久米島産菌根性担子菌の分離と同定: 沖縄地域学リポジトリ

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Title

久米島産菌根性担子菌の分離と同定

Author(s)

田邊, 俊朗; 小川和香奈

Citation

沖縄工業高等専門学校紀要 = Bulletin of Okinawa National

_{College of Technology(9): 13-20}

Issue Date

2015-03

URL

http://hdl.handle.net/20.500.12001/18712

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ஂ⡿ᓥ⏘⳦᰿ᛶᢸᏊ⳦ࡢศ㞳࡜ྠᐃ

*⏣㑔ಇᮁ1_{㸪ᑠᕝ࿴㤶ዉ}2 1_{⏕≀㈨※ᕤᏛ⛉㸪}2_{ᑓᨷ⛉㸦⌧ ಙᕞ኱Ꮫ኱Ꮫ㝔 ⥲ྜᕤᏛ⣔◊✲⛉㸧} ([email protected]) せ⣙ Ἀ⦖┴ஂ⡿ᓥ࡛཰✭ࡋࡓ⳦᰿ᛶᢸᏊ⳦࡟ࡘ࠸࡚ࠊ஧ḟ⳦⣒ࡢศ㞳ࠊ࠾ࡼࡧ rRNA 㑇ఏᏊෆࡢ ITS 㡿 ᇦࡢሷᇶ㓄ิゎᯒࢆ⾜ࡗࡓࠋ஧ḟ⳦⣒ࡀ⏕⫱ࡋ⳦ᰴࡢ༢㞳ࡀ࡛ࡁࡿᇵᆅ࡟ࡘ࠸᳨࡚ウࡋࡓ࡜ࡇࢁࠊࣅ ࣮ࣝ㓝ẕࢆྵࡴᇵᆅ࡛⏕⫱࣭༢㞳࡛ࡁࡿࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡗࡓࠋࡲࡓࠊᚓࡽࢀࡓ ITS 㡿ᇦሷᇶ㓄ิࢹ ࣮ࢱ࠿ࡽ BLAST ࡟ࡼࡿ┦ྠᛶゎᯒࢆ⾜ࡗࡓ࡜ࡇࢁࠊ࢔ࣥࢬࢱࢣ Cantharellus cibarius ࡟ 99.3%ࡢ┦ྠ ᛶࢆ♧ࡋࠊᴟࡵ࡚㏆࠸㢮⦕✀࡛࠶ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ

