JPH01320992A - Production of isoxazole-4-carboxylic acid and herbicide containing same - Google Patents

Abstract

PURPOSE:To obtain isoxazole-4-carboxylic acid in high efficiency using microorganisms by putting to culture, in a culture medium, isoxazole-4- carboxylic acid-productive bacteria classified as Streptomyces. CONSTITUTION:Isoxazole-4-carboxylic acid-productive bacteria classified as Streptomyces is put to culture in a culture medium followed by isolating isoxazole-4-carboxylic acid from the resultant cultured product, thus obtaining the objective isoxazole-4-carboxylic acid. Said bacteria is e.g. Streptomyces sp HS-1 strain (FERM No.10011). The culture is pref. made under aerobic conditions, esp. using a depth culture method. The collection of the objective isoxazole-4-carboxylic acid should be carried out by adsorption or partition column chromatography, gel filtration method or dialysis.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for producing isoxazole-4-carboxylic acid and a herbicide containing it as an active ingredient. More specifically, the present invention relates to a method for producing isoxazole-4-carboxylic acid using a microorganism belonging to the genus Streptomyces, and a herbicide containing isoxazole-4-carboxylic acid or a salt thereof as an active ingredient.

[Prior art and problems to be solved by the invention] Sasyl-4-carboxylic acid is Pan1zzi+L, (Ga
zz.

ChiIl, Ital, 77, 206.1947) and is a known compound. However, there have been no reports on a method for producing isoxazole-4-carboxylic acid using microorganisms or on the fact that isoxazole-4-carboxylic acid has herbicidal activity.

A large number of herbicides have been reported to date, and some of them have been put into practical use. However, there are still many unresolved problems in the field of herbicides.

[Means to solve the problem]

The present inventors isolated a large number of microorganisms from soil and searched for the substances produced by them, and found that certain microorganisms belonging to the genus Streptomyces produce isoxazole-4-carboxylic acid, which has strong herbicidal activity. They discovered this and completed the present invention.

That is, the present invention is an isoxazole-4-carboxylic acid-producing bacterium belonging to the genus Streptomyces that is cultured in a medium, and isoxazole-4-carboxylic acid is isolated from the culture. The present invention provides a method for producing an acid and a herbicide containing isoxazole-4-carboxylic acid or a salt thereof as an active ingredient.

The isoxazole-4-carboxylic acid-producing bacterium used in the present invention is strain N3-1, which was newly isolated from the soil at the National Institute of Preventive Health in Tokyo's Parts Ward. N3-
The mycological properties of one strain are as follows.

■, Morphobasic hyphae elongate and branch. When cultured with shaking in a liquid medium, fragmentation of hyphae is observed. Aerial mycelium grows on oatmeal agar, starch agar, etc., and the spore shape is good. The branching of the aerial hyphae is simple, with no axle branching, and the spore chain at the tip of the aerial hyphae is mainly linear. When observed using an electron microscope, the spores are oval or cylindrical in shape.
．． The size is 5 to 1.0 x 0.7 to 1.5 μm, and the surface is smooth. Spores usually form chains of 50 or more. Sporangium.

No motile spores or sclerotia were observed.

(2) Growth status on various media The growth status of the HS-1 strain on various media is shown in Table 1. Regarding color descriptions, the standards shown in parentheses are "
The one described in "Color Harmony Manual" was used. Observations were made after 14 to 21 days of culture at 28°C, r:
- Table 1 ■9 Physiological properties (1) Growth temperature range: Grows in a temperature range of 15 to 40°C on yeast/malt agar, and grows well at 25 to 35°C.

(2) Liquefaction of gelatin: Negative (3) Hydrolysis of starch: Positive (4) Reduction of nitrate: Positive (5) Peptonization of skim milk: Positive Coagulation of skim milk: Positive (6) Salt tolerance: 4% Na C/! It grows slightly in a medium containing it, but does not grow in a medium containing 5% or more.

(7) Production of melanin-like pigment: positive ■, availability of carbon source (using ISP N119 medium) (
1) Things to use: D-glucose, glycerol, L
-Arabinose.

D-xylose (2) Not used: D-mannitol, raffinose, myo-inositol, rhamnose (3) Used in question: D-fructose.

Sucrose ■, cell wall composition [method of 1ecker et al. (Appl, Microbi
As a result of the analysis by J.D. Ol, 13+ 236° 1965), diaminopimelic acid in the cell wall composition was of the LL type.

Based on the above characteristics, strain H3-1 belongs to the genus Streptomyces among actinomycetes, the color tone of the aerial hyphae is in the "Gray" series, the tip of the aerial hyphae is linear, the spore surface is smooth, and the underside color is pale yellow. ~ Yellowish brown in color, it can be summarized as a strain that produces a melanin-like pigment. The present inventors obtained the H5-1 strain from Streptomyces sp.
s sp, H3-],). In addition,
This strain has been entrusted to the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERMP-10011 (FERMP-10011) J.

