JPH0579077B2 - - Google Patents

Description

[Detailed description of the invention]

[Technical Field] The present invention relates to a novel antibiotic RK-634, a method for producing the same, and an agricultural and horticultural fungicide containing RK-634 as an active ingredient. [Background of the Invention] Conventionally, heavy metal compounds such as copper agents, mercury agents, and arsenic agents, as well as organochlorine agents and organic phosphate agents, have been used as disinfectants for agriculture and horticulture. These drugs are harmful to the human body, contaminate the soil, and remain in nature and act on animals and plants for long periods of time.Environmental pollution caused by these drugs has become a serious social problem, and their use is currently prohibited or restricted. . However, various plant diseases, including major diseases of rice, are showing an increasing trend due to the decrease in target drugs and the emergence of resistant bacteria. There is a strong desire for the development of new high-performance agricultural chemicals. [Object of the Invention] An object of the present invention is to provide a new antibiotic effective against various plant diseases and a method for producing the same. A further object of the present invention is to provide an agricultural and horticultural fungicide containing the above-mentioned antibiotic as an active ingredient. [Structure of the Invention] The antibiotic RK-634 of the present invention is a novel antibiotic that has the physical and chemical properties described below and has not been described in any literature, and is excellent against various plant diseases such as gray mold of cucumber and anthracnose of cucumber. It is an excellent agricultural and horticultural fungicide that exhibits a pest control effect, does not cause any chemical damage to plants, and does not have any effect on the human body when used. The present invention will be explained in detail below. <Production of antibiotic RK-634> (Microorganism used) The microorganism that produces the antibiotic RK-634 of the present invention is a bacterial species that belongs to the genus Streptomyces and has the ability to produce antibiotic RK-634. An example of such a microorganism is Streptomyces sp. RK-634 (hereinafter referred to as "RK-634 strain"), which has the above characteristics and is The antibiotic RK-634 of the invention can be advantageously produced and can be effectively used in the production method of the invention. In addition to natural and artificial mutant strains of the RK-634 strain mentioned above, all microorganisms belonging to the genus Streptomyces that have the ability to produce the antibiotic RK-634 described below can be used in the method of the present invention. . The RK-634 strain mentioned above is a soil actinomycete isolated from soil collected in Nago City, Okinawa Prefecture, and was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on June 3, 1989, and its microorganism accession number is FERM P-10771. RK-634 strain has the following mycological properties. 1 Morphological characteristics This strain, RK-634 strain, was cultured in a liquid medium containing 1% yeast extract and 1% glucose, and the cells were hydrolyzed with 6N hydrochloric acid at 110°C for 18 hours. Thin layer chromatography showed that L,L-diaminopimelic acid was detected. When observed under an electron microscope when grown on an agar plate, the aerial fungi are straight and gray in color, while the spores are cylindrical with a smooth surface. 2 Growth conditions on various media (27℃, 20 days culture, color tone is based on Descriptive Color Names Dictionary)
Names Dictionary) 1 Starch/Yeast Extract Agar Medium Growth: Good Aerobic Mycelium: Abundant Aerial Mycelium Color: 5ih (Lead Gray) Soluble Pigment: None 2 Yeast Extract/Malt Extract Agar Medium Growth: Good Aerial mycelium: Rich Aerial mycelium color: 7dc (dark pink) Soluble pigment: None 3 Oatmeal agar medium Growth: Poor Aerial mycelium: None Soluble pigment: None 4 Sucrose inorganic salt agar medium Growth: Good Aerobic mycelium: Rich Aerial mycelium color: 5fe (Thick) Soluble pigment: None 5 Sucrose nitrate agar growth: Good Aerobic Mycelium: Rich Aerial mycelium color: 3DC (Natural) Soluble pigment: None 6 Glucose/asparagine agar growth: Normal air Mycelia: Normal Aerial mycelium color: 2ba (pearl) Soluble pigment: None 7 Glycerol/asparagine agar growth: Good aerobic Mycelium: Rich Aerial mycelium color: 3ba, 5dc (pearl, gray) Soluble pigment: None 8 Nutrient agar medium Growth: Poor Aerial Hyphae: None Soluble pigment: None 9 Tyrosine agar medium Growth: Good Aerobic Hyphae: Abundant Aerial mycelium Color tone: C (gray) Soluble pigment: Dark brown 10 Peptone/yeast extract/iron agar medium Growth: Good atmosphere Mycelium: None Soluble pigment: None 3 Sugar usage D-glucose Use D-xylose 〃 L -Arabinose 〃 D-mannitol 〃 Inositol 〃 Raffinose Suspicious Sucrose 〃 D-fructose Not utilized 4 Other physiological properties This strain was positive for starch hydrolysis, skimmed milk coagulation and liquefaction, and melanin formation reactions. The reactions of gelatin liquefaction and soluble dye formation are negative. From the above, it is clear that this bacterium belongs to Streptomyces, and these properties can be determined according to Verge's Manual of Determinative.