࣮࣮࢟࣡ࢻ㸸 ITS 㡿ᇦࠊCantharellus cibarius ⥴ゝ Ἀ⦖┴ஂ⡿ᓥ࡟ࡣ࢟࢖ࣟࢼ࣮ࣂ࡜࿧⛠ࡉࢀࡿ㣗⏝࢟ࣀࢥࡀ⮬⏕ࡋ࡚࠾ࡾࠊᆅඖ࡛ࡣ⌋㔜ࡉࢀ࡚࠸ࡿࠋ ᫓࠿ࡽึኟ࡟࠿ࡅ࡚཰✭ࡉࢀ࡚࠸ࡿࡼ࠺࡛࠶ࡾࠊᆅඖ࡛ࡣኳ࡫ࡽ࡞࡝࡟ࡋ࡚㣗ࡉࢀ࡚࠸ࡿࠋஂ⡿ᓥฟ ㌟ࡢᮏ⛉Ꮫ⏕࣭௰ᮧῺᚿ㑻ࡣࠊಖㆤ⪅ࡢ▱ே࡟ࡇࡢ࢟࢖ࣟࢼ࣮ࣂࡢ◊✲ࢆ່ࡵࡽࢀࠊ࢟࢖ࣟࢼ࣮ࣂࡢ ⏕⫱࡟ᚲせ࡞᮲௳ࢆồࡵ࡚ஂ⡿ᓥࡢ᳜⏕࡞࡝ࢆㄪᰝࡋࠊ࢟࢖ࣟࢼ࣮ࣂ⮬⏕ᆅ࡜⪃࠼ࡽࢀࡿᒣᆅ㒊ࡣᴫ ࡡ࣐ࣜࣗ࢘࢟ࣗ࢘ࢶ᳜ᯘ࡛༨ࡵࡽࢀࡿࡇ࡜ࢆ▱ࡗࡓࠋࡉࡽ࡟ᆅඖ࡛ࡢ⪺ࡁྲྀࡾㄪᰝࢆ⥆ࡅࡓ࡜ࡇࢁࠊ ᖺࠎ཰✭᫬ᮇࡀ᪩ࡲࡗ࡚࠸ࡿࡼ࠺࡟ឤࡌࡿࠊ࡜ࡢゝ㉁ࢆᚓࡓࠋࡇࡢࡇ࡜࠿ࡽ࢟࢖ࣟࢼ࣮ࣂࡢ⮬⏕⎔ቃ ࡟⯆࿡ࢆぬ࠼ࡓ௰ᮧῺᚿ㑻࠾ࡼࡧලᚿሀ࿴ஓࠊᅧྜྷ୍┿ࠊ‪ᕝ┒ὒ㸦࠸ࡎࢀࡶ⏕≀㈨※ᕤᏛ⛉㸧ࡣࠊ ࣍ࢡࢺᰴᘧ఍♫ࢢ࣮ࣝࣉࡢ◊✲㈝ຓᡂ㸦㈈ᅋἲே࣍ࢡࢺ⏕≀⛉Ꮫ᣺⯆㈈ᅋᖹᡂ ᖺᗘ◊✲ዡບ㈹㸧ࢆ ཷࡅ࡚ࠊஂ⡿ᓥࡢ㐣ཤ ᖺศࡢẼ ኚືࢆㄪᰝࡋࡓࠋࡇࡢ⤖ᯝࠊ௰ᮧῺࡽࡣࠊ࢟࢖ࣟࢼ࣮ࣂ཰✭᫬ᮇ ࡣ㝆㞵ᚋࡢᛴ⃭࡞Ẽ పୗ࡜ࡢ㛵㐃ࡀ♧၀ࡉࢀࡿࡇ࡜ࠊࡍ࡞ࢃࡕ࢟࢖ࣟࢼ࣮ࣂᏊᐇయᙧᡂ࡟ࡣప ่ ⃭ࡀᚲせ࡞ࡢ࡛ࡣ࡞࠸࠿࡜⪃ᐹࡋ࡚࠸ࡿ>@_{ࠋࡲࡓ࢟࢖ࣟࢼ࣮ࣂ⮬యࡣࠊࡑࡢᏊᐇయᙧ≧࣭Ⰽ࣭≉᭷ࡢ} ᯝᐇ⮯࡞࡝࠿ࡽ࢔ࣥࢬࢱࢣ㢮⦕✀ࡀள⇕ᖏᛶẼೃ࡟㐺ᛂࡋࡓࡶࡢ࡜᥎ᐃࡉࢀࡿࠋ࢔ࣥࢬࢱࢣࡣ᪥ᮏ࡛ ࡣ࠶ࡲࡾ࡞ࡌࡳࡀ࡞࠸ࡶࡢࡢࠊḢ⡿࡛ࡣࡼࡃ▱ࡽࢀࡓඃⰋ࡞㣗⏝࢟ࣀࢥࡢ୍ࡘ࡛࠶ࡿࠋࡑࡢྡࡢ㏻ࡾ Ꮚᐇయࡣ࢔ࣥࢬⰍࢆ࿊ࡋࠊࡲࡓ࢔ࣥࢬᵝࡢ㤶ࡾࢆᣢࡘࠋ㣗࿡ࡀⰋ࠸ࡓࡵḢ⡿࡛ࡣேẼࡀ࠶ࡿࡀࠊᕷሙ ࡟ฟᅇࡿࡶࡢࡣኳ↛≀ࡤ࠿ࡾ࡛ၟᴗⓗ࡞ேᕤ᱂ᇵࡣ࡞ࡉࢀ࡚࠸࡞࠸>@_{ࠋࡇࢀࡣ࢔ࣥࢬࢱࢣࡀ࣐ࢶࢱࢣ} ࡸ࣍ࣥࢩ࣓ࢪ࡞࡝࡜ྠᵝ࡟⏕ࡁࡓᐟ୺࡜ࡋ࡚ᶞᮌࢆᚲせ࡜ࡍࡿ⳦᰿ᛶ࢟ࣀࢥ࡛࠶ࡿࡓࡵ࡟ࠊேᕤ᱂ᇵ ࡀᴟࡵ࡚㞴ࡋ࠸>@>@_{ࡇ࡜࡟ࡼࡿࡶࡢ࡛࠶ࡿࠋ࢔ࣥࢬࢱࢣࡢேᕤ᱂ᇵ࡟ࡘ࠸࡚ࡣ E. Danell ࡜ F.J. Canacho} ࡟ࡼࡿᏊᐇయᙧᡂࡢሗ࿌>@_{ࡀ࠶ࡿࡶࡢࡢࠊၟᴗ᥇⟬࣮࣋ࢫࡢேᕤ᱂ᇵࡣ⾜ࢃࢀ࡚࠸࡞࠸ࠋ} ⩻ࡗ࡚࢟࢖ࣟࢼ࣮ࣂࡶ࢔ࣥࢬࢱࢣྠᵝࠊ཰✭ࡍࡿࡤ࠿ࡾ࡛᱂ᇵ࡞࡝ࡣ᳨ウࡉࢀ࡚࠸࡞࠸ࠋ࡜ࡇࢁࡀ ㏆ᖺࠊஂ⡿ᓥࡣᒣᯘࡢ㛤ቧ࡜⥳ᆅ㐀ᡂࡀ㐍ࡳࠊᓥෆ࡛཰✭࡛ࡁࡿᏊᐇయ㔞ࡣࡲࡍࡲࡍῶᑡഴྥ࡛࠶ࡿࠋ ࡑࡢࡓࡵࠊ㑇ఏ㈨※ࢆಖᏲࡍࡿほⅬ࠿ࡽࡶ࢟࢖ࣟࢼ࣮ࣂࢆಖᏑ࣭ྠᐃࡍࡿᚲせᛶࡀㄆࡵࡽࢀࡿࠋࡑࡇ

