WO2019160075A1 - Method for obtaining sterilized cyst nematode - Google Patents
Method for obtaining sterilized cyst nematode Download PDFInfo
- Publication number
- WO2019160075A1 WO2019160075A1 PCT/JP2019/005512 JP2019005512W WO2019160075A1 WO 2019160075 A1 WO2019160075 A1 WO 2019160075A1 JP 2019005512 W JP2019005512 W JP 2019005512W WO 2019160075 A1 WO2019160075 A1 WO 2019160075A1
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- WIPO (PCT)
- Prior art keywords
- sterilized
- cyst
- cyst nematode
- nematode
- concentration
- Prior art date
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
Definitions
- the present invention relates to a method for obtaining a sterilized cyst nematode that can be used for drug screening and the like, and a sterilized cyst nematode obtained by the method.
- Cyst nematodes parasitize only on a specific host, and form cysts having environmental / drug resistance in the absence of the host, and can survive for a long time in that state. When the host comes close by planting or the like, it will hatch by detecting the hatching promoting substance derived from the host and infest the host.
- Potato Cyst Nematode (hereinafter also referred to as “PCN”) (scientific name: Globodera ⁇ rostochiensis) is a hard-to-control insect that infests only certain hosts such as solanaceous plants and infests potato roots. Has caused tremendous damage around the world. At present, no sufficient measures have been taken globally, and effective control agents have not been developed.
- cyst nematodes are parasitized by various filamentous fungi (Non-Patent Document 2), and in order to sterilize parasitic fungi, for example, surface sterilization is performed on cysts with sodium hypochlorite (Non-Patent Document 2). 2) Although surface sterilization is performed with methoxyethyl mercury chloride on eggs obtained from cysts (Non-patent Document 3), none of them is sufficient.
- JP 2011-173875 A Japanese Patent Laid-Open No. 9-2912 JP 62-255402 A JP2011-229443A
- an object of the present invention is to provide a sterilized cyst nematode that can establish sterilization of a cyst nematode and an in vitro large-scale proliferation system and can stably perform an evaluation test such as a hatch test.
- the present inventors have conducted intensive research on irrigating a host plant support (sand medium) at a concentration lower than usual and inoculating it with cyst nematode. As a result of further research, the inventors have found that a sterilized cyst nematode having the ability to form cysts encapsulating the next generation of eggs can be obtained. That is, the present invention relates to the following.
- the disinfectant includes a DMI agent.
- the DMI agent is tebuconazole.
- the method according to any one of [1] to [3], wherein the concentration of the bactericide is a concentration diluted by 2 to 800 times the effective concentration.
- the bactericidal agent further comprises streptomycin.
- cyst nematodes According to the method of the present invention, it is not necessary to use a mercury agent such as methoxyethyl mercury chloride, and cyst nematodes can be sterilized safely and easily without burdening the environment.
- the cyst nematode sterilized by the method of the present invention has cyst formation ability and can be passaged. Therefore, according to the present invention, an invitro culture system for cyst nematode was established.
- sterilized cyst nematodes can be proliferated in large quantities while maintaining and promoting sterility, and are necessary for, for example, large-scale screening for searching for hatching promoting substances. Aseptic cysts can be provided in large quantities.
- an egg obtained from a sterilized cyst nematode obtained by the method of the present invention it is not affected by a contaminated egg (parasitic egg) due to a parasitic bacterium parasitic on cyst nematode, and more accurately a candidate.
- the ability to promote drug hatching can be assessed.
- FIG. 1 is a graph showing a decrease in contaminated egg rate by aseptic processing of the present invention.
- the present invention relates to a method for obtaining a sterilized cyst nematode.
- sterilization includes reducing the number of parasitic fungus parasitic on cyst nematode or completely sterilizing, for example, confirming by reducing the rate of contaminated eggs by aseptic processing. Can do. If the contaminated egg rate is 0 to 15%, preferably 0 to 12%, it may be considered that the hatching test is not substantially affected.
- the method according to the present invention includes culturing cyst nematodes to be sterilized in a sand medium planted with a host plant, and irrigating the sand medium with a drug.
- the drug to be irrigated here may be a bactericidal agent against parasitic bacteria that are targets for sterilization, and can be appropriately selected according to the type of parasitic bacteria.
- Specific drugs used in the present invention include, for example, trifolin, fenarimol, oxpoconazole fumarate, pefazoate, prochloraz, triflumizole, cyproconazole, difenoconazole, fenbuconazole, hexaconazole, imibenco NMI, ipconazole, metconazole, microbutanyl, propiconazole, cimeconazole, tebuconazole, tetraconazole and other DMI agents (demethylation inhibitors: inhibitors of demethylase at position C14 in sterol biosynthesis); benzoates such as thiophanate methyl Imidazole fungicides; Guanidine fungicides such as iminotadine acetate and iminoctadine albecate; Polyene antifungals such as nystatin; Streptomycin, gentamicin, etc. Such as raw materials, and the like.
- Drugs can be used alone or in combination of two or more. When a combination of drugs is used, it is preferable to use a combination of an antifungal DMI agent and an antibacterial antibiotic, and it is particularly preferable to use a combination of tebuconazole and streptomycin.
- the concentration of the drug to be irrigated is a concentration that does not affect the growth of nematode and is usually thinner than the effective concentration of the drug to be used (the concentration of the registered prescription).
- the concentration of the drug to be irrigated is preferably a concentration diluted 2 to 800 times the effective concentration, more preferably a concentration diluted 5 to 160 times, particularly preferably a concentration diluted 10 to 80 times.
- the concentration of the registered prescription is about 133 to 800 ppm, but in the present invention, for example, it is 1 to 50 ppm, preferably 5 to 20 ppm, more preferably 10 ppm.
- the concentration of streptomycin is, for example, 0.05 ppm to 50 ppm, preferably 0.5 ppm to 5 ppm, more preferably 1 ppm.
- the cyst nematodes that can be targets for sterilization of the present invention include, for example, soybean cyst nematodes (Heterodera glycines), clover cyst nematodes (Heterodera trifolii), sugar beet nematodes (Heterodera schachtii), rice cyst nematodes (Heterodera oryzae), and okabo cyst nematodes.
- soybean cyst nematodes Heterodera glycines
- clover cyst nematodes Heterodera trifolii
- sugar beet nematodes Heterodera schachtii
- rice cyst nematodes Heterodera oryzae
- okabo cyst nematodes include, for example, soybean cyst nematodes (Heterodera glycines), clover cyst nematodes (Heterodera trifolii), sugar beet nematodes (Heterodera schachtii), rice cyst
- Heterodera plant parasitic cyst nematodes such as (Heterodera elachista), potato cyst nematodes (Globodera rostochiensis), potato white cyst nematodes (Globodera pallida), Globodera ⁇ ellingtonae, tobacco cyst nematodes (Globodera tabium acc) (Globodera hypolysi) and other plant parasitic cyst nematodes of the genus Globodera.
