effects, and as a result, they obtained new antibiotics, herbicidin E and F, and discovered that these have excellent bactericidal and herbicidal effects. The fungicidal herbicide of the present invention has an excellent bactericidal effect against a wide range of bacteria and filamentous fungi that parasitize and cause harmful effects on agricultural and horticultural crops. It also shows control effects against viral diseases such as cucurbit fungus, gray mold, and tobacco mosaic virus. In addition, it can be used to prevent the germination of seeds of higher plants, and when weeding rice fields after transplanting young seedlings and before the germination of weeds.
Regular weeds and broad-leaved weeds can be effectively controlled without causing any harm to transplanted paddy rice. On the other hand, regular weeds and broad-leaved weeds can be effectively exterminated by treating the stems and leaves of weeds in fields. In particular, herbicidin F exhibits an outstanding control effect on perennial weeds in paddy fields, and is also useful as a growth inhibitor for turfgrass. In other words, the active ingredient of the present invention can be used not only as a fungicide for agriculture and horticulture, but also for purposes such as weeding by appropriately selecting the application method, dosage, timing, location, etc. can be played separately or simultaneously. Herbicidin E and F substances, which are the active ingredients of the present invention, have the following physical and chemical properties. 1 Appearance

【table】 2 Melting point or decomposition point Herbicidin E 172-173℃ 〃 F 150-154℃ (decomposition) 3 Elemental analysis (%)

