JPH0310690A - Antibiotic substance rk-634, production thereof and agricultural and horticultural germicide - Google Patents

Abstract

NEW MATERIAL:An antibiotic substance RK-634 having the following physicochemical properties. Shape; white powder. Decomposition point; 129-131 deg.C. Elementary analysis (%); C, 61.28; H, 9.15; N, 3.37. Molecular weight; 1229. High- resolution fast atom bombardment mass spectrometry (HR-FABMS); (M+H)<+> m/z 1230.7390. Specific rotation; [alpha] =15.1 deg. (c=0.34, methanol). Solubility; soluble in methanol, slightly soluble in acetone and sparingly soluble in water, chloroform and ethyl acetate. Color reaction; positive to the Lemieux reaction and periodic acid-benzidine and false positive (yellow) to ninhydrin. Basic substance, etc. USE:An agricultural and horticultural germicide. PREPARATION:A strain, such as Streptomyces.sp.RK-634 (FERM P-10771). belonging to the genus Streptomyces is cultured at 25-35 deg.C preferably at about a neutral pH.

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 organic chlorine agents and organic phosphate agents, have been used as disinfectants for agriculture and horticulture, but these are all harmful to animals and humans. Environmental pollution caused by these chemicals has become a serious social problem, as they contaminate the soil, remain in nature and act on animals and plants for long periods of time, 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.

[Purpose 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 charcoal stain of cucumber. It is an excellent agricultural and horticultural fungicide that exhibits a pesticidal effect, does not cause any chemical damage to plants, and does not have any effect on the human body when applied.

The present invention will be explained in detail below.

<Production of antibiotic RK-634> (Microorganisms 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. As an example, Streptomyces sp.
634 (Streptomyces sp, RK
-634> (hereinafter referred to as "RK-634 strain"), which has the above characteristics and advantageously produces the antibiotic RK-634 of the present invention. This can be effectively used in the manufacturing method of the invention.

In addition, not only natural and artificial mutant strains of the above-mentioned RK-634 strain, but also all microorganisms belonging to the genus Streptomyces that have the ability to produce the antibiotic RK-634 described below may be used in the method of the present invention. I can do it.

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 No. 10771 (FERM
P-10771).

RK-634 strain has the following mycological properties.

1. Morphological characteristics This strain, strain RK-634, 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 mycelia are straight and gray in color, and the spores are cylindrical with a smooth surface.

2. Growth conditions on various media (cultured at 27°C for 20 days, color tones are shown in the Discrete Color Names Dictionary)
Names Dictionary))1)
Starch/Yeast Extract Agar Medium Growth: Good Aerial Mycelium: Rich Aerial Mycelium Color: 5ih (Lead Gray) Soluble Pigment: None 2) Yeast Extract/Malt Extract Agar Medium Growth: Good Aerial Mycelium: Rich Aerial Mycelium Color Near C ! c (dark pink) Soluble pigment: None 3) Oatmeal agar medium growth: Poor aerial hyphae; None Soluble pigment: None 4) Sucrose inorganic salt agar medium growth: Good aerial hyphae: Rich aerial hyphae color + 5fe (ash) Soluble pigment: None 5) Growth on sucrose nitrate agar medium: Good Aerial hyphae: Abundant Aerial hyphae color: 3 dc (natural) Soluble pigment: None 6) Growth on crucose-asparagine agar medium: Normal Aerial hyphae: Normal aerial hyphae color tone : 2ba (Pearl) Soluble pigment: None 7) Growth on glycerol/asparagine agar medium:
Good air hyphae: Abundant air hyphae Color tone: 3ba, 5dc (burl, gray) Soluble pigments: None 8) Nutrient agar medium growth: Poor air hyphae: None Soluble pigments: None 9) Tyrosine agar medium growth: Good air Hypha: Rich aerial mycelium Color tone: C (gray) Soluble pigment: Dark brown 10) Peptone, yeast extract, iron agar medium
Growth: Good Aerial Mycelia: None Soluble pigment: None 3 Utilization of Tong D-glucose D-xylose used L-arabinose L-rhamnose D-mannitol Inositol Raffinose Suspicious sucrose D-fructose Not used 4 , and other physiological properties. This strain shows positive reactions in starch hydrolysis, skim milk coagulation and liquefaction, and melanin formation, but negative reactions in gelatin liquefaction and soluble pigment formation.

From the above, it is clear that this bacterium belongs to Streptomyces, and these properties are summarized in the Vergeive Manual of Determinative Batteriology (
Bergey's Manual of Determinology
When searching for those described in Bacteriology, the closest relative is Streptomyces antibiotyphi.
antibioticus), Streptomyces novolidoensis (Strept.

myces noboritoensis).

