WO2019160075A1 - Méthode d'obtention de nématode à kyste stérilisé - Google Patents

Méthode d'obtention de nématode à kyste stérilisé Download PDF

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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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Prior art keywords
sterilized
cyst
cyst nematode
nematode
concentration
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PCT/JP2019/005512
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English (en)
Japanese (ja)
Inventor
琴美 林
裕子 宮代
恭子 鰐渕
貞子 小野
紀子 田林
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ホクサン株式会社
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Priority to JP2020500576A priority Critical patent/JP7254061B2/ja
Publication of WO2019160075A1 publication Critical patent/WO2019160075A1/fr

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    • 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

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  • 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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  • Life Sciences & Earth Sciences (AREA)
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Abstract

La présente invention aborde le problème de stérilisation de nématodes à kyste, l'établissement d'un système de propagation de masse in vitro pour les nématodes à kyste, et la fourniture de nématodes à kyste stérilisés qui permettent d'effectuer de manière stable des tests d'évaluation tels qu'un dosage d'éclosion des œufs. La présente invention concerne une méthode d'obtention de nématodes à kyste stérilisés, la méthode comprenant : la culture de nématodes à kyste, qui doivent être stérilisés, sur un milieu de sable dans lequel une plante hôte est plantée ; et l'irrigation du milieu de sable avec un médicament. Le médicament utilisé dans l'irrigation comprend un germicide à une concentration qui n'affecte pas la croissance des nématodes. La présente invention concerne également des nématodes à kyste stérilisés pouvant être obtenus par ladite méthode.
PCT/JP2019/005512 2018-02-16 2019-02-15 Méthode d'obtention de nématode à kyste stérilisé WO2019160075A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112868607A (zh) * 2021-03-01 2021-06-01 闫丰军 白蛾周氏啮小蜂繁育坏蛹病控制方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10510146A (ja) * 1994-11-21 1998-10-06 ザ ユニバーシティ オブ リーズ 改変されたプロテイナーゼインヒビター
JP2016520522A (ja) * 2013-03-15 2016-07-14 モンサント テクノロジー エルエルシー 線虫害虫防除のためのｎ−，ｃ−二置換アゾール

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10510146A (ja) * 1994-11-21 1998-10-06 ザ ユニバーシティ オブ リーズ 改変されたプロテイナーゼインヒビター
JP2016520522A (ja) * 2013-03-15 2016-07-14 モンサント テクノロジー エルエルシー 線虫害虫防除のためのｎ−，ｃ−二置換アゾール

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FOOT M. A.: "Laboratory rearing of potato cyst nematode; a method suitable for pathotyping and biological studies", NEW ZEALAND JOURNAL OF ZOOLOGY, vol. 4, no. 2, June 1977 (1977-06-01), pages 183 - 186, XP055634912 *
HANSEN E. ET AL.: "Differential expression patterns of the wound-inducible transgene wun1-uidA in potato roots following infection with either cyst or root knot nematodes", PHYSIOLOGICAL AND MOLECULAR PLANT PATHOLOGY, vol. 48, no. 3, March 1996 (1996-03-01), pages 161 - 170, XP055634910 *
KUSH工DA, ATSUHIKO ET AL: "Axenic Culture of Heterodera glycines on Milk-Vetch Roots Genetically Transformed by Agrobacterium rhizogenes", ANNUAL REPORT OF THE SOCIETY OF PLANT PROTECTION OF NORTH JAPAN, vol. 2002, no. 53, 2002, pages 297 - 300, XP055634919 *
TRIFONOVA Z. ET AL.: "Fungal parasitism of the cysts and eggs of the Globodera rostochiensis", JOURNAL OF AGRICULTURAL SCIENCES, vol. 48, no. 1, 2003, pages 103 - 110, XP055634913 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112868607A (zh) * 2021-03-01 2021-06-01 闫丰军 白蛾周氏啮小蜂繁育坏蛹病控制方法
CN112868607B (zh) * 2021-03-01 2022-12-16 闫丰军 白蛾周氏啮小蜂繁育坏蛹病控制方法

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