㔝
㔝⏕ࡁࡢࡇࡽศ㞳ࡋࡓ⳦㢮ࡼࡿ᳜≀ᐖ ࡢᢚไ㛵ࡍࡿ◊✲
㸦Studies on the inhibition of plant diseases by fungal strains isolated from wild mushrooms㸧
NGUYEN THI QUYET
2017
Doctor’s THESIS
Studies on the inhibition of plant diseases by fungal strains isolated from wild mushrooms
By
NGUYEN THI QUYET
The United Graduate School of Agricultural Sciences Tottori University (Laboratory of Plant Pathology, Faculty of Life and Environmental
Science, Shimane University)
March 2017
༤ኈㄽᩥ
㔝 㔝⏕ࡁࡢࡇࡽศ㞳ࡋࡓ⳦㢮ࡼࡿ᳜≀ᐖ ࡢᢚไ㛵ࡍࡿ◊✲
ࢢ࢙ࣥ ࢸ ࢙࢘ࢵࢺ
㫽ྲྀᏛᏛ㝔 㐃ྜ㎰Ꮫ◊✲⛉
㸦ᓥ᰿Ꮫ ⏕≀㈨※⛉Ꮫ㒊 ᳜≀⌮Ꮫ◊✲ᐊ㸧
2017 ᖺ 3 ᭶
1
┠┠ḟ
┠ḟ 1
ㅰ㎡ 4
➨1❶ ⥴ゝ 5
➨2❶ 㔝⏕ࡁࡢࡇࡽࡢ⳦㢮ࡢศ㞳ࢫࢡ࣮ࣜࢽࣥࢢ 7
➨1⠇ ᪥ᮏᅜෆ࡛᥇ྲྀࡋࡓ㔝⏕ࡁࡢࡇࡽࡢ⳦㢮ࡢศ㞳 7
1㸬ᮦᩱཬࡧ᪉ἲ 2㸬⤖ᯝ ➨2⠇ ศ㞳⳦ࡢᇵ㣴ࢁᾮࡀࢿ࠸ࡶࡕ⳦ࡢឤᰁ⾜ືཬࡰࡍᙳ㡪 11
1㸬ᮦᩱཬࡧ᪉ἲ 2㸬⤖ᯝ ➨3⠇ ࢫࢡ࣮ࣜࢽࣥࢢࡉࢀࡓศ㞳⳦ࡢྠᐃ 17
1㸬ᮦᩱཬࡧ᪉ἲ 2㸬⤖ᯝ ➨4⠇ ⪃ᐹ 19
➨3❶ O821⳦ᰴࡢ᳜≀ཎ⳦ᑐࡍࡿᢚไຠᯝࡘ࠸࡚ 21
➨1⠇ O821⳦ᰴࡢ≉ᚩཬࡧྠᐃ 21
1㸬ᮦᩱཬࡧ᪉ἲ 2㸬⤖ᯝ ➨2⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮࡼࡿࢿ࠸ࡶࡕ⳦ࡢᢚไຠᯝ㸦⇕ฎ⌮㸧 24
1㸬ᮦᩱཬࡧ᪉ἲ 2㸬⤖ᯝ ➨3⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮࡼࡿࢿ࠸ࡶࡕ⳦ࡢᢚไຠᯝ㸦ᇵ㣴᮲௳㸧 26
1㸬ᮦᩱཬࡧ᪉ἲ 2㸬⤖ᯝ ➨4⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮࡼࡿࢿ࠸ࡶࡕ⳦ࡢᢚไຠᯝ㸦⳦⣒⏕⫱㸧 28
1㸬ᮦᩱཬࡧ᪉ἲ 2㸬⤖ᯝ ➨5⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮࡼࡿ᳜≀ཎ⳦ࡢᢚไຠᯝ 31
1㸬ᮦᩱཬࡧ᪉ἲ 2㸬⤖ᯝ ➨6⠇ ⪃ᐹ 34
➨4❶ O821⳦ᰴࡢᇵ㣴ࢁᾮࡢ᳜≀యୖ࡛ࡢᩬᙧᡂࡢᢚไຠᯝ 36
➨1⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮࢿ࠸ࡶࡕ⳦ࡢΰྜ᥋✀ࡼࡿⓎᢚไຠᯝ 36 1㸬ᮦᩱཬࡧ᪉ἲ
2㸬⤖ᯝ
2
➨2⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮࡢࢿࡢ๓ฎ⌮ࡼࡿࢿ࠸ࡶࡕࡢⓎᢚไຠᯝ 39 1㸬ᮦᩱཬࡧ᪉ἲ
2㸬⤖ᯝ
➨3⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮࡼࡿ࢟ࣗ࢘ࣜᐖࡢⓎᢚไຠᯝ 41 1㸬ᮦᩱཬࡧ᪉ἲ
2㸬⤖ᯝ
➨4⠇ ⪃ᐹ 46
➨5❶ O821⳦ᰴࡢᇵ㣴ࢁᾮࡢࢿࡢᢠᛶㄏᑟ 47
➨1⠇ ࢿࡢᇵ㣴ࢁᾮฎ⌮ࡼࡿ㐣㓟Ỉ⣲ࡢ⏕ᡂ 47 1㸬ᮦᩱཬࡧ᪉ἲ
2㸬⤖ᯝ
➨2⠇ ࢿࡢᇵ㣴ࢁᾮฎ⌮ࡼࡿPBZ1㑇ఏᏊࡢⓎ⌧ 50 1㸬ᮦᩱཬࡧ᪉ἲ
2㸬⤖ᯝ
➨3⠇ ࢿࡢᇵ㣴ࢁᾮฎ⌮ࡼࡿࢧࣜࢳࣝ㓟ࡢ✚ 53 1㸬ᮦᩱཬࡧ᪉ἲ
2㸬⤖ᯝ
➨4⠇ ࢿࡢ࢝ࢱ࣮ࣛࢮࡢฎ⌮ࡀᇵ㣴ࢁᾮࡢάᛶ࠼ࡿᙳ㡪 55 1㸬ᮦᩱཬࡧ᪉ἲ
2㸬⤖ᯝ
➨5⠇ ⪃ᐹ 57
➨6❶ O821⳦ᰴࡢᇵ㣴ࢁᾮ⏕ᡂࡉࢀࡿᢚไ≀㉁ࡢศ㞳ཬࡧྠᐃ 59
➨1⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮ⏕ᡂࡉࢀࡿᢚไ≀㉁ࡢศ㞳 59 1㸬ᮦᩱཬࡧ᪉ἲ
2㸬⤖ᯝ
➨2⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮ⏕ᡂࡉࢀࡿᢚไ≀㉁ࡢྠᐃ 63 1㸬ᮦᩱཬࡧ᪉ἲ
2㸬⤖ᯝ
➨ 3 ⠇ O821 ⳦ᰴࡢᇵ㣴ࢁᾮ⏕ᡂࡉࢀࡿᢚไ≀㉁ࡼࡿࢿ࠸ࡶࡕ⳦ࡢឤᰁ⾜ືࡢ ᢚไຠᯝ
1㸬ᮦᩱཬࡧ᪉ἲ
2㸬⤖ᯝ
➨ 4 ⠇ O821 ⳦ᰴࡢᇵ㣴ࢁᾮࡽศ㞳ࡉࢀࡓᢚไ≀㉁ࡼࡿࢿ࡛ࡢࢿ࠸ࡶࡕࡢⓎ
ᢚไຠᯝ 1㸬ᮦᩱཬࡧ᪉ἲ
2㸬⤖ᯝ
65
67
3
➨ 5 ⠇ O821 ⳦ᰴࡢᇵ㣴ࢁᾮࡽศ㞳ࡉࢀࡓᢚไ≀㉁ࡢࢿࡢ๓ฎ⌮ࡼࡿࢿ࠸ࡶ
ࡕࡢⓎᢚไຠᯝ 1㸬ᮦᩱཬࡧ᪉ἲ
2㸬⤖ᯝ
➨6⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮࡽศ㞳ࡉࢀࡓᢚไ≀㉁ࡼࡿࢿ࡛ࡢ㐣㓟Ỉ⣲ࡢ⏕ᡂ
1㸬ᮦᩱཬࡧ᪉ἲ 2㸬⤖ᯝ
➨7⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮࡽศ㞳ࡉࢀࡓᢚไ≀㉁ࡼࡿࢿ࡛ࡢPBZ1㑇ఏᏊࡢⓎ⌧
1㸬ᮦᩱཬࡧ᪉ἲ 2㸬⤖ᯝ
➨8⠇ ⪃ᐹ 75
➨7❶ H921⳦ᰴࡢᇵ㣴ࢁᾮࡀࢿ࠸ࡶࡕ⳦ཬࡰࡍᙳ㡪 77
➨1⠇ H921⳦ᰴࡢᇵ㣴ࢁᾮࡀࢿ࠸ࡶࡕ⳦ࡢឤᰁ⾜ື࠼ࡿᙳ㡪 77
1㸬ᮦᩱཬࡧ᪉ἲ 2㸬⤖ᯝ ➨2⠇ H921⳦ᰴࡢᇵ㣴ࢁᾮࡼࡿࢿ࠸ࡶࡕ⳦ࡢᢚไຠᯝ 79
1㸬ᮦᩱཬࡧ᪉ἲ 2㸬⤖ᯝ ➨3⠇ H921⳦ᰴࡢྠᐃ 81
