JPH01275505A - Production of nematocide - Google Patents

Abstract

PURPOSE:To obtain the subject nematocide rich in alpha-terthienyl, having excellent nematocidal activity and containing active component suitable for mass- production, by culturing hair root of marigold infected with a specific microorganism and extracting the cultured product with an organic solvent. CONSTITUTION:A plant body of marigold which as a plant of family Compositae such as French or African marigold containing alpha-terthienyl having nematocidal activity is infected with Agrobacterium rhizogenes e.g. by incising the plant body. The produced transformed hair root of the marigold having good proliferability is cultured in a medium containing gibberellin as the sole plant hormone or absolutely free from plant hormone at 15-35 deg.C preferably under dark condition. The obtained cultured product is dried and extracted with an organic solvent such as n-hexane or acetone to obtain a substance rich in alpha- terthienyl exhibiting especially excellent nematocidal activity against root lesion nematocide. The substance is used as an active component for the objective nematocide.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application This invention relates to a method for producing a nematicide whose main active ingredient is α-terchenyl contained in marigold.

Conventional techniques As a countermeasure against nematode damage to crops, - The drugs used for boat fishing are synthetic drugs, which have been pointed out to cause environmental pollution and adverse effects on humans and livestock due to residual toxicity, and have poor long-lasting medicinal efficacy. There are many problems, and I can't find anything that satisfies me.

Marigold, which is a plant of the Asteraceae family, is known to contain α-terchenyl and its analogs, which are represented by the following formula and have nematocidal properties. [For example, L'Eculée des
Trapax Kimiku Death Bayes-Bass (Rec
Chimi
que des Pays-Bas) Volume 77 100
Pages 4 to 1009 (1958) and Volume 79, 38
2 to 390 (1959)) In addition, a typical method for chemically producing α-terchenyl is 2-terchenyl.
Synthesis method from acetylthiophene and trinotylsilane via thiophene silyl enol ether derivative (JP-A-52-118461, JP-A-52-118462)
(No. 3) is known, but it has not yet been put into practical use.

Problems to be Solved by the Invention The method of planting marigolds directly in fields to reduce the density of nematodes in the soil is widely known, and compared to chemicals, there is no concern about residual toxicity and the method is effective for a long time. Although it has the advantage of being sustainable, it is difficult to grow vegetables in fields planted with marigolds at the same time, so marigolds have been forced to fallow or reduce production.

The present invention improves the culture method of marigolds, and examines a method for mass-producing a nematicide obtained by extracting the culture.

Means for Solving the Problems In view of the above circumstances, the inventors of the present invention have conducted various tests and research on methods for mass-producing marigolds by tissue culture, and as a result, Agrobacterium rhizogenes (
Agrobacterium rhizogenes)
We found that the transformed hairy roots produced from marigolds infected with the above method had good growth properties, and that the hairy roots of marigolds treated in this way were grown in a medium containing only gibberellin as a plant hormone. Alternatively, by culturing it in a medium that does not contain it at all and extracting it with an organic solvent,
It has been found that a component can be obtained that exhibits particularly excellent nematocidal activity against Negusare nematodes.

Typical marigolds (T.
Agetes genus) is a French marigold (Tag
etes patula), African marigold (Tagetes erecta), Mexican marigold (Tagetes tenuifolia)
) etc.

In addition, methods for infecting marigolds with Agrobacterium rhizogenes include inoculating a sterilized marigold plant with a needle coated with the bacteria, or wounding the plant and injecting the bacteria. Possible methods include a method of coating, a method of immersing a part of the plant in a liquid medium containing bacteria, and a method of co-cultivating protoplasts obtained from the cells of the plant and bacteria.

After several weeks, marigolds infected by any of the above methods produce hairy roots in which a portion of the Ri plasmid of Agrobacterium rhizogenes is integrated into the plant's nuclear DNA. Treat the roots with antibiotics such as clahoran or carbenicillin at 0.1 to 1.0
g/1. sterilize by transplanting to a medium containing a certain amount of bacteria.

In this case, suitable media are those commonly used for plant tissue culture, such as Murashige-Skoog's medium, Linsmeyer-Skoog's medium, Gamborg's medium, or Nirachie's medium.

After culturing the hairy roots for several weeks using any of the above-mentioned media, the tips of the elongated hairy roots are cut off to a size of about 1 to 2 cm, and then subcultured into a medium of the same composition. Normally, hairy roots completely free of Agrobacterium rhizogenes can be obtained by carrying out the sterilization culture operation twice. Note that it is desirable to ground a portion of the hairy root and place it on a bacterial culture medium to confirm that there is no proliferation of bacteria. At this time, if bacterial growth is observed, it is necessary to repeat the sterilization operation.

The sterilized hairy roots thus obtained are propagated using a conventional liquid medium for plant tissue culture. At this time, regarding plant hormones, especially when using a medium without additives or a medium containing only gibberellin at a ratio of 10-4 to 10-7 mol/l, many components that exhibit nematicidal activity are included. There is. The culture conditions in the method of the present invention include a temperature of 15 to 35°C, preferably 20 to 30"C;
It is desirable to use light under dark conditions.

