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Developing long-term preservation method for hard-to-cryopreserve ectomycorrhizal basidiomycete cultures

Research Project

Project/Area Number 19K06088
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeMulti-year Fund
Section一般
Review Section Basic Section 39060:Conservation of biological resources-related
Research InstitutionTottori University

Principal Investigator

NAKAGIRI Akira  鳥取大学, 農学部, 教授 (70198050)

Co-Investigator(Kenkyū-buntansha) 遠藤 直樹  鳥取大学, 農学部, 助教 (20776439)
Project Period (FY) 2019-04-01 – 2022-03-31
Project Status Completed (Fiscal Year 2021)
Budget Amount *help
¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000)
Fiscal Year 2021: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2020: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2019: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Keywords菌株保存 / 凍結保存 / 菌根性担子菌 / 難保存性培養株 / 菌根形成 / 多孔性培養基剤 / 難保存性 / バーミキュライト / 菌根形成能
Outline of Research at the Start

真菌類の中でも菌株保存が特に困難である菌根性担子菌類の菌株の凍結保存法および凍結保存後の菌株の品質評価法を開発し、本菌群の長期保存法を確立することを目指す。
研究方法は、以下のとおりである。
① 凍結保存前の前培養法の改良:これまでに効果が認められている多孔性鉱物を培養基材とした手法を改良する。
② 凍結保存後の菌株の品質評価法の確立:宿主植物との共培養によって菌根形成能の保存性を評価する手法を確立する。
③ 汎用性の高い保存法の確立:多種多様な菌株を用い,①で改良した保存法の有効性を評価し、培地、凍結法、凍結保護剤、復元培養法などに改良を加え、菌株を長期間安定して保存できるプロトコルを開発する。

Outline of Final Research Achievements

This research aimed to develop and improve a useful cryopreservation method for cultures of mycorrhizal basidiomycetous fungi achieving high survival rates and maintaining hyphal growth rates and mycorrhiza forming ability in revived cultures. Our mycorrhiza synthesis method successfully generated artificial mycorrhizae on the root tips of Pinus densiflora seedlings and reproduced normal morphological characteristics identical to those of natural mycorrhizae, and the damage levels on fungal cultures caused by cryopreservation were assessed by the mycorrhiza formation rates on the root tips. Comparing and evaluating various preculturing methods for cryopreservation, we found "the Petri dish method" using vermiculites as culture substrate is more effective in achieving high survival rates and hyphal growth rates of revived cultures than the original "Tube method", whereas "the Agar disc method" is more efficient than "the Petri dish method" for maintaining mycorrhiza formation rates.

Academic Significance and Societal Importance of the Research Achievements

本研究の成果は、生態学的にも経済的にも重要な菌群である菌根性担子菌類の培養株を長期間安定的に保存する方法の開発・改良に貢献するものである。これまで本菌群は分離・培養が難しいだけでなく、その培養株の保存が困難なために、世界の菌株保存機関でもわずかな菌株が保存されているのみで、入手が困難なため活用されることが少なかったが、菌株の性状を維持できる凍結保存法を開発することによって、様々な研究分野において本菌群の多種多様な培養株を生物資源として活用することが可能になる。

Report

(4 results)
  • 2021 Annual Research Report   Final Research Report ( PDF )
  • 2020 Research-status Report
  • 2019 Research-status Report
  • Research Products

    (17 results)

All 2022 2021 2020 2019

All Journal Article (9 results) (of which Peer Reviewed: 8 results,  Open Access: 2 results) Presentation (8 results) (of which Int'l Joint Research: 3 results,  Invited: 1 results)

  • [Journal Article] Two new species of <i>Sistotrema</i> s.l. (<i>Cantharellales</i>) from Japan with descriptions of their ectomycorrhizae2022

    • Author(s)
      Sugawara, R., Shirasuka, N., Nagamune, K., Oguchi, K., Maekawa, N., Sotome, K., Nakagiri, A., Ushijima, S., Endo, N.
    • Journal Title

      Mycoscience

      Volume: 63 Issue: 3 Pages: 102-117

    • DOI

      10.47371/mycosci.2022.02.003

    • ISSN
      1340-3540, 1618-2545
    • Year and Date
      2022-05-31
    • Related Report
      2021 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Systematic revision of Hydnum species in Japan2022

    • Author(s)
      Sugawara, R., Maekawa, N., Sotome, K., Nakagiri, A., Endo, N.
    • Journal Title

      Mycologia

      Volume: 114 Issue: 2 Pages: 1-40

    • DOI

      10.1080/00275514.2021.2024407

    • Related Report
      2021 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Long-term cryopreservation of basidiomycete strains with Homolka's perlite protocol followed by storage in an ultra-low-temperature freezer and vapor-phase liquid nitrogen tank.2021

    • Author(s)
      Sato, M., Inaba, S., Okazaki, N., Nakagiri, A.
    • Journal Title

      Microb. Resour. Syst.

