JPH02275898A - Novel antibiotic substance kt-6291a and kt-6291b, microorganism capable of producing the same, its production and plant disease injury controlling agent - Google Patents

Abstract

NEW MATERIAL:An antibiotic substance KT-6291A, expressed by the formula and having the following physico-chemical properties. Molecular weight; 882. Composition formula; C41H74N19O11. Melting point; gradually browning from 170 deg.C. Specific rotatory power; [alpha] =+9.4 deg. (C=0.5, methanol). Color; white. Solubility; readily soluble in methanol, butanol and dimethyl sulfoxide and insoluble in ethyl acetate, chloroform and hexane. Color reaction;positive to the Dragendorff reaction, Rydon-Smith reaction and SAKAGUCHI reagent and negative to ninhydrin reaction. Basic, etc. USE:A plant disease injury controlling agent. PREPARATION:A strain, such as Bacillus.sp. KB-291, belonging to the Bacillus is cultured at pH5 to 8 and 18 to 42 deg.C, preferably in a liquid culture medium for 2 to 7 days.

Description

Detailed Description of the Invention [Object of the Invention] (Industrial Application Field) The present invention provides novel antibiotics KT-6291A and KT-6.
291B, microorganisms that produce them, production methods, and plant disease control agents containing them as active ingredients.

More specifically, the novel antibiotic KT-6291A of the present invention
and KT-6291B is Bacillus sp. KB-29
It can be obtained by culturing one strain of bacteria and collecting it from the culture solution.

(Prior art and problems to be solved by the invention) Conventionally,
Many peptide antibiotics are known as antibiotics produced by microorganisms belonging to the genus Bacillus, and the antibiotics KT-6291A and KT-6291B of the present invention
is novel and different from any known antibiotic in its physicochemical properties.

[Structure of the invention] (Means and effects for solving the problems) The novel antibiotics KT-629LA and KT-6291B of the present invention will be described below.
and its manufacturing method will be explained in detail.

KT-6 belonging to the genus Bacillus used in the present invention
The bacteria that produce 291A and KT-6291B include:
Bacillus sp. KB-291 (Bacillus sp. KB-291) was newly isolated from an airborne bacterium of the Ibaraki family by the present inventors.
llussp, KB-291) strain (rKB-291
The mycological properties of the 0 bacterial strains listed below are as follows.

A. Morphology (1) Shape and size: Bacillus 0.5-1. OX2. O ~ 3.0 μ (2) Polymorphic double or double series (3) Motility: Yes (peritrichous pen hairs) (4) Presence or absence of spores: Yes Spore shape: Oval spore formation site: Center ( 5) Durham staining: Positive (6) Acid-fastness: None B, Growth status (1) Slight growth on gravy agar plate culture, amorphous or diffuse, opaque and milky white (2) Slight growth on gravy agar slant culture surface , Opaque and milky white (3) Meat juice liquid culture Slightly grows, and as the culture days pass, it becomes slightly cloudy and the bacterial cells take on a milky color.

(4) Meat juice + 1% glucose culture Both plate culture and slant culture have good growth. The surface is raised and the contents are opaque and milky white. Growth is good in liquid culture. As the culture days pass, the bacteria become cloudy and the bacterial cells become milky white.

(5) Potato section Good growth, brown colonies formed (6) Ridmus milk When peptonized, lactose takes on a pink color.

(7) Grows at the puncture site of meat juice gelatin puncture. It grows particularly well on the surface and liquefies in layers.

C8 Physiological properties (1) Nitrate reduction: Yes (2) Denitrification reaction: No 3) MR test: Negative (4) VP test: Positive (5) Indole formation; No 6) Hydrogen sulfide formation: None 7) Starch hydrolysis: Positive (8) Citric acid hydrolysis: None 9) Nitrate utilization: Yes (10) Pigment production: Insoluble yellow pigment production in potato, sucrose, agar medium (11) Urease: None 12) Ketochrome oxidase: Negative (13) Casein decomposition: Positive (14) Gelatin decomposition: Positive (15) Growth pH: pH 5-8, optimum pH 6-7 (16)
) Growth temperature: 18-42℃, Optimum temperature: 28-32℃ (17) Attitude towards oxygen Bifacult anaerobic (18) Sodium chloride tolerance = 5% does not grow (19) O-F test: Glucose Produces acid more fermentatively.