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࡛ࠊᮏ◊✲࡛ࡣࠊࡲࡎ࢟࢖ࣟࢼ࣮ࣂࡀ⏕≀Ꮫⓗ࡟࡝ࡢࡼ࠺࡞⣔⤫ศ㢮ࡀ࡞ࡉࢀࡿࡢ࠿ࠊᮍྠᐃ࡛࠶ࡿ ⌧≧ࢆ㋃ࡲ࠼ࠊ࢟࢖ࣟࢼ࣮ࣂࡢᒓ✀ࡢྠᐃࢆ⾜࠺ࡇ࡜ࢆ┠ⓗ࡜ࡋࠊ'1$ ゎᯒࢆ⾜ࡗࡓࠋࡉࡽ࡟ࠊ࢟࢖ ࣟࢼ࣮ࣂࡣ༢㞳ࡸᇵ㣴᪉ἲࡀሗ࿌ࡉࢀ࡚࠸࡞࠸⌧≧ࢆ㋃ࡲ࠼ࠊᮏ◊✲࡛ࡣࠊ࢟࢖ࣟࢼ࣮ࣂࡢேᕤ᱂ᇵ ᢏ⾡ࢆ☜❧ࡍࡿࡓࡵࠊᏊᐇయ࠿ࡽ⳦⣒ࡢศ㞳᮲௳ࢆ᳨ウࡋࡓࠋ ᐇ㦂ᮦᩱ࡜᪉ἲ 1.1 Ꮚᐇయヨᩱ࠾ࡼࡧヨ⸆ ࢟࢖ࣟࢼ࣮ࣂᏊᐇయࡣࠊᮏ⛉Ꮫ⏕௰ᮧῺᚿ㑻ࡢಖㆤ⪅࡛࠶ࡿ௰ᮧῺ୍⏨Ặ࡜ࡑࡢ▱ே࡛࠶ࡿ௰ᮧ⚽ ኵẶࡼࡾࡈ౪୚࠸ࡓࡔ࠸ࡓࠋஂ⡿ᓥ࡛཰✭ࡋࡓ┤ᚋࡢᏊᐇయࢆ 4Υ࡟ಖࡗࡓࡲࡲ㍺㏦ࡋࠊ┤ࡕ࡟⳦⣒ ༢㞳᧯స࠾ࡼࡧᙧែほᐹ࡟౑⏝ࡋࡓࠋ୍㒊ࡣ-80Υࡲࡓࡣᾮయ❅⣲࡛෾⤖ಖᏑࡋࡓࠋ෾⤖ಖᏑヨᩱࡣࠊ ⷧ⭷ษ∦ࡢㄪ〇ࡸࢤࣀ࣒ DNA ᢳฟ࡟⏝࠸ࡓࠋ࢚ࣅ࢜ࢫ㘄ࡣ࢔ࢧࣄࣇ࣮ࢻ࢔ࣥࢻ࣊ࣝࢫࢣ࢔〇ࠊPDA ࡣ࿴ග⣧⸆〇ࠊ⢊ᮎࣁ࢖࣏ࢿࢵࢡࢫࡣࣁ࢖࣏ࢿࢵࢡࢫࢪࣕࣃࣥ〇ࠊ㓝ẕ࢚࢟ࢫࡣ Fluka BioChemika 〇ࠊ ᇵᆅ⏝ᐮኳࡣ࢖ࢼ࢜ࣉࢸ࢕࢝〇⤌⧊ᇵ㣴⏝ᐮኳࢆ⏝࠸ࡓࠋࡑࡢ௚ࡢヨ⸆ࡣ≉࡟᫂グࡋ࡞࠸㝈ࡾᕷ㈍≉ ⣭ࢆ⏝࠸ࡓࠋ 1.2 ᇵᆅ࡜ᇵ㣴 Ꮚᐇయ⤌⧊࠿ࡽࡢศ㞳ᇵ㣴࡟ࡣࠊ࢚ࣅ࢜ࢫᇵᆅ㸦2.0%ࢢࣝࢥ࣮ࢫࠊ0.5%࢚ࣅ࢜ࢫ㘄ࠊ2.0%ᐮኳ㸧ࠊ ࠾ࡼࡧኴ⏣὾⏣ᇵᆅ[6]_{㸦1.0%ࢢࣝࢥ࣮ࢫࠊ0.2%࢖࣮ࢫࢺ࢚࢟ࢫࠊ0.05%⢊ᮎࣁ࢖࣏ࢿࢵࢡࢫ}䒆_ࠊ1.5%ᐮ ኳ㸧ࠊ࣏ࢸࢺ࣭ࢹ࢟ࢫࢺ࣮ࣟࢫᐮኳᇵᆅ㸦0.4%࣏ࢸࢺ࢚࢟ࢫࢺࣛࢡࢺࠊ2.0%ࢹ࢟ࢫࢺ࣮ࣟࢫࠊ1.5%ᐮ ኳ㸧ࠊ↓ᰤ㣴ᐮኳᇵᆅ㸦1%ᐮኳ㸧ࡢ 4 ✀ࡢᖹᯈᇵᆅࢆ⏝࠸ࡓࠋ㝶కᚤ⏕≀ࡢ⏕⫱ᢚไࡢࡓࡵࠊྛᇵᆅ ࡟ࡣ 50 ȝJPO ࡢ࢔ࣥࣆࢩࣜࣥ࡜ 50 ȝJPO ࢫࢺࣞࣉࢺ࣐࢖ࢩࣥ࠾ࡼࡧ 500 ȝJPO ࡢάᛶⅣࢆຍ࠼ࡓࠋ