1㸬ᮦᩱཬࡧ᪉ἲ 2㸬⤖ᯝ ➨4⠇ H921⳦ᰴࡢᇵ㣴ࢁᾮࡀࢿ࡛ࡢࢿ࠸ࡶࡕࡢⓎ࠼ࡿᙳ㡪 84
1㸬ᮦᩱཬࡧ᪉ἲ 2㸬⤖ᯝ ➨5⠇ H921⳦ᰴࡢᇵ㣴ࢁᾮࡽࡢࢿ࠸ࡶࡕ⳦ࡢᢚไ≀㉁ࡢศ㞳 89
1㸬ᮦᩱཬࡧ᪉ἲ 2㸬⤖ᯝ ➨6⠇ ⪃ᐹ 91
➨8❶ ⥲ྜ⪃ᐹ 93
せ 97
Summary 99
ᘬ⏝ᩥ⊩ 107
ㄽᩥ┠㘓 110
Ꮫ⾡ㄽᩥ 110 101 69
71
73
4 ㅰㅰ㎡
ᮏ◊✲ࢆ㐍ࡵࡿ࠶ࡓࡾࠊ⤊ጞᑀ࡞ᚚᣦᑟᚚຓゝࢆ㡬ࡁࠊཪࠊᮏㄽᩥࡢᰯ㜀ࡢປࢆ࠾
ྲྀࡾ㡬࠸ࡓᓥ᰿Ꮫ⏕≀㈨※⛉Ꮫ㒊ᩍᤵ ୖ㔝 ㄔ༤ኈ῝ࡃឤㅰࡢពࢆ⾲ࡍࡿࠋࡲࡓࠊከࡃ ࡢᚚ༠ຊ᭷┈࡞ᚚຓゝࢆ㡬࠸ࡓᓥ᰿Ꮫ⌮(Ꮫ㛗) Ⲩ℩ ᴿ༤ኈࠊᓥ᰿Ꮫ⏕≀㈨※⛉
Ꮫ㒊ᩍᤵ ⸨ே༤ኈࠊ㫽ྲྀᏛᏛ㝔㐃ྜ㎰Ꮫ◊✲⛉ᩍᤵ ඣ⋢ᇶ୍ᮁ༤ኈࠊᓥ᰿Ꮫ⏕
≀㈨※⛉Ꮫ㒊ᩍᤵ ᮌཎ῟୍༤ኈ࡞ࡽࡧᓥ᰿Ꮫ⏕≀㈨※⛉Ꮫ㒊ຓᩍ ᯘ ᫀᖹ༤ኈᚰ
ࡼࡾᚚ♩⏦ࡋୖࡆࡿࠋO821⳦ᰴࡢᇵ㣴ࢁᾮ୰⏕⏘ࡉࢀࡿᢚไ≀㉁ࡢᵓ㐀ゎᯒ࠶ࡓࡾᚚ ᣦᑟᚚຓࢆ࠸ࡓࡔ࠸ࡓᓥ᰿Ꮫ⏕≀㈨※⛉Ꮫ㒊ຓᩍ ྜྷΎᜨ༤ኈ࡞ࡽࡧྡྂᒇ
ᏛᏛ㝔 ᆏཱྀె༤ኈᚰࡽឤㅰࡢពࢆ⾲ࡍࡿࠋࡲࡓࠊᮏ◊✲ࡢ㐙⾜࠶ࡓࡾࠊከ࡞
ᚚ༠ຊᚚຓゝࢆ㡬ࡁ ࡃぢᏲࡗ࡚ࡃࡔࡉࡗࡓᓥ᰿Ꮫ᳜≀⌮Ꮫ◊✲ᐊୖ⏣ຍዉࠊ⏣
ᮧ᭸ᏊࡢㅖẶཌࡃᚚ♩⏦ࡋୖࡆࡿࠋ
5
➨
➨1❶ ⥴ゝ
ࢿ࠸ࡶࡕࡣࠊ⣠ᯤཬࡧⓑⴥᯤඹࠊࢿࡢ᭱ࡶ㔜せ࡞ᐖࡢ 1 ࡘ࡛࠶ࡿ(Ou
1985)ࠋᮏࡣ⣒≧⳦ࡢᏊᄞ⳦㢮ᒓࡍࡿ Magnaporthe oryzae (Hebert) Barr (ୡ௦ࠊ
Pyricularia oryzae Cavara)ࡼࡗ࡚ᘬࡁ㉳ࡇࡉࢀࠊࢿࡢ㔞ࡸၟရ౯್ከ࡞ᙳ㡪ࢆ࠼
ࡿ(㒊1964㸹ྥ␊ࡽ2009)ࠋᮏ⳦ࡣࢿࡔࡅ࡛ࡣ࡞ࡃࠊࢥ࣒ࢠࠊ࣒࢜࢜ࢠཬࡧ࣡ࡶᐖ
ࢆᘬࡁ㉳ࡇࡍࡇࡀ▱ࡽࢀ࡚࠸ࡿ(Couch et al. 2005)ࠋ
ᮏࡢ㜵㝖ࡣࠊᢠᛶရ✀ࡢ⏝ࡸᏛྜᡂ㎰⸆ࡢ⏝ࡀ୍⯡ⓗ࡛࠶ࡿࡀࠊ㧗ᗘᢠᛶ ရ✀ࡢ⨯(Ohtaka et al. 2008)ࡸ㐣ᗘ࡞㎰⸆⏝ࡼࡿ⸆⪏ᛶ⳦ࡢฟ⌧ (ఀ⸨࣭ᒣཱྀ
1977)ࡀၥ㢟࡞ࡗ࡚࠸ࡿࠋᐇࠊ࠸ࡶࡕᢠᛶࢿရ✀ࡢ⨯ࡸࢱࣥࣃࢡྜᡂ㜼ᐖ
࡛࠶ࡿ࢝ࢫ࣐࢞ࢩࣥࡸ࣓ࣛࢽࣥྜᡂ㜼ᐖ࡛࠶ࡿ࢝ࣝࣉࣟࣃ࣑ࢻࠊࢪࢡࣟࢩ࣓ࢵࢺཬࡧ
ࣇ࢙ࣀ࢟ࢧࢽࣝᑐࡍࡿ⸆⪏ᛶ⳦ࡢฟ⌧ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡲࡓࠊ᭱㏆࡛ࡣࢫࢺࣟࣅࣝࣜ
ࣥ⣔⸆(QoI)ᑐࡍࡿ⸆⪏ᛶ⳦ࡢฟ⌧ࡀၥ㢟࡞ࡗ࡚࠸ࡿࠋࡑࡢࡓࡵࠊᑗ᮶ⓗࡣ
Ꮫྜᡂ㎰⸆ࡢ⏝㔞ࢆ㍍ῶࡋࡓ⎔ቃಖᆺࡢ㜵㝖ᢏ⾡㛤Ⓨࡀᚲせ࡞ࡗ࡚ࡁ࡚࠸ࡿࠋ
㏆ᖺࠊ᪥ᮏࡢ㎰ᴗ࠾ࡅࡿᐖ࣭㞧ⲡ㜵㝖ࡣᏛྜᡂ㎰⸆ࡢࡳ౫Ꮡࡋ࡞࠸⥲ྜⓗ࡞
ᐖ㜵㝖(⪔✀ⓗ㜵㝖ἲࠊ⏕≀ⓗ㜵㝖ἲࠊ≀⌮ⓗ㜵㝖ἲཬࡧᏛⓗ㜵㝖ἲࢆ⤌ࡳྜࢃࡏࡓ㜵 㝖ἲ)ࡀ᥎ዡࡉࢀ࡚࠸ࡿࠋ
ᚤ⏕≀ཬࡧᚤ⏕≀ࡀ⏕⏘ࡍࡿ 2 ḟ௦ㅰ⏘≀ࢆ⏝ࡋࡓ㜵㝖ἲࡣ⥲ྜⓗᐖ࣭㞧ⲡ㜵㝖
࠾ࡅࡿ㔜せ࡞᪉ἲࡢ1ࡘ࡛࠶ࡿࠋBacillus subtilisࡣࢺ࣐ࢺ㟷ᯤࠊ᰿⭉ⴎ (Asaka and Shoda 1996㸹Montealegre et al. 2003)ࠊ❧ᯤ(Phae et al. 1992)ཬࡧ⅊Ⰽࡧ(Sadfi-Zouaoui et
al. 2008)ࡸ࢝ࢺ࢘࢞ࣛࢩ (Lee et al. 2008)ࠊ㠀ཎᛶFusarium⳦ࡣࢧࢶ࣐ࣔࡘࡿ
ࠊ࣍࢘ࣞࣥࢯ࢘ⴎ (ᑠᕝ࣭㥖⏣ 1984㸹㒊࣭㉥ᆏ1997)ࠊPyricularia spp.ࡣࢯ࣐࣓ࣛ
㉥ⰍᩬⅬ(Arase et al. 1990)ࢆ㜵㝖࡛ࡁࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡲࡓࠊStreptomycesᒓ⳦ࡣ ᢠ⳦≀㉁ࢆ⏕⏘ࡍࡿࡇࡀ▱ࡽࢀ࡚࠾ࡾࠊࢧࢺ࢘ࢲࢥࣥ❧ᯤࠊࢿ⣠ᯤࠊࢳࢦ⅊Ⰽ
ࡧཬࡧࣈࣛࢼ⳦᰾ (Errakhi et al. 2007㸹Wan et al. 2008)ࠊࢿ᰿⭉ࢀࠊࢿ⣠ᯤࢀ
(Boukaew and Prasertsan 2014)ࠊⴎ (Chaiharn et al. 2009㸹Liu et al. 2009)ࠊ࢝ࣛࣔࣥࢪࣥ
࠾ࡅࡿ㟷ࡧ (Li et al. 2010)ࠊ࣒ࣖࣔⅣࡑࢆᢚไࡍࡿ(Palaniyandi et al. 2011)ࠋ
Trichoderma virens ࡀ⏕⏘ࡍࡿᢠ⳦௦ㅰ⏘≀ࡼࡗ࡚࢝ࣛࣥᩬⅬࡢ㜵㝖ࡀሗ࿌ࡉࢀࡓ
(Intana et al. 2005)ࠋᐇ㝿ࠊB. subtilisࠊ 㠀ཎᛶFusariumᒓ⳦ࠊ Trichoderma lignorum࡞
ࡢᚤ⏕≀ࡣࡑࢀࡒࢀ࣎ࢺ࣮࢟ࣛỈ(ฟග⯆⏘(ᰴ))ࠊ࣐ࣝ࢝ࣛࢺ(࢚࣮ࢨ⏕⛉◊(ᰴ))ࠊ ࢺࣜࢥࢹ࣐ࣝ⏕⳦(ᒣ㝧⸆ရ)ࡋ࡚㎰⸆Ⓩ㘓ࡉࢀࡓ(㥖⏣ 2003㸹ᑠᕝ 1998)ࠋ
ࢿ࠸ࡶࡕࡢ⏕≀ⓗ㜵㝖㛵ࡍࡿ◊✲ࡘ࠸࡚ࡶࠊ㠀ཎᛶBipolaris sorokinianaࠊ㠀
ཎᛶPyricularia oryzaeࠊB. subtilis IK-1080ࠊStreptomyces sp. PM5ࢆ⏝࠸ࡓ㜵㝖㛵ࡍࡿሗ࿌
ࡀ࠶ࡿ(Manandhar et al. 1998㸹⸨⏣ࡽ1990㸹⏣ཱྀࡽ2003㸹Prabavathy et al. 2006)ࠋ᭱㏆࡛ࡣࠊ
ࢿⴥୖ⏕ᜥࡋ࡚࠸ࡿⴥ㠃⳦ࢆ⏝ࡋࡓࢿ࠸ࡶࡕ⳦ࡢ㜵㝖㛵ࡍࡿ◊✲(Kawamata
et al. 2004㸹Ohtaka et al. 2008)ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡲࡓࠊࢿⴥୖ࡛Ⓨⱆࡋࡓࢿ࠸ࡶࡕ⳦
6
⬊Ꮚࡢ╔ࢆᢚไࡍࡿࡇ࡛ࢿ࠸ࡶࡕࡢⓎࢆᢚไࡍࡿ⳦ࡸ᳜≀⏕⫱ಁ㐍⣽⳦ཬࡧศ 㞳ࡋࡓ⳦ࡢᇵ㣴ࢁᾮࡼࡿࢿ࠸ࡶࡕࡢᢚไຠᯝࡘ࠸࡚ࡢሗ࿌ࡶ࠶ࡿ(Shimoi et al.
