JP7254061B2 - How to obtain sterile cyst nematodes - Google Patents

Description

The present invention relates to a method for obtaining sterilized cyst nematodes that can be used for drug screening and the like, and sterilized cyst nematodes obtained by the method.

Cyst nematodes parasitize only a specific host, form cysts with environmental and drug resistance in the absence of a host, and can survive for a long time in that state. When a host comes to exist nearby due to planting, etc., it senses the hatching promoting substance derived from the host and hatches and parasitizes the host. For example, Potato Cyst Nematode (hereinafter also referred to as “PCN”) (scientific name: Globodera rostochiensis) is a difficult-to-control pest that parasitizes only certain hosts such as solanaceous plants and parasitizes potato roots. causing enormous damage around the world. At present, sufficient countermeasures have not been taken worldwide, and effective control agents have not been developed. In recent years, it has been reported that a potato cyst nematode (Globodera pallida), which belongs to the same genus as PCN, has occurred in Japan (Hokkaido), and that a new species of Globodera ellingtonae has been discovered in the United States (Oregon State) (non Patent document 1). Countermeasures against these new cyst nematodes are also required.

As one method for controlling cysto nematodes, attempts have been made to apply a hatching-promoting substance to soil where the host has not been planted, promote the hatching of the eggs in the cysts, and starve the larvae of the cysto nematodes. (Patent Documents 1 to 3).

On the other hand, cyst nematodes are parasitized by various filamentous fungi (Non-Patent Document 2). 2) Eggs obtained from cysts have been surface sterilized with methoxyethylmercuric chloride (Non-Patent Document 3), but none of them are sufficient.

JP 2011-173875 A Japanese Patent Application Laid-Open No. 9-2912 JP-A-62-255402 JP 2011-229443 A

Zafar A. et al., (2012). "Description of Globodera ellingtonae n. sp. (Nematoda: Heteroderidae) from Oregon". Journal of Nematology 44(1), pages 40-57. Trifonava, Z. and Karadjova, J. (2003). "Fungal parasitism of the cysts and eggs of the Globotera rostochiensis". Journal of Agricultural Sciences Vol. 48, No.1, pages 103-110. Foot, M.A., (1977). "Laboratory rearing of potato cyst nematode; a method suitable for pathotyping and biological studies". New Zealand Journal of Zoology, Vol. 4, pages 183-186.

In the search for new hatching-enhancing substances for cyst nematodes, it is necessary to conduct hatching tests on cyst nematode eggs. In general, cyst nematodes collected from the field (field) are inoculated into potatoes in a greenhouse, and eggs obtained by proliferating are used as test materials. Some of the eggs are contaminated, and if they are repeatedly propagated in the greenhouse, the contamination rate will increase. came to. That is, an object of the present invention is to sterilize cyst nematodes, establish an in vitro mass proliferation system, and provide sterilized cyst nematodes that can stably perform evaluation tests such as hatching tests.

In the course of extensive research to solve such problems, the present inventors irrigated the support (sand medium) of the host plant with a fungicide at a concentration lower than usual, and inoculated it with cyst nematodes. found that a sterilized cyst nematode having cyst-forming ability encapsulating the next-generation egg can be obtained, and as a result of further research, the present invention was completed. That is, the present invention relates to the following.

[1] A method for obtaining a sterilized cyst nematode, comprising:
culturing the cyst nematode to be sterilized in a sand medium inoculated with the host plant; and
irrigating the sand medium with the agent;
The above method, wherein the irrigating agent comprises a fungicide at a concentration that does not affect nematode growth.
[2] The method according to [1] above, wherein the disinfectant comprises a DMI agent.
[3] The method according to [2] above, wherein the DMI agent is tebuconazole.
[4] The method according to any one of the above [1] to [3], wherein the concentration of the fungicide is 2 to 800 times the effective concentration.
[5] The method according to any one of [2] to [4] above, wherein the bactericidal agent further contains streptomycin.

[6] The method according to any one of [1] to [5], wherein the cyst nematode is potato cyst nematode (Globodera rostochiensis).
[7] A sterilized cyst nematode having cyst-forming ability, obtained by the method according to any one of [1] to [6] above.
[8] A sterilized cyst nematode having cyst-forming ability.
[9] The sterilized cyst nematode according to [7] or [8], which is potato cyst nematode (Globodera rostochiensis).
[10] The sterilized cyst nematode according to any one of the above [7] to [9], which is used for screening of hatching promoting substances.

According to the method of the present invention, it is possible to sterilize cyst nematodes safely and easily without using a mercury agent such as methoxyethylmercuric chloride and without imposing a burden on the environment. In addition, the cyst nematode sterilized by the method of the present invention has the ability to form cysts and can be subcultured. Therefore, according to the present invention, an in vitro culture system for cyst nematode was established. According to the present invention, it is possible to proliferate sterilized cyst nematodes in large quantities while maintaining and promoting sterility. A large amount of sterile cysts can be provided. That is, by using eggs obtained from sterilized cyst nematode obtained by the method of the present invention, it is not affected by contaminated eggs (parasitic eggs) due to parasitic fungi parasitic on cyst nematodes, and more accurate candidates The ability of agents to promote hatching can be evaluated.

