JPH0248234B2 - KOSEIBUTSUSHITSUNOSEIZOHO - Google Patents

Description

[Detailed description of the invention]

The present invention is based on the formula The present invention relates to a method for producing a compound represented by the formula [wherein R represents hydrogen, a halogen atom, a lower alkyl group, or a hydroxymethyl group] or an acid salt thereof. As a result of various studies, the present inventors have discovered that the mildeiomycin-producing bacterium belonging to the genus Streptovaartecillium has a formula of When cultured in a medium containing a compound represented by [wherein R represents hydrogen, a halogen atom, a lower alkyl or a hydroxymethyl group], the compound represented by the above formula () (hereinafter referred to as compound ())
It is sometimes called. or its acid salt can be obtained, especially when cultured in a medium containing 3mM or more of the N-methyl compound, the production of compound () or its acid salt is good, and furthermore, compound () is an excellent plant bactericidal and miticide. The present invention was completed based on these findings. That is, the present invention provides: (1) Cultivating a mildeiomycin-producing bacterium belonging to the genus Streptovaartecillium and having the ability to convert compound () into compound () in a medium containing compound (); (2) A method for producing a compound () or its acid salt, characterized by producing and accumulating the compound () or its acid salt in a substance, and (2) culturing in a medium containing 3mM or more of an N-methyl compound. The present invention relates to the manufacturing method described in (1) above. In the above formula, bromine, chlorine, iodine, and fluorine are used as the halogen atom represented by R.
The alkyl group is preferably a linear or branched one having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and the like. Compound()
Those in which R is a hydrogen atom can be produced at low cost and are advantageous in industrial production. Moreover, the compound of the present invention includes not only the free compound () but also its acid salt. Such acid salts include
For example, salts with inorganic acids such as hydrochloric acid, sulfuric acid, and nitric acid, or organic acids such as formic acid, acetic acid, and butyric acid are used, and salts with hydrochloric acid, sulfuric acid, formic acid, and the like are particularly preferred. Since the compound of the present invention has an asymmetric carbon atom, it is thought that it can take various three-dimensional structures. ) shows the same three-dimensional structure. The mildeiomycin-producing bacteria used for the production of compound () include strains belonging to the actinomycete Streptoverticillium rimofaciens, particularly Streptomyces rimofaciens B-98891 [Deposit number: IFO13592 (FERM -P2549)] is famous [The Journal of Antibiotics, No. 31].
Vol. 6, pp. 511-518, 1978] is an example of a bacterium that can be effectively used for the purpose of carrying out the present invention.
As a general characteristic of actinomycetes, their mycological or physiological properties easily mutate, so various mutant strains or species can be easily obtained both artificially and naturally. These mutant strains and mutant species can also be used for the purpose of the present invention, as long as they do not lose the property of producing the compound () or its acid salt. The production of compound () or its acid salt is carried out by culturing mildeiomycin-producing bacteria in a medium containing compound ().
In general, a medium is used in which a compound () is added in addition to a carbon source, a nitrogen source, and inorganic salts that can be assimilated by microorganisms, and if necessary, trace amounts of effective substances such as micronutrients and growth-promoting factors. In general, carbon sources include
For example, glucose, sucrose, molasses, starch, dextrin, glycerin, sorbitol, etc. are used, and nitrogen sources include, for example, meat extract, soybean flour, corn, staple, liquor, etc.
In addition to organic nitrogen sources such as casein, peptone, and cottonseed meal, inorganic salts such as ammonium salts and nitrates are used, and these may be used alone or in combination. Examples of the compound () include cytosine when R=H, cytidine, cytidylic acid, etc. that are decomposed in the medium to produce cytosine, as well as nucleic acids or their hydrolysates, or many microbial cells containing them. , agricultural and fisheries resources are used. When R=halogen atom, 5-halogenocytosine or 5-halogenocytidine, 5-halogenocytidylic acid, etc., which are decomposed in the medium to produce 5-halogenocytosine, are used.
When R=alkyl group, i.e. 5-alkylcytosine or 5-alkylcytidine, which is decomposed in the medium to produce 5-alkylcytosine, 5
-Alkylcytidylic acid etc. are used, and 5
