JPH0581561B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD This invention relates to a method for producing a nematicide whose main active ingredient is α-terthienyl contained in marigold. Conventional technology Generally used nematicides are synthetic drugs, and they have problems such as environmental pollution due to residual toxicity and negative effects on humans and livestock, as well as poor sustainability of medicinal efficacy. I can't find anything that satisfies me. Marigold, a plant of the Asteraceae family, has nematicidal properties.

It is known to contain α-terthienyl and its related substances. [For example, Recueille des Traveaux Chimiques]
des Pays-Bas) Vol. 77, pp. 1004 to 1009 (1958) and Vol. 79, pp. 382 to 390 (1959)] Method for reducing nematode density in soil by planting marigolds directly in fields is widely known,
Compared to chemicals, it has the advantage of not having to worry about residual toxicity and its effects last for a long time, but on the other hand, it is difficult to grow vegetables at the same time in fields planted with marigolds, so marigolds are forced to fallow or reduce production during that time. It had been. Although methods for chemically synthesizing α-terthienyl have also been studied (for example, Japanese Patent Application Laid-Open No. 118462/1983), they have not yet been put to practical use. Means for Solving the Problems In view of the above circumstances, the present inventors have conducted various experiments and research on methods for mass-producing marigolds by tissue culture. Culture exhibiting particularly strong nematocidal activity is achieved by inducing callus in a callus-forming medium containing auxin and cytokinin, and then growing it in a growth medium containing auxin at a low concentration of 0.01 mg/ to 1 mg/. By extracting this with organic solvents such as n-hexane, acetone, and acetonitrile, we discovered a nematicide with excellent insecticidal performance that is also effective against root rot nematodes. Ta. Typical marigolds (Tagetes genus) suitable for carrying out the method of the present invention include French marigolds (Tagetes patula), African marigolds (Tagetes erecta), etc. In the present invention, these marigold leaves, stems, After tissue pieces collected from roots, buds, etc. are sterilized by a conventional method, callus formation is induced in a callus formation medium containing auxin or auxin and cytokinin as a plant hormone. Note that typical auxins suitable for carrying out the present invention include indoleacetic acid, naphthaleneacetic acid, 2,4
- dichlorophenoxyacetic acid, etc. Typical cytokinins include kinetin, benzyladenine, zeatin, etc. As a callus formation medium, basic media such as Murashige-Skoog's medium, Linsmeyer-Skoog's medium, Gamborg's medium, Nitsuchie's medium, etc.
In addition to the above-mentioned plant hormones, it is preferable to add an appropriate amount of carbon sources such as sucrose and glucose.
~5% by weight, agar 0.5-1.0% by weight, naphthalene acetic acid as auxin 0.01-20mg/, benzyladenine as cytokinin 0-20mg/
In the case of a medium added within this range, good callus formation is observed after 2 to 3 weeks of culture. The conditions for callus culture are the same as for normal plant tissue culture, and the temperature is 15-35℃, preferably 20-30℃.
°C and PH are suitably in the range of 4 to 8, preferably 5 to 6. The callus-induced marigolds are subsequently grown in a growth medium containing the same auxin or auxin and cytokinin with other additives as described above, but in the practice of the invention a culture with particularly strong nematicidal potency is used. The amount of auxin added in the growth medium was 0.01 mg/
The concentration should be as low as 1 to 1 mg/ml, and cytokinin should also be absent or at a low concentration of 3 mg/ml or less. When the amount of auxin and cytokinin added to the growth medium exceeds a predetermined amount, the efficacy of the nematicide obtained by extracting the culture is significantly reduced. In addition, as a method of propagation culture, either static culture using a fixed medium containing agar or shaking culture using a liquid medium excluding agar is possible. In the extraction process of cultured marigolds, dried marigolds are extracted using organic solvents such as n-hexane, acetone, and acetonitrile. For example, lightly crush the dried culture and grind it for 10 minutes.
