JPH0819131B2 - Novel substance PF1018 substance, its production method and disinfectant - Google Patents

Description

The present invention relates to a novel substance PF1018 substance and its production method,
And pesticides.

2. Description of the Related Art Conventionally, various physiologically active substances produced by microorganisms have been known, but since many substances having insecticidal activity have not been found, the creation of new insecticidal substances has always been demanded. There is. An object of the present invention is to provide a novel substance PF1018 substance, a method for producing the same, and an insecticide containing the PF1018 substance as an active ingredient.

Means for Solving the Problem The gist of the first aspect of the present invention is that a novel substance PF1018 is used.
In substances and their salts. The physicochemical properties of the PF1018 substance according to the present invention are as follows.

(1) Color and shape: pale yellow granular crystals (2) Elemental analysis: Calculated as C ₂₈ H ₃₅ NO ₃ · H ₂ O C 74.47%, H 8.26%, N 3.10% Actual measurement C 75.08%, H 8.06% , N 3.38% (3) Mass spectrum (FD-MS): m / z 433 (M ⁺ ) (4) Melting point: 182-184 ℃ (5) Specific rotation: ▲ [α] ²⁴ _D ▼ ＝ -185 ° ( c 1.0, CHCl
₃ ) (6) Ultraviolet and visible absorption spectrum λ _{max nm} (▲ E ^1% _1cm ▼) [MeOH]: 204 (393), 251 (294), 320 (355) [0.1N HCl-MeOH]: 204 (215), 235 (229) , 332 (471) 358 (sh 280) [0.1N NaOH-MeOH]: 213 (992), 254 (326), 316 (340) (7) Infrared absorption spectrum (KBrcm ^{- 1} ): 3410,2960,2925,2870,1710,1640,1580,1
430,1380,1360,1330,1315,1290,1245,1230,1165,1125,1
075,1040,1010,1000,950,920,890,870,845,805,775,76
0,720 (8) ¹ H NMR spectrum: shown in FIG.

(9) ¹³ C NMR spectrum: shown in FIG.

(10) Solubility: chloroform, ethyl acetate, acetone,
Soluble in methanol, not water.

(11) Distinction between basic, acidic and neutral: acidic substance Further research based on these physicochemical properties showed that PF
The 1018 substance was found to have the following chemical structure: The gist of the second aspect of the present invention is a method for producing a PF1018 substance by culturing a PF1018 substance-producing bacterium belonging to an incomplete bacterium and collecting the PF1018 substance from the culture.

An example of the PF1018 substance-producing bacterium used in the present invention is the PF1018 strain newly isolated from soil in Omachi City, Nagano Prefecture.

1. Bacteriological properties of PF1018 substance-producing bacteria: potato-glucose agar medium (PDA), potato-carrot agar medium (PCA), cornmeal agar medium (CMA)
The growth state of the PF1018 strain was examined in various types of medium, and the same growth state was shown in all the medium. Growth rate at 25 ° C for 7 days
It was 20 to 22 mm for 10 mm for 14 days, but did not grow at 37 ℃. Growth at pH 5-7 is good. The surface of the community is flat and fluffy in white to gray, but becomes black as the conidia of brown to black are settled. The back of the settlement is also orange at first, but it becomes black with conidia formation. No soluble pigment is produced.

The observation results under the microscope are described below. Conidia peduncle is colorless, singulates from aerial hyphae, unbranched, and shows cuboid or ampoule type. Conidia are unicellular and smooth. The size is 6.0-8.4 × 3.6-4.4 μm, and its shape is elliptical. The conidia is of the Aleuro type, with conidia singly on the tip and sides of the conidia peduncle and not linked. The upper part is round and the base is cut.

From the above bacteriological characteristics, the PF1018 strain was identified as MB Ellis [Dematiaceous Hyphomycetes.75-77, CM
I., Kew (1971)] described Humico spp.
The present inventors named this bacterium as Humicola sp. PF1018 (Humicola sp. PF1018). Initially, this strain was entrusted to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Microindustrial Research Institute No. 10365 (FERM P-10365), but now it has been converted to an international deposit and has been approved by the Microindustrial Research Institute. Commissioned as No. 2627 (FERM BP-2627).

The PF1018 strain is likely to change its properties as seen in other fungi. For example, mutant strains (naturally occurring or inducible) derived from the PF1018 strain, transzygotes or genetic recombinants that produce the PF1018 substance can all be used in the present invention.