FIG. 1 is a graph showing the reduction in contaminated egg rate by the aseptic process of the present invention.

The present invention relates to a method of obtaining sterile cyst nematodes. Here, "sterilization" includes reducing the number of parasitic fungi parasitic on cyst nematodes or completely sterilizing them, for example, confirming by reducing the rate of contaminated eggs by aseptic treatment can be done. An egg contamination rate of 0-15%, preferably 0-12%, can be said to have substantially no effect on hatching tests.

The method of the present invention involves culturing the cyst nematodes to be sterilized in a sand medium inoculated with host plants, and irrigating the sand medium with a drug. The drug to be irrigated here may be a bactericidal agent against the parasitic fungus that is the target of sterilization, and can be appropriately selected according to the type of the parasitic fungus. Specific drugs used in the present invention include, for example, trifoline, fenarimol, oxpoconazole fumarate, pefurazoate, prochloraz, triflumizole, cyproconazole, difenoconazole, fenbuconazole, hexaconazole, imibenco DMI agents (demethylation inhibitors: inhibitors of C14 demethylase in sterol biosynthesis) such as Nazol, Ipconazole, Metconazole, Mycrobutanil, Propiconazole, Simeconazole, Tebuconazole, Tetraconazole; imidazole-based fungicides; guanidine-based fungicides such as iminoctadine acetate and iminoctadine albesilate; polyene antifungal agents such as nystatin; antibiotics such as streptomycin and gentamicin.

The drug can be used singly or in combination of two or more. When drugs are used in combination, it is preferable to use a combination of an antifungal DMI agent and an antibacterial antibiotic, and particularly preferably tebuconazole and streptomycin.

The concentration of the drug to be irrigated is a concentration that does not affect the growth of nematodes, and is usually lower than the effective concentration of the drug used (concentration of registered prescription). The concentration of the drug to be irrigated is preferably a 2- to 800-fold dilution of the effective concentration, more preferably a 5- to 160-fold dilution, and particularly preferably a 10- to 80-fold dilution.

For example, when tebuconazole is used as a drug, its registered prescription concentration is about 133 to 800 ppm, but in the present invention, it is, for example, 1 ppm to 50 ppm, preferably 5 ppm to 20 ppm, more preferably 10 ppm. When tebuconazole and streptomycin are used in combination, the streptomycin concentration is, for example, 0.05 ppm to 50 ppm, preferably 0.5 ppm to 5 ppm, more preferably 1 ppm.

Cyst nematodes that can be targeted for sterilization in the present invention include, for example, Heterodera glycines, Heterodera trifolii, Heterodera schachtii, Heterodera oryzae, and Heterodera oryzae. Plant-parasitic cyst nematodes of the genus Heterodera, such as (Heterodera elachista), as well as potato cyst nematodes (Globodera rostochiensis), potato cyst nematode (Globodera pallida), Globodera ellingtonae, tobacco cyst nematode (Globodera tabaccum), mugwort nematode plant-parasitic cyst nematodes of the genus Globodera, such as (Globodera hypolysi). In the aseptic process of the present invention, it is preferable to target potato cyst nematodes.

The incubator used in the method of the present invention is not particularly limited as long as it is a vessel used for culturing cyst nematodes, and may be, for example, a glass or plastic vessel. The size of the container is not particularly limited, but it may be about 100 ml to 1000 ml. About 3 plants are planted and used for culture. Seedlings of the host plant are preferably aseptically cultured seedlings.
The sand medium used in the method of the present invention is not particularly limited as long as it is for sand cultivation used in agriculture and gardening, such as sand soil.

Moreover, the host plant used in the method of the present invention is the host plant of the cyst nematode, which is the target of sterilization. For example, when potato cyst nematode is targeted, the host plant is a solanaceous plant, preferably potato.
The culture conditions for culturing cyst nematodes in the method of the present invention were 20° C., 16 hours light period/day, and the lower part of the culture vessel was covered with aluminum foil to shield the medium from light.

The present invention, in one aspect, relates to a sterilized cyst nematode having the ability to form cysts.
Since the sterilized cyst nematode according to the present invention has cyst-forming ability and can be subcultured, it can be used for various experiments and, for example, for screening of hatching-promoting substances.

EXAMPLES The present invention will be described in more detail below using examples, but the present invention is not limited to these examples.