-Hydroxymethylcytosine or a compound that produces it in the medium, etc. are used. When adding compounds (), or when adding compounds that produce these compounds () in the medium, the concentration of the compound () is 0.01 to 0.01.
It is preferably used at 1.0% (weight/volume), more preferably from 0.03 to 0.5% (weight/volume).
The most effective time to add these to the medium is before the start of culture, but they may also be added at some point during culture. Furthermore, in order to improve the production of compound (), it is preferable to have an N-methyl compound present in the medium. As such an N-methyl compound, a compound having one or more nitrogen atoms substituted with 1 to 4 methyl groups in the molecule is used. Among these, for example, those having one nitrogen atom substituted with a methyl group in the molecule are preferable, and in particular, a trimethylammonio group with a nitrogen atom substituted with three methyl groups:
Quaternary ammonium _{salts with -N+(CH3)3 are} preferred. Also, compounds that can be converted to >N-CH ₃ in the culture medium, such as N,N-methylenebisacrylamide, are also used as N-methyl compounds.
The molecular weight of the N-methyl compound is usually 50-1000, preferably 90-130. Although it can be used regardless of whether it is water-soluble or water-insoluble, easily water-soluble N-methyl compounds are easily used. As a specific example, for example, N
-Methyl acid amide, N-methylamino compound, N
-Methylamine, N-methylammonium, N,N-methylenebisacrylamide, etc. are used. Examples of N-methyl acid amides include N,N-dimethylacetamide and N-methylacrylamide; examples of N-methylamino compounds include N-methylurea, 1,1,3,3-tetramethylurea, and -dimethylaminoethanol, etc.; N-methylamines include trimethylamine, dimethylamine, etc.; N-methylammonium includes lecithin,
Choline, betaine, tetramethylammonium and the like are advantageously used. Good results are obtained with N-methylammonium, especially choline, betaine and tetramethylammonium. These N-methyl compounds may be used alone or in combination of two or more. Another method is to add a large amount of N-methyl compound-containing substances, such as sugar beet molasses containing betaine, soybean flour containing lecithin, or chicken eggs containing choline and lecithin, to the medium to contain 3mM or more of N-methyl compounds. Also included in the method of the present invention. The concentration of the N-methyl compound contained in the medium may be appropriately selected within a range that does not inhibit the growth of the microorganisms used.
It is usually contained at 3mM or more. Preferably 4mM
Addition of 7 to 200mM, more preferably 7 to 50mM, is effective, and if it exceeds 200mM, the rate of extension of the effect will slow down. Furthermore, the amount of natural products containing N-methyl compounds to be added may be selected so that the concentration of the N-methyl compounds contained falls within the above-mentioned range, but a larger amount than the conventional amount would be used. Therefore, if natural products are added alone, the growth of microorganisms will be significantly affected by the influence of other ingredients, and the production of mildeiomycin may be inhibited.
In that case, it is preferable to use an N-methyl compound in combination. The easiest and most effective way to add these N-methyl compounds to the medium is before inoculating the medium with microorganisms, but they can also be added as appropriate during cultivation. Although culture may be carried out by surface culture, it is usually rational to use deep aeration culture. When using the deep culture method, the liquid nature of the medium is preferably slightly acidic or slightly alkaline, and the culture temperature is desirably maintained at 15 to 40°C, particularly 24 to 34°C. However, it goes without saying that these culture conditions should be selected as appropriate to obtain preferable results depending on the type of bacterial strain used, external conditions, etc. Usually 4 days~
If cultured for 14 days, a culture solution containing a significant amount of compound () can be obtained;
In order to collect microorganisms, separation and collection means commonly used for collecting metabolites of microorganisms from their culture fluids are appropriately used. For example this compound ()
Since it is a water-soluble basic substance and is mainly contained in the culture solution, the bacterial cells are first removed by means such as filtration or centrifugation. The obtained supernatant liquid is treated with a suitable adsorbent such as activated carbon, adsorbent resin, cation exchange resin,
After adsorbing the target substance by contacting it with an adsorbent such as activated alumina, silica gel, or molecular sieve,
