JPH0286784A - Antibiotic rs-10, predetermined thereof and sterilizer for agriculture and horticulture - Google Patents

Abstract

NEW MATERIAL: An antibiotic RS-10 having the below physicochemical properties. Decomposition point: 230-232°C; [α]²⁰D=+9.2° (C=0.5%, H₂O); solubility: easily soluble in water, slightly soluble in methanol, acetone, insoluble in chloroform, benzene, ethyl acetate; mol. wt.: 822 (measured by SIMS spectrum); molecular formula: C₂₉H₄₂N₈O₁₈S; color reaction: positive for ninhydrin, REMYU, Lydon-smith, periodic acid-benzidine reactions; negative for anthrone and FeCl₃ reactions; substance color: white powder, etc.

USE: A microbicide for agriculture and horticulture.

PREPARATION: This antibiotic is prepared by culturing an antibiotic RS-10- producing fungus belonging to the genus Streptomyces [preferably Streptomyces S.P.-10 (FERM P-10219)].

COPYRIGHT: (C)1990,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a novel antibiotic RS-10, a method for producing the same, and an agricultural and horticultural fungicide containing RS-10 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.

[Object of the Invention] An object of the present invention is to provide a novel 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 RS-10 of the present invention is a novel antibiotic that has the physical and chemical properties described below and has not been published in any literature, and has excellent control effects against various plant diseases such as cucumber gray mold and cucumber charcoal stain. and does not cause any phytotoxicity to the plant body,
It is an excellent agricultural and horticultural fungicide that does not have any effect on the human body when applied.

The present invention will be explained in detail below.

<Production of antibiotic RS-10> (Microorganisms used) The microorganism that produces antibiotic RS-10 of the present invention is a species of bacteria that belongs to the genus Streptomyces and has the ability to produce antibiotic RS-10.

As an example, Streptomyces SB RS
-I Q (Streptomyces sp,
RS-10) (hereinafter referred to as "RS-10 strain"), which has the above characteristics and advantageously produces the antibiotic RS-10 of the present invention, This can be effectively used in the production method of the present invention.

In addition, not only natural and artificial mutant strains of the above-mentioned RS-10 strain, but also all microorganisms belonging to the genus Streptomyces and having the ability to produce the antibiotic RS-10 described below can be used in the method of the present invention. can.

The above strain RS-10 is a soil actinomycete isolated from soil collected in Guangxi Province, China, and was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on August 20, 1988, and its microorganism accession number is FER No. 10.219 (FER
? vf P-10, 219).

RS-10 strain has the following mycological properties.

1. Morphological characteristics This strain, RS-10 strain, was prepared with 1% yeast extract,
Cultured in a liquid medium containing 1% glucose, the cells were
Thin layer chromatography of the product hydrolyzed with N-hydrochloric acid at 110° C. for 18 hours detected L,L-diaminopimelic acid, but not meso-diaminopimelic acid. When observed under an electron microscope when grown on an agar plate, the aerial mycelia have a spiral shape, and the spore surface is smooth and egg-shaped. A large number of spore chains are formed at the tip of the aerial hyphae.