࡯ࡰࢥࣥࢱ࣑ࢿ࣮ࢩࣙࣥࡀ↓࠸࡜⪃࠼ࡽࢀࡿᏊᐇయ୰ᚰ㒊࠿ࡽ⤌⧊ࢆ࣓ࢫ࡛⣙ 5 mm × 5 mm × 2 mm ࡟ษࡾฟࡋࠊษ∦ࢆྛᇵᆅୖ࡟ᐦ╔ࡉࡏ㟼⨨ࡋࡓࠋࣃࣛࣇ࢕࣒࡛ࣝࢩ࣮ࣕࣞ࡜ࣇࢱࢆᅛᐃࡋ 20Υ ࡛ᇵ㣴ࡋࡓࠋ⏕㛗ࡋࡓ⳦⣒ࡣࠊᇵᆅࡈ࡜ 1 cm ゅ࡟ษࡾฟࡋࠊ᪂ࡋ࠸ᇵᆅ࡟㟼⨨ࡋ࡚⥅௦ࡋࡓࠋ 1.3 ⫗║࠾ࡼࡧ㢧ᚤ㙾࡟ࡼࡿᙧែほᐹ ࢚ࣅ࢜ࢫᇵᆅୖ࡟⏕⫱ࡋࡓ⳦⣒యࢆᇵᆅࡈ࡜ゎ๗⏝࣓ࢫ࡛ⷧ⭷≧࡟ษࡾฟࡋࠊࢫࣛ࢖ࢻࢢࣛࢫ࡜࢝ ࣂ࣮ࢢࣛࢫࡢ㛫࡟ᣳࡳ㎸ࢇ࡛ࠊ⳦⣒యᇵᆅษ∦ࢆṇ❧ගᏛ㢧ᚤ㙾࡟ࡼࡾほᐹࡋࡓࠋ≉࡟ᢸᏊ⳦㢮≉᭷ ࡢࢡࣛࣥࣉ⤖ྜࡢ᭷↓ࢆほᐹࡋࡓࠋ 1.4 ࢤࣀ࣒ DNA ࡢᢳฟ࠾ࡼࡧ DNA ሷᇶ㓄ิࡢゎᯒ