(Culture method and purification method) To obtain the antibiotic RK-634 of the present invention, the above-mentioned 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 culturing, a spore suspension or culture solution of the above-mentioned producing bacteria may be inoculated into a medium and culture with aeration and stirring may be performed.

The nutrient source of the medium is not particularly limited (carbon sources, nitrogen sources, etc. commonly used for culturing microorganisms can be contained in the medium. Carbon sources include starch,
Dextrin, glycerin, glucose, sucrose, galactose, inositol, mannitol, etc. Nitrogen sources include peptone, soy flour, meat extract,
Rice bran, wheat gluten, urea, cornstarch liquor, ammonium salts, nitrates, and other organic or inorganic nitrogen compounds are used. In addition, inorganic salts such as common salt, phosphates, potassium, calcium, zinc, manganese, iron, and other metal salts may be added as appropriate, and animal, vegetable, mineral oils, etc. may be added as antifoaming agents if necessary. may be added. Culture conditions such as culture temperature and culture time are selected to be suitable for the growth of the bacteria used and to maximize the production of -634 in R'.

For example, the pH of the medium should be between 4 and 9, especially around neutrality.
The appropriate culture temperature is preferably about 25 to 35°C. However, these culture conditions such as 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 and external conditions. Needless to say, it is. 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, any of the methods that utilize the solubility difference between RK-634 and impurities, the ionic binding force difference, the adsorption affinity difference, and the molecular weight difference may be used alone or , used in appropriate combinations or repeatedly. Specifically, RK-
634 is mostly present in the culture filtrate. When the culture solution is purified using a combination of various ion exchange chromatography, gel filtration chromatography, adsorption chromatography, liquid chromatography, cellulose partition chromatography, etc., a fraction containing RK-634 and other active ingredients can be obtained. It will be done. The powder obtained by freeze-drying both components is further purified by high performance liquid chromatography (e.g., developed with Nucleozil 5CI 8.50% methanol) to obtain a purified white powder of RK-634.

The physicochemical and biological properties of RK-634 thus obtained are as follows.

<Physicochemical and biological properties of antibiotic RK-634> Shape: White powder Decomposition point: 129-131°C Elemental analysis: Charcoal! 61.28% Hydrogen 9.15% Nitrogen 3
．． Calculated value as 37% C63H11, N3O20 Carbon 61.4
9% Hydrogen 9.09% Nitrogen 3.41% Molecular weight: 1229 HR-FABMS (M+H)” m/z; 123
0.7890 Specific optical rotation: [α] = +15.1”
(co, 34 methanol) (shown in Figure 1) 227 shoulders (228L232 (241), 240 shoulders (1
68) Infrared absorption spectrum: in KBr (shown in Figure 2) 990.1060.1120.1180.1250.1
290.1370.1455.1600.1630.1
710-20.2930-70.3400 cm-' Solubility: Soluble in methanol, slightly soluble in acetone, poorly soluble in water, chloroform, ethyl acetate Color reaction: positive; Lemieux, periodic acid-benzidine false positive: ninhydrin (Yellow) Distinction between basic, acidic, and neutral: basic substance pK'a;
4.9 Bacterial resistance spectrum (in 70% methyl cellosolve): Verification was performed using the usual agar plate dilution method.

Staphylococcus aureus
FD809P12.5 Bacillus subtilis (Bacillus 5ubtilis) NIHJ
PC121912,5 Escherichia coli >100 (ε5
cherichia coli) K-121FO3
301 Salmonella typhimurium
TV119〉100 Cochliobolus mlyabeanus
IFO52773,2 Alternaria mati (1 to 1ternaria mati) IFO
898412,5 Botrytis cinerea IFO536
51,6 Colletotrichum lagenarium (Colle, totrichuITIIagenar)
ium) IFO75131,6 Rhizactonia 5olani 1F06
2586 Glomerella singulate IF
O97675 Pyricularia oryzae IFO
59946,25 Trichophyton mentagrophytes 6.25 (T
richophyton mentagrophyte
s) IFO6202 Aspergillus oryzae
IF [152395 Penicillium chrysogenum
00 Saccharomyces cerevisiae 1.6
) IFO304 <Comparison with other substances> This substance is azalomycin,
Although it is thought to be a substance related to copiamycin, guanidyl fungin, and niphimycin, there is no substance with a molecular formula that matches this substance, so RK-634 has been designated as a new antibiotic. concluded.

(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 technical field. It can be applied by formulating it into any dosage form such as granules, powders, emulsions, wettable powders, tablets, oils, sprays, atomizers, etc. using solid carriers, liquid carriers, emulsifying dispersants, etc. 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,
Examples include acetone, dimethylformamide, ethylene glycol, and the like. In addition, 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 sulfonate, quaternary ammonium salt, polyalkylene oxide, etc. can be blended as appropriate.