(20) Catalase: Positive (21) Tyrosine decomposition: Negative, presence or absence of acid production from carbon source L-arabinose + D-xylose + D-glucose + D-mannose + D-fructose + D-galactose + maltose + Sucrose + Lactose + Trehalose + D-Sorvit D-Mannit + Inosit Glycerin + Starch + Basal medium composition' (NH4) * HP 041
g, potassium chloride 0.2g, magnesium sulfate 0.2g
, yeast extract 0.2g, various sugars 5g, agar 15g, bromcresol purple o, 008g and distilled water 1000g
H.I.

The mycological properties of the KB-291 strain were determined using the Bersey's Manual on Determinative Bacteriology (
According to the description of KB-291 (8th edition), based on its mycological properties, physiological properties, and characteristics of acid production from carbon sources,
The strain is Bacillus sp. KB-291 (Bacillus sp. KB-291).
sp, KB-291) and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, as Fiber Science and Technology Research Institute No. 10395.

In addition, as the microorganism of the present invention, the KB-291 strain is a bacterial species belonging to the genus Bacillus, as well as natural and artificial mutant strains, and the antibiotic KT-629IA and KT-629, which will be described later,
All microorganisms capable of producing 91B are included in the microorganisms of the present invention.

In carrying out the present invention, antibiotic KT-6291A and KT-6291B-producing bacteria belonging to the genus Bacillus,
It can be cultured using conventional methods for producing antibiotics. For industrially advantageous production, aerobic agitation culture may be performed in a medium containing various nutrients under aerobic conditions before production.

The culture conditions and composition of the medium may be selected from those used in the production of general antibiotics. That is, the medium contains a carbon source, a nitrogen source, inorganic salts, and vitamins, precursors, etc. as necessary. Examples of carbon sources that may be added include:
Glucose, sucrose, starch, molasses, potatoes, etc.
Used alone or as a mixture, nitrogen sources include, for example, ammonium salts, nitrates, peptone, soybean flour, corn stew liquor, malt extract, etc., or mixtures thereof. Further, if necessary, an antifoaming agent such as silicone oil, soybean oil, or surfactant may be added.

The medium is preferably a liquid medium, and the pH of the medium is 5.0 to 8.
The culture temperature is preferably adjusted to 18 to 42°C, and the culture period is usually 2 to 7 days. Note that the optimum culture conditions may be selected depending on the characteristics of the producing bacteria used.

After completion of the culture, the antibiotics KT-6291A and KT-6291B can be separated and collected from the culture solution in accordance with the usual method for separating and collecting antibiotics from the culture, i.e., by using various organic solvents. Antibiotics KT-6291A and/or KT-6291B can be obtained by appropriately combining extraction methods, chromatography using various active adsorbents, etc.
Collect.

(Examples of the Invention) Hereinafter, the present invention will be explained in more detail with reference to Examples, but these Examples are not intended to limit the scope of the present invention in any way.

Example Bacillus sp. KB-291 strain was used as a seed medium, i.e.
Soluble starch 2%, sucrose 1%, yeast extract 0.5
% and peptone 0.5% (pH 7,0)
Cultured at 28°C for 24 hours, the resulting culture solution was inoculated into a fermentation medium (pH 7.0) containing 2% soluble starch, 1% sucrose and 0.5% yeast extract,
The cells were cultured at 28°C for 4 days.

N-butanol was extracted with stirring into the obtained culture solution II2 to obtain an n-pukunol extraction section. The n-buknol extracted fraction was concentrated to dryness under reduced pressure, then dissolved in methanol, applied to a Sephadex LH-20 column, and eluted with 100% methanol. Both active components were concentrated to dryness under reduced pressure to form powder 1.
50 mg was obtained.

The obtained powder was dissolved in methanol and YMC-Pack
Antibiotics KT-62918, KT-
It was eluted in the order of 6291A.