࢟࢖ࣟࢼ࣮ࣂࡢᏊᐇయ࠿ࡽ TaKaRa Gen ࡜ࡿࡃࢇ㓝ẕ⏝㸦ࢱ࢝ࣛࣂ࢖࢜㸧࡛ᢳฟࡋࡓࢤࣀ࣒ DNA ࢆ 㗪ᆺ࡜ࡋ࡚⏝࠸ࠊPCR ἲ࡟ࡼࡾ 5.8S ࢆྵࡴ ITS1 ࡜ ITS2 㡿ᇦࢆቑᖜࡋࡓࠋPCR ࡣࠊ0.5 units ࡢ TaKaRa LA TaqTM_{㸦ࢱ࢝ࣛࣂ࢖࢜㸧ࠊ0.2 mM ࡢྛ dNTPࠊ2 ȝO ࡢ 10×PCR GC BufferI ࠾ࡼࡧ 1 ȝ0 ࡢྛࣉࣛ࢖࣐}

࣮ࢆྵࡴ 20 ȝO ࡢ⁐ᾮ୰࡛⾜ࡗࡓࠋࢧ࣮࣐ࣝࢧ࢖ࢡ࣮ࣛࡣ PTC 1196 ᆺ㸦BIO-RAD㸧ࢆ౑⏝ࡋࠊ94Υ࡛ 1 ศ㛫⇕ኚᛶࡋࡓᚋࠊ94Υ30 ⛊㸦ኚᛶ㸧ࠊ45Υ30 ⛊㸦࢔ࢽ࣮ࣜࣥࢢ㸧ࠊ72Υ2 ศ㸦ఙ㛗㸧ࢆ 30 ᅇ ⧞ࡾ㏉ࡋࠊ᭱ᚋ࡟ 72Υ࡛ 10 ศ㛫ఙ㛗཯ᛂࢆ⾜ࡗࡓࠋPCR ⏘≀ࡣ TaKaRa SUPRECTM_{-PCR㸦ࢱ࢝ࣛࣂ}