2. Growth conditions on various media (27°C, 20 days culture, color tone is Disclitiv Color Names f
Eve'/-1+! 7 (Descriptive
(according to “olorNames Dictionary”) I) Starch yeast agar medium Growth: Good Aerial hyphae: Abundant Aerial hyphae Color tone: 2fe (covert gelai) Surface color tone
:2gc (bamboo) Soluble color S: None 2) Yeast extract/malt extract agar medium growth:
Good air mycelium: Abundant air mycelium Color tone: 3cb+) fe (sand + ash) Surface color tone: 21c (honey gold) Soluble pigment:
None 3) Growth on oatmeal agar: Good Aerobic mycelium: Small amount Aerobic mycelium Color tone: C (light gray) Surface color tone: B (Oyster white) Soluble pigment: None 4) Growth on starch/inorganic salt agar medium: Poor air hyphae: Small amount air hyphae Color tone: 2ec (oatmeal) Surface color tone:
2gc (Bamboo) Soluble pigment: None 5) Tyrosine agar medium Growth: Good Aerial mycelium: Abundant Aerial mycelium Color a: 3dc (Natural) Surface color: 3
gc (Light Dan) Soluble pigment: None 6) Sucrose nitrate agar medium Growth: Normal aerial hyphae: Normal aerial hyphae Color tone: 3cb (Sand) Surface color tone: 3dc (Natural) Soluble pigment: None 7) Glucose/Asparagine Agar medium growth: Poor air mycelium: Small amount of air Mycelial color tone: 2cb (eye poly tint) Surface color tone: 2dc (natural) Soluble color S: None 8) Glycerol/asparagine agar medium Growth: Poor air mycelium: Small amount of air Mycelium color tone: 2cb (Ipoly Tint) Surface color tone: 2dc (Natural) Soluble pigment: None 9) Nutrient agar medium growth: Poor Aerial mycelium: Small amount Aerial mycelium color tone: 2cb (Ipoly Tint) Surface color tone: 2dc (Natural) Soluble color S: None 10) Peptone/yeast extract/iron agar medium Growth: Good Aerial mycelium: None Aerial mycelial color Surface color: 28C (bamboo) Soluble pigment: None 3. Sugar utilization D-glucose Inositol with good growth D-mannitol with good growth D-galactose with good growth Maltose with good growth D-fructose with good growth Sorbitol with normal growth D-xylose with good growth L-arabinose with good growth Sucrose with no growth L-rhamnose with no growth Raffinose without growth Lactose does not grow. Melibiose does not grow. Sorbose does not grow. Reactions for reduction, hydrolysis of cellulose, and formation of melanin-like pigments are negative.

From the above, it is clear that this bacterium belongs to Streptomyces, and these properties are summarized in Verge's Manual of Determinative Bacteriology (
Bergey's! , Annual of Dete
rminative Bacteriology) and International Journal of Systematic Bacteriology (Internationala
l Journal of Systematlc Ba
cteriology) If you search for what is written in your own words,
Its closest relative is Streptomyces lividans (S
treptomyces l1vidans),
This bacterium is identified as this species.

(Culture method and purification method) To obtain the antibiotic RS-10 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, 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, calactose, inositol, mannitol, etc. Nitrogen sources include peptone, soy flour, meat extract,
Rice bran, wheat gluten, urine L corn staple 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. The 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 RS-10.

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 RS-10 from the culture thus obtained, it can be collected using appropriate means commonly used for collecting metabolites. For example, means that utilizes the solubility difference between RS-10 and impurities, means that utilizes the difference in ionic bond strength,
Either the means that utilizes the difference in adsorption affinity or the means that utilizes the difference in molecular weight, each alone or in appropriate combination,
Or used repeatedly. Specifically, RS-10
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 RS-10 and other active ingredients can be obtained. . The powder obtained by freeze-drying these two fractions was further subjected to high performance liquid chromatography (for example, the old gh-Carbon OD S).

0.02% formic acid-water system).
A purified white powder of 10 is obtained.

The physicochemical properties and biological properties of RS-10 thus obtained are as follows.

Physicochemical and biological properties of antibiotic RS-10 Decomposition point = 230-232°C Original cumulative analysis: Carbon 40.43% Hydrogen 5.45% Nitrogen: i1
2.82% Sulfur 3.32% Specific optical rotation: [α]D-19.2° (C=0.5%H
2O) Infrared absorption spectrum: 3350.2900.1650
．． 1570.1530.1380.1210.1130
．． 1070.760.540 cm-' Solubility: Easily soluble in water, slightly soluble in methanol and acetone, insoluble in chloroform, benzene, and ethyl acetate Molecule M: 822 (based on S1MS mass spectrum) (Molecular formula: C29H42N801115) Color reaction :
Ninhydrin, Lemieux, Lydon-Smith, positive for periodic acid-benzidine reaction, negative for Anthrone, FeCβ3 reaction Color of substance: White powder Thin layer chromatography: Merck & Co., USA thin layer plate 0.25 m++n Silica gel solvent Butanol: Pyridine :Acetic acid:Water=10:10:
3+12 Rf value 0.29 Solvent Butanol:methanol:water=2+1:2 Rf value 0.23 Antibacterial spectrum: Shown by disk assay on agar plate with appropriate sound.