【table】 4 Ultraviolet absorption spectrum

[Table] 5 Molecular weight Herbicidin E 537 (FD mass spectrum) Herbicidin F 535 (FD mass spectrum) 6 Solubility Herbicidin E: Soluble in hot water and warm methanol Herbicidin F: Water, methanol, ethanol, acetone, ethyl acetate, methylene chloride Soluble in 7 Stability Both Herbicidin E and F are unstable in acids and alkalis 8 Silica gel chromatography On a silica gel plate for thin layer chromatography (glass plate, containing a fluorescent indicator, manufactured by Merck & Co.), chloroform:methanol ( 7:3) and observed under ultraviolet light, the R _f values of herbicidin E and F substances are 0.69 and 0.72, respectively. Herbicidin E and F substances, which are the active ingredients of the present invention, can be obtained by culturing microorganisms belonging to the Streptomyces stratum that can produce these substances, and producing herbicidin E and F substances in the culture.
Manufactured by collecting these. A specific example of a microorganism that can produce herbicidin E and F substances is Streptomyces saganonensis No. 4075, and the mycological properties of this strain have already been described in the Journal of Antibiotics. ) Vol. 29, p. 863 (1976), and this bacterial strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, and its microbial accession number is Microbiological Research Institute No. 1821. In addition, Streptomyces saganonensis M403, an artificially mutated version of this strain,
M403R and m17-403 strains have even better productivity of herbicidin E and F, and these bacteria have also been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, and their deposit numbers are respectively No. 4974, No. 4975, and No. 4976. As seen in other Streptomyces strains, the bacterial strain used in the present invention is susceptible to changes in properties, such as exposure to ultraviolet rays, X-rays, high frequency,
It can be mutated by artificial mutagenesis methods using radiation, chemicals, etc., and strains of this bacterium that have been mutated by artificial methods are, of course, microorganisms belonging to the genus Streptomyces, and have no ability to produce herbicidin E and F substances. Any substance having the following can be used as the active ingredient according to the present invention. As a method for culturing production bacteria for producing herbicidin E and F substances, the above-mentioned bacterial strain is cultured in a medium containing nutrients that can be used by ordinary microorganisms. As the nutrient source, any known nutrient source conventionally used for culturing Streptomyces bacteria can be used. For example, glucose, sucrose, starch, glycerin, starch syrup, molasses, soybean oil, etc. can be used as the carbon source. Further, as the nitrogen source, soybean flour, wheat germ, meat extract, peptone, yeast cells, corn steep liquor, ammonium sulfate, sodium nitrate, etc. can be used. other than this,
Calcium carbonate, salt, potassium chloride, as needed.
In addition to adding inorganic salts such as phosphates, organic and inorganic substances that aid the growth of bacteria and promote the production of herbicidin E and F substances can be added as appropriate. As for the culture method, the liquid culture method, especially the deep culture method, is most suitable, as is the case with general antibiotic production methods. Cultivation is carried out under aerobic conditions, and the appropriate temperature for cultivation is 22-30°C, but in many cases 24°C
Culture at around ℃. The production of herbicidin E and F reaches its maximum value in 5 to 10 days in both shaking culture and tank culture. The following method is used for assaying herbicidin E and F substances. Silica gel plate for thin layer chromatography (glass plate, containing fluorescent indicator, manufactured by Merck & Co.),
Add 5 microliters of a sample prepared by appropriately diluting the culture solution, and add chloroform:methanol (7:3).
Developed in a mixed solvent of , and measured the ultraviolet absorption using a dual-wavelength chromatography scanner (sample wavelength 258 nm, reference wavelength 380 nm) along with a spot of each standard. When collecting herbicidin E and F substances from cultures, adsorbents such as activated carbon, alumina, and silica gel, synthetic adsorbents such as Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation), and other ion exchange resins and ion exchange Although gel filters and the like are used, the collection method shown below is the most efficient. Cells and other solid matter are separated from the culture solution using a supernatant such as diatomaceous earth, and then the active ingredients in the solution are adsorbed onto Diaion HP-20. After washing the resin part with water, it is eluted using hydrous alcohol. The active fraction in the eluate is concentrated under reduced pressure to distill off the alcohol. The active ingredient is extracted from the obtained concentrate using butanol, methylene chloride, ethyl acetate, etc., and the respective organic solvents are distilled off to obtain a crude powder of the active ingredient. Among the herbicidins, Substance F can be obtained in a crystalline form by recrystallizing the above-mentioned coarse powder with methanol, while Substance E can be obtained in a crystalline form by recrystallizing with methanol after silica gel chromatography. Next, a method for producing Herbicidin E and F will be explained using reference examples. Reference example 1 Streptomyces saganonensis strain M403 was mixed with 2% soluble starch (Lastargen FK),
Soy flour 2%, glucose 1%, KH ₂ PO ₄ 1%,
_CaCO3 0.3%, Diceform CB442 0.02% PH
3 days with preculture medium adjusted to 6.5, and 1
2% soluble starch, 8% soybean flour, 1% glucose, ₁ % _KH2PO4 , _0.3% CaCO3.
%, NH ₄ Cl 0.5%, MgSO ₄ 7H ₂ O 0.02%,
ZnSO ₄・7H ₂ O0.1%, Disform CB442 0.02
% of the main culture medium was transferred with a volume equivalent to 3% of the seed culture broth. The main culture tank has an internal pressure of 1Kg/cm ²
Maintain the temperature at a constant temperature and aerate at an equal rate per minute to remove dissolved oxygen.
Stirring was performed to adjust the concentration to 2 ppm. The culture temperature is
The temperature was kept at 30°C for 2 days, and the temperature was lowered to 24°C from the 3rd day, and the culture was continued for 13 days while adding glucose to maintain the glucose concentration in the culture solution at 1%. Add 10kg of supernatant (Celite) to the culture solution obtained here and filter it under pressure to obtain a solution of 85%
was adsorbed on a column containing Diaion HP-20 8.5 using SV4, and the column was washed with 80% water using SV4, followed by 10% methanol 80%.
Wash with SV4. Then 50% methanol 80
Elute herbicidin A and B with SV2 at SV2, followed by elution of herbicidin E material with 90% methanol 20 at SV2. Here, the total number of fractions containing substance E is 10. This was heated to 50℃ under reduced pressure.
Concentrate to 0.5. This concentrated solution is extracted five times with 0.5 methylene chloride to obtain extract 2.5. After dehydrating this with 250 g of Na ₂ SO ₄ , methylene chloride was distilled off to obtain 18.5 g of an oil. This is silica gel 250 filled with chloroform.