࢖࢜㸧࡟ࡼࡾࠊṧ␃ࡋ࡚࠸ࡿࣉࣛ࢖࣐࣮࠾ࡼࡧ dNTPs ࢆ㝖ཤࠊࢲ࢖ࣞࢡࢺࢩ࣮ࢣࣥࢫࡢࢸࣥࣉ࣮ࣞࢺ

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2.5 ┦ྠᛶ᳨⣴࡜⣔⤫ゎᯒ

࢟࢖ࣟࢼ࣮ࣂࡢ DNA ሷᇶ㓄ิゎᯒࡢ⤖ᯝࠊ࢟࢖ࣟࢼ࣮ࣂࡢ ITS 㡿ᇦࡣ࠸ࡃࡘ࠿ࡢ Cantharellus ᒓࡢ ITS 㡿ᇦ࡜㧗࠸┦ྠᛶࢆ♧ࡋࡓࠋࡇࡢ୰࡛ࡶࠊ࢔ࣥࢬࢱࢣ Cantharellus cibarius ࡢ㓄ิࢹ࣮ࢱ࡜ࡣ 99.3% ࡢ┦ྠᛶࢆ♧ࡋࡓ㸦ᅗ 6㸧ࠋ ࡲࡓࠊClustalW ࡟ࡼࡿ⣔⤫ゎᯒࢆ⾜࠸ࠊసᡂࡋࡓ↓᰿⣔⤫ᶞࢆᅗ 7 ࡟♧ࡋࡓࠋ Query: 1 ttctctgacctgaggtaacccatagtaatgatgatgataatgataaattgctagttgaag 60 Sbjct: 1398 ttctctgacctgaggtaacccatagtaatgatgatgataatgataaattgctagttgaag 1339 Query: 61 tccaatcacgcatgcatcatgacggtcatcggacgccacgaacagagctgcccttgcgcc 120 Sbjct: 1338 tccaatcacgcatgcatcatgacggtcatcggacgccacgaacagagctgcccttgcgcc 1279 Query: 121 cccaatgcagccccaaagtccttcaacccagccttgttggctagactgaaaaccccaatc 180 Sbjct: 1278 cccaatgcagccccaaagtccttcaacccagccttgttggctagactgaaaaccccaatc 1219 Query: 181 aaagcacctatgacggtcaagggcttgccatagcgcagagcctgcgtccccaatgcatgc 240 Sbjct: 1218 aaagcacctatgacggtcaagggcttgccatagcgcagagcctgcgtccccaatgcatgc 1159

Query: 241 ttgaaagtccaatccacacaacgcttgattttaaggcaagccagagcaatatcaccccaa 300 Sbjct: 1158 ttgaaagtccaatccacacaacgcttgattttaaggcaagccagagcaatattaccccaa 1099 Query: 301 gcaagcccaatccccagctaaccaacggtcacactggatcagttctcattaccctcagcc 360 Sbjct: 1098 gcaagcccaatccccagctaaccaacggtcacactggatcagttctcattaccctcagcc 1039 Query: 361 agagatacccttgtcttcatcctccgccaccgctttggaatggactggaagggtgccgtt 420 Sbjct: 1038 agagatacccttgtcttcatcctccgccaccgctttggaatggactggaagggtgccgtt 979 Query: 421 tgcgttcaaagactcgatgattcgcttggtgttagattagaagtgccatgcaactcacac 480 Sbjct: 978 tgcgttcaaagactcgatgattcgcttggtgttagattagaagtgccatgcaactcacac 919 Query: 481 cagttatcgcagttcactgcgttcttcatcgatgcaagagcctagagatccattgttgag 540 Sbjct: 918 cagttatcgcagttcactgcgttcttcatcgatgcaagagcctagagatccattgttgag 859

Query: 541 agttatctaaaaacccattggaaaaactcagcttgatgttatagacctaatcagtcaaga 600 Sbjct: 858 agttatctaaaaacccattgg-aaaactcagcttgatgttatagacctaatcagtcaaga 800