For example, the target substance can be eluted using a water-containing solution of a water-soluble organic solvent such as acetone, methanol, ethanol, propanol, or butanol, or an aqueous solution of an acid, an alkali, a buffer, or an inorganic salt or an organic salt. After carrying out an appropriate combination of these separation means, the effective fraction is concentrated and powdered, so that the compound () can be collected in a free state or a salt state. In addition, the compound () used as a raw material is
For example, the Journal of the American Chemical Society
American Chemical Society) Vol. 56, p. 134 -
139 pages and Journal of Labeled Compounds
It can be produced by the method described in Vol. 9, No. 3, pages 475-482, or a method similar thereto. The plant fungicidal and acaricide of the present invention contains the compound () as an active ingredient. The compound () may be used alone or in combination with other agents that can be applied to plants. As the dosage form, various dosage forms of known plant fungicides can be appropriately employed. For example, it can be dissolved or dispersed in a liquid carrier (e.g. a solvent), or mixed with or adsorbed onto a suitable solid carrier (e.g. diluent, filler), and if required further coated with e.g. curing agent, dispersing agent, suspending agent, spreading agent, penetrating agent, wetting agent, mucilage agent,
Stabilizers are added and used in the form of oils, emulsions, wettable powders, powders, tablets, granules, sprays, etc. The appropriate concentration of the main active ingredient () is approximately 1 to 70% for emulsions, hydrating agents, etc., and approximately 0.01 to 10% for oils, powders, etc., depending on the purpose of use. These concentrations may be changed as appropriate. In addition, when using emulsions, hydrating agents, etc., dilute or increase the amount with water as appropriate (for example, 100 to 10,000
It is best to double the amount and distribute it. The solvent used in the plant fungicidal and acaricide of the present invention includes, for example, water, alcohols (such as methyl alcohol, ethyl alcohol, and ethylene glycol), and ethers (such as dioxane,
tetrahydrofuran, cellosolve, etc.), aliphatic hydrocarbons (e.g. gasoline, kerosene, kerosene, fuel oil, machine oil, etc.), aromatic hydrocarbons (e.g. benzene, toluene, xylene, solvent naphtha, methylnaphthalene, etc.) Other organic bases (e.g., pyridine, aldehyde collidine, etc.), halogenated hydrocarbons (e.g., chloroform, carbon tetrachloride, etc.), acid amides (e.g., dimethylformamide, etc.), esters (e.g., ethyl acetate, butyl acetate, etc.) , glycerin esters of fatty acids, etc.), nitriles (eg, acetonitrile), and the like, and one or a mixture of two or more of these are used. Diluents and bulking agents include vegetable powders (such as soybean flour, tobacco flour, wheat flour, wood flour, etc.), mineral powders (such as kaolin, bentonite, clays such as acid clay, talcs such as talc powder and waxite powder, silica such as diatomaceous earth and mica powder), as well as alumina, sulfur powder, activated carbon, etc.
One or a mixture of two or more of these may be used. In addition, surfactants used as emulsifiers, spreaders, penetrants, dispersants, etc. include soaps, sulfuric acid esters of higher alcohols, alkylsulfonic acids, alkylarylsulfonic acids, quaternary ammonium salts, Alkylamines, fatty acid esters, polyalkylene oxides, anhydrosorbitols, etc. can be widely used.
In addition, casein, gelatin, starch, alginic acid, agar, polyvinyl alcohol,
Pine oil, bran oil, bentonite, cresol soap, etc. can also be used. In addition to compounds (), other types of fungicides (e.g., copper-based fungicides, mercury-based fungicides, organic sulfur-based fungicides, phenolic fungicides, etc.), herbicides, and insecticides (organic chlorine-based fungicides, organic Phosphorus-based insecticides, natural insecticides, etc.) and other acaricides, nematicides, plant growth regulators, synergists, attractants, repellents, fragrances, plant nutrients, fertilizers, etc., are mixed and used. You can also do that. The amount of the plant fungicidal and acaricide of the present invention to be used, the combination with other types of chemicals, and the mixing ratio thereof will depend on the type of crop to be applied, growth stage, growth conditions, cultivation method, type of disease, and disease onset. Although it varies depending on various conditions such as the condition, the time of application of the drug, the accompanying environmental conditions, the method of application, and the economical use of the drug, the amount may normally be about 1 to 300 g per 10 ares. As for the concentration used, it is suitable that the final concentration containing the compound () of the present invention is in the range of 1 μg/ml to 10 mg/ml. Furthermore, the fungicidal and acaricide of the present invention may be used by directly spraying it on the surface of crops or by irrigating it into the soil. That is, as long as it is used safely and effectively on crops, no restrictions will be placed on the present invention, regardless of the amount, concentration, or method of use. The compound () or its acid salt obtained by the method of the present invention is used, for example, as a fungicidal and acaricide for plants, and is effective against various plant diseases, and is also useful in combination with an acaricidal effect. For example, food crops (e.g. barley), specialty crops (e.g.