- the incubator used in the method of the present invention is not particularly limited as long as it is a container used for cyst nematode culture.
- it may be a glass or plastic container.
- the size of the container is not particularly limited, it may be about 100 ml to 1000 ml, and a sand culture medium is added to this in an amount of about 1/5 to 1/3 of the container amount, and 1 seedling of the host plant is added. About 3 plants are planted and used for culture.
- the seedling of the host plant is preferably a cultured seedling that has been sterilized.
- the sand culture medium used in the method of the present invention is not particularly limited as long as it is for sand cultivation used for agriculture and horticulture such as sand and earth.
- the host plant used in the method of the present invention is a cyst nematode host plant that is a target for sterilization.
- the host plant is a solanaceous plant, preferably potato.
- the culture conditions for cultivating 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 and shielded from light so that the medium did not receive light.
- the present invention relates to a sterilized cyst nematode having cyst forming ability. Since the sterilized cyst nematode according to the present invention has cyst-forming ability and can be passaged, it can be used for various experiments, for example, for screening for hatching promoting substances.
- the surviving larvae were agar or julan gum medium (composition: MS medium (manufactured by Wako Pure Chemical Industries, Ltd.)), MS Vitamin Solution (manufactured by Sigma), aseptically planted potato seedlings (variety: make-in) ), 30% sucrose, agar 0.8% or gellan gum 0.2%) and cultured at 20 ° C. for 75 days. The presence or absence of cyst formation was observed 30 days after the larva inoculation. The percentage of cultured seedlings contaminated with parasitic bacteria was defined as the contamination rate (%). The results are shown in Table 1.
- a fungicide that combines tebuconazole (10 ppm to 100 ppm) and streptomycin (1 ppm) to sand culture medium (lawn sand (Tokai Gravel Co., Ltd.)) or cell culture soil (Hokusan Co., Ltd.) Irrigated. From the 30th day after the drug irrigation, the presence or absence of contamination of the cultured seedling and the presence of cyst formation were confirmed. The test was repeated three times. The results are shown in Table 2.
- larvae hatched from aseptically treated cysts obtained from Example 2 were washed with sterilized water, and then a potato cultured seedling (variety: varieties :) using a sand medium irrigated with tebuconazole (10 ppm) and streptomycin (1 ppm) as a support.
- a container (1 L plastic container) in which two strains of make-in were planted was inoculated with 900 heads per container. Cyst formation was confirmed about one month after inoculation.
- the sterilized cyst nematode having cyst-forming ability provided by the present invention can eliminate the influence on the hatching by the parasitic bacteria, drug screening such as hatching-promoting substances and nematocides, mechanism analysis of hatching induction, It can be used to screen nematode parasitic microorganisms (filamentous fungi, bacteria, etc.) for the development of microbial pesticides, to analyze the parasitic ecology and dynamics of nematodes in host plants, and to elucidate the mechanisms for detecting trace substances such as hatching-promoting substances. , Can play a big role in these.
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Abstract
The present invention addresses the problem of sterilizing cyst nematodes, establishing an in-vitro mass propagation system for cyst nematodes, and providing sterilized cyst nematodes that allow evaluation tests such as an egg hatch assay to be stably conducted. The present invention relates to a method for obtaining sterilized cyst nematodes, the method including: culturing cyst nematodes, which are to be sterilized, on a sand medium in which a host plant is planted; and irrigating the sand medium with a drug. The drug used in the irrigation includes a germicidal at a concentration that does not affect the growth of the nematodes. The present invention also relates to sterilized cyst nematodes obtained by said method.
Description
本発明は、薬剤スクリーニングなどに利用可能な無菌化シストセンチュウを得る方法および該方法で得られた無菌化シストセンチュウに関する。
The present invention relates to a method for obtaining a sterilized cyst nematode that can be used for drug screening and the like, and a sterilized cyst nematode obtained by the method.
シストセンチュウは、特定の宿主にのみ寄生し、宿主が存在しない状態では環境・薬剤耐性を有するシストを形成し、その状態で長期間生存することができる。作付けなどにより宿主が近くに存在するようになると、宿主由来の孵化促進物質を感知して孵化し、宿主に寄生する。例えば、ジャガイモシストセンチュウ（Potato Cyst Nematode：以下、「ＰＣＮ」とも記す）（学名：Globodera rostochiensis）は、ナス科植物などの特定の宿主にのみ寄生し、ジャガイモの根に寄生する難防除害虫であり、世界中で甚大な被害を発生させている。未だ世界的に十分な対策がなされておらず、効果的な防除剤の開発がなされていないのが現状である。また近年、日本（北海道）でＰＣＮと同属のジャガイモシロシストセンチュウ（Globodera pallida）が発生したことが報告され、米国（オレゴン州）では新種のGlobodera ellingtonaeが発見されたことが報告されている（非特許文献１）。これらの新しいシストセンチュウに対する対策も必要となる。
Cyst nematodes parasitize only on a specific host, and form cysts having environmental / drug resistance in the absence of the host, and can survive for a long time in that state. When the host comes close by planting or the like, it will hatch by detecting the hatching promoting substance derived from the host and infest the host. For example, Potato Cyst Nematode (hereinafter also referred to as “PCN”) (scientific name: Globodera 寄生 rostochiensis) is a hard-to-control insect that infests only certain hosts such as solanaceous plants and infests potato roots. Has caused tremendous damage around the world. At present, no sufficient measures have been taken globally, and effective control agents have not been developed. In recent years, it has been reported that potato white cyst nematode (Globodera pallida), which belongs to the same genus as PCN, has occurred in Japan (Hokkaido), and a new species of Globodera ellingtonae has been discovered in the United States (Oregon) (non-) Patent Document 1). Measures against these new cyst nematodes are also required.
シストセンチュウの防除法の１つとして、宿主が作付けされていない土壌に孵化促進物質を施用し、シスト中の卵の孵化を促進させて、シストセンチュウの幼虫を餓死させることなどが試みられている（特許文献１～３）。
As one of the control methods for cyst nematodes, attempts have been made to apply hatching-promoting substances to soil where the host is not planted, to promote the hatching of eggs in the cysts, and to starve the cyst nematode larvae, etc. (Patent Documents 1 to 3).