Query: 601 acaggggcaccgcgtgtaggtctaggtccatgccgggcc-aaaacagatatgaccccctc 659 Sbjct: 799 acaggggcaccgcgtgtaggtctaggtccatgccgggccaaaaacagatatgaccccctc 740

Query: 660 gacttacatcaattcgtcgaggcccataacttcagacgaatggggcacaacggaa 714 Sbjct: 739 gacttacatcaattcgtcgaggcccataacttcagacg-attgggcacaacggaa 686

ᅗ 6㸬࢟࢖ࣟࢼ࣮ࣂ࡜࢔ࣥࢬࢱࢣ Cantharellus cibarius ࡢ ITS 㡿ᇦࡢሷᇶ㓄ิẚ㍑ Query ࡣ࢟࢖ࣟࢼ࣮ࣂࡢ㓄ิࢆ Sbjct ࡣ Cantharellus cibarius ࡢ㓄ิࢆ♧ࡋ࡚࠸ࡿࠋ

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ㅰ㎡ ᮏ◊✲ࢆጞࡵࡿ࡟࠶ࡓࡾࠊ㈗㔜࡞ヨᩱ㸦࢟࢖ࣟࢼ࣮ࣂᏊᐇయ㸧ࢆᛌࡃᥦ౪ࡋ࡚㡬࠸ࡓ௰ᮧῺ୍⏨Ặ ࡜௰ᮧ⚽ኵẶ࡟῝⏒࡞ࡿឤㅰࡢពࢆ⾲ࡋࡲࡍࠋ ࡲࡓᮏ◊✲ࡢ㐙⾜࡟࠶ࡓࡾࠊᛌࡃᏊᐇయࡢⷧ⭷ษ∦ࡢస〇ἲࢆࡈᩍ♧࠸ࡓࡔࡁࠊࡲࡓᐇ㝿࡟స〇ࡋ ࡚㡬࠸ࡓᒣᇛ⚽அඛ⏕㸦⌧ ⌰⌫኱Ꮫᩍᤵ㸧࡟῝ࡃឤㅰࡢពࢆ⾲ࡋࡲࡍࠋ ࡲࡓᮏ◊✲ࡢ㐙⾜࡟࠶ࡓࡾࠊ࢟࢖ࣟࢼ࣮ࣂࡢ U'1$ ሷᇶ㓄ิゎᯒ࡟㛵ࡋ࡚ᵝࠎ࡞ࡈᣦᑟࡈຓゝࢆ㈷ࡾ ࡲࡋࡓ☾ᮧᑦᏊඛ⏕࡟῝ࡃឤㅰࡢពࢆ⾲ࡋࡲࡍࠋ ࡲࡓᮏ◊✲࡟࠶ࡓࡾࠊ኱ኚࡲࡌࡵ࡟ᐇ㦂ࡑࡢ௚࡟࠾࠸࡚๰㐀◊✲ࢆᒚಟࡋࡓᮏ⛉㸲ᖺ⏕࡛࠶ࡗࡓ ௰ᮧῺᚿ㑻ྩࠊලᚿሀ࿴ஓྩࠊᅜྜྷ୍┿ྩࠊ‪ᕝ┒ὒྩ࡟ཌࡃᚚ♩⏦ࡋୖࡆࡲࡍࠋ ᮏ◊✲ࡢ୍㒊ࡣࠊ㈈ᅋἲே࣍ࢡࢺ⏕≀⛉Ꮫ᣺⯆㈈ᅋᖹᡂ ᖺᗘ◊✲ዡບ㈹࡟ࡼࡾ㐙⾜ࡍࡿࡇ࡜ࡀ࡛ ࡁࡲࡋࡓࠋཌࡃᚚ♩⏦ࡋୖࡆࡲࡍࠋ ᘬ⏝ᩥ⊩ [1] ௰ᮧῺ ᚿ㑻ࠊලᚿሀ ࿴ஓࠊᅜྜྷ ୍┿ࠊ‪ᕝ ┒ὒࠊᣦᑟᩍဨ㸸⏣㑔 ಇᮁࠊἈ⦖┴ஂ⡿ᓥ࡟⏘ ࡍࡿ࢟ࣀࢥ㸦࢟࢖ࣟࢼ࣮ࣂ㸧࡟ࡘ࠸࡚ࠊ᪥ᮏ㎰ⱁ໬Ꮫ఍ 2008 ᖺᗘ኱఍ㅮ₇せ᪨㞟ࠊࢪࣗࢽ࢔㎰ ⱁ໬Ꮫ఍ࠊp.12ࠊ(2008).