tobacco), fruit trees (e.g. apples), forest trees (e.g. oaks), vegetables (e.g. cucumbers, Japanese cucumbers, green peppers,
powdery mildew on tomatoes), petals (e.g. roses),
For example, it is particularly effective against diseases of tomatoes, potatoes, etc. Furthermore, there are no problems with toxicity (eg, acute toxicity, irritation of the eye mucous membranes and skin, fish poisoning, etc.) and it is extremely safe. Example 1 500 ml of a medium consisting of 1% glucose, 0.3% yeast extract, and 0.5% peptone (weight/volume) was adjusted to pH 7 with a 20% aqueous solution of caustic soda, then dispensed into two Sakaguchi flasks, sterilized, and then injected with streptovertebrate. Streptoverticillium rimofaciens IFO13592
After inoculating the slant culture of (FERM-P2549),
Culture was carried out on a reciprocating shaker incubator at 28°C for 48 hours. 50
10% glucose, 3% Proflo (manufactured by Trailers), 1% cornstarch liquor in a fermenter,
Salt 0.5%, calcium carbonate 0.5%, ferrous sulfate
0.001%, antifoaming agent (Actocor, manufactured by Takeda Pharmaceutical)
Medium 30 consisting of 0.05% (weight/volume) was adjusted to pH 7 with 20% caustic soda aqueous solution, sterilized, and inoculated with 500 ml of the previously cultured Sakaguchi flask culture, and the aeration volume was
A seed culture was obtained by culturing at 28° C. for 48 hours at 1 VVM (aeration volume per minute per unit liquid volume) and a stirring speed of 100 rpm. After preparing and sterilizing the above seed culture medium 100 with 100 g of cytosine added to a 200-volume fermenter, the above seed culture 10 was transplanted, and the aeration volume was 0.5 VVM and the stirring rotation speed.
The cells were cultured at 200 rpm and a temperature of 28°C for 5 days. Take 30% of the culture fluid thus obtained, add 20% of water and 1kg of Hyflo Supercel (manufactured by Johns Manville), and filter it to obtain 45% of the filtrate.
This was passed through a column filled with activated carbon for chromatography (manufactured by Takeda Pharmaceutical Co., Ltd.) No. 6. column
After washing with 20% water, 7% isobutanol water 30%
The effective fractions were collected and passed through a 6 ion exchange resin, Amberlite IRC50 (manufactured by Rohm and Haas) H ⁺ type column, and after washing the column with 20 ml of water, 2% ammonia was added. It eluted with water. Effective fractions of the eluate were collected and concentrated under reduced pressure to obtain a 0.4 concentration solution, and the insoluble materials contained therein were filtered off. The filtrate was added dropwise to 3.5 ml of ethanol, the resulting precipitate was collected, and when dried, the R=H compound ()
15 g of coarse powder was obtained. Example 2 5 g of the coarse powder of the compound obtained in Example 1 was dissolved in 30 ml of water, and this was added to the ion exchange resin Amberlite.
Pass through a column filled with CG-50 (manufactured by Rohm & Haas) 1 and buffered with 0.3M lithium borate buffer (PH8.8), then use the same buffer to flow out the effective fraction. did. The effective fraction obtained is 50
ml activated carbon for chromatography (manufactured by Takeda Pharmaceutical)
After washing the column with 200 ml of water, it was eluted with 5% isobutanol water.
Effective fractions were collected and concentrated under reduced pressure, and the concentrated solution was dropped into ethanol to precipitate. When this precipitate was collected and dried, 1.2 g of R=H compound () was obtained. Example 3 4 g of the coarse powder of the compound obtained in Example 1 was dissolved in water, and ion exchange resin Amberlite CG-50 1.8
Chromatography was carried out in the same manner as in Example 2 using the same column, and effective fractions were collected. 40ml of this
Pass the liquid through an activated carbon column for chromatography to adsorb it, wash with 150 ml of water, and then add 20% acetone water containing 0.5% formic acid (both weight/volume).
The effective fractions were collected, concentrated under reduced pressure, and added dropwise to ethanol. The precipitate was collected and dried, and the formate of the compound () with R=H
1.0g was obtained. The typical physical and chemical properties of this product were as follows. Melting point 227℃ (decomposition) Optical rotation [α] ²² _D = +71.3゜ (c = 0.5, water) Ultraviolet absorption λ ^H2O _nax = 268nm, E ¹ % _1cn = 163 Elemental analysis value C = 39.12%, H = 6.47%, N=18.93% Example 4 When culturing according to the method of Example 1, when cytosine was added to the medium at various concentrations listed in Table 1 below,
The compound () with R=H was obtained in the amounts shown in Table 1.