2010㸹Lucas et al. 2009㸹Naureen et al. 2009㸹Yu et al. 2013)ࠋ
୍᪉ࠊࡁࡢࡇ㢮ࡣ᳜≀ཎ⳦ࢆᢚไࡍࡿ⳦ࡀᏑᅾࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿࠋ࠼ࡤࠊࢥ
࣒ࢠࡢⴥࡽศ㞳ࡉࢀࡓIrpex lacteusࡣ2✀ࡢⓎᛶᢠ⳦≀㉁࡛࠶ࡿ5-pentyl-2-furaldenhyde
5-(4-pentenyl)-2-furaldehydeࢆ⏕⏘ࡋࠊF. oxysporum f. sp. lycopersis ཬࡧOidium sp.ࡢ⏕⫱
ࢆᢚไࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ(Koitabashi et al. 2004㸹Koitabashi 2005)ࠋࡲࡓࠊⓎᛶᢠ⳦
≀㉁5-pentyl-2-furaldehydeࢆ⏕⏘ࡍࡿOxyporus latemarginatus EF069ࡣAlternaria alternataࠊ Botrytis cinereaࠊColletotrichum gloeosporioidesࠊF. oxsporum f. sp. lycopersiciཬࡧRhizoctonia
solani ࡢ⳦⣒⏕⫱ࢆ㜼ᐖࡋࠊB. cinerea ࡼࡗ࡚ᘬࡁ㉳ࡇࡉࢀࡿ✭ᚋࡢᯝᐇࡢ⭉ᩋࡸ R.
solani ࡼࡗ࡚ᘬࡁ㉳ࡇࡉࢀࡿ⬌⼖⹒ࡢࣜࢰࢡࢺࢽ᰿⭉ࡢ㐍ᒎࢆຠᯝⓗῶᑡࡉࡏࡿ
ࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ(Lee et al. 2009)ࠋࡉࡽࠊLentinula edodesࡢᇵ㣴ࢁᾮCryptococcus laurentiiࢆࣜࣥࢦฎ⌮ࡍࡿࡼࡗ࡚Penicillium expansumࡀ㜵㝖ࡉࢀ(Tolaini et. al. 2010)ࠊ Sterium ostreaࡢᇵ㣴ࢁᾮࡣC. gloeosporioides ࠊCochliobolus miyabeanusཬࡧB. cinerea ᑐ ࡍࡿᢚไຠᯝࢆ♧ࡍࡇࡀሗ࿌ࡉࢀࡓ(Imtiaj and Lee 2007㸹Imtiaj et al. 2007)ࠋୡ⏺ࡢࠕࡁࡢ ࡇࠖࡣ14✀⛬ᗘゝࢃࢀ࡚࠸ࡿࡀࠊྠᐃࡉࢀ࡚࠸ࡿࡢࡣ10%⛬ᗘ࡛࠶ࡾࠊᮍࡔᮍྠᐃ ࡢ≀ࡀከ࠸(Wasser 2002)ࠋࡇࢀࡽࡢࠕࡁࡢࡇࠖࡣࠕᐤ⏕ཪࡣඹ⏕ࡍࡿ⳦㢮ࠖࡀᏑᅾࡋࠊ
ᐖ㜵㝖⏝ྍ⬟࡞ᮍ▱ࡢ᭷⏝࡞⳦ࡶᏑᅾࡋ࡚࠸ࡿ⪃࠼ࡽࢀࡿࠋ
ࡑࡇ࡛ࠊᮏ◊✲࡛ࡣ᪥ᮏࡢࡉࡲࡊࡲ࡞ሙᡤ⏕ᜥࡍࡿࠕ㔝⏕ࡁࡢࡇࠖࢆ᥇ྲྀࡋࠊࠕࡁࡢࡇ
ᐤ⏕ཪࡣඹ⏕ࡍࡿ⳦㢮ࠖࢆศ㞳ࡋ࡚ࠊࢿ࠸ࡶࡕ⳦➼ࡢ᳜≀ཎ⳦ࡢ㜵㝖⏝ྍ⬟࡞
⳦㢮ࢆ᥈⣴ࡍࡿ┠ⓗ࡛◊✲ࢆ⾜ࡗࡓࠋᚓࡽࢀࡓᡂᯝࡣᏛㄅ࡞ᢞ✏(Nguyen et al. 2015㸹 Nguyen et al. 2016 a㸹Nguyen et al. 2016 b㸹Nguyen et al. 2017)ࡍࡿඹࠊ᪥ᮏ᳜≀⌮Ꮫ
(Nguyen ࡽ 2011㸹2012 a㸹2012 b㸹2012 c㸹2013㸹ୖ㔝ࡽ2014㸹Nguyen ࡽ2014㸹2015㸹2016)
ཱྀ࡛㢌ཬࡧ࣏ࢫࢱ࣮Ⓨ⾲ࡋࡓࠋᮏㄽᩥࡣࡇࢀࡽᮍⓎ⾲ࡢᡂᯝࡶຍ࠼࡚ࡲࡵࡓࡶࡢ࡛࠶
ࡿࠋ
7
➨
➨2❶ 㔝⏕ࡁࡢࡇࡽࡢ⳦㢮ࡢศ㞳ࢫࢡ࣮ࣜࢽࣥࢢ
᪥ᮏࡣከࡃࡢࡁࡢࡇࡀ⏕ᜥࡋ࡚࠸ࡿࡀࠊᮍྠᐃࡢࡶࡢࡀከࡃᏑᅾࡍࡿࡇࡀ▱ࡽࢀ࡚
࠸ࡿࠋࡇࢀࡲ࡛ࡢࠕࡁࡢࡇࠖࢆ⏝࠸ࡓᐖ㜵㝖㛵ࡍࡿඛ⾜◊✲ࡋ࡚ࡣࠊ㣗⏝ࡁࡢࡇࡸࡑ
ࢀࡽࡢᗫ⳦ᗋࢆ⏝࠸ࡓ◊✲ࡀ࠶ࡿ(Parada et al. 2012㸹Nishino et al. 2013㸹Oka et al. 2015)ࠋࡋ
ࡋࠊ㔝እ࡛᥇ྲྀࡋࡓࠕ㔝⏕ࡁࡢࡇࠖࡽศ㞳ࡋࡓࠕᐤ⏕ཪࡣඹ⏕ࡋ࡚࠸ࡿ⳦㢮ࠖࡢᇵ㣴ᾮ
ࢆ⏝࠸࡚ࠊࢿ࠸ࡶࡕࡢ㜵㝖⏝ྍ⬟࡞ᚤ⏕≀ࢆ᥈⣴ࡍࡿ◊✲ࡣ࡞ࡗࡓࠋࡑࡇ࡛ᮏ❶
࡛ࡣࠊ᪥ᮏᅜෆ࡛᥇ྲྀࡋࡓ㔝⏕ࡁࡢࡇࡽࡢࠕᐤ⏕ཬࡧඹ⏕ࡋ࡚࠸ࡿ⳦㢮ࠖࡢศ㞳ࠊࢿ
࠸ࡶࡕࡢᢚไ⏝ྍ⬟࡞⳦ࡢ᥈⣴ࡘ࠸࡚㏙ࡿࠋ
➨1⠇ ᪥ᮏᅜෆ࡛᥇ྲྀࡋࡓ㔝⏕ࡁࡢࡇࡽࡢ⳦㢮ࡢศ㞳 1㸬ᮦᩱཬࡧ᪉ἲ
a) ࡁࡢࡇࡢ᥇ྲྀ
ᓥ᰿ࠊ㫽ྲྀࠊᗈᓥࠊ㜰ࠊ⚟ཬࡧ⇃ᮏ┴ෆ࡛ࡁࡢࡇ(Ꮚᐇయ)ࢆࡑࢀࡒࢀ7ࠊ8ࠊ10ࠊ5ࠊ 4ཬࡧ12ಶࡢྜィ46ಶయ᥇ྲྀࡋࡓࠋ᥇ྲྀࡋࡓࡁࡢࡇࡣ⁛⳦ࢧࣥࣉࣝࣂࢵࢢ(Thermo Fisher Scientific Waltham, Massachusetts, USA)ධࢀ࡚ࠊᐇ㦂ᐊෆ࡛┿ࢆࡾࠊグ㘓ࡋࡓ(Fig. 2-1)ࠋ b) ᇵᆅࡢㄪ〇ཬࡧᇵ㣴᮲௳
ࢪ࣭ࣕ࢞ࣔࢫࢡ࣮ࣟࢫᐮኳᇵᆅ(PSA ᇵᆅ)ࢆ⏝࠸ࡓࠋ⓶ⱆࢆྲྀࡾ㝖࠸ࡓࢪࣕ࢞ࣔ
(200 g)ࢆษࡾࡋࠊ␃Ỉ800 mlࢆධࢀࡓ1 Lᐜ୕ゅࣇࣛࢫࢥෆຍ࠼ࡓࠋࡑࡢᚋࠊ
࣑࡛ࣝ࣍ࣝࢆࡋ࡚ࠊ࣮࢜ࢺࢡ࣮ࣞࣈ(105Υࠊ40ศ㛫)ࢆ⏝࠸࡚ຍ⇕ࡋࡓࠋຍ⇕ᚋࠊ↦Ồ
ࢆ࣮࢞ࢮ࡛ࢁ㐣ࡋࠊ␃Ỉ࡛1 L࣓ࢫࢵࣉࡋࡓࠋࡑࡢᚋࠊ300 mlᐜ୕ゅࣇࣛࢫࢥ⢊ᮎ ᐮኳ(4 g)ࠊࢫࢡ࣮ࣟࢫ(4 g) ཬࡧ↦Ồ(200 ml) ࢆຍ࠼࡚࣑࡛ࣝ࣍ࣝࢆࡋࡓࠋᇵᆅࡣ
121Υࠊ20ศ㛫࣮࢜ࢺࢡ࣮ࣞࣈ⁛⳦ᚋᐇ㦂⏝࠸ࡓࠋ
PSAᇵᆅࡣ㟁Ꮚࣞࣥࢪ࡛⁐ゎࡋࡓᚋࠊ200 mlࡢᇵᆅࢡ࣒ࣟࣛࣇ࢙ࢽࢥ࣮ࣝ(20 ppm)ࢆῧ ຍࡋࡓࠋῧຍᚋࠊࣉࣛࢫࢳࢵࢡࢩ࣮ࣕࣞ(┤ᚄ9 cm)⣙20 mlࡎࡘศὀࡋ࡚ࠊᅛࡵࡓࡶࡢࢆ
ᐇ㦂⏝࠸ࡓࠋ
c) ࡁࡢࡇࡽࡢ⳦㢮ࡢศ㞳ἲ
ࡁࡢࡇࡽࡢ⳦㢮ࡢศ㞳ࡣ⤌⧊ศ㞳ἲࢆ⏝࠸ࡓࠋ5 mm ᅄ᪉ษࡗࡓࡁࡢࡇࡢ⤌⧊ࢆ
80 %࢚ࢱࣀ࣮ࣝ2ศ㛫ᾐₕᚋࠊ1 %ḟளሷ⣲㓟ࢼࢺ࣒ࣜ࢘Ỉ⁐ᾮ2-3ศ㛫ᾐₕࡋࠊ⁛⳦