Although the H3=1 strain is susceptible to changes in its properties as seen in other actinomycetes, it is the H3-1 strain or a mutant strain derived from this strain (naturally occurring or induced).

Any transconjugant or genetically recombinant having the ability to produce isoxazole-4-carboxylic acid can be used in the present invention.

In the present invention, the above-mentioned bacteria are cultured in a medium containing nutrients that can be used by ordinary microorganisms. As a nutrient source, known ones conventionally used for culturing actinomycetes can be used; for example, as a carbon source, glucose, starch syrup, dextrin,
Starch, sucrose, I! dense.

Animal/vegetable oils, etc. can be used. Also, soybean flour, wheat germ, cornstap liquor, and cottonseed meal as nitrogen sources.

Meat extract, peptone, yeast extract, ammonium sulfate,
Sodium nitrate, urea, etc. can be used. In addition, it is effective to add inorganic salts capable of producing sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid, and other ions as required. It also helps the growth of bacteria and isoxazole-4-
Organic and inorganic substances that promote the production of carboxylic acid can be added as appropriate.

The most suitable culture method is a culture method under aerobic conditions, especially a deep culture method. The appropriate temperature for culturing is 25-35°.
However, in most cases, it is cultured at around 28°C. The culture period for producing isoxazole-4-carboxylic acid varies depending on the medium and culture conditions, but the accumulation usually reaches its maximum within 2 to 7 days in both shaking culture and tank culture. Therefore, the culture may be stopped when the amount of isoxazole-4-carboxylic acid accumulated during the culture reaches its maximum.

The amount of isoxazole-4-carboxylic acid produced in the culture solution can be quickly measured using well-known methods such as thin layer chromatography and high performance liquid chromatography.

When collecting the isoxazole-4 carboxylic acid obtained in this way from the culture, conventional separation methods utilizing its properties such as filtration, solvent extraction, adsorption or distribution column chromatography, and gel filtration can be used. Extraction and purification can be carried out by methods such as dialysis, dialysis, precipitation, etc. alone or in appropriate combinations. For example, isoxazole-4-carboxylic acid can be extracted into an organic solvent layer by acidifying the pH of the culture filtrate and extracting with an organic solvent that is immiscible with water, such as butanol or ethyl acetate. Furthermore, it can be extracted from the cultured bacterial cells with a mixture of acetate/water or methanol/water.

For further purification, use silica gel (Wakogel C-20
Purification may be carried out by chromatography or crystallization using an adsorbent such as alumina or Sephadex LH-20 (manufactured by Pharmacia).

Next, the physicochemical properties of isoxazole-4-carboxylic acid (or its sodium salt) are shown below.

1. Appearance: Colorless crystals 2. Elemental analysis (as sodium salt, C411□N0Ja) HN measurement value (χ) 35.16 1.84 10.59
Calculated value (2) 35.56 1.48 10.37
3. Mass spectrum: Measured value 113.0113 Calculated value C,83NO3113,01134, Molecular formula:
C4113NO3 5, specific optical rotation: [α) :”=0” (c=1.0
+ MeOH) 6, melting point: 123°C 7, ultraviolet absorption spectrum (methanol solution): 8 as shown in Figure 1, infrared absorption spectrum (KBr method): 9 as shown in Figure 2, 'H-N MR spectrum (400MHz
.

CD, 00. Sodium salt): δ 8.57 (IH, S), 8.92 (LH, 5) 1
0, '3C-NMR spectrum (100MHz.

CD, OD, sodium salt): 6122.2(S), 151.0(d).

161.5 (d), 168.3 (S) 11, high pressure filter paper electrophoresis (pit 6.4.3500V, 15 minutes): Rrn (glutamic acid) = 1.4 12, color reaction: positive; ninhydrin reagent, Lemieux reagent.

Negative: Based on the properties of the 10% sulfuric acid reagent or higher, it was determined that the material collected from the culture was isoxazole-4-carboxylic acid.

In addition, in an elephant sexual toxicity test using sodium salt of isoxazole-4-carboxylic acid, 200 m
Both oral administration and intravenous injection at 100 g/kg showed survival.

In order to obtain a herbicide containing isoxazole-4-carboxylic acid or its salt as an active ingredient obtained by the above production method, it may be mixed with other carriers or auxiliary agents as necessary and used as a herbicide. It is preferable to use the preparation in the form of a powder, two grains, a wettable powder, an emulsion, a liquid, or an aqueous solution. Examples of solid carriers include talc, bentonite, clay, kaolin, diatomaceous earth, vermiculite, and calcium carbonate, and examples of liquid carriers include water, alcohol, ethyl acetate, acetone, dimethyl sulfoxide, and cyclohexanone.