After the cultured marigold hairy roots are dried,
Extract using an organic solvent such as n-hexane, acetone, or acetonitrile.

For example, lightly crush dry hairy roots,
It may be immersed in 0 times the amount of n-hexane, stirred for 30 minutes to several hours, and filtered to remove solids.

When using the nematicide obtained by the method 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 bulking agent may be added thereto. It can be made into a solid preparation, or it can be made into an aqueous solution or emulsion by adding water, an emulsifier, etc.

The method of the present invention will be specifically explained below using Examples and Comparative Examples.

Examples 1 and 2 and Comparative Examples 1 and 2 After sterilizing French marigold (variety name: Bolero) seeds, they were placed on a Murashige-Skoog medium and allowed to germinate to obtain sterile seedlings. Agrobacterium rhizogenes ATCC1
5834 strain was cultured in YEB medium, and this bacteria was attached to the tip of a sterilized needle and pierced into the above-mentioned sterile seedlings to infect them.

Approximately 2 weeks later, hairy roots appeared near the inoculated area, so these were cut out and placed on a Murashige-Skoog medium containing 0.1 g/ρ of Krahoran, and cultured for sterilization at 25°C. After culturing for 3 weeks, the tips of the elongated hairy roots approximately 1 to 2 cm were placed in a medium with the same composition as above, and further cultured for 3 weeks.
The cells were cultured for sterilization under the same culture conditions for weeks. Thereafter, approximately 2 cm of the tips of the cultured hairy roots were placed on Murashige-Skoog medium (p
F15.0), mash the remaining part, YEB
It was confirmed whether sterilization was sufficient by placing it on a culture medium and checking the state of bacterial growth. Culture is carried out using a rotating shaker incubator at a rotation speed of 11 Or
pm and a temperature of 25°C in the dark. After two weeks of culture, in Example 1, some of the hairy roots were subcultured on Murashige-Skoog medium without plant hormones, and in Example 2, some of the same hairy roots were subcultured with gibberellin. (
The cells were subcultured into Murashige-Skoog medium containing GAs) 3 xto-b mol/l and cultured for an additional 3 weeks under the same conditions as above. The proliferation rate was 100 times higher in the dry weight ratio in the case without plant hormones, and 90 times in the case in the case containing gibberellin.

Next, these hairy roots were dried under sterile conditions and lightly crushed. After that, n-hexane was mixed at a ratio of 5,011 parts per 1 g of dry hairy roots, extraction was performed for 30 minutes, and the solid matter was filtered off. An extract was obtained.

Further, as Comparative Example 1, an extract was obtained from the roots of French marigold (variety name: Bolero) grown from seeds by natural cultivation methods in the same manner as described above.

Analysis of α-terchenyl, the main active component of the extract,
The analysis was carried out by high performance liquid chromatography using α-terchenyl manufactured by Fururikisha as a standard.

The results were as shown in Table 1.

Table 1 Content of α-terchenil Next, a nematicidal test was conducted using the extract obtained by the above method.

As a nematode, Pratylench nematode
uspenetrans) and Caenorhabditis elega
(hereinafter abbreviated as C and elegans) were used.

Pratylenchus pen
The test using (etrans) is as follows. That is, 50μ2 of each of the extract, its diluted solution, and pure n-hexane as a control were dropped onto separate slide glasses and air-dried.
enetrans) in deionized water by Bellman's method. !
(There are 20 to 40 nematodes in this.) It was dropped onto the slide glass mentioned above, and the slide glass was placed in a petri dish that was used as a greenhouse, and left undisturbed for 8 hours in a lighted incubator at 25"C. Afterwards, microscopic observation was performed to determine whether the nematodes were alive or dead.

The test using C. elegans is generally similar; 20 μl each of the extract, its diluted solution, and pure n-hexane as a control were dropped onto separate glass slides, air-dried, and then NG medium was added. Transfer 20 to 100 C. elegans cultured using E. coli as bait, and add 30μ of NG medium on top! In addition, the slide glass was placed in a petri dish that was used as a greenhouse, and after being allowed to stand for 8 hours in a lighted incubator at 25°C, microscopic observation was performed to determine whether the nematodes were alive or dead.

In addition, α-terchenyl manufactured by Fururikisha was used as the extract of Comparative Example 1 and Comparative Example 2, and the same n as in Example 1 was used.
- A hexane solution concentration was prepared and a nematicidal test was conducted in the same manner as above.

The results of these nematocidal tests are shown in Table 2.

Table 2 Nematicidal test (survival rate 2%) Effect of invention

Claims (1)

[Claims] (1) Agrobacterium rhizogenes on marigolds
A method for producing a nematicide, which comprises the steps of infecting the treated marigold hairy roots, and extracting the thus obtained culture with an organic solvent.