2. Cultivation method of PF1018 substance-producing bacterium The PF1018 substance-producing bacterium belonging to the imperfect bacterium is cultured in a medium containing nutrients that can be used by ordinary microorganisms. As the nutrient source, known ones conventionally used for culturing fungi can be used. For example, as the carbon source, glucose, starch syrup, dextrin, starch, molasses, animal / vegetable oil, etc. may be used. As the nitrogen source, soybean flour, wheat germ, corn steep liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate,
Urea or the like may be used. If necessary, sodium,
It is effective to add inorganic salts capable of forming potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid and other ions. In addition, organic and inorganic substances that help the growth of bacteria and promote the production of PF1018 substance can be added appropriately.

As the culture method, the culture method under aerobic conditions, especially the submerged culture method is most suitable. A suitable temperature for culturing is 23 to 30 ° C, but in most cases culturing is performed at around 26 ° C. The production of the PF1018 substance varies depending on the medium and culture conditions, but the maximum accumulation is usually reached in 2 to 7 days in both shaking culture and tank culture. When the accumulated amount of PF1018 substance in the culture reaches the maximum, stop the culture and isolate and purify the target substance from the culture.

3. Method for Purifying PF1018 Substance In collecting the PF1018 substance obtained by the present invention from a culture, a conventional separation means utilizing its properties,
For example, a solvent extraction method, an ion exchange resin method, an adsorption or distribution column chromatography method, a gel filtration method, a dialysis method, a precipitation method and the like can be used alone or in combination to perform extraction and purification. For example, the PF1018 substance is extracted from the cultured cells with acetone-water, methanol-water, ethyl acetate or the like. If the PF1018 substance accumulated in the culture solution is extracted with an organic solvent immiscible with water, such as butanol or ethyl acetate, the PF1018 substance is extracted into the organic solvent layer.

To further purify the PF1018 substance, silica gel (Wakogel C-200, Wako Pure Chemical Industries, Ltd.), adsorbent such as alumina, Sephadex LH-20 (Pharmacia), Toyopearl HW-40 (Tosoh Corporation) It is advisable to carry out chromatography using a product manufactured by Mitsui Chemicals, Inc.

The PF1018 substance thus produced in the culture can be separated in free form, as the PF1018 substance itself, and the solution containing the PF1018 substance or its concentrate can be treated with a base, for example sodium hydroxide, water. For example, extraction, separation or with an alkali metal compound such as potassium oxide, an alkaline earth metal compound such as calcium hydroxide or magnesium hydroxide, an inorganic base such as ammonium salt, or an organic base such as ethanolamine, triethylamine or dicyclohexylamine. When processed during the operation of each step of purification, the PF1018 material transforms into its corresponding salt form and is separated. Another PF manufactured in this way
The salt of substance 1018 can be converted into a free form by a conventional method. Further, the PF1018 substance obtained in a free form may be converted into the corresponding salt thereof by a conventional method with the above base. Therefore, the salts thereof as described above as well as the PF1018 substance are included in the scope of the present invention.

The gist of the third invention is an insecticide containing the PF1018 substance as an active ingredient.

The pests for which the PF1018 substance of the present invention is particularly effective include Lepidoptera such as Spodoptera litura, Plutella xylostella, Culicidae, Weevil, Coleoptera such as Chrysomelidae, Diptera such as Musca domestica, Culex pipiens, Thysanoptera, and web wing of cockroaches, etc. Eyes, aphids, planthoppers, leafhoppers, stink bugs and other hemiptera, other orthoptera, spider mites and the like.

When the compound of the present invention is used as an active ingredient of an insecticide, it may be used as it is, but usually a suitable solid carrier,
Mixture with liquid carrier, gaseous carrier, surfactant, dispersant and other formulation aids, bait etc., emulsion, liquid, wettable powder, powder, granule, oil, aerosol, flowable, poison bait, etc. It can be used as a dosage form.

Examples of the solid carrier include talc, bentonite, clay, kaolin, diatomaceous earth, ba-miculite, white carbon, calcium carbonate and the like. Examples of the liquid carrier include alcohols such as methanol, n-hexanol, ethylene glycol and cellosolve, ketones such as acetone, methyl ethyl ketone and cyclohexanone, aliphatic hydrocarbons such as kerosene and kerosene, benzene, toluene, xylene and methylnaphthalene. Such as aromatic hydrocarbons, dichloroethane, trichloroethylene, halogenated hydrocarbons such as carbon tetrachloride, diethyl ether, dioxane, ethers such as tetrahydrofuran,
Examples thereof include esters such as ethyl acetate, nitriles such as acetonitrile and isobutyronitrile, acid amides such as dimethylformamide and dimethylacetamide, vegetable oils such as soybean oil and cottonseed oil, dimethyl sulfoxide and water. Examples of the gaseous carrier include LPG, CFC gas, air, nitrogen, carbon dioxide gas, dimethyl ether and the like.