一方、シストセンチュウは種々の糸状菌によって寄生されており（非特許文献２）、寄生菌を殺菌するために、例えば、シストに対して次亜塩素酸ナトリウムで表面殺菌を行ったり（非特許文献２）、シストから得られた卵に対してメトキシエチル水銀塩化物で表面殺菌を行ったりしているが（非特許文献３）、いずれも十分なものではなかった。
On the other hand, cyst nematodes are parasitized by various filamentous fungi (Non-Patent Document 2), and in order to sterilize parasitic fungi, for example, surface sterilization is performed on cysts with sodium hypochlorite (Non-Patent Document 2). 2) Although surface sterilization is performed with methoxyethyl mercury chloride on eggs obtained from cysts (Non-patent Document 3), none of them is sufficient.
シストセンチュウの新たな孵化促進物質の探索においては、シストセンチュウの卵の孵化試験を行う必要がある。一般に、野外（畑）から採取したシストセンチュウを温室にてジャガイモに接種、増殖して得た卵を試験材料として用いているが、本発明者らは、野外から採取したシストセンチュウには菌に汚染しているものがあり、これを温室で増殖を繰り返すと、汚染率が上がってしまい試験材料の卵に汚染卵が含まれると孵化試験等の評価系に大きな影響を及ぼすとの問題を認識するに至った。すなわち、本発明の課題は、シストセンチュウの無菌化とin vitroの大量増殖系を確立し、孵化試験などの評価試験を安定的に実施できる無菌化シストセンチュウを提供することにある。
In searching for a new substance for promoting the incubation of cyst nematodes, it is necessary to conduct a hatch test of cyst nematode eggs. In general, eggs obtained by inoculating and growing cyst nematodes collected from the field (field) in potatoes in a greenhouse are used as test materials. Recognize that some of them are contaminated, and if this is repeated in the greenhouse, the contamination rate will increase, and if contaminated eggs are contained in the eggs of the test material, it will greatly affect the evaluation system for hatching tests, etc. It came to do. That is, an object of the present invention is to provide a sterilized cyst nematode that can establish sterilization of a cyst nematode and an in vitro large-scale proliferation system and can stably perform an evaluation test such as a hatch test.
本発明者らは、かかる課題を解決するために鋭意研究を重ねる中で、通常よりも薄い濃度で殺菌剤を宿主植物の支持体（砂培地）に灌注し、これにシストセンチュウを接種することで次世代の卵を内包したシスト形成能を有する無菌化シストセンチュウを得ることができることを見出し、さらに研究を進めた結果、本発明を完成するに至った。すなわち本発明は、以下に関する。
In order to solve such problems, the present inventors have conducted intensive research on irrigating a host plant support (sand medium) at a concentration lower than usual and inoculating it with cyst nematode. As a result of further research, the inventors have found that a sterilized cyst nematode having the ability to form cysts encapsulating the next generation of eggs can be obtained. That is, the present invention relates to the following.
［１］ 無菌化シストセンチュウを得る方法であって、
宿主植物を植えた砂培地で無菌化すべきシストセンチュウを培養すること、および、
砂培地に薬剤灌注することを含み、
灌注する薬剤が、センチュウの生育に影響を及ぼさない濃度の殺菌剤を含む、前記方法。
［２］ 殺菌剤が、ＤＭＩ剤を含む、前記［１］に記載の方法。
［３］ ＤＭＩ剤が、テブコナゾールである、前記［２］に記載の方法。
［４］ 殺菌剤の濃度が、有効濃度の２倍～８００倍希釈した濃度である、前記［１］～［３］のいずれか一項に記載の方法。
［５］ 殺菌剤が、さらにストレプトマイシンを含む、前記［２］～［４］のいずれか一項に記載の方法。 [1] A method for obtaining a sterilized cyst nematode,
Culturing cyst nematodes to be sterilized in a sand medium planted with the host plant; and
Including drug irrigation in a sand medium,
The method, wherein the agent to be irrigated comprises a concentration of a fungicide that does not affect nematode growth.
[2] The method according to [1], wherein the disinfectant includes a DMI agent.
[3] The method according to [2] above, wherein the DMI agent is tebuconazole.
[4] The method according to any one of [1] to [3], wherein the concentration of the bactericide is a concentration diluted by 2 to 800 times the effective concentration.
[5] The method according to any one of [2] to [4], wherein the bactericidal agent further comprises streptomycin.
宿主植物を植えた砂培地で無菌化すべきシストセンチュウを培養すること、および、
砂培地に薬剤灌注することを含み、
灌注する薬剤が、センチュウの生育に影響を及ぼさない濃度の殺菌剤を含む、前記方法。
［２］ 殺菌剤が、ＤＭＩ剤を含む、前記［１］に記載の方法。
［３］ ＤＭＩ剤が、テブコナゾールである、前記［２］に記載の方法。
［４］ 殺菌剤の濃度が、有効濃度の２倍～８００倍希釈した濃度である、前記［１］～［３］のいずれか一項に記載の方法。
［５］ 殺菌剤が、さらにストレプトマイシンを含む、前記［２］～［４］のいずれか一項に記載の方法。 [1] A method for obtaining a sterilized cyst nematode,
Culturing cyst nematodes to be sterilized in a sand medium planted with the host plant; and
Including drug irrigation in a sand medium,
The method, wherein the agent to be irrigated comprises a concentration of a fungicide that does not affect nematode growth.
[2] The method according to [1], wherein the disinfectant includes a DMI agent.
[3] The method according to [2] above, wherein the DMI agent is tebuconazole.
[4] The method according to any one of [1] to [3], wherein the concentration of the bactericide is a concentration diluted by 2 to 800 times the effective concentration.
[5] The method according to any one of [2] to [4], wherein the bactericidal agent further comprises streptomycin.
［６］ シストセンチュウが、ジャガイモシストセンチュウ（Globodera rostochiensis）である、前記［１］～［５］のいずれか一項に記載の方法。
［７］ 前記［１］～［６］のいずれか一項に記載の方法によって得られた、シスト形成能を有する無菌化シストセンチュウ。
［８］ シスト形成能を有する無菌化シストセンチュウ。
［９］ ジャガイモシストセンチュウ（Globodera rostochiensis）である、前記［７］または［８］に記載の無菌化シストセンチュウ。
［１０］ 孵化促進物質のスクリーニング用である、前記［７］～［９］のいずれか一項に記載の無菌化シストセンチュウ。 [6] The method according to any one of [1] to [5], wherein the cyst nematode is a potato cyst nematode (Globodera rostochiensis).
[7] A sterilized cyst nematode having a cyst-forming ability, obtained by the method according to any one of [1] to [6].
[8] A sterilized cyst nematode having cyst forming ability.
[9] The sterilized cyst nematode according to the above [7] or [8], which is a potato cyst nematode (Globodera rostochiensis).
[10] The sterilized cyst nematode according to any one of [7] to [9], which is used for screening for a hatching promoting substance.