The antibiotic KT-6291A thus obtained is a novel antibiotic having the physicochemical properties described below.

(1) Molecular weight: 882 (according to FAB mass spectrum) (2) Compositional formula: C41H14N10011 (high resolution F
(according to AB mass spectrum) (3) Melting point: Gradual browning from 170°C (4) Specific optical rotation: [α] Otsu' = +9.4' (C = 0.
5. Methanol) (5) Color of substance: white (6) Ultraviolet absorption spectrum: 210 nm Shown in FIG.

(7) Infrared absorption spectrum: 2Ham-' 3300, 2940°2860
．． 1740, 1660°1630.1550,1450°1380.1210.1140°840, 800. 720 shown in Figure 2.

(8) Solubility in solvents: Easily soluble in methanol, butanol, dimethyl sulfoxide, insoluble in ethyl acetate, chloroform, hexane (9) Color reaction: Positive for Dragendorff reaction, Lydon-Smith reaction,
Positive for Sakaguchi's reagent, negative for ninhydrin reaction (10) Distinction between acidic, neutral, and basic: Basic (11) Proton nuclear magnetic resonance spectrum: Shown in Figure 3.

(12) C-13 nuclear magnetic resonance spectrum: Shown in FIG.

(13) Rf value :Silica gel thin layer chromatography (HPTLC Kieselge manufactured by Merck & Co., Ltd. 60) n-butanol:acetic acid:water (65 10:25) Rf value 0.50 (14) Acid hydrolysis Hydrolyze with 26N hydrochloric acid for 16 hours and aspartic acid, valine, and thread. Onine, allothreonine, alanine in a molar ratio of 1:2:l:l:l. It is a substance that

At the same time, in the early stage of hydrolysis, guar is present at the terminal. β- with a molecular weight of 315 and a nidyl group Gives hydroxy fatty acids and hydrolyzes them At the end, it dehydrates and leaves guani at the end. Molecular weight 297 α, β- with zyl group It is a substance that provides unsaturated fatty acids. Ru.

Furthermore, based on the results of the above-mentioned physicochemical properties and spectral analysis, the chemical structure of the new antibiotic KT-6291A was identified as follows.

The antibiotic KT-6291B thus obtained is a novel antibiotic having the physicochemical properties described below.

(1) Molecular weight: 946 (according to FAB mass spectrum) (2) Compositional formula: C4sH,4N 100□ (according to high-resolution FAB mass spectrum) (3) Melting point: Gradual browning from 160°C (4) Specific optical rotation: [α]D25=+18.2°(C=
0.5, methanol) (5) Color of substance: white (6) Ultraviolet absorption spectrum: LH::'= 212 nm, 228 nm, 27
8 nm as shown in FIG.

(7) Infrared absorption spectrum: cm-' 3300.2900°28
60.1740,1660°1520.1200.1140 800, 720 Shown in Figure 6.

(8) Solubility in solvents: Easily soluble in methanol, butanol, dimethyl sulfoxide, insoluble in chloroform and hexane (9) Color reaction: Positive for Dragendorff reaction, Lydon-Smith reaction,
Positive for Bauli reagent and Itame reagent, negative for ninhydrin reaction (10) Distinction between acidic, neutral, and basic: Basic (11) Proton nuclear magnetic resonance spectrum: Shown in FIG.

(12) C-13 nuclear magnetic resonance spectrum; shown in FIG.

(13) Rf value :Silica gel thin layer chromatography (HPTLC Kieselge manufactured by Merck & Co., Ltd. 60) n-butanol:acetic acid:water (65 10:25) Rf value 0.57 (14) Acid hydrolysis = Hydrolyzed with 6N hydrochloric acid for 16 hours and aspartic acid, valine, and thread. Onin, Allothreonine, Arani and tyrosine l:1:1:l:1 It is a substance containing in a molar ratio of .

At the same time, in the early stage of hydrolysis, guar is present at the terminal. β- with a molecular weight of 315 and a nidyl group Gives hydroxy fatty acids and hydrolyzes them At the end, it is dehydrated and guanidine is added to the end. α,β- with a molecular weight of 297 It is a substance that provides unsaturated fatty acids.