Botrytis cinerea 40m+n (Botr
ytis cinerea) Colletotrichum lagenarium 2O City (Colletotrichum Iag
enarium) Pyricularia oryzae 0 (, Alternaria mali) Xanthomonas orvzae 0 (Escherichia coli)
coli) Staphylococcus aureus 0 (Staphylococcus aureus) Candida albicans 0 (Candida albicans) Acid hydrolysis: 5
．． 7NHCj! After acid hydrolysis at 110°C for 20 hours, amino acid analysis detected serine and cystathionine.

Comparison with other substances In the UV spectrum, antibiotics that have absorption at 2621 nm in medium and acidic conditions and 285 nm in alkaline conditions include nucleoside antibiotics that have a uracil nucleus. especially,
The UV absorptions of Nijimycin B, C, and D and RS10 are the same. However, RS-10 and Nijimycin B+, B2
, C+, C2, D+, and D2 were directly compared by HPLC, but RS-10 did not match these nijmycins. Nijimycin only contains cystathionine as an amino acid, but RS-10 contains cystathionine and serine, and there are no known nucleoside antibiotics containing these two amino acids. Therefore, it was concluded that RS-10 is a new antibiotic.

(Agricultural and horticultural fungicide containing RS-10 as an active ingredient) When using the active ingredient of the present invention as an agricultural and horticultural fungicide,
Generally, granules, powders, emulsions, wettable powders, tablets, oils, sprays, and atomized formulations are prepared using suitable solid carriers, liquid carriers, emulsifying dispersants, etc., in the same way as agricultural chemical formulations known in the art. It can be formulated into any dosage form and applied.

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, ethanolopacetone, dimethylformamide, Examples include ethylene glycol. In addition, adjuvants commonly used in formulations, such as higher alcohols such as ethenopepolyoxyethylene, alkyl, allylethenopearkyl, allyl, polyethylene, glycol ether, alkyl, allyl, sorbitan, dried nolaurate, alkyl - Allyl sulfonate, quaternary ammonium salt, polyalkylene oxide, etc. can be blended as appropriate.

The blending ratio of active ingredients is 10 to 10 for emulsions and hydrating agents.
Approximately 90% is appropriate, and for powders, oils, etc., 0.
Approximately 1 to 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%
, potassium phosphate 0. 3 consisting of a composition of 5% OO
The above RS-10 strain was inoculated into a 6β medium and incubated at 27°C.
The culture was carried out with shaking for 6 hours. The filtrate 27β obtained by filtering this culture solution was transferred to a DO~rex-1 column (7,5X 70
cm) to adsorb the active substance. Next, after washing with 151 g of water, the active substance was eluted with 1N acetic acid of 6β.

The eluate was neutralized with pyridine, concentrated, applied to the top of an IRC-50 column (5.7 x 30 cm), and eluted with water.

The eluate was fractionated, and the active fractions were collected and concentrated, then applied to the top of a Diaion HP2O column (7.5×35 cm) and eluted with water. The eluate was fractionated, and the active fraction was collected, concentrated, and lyophilized to obtain 14 g of crude powder of this substance.

Dissolve 5 g of this 14 g in a small amount of solution (composition of butanol: methanol: water = 2:1:2), add it to the top of a cellulose column (3.8 x 80 cm), elute with a solution of the same composition, and fractionate. did. The fraction amount is 18+r+j! The active substance appeared in fraction No. 88-140, which was concentrated and freeze-dried to obtain 415 mg of powder. The substance was further purified by performing high performance liquid chromatography using this powder. The column is Nucleosil H
Using Carbon○DS (diameter length 20 mm, length 25 cm), the solvent was a 0.02% formic acid-water system.
Flow rate9. Omβ/min.

Pressure 180 kg/c++! If you do this, the development time will be 16~
Active substance appears at 18 minutes. This active fraction was repeatedly collected, concentrated, and then lyophilized to obtain 60 mg of pure white powder of this substance.