［７］ 前記［１］～［６］のいずれか一項に記載の方法によって得られた、シスト形成能を有する無菌化シストセンチュウ。
［８］ シスト形成能を有する無菌化シストセンチュウ。
［９］ ジャガイモシストセンチュウ（Globodera rostochiensis）である、前記［７］または［８］に記載の無菌化シストセンチュウ。
［１０］ 孵化促進物質のスクリーニング用である、前記［７］～［９］のいずれか一項に記載の無菌化シストセンチュウ。 [6] The method according to any one of [1] to [5], wherein the cyst nematode is a potato cyst nematode (Globodera rostochiensis).
[7] A sterilized cyst nematode having a cyst-forming ability, obtained by the method according to any one of [1] to [6].
[8] A sterilized cyst nematode having cyst forming ability.
[9] The sterilized cyst nematode according to the above [7] or [8], which is a potato cyst nematode (Globodera rostochiensis).
[10] The sterilized cyst nematode according to any one of [7] to [9], which is used for screening for a hatching promoting substance.
本発明の方法によれば、メトキシエチル水銀塩化物などの水銀剤を用いる必要がなく、環境に負荷をかけることなく、安全で容易にシストセンチュウを無菌化することができる。また、本発明の方法により無菌化されたシストセンチュウは、シスト形成能を有し、継代することができる。したがって、本発明によって、シストセンチュウのin vitro培養系が確立された。本発明によれば、無菌化シストセンチュウを、無菌状態を維持、推進しながら、大量に増殖することが可能であり、例えば、孵化促進物質の探索のための大規模スクリーニングを行う場合などに必要となる無菌シストを大量に提供することができる。すなわち、本発明の方法により得られた無菌化シストセンチュウから得られた卵を用いることにより、シストセンチュウに寄生する寄生菌による汚染卵（寄生卵）の影響を受けることがなく、より正確に候補薬剤の孵化を促進する能力を評価することができる。
According to the method of the present invention, it is not necessary to use a mercury agent such as methoxyethyl mercury chloride, and cyst nematodes can be sterilized safely and easily without burdening the environment. The cyst nematode sterilized by the method of the present invention has cyst formation ability and can be passaged. Therefore, according to the present invention, an invitro culture system for cyst nematode was established. According to the present invention, sterilized cyst nematodes can be proliferated in large quantities while maintaining and promoting sterility, and are necessary for, for example, large-scale screening for searching for hatching promoting substances. Aseptic cysts can be provided in large quantities. That is, by using an egg obtained from a sterilized cyst nematode obtained by the method of the present invention, it is not affected by a contaminated egg (parasitic egg) due to a parasitic bacterium parasitic on cyst nematode, and more accurately a candidate. The ability to promote drug hatching can be assessed.
本発明は、無菌化シストセンチュウを得る方法に関する。ここで「無菌化」とは、シストセンチュウに寄生する寄生菌の数を減ずるか、完全に滅菌することを包含するものであり、例えば、無菌処理によって汚染卵率を低減することによって確認することができる。汚染卵率が、０～１５％、好ましくは０～１２％であれば、孵化試験に実質的に影響を与えないとし得る。
The present invention relates to a method for obtaining a sterilized cyst nematode. Here, “sterilization” includes reducing the number of parasitic fungus parasitic on cyst nematode or completely sterilizing, for example, confirming by reducing the rate of contaminated eggs by aseptic processing. Can do. If the contaminated egg rate is 0 to 15%, preferably 0 to 12%, it may be considered that the hatching test is not substantially affected.
本発明に係る方法は、宿主植物を植えた砂培地で無菌化すべきシストセンチュウを培養すること、および、砂培地に薬剤灌注することを含む。ここで灌注する薬剤は、無菌化の標的となる寄生菌に対する殺菌剤であればよく、寄生菌の種類に応じて適宜選択することができる。本発明において用いられる具体的な薬剤としては、例えば、トリホリン、フェナリモル、オキスポコナゾールフマル酸塩、ペフラゾエート、プロクロラズ、トリフルミゾール、シプロコナゾール、ジフェノコナゾール、フェンブコナゾール、ヘキサコナゾール、イミベンコナゾール、イプコナゾール、メトコナゾール、ミクロブタニル、プロピコナゾール、シメコナゾール、テブコナゾール、テトラコナゾールなどのＤＭＩ剤（脱メチル化阻害剤：ステロール生合成におけるC14位の脱メチル化酵素の阻害剤）；チオファネートメチルなどのベンゾイミダゾール系殺菌剤；イミノクタジン酢酸塩、イミノクタジンアルベシル酸塩などのグアニジン系殺菌剤；ナイスタチンなどのポリエン系抗真菌薬；ストレプトマイシン、ゲンタマイシンなどの抗生物質などが挙げられる。
The method according to the present invention includes culturing cyst nematodes to be sterilized in a sand medium planted with a host plant, and irrigating the sand medium with a drug. The drug to be irrigated here may be a bactericidal agent against parasitic bacteria that are targets for sterilization, and can be appropriately selected according to the type of parasitic bacteria. Specific drugs used in the present invention include, for example, trifolin, fenarimol, oxpoconazole fumarate, pefazoate, prochloraz, triflumizole, cyproconazole, difenoconazole, fenbuconazole, hexaconazole, imibenco NMI, ipconazole, metconazole, microbutanyl, propiconazole, cimeconazole, tebuconazole, tetraconazole and other DMI agents (demethylation inhibitors: inhibitors of demethylase at position C14 in sterol biosynthesis); benzoates such as thiophanate methyl Imidazole fungicides; Guanidine fungicides such as iminotadine acetate and iminoctadine albecate; Polyene antifungals such as nystatin; Streptomycin, gentamicin, etc. Such as raw materials, and the like.
薬剤は、１種または２種以上を組み合わせて用いることができる。薬剤を組み合わせて用いる場合、抗真菌性のＤＭＩ剤と抗細菌性の抗生物質とを組み合わせて用いることが好ましく、特に、テブコナゾールとストレプトマイシントを組み合わせて用いることが好ましい。
Drugs can be used alone or in combination of two or more. When a combination of drugs is used, it is preferable to use a combination of an antifungal DMI agent and an antibacterial antibiotic, and it is particularly preferable to use a combination of tebuconazole and streptomycin.
灌注する薬剤の濃度は、センチュウの生育に影響を及ぼさない濃度であり、通常、用いる薬剤の有効濃度（登録処方の濃度）より薄い。灌注する薬剤の濃度は、好ましくは、有効濃度の２倍～８００倍希釈した濃度、より好ましくは、５倍～１６０倍希釈した濃度、とくに好ましくは、１０倍～８０倍希釈した濃度である。
The concentration of the drug to be irrigated is a concentration that does not affect the growth of nematode and is usually thinner than the effective concentration of the drug to be used (the concentration of the registered prescription). The concentration of the drug to be irrigated is preferably a concentration diluted 2 to 800 times the effective concentration, more preferably a concentration diluted 5 to 160 times, particularly preferably a concentration diluted 10 to 80 times.