Furthermore, based on the results of the above-mentioned physicochemical properties and spectral analysis, the chemical structure of the new antibiotic KT-6291B was identified as follows.

Next, antibiotics KT-6291A and KT-6291B
Describe the biological activity of Antibiotic KT-62
91A and KT-6291B show a strong growth inhibitory effect on some yeast bacteria, Durum-positive bacteria, and filamentous fungi.The measurement of the minimum inhibitory concentration (MIC) was performed using the antibiotic KT-6291B.
Agar plate dilution method containing 91A or KT-6291B was used.

The results are shown below.

Takumi Summer 1st January Minimum Inhibitory Concentration (77ml) T-6291A KT-6291B lagenarium aeruginosa IFO-3923) IFO-3
5131 Antibiotics KT-6291A and KT-6291B are
It can be used as an agricultural fungicide because it exhibits excellent antibacterial activity against various plant pathogenic bacteria, especially filamentous fungi.

When KT-6291A and KT-6291B are used as agricultural fungicides, they can be prepared into granules, powders, or emulsions using appropriate solid carriers, emulsifying dispersants, etc. in the same way as agricultural chemical formulations known in the art. It can be formulated into any formulation such as wettable powders, tablets, oils, sprays, atomizers, etc. As the solid carrier, clay, kaolin, bentonite, acid clay, diatomaceous earth, calcium carbonate, nitrocellulose, starch, gum arabic, etc. can be used. Also,
Water, methanol, ethanol, dimethylformamide, ethylene glycol, etc. can be used as liquid carriers.

In addition, adjuvants commonly used in formulations, such as sulfuric esters of higher alcohols, polyoxyethylene alkylaryl ethers and their sulfonates, alkylaryl sorbicune monolaurate, alkylaryl sulfonates, alkylaryl polyethylene glycols, etc. Ether, formalin condensate of sodium dinaphthylmethane disulfonate, alkyldimethylbenzyl ammonium chloride, etc. can be appropriately blended.

In addition, the drug of the present invention may be used with other fungicides, herbicides, insecticides,
It can be used by appropriately mixing it with fertilizers and soil conditioners.

The present invention will be specifically explained below using manufacturing examples, but the present invention is not limited thereto.

In addition, "parts" in the formulation examples represent parts by weight.

Formulation example 1 (hydrating powder) Antibiotic KT-6291A or KT-6291B10
10 parts of a polyoxyethylene alkylaryl ether sulfonate, 2 parts of a formalin condensate of sodium dinaphthylmethane disulfonate, and 78 parts of clay were mixed and ground to obtain 100 parts of a wettable powder.

Formulation example 2 (liquid) Antibiotic KT-6291A or KT-6291BIO
20 parts of dimethylformamide, 10 parts of ethylene glycol, 10 parts of alkyldimethylbenzyl ammonium chloride, and 50 parts of methanol were mixed and dissolved to make a solution of 100 parts.
I got the department.

Formulation example 3 (hydrating powder) N-butanol was added to the culture solution lI2 of this example shown above and extracted with stirring, and the obtained n-butanol fraction was applied to a column of Sephadex LH-20, and the active fraction was The mixture was concentrated to dryness under reduced pressure to obtain 1150 mg of a mixture of KT-6291A and KT-6291B [KT-6291A/KT-6291B (weight ratio = 10:1)].

10 parts of this mixture, 10 parts of polyoxyethylene alkylaryl ether sulfonate, 2 parts of formalin condensate of sodium dinaphthylmethane disulfonate, 7 parts of clay
8 parts were mixed and ground to obtain 100 parts of a wettable powder.

Next, the effect of controlling various plant diseases by the fungicide of the present invention will be specifically explained using test examples.