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

Example 2 (Emulsion) 10.8 parts of RS-10 parts of ethylene glycol, 20 parts of dimethylformamide, alkyl dimethylbenzyl.
10 parts of ammonium chloride and 52 parts of methanol are mixed and dissolved to obtain 100 parts of an emulsion.

Example 3 (Powder) RS-10.0.2 parts, calcium stearate 0.5
250 parts of clay and 49.3 parts of clay were mixed and ground to obtain 100 parts of a powder.

Example 4 (granules) RS-10, 10 parts, starch 15 parts, bentonite 7
2 parts and lauryl alcohol sulfate) IJ
100 parts of granules are obtained by mixing and pulverizing 3 parts of um salt.

Test Example 1 (Cucumber Botrytis Botrytis Control Test) Cucumber (
Variety: Sumo Hanpaku) was sown and the seedlings grew for 15 days.
A hydrating powder prepared according to Example 1 was diluted to a predetermined concentration and sprayed, followed by air drying.

Botrytis ciner
ea) was cultured on potato glucose agar plate medium, and the spores induced by irradiation with BLB light were
It was suspended in a 1% solution of 0% yeast extract.

After the chemical solution dries, move the cucumbers to an inoculation box, and spray the suspension with a spray gun at one dose per 10 young cucumber seedlings.
0cc spray inoculation.

After inoculation, cucumber seedlings were left under constant temperature and humidity conditions, and
The degree of onset of the disease was examined a day later.

In addition, the control value was calculated by the following method.

(Incidence Index) (Severity of Infection) 0 No onset 1 Slight lesions observed.

2. The diseased area is less than 10%.

The disease area is 10% or more and 20% or less. I can't stand it.

The disease area is 20% or more and 30% or less. I can't stand it.

The affected area is 30% or more and 40% or less. I can't stand it.

The affected area is more than 40%.

The results are shown in the table below.

*Methyl 1-(7” tylcarbamoyl)-2-benzimidazolecarbamate (effect of the invention) From the above test results, the agricultural and horticultural fungicide of the present invention is extremely effective against the above plant diseases even at low concentrations. It was clarified that it would provide a highly safe agricultural chemical that showed high pest control value and no phytotoxicity.

The antibiotic RS-10 of the present application is the same as the substance referred to as "'Antibiotic 5R-10" in the Chinese patent application corresponding to the present application.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the ultraviolet absorption spectrum of the antibiotic RS-10 of the present invention, and FIG. 2 is a diagram showing the ultraviolet absorption spectrum of the antibiotic RS-10 of the present invention.
10 is a diagram showing the infrared absorption spectrum of No. 10. Figure 1 Wavelength (nm)

Claims (4)

[Claims] (1) Antibiotic RS-10 with the following physical and chemical properties
. Decomposition point: 230-232°C Elemental analysis: Carbon 40.43% Hydrogen 5.45% Nitrogen 12.
82% sulfur 3.32% Specific rotation: [α]_D=+9.2° (C=0.5%H_
2O) Ultraviolet absorption spectrum: ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Infrared absorption spectrum: 3350, 2900, 1650
, 1570, 1530, 1380, 1210, 1130
, 1070, 760, 540cm^-^1 Solubility: Easily soluble in water, slightly soluble in methanol and acetone, insoluble in chloroform, benzene, and ethyl acetate Molecular weight: 822 (according to SIMS mass spectrum) (Molecular formula: C_2_9H_4_2N_8O_1_8S) Color reaction : Ninhydrin, Lemieux, Lydon-Smith, positive for periodic acid-benzidine reaction, Anthrone,
Color of negative substance for FeCl_3 reaction: white powder (2) Streptomyces
1. A method for producing antibiotic RS-10, which comprises culturing antibiotic RS-10-producing bacteria belonging to the genus, and separating and collecting antibiotic RS-10 from the culture. (3) The antibiotic RS-10 producing bacteria is Streptomyces
SB RS-10 (Streptomycesp
．． RS-10).The manufacturing method according to claim (2). (4) An agricultural and horticultural fungicide characterized by containing antibiotic RS-10 as an active ingredient.