例えば、薬剤としてテブコナゾールを用いる場合、その登録処方の濃度は、１３３～８００ｐｐｍ程度であるが、本発明においては、例えば、１ｐｐｍ～５０ｐｐｍ、好ましくは、５ｐｐｍ～２０ｐｐｍ、より好ましくは、１０ｐｐｍである。テブコナゾールとストレプトマイシンを組み合わせて用いる場合、ストレプトマイシンの濃度は、例えば、０．０５ｐｐｍ～５０ｐｐｍ、好ましくは、０．５ｐｐｍ～５ｐｐｍ、より好ましくは、１ｐｐｍである。
For example, when tebuconazole is used as a drug, the concentration of the registered prescription is about 133 to 800 ppm, but in the present invention, for example, it is 1 to 50 ppm, preferably 5 to 20 ppm, more preferably 10 ppm. When tebuconazole and streptomycin are used in combination, the concentration of streptomycin is, for example, 0.05 ppm to 50 ppm, preferably 0.5 ppm to 5 ppm, more preferably 1 ppm.
本発明の無菌化の標的となり得るシストセンチュウは、例えば、ダイズシストセンチュウ（Heterodera glycines）、クローバーシストセンチュウ（Heterodera trifolii）、テンサイシストセンチュウ（Heterodera schachtii）、イネシストセンチュウ（Heterodera oryzae）、オカボシストセンチュウ（Heterodera elachista）などのヘテロデラ属（Heterodera）の植物寄生性シストセンチュウや、ジャガイモシストセンチュウ（Globodera rostochiensis）、ジャガイモシロシストセンチュウ（Globodera pallida）、Globodera ellingtonae、タバコシストセンチュウ（Globodera tabaccum）、ヨモギシストセンチュウ（Globodera hypolysi）などのグロボデラ属（Globodera）の植物寄生性シストセンチュウが挙げられる。本発明の無菌処理においては、ジャガイモシストセンチュウを標的にする場合が好ましい。
The cyst nematodes that can be targets for sterilization of the present invention include, for example, soybean cyst nematodes (Heterodera glycines), clover cyst nematodes (Heterodera trifolii), sugar beet nematodes (Heterodera schachtii), rice cyst nematodes (Heterodera oryzae), and okabo cyst nematodes. Heterodera plant parasitic cyst nematodes such as (Heterodera elachista), potato cyst nematodes (Globodera rostochiensis), potato white cyst nematodes (Globodera pallida), Globodera 、 ellingtonae, tobacco cyst nematodes (Globodera tabium acc) (Globodera hypolysi) and other plant parasitic cyst nematodes of the genus Globodera. In the aseptic processing of the present invention, it is preferable to target potato cyst nematodes.
本発明の方法において用いる培養器は、シストセンチュウの培養に用いられる容器であれば特に限定されないが、例えば、ガラス製またはプラスチック製の容器であってよい。容器の大きさは、特に限定されないが、１００ｍｌ～１０００ｍｌ程度の大きさであればよく、これに砂培地を容器量の１／５～１／３程度の量を入れ、宿主植物の苗を１～３本程度植えて培養に用いる。宿主植物の苗は、無菌培養された培養苗であることが好ましい。
本発明の方法において用いる砂培地は、砂土等、農業・園芸用に用いる砂栽培用のものであれば特に限定されない。 The incubator used in the method of the present invention is not particularly limited as long as it is a container used for cyst nematode culture. For example, it may be a glass or plastic container. Although the size of the container is not particularly limited, it may be about 100 ml to 1000 ml, and a sand culture medium is added to this in an amount of about 1/5 to 1/3 of the container amount, and 1 seedling of the host plant is added. About 3 plants are planted and used for culture. The seedling of the host plant is preferably a cultured seedling that has been sterilized.
The sand culture medium used in the method of the present invention is not particularly limited as long as it is for sand cultivation used for agriculture and horticulture such as sand and earth.
本発明の方法において用いる砂培地は、砂土等、農業・園芸用に用いる砂栽培用のものであれば特に限定されない。 The incubator used in the method of the present invention is not particularly limited as long as it is a container used for cyst nematode culture. For example, it may be a glass or plastic container. Although the size of the container is not particularly limited, it may be about 100 ml to 1000 ml, and a sand culture medium is added to this in an amount of about 1/5 to 1/3 of the container amount, and 1 seedling of the host plant is added. About 3 plants are planted and used for culture. The seedling of the host plant is preferably a cultured seedling that has been sterilized.
The sand culture medium used in the method of the present invention is not particularly limited as long as it is for sand cultivation used for agriculture and horticulture such as sand and earth.
また、本発明の方法において用いる宿主植物は、無菌化の標的となるシストセンチュウの宿主植物である。例えば、ジャガイモシストセンチュウを標的とした場合、宿主植物は、ナス科植物であり、好ましくは、ジャガイモである。
本発明の方法においてシストセンチュウを培養する際の培養条件は、２０℃・１６時間明期／日とし、培地に光が当たらないように、培養容器の下部はアルミホイルで覆い遮光した。 The host plant used in the method of the present invention is a cyst nematode host plant that is a target for sterilization. For example, when targeting potato cyst nematodes, the host plant is a solanaceous plant, preferably potato.
The culture conditions for cultivating 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 and shielded from light so that the medium did not receive light.
本発明の方法においてシストセンチュウを培養する際の培養条件は、２０℃・１６時間明期／日とし、培地に光が当たらないように、培養容器の下部はアルミホイルで覆い遮光した。 The host plant used in the method of the present invention is a cyst nematode host plant that is a target for sterilization. For example, when targeting potato cyst nematodes, the host plant is a solanaceous plant, preferably potato.
The culture conditions for cultivating 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 and shielded from light so that the medium did not receive light.
本発明は、一態様において、シスト形成能を有する無菌化シストセンチュウに関する。
本発明に係る無菌化シストセンチュウは、シスト形成能を有しており、継代可能であることから、各種の実験用として、また、例えば、孵化促進物質のスクリーニング用として用いることができる。 In one aspect, the present invention relates to a sterilized cyst nematode having cyst forming ability.
Since the sterilized cyst nematode according to the present invention has cyst-forming ability and can be passaged, it can be used for various experiments, for example, for screening for hatching promoting substances.
本発明に係る無菌化シストセンチュウは、シスト形成能を有しており、継代可能であることから、各種の実験用として、また、例えば、孵化促進物質のスクリーニング用として用いることができる。 In one aspect, the present invention relates to a sterilized cyst nematode having cyst forming ability.