It may be immersed in ~100 times the amount of n-hexane, stirred for 30 minutes to several hours, and filtered to remove solids. When using the nematicide of the present invention, the organic solvent may be vaporized and dissipated during spraying, but the organic solvent may be removed from the extract in advance and a known filler added to it to form a solid agent. Alternatively, water, an emulsifier, etc. can be added to form an aqueous solution or emulsion. Analysis of the nematocide of the present invention by high-performance liquid chromatography revealed that it was very similar to the marigold root extract used for culture, and contained α-terthienyl and related substances. Examples and effects of the method of the present invention will be specifically described below based on test examples. In addition, the nematicidal test method in these test examples is as follows:
After dropping 20 to 50μ of the extract onto a glass slide and removing the solvent by air drying, 20μ of the same species of nematode in deionized water or nematode culture medium was placed on top of it.
Place ~100 fish and copy them in a charrette in a damp room.
The survival status of the nematodes was determined by placing them in a lighted incubator kept at a temperature of 25°C and observing them with a microscope at regular intervals. Example 1 and Comparative Example 1 Cotyledons of sterile seedlings of French marigold (variety name: Bolero) that have been germinated for 2 weeks are cut into approximately 5 mm square pieces, and 3 weights of sucrose is added to Murashige-Skoog basic medium. %, agar 0.8% by weight, naphthalene acetic acid 0.1 mg/, benzyladenine 0.1 mg/
was added and placed on a callus-forming medium sterilized by a conventional method. Cult induction was performed under continuous illumination while keeping the temperature at 25°C in this state, and after one month, the culture was subcultured to a growth medium with a composition excluding benzyladenine from the callus formation medium, and cultured again under the same conditions for one month. and multiplied. Thereafter, the cells were passaged twice at one-month intervals for propagation. The culture thus obtained was a dark green, very hard callus, and an approximately 15-fold increase in production was observed with each multiplication. Next, this culture is dried at room temperature under sterile conditions,
The lightly crushed and dried culture was mixed with 50 ml of n-hexane per gram of the culture, extracted for 30 minutes, and the solid matter was filtered off to obtain an extract. A nematicidal test was conducted using the extract thus obtained. Nematicidal tests include Pratylenchus penetrans and Caenorhabditis elegans.
C. elegans) was used. Pratylenchus penetrans
The test using is as follows. That is, 50μ of each of the extract, its diluted solution, and pure n-hexane as a control were dropped onto separate slide glasses and air-dried, and Pratylenchus penetrans cultured in Roussin callus was placed on top of the slide glass using the Bellman method. 50μ of each sample (there are 20 to 40 nematodes) in deionized water was dropped, and the glass slides were placed in a shear dish made into a humid chamber, and placed in a lighted incubator at 25°C. After standing for 8 hours, microscopic observation was performed to determine whether the nematodes were alive or dead. The test using C. elegans is roughly the same; 20μ of each of the extract and its diluted solution and pure n-hexane as a control were dropped onto separate glass slides and air-dried, and then E. coli was placed on top of the slide glass in NG medium. Transfer 20 to 100 C. elegans cultured using C. elegans as bait, add 30μ of NG medium on top, place the slide glass in a shear dish with a humid chamber, and incubate for 25 minutes.
After being allowed to stand for 8 hours in an illuminated incubator at ℃, microscopic observation was performed to determine whether the nematodes were alive or dead. As a comparative example, the roots of French marigold (cultivar name: Bolero) were dried using natural cultivation methods, and the extract and its diluted solution were obtained by performing the same extraction process as above, and C. elegans was used. A nematocidal test was conducted. The test results are shown in Table 1, and the extract obtained by the method of the present invention was found to have strong nematicidal activity comparable to that of the extract obtained from naturally cultivated roots.