[2] Wang, Y. and Hall, I. R., Edible ectomycorrhizal mushrooms: challenges and achievements, Can. J. Bot.,

82, p.1063-1073 (2004).

[3] Danell, E., Alstroem, S. and Ternstroem, A., Pseudomonas fluorescens in association with fruit bodies of the ectomycorrhizal muchroom Cantharellus cibarius, Mycological Research, 97, p.1148-1152 (1993). [4] Danell E., Cantharellus cibarius: Mycorrhiza formation and Ecology, Comprehensive Summaries of

Uppsala Dissertations from the Faculty of Science and Technology, 35, p.1-75 (1994).

XSGDWHGLQKWWSZZZ-P\NRSDWVOXVH1HZZHEVLWHP\FRUUKL]DNDQWDUHOOILOHUWH[WHUUWIKWP [5] Danell, E. and Canacho, F. J., Successful cultivation of the golden chanterelle, Nature, 385, p.303 (1997). [6] Ohta A., A new medium for mycelial growth of mycorrhizal fungi, Trans. Myc. Soc. Japan, 33,

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[7] Gardes M., White T.J., Fortin J.A., Bruns T.D., Taylor J.W., Identification of indigenous and introduced symbiotic fungi in ectomycorrhizae by amplification of nuclear and mitochondrial ribosomal DNA. Can. J.

Bot., 69, p.180- 190 (1991) .

[8] Altschul S.F., Madden T.L., Schaffer A.A., Zhang J., Zhang Z., Miller W., and Lipman D.J., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, p.3389-3402. (1997)

[9] Thomopson J.D., Higgins D.G., and Gibson T.J., CLUSTALW. Nucleic Acids Res. 22, p.4673-4680 (1994).

[10] Saitou N. and Nei M., The neighbor-joining method: a new method for reconstructing phylogenetic trees.

Mol. Biol. Evol., 4(4), p.406-425(1987).

[11] Sugita T, Nishikawa A, Ikeda R, Shinoda T: Identification of medically relevant Trichosporon species based on sequences of internal transcribed spacer regions and construction of a database for Trichosporon identification. J. Clin. Microbiol., 37, p.1985-1993(1999)

[12] Soukupova,L., Hrselova,H., Gryndlerova,H., Merhautova,V. and Gryndler,M: Alkali-extractable soil organic matter: An important factor affecting the mycelial growth of ectomycorrhizal fungi. Agric.,

Ecosyst. Environ., Appl. Soil Ecol. 40 (1), p.37-43 (2008)

Isolation and Identification of

a Mycorrhizal Mushroom from Kumejima island

*Toshiaki TANABE1_{㸪Wakana OGAWA}2

1_{Dapartment of Bioresources and Engineering,}

2_{Creative Systems Engineering Advanced Course㸦Present Affiliation: Interdisciplinary Graduate School of}

Science and Technology, Shinshu University㸧 ([email protected])

Abstract

A mycorrhizal mushroom was collected from Kiumejima island in Okinawa prefecture. Isolation of its secondary hyphae was carried out. An optimum solid medium for growing of this fungus was investigated. The secondary hyphae grew only on the medium containing brewer's yeast and was isolated. The DNA sequence of the ITS region in the rRNA gene of this fungus was analyzed. Homology search of the obtained DNA sequence with BLAST was carried out. The DNA sequence of the ITS region of this fungus was shown high homology, 99.3%, with that of Cantharellus cibarius. It was suggested that this fungus could be a species extremely close to C. cibarius.