[Test Example 1] Study of sterilization of potato cyst nematodes (1)
Potato cyst nematode (PCN) larvae (cysts) proliferated in potato seedlings (variety: May Queen) normally grown in a greenhouse. Ethanol (treatment concentration 70%), sodium hypochlorite (treatment concentration 2.0-0.002%), PPM (trademark) (PLANT PRESERVATIVE MIXTURE, Plant Cell Technology Co.) (treatment concentration 0.2 to 0.00025%) and slightly acidic electrolyzed water (treatment concentration 0.2 to 0.00025%) were used to sterilize the surface for 2 minutes. Larvae subjected to surface sterilization were observed under a microscope to confirm the presence or absence of survival. In addition, the surviving larvae were aseptically planted with potato culture seedlings (variety: May Queen) on agar or dulan gum medium (composition: MS medium (manufactured by Wako Pure Chemical Industries, Ltd.), MS Vitamin Solution (manufactured by Sigma) ), 30% sucrose, 0.8% agar or 0.2% gellan gum) at 20°C for 75 days. The presence or absence of cyst formation was observed from 30 days after larval inoculation. The percentage of cultured seedlings contaminated with parasitic fungi was defined as the contamination rate (%). Table 1 shows the results.

As shown in Table 1, it was clarified that surface treatment with sodium hypochlorite can remove parasites while maintaining PCN survival. However, as a result of further investigation, it was found that larvae treated with sodium hypochlorite did not form cysts and could not be used for hatching tests.

[Test Example 2] Study of sterilization of potato cyst nematodes (2)
PCN larvae (cysts) proliferated in potato seedlings (variety: May Queen) normally cultivated in a greenhouse were immersed in sterilized water for 3 days, then immersed in potato root exudate, and 6-10 days later, they migrated out from the cysts. hatched larvae) were cultured at 20° C. for 75 days in a sand medium or cell medium in which potato culture seedlings (variety: May Queen) were aseptically planted. On the first day of culture, a fungicide consisting of a combination of tebuconazole (10 ppm to 100 ppm) and streptomycin (1 ppm) was applied to the sand medium (grass sand (manufactured by Tokai Gravel Co., Ltd.)) or cell culture soil (manufactured by Hokusan Co., Ltd.). irrigated. From the 30th day after irrigation with the chemical, the presence or absence of contamination of the cultured seedlings with parasitic fungi and the presence or absence of cyst formation were confirmed. The test was done in triplicate. Table 2 shows the results.

[Test Example 3] Recovery and passage of aseptic cysts Rate of contaminated eggs in 10 cysts recovered from PCN grown in potato seedlings (variety: May Queen) normally grown in a greenhouse, and recovery at the same time as the cysts The cysts thus obtained were treated in the same manner as in Test Group 1 of Test Example 2, and the rate (%) of contaminated eggs in the cysts formed after culturing was calculated. In addition, the contaminated egg rate (%) was calculated by counting the total number of eggs (healthy eggs + contaminated eggs) under a microscope using a nematode calculator (per 1 ml) for the eggs obtained from the collected cysts. . The results are shown in Tables 3 and 4.

As shown in Table 3, the contamination egg rate of cysts recovered from PCN grown in conventionally cultivated potato seedlings was 17.3-32.3%, while as shown in Table 4, the aseptic egg rate of the present invention The percentage of cyst-contaminated eggs obtained by the treatment was 4.5-10.1%, demonstrating a significant reduction in parasitic-contaminated eggs (Fig. 1).
The sterilization rate (%) (reduction rate of contaminated eggs) by the aseptic treatment of the present invention was about 47.8 to 82.0%, and sterilization was achieved very effectively.

Further, the larvae hatched from the aseptically treated cysts obtained in Example 2 were washed with sterilized water. 900 heads per container were inoculated into a container (1 L plastic container) in which 2 strains of May Queen were planted. About one month after inoculation, cyst formation was confirmed.
By subculturing sterilized cyst nematodes having cyst-forming ability while performing the aseptic treatment of the present invention in this way, it is possible to significantly reduce the rate of contaminated eggs, and it can be expected that parasitic fungi will be completely sterilized. .

Since the sterilized cyst nematodes having cyst-forming ability provided by the present invention can eliminate the influence of parasites on hatching, screening of drugs such as hatching promoting substances and nematocides, analysis of the mechanism of hatching induction, It can be used for screening of parasitic nematode microorganisms (filamentous fungi, bacteria, etc.) for the development of microbial pesticides, analysis of the parasitic ecology and dynamics of nematodes in host plants, and elucidation of the mechanism of detecting trace substances such as hatching promoting substances. , can play a major role in these.

Claims (6)

A method of obtaining a sterile cyst nematode, comprising:
culturing the cyst nematode to be sterilized in a sand medium inoculated with the host plant; and
irrigating the sand medium with the agent;
The above method, wherein the irrigating agent comprises a fungicide at a concentration that does not affect nematode growth. 2. The method of claim 1, wherein the disinfectant comprises a DMI agent. 3. The method of claim 2, wherein the DMI agent is tebuconazole. The method according to any one of claims 1 to 3, wherein the concentration of the fungicide is a 2- to 800-fold diluted concentration of the effective concentration. The method of any one of claims 2-4, wherein the bactericidal agent further comprises streptomycin. A method according to any one of claims 1 to 5, wherein the cyst nematode is the potato cyst nematode (Globodera rostochiensis).

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