[Table] Furthermore, as a result of analyzing the extracts of this example and comparative example using α-terthienyl manufactured by Fluka by high-performance liquid chromatography, it was found that the extracts of this example had α-terthienyl at a rate of 0.5 μg/ml -terthienyl, and the extract of the comparative example contained 0.4 μg/ml α-terthienyl. Comparative Examples 2 and 3 In Example 1, French marigold cotyledons were mixed with 3 sucrose in Murashige-Skoog's basal medium.
Weight%, agar 0.8% by weight, naphthalene acetic acid 3mg/
Callus was induced using a callusization medium supplemented with 3 mg of benzyladenine, followed by the same culture treatment using a growth medium with the same components and concentrations as the callus medium, and the culture was extracted in the same manner as above. We conducted a nematicidal test against the nematode nematodes. (Comparative Example 2) In addition, naphthalene acetic acid and benzyladenine were added at 3 mg/10 and 10
A similar culture was carried out at mg/mg/ml, and the extract of the culture thus obtained was subjected to a nematicidal test against C. elegans. (Comparative Example 3) The test results are shown in Table 2, and the nematicidal activity of the extract obtained from marigold tissue cultured in a growth medium with a high auxin concentration was extremely weak.

[Table] Examples 2 to 4 and Comparative Example 4 Cotyledons (Example 2) two weeks after germination of French marigold (variety name: Bolero) were collected and sterilized with 70% ethanol and antiformin solution according to the conventional method. After that, the cells were thoroughly washed with sterilized purified water, and 3% by weight of sucrose, 0.8% by weight of agar, and 1mg of naphthalene acetic acid were added to Murashige-Skoog's basal medium, and placed on a callusization medium sterilized by a conventional method. Callus was also induced on the marigold roots (Example 3) and buds (Example 4) under the same conditions. All cultures were performed under continuous illumination at a temperature of 25°C, and after one month, the cultures were subcultured to a growth medium with the same composition as above, and further grown for one month under the same culture conditions, and then for another month. The cells were propagated by being passaged twice at regular intervals, and the resulting multiplications were all yellow-green to brown calluses with a large number of short roots differentiated. The mixture was dried under water, crushed lightly, and extracted for 30 minutes by adding 50 ml of n-hexane per 1 g. The solid matter was filtered off to obtain an extract. As a result of analysis using high performance liquid chromatography,
The extract in each example had an α of 0.04 μg/ml.
-Contained terthienyl. Therefore, α-terthienyl (manufactured by Fluka) synthesized as Comparative Example 4 was added to an n-hexane solution.
Samples were prepared in which 0.04 μg/ml was added, and a nematicidal test was conducted together with each of the above-mentioned extracts. The results of these tests are shown in Table 3, and marigolds cultured from any tissue site have the same nematocidal properties and have the same concentration of α-terthienyl. Even if there is, the extract from marigold culture is synthetic α
- It was found that the nematicide was clearly stronger than those containing terthienyl.

[Table] Examples 5 and 6 Cotyledons of French marigold [variety name: Bolero (Example 5)] and African marigold [variety name: Orange Hawaii (Example 6)] were collected two weeks after germination, and After sterilizing with ethanol and antiformin solution, washing with sterile purified water,
These were added to Murashige-Skoog's basic medium with 3% by weight of sucrose, 0.8% by weight of agar, and 1% by weight of naphthalene acetic acid.
mg/mg/benzyladenine and 3 mg/mg/benzyladenine were added, and the mixture was placed on a sterilized callus-forming medium using a conventional method to induce callus. Both callus-forming cultures were performed at a temperature of 25° C. under continuous illumination, and after one month, the resulting cultures were subcultured to a growth medium with the same composition as above, and further grown for one month under the same culture conditions. Thereafter, the cells were grown again for one month using a medium with the same composition and under the same conditions. All of the cultures thus obtained were yellow-green callus, and the production amount increased by about 20 times due to each multiplication. The culture was dried under aseptic conditions at room temperature, lightly crushed, mixed with 50 ml of n-hexane per 1 g, extracted for 30 minutes, and solids were filtered off to obtain an extract. Using the thus obtained extract and its diluted solution, a nematicidal test was conducted in the same manner as in the previous example. The test results are shown in Table 4, and the extracts obtained from the cultures of both French marigold and African marigold exhibited excellent nematocidal properties.

【table】

[Table] Examples 7 and 8 Cotyledons of French marigold (variety name: Bolero) were collected two weeks after germination, and 70% of the cotyledons were collected according to a conventional method.
% ethanol and antiformin solution, thoroughly washed with sterile purified water, and added to Murashige-Skoog's basic medium with 3% sucrose, 0.8% agar, and
Indole acetic acid 1 mg/, benzyladenine 1
3% by weight of sucrose, 0.8% by weight of agar, and 1% by weight of naphthalene acetic acid in a medium (Example 7) and Murashige-Skoog's basic medium, which were sterilized by a conventional method.
The cells were placed in a medium (Example 8) which had been sterilized by a conventional method to which 1 mg/mg/mg of kinetin had been added, and callus was induced. Both cultures were performed at a temperature of 25°C under continuous illumination.
The cultures obtained after one month were each subcultured into a medium with the same composition as above, and propagated under the same culture conditions for an additional month, and then subcultured twice at one-month intervals for propagation. The cultures obtained in this way were both yellow-green calluses, which were dried under sterile conditions at room temperature, crushed lightly, and extracted for 30 minutes by adding 50 ml of n-hexane per gram. The solid matter was filtered off to obtain an extract. A nematicidal test was conducted in the same manner as shown in Example 1 using the above extract and its diluted solution. The test results are shown in Table 5, and the extracts of these cultures were found to have excellent nematocidal properties, regardless of the types of auxin and cytokinin.

【table】

【table】

【table】

Claims (1)

[Scope of Claims] 1. Callus is induced from marigolds using a callus-forming medium containing auxin or auxin and cytokinin, and then tissue culture is performed using a growth medium in which auxin is regulated at a low concentration of 0.01 mg/ to 1 mg/, A method for producing a nematicide, which comprises extracting the culture obtained by the above treatment with an organic solvent. 2. The method according to claim 1, wherein the type of marigold is French marigold. 3. The method according to claim 1, wherein the type of marigold is African marigold.