Examples include xylene. In addition, in order to improve emulsification and/or wettability, for example, alkyl sulfate esters,
Surfactants such as alkyl sulfonates, polyoxyethylene glycol ethers, and polyhydric alcohol esters are used, and auxiliary agents for improving the dispersibility of water cooling agents may also be used.

The content of isoxazole-4-carboxylic acid in the herbicide of the present invention is, for example, 10 to 80% when used as a hydrating agent.
Appropriate parts by weight are 10 to 50 parts by weight when used as an emulsion, 2 to 10 parts by weight when used as a powder, and 2 to 20 parts by weight when used as a granule.

The herbicide of the present invention can be used in rice fields, fields, orchards, greenhouses, and forest areas.

Other herbicides and fungicides are used to kill weeds in non-agricultural land, depending on the respective conditions.

It can be used in combination with pesticides and/or fertilizers. In particular, it exhibits a strong herbicidal effect when sprayed on the foliage of weeds. The range of effective weeds is wide;
-Exhibits high herbicidal effects against annual and perennial monocotyledonous and dicotyledonous weeds. In addition, weeds can be controlled during germination by soil treatment.

〔Example〕

Examples of the present invention are shown below, but these are not intended to limit the present invention. Of course, many variations and modifications not exemplified here may be used.

Example 1 Starch 1.0%, glucose 1.0% as seed medium.

1.0% wheat germ, 1.0% cottonseed meal, 1.0% soybean flour.

Magnesium sulfate 0.05%, dipotassium hydrogen phosphate 0
．． A medium containing 2% calcium carbonate and 0.2% calcium carbonate (no pH adjustment) was used. In addition, starch syrup 2 is used as a production medium.
．． 0%, soybean oil 1.0%, gluten meal 1.0%, wheat germ 1.0%, cottonseed meal 0.5%, calcium carbonate 0.
1%, magnesium sulfate (heptahydrate) 0.1%, ferrous sulfate (heptahydrate) 0.0005%, and cobalt chloride (hexahydrate) 0.0005% (adjusted to pH 7.0). A medium was used.

A 100 ml Erlenmeyer flask containing 20 ml of the seed medium was sterilized at 120°C for 30 minutes, and Streptomyces sp. MS-1 strain (FERM P-
10011) was cultured on slanted agar, inoculated with 2 to 3 platinum loops, cultured with shaking at 28°C for 3 days, and used as a type 1 culture. In addition, a 22-volume Erlenmeyer flask into which the aforementioned seed medium 320 was dispensed was sterilized at 120"C for 30 minutes, inoculated with the aforementioned first seed culture 20!d, and cultured with shaking at 28°C for 1 day. This was a second type of culture.Next, it was incubated at 120°C for
A 502-capacity jar fermenter containing 3542 production medium sterilized for 0 minutes was inoculated with 320 d of the above-mentioned second type culture, and stirred (initial rotation at 250 rpm for 24 hours and then at 400 rpm).
The cells were cultured at 28°C with aeration (201 rpm) for 4 days. After the culture was completed, diatom was added as a filter aid and filtered.

Hydrochloric acid was added to the obtained culture filtrate 551 to adjust the pH to 2.5, followed by extraction with 30ffi of ethyl acetate. This extract is 30! After adjusting the pH to 8.5 by adding 20ffi of water, the active ingredients were extracted by reverse extraction.Then, this extract 201 was
After adjusting to H2.5, add 10I of ethyl acetate again! , was extracted. This extract was concentrated to dryness to give an oily crude substance of about 23
I got g.

Next, 23 g of the obtained crude fluid was applied to a 1.52 ml silica gel column (Wako Gel C-200), and 2.0 g of chloroform was added.
After washing with 52 and developing with a mixed solvent of chloroform-methanol 50:1, the fraction was 300.1 and the fraction was 2.
An active fraction containing isoxazole-4-carboxylic acid was obtained. Both fractions were collected and concentrated to yield 6.2 g of oil.

Next, 6.2 g of the obtained oily substance was dissolved in a small amount of methanol, and this was applied to a Sephadex LH-20 (2,0Q) column filled with methanol in advance and developed with methanol. Fraction Nα62~
72 active fractions were obtained. These two parts were collected and concentrated to dryness to obtain 4.3 g of crude powder of sodium salt of isoxazole-4-carboxylic acid.