Bacteriology (Bergey's Manual of
When searching for those described in Determi native Bacteriorogy, the closest relative is Streptomyces antebiotex.
antibioticus) and Streptomyces noboritoensis. (Culture method and purification method) In obtaining the antibiotic RK-634 of the present invention,
The antibiotic-producing bacteria belonging to the genus Streptomyces can be cultured by a conventional method for producing antibiotics. The form of culture may be liquid culture or solid culture, and for industrially advantageous culture,
A spore suspension or culture solution of the producing bacteria may be inoculated into a medium and cultured with aeration and stirring. The nutrient source of the medium is not particularly limited, and the medium may contain carbon sources, nitrogen sources, and other sources commonly used for culturing microorganisms. Carbon sources include starch, dextrin, glycerin, glucose, sucrose, galactose, inositol, mannitol, etc. Nitrogen sources include peptone, soybean flour, meat extract, rice bran, wheat gluten, urea, cornstap liquor, and ammonium salts. , nitrates and other organic or inorganic nitrogen compounds are used. In addition, inorganic salts such as common salt, phosphates, potassium, calcium,
Metal salts such as zinc, manganese, iron, etc. may be added as appropriate, and if necessary, animal, vegetable, or
Mineral oil etc. may be added. Culture conditions such as culture temperature and culture time are suitable for the growth of the bacteria used, and RK
The conditions are chosen such that the production of -634 is the highest.
For example, the pH of the culture medium should preferably be 4 to 9, especially around neutrality, and the optimum culture temperature should be about 25 to 35°C. but,
These culture conditions, such as the culture composition, hydrogen ion concentration of the medium, culture temperature, and stirring conditions, should be adjusted as appropriate to obtain favorable results depending on the type of bacterial strain used, external conditions, etc. Needless to say. In order to obtain RK-634 from the culture thus obtained, it can be collected using appropriate means commonly used for collecting metabolites. For example, each of the methods that utilizes the solubility difference between RK-634 and impurities, the ionic bond strength difference, the adsorption affinity difference, and the molecular weight difference may be used alone or , used in appropriate combinations or repeatedly. Specifically, most of RK-634 is present in the culture filtrate. When the culture solution is purified by a combination of various ion exchange chromatography, gel filtration chromatography, adsorption chromatography, liquid chromatography, cellulose partition chromatography, etc., RK-634 and other active ingredients are found. fractions are obtained. The powder obtained by freeze-drying this fraction was further subjected to high-performance liquid chromatography (for example, Nucleozil 5C ₁₈ , 50
% methanol system) and purified by RK-
634 refined white flour powder is obtained. The physicochemical properties and biological properties of RK-634 thus obtained are as follows. <Physical and biological properties of antibiotic RK-634> Shape: White powder Decomposition point: 129-131℃ Elemental analysis: Calculated as carbon 61.28% hydrogen 9.15% nitrogen 3.37% C ₆₃ H ₁₁₁ N ₃ O ₂₀ Value Carbon 61.49% Hydrogen 9.09% Nitrogen 3.41% Molecular weight: 1229 HR−FABMS (M+H) ⁺ m/z;
1230, 7890 Specific rotation; [α] ²⁰ _D = 15.1° (c0.34 methanol) Ultraviolet absorption spectrum: λ ^McOH _nax (E1% 1cm) (shown in Figure 1) 227 shoulder (228), 232 (241) , 240 shoulder (168) Infrared absorption spectrum: in KBr (shown in Figure 2) 990, 1060, 1120, 1180, 1250,
1290, 1370, 1455, 1600, 1630,
1710-20, 2930-70, 3400 cm ^-1 Solubility: Soluble in methanol, slightly soluble in acetone, slightly soluble in water, chloroform, and ethyl acetate Color reaction: Positive; Remieux, periodic acid-benzidine, Sakaguchi coagulation positive ; Ninhydrin (yellow) Distinction between basic, acidic, and neutral: Basic substance pKa′; 4.9 (in 70% methyl cellosolve) Bacterial resistance spectrum: Tested using the usual agar plate dilution method.