ml column, and with chloroform 1
Wash with SV1, followed by SV1 with chloroform 1 containing 1% methanol. It is then eluted with chloroform 2 containing 2% methanol at SV1. The effective fraction 2 was distilled off at 50°C under reduced pressure, the residue was dissolved in 10 ml of ethyl acetate, 50 ml of n-hexane was added, and the mixture was refrigerated.
600 mg of substance was obtained in crystalline form. Reference example 2 Streptomyces saganonensis m17−
The 403 strain was cultured for 5 days in the same manner as in Reference Example 1.
However, the main culture medium contains 2% soluble starch, 2% defatted soybean flour, 1% glucose, 1% KH ₂ PO ₄ ,
CaCO ₃ 0.3%, fresh yeast 2%, NH ₄ Cl 0.5%,
_MgSO4・_7H2O0.02 %, _ZnSO4・_7H2O0.1 %, PH
It is 6.5. 2.4 kg of celite is added to the broth 30 obtained here, and pressure filtration is performed to obtain a liquid 20.
This was adsorbed onto a column packed with 2 parts of Diaion HP-20 (SV5), washed with 10 parts of water (SV5), then washed with 10 parts of 40% methanol (SV5), and then washed with 45% methanol. Elute (SV3) to obtain G substance containing category 15. Next, elute with 70% methanol (SV3) to obtain F substance containing category 20. The F substance-containing fraction is concentrated to 3 at 45°C under reduced pressure. This was extracted three times with methylene chloride in step 3 to obtain the extract in step 9. After dehydration with 1 kg of Glauber's salt, methylene chloride was distilled off, dissolved in 500 ml of methanol, decolorized with 5 g of charcoal powder, and concentrated to obtain 12 g of herbicidin F substance as crystals. In order to prepare the fungicidal herbicide of the present invention, the herbicidin E and F substances thus obtained are diluted with a carrier, either alone or as a mixture, and if necessary, other adjuvants are added to form powders, coarse powders,
It can be prepared into granules, fine granules, wettable powders, aqueous solutions, liquids, etc. Of course, the purification can be stopped at any stage and the crude product can be used as an active ingredient. A carrier is defined as a synthetic or natural inorganic or organic substance that is mixed into a fungicidal herbicide to aid in the access of the active ingredient to the plant or to facilitate storage, transport, or handling of the active ingredient. means. Suitable solid carriers include inorganic substances such as clay, talc, diatomaceous earth, kaolin, bentonite, calcium carbonate and synthetic calcium silicates, natural and synthetic resins such as coumaron resins, alkyd resins and polyvinyl chloride, carnauba wax, paraffin wax. Examples include wax, etc., shells of nuts such as walnuts and nuts, and soybean flour. Examples of suitable liquid carriers include, for example, water,
Alcohols such as ethanol, isopropanol, and ethylene glycol; glycol ethers such as ethylene glycol monophenyl ether and diethylene glycol monoethyl ether; ketones such as acetone, methyl isobutyl ketone, cyclohexanone, acetophenone, and isophorone;
Contains ethers such as tetrahydrofuran, dioxane, aromatic hydrocarbons such as benzene, toluene, xylene, methylnaphthalene, chlorinated hydrocarbons such as trichlorethylene, carbon tetrachloride, kerosene, light oil or aromatic hydrocarbons. Examples include low boiling point and medium/high boiling point petroleum fractions. Suitable propellants include, for example, chlorofluorocarbon gas, liquefied petroleum gas, methyl ether, and vinyl chloride monomer. Surfactants used for purposes such as emulsification, dispersion, wetting, and spreading may be ionic or nonionic. Suitable anionic surfactants include, for example, sodium or calcium salts of ligninsulfonic acid, sodium or potassium salts of oleic acid, sodium laurylsulfonate,
Examples include sodium or calcium salts of dodecylbenzenesulfonic acid. Suitable cationic surfactants include, for example, higher aliphatic amines, higher aliphatic amine ethylene oxide condensates, and the like. Suitable nonionic surfactants include, for example, glycerol of fatty acids, sucrose esters of fatty acids, ethylene oxide condensates of higher aliphatic alcohols, ethylene oxide condensates of higher fatty acids, ethylene oxide condensates of alkylphenols or alkylnaphthols. and copolymers of ethylene oxide and propylene oxide. The fungicidal herbicides of the present invention may also contain other ingredients, such as gelatin, gum arabic, casein, polyvinyl alcohol, protective colloids such as carboxymethyl cellulose, thixotropic agents such as sodium polyphosphate, bentonite, and the like. The fungicidal herbicide of the present invention can be used with other herbicides such as 2,
4,6-Trichlorophenyl-4'-nitrophenyl ether; 2,4-dichlorophenyl-3'-methoxy-4'-nitrophenyl ether; 5-tert
-Butyl-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazolin-2-one; S-(4-chlorobenzyl)
N,N-diethylthiol carbamate; S-ethyl-hexahydro-1H-azepine-1-carbothioate; 2-chloro-2',6'-diethyl-
N-(butoxymethyl)acetanilide; 2-chloro-2',6'-diethyl-N-(propoxyethyl)acetanilide; 2-methylthio-4,6-
Bisethylamino-S-triazine; 2-methylthio-4-ethylamino-6-(1,2-dimethylpropylamino)-S-triazine; 1-(α,
α-dimethylbenzyl)-3-(p-tolyl)urea; 3-isopropyl-1H-2,1,3-benzothiadiazin-4(3H)-one-2,2-dioxide or fungicide, insecticide It can be mixed with plant growth regulators, fertilizers, etc. to expand the scope of application and save labor. Examples of formulations of the fungicidal herbicide of the present invention are given below. In the text, all parts simply refer to parts by weight. Formulation Example 1 Powder 5 parts of Herbicidin E and 95 parts of clay are uniformly mixed and pulverized to obtain a powder. Formulation Example 2 Wettable powder 50 parts of Herbicidin F, 45 parts of clay, 3 parts of sodium dodecylbenzenesulfonate and 2 parts of polyvinyl alcohol are uniformly mixed and pulverized to obtain a wettable powder. Formulation Example 3 Water Solvent 50 parts of Herbicidin E, 2 parts of polyoxyethylene nonyl phenyl ether, 10 parts of synthetic silicic acid, and 38 parts of ammonium sulfate are uniformly mixed to obtain a water solvent. to obtain an aqueous solvent. Formulation Example 4 Liquid Formulation 10 parts of Herbicidin F, 2 parts of sodium lauryl sulfate, and 88 parts of methanol are uniformly dissolved to obtain a liquid form. Next, the effects of the fungicidal herbicide of the present invention will be explained by giving test examples. Test Example 1 Kiyu Cannabis Disease Control Test A clay pot with a diameter of 9 cm was filled with soil, and young Kiyu cucumber seedlings (variety: Sagami Hanshiro) at the cotyledon stage were transplanted one by one and cultivated in a greenhouse until the single true leaf stage. A chemical solution of a predetermined concentration was sprayed onto the leaves using a spray gun. 24 hours after spraying, the plants were spray-inoculated with a spore suspension of Colletotrichum lagenarium, left in a humid room at a temperature of 20-22°C and 95% or higher for 20 hours, and then transferred to a greenhouse at 24-26°C. After 3 days, the number of lesions formed on the leaves of the test plants was investigated. The results are shown in Table 1.