Test Example 1 (Control test against gray mold of cucumber) After germination of cucumber (variety: Tokiwa Hikari No. 3 P type), young seedlings grown for 10 days were cut from the stem, and spores of the gray mold of cucumber (Botrytiscinerea) were added to the cotyledon surface. The suspension was placed at 304 ml. A paper disk for antibiotic assay (diameter 8 mm) was placed on top of the spore suspension, and the hydrating powder 9ON! prepared according to Formulation Example 1 was added. 0 and then 20 cotyledons.
The plants were left under conditions of 90% relative humidity and 90% relative humidity, and the degree of lesion development on the underside of the cotyledons was examined after 3 days.

In addition, the inhibition of lesion progression was calculated by the following method.

Lesion progression inhibition rate (%) = × 100 (Water is placed in place of the preparation) The results are shown below.

KT-6291A 100 100
None 50 1 (1 (l None 25
75 None KT-62918100100 None 50 100 None 2530 None Benlate 250 -100 None Test Example 2 (Cucumber anthracnose control test) One cucumber per pot in a synthetic resin pot with a diameter of 6 cm (variety: Tokiwa Hikari No. 3) P type) seeds are planted and grown in a greenhouse. A hydrating powder prepared according to Formulation Example 1 was added to a pot grown at the 1-2 leaf stage at a dose of 1011 d per plant! Sprayed using a spray gun. After air-drying, anthrax Bacillus chieuri (C
olletotrichumlagenarium) spore suspension (1.2xlO' spores per liter) was spray-inoculated and left in the dark at a relative humidity of 90% and a temperature of 25°C for 2 days, then transferred to a greenhouse, and after 5 days, The lesions on the leaves were investigated and the control value was calculated using the following formula.

Control value (%) = The results are shown below.

T-6291A 100 92
None 5080 None KT-6291B 100 96
None 5075 None Daconiflu Ioo (191 Pear Test Example 3 (Control test against rice blast) Rice paddy (variety: Koshihikari) was sown directly into synthetic resin pots with a diameter of 6 cm, 10 plants/pot), and Formulation Example 1 was applied at the 4-leaf stage. A hydrating powder prepared according to the method was diluted to a predetermined concentration and sprayed onto the complement at 50 mf per pot using a spray gun. After air-drying, rice blast fungus (Pyricular
Ia oryzae) spore suspension (30 spores per 100x microscope field) was spray-inoculated, and the relative humidity was 90%.
After leaving it in the dark at a temperature of 25℃ for one day, it was transferred to a greenhouse.
After 5 days, lesions on the leaves were investigated, and the control value was determined using the formula shown in Test Example 2.

The results are shown below.

KT-6291A 100 95
None 5070 None Loveside 500 98 None From the above results, the drug containing the antibiotic KT-6291A of the present invention as an active ingredient shows an extremely high control value against various plant diseases, and no phytotoxicity is observed. It has been shown that it is effective as an agricultural fungicide.

Test Example 4 The lesion progression inhibition rate was determined in the same manner as Test Example 1, except that a hydrating powder prepared according to Formulation Example 3 was used instead of the hydrating powder prepared according to Formulation Example 1. .

The results are shown below.

KT-6291A, B 100. 10
0 mixture) 50 1
00 None 2575 None Benlate 250 100 None From the above results, the antibiotic KT-6291A of the present invention
It has been clarified that drugs containing KT-6291B as an active ingredient are effective as agricultural fungicides, showing extremely high control value against various plant diseases and causing no phytotoxicity. Ta.

[Effects of the Invention] According to the present invention, novel antibiotics KT-6291A and KT-62 exhibit excellent effects against various plant pathogenic filamentous fungi.
91B can be provided.

[Brief explanation of drawings]

Figures 1, 2, 3 and 4 respectively show the ultraviolet absorption spectrum, infrared absorption spectrum, proton nuclear magnetic resonance spectrum and C-
13 nuclear magnetic resonance spectra, and FIGS. 5, 6, 7, and 8 respectively show the antibiotic KT-6291B.
The ultraviolet absorption spectrum, the infrared absorption spectrum, the proton nuclear magnetic resonance spectrum, and the C-13 nuclear magnetic resonance spectrum are shown. Procedural amendment (formality) January 24, 1999

Claims (1)