ỈࡼࡿὙίᚋࢁ⣬࡛Ỉศࢆ㝖ཤᚋࠊ20 ppmࡢࢡ࣒ࣟࣛࣇ࢙ࢽࢥ࣮ࣝࢆྵࡴPSAᇵᆅ
⨨ᗋࡋࡓࠋ⨨ᗋᚋࡣࠊ26Υࡢᜏ ᐊ࡛ᇵ㣴ࡋࠊࡁࡢࡇࡢ⤌⧊࿘㎶ࡽ⏕⫱ࡋ࡚ࡁࡓ⳦⣒ࢆศ 㞳ࡋࠊᐇ㦂⏝࠸ࡓࠋศ㞳ࡉࢀࡓ⳦ᰴࡣࠊPSAᩳ㠃ᇵᆅ࡛ಖᏑࡋࡓࠋࡲࡓࠊ㛗ᮇ㛫ⓗಖᏑ ࡍࡿࡓࡵ⁛⳦ࢁ⣬(┤ᚄ6 mm)ࢆ⨨ᗋࡋࡓPSAᇵᆅศ㞳ࡋࡓ⳦ᰴࢆ⛣᳜ࡋࡓࠋ⳦⣒ࡀ⏕
⫱ࡋࡓࢁ⣬ࢆྲྀࡾฟࡋࠊ⁛⳦ࢩ࣮ࣕࣞෆ࡛⇱ᚋ̺20 Υࡢ෭ᗜ࡛ಖᏑࡋࡓࠋ
8 2㸬㸬⤖ᯝ
᪥ᮏࡢᓥ᰿ࠊ㫽ྲྀࠊᗈᓥࠊ㜰ࠊ⚟ཬࡧ⇃ᮏࡢྛ┴࡛᥇ྲྀࡋࡓ46ಶయ(Fig. 2-1)ࡢࡁࡢ ࡇࡽ⳦ࢆศ㞳ࡋࡓࠋࡑࡢ⤖ᯝࠊᓥ᰿ࠊ㫽ྲྀࠊᗈᓥࠊ㜰ࠊ⚟ཬࡧ⇃ᮏ࡛ࡑࢀࡒࢀ 17ࠊ
11ࠊ9ࠊ15ࠊ13ཬࡧ40⳦ᰴࡢྜィ105⳦ᰴࡀศ㞳ࡉࢀࡓ(Table 2-1)ࠋ
9
Fig. 2-1. Collection of fresh fruiting bodies of wild mushrooms from a paddy field in Japan.
10 Shimane 㻌
Tottori 㻌 Hiroshima 㻌 Osaka 㻌 Fukui 㻌 Kumamoto 㻌
17㻌 11㻌 9㻌 15㻌 13㻌 40㻌
Place (Prefecture) Number of isolated fungi strains㻌 Table 2-1. The number of isolated fungi strains from fresh fruiting body of wild mushrooms㻌
11
➨
➨2⠇ ศ㞳⳦ࡢᇵ㣴ࢁᾮࡀࢿ࠸ࡶࡕ⳦ࡢឤᰁ⾜ືཬࡰࡍᙳ㡪 1. ᮦᩱཬࡧ᪉ἲ
a) ౪ヨ⳦ཬࡧᇵ㣴᮲௳
ࢿ࠸ࡶࡕ⳦ࡋ࡚㛗 69-150ᰴ(࣮ࣞࢫ007ࠊࢥࢩࣄ࢝ࣜᑐࡋ࡚ぶᛶ⳦)ࢆ⏝࠸ࡓࠋ
ࢿ࠸ࡶࡕ⳦ࡣணࡵヨ㦂⟶(φ 1.8×18 cm)20 mlศὀࡋࡓPSAᩳ㠃ᇵᆅ᳜࠼ࡅ࡚࠾
࠸ࡓࠋ⳦ࡣPSAᇵᆅࢆ⣙20 mlศὀࡋࡓࣉࣛࢫࢳࢵࢡࢩ࣮ࣕࣞ(┤ᚄ7 cm) (As one, Osaka,
Japan)⛣᳜ࡋࠊᬯ㯮ୗࠊ26Υ࡛⣙14᪥㛫ᇵ㣴ࡋࡓࡶࡢࢆᐇ㦂⏝࠸ࡓࠋࡲࡓࠊ⣙20 mlࡎ
ࡘࣉࣛࢫࢳࢵࢡࢩ࣮ࣕࣞ(┤ᚄ7 cm)ศὀࡋ࡚ᅛࡵࡓ⡿ࡠᐮኳᇵᆅ(⡿ࡠ50 gࠊᐮኳ20
g/Ỉ1 L)࡛⣙14᪥㛫ᇵ㣴ࡋࡓࠋࡑࡢᚋࠊࢩ࣮ࣕࣞ⁛⳦Ỉࢆὀࡂࠊࣛࣂ࣮ࢫࣃࢳࣗࣛࢆ⏝
࠸࡚ࠊ⳦ྀࡢẼ୰⳦⣒ࢆ㝖ཤࡋࡓࠋBlack light blue (BLB) (FL20s BL-B; Panasonic, Osaka, Japan)
ࢆ↷ᑕࡋ࡚ࠊ⬊Ꮚࢆᙧᡂࡉࡏࡓࠋ⬊Ꮚᙧᡂᚋࠊࢩ࣮ࣕࣞ⁛⳦Ỉࢆὀࡂࠊ⬊Ꮚࢆ⁛⳦Ỉ
ᠱ⃮ࡉࡏࡓࠋࡇࡢᠱ⃮ᾮࡣ2㔜ࡢࢸࢵࢩ࣮ࣗ࣌ࣃ࣮࡛ࢁ㐣ࡋࠊ⳦⣒∦➼ࢆ㝖ཤᚋࠊ㐲ᚰศ 㞳(1600×gࠊ10ศ㛫)(ࢸ࣮ࣈࣝࢺࢵࣉ㐲ᚰᶵ4000ࠊஂಖ⏣ၟᰴᘧ♫)ࡋࠊ⬊Ꮚࢆᅇᚋࠊ
⏕≀᳨ᐃ⏝࠸ࡓࠋ
➨2❶ࠊ➨1⠇࡛ศ㞳ࡋࡓ⳦(105⳦ᰴ)ࢆ⏝࠸ࡓࠋணࡵࠊศ㞳⳦ࡣୖグྠᵝࡢPSAᩳ㠃 ᇵᆅ᳜࠼ࡅ࡚࠾࠸ࡓࠋࡑࢀࡒࢀPSAᩳ㠃ᇵᆅࡽࢆ⣙20 mlศὀࡋࡓࣉࣛࢫࢳࢵࢡࢩ
࣮ࣕࣞ(┤ᚄ7 cm)ศ㞳⳦ࢆ⛣᳜ࡋࠊᬯ㯮ୗࠊ26Υ࡛⣙14᪥㛫ᇵ㣴ࡋࡓࡶࡢࢆᐇ㦂⏝࠸
ࡓࠋ
b) ศ㞳⳦ࡢᇵ㣴ࢁᾮࡢㄪ〇⏕≀᳨ᐃἲ
ศ㞳⳦ࡢᇵ㣴ࢁᾮࡢㄪ〇ࡣࢪ࣭ࣕ࢞ࣔࢫࢡ࣮ࣟࢫ(PS)ᾮయᇵᆅࢆ⏝࠸ࡓࠋPS ᾮయᇵ ᆅࡣヨ㦂⟶(φ 3×20 cm)20 mlࡎࡘศὀࡋࠊ121Υ࣭20ศ㛫࣮࢜ࢺࢡ࣮ࣞࣈ⁛⳦ᚋᐇ㦂
⏝࠸ࡓࠋศ㞳⳦ࡣணࡵPSAᇵᆅ࡛ᇵ㣴ᚋࠊ7 mmࢥࣝࢡ࣮࣮࡛࣎ࣛ⳦ྀࢆࡃࡾᢤࡁࠊPSᾮ యᇵᆅ(20 ml)1∦ࡎࡘ⛣᳜ࡋࡓࠋ⛣᳜ᚋࠊ7᪥㛫ࠊ120ᅇ㌿/ศ࡛┞ᇵ㣴ࡋࡓࠋ┞ᇵ㣴 ᚋࠊᇵ㣴ᾮࡣ⁛⳦ࢁ㐣ࣇࣝࢱ࣮(0.22 μm)(RephiLe Bioscience, Boston, USA)࡛ࢁ㐣ࡋࡓࠋᑐ
↷༊ࡋ࡚ࠊ↓᥋✀ࡢPSᾮయᇵᆅࢆྠᵝࡢ᪉ἲ࡛ㄪ〇ࡋࡓࡶࡢࢆ⏝࠸ࡓࠋ
ᇵ㣴ࢁᾮࡢ⏕≀άᛶࡣࠊࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆཬࡧ╔ᙧᡂࡢᢚไࡼࡾㄪᰝࡋࡓࠋ
ୖグࡢ᪉ἲ࡛ㄪ〇ࡋࡓࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚࢆྛᇵ㣴ࢁᾮ 1×105spores/ml ࡞ࡿࡼ࠺
ᠱ⃮ࡉࡏࡓࠋᑐ↷༊ࡋ࡚ࠊPS ᾮయᇵᆅࡢࢁᾮࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚࢆᠱ⃮ࡉࡏࡓࡶࡢ
ࢆ⏝࠸ࡓࠋㄪ〇ࡋࡓᠱ⃮ᾮࡣ‵ᐊࡋࡓࣉࣛࢫࢳࢵࢡࢣ࣮ࢫෆ୪ࡓࢫࣛࢻ࢞ࣛࢫ
ୗࡋࡓࠋࢫࣛࢻ࢞ࣛࢫࡣ2ᯛ⏝ពࡋࠊ30 μlࡎࡘィ6ᡤୗࡋࡓࠋୗᚋࠊᬯ㯮ୗࠊ 26ΥࡢேᕤẼ㇟ჾ(LIB-302(H), IWAKI)ෆ࡛ᇵ㣴ࡋࡓࠋᇵ㣴24㛫ᚋࠊගᏛ㢧ᚤ㙾ୗ࡛ࢿ
࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆᩘཬࡧ╔ჾᙧᡂᩘࢆㄪᰝࡋࡓࠋ⬊ᏊⓎⱆᩘࡣࠊ1ᡤᙜࡓࡾ⬊Ꮚ50 ಶ୰ࡢⓎⱆ⬊Ꮚᩘࢆィᩘࡋࠊ⬊ᏊⓎⱆ⋡ࢆ⟬ฟࡋࡓࠋ╔ჾᙧᡂᩘࡣ1ᡤᙜࡓࡾⓎⱆ⬊Ꮚ 50ಶ୰ࡢ╔ჾᙧᡂ⬊Ꮚᩘࢆィᩘࡋࠊ╔ჾᙧᡂ⋡ࢆ⟬ฟࡋࡓࠋᐇ㦂ࡣ3ᅇ⧞ࡾ㏉ࡋ࡚ࠊ
⾜࠸ࠊྜィ900⬊Ꮚࢆほᐹࡋࡓࠋ
࣮࢜ࢺࢡ࣮ࣞࣈࢆ⏝࠸࡚ࠊ㑅ᢤࡉࢀࡓศ㞳⳦ࡢᇵ㣴ࢁᾮࢆ⇕ฎ⌮ࡋ(121Υࠊ20ศ)ࠊୖグ
12
ྠᵝࡢ᪉ἲ࡛ࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆ⋡ཬࡧ╔ᙧᡂ⋡ࢆㄪᰝࡋࡓࠋ