【table】

[Table] Test Example 2: Weed control test using foliage treatment A clay pot with a diameter of 9cm was filled with sandy loam, and seeds of Japanese radish, Japanese grasshopper, and crabgrass, representative of narrow-leaved plants, and radish and tomato, representative of broad-leaved plants, were sown to approx. The soil was covered with about 3 to 5 mm of soil. The pots were buried in vermiculite packed in a bench in the greenhouse, water was supplied indirectly through the vermiculite, and the sown plants were allowed to grow for about two weeks, and the predetermined concentration of the drug prepared in a hydrating powder was applied. The chemical solution was sprayed directly onto the stems and leaves of the plants using a spray gun. The progress after spraying was observed, and an investigation was conducted on the 7th day. The results are shown in Table 2. The herbicidal effect was determined based on the following criteria. Dead stem and leaf area is 0-10% of the non-sprayed area Judgment 0 〃 10-30% 1 〃 30-50% 2 〃 50-70% 3 〃 70-90% 4 〃 90-100% 5

【table】

[Table] Test Example 3 Pre-emergence soil treatment test for field weeds A clay pot with a diameter of 9 cm was filled with sandy loam soil, and each seed of Japanese sweetberry, Japanese millet, and crabgrass as representatives of narrow-leaved plants, and radish and tomato as representatives of broad-leaved plants were added. The seeds were sown and covered with soil to a depth of 3 to 5 mm. Immediately after covering the soil, a suspension of the test chemical was applied to the soil surface using a pipette, and then transferred to a greenhouse. Two weeks after treatment, the herbicidal effect on each weed was observed. The results are shown in Table 3. Note that the criteria for determining effectiveness are the same as in Test Example 2.

[Table] Test Example 4 Paddy field weed flooded soil treatment test A plastic pot with a surface area of 200 cm ² and a depth of 10 cm was filled with paddy soil, and paddy rice (variety: Jinnanfeng) was prepared.
One seedling at the 2.5-leaf stage was transplanted, and two tubers of each of Prunus japonicum, Cyperus japonica, Kurogwai, and Omodaka were transplanted as representatives of perennial weeds, and seeds of Japanese grasshopper and Firefly were planted as representatives of narrow-leaved weeds, and then seeds of paddy rice were transplanted. Mix well with the soil. Furthermore, one growing stock of Pinus vulgaris is transplanted, and finally, the seeds of Japanese cypress, azalea, and sycamore, representative of broad-leaved weeds, are thoroughly mixed with the soil. The pots were cultivated in a greenhouse for 3 days in a paddy field state, and after the plants took root, the soil was treated with a suspension of the test chemical under water-logging conditions. Three weeks after the treatment, the herbicidal effect on each weed and the chemical damage to paddy rice were observed and determined. The results are shown in Table 4. Note that the criteria for determining effectiveness are the same as in Test Example 2.

【table】

【table】

Claims (1)

[Claims] 1. A bactericidal herbicide containing the new antibiotic herbicidin E and/or F as an active ingredient.