[Table] <Comparison with other substances> This substance is considered to be related to azalomycin, copiamycin, guanidyl fungin, and niphimycin; Since no substance has a matching molecular formula,
It was concluded that RK-634 is a new antibiotic. (Agricultural and Horticultural Fungicide Containing RK-634 as an Active Ingredient) When using the active ingredient of the present invention as an agricultural and horticultural fungicide, it is generally necessary to use an appropriate pesticide formulation similar to that known in the art. Granules, powders, emulsions, etc. using solid carriers, liquid carriers, emulsifying dispersants, etc.
It can be formulated and applied in any dosage form such as wettable powders, tablets, oil solutions, sprays, and aerosols. These carriers include clay, kaolin, bentonite, acid clay, diatomaceous earth, calcium carbonate, solid carriers such as nitrocellulose, starch, gum arabic, etc., and liquid carriers such as water, methanol, ethanol, acetone, dimethylformamide, and ethylene. Examples include glycol and the like. Also,
Adjuvants commonly used in formulations, such as sulfuric esters of higher alcohols, polyoxyethylene alkyl allyl ether, alkyl allyl polyethylene glycol ether, alkyl allyl sorbitan monolaurate, alkyl allyl sorbitan monolaurate, Sulfonates, quaternary ammonium salts, polyalkylene oxides, and the like can be appropriately blended. The appropriate blending ratio of the active ingredient is about 10 to 90% for emulsions and wettable powders, and about 0.1 to 10% for powders, oils, etc. However, depending on the purpose of use, these The concentration may be increased or decreased as appropriate. In addition, the drug of the present invention may be used with other fungicides, herbicides,
It can be used by appropriately mixing with pesticides, fertilizer substances, and soil conditioners. The present invention will be specifically explained below using production examples, working examples, and test examples, but the present invention is not limited thereto. In addition, "%" and "part" represent weight % and weight part, respectively. Production example Glucose 2%, soluble starch 1%, meat extract 0.1%, dried yeast 0.4%, soy flour 2.5%, salt 0.2
The RK-634 strain was inoculated into a medium containing 0.005% dibasic potassium phosphate, and the mixture was incubated at 27°C.
The cells were cultured with shaking for 96 hours. Filter this culture solution,
It was divided into filtrate 5 and bacterial cells. After extracting the bacterial cells with 80% acetone in step 5, concentrate and remove the acetone.
After concentrating to 1.5, extract the mixture with the filtrate twice using 5 portions of ethyl acetate, and discard the extract. The lower layer is extracted twice using butanol 3 and 2, and the active substance is completely transferred to butanol. Butanol is concentrated under reduced pressure to obtain syrup. After this, chromatography is performed using a silica gel column (45φ x 400H) packed with dichloromethane. Dichloromethane-methanol ratio 30:1 → 20:1
→ When changing the ratio to 15:1, activity begins to appear at 15:1. The active part is collected and concentrated, and chromatographed again using a silica gel column (30φ x 500H) packed with butanol-methanol-water (8:1:1). Butanol-methanol-water 8:1:1
→If you change it to 5:1:1→4:1:2, it will be 4:
Activity begins to appear at a ratio of 1:2. The active portion was collected and concentrated under reduced pressure to obtain 1.5 g of syrup. 0.4 g of the sample was used for chromatography using a Sephadex LH-20 column (25φ x 1200H) packed with methanol.