c) ࣒࢜࢜࢜ࢠࡢศ㞳⳦ࡢᇵ㣴ࢁᾮࡢ๓ฎ⌮ࡀࢿ࠸ࡶࡕࡢᩬᙧᡂ࠼ࡿᙳ㡪 ౪ヨ᳜≀ࡋ࡚࣒࢜࢜ࢠ(ရ✀:࣡ࢭࢻࣜᅄ᮲)ࢆᐇ㦂⏝࠸ࡓࠋ✀Ꮚࡣࢢ࣮ࣜࣥࢯࣝ (❅
⣲0.9 gࠊ⇥㓟1.1 gࠊ࢝ࣜ1.0 g/3.3 kg)(ฟ㞼ࢢ࣮࢚࣏ࣜࣥࢵࢡᰴᘧ♫)ࢆຍ࠼ࡓࢩ࣮ࢻࣜࣥ
ࢢࢣ࣮ࢫࠊ1ࢣ࣮ࢫ20⢏ࡎࡘ✀ࡋࡓࠋそᅵࡣࢢ࣮ࣜࣥࢯࣝそᅵ(ฟ㞼ࢢ࣮࢚ࣜࣥ
࣏ࢵࢡᰴᘧ♫)ࢆ⏝࠸ࡓࠋ✀ᚋࠊ࢞ࣛࢫᐊෆ࡛ 2ⴥᮇࡲ࡛⏕⫱ࡉࡏࡓࡶࡢࢆᐇ㦂⏝࠸
ࡓࠋ
2ⴥᮇࡢ࣒࢜࢜ࢠࢆࢩ࣮ࢻࣜࣥࢢࢣ࣮ࢫࡽ᰿ࢆษ᩿ࡋ࡞࠸ࡼ࠺ᢤࡁྲྀࡾࠊᅵተࢆ㝖ཤ ࡋࡓࠋࡑࡢᚋࠊ‵ࡽࡏࡓ࢟ࢵࢳ࣮ࣥ࣌ࣃ࣮ࢆᘬ࠸ࡓࣉࣛࢫࢳࢵࢡࢣ࣮ࢫ(2.5 cm× 20 cm×10 cm)7ಶయࡎࡘ୪ࡓࠋୖグࡢ᪉ἲ࡛ㄪ〇ࡋࡓศ㞳⳦ࡢᇵ㣴ࢁᾮࢆྛฎ⌮5 mlࡎࡘ࣒࢜࢜
ࢠᄇ㟝ฎ⌮ࡋࡓࠋᑐ↷༊ࡋ࡚↓᥋✀ࡢPSᾮయᇵᆅࢆᄇ㟝ฎ⌮ࡋࡓࠋ24㛫ᚋࠊࢿ࠸
ࡶࡕ⳦ࡢᠱ⃮ᾮ1×105spores/mlࢆᄇ㟝᥋✀ࡋࡓࠋࣉࣛࢫࢳࢵࢡࢣ࣮ࢫࡣࡩࡓࢆ㛢ࡵࠊࢭࣟ
ࣁࣥࢸ࣮ࣉ࡛ᐦ㛢ࡋࠊேᕤẼ㇟ჾෆ(LIB-302(H), IWAKI)ධࢀࠊᬯ㯮ୗ࡛24㛫ᇵ㣴ᚋࠊ 12㛫ග᮲௳ୗಖᣢࡋࡓࠋ᥋✀3᪥ᚋࠊ࠸ࡶࡕᩬࡢᙧᡂࢆほᐹࡋࡓࠋ
2㸬⤖ᯝ
ศ㞳⳦(105⳦ᰴ)ࡢᇵ㣴ࢁᾮࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚࡢΰྜᠱ⃮ᾮࢆࢫࣛࢻ࢞ࣛࢫ
ୗࡋ࡚ࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆ⋡ཬࡧ╔ჾᙧᡂ⋡ࢆㄪᰝࡋࡓࠋࡑࡢ⤖ᯝࠊᇵ㣴ࢁᾮࡀ ᚓࡽࢀࡓ90⳦ᰴ୰20⳦ᰴ࡛ᑐ↷༊ẚ㍑ࡋ࡚ࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆࡀ 50%௨ୖᢚไ ࡉࢀࡓࠋᑐ↷༊ࡢ⬊ᏊⓎⱆ⋡ࡣ90.9 ± 5.7%࡛࠶ࡗࡓ(Fig. 2-2)ࠋศ㞳⳦ࡢᇵ㣴ࢁᾮ༊࡛ࡢ⬊Ꮚ
Ⓨⱆ⋡ࡣࠊF5ࠊ K7ࠊK24ࠊK36ࠊO1ࠊS5ཬࡧS8 ࡀࡑࢀࡒࢀ1.8 ± 5.3%ࠊ 12.4 ± 21.0%ࠊ 34.7 ± 35.5%ࠊ 18.7 ± 7.3%ࠊ2.9 ± 5.9%ࠊ0.9 ± 2.0%ཬࡧ 27.3 ± 19.2%࡛࠶ࡗࡓࠋ୍᪉ࠊF1ࠊ
F2ࠊ F3ࠊ H2ࠊ H5ࠊ H7ࠊ H9ࠊ K1ࠊ K9ࠊ K21ࠊ O3ࠊ O6ཬࡧ S9 ࡢᇵ㣴ࢁᾮ༊࡛ࡢ
⬊ᏊⓎⱆ⋡ࡣ0%࡛࠶ࡗࡓ(Fig. 2-2)ࠋ
ᑐ↷༊ẚ㍑ࡋ࡚ࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆࡀ 50%௨ୖᢚไࡉࢀࡓ 20 ศ㞳⳦(F1ࠊF2ࠊ F3ࠊF5ࠊH2ࠊH5ࠊH7ࠊH9ࠊK1ࠊK7ࠊK9ࠊK21ࠊK24ࠊK36ࠊO1ࠊO3ࠊO6ࠊS5ࠊS8ཬࡧ S9) ࡢᇵ㣴ࢁᾮࢆ⇕ฎ⌮ࡋࠊࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆࡢᢚไຠᯝࢆㄪᰝࡋࡓࠋࡑࡢ⤖ᯝࠊ3ࢢ
࣮ࣝࣉศ㢮ࡉࢀࡓࠋ➨1ࢢ࣮ࣝࣉࡣF3ࠊH2ࠊK21ࠊK24 ཬࡧS8⳦ᰴ࡛࠶ࡿࠋࡇࢀࡽࡢศ 㞳⳦ࡢᇵ㣴ࢁᾮࡣࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆཬࡧ╔ჾᙧᡂඹᢚไࡋ࡞ࡗࡓ(Fig. 2- 3)ࠋḟࠊ➨2ࢢ࣮ࣝࣉࡣF2ࠊF5ࠊH9ࠊK1ࠊO1ࠊO3ࠊO6ࠊS5ཬࡧS9⳦ᰴ࡛࠶ࡾࠊࢿ࠸
ࡶࡕ⳦ࡢឤᰁ⾜ືࢆᢚไࡋࡓ(Fig. 2-3)ࠋ➨3ࢢ࣮ࣝࣉࡣF1ࠊH5ࠊH7ࠊK7ࠊK9ཬࡧK36
⳦ᰴ࡛࠶ࡾࠊ⬊ᏊⓎⱆࢆᢚไࡋ࡞ࡗࡓࡀࠊ╔ჾᙧᡂࢆᢚไࡋࡓ(Fig. 2-3)ࠋ
ࡑࡇ࡛ࠊୖ㏙ࡢᐇ㦂࡛⇕Ᏻᐃ࡞ᢚไ≀㉁ࢆྵࡴ⪃࠼ࡽࢀࡿF2ࠊF5ࠊH9ࠊK1ࠊO1ࠊO3ࠊ O6ࠊS5ཬࡧS9ศ㞳⳦ࡢᇵ㣴ࢁᾮࢆ࣒࢜࢜ࢠ๓ฎ⌮ࡋࠊ24㛫ᚋࠊࢿ࠸ࡶࡕ⳦ࡢᄇ 㟝᥋✀ࢆ⾜࠸ࠊ࠸ࡶࡕᩬࡢᙧᡂࢆほᐹࡋࡓࠋࡑࡢ⤖ᯝࠊᑐ↷༊࡛ࡣࠊ࣒࢜࢜ࢠࡢⴥࡀ㯤Ⰽ
ኚࡋࠊ⅊Ⰽࡢ࠸ࡶࡕᩬࡀయᙧᡂࡉࢀ࡚࠸ࡓ(Fig. 2-4 A)ࠋ୍᪉ࠊO1䚸O3䚸S5ཬ䜃 S9
13
ศ㞳⳦ࡢᇵ㣴ࢁᾮฎ⌮༊࡛ࡣࠊ࡞≧ែࡢ࣒࢜࢜ࢠࡢⴥࡀほᐹࡉࢀࠊⓎ⋡ࡣࡑࢀࡒࢀ
19.0 ± 4.1%ࠊ26.2 ± 4.1%ࠊ28.6 ± 7.1%ཬࡧ9.5 ± 4.1%࡛࠶ࡾࠊ࠸ࡶࡕᩬࡢᙧᡂࡣᑐ↷༊
ẚ㍑ࡋ࡚ῶᑡࡋ࡚࠸ࡓ(Fig. 2-4 AࠊB)ࠋࡋࡋࠊF2ࠊF5ࠊH9ࠊK1ཬࡧO6⳦ᰴࡢᇵ㣴ࢁᾮࡣ
ࢿ࠸ࡶࡕࡢᩬᙧᡂࢆᢚไ࡛ࡁ࡞ࡗࡓ(Fig. 2-4 B)ࠋ
14
Percentage of spore germination (%)PercenPercentage of spore germination (%)PercenPercentage of spore germination (%) PercenPercentage of spore germination (%) ntage of spore germination (%)Percenntageofsporegermination(%)Percen%) 100
90 80 70 60 50 40 30 20 10 0 CLPSB
F1 F2 F3 F4 F5 F6 F7 F8 F9F10 F11F12 F13 H1 H2 H3 H4 H5 H6 H7 H8 H9 K1
CL PSB F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 H1 H2 H3 H4 H5 H6H7 H8 H9 K1
K2 K3 K4 K5 K6 K7 K8 K9 K10 K11 K12 K13 K14 K15 K16 K17 K18 K19 K20K21 K22 K23 K24