Examples of surfactants and dispersants for emulsification, dispersion, spreading and the like include alkyl sulfates, alkyl (aryl) sulfonates, polyoxyalkylene alkyl (aryl) ethers, polyhydric alcohol esters,
Lignin sulfonate and the like are used.

Further, as an auxiliary agent for improving the properties of the preparation, for example, carboxymethyl cellulose, gum arabic, polyethylene glycol, calcium stearate, etc. are used.

The above-mentioned carrier, surfactant, dispersant and auxiliary agent may be used alone or in combination as necessary. The content of the active ingredient in these preparations is usually 1-50 parts by weight for emulsions, 0.3-25 parts by weight for powders, and usually for wettable powders.
1-90 parts by weight, and usually 0.5-10 parts by weight for granules are suitable.

These formulations are used as they are or after being diluted. Further, it can be used by mixing with other insecticides, acaricides, fungicides, herbicides, plant growth regulators, fertilizers, soil conditioners, synergists and the like.

Examples Production examples, formulation examples, and test examples of the present invention are shown below, but the present invention is not limited to these exemplifications.

Production Example 1. As a seed medium, a medium having a composition of 2.0% starch, 1.0% glucose, 0.6% wheat germ, 0.5% polypeptone, 0.2% soybean flour, 0.3% yeast extract, and 0.1% calcium carbonate was used. As a production medium, starch 2.0%, glucose 2.0%, soybean flour 1.0%, wheat germ 1.0%, meat extract 0.5%, sodium chloride 0.2%, calcium carbonate 0.3.
%, Magnesium sulfate (7-hydrate) 0.1%, zinc sulfate (7
A medium having a composition of 0.001% (water salt) was used. The pH before sterilization was adjusted to pH 7.0 before use.

1 ml of a 100 ml Erlenmeyer flask in which 20 ml of the seed medium was dispensed.
Sterilize at 20 ℃ for 30 minutes, then add Fumicola, SP, PF
Strain agar culture of 1018 strain (FERM BP-2627) was inoculated with 2 to 3 platinum loops and shake-cultured at 26 ° C. for 5 days to prepare a first seed culture. Then, the 500 ml Erlenmeyer flask into which the seed medium (80 ml) was dispensed was sterilized at 120 ° C for 30 minutes, and the first seed culture (4 ml) was performed.
Was inoculated and cultured at 26 ° C. for 3 days with shaking to give a second seed culture.

50L containing 35L production medium sterilized in advance at 120 ℃ for 30 minutes
Each of the 2nd seed cultures described above was applied to 2 Yar fermenters.
400 ml of each was inoculated, and aeration (20 L / min) and stirring (initial 250 rpm, 400 rpm after 41 hours) were cultivated at 26 ° C for 5 days. After the completion of the culture, diatomaceous earth was added as a filter aid and filtered to obtain a filtrate and cells.

60% acetone water (50 L) was added to the cells, and the cells were filtered for 1 hour, and the cells were filtered to obtain a cell extract. For the cell extract, 22 L of concentrated solution was prepared by distilling off acetone under reduced pressure. The PF1018 substance was extracted from this concentrated solution with ethyl acetate (20 L × 2), and the ethyl acetate layer was concentrated to give an oily substance (16 g). This oily substance was placed on the top of a silica gel column (700 g) and subjected to chromatography using hexane-acetone (4: 1) as a developing solvent, and the fraction containing the PF1018 substance was concentrated to dryness to give a brown oily substance (320 mg). was gotten. Then, this oily substance was placed on the top of a silica gel column (20 g) and subjected to chromatography using chloroform-methanol (100: 1) as a developing solvent. Crude PF10 obtained
The 18 substances (233 mg) were further purified by Sephadex LH-20 (600 ml) column chromatography using methanol as a developing solvent to obtain a pale yellow oily substance (216 mg). Chloroform (120 ml) of this pale yellow oily substance
Dissolved in water and washed with 0.01 N hydrochloric acid (120 ml), the chloroform layer was concentrated to dryness, and the residue was dissolved in methanol (5 ml) and concentrated to dryness again to give pure PF1018 substance as a pale yellow powder (176 m
g). This powder was dissolved in methanol and recrystallized to obtain 110 mg of pale yellow granular crystals of PF1018 substance.

Formulation Example 1. Emulsifier 20 parts by weight of the compound of the present invention, N, N dimethylformamide 20
10 parts by weight of polyoxyethylene alkylallyl ether was added to 50 parts by weight of xylene and 10 parts by weight of polyoxyethylene alkyl allyl ether to uniformly mix and dissolve to obtain an emulsion.