Since the sterilized cyst nematode according to the present invention has cyst-forming ability and can be passaged, it can be used for various experiments, for example, for screening for hatching promoting substances.
以下、本発明について、さらに詳細に実施例を用いて説明するが、本発明はこれらの実施例に限定されるものではない。
Hereinafter, the present invention will be described in further detail using examples, but the present invention is not limited to these examples.
［試験例１］ジャガイモシストセンチュウの無菌化の検討（１）
温室内で通常栽培したジャガイモ苗（品種：メークイン）において増殖したジャガイモシストセンチュウ（ＰＣＮ）の幼虫（シスト3日間滅菌水に浸漬し、続いてジャガイモ根浸出液に浸漬処理し、６～１０日後にシストから遊出してきた孵化幼虫を使用）に対し、エタノール（処理濃度７０％）、次亜塩素酸ナトリウム（処理濃度２．０～０．００２％）、ＰＰＭ（商標）（PLANT PRESERVATIVE MIXTURE、Plant Cell Technology社）（処理濃度０．２～０．０００２５％）、微酸性電解水（処理濃度０．２～０．０００２５％）の夫々を用いて、２分間表面殺菌を行った。表面殺菌を行った幼虫を顕微鏡下で観察し、生存の有無を確認した。また、生存していた幼虫を、無菌的にジャガイモ培養苗（品種：メークイン）を植えた寒天若しくはジュランガム培地（組成：MS培地（和光純薬工業(株)製）、MS Vitamin Solution (Sigma社製)、30％ショ糖、寒天0.8％若しくはジェランガム0.2％）において２０℃で７５日間培養した。幼虫接種の３０日後からシスト形成の有無の観察した。寄生菌に汚染された培養苗の割合を汚染率（％）とした。結果を表１に示す。 [Test Example 1] Examination of sterilization of potato cyst nematode (1)
Potato cyst nematode (PCN) larvae grown in potato seedlings (variety: make-in) cultivated in a greenhouse (soaked in sterilized water for 3 days, followed by soaking in potato root exudate and cysts 6-10 days later ) Using ethanol (treatment concentration 70%), sodium hypochlorite (treatment concentration 2.0-0.002%), PPM (trademark) (PLANT PRESERVATIVE MIXTURE, Plant Cell) Technology) (treatment concentration: 0.2 to 0.00025%) and slightly acidic electrolyzed water (treatment concentration: 0.2 to 0.00025%) were used for surface sterilization for 2 minutes. The surface sterilized larvae were observed under a microscope to confirm the presence or absence of survival. In addition, the surviving larvae were agar or julan gum medium (composition: MS medium (manufactured by Wako Pure Chemical Industries, Ltd.)), MS Vitamin Solution (manufactured by Sigma), aseptically planted potato seedlings (variety: make-in) ), 30% sucrose, agar 0.8% or gellan gum 0.2%) and cultured at 20 ° C. for 75 days. The presence or absence of cyst formation was observed 30 days after the larva inoculation. The percentage of cultured seedlings contaminated with parasitic bacteria was defined as the contamination rate (%). The results are shown in Table 1.
温室内で通常栽培したジャガイモ苗（品種：メークイン）において増殖したジャガイモシストセンチュウ（ＰＣＮ）の幼虫（シスト3日間滅菌水に浸漬し、続いてジャガイモ根浸出液に浸漬処理し、６～１０日後にシストから遊出してきた孵化幼虫を使用）に対し、エタノール（処理濃度７０％）、次亜塩素酸ナトリウム（処理濃度２．０～０．００２％）、ＰＰＭ（商標）（PLANT PRESERVATIVE MIXTURE、Plant Cell Technology社）（処理濃度０．２～０．０００２５％）、微酸性電解水（処理濃度０．２～０．０００２５％）の夫々を用いて、２分間表面殺菌を行った。表面殺菌を行った幼虫を顕微鏡下で観察し、生存の有無を確認した。また、生存していた幼虫を、無菌的にジャガイモ培養苗（品種：メークイン）を植えた寒天若しくはジュランガム培地（組成：MS培地（和光純薬工業(株)製）、MS Vitamin Solution (Sigma社製)、30％ショ糖、寒天0.8％若しくはジェランガム0.2％）において２０℃で７５日間培養した。幼虫接種の３０日後からシスト形成の有無の観察した。寄生菌に汚染された培養苗の割合を汚染率（％）とした。結果を表１に示す。 [Test Example 1] Examination of sterilization of potato cyst nematode (1)
Potato cyst nematode (PCN) larvae grown in potato seedlings (variety: make-in) cultivated in a greenhouse (soaked in sterilized water for 3 days, followed by soaking in potato root exudate and cysts 6-10 days later ) Using ethanol (treatment concentration 70%), sodium hypochlorite (treatment concentration 2.0-0.002%), PPM (trademark) (PLANT PRESERVATIVE MIXTURE, Plant Cell) Technology) (treatment concentration: 0.2 to 0.00025%) and slightly acidic electrolyzed water (treatment concentration: 0.2 to 0.00025%) were used for surface sterilization for 2 minutes. The surface sterilized larvae were observed under a microscope to confirm the presence or absence of survival. In addition, the surviving larvae were agar or julan gum medium (composition: MS medium (manufactured by Wako Pure Chemical Industries, Ltd.)), MS Vitamin Solution (manufactured by Sigma), aseptically planted potato seedlings (variety: make-in) ), 30% sucrose, agar 0.8% or gellan gum 0.2%) and cultured at 20 ° C. for 75 days. The presence or absence of cyst formation was observed 30 days after the larva inoculation. The percentage of cultured seedlings contaminated with parasitic bacteria was defined as the contamination rate (%). The results are shown in Table 1.
表１に示すとおり、次亜塩素酸ナトリウムによる表面処理によって、ＰＣＮの生存を維持しつつ、寄生菌を除去することができることが明らかになった。しかしながら、さらに検討を進めた結果、次亜塩素酸ナトリウム処理した幼虫からはシストが形成されず、孵化試験に利用することができないことが判明した。
As shown in Table 1, it was revealed that surface treatment with sodium hypochlorite can remove parasitic bacteria while maintaining the survival of PCN. However, as a result of further investigation, it was found that cysts were not formed from larvae treated with sodium hypochlorite and could not be used for hatching tests.