K25 K26K27 K28 K29 K30 K31 K32 K33 K34 K35 K36 O1 O2 O3 O4 O5O6 O7 O8 O9 O10
O11 O12 O13 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 T1 T2 T3 T4 T5 T6 T7 T8
ntage of spore germination (%)Percen
100 90 80 70 60 50 40 30 20 10 0
ntage of spore germination (%Percen%) 100 90 80 70 60 50 40 30 20 10 0
Percentageof spore germination (%)
)100
90 80 70 60 50 40 30 20 10 0
K2 K3 K4 K5 K6 K7 K8 K9 K10 K11 K12 K13 K14 K15 K16 K17 K18 K19 K20 K21K22 K23 K24 0
0 0 0 0 0 0 0 0 0 0
a a a
a
a a
a CL
CL a
a
a a a a
a
a a
a
a
a
a
a a a
a a
2 F13
a a
a a
a a
a
a
a a
a a
a F2 F
a a
a a
a a
a a
a
H8 H9 H8 H9 a
a
a a
a
a
a a a
a
a
a
a a
a
a a
a a
a a
ab PSB F
L PSB F L P L P
abc
abc bc
bc c c
c
c
c c
c
6 c c c
c
c c
0 21c c
c c
c
c
a
Fig. 2-2. Culture filtrates of fungi isolated from wild mushrooms influence the infection behaviors of Magnaporthe oryzae. Data were obtained from the results of 6 experiments of 3 replicates and expressed as mean ± SD. Means followed by different letters are significantly different according to the Scheffe’s test (p < 0.05).
15
Fig. 2-3. Inhibition by heat-treated culture isolates on the infection behaviors of Magnaporthe oryzae.
The spore germination (A) and appressorium formation (B) were determined after 24 h incubation. Data were obtained from the results of 6 experiments of 3 replicates and expressed as mean ± SD. Means followed by different letters are significantly different according to the Scheffe’s test (p < 0.05).
Percentage of spore germination (%)
100 90 80 70 60 50 40 30 20 10
0 CL PSB K1 K7 K9 K21 K24 K36 F1 F2 F3 F5 H2 H5 H7 H9 O1O3 O6 S5 S8 S9
)
1000 900 800
a a a a a
ab a abc bcabcd abc
bcd b d cd
e
e e e
e e e e e
abc
Percentage of appressorium formation (%) n(%)
CL PSB K1 K7 K9 K21K24 K36 F1 F2 F3 F5 H2 H5 H7 H9 O1O3 O6 S5 S8 S9
Percentage of appressoriumformationn(%) 100 90 80 70 60 50 40 30 20 10 0
a
a a a
a
a b
c c c c c c c c c c c c c c c
A
B
16
Fig. 2-4. Inhibition by culture filtrates of㻌 selected isolates on lesion formation by Magnaporthe oryzae in barley leaves. (A) Blast lesion formation by M. oryzae. (B) Percentage of blast lesion formation by M. oryzae. Data were obtained from the results of 10 experiments of 3 replicates and expressed as mean ± SD. Means followed by different letters are significantly different according to the Scheffe’s test (p < 0.05).