When 4.3 g of the obtained crude powder was extracted with a mixed solvent of 100 d of 0.05N hydrochloric acid and 100 trrl of ethyl acetate, isoxazole-4-carboxylic acid was extracted as a free acid in the ethyl acetate layer. After concentrating this extract to dryness, it was dissolved in chloroform and allowed to stand in a cool place to precipitate crystals. The precipitated crystals were collected by filtration and dried to give 2.8g.
crystals were obtained. Furthermore, 2.0 g of free acid crystals of isoxazole-4-carboxylic acid were obtained by recrystallizing from chloroform in the same manner as above.

Formulation Example 1 (Liquid) Isoxazole-4-carboxylic acid 30 parts Polyoxyethylene lauryl ether 10 parts Polyoxyethylene alkylamine 20 parts Sodium phosphate 1 part Disodium phosphate 4 parts Water
Mix 35 parts thoroughly to form a solution.

Formulation example 2 (hydrating powder) Isoxazole-4-carboxylic acid 30 parts Sodium dodecylbenzenesulfonate 5 parts Higher alcohol sodium sulfate 10 parts Polyvinyl alcohol 2 parts Diatomaceous earth
Mix 33 parts thoroughly and crush to obtain a wettable powder.

Test Example 1 The herbicidal effect of isoxazole-4-carboxylic acid (sodium salt, hereinafter the same) applied to field foliage was investigated in a pot test.

Fill a plastic pot with a diameter of 6 cm with field soil, and place it in a plastic pot with a diameter of 6 cm.
+Ech i noch Ioa anus uJ
Setaria viridi), Setaria viridi
s) and Japanese knotweed (h 11 Mingatsu M” mutual ■ 1)
seeds were sown. When it grows to a predetermined size, Tween-2 is added to an aqueous solution of isoxazole-4-carboxylic acid at a predetermined concentration (10, 50, 250, 1000 ppm).
0 (polyoxyethylene sorbitan monolaurate) was added to 1000 ppts, and 100 ff
Spraying treatment was carried out using a sprayer at an amount of water of i/10 are.

The results of the investigation 7 days after treatment are shown in Table 2. As a control, the same operation was performed using alloxydim instead of isoxazole-4-carboxylic acid. The degree of herbicidal effect was expressed using the following 6-level index evaluation method.

O: No effect 1; Effect of about 20 parts 2: Effect of about 40 parts 3; Effect 4 of about 60 parts; Effect 5 of about 80 parts; Complete withering Table 2 7I + Xydim" 103430 *Alloxydim: 5ethyl 3- ( 1-(al
lyloxyan+1no)butylidene)
6+6-dia+ethyl-2+4-dioxycy
Clohexanecarboxylate Test Example 2 The weed-inhibiting effect of isoxazole-4-carboxylic acid in upland soil treatment was investigated by a pot test.

Fill a plastic pot with a diameter of 6 cm with field soil, and add the Japanese crabgrass (Rit±m匡clearis) + Japanese poppy grass■
Maru-kuni-ran-an-ro-o■'' Rainbow■) and Yaenari (Phaseo
lus radiatus) and covered with soil.

100 jl! of an aqueous solution of isoxazole-4-carboxylic acid at a predetermined concentration (100, 500, 2500 ppm).
The soil was treated with a sprayer using a water amount of /10 are. The results of the investigation 14 days after treatment are shown in Table 3. As a control, the same operation was performed using alachlor instead of isoxazole-4-carboxylic acid. The degree of weed control effect was expressed using the following 6-level index evaluation method.

0; No effect 1; Approximately 20% effect 2; Approximately 40% effect 3; Approximately 60% effect 4; Approximately 80% effect 5; Complete germination suppression
0*Alachlor: 2-chloro-2
+6-diethyl-N-(methoxyme
thy 1) acetaniide [Effects of the Invention] According to the present invention, isoxazole-4-carboxylic acid can be efficiently produced using microorganisms. Also,
The herbicide of the present invention containing this compound as an active ingredient exhibits a strong herbicidal effect in foliage treatment, and can be considered to be used as a foliage treatment herbicide. In addition, since it is effective in soil treatment, it can be used as a soil treatment herbicide that suppresses weed germination without causing chemical damage to crops by treating the soil just before or after sowing crops.

[Brief explanation of the drawing]

FIG. 1 is an ultraviolet absorption spectrum of isoxazole-4-carboxylic acid in a methanol solution. FIG. 2 is an infrared absorption spectrum of isoxazole-4-carboxylic acid in potassium bromide tablets.

Claims (2)

[Claims] (1) Isoxazole belonging to the genus Streptomyces
1. A method for producing isoxazole-4-carboxylic acid, which comprises culturing 4-carboxylic acid-producing bacteria in a medium and isoxazole-4-carboxylic acid is isolated from the culture. (2) A herbicide containing isoxazole-4-carboxylic acid or its salt as an active ingredient.