【table】

[Table] Example 5 Streptoverticillium limofaciens was grown in the same manner as in Example 1.
rimofaciens) IFO13592 (FERM-P2549) was used as a seed culture, but in Example 1,
While cytosine was added to a 200 volume fermenter,
In this example, 100 g of 5-bromocytosine was added. Culture was carried out using a 200 volume fermenter under the same conditions as in Example 1, and approximately 100 culture solutions were obtained.
Take 30% of the culture solution obtained in this way, add 20% water and 1kg of Hyflo Super Cell (manufactured by Johns Manville) to obtain a 45% filtrate, and filter this with activated carbon for chromatography (Takeda Pharmaceutical Co., Ltd.) of 6. The solution was passed through a column packed with a 20 columns
After washing with water, elution was performed using 30% 7% isobutanol water, the effective fractions were collected, and the ion exchange resin Amberlite IRC-50 (manufactured by Rohm and Haas) H ⁺ type was collected. The column was washed with 20ml of water and then eluted with 2% aqueous ammonia. The effective fractions after elution were collected and concentrated under reduced pressure to obtain a 0.4% concentrated solution, and the insoluble matter contained therein was filtered off. The filtrate was dropped into 3.5 g of ethanol, and the resulting precipitate was collected and dried to obtain 10.5 g of coarse powder. When 5 g of this crude powder was repurified in the same manner as in Example 2, 1.8 g of R=Br compound ()
was gotten. Example 6 Streptoverticillium limofaciens was grown in the same manner as in Example 1.
rimofaciens) IFO13592 (FERM-P2549) was used as a seed culture, but instead of the cytosine added in Example 1, 100 g of 5-methylcytosine was added in this example. Culture was carried out using a 200 volume fermenter under the same conditions as in Example 1, and approximately 100 culture solutions were obtained.
30 was taken from the culture solution obtained in this way, and a purification procedure similar to that shown in Example 5 was carried out to obtain R.
1.5 g of the compound () powder of =CH ₃ was obtained. The free compounds () obtained in Examples 2, 5, and 6 above exhibited the following properties. (1) Solubility It is easily soluble in water, but poorly soluble in other organic solvents. (2) Color reaction Sakaguchi reaction, potassium permanganate reagent reaction is positive, phthalic acid, aniline reagent, ninhydrin reagent reaction is false positive, Ehritz reaction, Dragendorff reaction, Burton reaction is negative. (3) Thin layer chromatography The thin layer chromatography Rf values of the compounds obtained in Examples 2, 5, and 6 are collectively shown in Table 2.