The remaining syrup was similarly chromatographed, yielding 105 mg of almost single RK-634 substance. Finally, by high performance liquid chromatography using a reverse phase column Nucleosil 5C ₁₈ (20φ x 300H), RK-
A single peak of 634 substances appears. This was collected, concentrated under reduced pressure, and lyophilized to obtain 43 mg of a colorless white powder. Example 1 (Wettable powder) RK-634, 10 parts, sodium lauryl sulfate 5
1 part, dinaphthylmethane disulfonic acid soda formalin condensate, 2 parts, and 83 parts of clay were mixed and ground to obtain 100 parts of a wettable powder. Example 2 (Emulsion) RK-634, 8 parts, ethylene glycol 10 parts,
20 parts of dimethylformamide, 10 parts of alkyl dimethylbenzyl ammonium chloride and 52 parts of methanol are mixed and dissolved to obtain 100 parts of an emulsion. Example 3 (Powder) RK-634, 0.2 parts, calcium stearate 0.5
50 parts of talc and 49.3 parts of clay were mixed and ground to obtain 100 parts of powder. Example 4 (Powder) 10 parts of RK-634, 15 parts of starch, 72 parts of bentonite, and 3 parts of sodium salt of lauryl alcohol sulfate were mixed and ground to obtain 100 parts of a powder. Test example (1) Control test against gray mold of cucumber (a)
We used single-leaf stage cucumbers (Sagami Hanshiro) grown one pot per plant for 13 days at a minimum of 20°C at night, 60% humidity, and supplemented with a mercury lamp so that the daylight hours were 12 hours a day. , 3 consecutive per ward. (b) Application of leaf liquid The spray liquid was prepared by dissolving the test chemical in acetone, adding distilled water, and adding one drop of a spreading agent. The final drug concentration is
The final concentration of acetone was adjusted to 500 ppm or less. In addition, a 5% acetone aqueous solution was used as the spray liquid for the untreated area. C. Inoculation and disease onset The inoculum was a botrytis fungus Botrytis with a diameter of 0.6 mm placed in the center of the PSA plate medium for spore inoculation.
cinerea bacterial flora was transplanted and cultured at 20° C. for 5 to 7 days, and the spores formed were suspended in a 1% yeast extract and 5% glucose solution. (Spore concentration of inoculum: 5×10 ⁵ /ml ~ 1×
10 ⁶ /ml). Vaccination is carried out in a polyvinyl inoculation box.
The test was carried out by uniformly spraying 20 cc per 50 test plants using a chromatograph sprayer. Furthermore, inoculation of bacterial flora was performed by culturing for 3 days in the same manner and directly attaching the bacterial flora to the true leaves of test plants. After inoculation, spray enough water into the inoculation box and keep the humidity at 90%.
The specimens were left in the dark at 20°C for 72 hours to develop the disease. D. Survey and calculation of control value The diameter of the lesions was measured. The control value (%) was calculated using the following formula. Control value = [1 - Disease onset diameter of test plot / Disease onset diameter of untreated plot] x 100 (%) (2) Control test against cucumber anthracnose Test plant After sowing, plant in an air-conditioned greenhouse (maximum 28℃ during the day, minimum temperature at night) 20
℃, humidity 60%, and sunlight hours supplemented with a mercury lamp to provide 12 hours of sunlight per day) for 2 weeks.
Seedlings of true-leaf single-leaf stage cucumber (Sagami Hanshiro) were planted one per pot, with three rows per area. B. Application of chemicals The spray solution was prepared by dissolving the test chemicals in acetone, adding distilled water, and adding one drop of a spreading agent. The final drug concentration is
The final concentration of acetone was adjusted to 500 ppm or less. To apply the chemicals, the test plants were placed on a turntable and sprayed with a spray gun while rotating. Thereafter, it was air-dried for 2 hours at room temperature. As a control drug, 750 ppm of Daconyl hydrating agent was normally used, and as a control, a 5% aqueous acetone solution was used. C. Inoculation and disease onset The inoculum was the anthrax bacterium Colletotrichum Iagenarium grown in PSA pilate medium at 20℃ for 10 days.