100 90 80 70 60 50 40 30 20 10 0
CL PSB F2 F5 H9 K1 O1 O3 O6 S5 S9 8
7 6 5 4 3 2 1
Percentage of blast lesion formation(%)
000 00 10 )(%(%) 9 B A
CL PSB F2 F5 H9 K1 O1 O3 O6 S5 S9
a a a a a
a a
b
b b
b
17
➨
➨3⠇ ࢫࢡ࣮ࣜࢽࣥࢢࡉࢀࡓศ㞳⳦ࡢྠᐃ 1㸬ᮦᩱཬࡧ᪉ἲ
a)౪ヨ⳦
࣒࢜࢜ࢠ࡛ᢚไάᛶࢆ♧ࡋࡓO1䚸O3䚸S5ཬ䜃 S9⳦ᰴࢆᐇ㦂⏝࠸ࡓࠋศ㞳⳦ࡣ➨2❶ࠊ
➨2⠇ࠊ1a)࡛㏙ࡓ᪉ἲࡼࡾ‽ഛࡋࡓࠋ
b)DNAࡢᢳฟཬࡧITS㡿ᇦࡢࢩ࣮ࢡ࢚ࣥࢫゎᯒ
DNAᢳฟࡣSuzuki et al. (2006) ࡢ᪉ἲᚑࡗ࡚⾜ࡗࡓࠋTE(10 mM Tris-ClࠊpH 8.0ࠊ1 mM
EDTA)ࠊPCRࢳ࣮ࣗࣈ(PCR-02-CࠊAXYGEN)20 μl ࡎࡘศὀࡋࡓࠋPSAᇵᆅ࡛1㐌㛫ᇵ
㣴ࡋࡓ⳦ྀ⾲㠃ࢆ⁛⳦ࡋࡓࢳࢵࣉ࡛᧿ࡾྲྀࡾࠊࡈࡃᑡ㔞TEᠱ⃮ࡋࡓࠋ㟁Ꮚࣞࣥࢪ(500 W)
࡛30⛊ࢆ2ᅇ⧞ࡾ㏉ࡋࡓᚋࠊ㐲ᚰศ㞳(20,600×gࠊ4Υࠊ15ศ㛫) (ࢸ࣮ࣈࣝࢺࢵࣉ㐲ᚰᶵ3500ࠊ
ஂಖ⏣ၟᰴᘧ♫)ࡋࡓࠋୖΎࢆ᪂ࡋ࠸࣐ࢡࣟࢳ࣮ࣗࣈ⛣ࡋࠊDNAᢳฟᾮࡋ࡚⏝࠸
ࡓࠋPCRᛂᾮ(⁛⳦Ỉ34.75 μlࠊ10×Buffer 5 μlࠊ2.5mM dNTPs 4 μlࠊTaq DNA polymerase 0.25 μl) ࢆ స ᡂ ࡋ ࡚ ࠊ ࣉ ࣛ ࣐ ࣮ITS1(5’-TCCGTAGGTGAACCTGCGG-3’)ཬ ࡧ ITS4(5’-
TCCTCCGCTTATTGATATGC-3’)ࢆ⏝࠸࡚PCRࢆ⾜࠸ࠊITS㡿ᇦࢆቑᖜࡋࡓࠋ95Υࠊ5ศ㛫
ࡢ⇕ኚᛶᚋࠊ94Υ࡛1ศ㛫ࠊ62Υ࡛1ศ㛫ࠊ72Υ࡛1ศ㛫ࢆ30ࢧࢡࣝ⧞ࡾ㏉ࡋࠊ᭱ᚋ
72Υ࡛5ศ㛫ࡉࡏࡓࠋ㟁ẼὋື࡛DNAࡀቑᖜࡉࢀ࡚࠸ࡿࡇࢆ☜ㄆࡋࡓࠋ☜ㄆᚋࠊDNA
⢭〇࢟ࢵࢺ(RBC Bioscience)ࢆ⏝࠸࡚ࠊDNAࡢ⢭〇ࢆ⾜ࡗࡓࠋቑᖜࡉࢀࡓDNAࢆ࣑࢝ࢯࣜ
࡛ษࡾᢤࡁࠊDF Buffer(500 μl)ධࢀࠊ10ศ↦(56Υ)ࡋ࡚ࠊࢤࣝࢆ⁐ゎࡋࡓࠋCollection ࢳ
࣮ࣗࣈ DF Column ࡢࢭࢵࢺ⛣ࡋࠊ20,600×g ࡛ 30 ⛊㐲ᚰࡋࡓࠋᗫᾮࢆ㝖ཤࡋࠊWash
Buffer(600 μl)ࢆຍ࠼ࠊ20,600×g࡛30⛊㐲ᚰࡋ࡚ࠊᗫᾮࢆ㝖ཤᚋࠊᗘ20,600×g࡛2ศ㐲ᚰ ࡋࡓࠋDF Columnࢆ1.5 mlࢳ࣮ࣗࣈ⛣ࡋࠊElutionᾮ(10 μl)ࢆຍ࠼ࠊ2ศ㛫㟼⨨ᚋࠊ20,600×g
࡛2ศ㐲ᚰࡋࠊ⢭〇ࡉࢀࡓDNAࢆ⁐ฟࡋࡓࠋ
ࡑࡢᚋࠊࢩ࣮ࢡ࢚ࣥࢫᛂࢆ⾜ࡗࡓࠋ⁛⳦Ỉ(6.8 μl)ࠊࢩ࣮ࢡ࢚ࣥࢫࣉ࣑ࣞࢵࢡࢫ(0.2 μl)ࠊ 5×ࣂࢵࣇ࣮(1 μl)ࠊࣉ࣐࣮ࣛ(3.2 pmol)(1 μl)ࠊDNA(1 μl)ࢆΰྜࡋࠊᛂࢆ⾜ࡗࡓࠋᛂ ᮲௳ࡣ96Υࠊ2ศ㛫ࡢ⇕ኚᛶᚋࠊ96Υ࡛10⛊ࠊ50Υ࡛5⛊ࠊ60Υ࡛3ศ30⛊ࢆ25ࢧ
ࢡࣝ⧞ࡾ㏉ࡋࠊ᭱ᚋ10ΥࡋࡓࠋᛂࡢᚋࠊDNA⢭〇ࢆᗘ⾜ࡗࡓࠋ᭱ึ125 mM EDTA(1 μl)ࠊ3 M NaOAC(1 μl)ࠊ࢚ࢱࣀ࣮ࣝ(25 μl)ࢆຍ࠼ࠊ4Υࠊ20,600×g࡛15ศ㛫㐲ᚰࡋࡓࠋ㐲ᚰ ᚋࠊୖΎࢆᗫᲠࡋࠊ70%࢚ࢱࣀ࣮ࣝ(25 μl)ࢆຍ࠼ࠊᗘ4Υࠊ20,600×g࡛15ศ㛫㐲ᚰࡋࠊୖ
Ύࢆ㝖ཤᚋࠊHiDi ࣒࣑࣍ࣝࢻ(20 μl)⁐ゎࡋࡓࠋࡑࡢᚋࠊDNA ࢩ࣮ࢡ࢚ࣥࢧ࣮(ABI PRISM®3100̺Avant, ࢪࣗࢿࢸࢵࢡࢼࣛࢨ, Applied Biosystems)࡛ゎᯒᚋBLAST᳨
⣴(http://www.ddbj.nig.ac.jp/Welcome-j.html)ཬࡧGENETYXࢆ⏝࠸࡚ࠊ┦ྠᛶࢆㄪᰝࡋࡓࠋ
2㸬⤖ᯝ
࣒࢜࢜ࢠ࡛ࢿ࠸ࡶࡕࡢᩬᙧᡂࢆᢚไࡋࡓO1䚸O3䚸S5ཬ䜃 S9 ⳦ᰴࡢྠᐃࢆITS㡿 ᇦࡢࢩ࣮ࢡ࢚ࣥࢫゎᯒࡼࡾ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊS9䛿Annulohypoxylon sp.䚸S5䛿Nigrospora sp.ཬ䜃O1䛸O3 䛿Penicillium sp. 䛸㧗䛔┦ྠᛶ䜢♧䛧䛯 (Table 2-2)䚹
18
㻌 㻌 Table 2-2. Identification of fungi isolated from mushroom by sequences of rDNA ITS regions O1 Penicillium sp.
O3 Penicillium sp.
S5 Nigrospora sp.
S9 Annulohypoxylon sp.