【table】

[Table] (4) Melting point All compounds gradually carbonize from around 230°C and do not show a clear melting point. (5) Specific rotation The optical rotations are listed in Table 3.

[Table] (6) Contained elements and elemental analysis values are summarized in Table 4. The elemental analysis values for each compound are shown, and the measured values almost matched the calculated values.

【table】

[Table] (7) Ultraviolet absorption spectrum Spectra obtained when water, 1/10N NaOH, and 1/10N HCl were used as solvents were obtained, and the maximum value λ _nax and E ¹ % _1cn when water was used as the solvent were obtained. 5.

[Table] (8) Nuclear magnetic resonance spectrum Table 6 shows the δ values (ppm) obtained from the ¹³ C-NMR spectrum measured in heavy water.

【table】

[Table] (9) High performance liquid chromatography column; inner diameter 2.1mm x 450mm, packing material: Hitachi ion exchange resin #2610, transfer tank: 0.3M lithium borate buffer (PH9.02), temperature 45℃ When performing chromatography at a pressure of 90 to 110 Kg/cm ² and a flow rate of 0.6 ml/min to detect absorption at 254 nm, the retention time of the compound of Example 2 was 9.5 minutes, and the retention time of the compound of Example 5 was 12.5 minutes, Example 6
The retention time of the compound is 10.5 minutes. (10) Biological activity Compound () by agar dilution method (in vitro)
The antibacterial spectrum of is shown in Table 7. That is, the compound () is a type of bacteria,
It is found that it exhibits antibacterial activity against plant pathogenic fungi and some yeasts. In particular, the compound of Example 5 showed extremely strong antibacterial activity against some bacteria.