Formulation Example 2. Wettable powder 25 parts by weight of the compound of the present invention, 30 parts by weight of clay, diatomaceous earth
35 parts by weight, 3 parts by weight of calcium ligninsulfonate and 7 parts by weight of polyoxyethylene alkylallyl ether were uniformly mixed and pulverized to obtain a wettable powder.

Formulation Example 3. Powder A powder was obtained by uniformly mixing 2 parts by weight of the compound of the present invention, 60 parts by weight, 37 parts by weight of talc, and 1 part by weight of calcium stearate.

Formulation Example 4. Granules 5 parts by weight of the compound of the present invention, 40 parts by weight of bentonite, 53 parts by weight of talc, 2 parts by weight of calcium lignin sulfonate are uniformly pulverized and mixed, and water is added and kneaded well, and then granulated and dried. To obtain granules.

Test Example 1. Efficacy test against diamondback moth (Plutella xylostella) larvae A solution prepared according to Formulation Example 1 and diluted to 1000 ppm with water containing a surfactant (0.05%) was used to prepare 10 1st larvae of the diamondback moth.
It was immersed for 0 seconds. After the treatment, the insects were released into a 9 cm-diameter plastic cup containing cabbage leaf pieces (5 cm × 5 cm) and left in a constant temperature room at 25 ° C. The number of live and dead insects after 2 days was investigated, and the mortality rate was calculated from the following formula.

Mortality rate (%) = (number of dead insects / number of test insects) x 100 The result was a 100% mortality rate.

Test Example 2. Efficacy test for adult female of Tetranychus cinnabarinus (Tetranychus cinnabarinus) Ten adult females of the Japanese red spider mite were inoculated on primary leaves of Phellinus linteus grown in a plastic pot having a diameter of 6 cm.
One day after the inoculation, a solution was prepared according to Preparation Example 2 and 10 ml of a chemical solution diluted with water containing a surfactant (0.05%) to 100 ppm was sprayed in a spray tower. After the treatment, it was left in a thermostatic chamber at 27 ° C. The number of live and dead insects after 1 day was investigated, and the death rate was calculated from the formula of Test Example 1. The result was 100% mortality.

Test Example 3. Efficacy test against adult housefly (Musca domestica) females On the back of the chest of 10 adult housefly female anesthetized houseflies,
An acetone solution (1.0 μg / μl) of the compound of the present invention was applied locally by 1 μl using a microsyringe. After treatment,
Diameter 9 cm containing absorbent cotton soaked with sucrose liquid as bait
The insects were released in a plastic cup, and left in a thermostatic chamber at 25 ° C. The number of live and dead insects after 1 day was investigated, and the death rate was calculated from the formula of Test Example 1. The result was 100% mortality.

Effect of the Invention The PF1018 substance of the present invention has a strong insecticidal activity as described above. Based on this property, the PF1018 substance of the present invention can be used as an insecticide or a raw material for conversion to it.

[Brief description of drawings]

Figure 1: PF1018 substance in methanol (20 μg / ml, solid line),
The ultraviolet and visible absorption spectra in acidic methanol (20 μg / ml, dashed line) and in basic methanol (20 μg / ml, alternate long and short dash line) are shown. Fig. 2: Infrared absorption spectrum of PF1018 substance in potassium bromide tablet. Fig.3: 400MHz ^{1 of} PF1018 substance in deuterated chloroform solution
1 shows a 1 H NMR spectrum. Fig.4: 100MHz in deuterated chloroform solution of PF1018 substance
¹³ shows a ¹³ C NMR spectrum.

Front page continuation (72) Shinji Miyamichi 760 Meiji Seika Co., Ltd., Meiji Seika Co., Ltd., 760 Shimooka-cho, Kohoku-ku, Yokohama, Kanagawa Prefecture In-house Pharmaceutical Research Institute (72) Inventor Katsu Shimura 760 Meiji Seika Co., Ltd., Kohoku-ku, Yokohama-shi, Kanagawa Prefecture Meiji Seika Co., Ltd. (72) Inventor Shigeharu Inoue 760 Meiji Seika Co., Ltd. Meiji Seika Co., Ltd.

Claims (3)

[Claims] 1. A novel substance PF1018 substance represented by the following structural formula and salts thereof. 2. A PF10 belonging to the genus Humicola.
A method for producing a PF1018 substance, which comprises culturing an 18 substance-producing bacterium and collecting the PF1018 substance from the culture. 3. An insecticide containing the PF1018 substance as an active ingredient.