spores were suspended in sterile water and 1 drop of spreading agent was added to 50 ml to make the inoculum (spore concentration of the inoculum: 15×
10 ⁵ /ml). Inoculation was carried out by uniformly spraying 20 ml of the inoculum solution per 50 test plants using a chromatography sprayer in a polyvinyl chloride inoculation box. After inoculation, spray water into the inoculation box and leave it in the dark at 25℃ with a humidity of 90% or higher for 24 hours, then move the plants to a flooded open shelf and leave them for 72 hours at a humidity of 70% and a temperature of 25℃. Made me sick. During this period of illness, use a mercury lamp to
Artificial lighting was provided for 12 hours a day. D. Investigation and calculation of control value The total number of lesions on the true leaves of plants was measured, and the control value (%) was calculated using the following formula using the total number of lesions in each section. B. Control value = [1 x total number of lesions in the test area / total number of lesions in the untreated area] x 100 (%) The total number of lesions in the untreated area is 200 to 300, and the control value of the control agent is 95% or more. The test conditions were adjusted accordingly. (3) Control test against rice sesame leaf blight Test plant Rice (Aichi), 10 plants per pot, grown in an air-conditioned greenhouse (maximum 28℃ during the day, minimum 20℃ at night, 60% humidity) for 3 weeks after sowing Asahi) was used. Application of chemicals The spray solution was prepared by dissolving the test chemicals in acetone, adding distilled water, and adding one drop of a spreading agent. The final drug concentration is
The final concentration of acetone was adjusted to 500 ppm and 5%. As a control drug, triazine hydrating agent
250ppm was used. The test plants were placed on a turntable and sprayed with a spray gun while rotating. Thereafter, it was air-dried at room temperature. C. Inoculation and disease investigation The inoculum was the sesame leaf blight fungus Cochliobolus miyabeanus, which had been cultured on a PSA plate medium at 28℃ for 2 weeks.
spores were used, and the spore concentration was 15×10 ⁴ to 2×10 ⁵ /ml.
And so. Inoculation was performed using an airbrush in an inoculation box that had been kept at a humid temperature. After inoculation, the plants were placed in a dark, humid room at 25°C for 24 hours, and then the plants were taken out and allowed to develop disease under artificial illumination with a mercury lamp at 25°C for 12 hours a day. Lesions were measured 4 days later. D. Control value The control value (%) was calculated using the following formula. Control value = [1 x total number of lesions in test area/total number of lesions in untreated area] x 100 (%) The above results are shown below. Concentration Control value (ppm) (%) Gray Anthracnose Sesame leaf mold Blight 100 97 82 81 75 92 77 79 50 88 60 75 25 0 43 64 (Effect of the invention) From the above test results, the present invention This agricultural and horticultural fungicide shows high control value against the above plant diseases,
It has become clear that an excellent agrochemical can be provided.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the ultraviolet absorption spectrum of the antibiotic RK-634 of the present invention, and FIG. 2 is a diagram showing the infrared absorption spectrum of the antibiotic RK-634.

Claims (1)

[Claims] 1. Antibiotic RK- having the following physical and chemical properties
634. Shape: White powder Decomposition point: 129-131℃ Elemental analysis: Carbon 61.28% Hydrogen 9.15% Nitrogen 3.37% C ₆₃ H ₁₁₁ N ₃ O Calculated value as ₂₀ Carbon 61.49% Hydrogen 9.09% Nitrogen 3.41% Molecular weight: 1229 HR− FABMS(M+H) ⁺ m/z;
1230, 7890 Specific rotation; [α] ²⁰ _D = +15.1° (c0.34 methanol) Ultraviolet absorption spectrum: λ ^MeOH _nax (E1% 1cm) 227 shoulder (228), 232 (241), 240 shoulder (168 ) Infrared absorption spectrum: 990, 1060, 1120, 1180, 1250, in KBr
1290, 1370, 1455, 1600, 1630,
1710-20, 2930-70, 3400 cm ^-1 Solubility: Soluble in methanol, slightly soluble in acetone, slightly soluble in water, chloroform, and ethyl acetate Color reaction: Positive; Remieux, periodic acid-benzidine, Sakaguchi coagulation positive ; Ninhydrin (yellow) Distinction between basic, acidic, and neutral: Basic substance pKa′; 4.9 (in 70% methyl cellosolve) 2. A method for producing antibiotic RK-634, which comprises separating and collecting antibiotic RK-634 from a culture. 3 The antibiotic RK-634 producing bacterium is Streptomyces sp.
-634) The manufacturing method according to claim 2. 4. An agricultural and horticultural fungicide characterized by containing antibiotic RK-634 as an active ingredient.