[Table] Test medium A: 3.0% sucrose, 0.2% L-asparagine,
NH ₄ NO ₃ 0.3%, KH ₂ PO ₄ 0.1%, MgSO ₄ .
7H ₂ O 0.1%, Vercenol (Dow Chemical Iron Chelate Compound, Iron Sodium; Contains 57.04% Ethanol/Ethylenediamine Triacetate) 0.001%, Powdered Agar
1.5% (PH7) Add the following vitamins to the culture medium at the final concentrations listed before use. Thiamine 1μg/ml, riboflavin 1μ
g/ml, calcium pantothenate 1 μg/ml,
Niacin 1μg/ml, biotin 0.005μg/ml,
Folic acid 0.5μg/ml, pyridoxine hydrochloride 2μg/
ml, para-aminobenzoic acid 0.5μg/ml, cyanocobalamin 0.0002μg/ml B: Ehrlitsuch meat extract 0.5%, polypeptone 0.5%, NaCl 0.5%, powdered agar 1.5% (PH7) C: Glycerin 3.0%, Ehrlitsuch meat extract
0.5%, polypeptone 0.5%, NaCl 0.5%, powdered agar 1.5% (PH7) (11) Toxicity The acute toxicity of the compounds of Examples 2, 5, and 6 using mice is as follows. LD ₅₀ (mg/Kg) Intravenous injection >100 Intraperitoneal administration >200 Subcutaneous administration >200 Example 7 Similar to Example 1, Streptoverticillium
rimofaciens) IFO13592 (FERM-P2549) to obtain a seed culture. Glucose 10%, cottonseed germ powder (trade name: Proflo, manufactured by Trailers) 3
%, cornstarch liquor 1%, salt 0.5%,
Calcium carbonate 0.5%, ferrous sulfate 0.001%, antifoaming agent (trade name Actocol, manufactured by Takeda Pharmaceutical) 0.05%
A medium with a composition of (weight/volume) is used as a basic medium, and various additives described below are added to it, and 20%
After adjusting the pH to 7 with aqueous caustic soda solution, add 25 ml each.
Dispense into a 200ml Erlenmeyer flask. As shown in Table 8, R=H in compound (), Br,
Four types of compounds corresponding to CH ₃ and I are respectively added, and in each case, choline is added as an N-methyl compound and cases are set where it is not added, to prepare a total of 8 types of media. Transfer 1 ml of the seed culture to each of the 8 types of media and culture on a rotary shaker (rotation speed 200 rpm) at 28°C for 5 days.
The culture solution was centrifuged at 5000 g to remove the bacterial cells and obtain supersemen, which was diluted 50 times and subjected to high performance liquid chromatography to determine the R= When H is added, the compound () (R=H) is added, and when R=Br in the compound () is added, the compound () (R=
Br), when R=CH ₃ in the compound () is added, the compound () (R=CH ₃ ), and when R=I in the compound () is added, the compound () (R
=I) is quantified by the method described below. A 45 cm column was packed with Hitachi 2610, a cation exchange resin, and installed in a high performance liquid chromatography device (Waters, Model 6000A). Drain the M boric acid-lithium hydroxide buffer (PH9.02). When 25μ of culture dilution was injected into this, compound () (R=H) was dissolved in 9.5 minutes, compound () (R=
Br) is 12.5 minutes, and compound () (R=CH ₃ ) is 10.5 minutes.
The compound () (R=I) is released with a retention time of 20.5 minutes, and its ultraviolet wavelength absorption at 254 nm is detected by a UV detector, and quantified from the maximum value of the absorption curve recorded by a self-recording device.

【table】

[Table] * Quantitative value calculated from the maximum value of the 254 nm ultraviolet absorption curve of compound () using high performance liquid chromatography Resin: Hitachi 2610 cation exchange resin, column length: 45 cm, column heat retention: 45°C , Eluent: 0.3M boric acid-lithium hydroxide buffer (PH9.02) Outflow rate: 0.6ml per minute Under these conditions, the product was subjected to high performance liquid chromatography, and the retention time was as follows. Product, Compound () (R=H, Br, _CH3 and I respectively) R=H 9.5 min; R=Br 12.5 min; R= _CH3 10.5
minutes; R=I 20.5 minutes Example 8 In the same manner as in Example 7, N-
Using trimethylamine as a methyl compound,
The effect of the addition was measured and the results are shown in Table 9.

[Table] Quantitative values calculated from [Table] The conditions were as described in Example 7. Example 9 Similar to Example 1, Streptoverticillium limofaciens (Streptoverticillium
rimofaciens) IFO13592 (FERM-P2549) to obtain a seed culture. In a 200ml Erlenmeyer flask,
Glucose 10%, Cottonseed Germ Powder (Product Name: Proflo,
Trailers Inc.) 3%, corn staple liquor 1%, salt 0.5%, calcium carbonate 0.5%, ferrous sulfate 0.001%, antifoaming agent (trade name: Actocol,
25 ml of a medium having a composition of 0.05% (weight/volume) (manufactured by Takeda Pharmaceutical) was adjusted to pH 7 with a 20% aqueous solution of caustic soda and dispensed, and this was used as the basic medium. As shown in Table 10, a culture comparison test was carried out with and without adding something corresponding to R=CH ₂ OH in compound (), that is, 5-hydroxymethylcytosine, as an additive. Seed culture solution 1
ml was transplanted, and cultured on a rotary shaker (rotation speed 200 rpm) at 28°C for 5 days, and a compound corresponding to R=CH ₂ OH in the product compound (・Letters, Tetrahedron Letters
No. 444277-4280 (1978)) was quantified by high performance liquid chromatography in the same manner as in Example 8. Mildeiomycin was detected at a retention time of 5.6 minutes by high performance liquid chromatography under these conditions, and the quantitative results are shown in Table 10.