［試験例２］ジャガイモシストセンチュウの無菌化の検討（２）
温室内で通常栽培したジャガイモ苗（品種：メークイン）において増殖したＰＣＮの幼虫（シスト3日間滅菌水に浸漬し、続いてジャガイモ根浸出液に浸漬処理し、６～１０日後にシストから遊出してきた孵化幼虫を使用）を、無菌的にジャガイモ培養苗（品種：メークイン）を植えた砂培地またはセル培土において２０℃で７５日間培養した。培養初日に、砂培地（芝生用目砂（東海砂利(株)製））またはセル培土（ホクサン(株)製）に対し、テブコナゾール（１０ｐｐｍ～１００ｐｐｍ）およびストレプトマイシン（１ｐｐｍ）を組み合わせた殺菌剤を灌注した。薬剤灌注後３０日目から寄生菌による培養苗の汚染の有無とシスト形成の有無を確認した。試験は３反復行った。結果を表２に示す。
[Test Example 2] Examination of sterilization of potato cyst nematode (2)
PCN larvae grown in potato seedlings (variety: makein) cultivated normally in a greenhouse (soaked in sterilized water for 3 days with cysts, then dipped in potato root exudate, and emanated from cysts after 6-10 days Hatching larvae were used) and cultured for 75 days at 20 ° C. in a sand medium or cell culture medium in which aseptically planted potato seedlings (variety: make-in) were planted. On the first day of culture, a fungicide that combines tebuconazole (10 ppm to 100 ppm) and streptomycin (1 ppm) to sand culture medium (lawn sand (Tokai Gravel Co., Ltd.)) or cell culture soil (Hokusan Co., Ltd.) Irrigated. From the 30th day after the drug irrigation, the presence or absence of contamination of the cultured seedling and the presence of cyst formation were confirmed. The test was repeated three times. The results are shown in Table 2.
温室内で通常栽培したジャガイモ苗（品種：メークイン）において増殖したＰＣＮの幼虫（シスト3日間滅菌水に浸漬し、続いてジャガイモ根浸出液に浸漬処理し、６～１０日後にシストから遊出してきた孵化幼虫を使用）を、無菌的にジャガイモ培養苗（品種：メークイン）を植えた砂培地またはセル培土において２０℃で７５日間培養した。培養初日に、砂培地（芝生用目砂（東海砂利(株)製））またはセル培土（ホクサン(株)製）に対し、テブコナゾール（１０ｐｐｍ～１００ｐｐｍ）およびストレプトマイシン（１ｐｐｍ）を組み合わせた殺菌剤を灌注した。薬剤灌注後３０日目から寄生菌による培養苗の汚染の有無とシスト形成の有無を確認した。試験は３反復行った。結果を表２に示す。
PCN larvae grown in potato seedlings (variety: makein) cultivated normally in a greenhouse (soaked in sterilized water for 3 days with cysts, then dipped in potato root exudate, and emanated from cysts after 6-10 days Hatching larvae were used) and cultured for 75 days at 20 ° C. in a sand medium or cell culture medium in which aseptically planted potato seedlings (variety: make-in) were planted. On the first day of culture, a fungicide that combines tebuconazole (10 ppm to 100 ppm) and streptomycin (1 ppm) to sand culture medium (lawn sand (Tokai Gravel Co., Ltd.)) or cell culture soil (Hokusan Co., Ltd.) Irrigated. From the 30th day after the drug irrigation, the presence or absence of contamination of the cultured seedling and the presence of cyst formation were confirmed. The test was repeated three times. The results are shown in Table 2.
［試験例３］無菌処理シストの回収および継代
温室内で通常栽培したジャガイモ苗（品種：メークイン）において増殖したＰＣＮから回収されたシスト１０粒中の汚染卵率、および、前記シストと同時に回収されたシストを試験例２の試験区１と同様に処理して、培養後に形成されたシスト中の汚染卵率（％）を算出した。なお、汚染卵率（％）は、回収されたシストから得られた卵について、センチュウ計算板（１ｍｌあたり）を用い、顕微鏡下で全卵数（健全卵＋汚染卵）を計数して算出した。結果を表３および表４に示す。 [Test Example 3] Recovery and passage of aseptically treated cysts Contaminated egg rate in 10 cysts recovered from PCN grown in potato seedlings (variety: make-in) normally grown in a greenhouse, and recovered simultaneously with the cysts The treated cysts were treated in the same manner as in test group 1 of Test Example 2, and the contaminated egg rate (%) in the cysts formed after culturing was calculated. The contaminated egg rate (%) was calculated by counting the total number of eggs (healthy eggs + contaminated eggs) under a microscope using the nematode calculation plate (per ml) for the eggs obtained from the collected cysts. . The results are shown in Table 3 and Table 4.
温室内で通常栽培したジャガイモ苗（品種：メークイン）において増殖したＰＣＮから回収されたシスト１０粒中の汚染卵率、および、前記シストと同時に回収されたシストを試験例２の試験区１と同様に処理して、培養後に形成されたシスト中の汚染卵率（％）を算出した。なお、汚染卵率（％）は、回収されたシストから得られた卵について、センチュウ計算板（１ｍｌあたり）を用い、顕微鏡下で全卵数（健全卵＋汚染卵）を計数して算出した。結果を表３および表４に示す。 [Test Example 3] Recovery and passage of aseptically treated cysts Contaminated egg rate in 10 cysts recovered from PCN grown in potato seedlings (variety: make-in) normally grown in a greenhouse, and recovered simultaneously with the cysts The treated cysts were treated in the same manner as in test group 1 of Test Example 2, and the contaminated egg rate (%) in the cysts formed after culturing was calculated. The contaminated egg rate (%) was calculated by counting the total number of eggs (healthy eggs + contaminated eggs) under a microscope using the nematode calculation plate (per ml) for the eggs obtained from the collected cysts. . The results are shown in Table 3 and Table 4.
表３に示すとおり、通常栽培したジャガイモ苗において増殖したＰＣＮから回収されたシストの汚染卵率は、１７．３～３２．３％であった一方で、表４に示すとおり、本発明の無菌処理によって得られたシストの汚染卵率は、４．５～１０．１％であり、寄生菌による汚染卵が著しく減少したことが明らかになった（図１）。
本発明の無菌処理による無菌化率（％）（汚染卵の減少割合）は、約４７．８～８２．０％であり、極めて有効に無菌化が達成できた。 As shown in Table 3, while the contaminated egg rate of cysts recovered from PCN grown in cultivated potato seedlings was 17.3 to 32.3%, as shown in Table 4, the sterility of the present invention The contaminated egg rate of the cysts obtained by the treatment was 4.5 to 10.1%, and it was revealed that the contaminated eggs due to the parasitic bacteria were remarkably reduced (FIG. 1).
The sterilization rate (%) by the sterilization treatment of the present invention (reduction rate of contaminated eggs) was about 47.8 to 82.0%, and sterilization could be achieved very effectively.