[Claims] 1. Novel antibiotic KT-62 represented by structural formula (I)
91A ▲ Contains mathematical formulas, chemical formulas, tables, etc. ▼ (I) 2. KT-6, a new antibiotic with the following physical and chemical properties
291A substance (1) Molecular weight: 882 (2) Compositional formula: C_4_1H_7_4N_1_0O_1
_1 (3) Melting point: Gradually browns from 170℃ (4) Specific optical rotation: [α]D^2^5=+9.4° (C=
0.5, methanol) (5) Color of substance: white (6) Ultraviolet absorption spectrum: λ^M^e^O^H_m_a_x=210nm (7)
Infrared absorption spectrum: ν^K^B^r_m_a_xcm^-^13300,
(8) Solubility in solvents: Easily soluble in methanol, butanol, dimethyl sulfoxide, ethyl acetate, chloroform, hexane Insoluble (9) Color reaction: Positive for Dragendorff reaction, Lydon-Smith reaction and Sakaguchi reagent, negative for ninhydrin reaction (10) Distinction between acidic, neutral and basic: Basic (11) R
f value: Silica gel thin layer chromatography (HPTLC Kieselgel 60 manufactured by Merck & Co.) n-butanol:acetic acid:water (65: 10:25) Rf value 0.50 (12) Acid hydrolysis: Hydrolysis with 6N hydrochloric acid for 16 hours Then, it is a substance containing aspartic acid, valine, threonine, allothreonine, and alanine in a molar ratio of 1:2:1:1:1. At the same time, it is a substance that gives a β-hydroxy fatty acid with a molecular weight of 315 with a guanidyl group at the end at the initial stage of hydrolysis, and dehydrates to give an α, β unsaturated fatty acid with a molecular weight of 297 and a guanidyl group on the end after hydrolysis. 3. Novel antibiotic KT-629 represented by structural formula (II)
1B ▲ Contains mathematical formulas, chemical formulas, tables, etc. ▼ (II) 4. KT-6, a new antibiotic with the following physical and chemical properties
291B substance (1) Molecular weight: 946 (2) Compositional formula: C_4_5H_7_4N_1_0O_1
_2 (3) Melting point: Gradually browning from 160℃ (4) Specific optical rotation: [α]D^2^5 = +18.2° (C
=0.5, methanol) (5) Color of substance: white (6) Ultraviolet absorption spectrum: λ^M^e^O^H_m_a_x=212 nm, 22
8nm, 278nm (7) Infrared absorption spectrum: ν^K^B^r_m_a_xcm^-^13300,
2900, 2860, 1740, 1660, 1520, 1200, 1140, 800, 720 (8) Solubility in solvents: Easily soluble in methanol, butanol, dimethyl sulfoxide, insoluble in chloroform and hexane (9) Color reaction: Dragendorff Reaction, positive for Lydon-Smith reaction, positive for Bauli reagent and Sakaguchi reagent, negative for ninhydrin reaction (10) Distinction between acidic, neutral, and basic: basic (11) R
f value: Silica gel thin layer chromatography (HPTLC Kieselgel 60 manufactured by Merck) n-butanol:acetic acid:water (65: 10:25) Rf value 0.57 (12) Acid hydrolysis: Hydrolysis with 6N hydrochloric acid for 16 hours Then, it is a substance containing aspartic acid, valine, threonine, allothreonine, alanine, and tyrosine in a molar ratio of 1:1:1:1:1. At the same time, in the early stage of hydrolysis, it gives a β-hydroxy fatty acid with a molecular weight of 315, which has a guanidyl group at the end, and at the end of hydrolysis, it dehydrates to give an α,β-unsaturated fatty acid with a molecular weight of 297, which has a guanidyl group at the end. be. 5. Bacillus sp. KB-291 strain having productivity of antibiotics KT-6291A and KT-6291B belonging to the genus Bacillus. 6. KT-6291A and KT-6 belonging to the genus Bacillus
291B substance-producing bacteria were cultured, and KT-6 was obtained from the culture.
KT-6291A and/or KT-291A and/or KT-291A and/or KT-6291B substances are collected.
6291B manufacturing method. 7. Antibiotic KT-6291A and/or KT-62
A plant disease control agent containing 91B as an active ingredient.