      Volume: 37 Pages: 13-20

    • NAID

      40022635722

    • Related Report
      2021 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] 特集 きのこの育種と遺伝資源 きのこ遺伝資源の保存2021

    • Author(s)
      中桐 昭
    • Journal Title

      アグリバイオ

      Volume: 5 Pages: 1022-1026

    • Related Report
      2021 Annual Research Report
  • [Journal Article] Mycorrhizal synthesis, morpho-anatomical characterization of mycorrhizae, and evaluation of mycorrhiza-forming ability of Hydnum albidum-like species using monokaryotic and dikaryotic cultures.2021

    • Author(s)
      Sugawara R, Sotome K, Maekawa N, Nakagiri A, Endo N.
    • Journal Title

      Mycorrhiza

      Volume: 31 Issue: 3 Pages: 349-359

    • DOI

      10.1007/s00572-021-01024-7

    • Related Report
      2020 Research-status Report
    • Peer Reviewed
  • [Journal Article] Vermiculite as a culture substrate greatly improves the viability of frozen cultures of ectomycorrhizal basidiomycetes.2020

    • Author(s)
      Sato, M., Inaba, S., Noguchi, M., Nakagiri, A.
    • Journal Title

      Fungal Biology

      Volume: 124 Issue: 8 Pages: 742-751

    • DOI

      10.1016/j.funbio.2020.05.002

    • Related Report
      2020 Research-status Report
    • Peer Reviewed
  • [Journal Article] Four mycelial strains of Entoloma clypeatum species complex form ectomycorrhiza-like roots with Pyrus betulifolia seedlings in vitro, and one develops fruiting bodies 2 months after inoculation2020

    • Author(s)
      Shishikura, M., Takemura, Y., Sotome, K., Maekawa, N., Nakagiri, A., Endo, N.
    • Journal Title

      Mycorrhiza

      Volume: 31 Issue: 1 Pages: 31-42

    • DOI

      10.1007/s00572-020-00994-4

    • Related Report
      2020 Research-status Report
    • Peer Reviewed
  • [Journal Article] First successful isolation of Entoloma clypeatum species complex from basidiospores2019

    • Author(s)
      Shishikura Manami、Sugawara Ryo、Takemura Yoshihiro、Sotome Kozue、Maekawa Nitaro、Nakagiri Akira、Endo Naoki
    • Journal Title

      Mycoscience

      Volume: - Issue: 4 Pages: 221-227

    • DOI

      10.1016/j.myc.2019.02.011

    • NAID

      40021955238

    • Related Report
      2019 Research-status Report
    • Peer Reviewed
  • [Journal Article] Establishment of monokaryotic and dikaryotic isolates of Hedgehog mushrooms (Hydnum repandum and related species) from basidiospores.2019

    • Author(s)
      Sugawara R, Yamada A, Kawai M, Sotome K, Maekawa N, Nakagiriag A, Endo N.
    • Journal Title

      Mycoscience

      Volume: 60 Issue: 3 Pages: 201-209

    • DOI

      10.1016/j.myc.2019.02.007

    • NAID

      40021933423

    • Related Report
      2019 Research-status Report
    • Peer Reviewed
  • [Presentation] Lactarius akahatsu菌株の凍結保存に有効なバーミキュライト法の開発.2021

    • Author(s)
      麻生 侑希・北岡 静・山本 樹・前川二太郎・中桐 昭・遠藤 直樹
    • Organizer
      日本微生物資源学会第27回大会
    • Related Report
      2021 Annual Research Report
  • [Presentation] 亜高山帯森より採取されたHydnum repando-orientale類似菌の分類学的精査2021

    • Author(s)
      菅原 遼・前川二太郎・中桐 昭・早乙女梢・遠藤直樹
    • Organizer
      日本菌学会第65回大会
    • Related Report
      2021 Annual Research Report
  • [Presentation] 日本産テングタケ属コタマゴテングタケ列種の分類に関する研究2021

    • Author(s)
      西村光世・糟谷大河・早乙女梢・中桐 昭・前川二太郎・遠藤直樹
    • Organizer
      日本菌学会第65回大会
    • Related Report
      2021 Annual Research Report
  • [Presentation] Collection and Preservation of Mushroom Cultures.2021

    • Author(s)
      Nakagiri, A.
    • Organizer
      The 4th International Workshop on Mushroom Biology and Technology
    • Related Report
      2021 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] 交配和合性に基づく日本産カノシタ属(Hydnum L.)種の再評価2020

    • Author(s)
      菅原遼,前川二太郎,中桐昭,早乙女梢,遠藤直樹
    • Organizer
      日本菌学会第64回大会(大阪)
    • Related Report
      2020 Research-status Report
  • [Presentation] Hydnum 属菌の外生菌根形態の多様性とその分類形質としての評価2020

    • Author(s)
      菅原遼,早乙女梢,中桐昭,前川二太郎,遠藤直樹
    • Organizer
      菌根研究会 2020 年度大会(JCOM2020,信州)
    • Related Report
      2020 Research-status Report
  • [Presentation] Success in artificial root colonizations and fruit body formations of Entoloma clypeatum with Pyrus betulaefolia2019

    • Author(s)
      Shishikura Manami、Sugawara Ryo、Takemura Yoshihiro、Sotome Kozue、Maekawa Nitaro、Nakagiri Akira、Endo Naoki
    • Organizer
      The 10th International Workshop on Edible Mycorrhizal Mushrooms
    • Related Report
      2019 Research-status Report
    • Int'l Joint Research
  • [Presentation] Ectomycorrhization of monokaryotic and dikaryotic strains of hedgehog mushrooms (Hydnum L.) with pine seedlings in vitro2019

    • Author(s)
      Sugawara Ryo、Yamada Akiyoshi、Kawai Masataka、Sotome Kozue、Maekawa Nitaro、Nakagiri Akira、Endo Naoki
    • Organizer
      The 10th International Workshop on Edible Mycorrhizal Mushrooms
    • Related Report
      2019 Research-status Report
    • Int'l Joint Research

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Published: 2019-04-18   Modified: 2023-01-30  

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