本発明の無菌処理による無菌化率（％）（汚染卵の減少割合）は、約４７．８～８２．０％であり、極めて有効に無菌化が達成できた。 As shown in Table 3, while the contaminated egg rate of cysts recovered from PCN grown in cultivated potato seedlings was 17.3 to 32.3%, as shown in Table 4, the sterility of the present invention The contaminated egg rate of the cysts obtained by the treatment was 4.5 to 10.1%, and it was revealed that the contaminated eggs due to the parasitic bacteria were remarkably reduced (FIG. 1).
The sterilization rate (%) by the sterilization treatment of the present invention (reduction rate of contaminated eggs) was about 47.8 to 82.0%, and sterilization could be achieved very effectively.
さらに実施例２から得られた無菌処理シストから孵化した幼虫を滅菌水で洗浄した後、テブコナゾール(10 ppm)およびストレプトマイシン（1 ppm）を灌注した砂培地を支持体とし、ジャガイモ培養苗（品種：メークイン）を２株植えた容器（１Ｌプラスチック容器）に、１容器あたり９００頭を接種した。接種約１ヵ月後にシスト形成が確認できた。
このようにシスト形成能を有する無菌化シストセンチュウを本発明の無菌処理を行いながら継代することによって、汚染卵率を著しく低減することが可能となり、寄生菌を完全に滅菌することが期待できる。 Further, larvae hatched from aseptically treated cysts obtained from Example 2 were washed with sterilized water, and then a potato cultured seedling (variety: varieties :) using a sand medium irrigated with tebuconazole (10 ppm) and streptomycin (1 ppm) as a support. A container (1 L plastic container) in which two strains of make-in were planted was inoculated with 900 heads per container. Cyst formation was confirmed about one month after inoculation.
By passing the sterilized cyst nematode having cyst-forming ability in this way while performing the sterilization treatment of the present invention, it becomes possible to remarkably reduce the contaminated egg rate, and it can be expected to completely sterilize the parasitic bacteria. .
このようにシスト形成能を有する無菌化シストセンチュウを本発明の無菌処理を行いながら継代することによって、汚染卵率を著しく低減することが可能となり、寄生菌を完全に滅菌することが期待できる。 Further, larvae hatched from aseptically treated cysts obtained from Example 2 were washed with sterilized water, and then a potato cultured seedling (variety: varieties :) using a sand medium irrigated with tebuconazole (10 ppm) and streptomycin (1 ppm) as a support. A container (1 L plastic container) in which two strains of make-in were planted was inoculated with 900 heads per container. Cyst formation was confirmed about one month after inoculation.
By passing the sterilized cyst nematode having cyst-forming ability in this way while performing the sterilization treatment of the present invention, it becomes possible to remarkably reduce the contaminated egg rate, and it can be expected to completely sterilize the parasitic bacteria. .
本発明によって提供されるシスト形成能を有する無菌化シストセンチュウは、寄生菌による孵化への影響を排除することができるため、孵化促進物質や殺センチュウ剤などの薬剤スクリーニング、孵化誘導のメカニズム解析、微生物農薬開発に向けたセンチュウ寄生性微生物（糸状菌、細菌など）のスクリーニング、宿主植物中のセンチュウの寄生生態や動態の解析、孵化促進物質などの微量物質検出のメカニズム解明に利用することができ、これらにおいて大きな役割を担い得るものである。
Since the sterilized cyst nematode having cyst-forming ability provided by the present invention can eliminate the influence on the hatching by the parasitic bacteria, drug screening such as hatching-promoting substances and nematocides, mechanism analysis of hatching induction, It can be used to screen nematode parasitic microorganisms (filamentous fungi, bacteria, etc.) for the development of microbial pesticides, to analyze the parasitic ecology and dynamics of nematodes in host plants, and to elucidate the mechanisms for detecting trace substances such as hatching-promoting substances. , Can play a big role in these.
Claims (10)
- 無菌化シストセンチュウを得る方法であって、
宿主植物を植えた砂培地で無菌化すべきシストセンチュウを培養すること、および、
砂培地に薬剤灌注することを含み、
灌注する薬剤が、センチュウの生育に影響を及ぼさない濃度の殺菌剤を含む、前記方法。 A method for obtaining a sterilized cyst nematode,
Culturing cyst nematodes to be sterilized in a sand medium planted with the host plant; and
Including drug irrigation in a sand medium,
The method, wherein the agent to be irrigated comprises a concentration of a fungicide that does not affect nematode growth. - 殺菌剤が、ＤＭＩ剤を含む、請求項１に記載の方法。 The method according to claim 1, wherein the bactericidal agent comprises a DMI agent.
- ＤＭＩ剤が、テブコナゾールである、請求項２に記載の方法。 The method according to claim 2, wherein the DMI agent is tebuconazole.
- 殺菌剤の濃度が、有効濃度の２倍～８００倍希釈した濃度である、請求項１～３のいずれか一項に記載の方法。 The method according to any one of claims 1 to 3, wherein the concentration of the bactericide is a concentration diluted by 2 to 800 times the effective concentration.
- 殺菌剤が、さらにストレプトマイシンを含む、請求項２～４のいずれか一項に記載の方法。 The method according to any one of claims 2 to 4, wherein the bactericide further comprises streptomycin.
- シストセンチュウが、ジャガイモシストセンチュウ（Globodera rostochiensis）である、請求項１～５のいずれか一項に記載の方法。 The method according to any one of claims 1 to 5, wherein the cyst nematode is a potato cyst nematode (Globodera rostochiensis).
- 請求項１～６のいずれか一項に記載の方法によって得られた、シスト形成能を有する無菌化シストセンチュウ。 A sterilized cyst nematode having a cyst-forming ability obtained by the method according to any one of claims 1 to 6.
- シスト形成能を有する無菌化シストセンチュウ。 Sterilized cyst nematode with cyst formation ability.
- ジャガイモシストセンチュウ（Globodera rostochiensis）である、請求項７または８に記載の無菌化シストセンチュウ。 The sterilized cyst nematode according to claim 7 or 8, which is a potato cyst nematode (Globodera rostochiensis).
- 孵化促進物質のスクリーニング用である、請求項７～９のいずれか一項に記載の無菌化シストセンチュウ。 10. The sterilized cyst nematode according to any one of claims 7 to 9, which is used for screening for a hatching promoting substance.
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| JPH10510146A (en) * | 1994-11-21 | 1998-10-06 | ザ ユニバーシティ オブ リーズ | Modified proteinase inhibitors |
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| CN112868607A (en) * | 2021-03-01 | 2021-06-01 | 闫丰军 | Method for controlling bad pupa disease of Chouioia cunea Yang breeding |
| CN112868607B (en) * | 2021-03-01 | 2022-12-16 | 闫丰军 | Method for controlling bad pupa disease of Chouioia cunea Yang breeding |
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