[Table] Example 10 A wettable powder is obtained by mixing 10 parts of the compound of Example 2, 2 parts of sodium lignin sulfonate, 3 parts of polyoxyethylene alkylaryl ether, and 85 parts of clay. Dilute this agent 500 to 1000 times with water and spray it uniformly on agricultural and horticultural crops at a rate of 100 to 200 parts per 10 acres. Example 11 0.5 part of the compound of Example 2 and 99.5 parts of clay are mixed to obtain a powder. 3 to 5 kg of this drug per 10a
Spray directly. Example 12 10 parts of the compound of Example 3, 5 parts of polyoxyethylene alkylaryl ether, and 85 parts of lactose were mixed and stirred to form a water solvent, and diluted with water to the desired concentration.
Spray evenly on 100 agricultural and horticultural crops per 10a. Example 13 An aqueous solution prepared by mixing 50 parts of the compound of Example 5, 5 parts of methanol, 5 parts of amine stearate and 40 parts of water is diluted with water and sprayed in the same manner as in Example 10. Example 14 0.5 parts of the compound of Example 6, 5 parts of gum arabic,
A granule prepared by mixing and granulating 30 parts of bentonite and 64.5 parts of talc is directly applied in an amount of 3 to 5 kg per 10 a. Test Example 1 A tomato late blight control test was conducted. Tomatoes (variety Daiso Soju) were grown in 12 cm pots, and the seedlings were used for 30 days. Two replicate tests were conducted with 4 pots per section. A sufficient amount of the chemical solution was sprayed, and two days later, a zoospore suspension of Phytophthora tomato was inoculated by spraying. The plants were kept in a greenhouse for 4 days and the lesion area ratio (on the 3rd and 4th leaves) was investigated. The results are shown in Table 11.

[Table] Test Example 2 Iwasa et al. [The Journal of Antibiotics,
Vol. 31, No. 6, p. 512, 1978], the control effect on powdery mildew of barley was investigated. All of the compounds of Examples 2, 5, and 6 were found to be extremely effective. Test Example 3 A miticidal effect test was conducted. Female adult of the two-spotted spider mite (Totranychus urticae) on the true leaves of green bean seedlings (5 days after germination) planted in pots (9 cm in diameter)
After about 50 animals were inoculated, 20 ml of the test solution was sprayed per pot using a spray gun. After spraying, the pot was placed in a Phytotron (28℃), and the number of surviving mites on the leaves on the 5th and 7th day was investigated, and the reduction rate relative to the number of mites immediately before spraying (= number of test mites - number of surviving mites) / number of test mites x 100). Table 12 shows that all of them were extremely effective. At the same time, chemical damage to green beans was observed, but was not observed in all cases.

[Table] *: Indicates the rate of increase.

Claims (1)

[Scope of Claims] 1 Belongs to the genus Streptuvatecilium and has the formula A compound [hereinafter referred to as compound ()] represented by [wherein R represents hydrogen, a halogen atom, a lower alkyl or a hydroxymethyl group] is represented by the formula A mildeiomycin-producing bacterium that has the ability to convert into a compound represented by [the symbols in the formula have the same meanings as above] [hereinafter referred to as compound ()] is cultured in a medium containing compound () to produce a culture. A method for producing the compound, which comprises producing and accumulating the compound () or its acid salt in the compound. 2. Claim 1, characterized in that the culture is carried out in a medium containing 3mM or more of an N-methyl compound.
Manufacturing method described in section.