The blending ratio of the active ingredients is 10~ for emulsions and hydrating agents.
Approximately 90% is appropriate, and for powders, oils, etc., 0.
Approximately 1-10% is appropriate, but these concentrations may be increased or decreased as appropriate depending on the purpose of use.

In addition, the drug of the present invention may be used with other fungicides, herbicides, insecticides,
It can be used by appropriately mixing with 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
%, dry yeast 0.4%, soy flour 2.5%, salt 0.2%
, 7β consisting of 0.005% potassium phosphate
The RK-634 strain was inoculated into a medium of
Cultured with shaking for hours. This culture solution was filtered and divided into filtrate 51 and bacterial cells. The bacterial cells are extracted with 5 liters of 80% acetone, concentrated to remove the acetone and concentrated to 1.5β, then combined with the filtrate and extracted twice with ethyl acetate 5β, and the extract is discarded. The bottom layer is 3jl of butanol! and 2I
! The active substance is completely transferred to butanol after two extractions with . Butanol is concentrated under reduced pressure to obtain Shirabu. Thereafter, chromatography is performed using a silica gel column (45φ x 400H) packed with dichloromethane. Dichloromethane-methanol ratio 30:1 → 20:1 → 15
: When changing to 1, the activity starts to appear when 15=1. The active portion was collected and concentrated and diluted with butanol-methanol-water (8:
Silica gel column (30φX) packed with 1:1)
Perform chromatography again using 500H). Butanol
Methanol-water 8:l:1→5:1:1→4:1:2
When changing to 4:1:2, activity begins to appear.

The active parts were collected and concentrated under reduced pressure to obtain 1.5 g of silica.

5ep filled with methanol using 0.4g of it
hadex L) 1-20 column (25φx1200H
). Chromatography was performed on the remaining Shirabu in the same way, and almost a single RK-634 substance 105
mg was obtained. Finally, the reverse phase column Nucleosil
By high performance liquid chromatography using 5C+a (20φ x 300H), 82% methanol, 8.5
RK-6 at 26 minutes with mf/+++in deployment
A single peak of 34 substances appears. This was collected, concentrated under reduced pressure, and lyophilized to obtain 43 mg of a colorless white powder.

Example 1 (hydrating powder) RK-634, 10 parts, sodium lauryl sulfate 5 parts,
2 parts of dinaphthylmethane disulfonic acid soda formalin condensate and 83 parts of clay are mixed and ground to obtain 100 parts of a wettable powder.

Example 2 (Emulsion) 8 parts of RK-634, 10 parts of ethylene glycol, 20 parts of dimethylformamide, 10 parts of alkyl dimethylbenzyl ammonium chloride and 52 parts of methanol were mixed and dissolved to obtain 100 parts of an emulsion.

Example 3 (powder) RK-634, 1112 parts, calcium stearate 0
．． 5B, 50 parts of talc, and 49.3 parts of clay were mixed and ground to obtain 100 parts of a powder.

Example 4 (Granules) 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 granules.

Test example (1) Control test against botrytis botrytis blight
A leaf-leaf stage cucumber (Sumo Hanshiro) grown in one pot was grown for 13 days in a climate with 60% humidity and 12 hours of sunshine per day (supplemented with a mercury lamp).
Continuous.

Application of chemical solution The spray solution is prepared by dissolving the test drug in acetone, adding distilled water,
One drop of spreading agent was added. The final drug concentration was 500 ppm, and the final acetone concentration was adjusted to 5% or less.

Furthermore, 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 with a diameter of 0.6 mm placed in the center of the PSA Bray medium for spore inoculation.
cinerea bacterial flora pieces were transplanted and grown for 20 days for 5 to 7 days.
The spores formed through culture at 0.degree. C. were suspended in a 1% yeast extract and 5% glucose solution. (Spore concentration of inoculum: 5X10'/d~IXI
O'/m1). Vaccination is carried out in a polyvinyl inoculation box.
A 20 cc amount per 50 test plants was uniformly sprayed using a chromatograph sprayer.

In addition, for bacterial flora inoculation, the bacterial flora that had been similarly cultured for 3 days was applied directly to the true leaves of the test plants.

After inoculation, spray enough water into the inoculation box and keep the humidity at least 90%.
The cells were left in the dark at 20°C for 72 hours to develop the disease.

2. Investigation 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 charcoal stain disease A. After sowing the test plants, in an air-conditioned greenhouse (maximum 28℃ during the day, nighttime) Minimum 20℃,
Using true-leaf stage cucumber seedlings (Sagami Hanshiro) planted one pot per pot, grown for two weeks in a humidity of 60% and sunlight supplemented with a mercury lamp so that the sunlight was 12 hours a day. -3 consecutive wards.

To prepare the spray solution for applying the drug, dissolve the test drug in acetone, add distilled water,
One drop of spreading agent was added. The final concentration of the drug was 500 ppm.
The final concentration of acetone was adjusted to 5% 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 reference drug, usually 750 ppm of Daconyl Permanent Agent and 5% as a control.
An acetone aqueous solution was used.

C. Inoculation and disease onset The inoculum was C01letOtrichumIag, which was grown on a PSA plate medium at 20°C for 10 days.
enarium spores were suspended in sterile water and
One drop of a spreading agent was added to the inoculum to serve as an inoculum (spore concentration of the inoculum: l 5 x 10'/-).

Inoculation is carried out in a polyvinyl chloride inoculation box with 50 test plants.
For each strain, 20 mj' of the inoculum solution was uniformly sprayed using a chromatograph sprayer.

After inoculation, spray water into the inoculation box and keep the humidity at least 90% at 25
After being left in the dark for 24 hours, the plants were transferred to a flooded open shelf and left at a humidity of 70% and a temperature of 25°C for 72 hours to develop the disease. During this period of illness, artificial lighting was provided with a mercury lamp for 12 hours a day.

2. 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.

Control value = [1 - total number of lesions in the test area / total number of lesions in the untreated area) x 100 (9) In addition, the total number of lesions in the untreated area was 200 to 300, and the control value of the control agent was tested to be 95% or more. Adjusted conditions.

(3) Control test against rice sesame leaf blight (A), 3 weeks after sowing test plants in an air-conditioned greenhouse (maximum 28℃ during daytime, minimum 20℃ at night)
A rice plant (Aichi Asahi), grown in a temperature of 10°C and 60% humidity, was used.

To prepare the spray solution for applying the drug, dissolve the test drug in acetone, add distilled water,
One drop of spreading agent was added. Final drug concentration is 500ppm
The final concentration of acetone was adjusted to 5%. As a control drug, 250 ppm of triazine perpetuating agent 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 a sesame leaf blight fungus Cochliobolus miyabe cultured at 28°C for 2 weeks in a PSA plate medium.
Anus spores are used, and the spore concentration is 15X104 ~
It was set as 2×10'/-.

Inoculation was performed using an airbrush in an inoculation box that had been previously set up in a greenhouse. After inoculation, the plants were placed in a dark greenhouse at 25°C for 24 hours, and then the plants were taken out and placed under artificial illumination with a mercury lamp at 25°C for 12 hours a day to develop the disease. Lesions were measured 4 days later.

2. Control value The control value (%) was calculated using the following formula.

Control value = [l-total number of lesions in test plot/ Total number of lesions in untreated area) x 100 (%) The above results are shown below.

100 97 82 81
75 92 77 795
0 88 60 7525
0 43 64 (Effect of the invention) From the above test results, it became clear that the agricultural and horticultural fungicide of the present invention exhibited a high control value against the above plant diseases and could provide an excellent agricultural chemical. .

[Brief explanation of drawings]

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 ultraviolet absorption spectrum of the antibiotic RK-634 of the present invention.
634 is a diagram showing an infrared absorption spectrum of 634. Figure 1 Ultraviolet absorption spectrum (in MeOH) Wavelength (nm)

Claims (1)

[Claims] (1) Antibiotic RK-63 having the following physicochemical properties
4. Shape: White powder Decomposition point: 129-131°C Elemental analysis: Carbon 61.28% Hydrogen 9.15% Nitrogen 3.3
Calculated value as 7% C_6_3H_1_1_1_1N_3O_2_0 Carbon 61.49% Hydrogen 9.09% Nitrogen 3.41% Molecular weight: 1229 HR-FABMS (M+H)^+m/z; 1230.7
890 Specific rotation: ▲There are mathematical formulas, chemical formulas, tables, etc.▼=
+15.1° (c0.34 methanol) Ultraviolet absorption spectrum: ▲ Contains mathematical formulas, chemical formulas, tables, etc. ▼ 227 shoulder (228), 232 (241), 240 shoulder (1
68) Infrared absorption spectrum: 990, 1060, 1120, 1180, 1250, 1 in KBr
290, 1370, 1455, 1600, 1630, 1
710-20, 2930-70, 3400cm^-^1 Solubility: Soluble in methanol, slightly soluble in acetone, poorly soluble in water, chloroform, and ethyl acetate Color reaction: Positive; Lemieux, periodic acid-benzidine coagulation positive ; Ninhydrin (yellow) Distinction between basic, acidic, and neutral: Basic substance pK'a; 4.9 (in 70% methyl cellosolve) (2)
A method for producing antibiotic RK-634, which comprises culturing antibiotic RK-634-producing bacteria belonging to the genus Streptomyces, and separating and collecting antibiotic RK-634 from the culture. (3) The antibiotic RK-634 producing bacteria is Streptomyces sp.
sp. RK-634) The manufacturing method according to claim (2). (4) An agricultural and horticultural fungicide characterized by containing antibiotic RK-634 as an active ingredient.