Isolate Candidate fungus Sampling location Sampling year Japan (Osaka)
Japan (Osaka) Japan (Shimane) Japan (Shimane)
2010 2010 2009 2009
19
➨
➨4⠇ ⪃ᐹ
ᓥ᰿ࠊ㫽ྲྀࠊᗈᓥࠊ㜰ࠊ⚟ཬࡧ⇃ᮏࡢྛ┴࡛᥇ྲྀࡋࡓ46ಶయࡢࡁࡢࡇࡽࡣPSAᇵ ᆅࢆ⏝࠸ࡓ⤌⧊ศ㞳ἲࡼࡾ105⳦ᰴࡀศ㞳ࡉࢀࡓ(Table 2-1)ࠋ᥇ྲྀࡋࡓࡁࡢࡇࡘ࠸࡚ࡢ ヲࡋ࠸ྠᐃࡣ⾜ࢃ࡞ࡗࡓࡀࠊࢧࣝࣀࢥࢩ࢝ࢣࠊ࣒ࣛࢧ࢟ࢦ࣒ࢱࢣࠊࢸࣥࢢࢱࢣᒓࡸከᏍ⳦
┠ࡢࡁࡢࡇ࡞࡛࠶ࡿ⪃࠼ࡽࢀࡿࠋศ㞳ࡋࡓ 105 ⳦ᰴࡢ⳦⣒⏕⫱ཬࡧ⳦ྀࡢᙧែࢆㄪᰝ ࡍࡿࠊ⳦⣒⏕⫱ࠊศ㞳⳦ࡀ⏕⏘ࡋࡓⰍ⣲ࡸ⳦ྀࡢᙧែࡁ࡞㐪࠸ࡀぢࡽࢀࠊ✀㢮ࡢ␗࡞
ࡿࡁࡢࡇᐤ⏕ཪࡣඹ⏕ࡍࡿ⳦㢮ࡀศ㞳ࡉࢀ࡚࠸ࡿ⪃࠼ࡽࢀࡓࠋࡲࡓࠊࡁࡢࡇࡀ⏕ᜥࡋࡓ ሙᡤࡢ⎔ቃ᮲௳ࡢ㐪࠸ࡼࡾࠊࡇࡢࡼ࠺ከᵝ࡞⳦ࡀศ㞳ࡉࢀࡓ⪃࠼ࡽࢀࡓࠋࡇࢀࡲ࡛
㣗⏝ࡁࡢࡇࢆ⏝࠸ࡓ᳜≀ཎ⳦ࡢ㜵㝖㛵ࡍࡿሗ࿌ࡣ࠶ࡿࠋࡋࡋࠊ㔝⏕ࡢࡁࡢࡇࢆ⏝࠸࡚ࠊ ࠕࡁࡢࡇᐤ⏕ཪࡣඹ⏕ࡍࡿ⳦㢮ࠖࢆ᳜≀ཎ⳦ࡢ㜵㝖⏝ࡋࡓࡣⓙ↓࡛࠶ࡾࠊᅇࡢ ᐇ㦂ࡣ㜵㝖⏝ྍ⬟࡞᪂ࡓ࡞⳦ࡢⓎぢࡘ࡞ࡀࡿྍ⬟ᛶࡀ࠶ࡿࠋ
ࡑࡇ࡛ࠊPSᾮయᇵᆅࢆ⏝࠸࡚ㄪ〇ࡋࡓศ㞳⳦(105⳦ᰴ)ᇵ㣴ࢁᾮࡢࢫࢡ࣮ࣜࢽࣥࢢࢆ⾜ࡗ ࡓࠋࡑࡢ⤖ᯝࠊࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆཬࡧ╔ჾᙧᡂࢆᢚไࡍࡿ20⳦ᰴࡀࢫࢡ࣮ࣜࢽ
ࣥࢢࡉࢀࡓࠋ㑅ᢤࡉࢀࡓ20ศ㞳⳦ࡢᇵ㣴ࢁᾮࢆ⇕ฎ⌮ࡋࠊࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆࡢᢚ ไຠᯝࢆㄪᰝࡋࡓࠋࡑࡢ⤖ᯝࠊF2ࠊF5ࠊH9ࠊK1ࠊO1ࠊO3ࠊO6ࠊS5ཬࡧS9ศ㞳⳦ࡢᇵ㣴ࢁ
ᾮࡀⴭࡋࡃࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆཬࡧ╔ჾᙧᡂࢆᢚไࡋࡓࠋ᳜≀ཎ⳦ࡢ⣽⬊ቨཬ ࡧ⣽⬊⭷ࢲ࣓࣮ࢪࢆ࠼ࡿ≀㉁ࡋ࡚ࠊ࢟ࢳࢼ࣮ࢮཬࡧβ-1, 3ࢢࣝ࢝ࢼ࣮ࢮࡢࡼ࠺࡞㓝⣲
ࡀ▱ࡽࢀ࡚࠾ࡾࠊࡑࢀࡽࢆ⏕⏘ࡍࡿᚤ⏕≀ࡀᏑᅾࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ (Lorito et al.
1994㸹Shoresh and Harman 2010㸹Sakdapetsiri et al. 2016)ࠋࡋࡋࠊF2ࠊF5ࠊH9ࠊK1ࠊO1ࠊO3ࠊ
O6ࠊS5ཬࡧ S9 ศ㞳⳦ࡢᇵ㣴ࢁᾮ୰⏕⏘ࡉࢀࡿᢚไ≀㉁ࡣ⇕Ᏻᐃ࡛ࠊࡇࢀࡽࡢ⳦ᰴࡀ⏕
⏘ࡍࡿ≀㉁ࡣ࢟ࢳࢼ࣮ࢮཬࡧβ-1, 3ࢢࣝ࢝ࢼ࣮ࢮࡢࡼ࠺࡞≀㉁࡛ࡣ࡞࠸ࡇࡀ♧ࡉࢀࡓࠋ୍
᪉ࠊF3ࠊH2ࠊK21ࠊK24 ཬࡧS8ศ㞳⳦ࡢᇵ㣴ࢁᾮᏑᅾࡍࡿᢚไ≀㉁ࡣ⇕Ᏻᐃ࡛࠶ࡿࡇ
ࡽࠊ࢟ࢳࢼ࣮ࢮཬࡧβ-1, 3ࢢࣝ࢝ࢼ࣮ࢮࡢࡼ࠺࡞≀㉁࡛࠶ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋࡉࡽ
ࠊ⇕ฎ⌮ࡋࡓF1ࠊH5ࠊH7ࠊK7ࠊK9ཬࡧK36ศ㞳⳦ࡢᇵ㣴ࢁᾮࡣ⬊ᏊⓎⱆࢆᢚไࡋ࡞
ࡗࡓࡀࠊ╔ჾᙧᡂࢆᢚไࡋࡓࠋࡇࡢࡇࡽࠊࡇࢀࡽࡢศ㞳⳦ࡢᇵ㣴ࢁᾮ୰ࡣ⇕Ᏻᐃ
࡞ᢚไ≀㉁⇕Ᏻᐃ࡞ᢚไ≀㉁ࡢ୧᪉ࡀྵࡲࢀ࡚࠸ࡿྍ⬟ᛶࡀ࠶ࡿࠋࡇࢀࡲ࡛ࠊ㔝⏕ࡁࡢࡇ
࡛࠶ࡿHypholoma fasciculare ཬࡧCantharellus cibariusࡀBacillus cereusࠊ Bacillus subtilis ཬ
ࡧStaphylococcus aureusᑐࡋ࡚ᢚไຠᯝࢆ♧ࡍᢠ⳦≀㉁ࢆ⏕⏘ࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ
(Barros et al. 2008)ࠋᚤ⏕≀ࡣ⮬↛⏺࡛ᵝࠎ࡞ື≀ࠊ᳜≀ࡸࡢᚤ⏕≀ඹ⏕ࡸᐤ⏕ࡢࡼ࠺࡞
┦㛵ಀࢆಖࡕ࡞ࡀࡽࠊ⏕Ꮡࡋ࡚࠸ࡿࠋ≉ࠊࡢᚤ⏕≀ࡢ⏕Ꮡ➇தࡣ⃭ࡋࡃࠊ⮬㌟ࢆ
ࡢᚤ⏕≀ࡽᏲࡿࡓࡵᛶ㉁ࡢ␗࡞ࡿከᵝ࡞2ḟ௦ㅰ⏘≀ࢆ⏕⏘ࡋ࡚࠸ࡿ⪃࠼ࡽࢀࡿࠋ 㻌 䜸䜸䝮䜼䛷䛾๓ฎ⌮䛻䛚䛔䛶䜒ᢚไຠᯝ䜢♧䛧䛯 O1䚸O3䚸S5 ཬ䜃 S9 ⳦ᰴࡢྠᐃࢆ⾜ࡗࡓ
⤖ᯝࠊS9䛿Annulohypoxylon sp.䚸S5䛿Nigrospora sp.ཬ䜃O1䛸O3 䛿Penicillium sp.䛸㧗䛔┦
ྠ ᛶ 䜢 ♧ 䛧 䛯 䚹Penicillium spp.䛜 ᵝ 䚻 䛺 ᳜ ≀ ཎ ⳦ 䜢 ᢚ ไ 䛩 䜛 䛣 䛸 䛿 䜘 䛟 ▱ 䜙 䜜 䛶 䛔 䜛 䚹 Penicillium spp.䛜⏕⏘䛩䜛᳜≀ཎ⳦䛾ឤᰁ䜢ᢚไ䛩䜛≀㉁䛸䛧䛶Mycophenolic acid䚸Patulin䚸 3-omethylfuniconeཬ䜃 Decumbenone A㻌 䛜▱䜙䜜䛶䛔䜛(Fujii et al. 2002䠗Nicoletti and Stefano
20
2012)䚹䜎䛯䚸Nigrospora sphaerica (Sacc.) E.W. Mason㻌 䛛䜙ศ㞳䛥䜜䛯 Phomalactone 䛿᳜≀
ཎ⳦䛾ឤᰁ⾜ື䜢ᢚไ䛧(Kim et al. 2001)䚸Nigrospora oryzae (Berk. & Br.) Petch 䛾ᇵ㣴䜝ᾮ䛻
⏕⏘䛥䜜䜛Nigrosporin Aཬ䜃B䛻䛿 ᳜≀ẘᛶ䛜䛒䜛䛣䛸䛜▱䜙䜜䛶䛔䜛(Tanaka et al. 1997)䚹䛧 䛛䛧䚸ᮏ◊✲䛻⏝䛔䛯Nigrospora sp.䛾ᇵ㣴䜝ᾮ䛿䜸䜸䝮䜼䛻ᑐ䛧䛶䚸ẘᛶ䜢♧䛥䛺䛛䛳䛯䚹䛥䜙䛻䚸
Annulohypoxylon sp. 䛾ᇵ㣴䜝ᾮ䛻䛴䛔䛶䛿䚸᳜≀ཎ⳦䛾㜵㝖䛻⏝䛥䜜䛯䛜䛣䜜䜎䛷ሗ࿌
䛥䜜䛶䛔䛺䛔䚹Annulohypoxylon sp.䛿Xylariaceae ⛉䛻ᒓ䛧䛶䛔䜛䛣䛸䛜▱䜙䜜䛶䛔䜛䚹Xylariaceae 䛻䛿36௨ୖ䛾ᒓ䛜ྵ䜎䜜䛶䛚䜚䚸ᑡ䛺䛟䛸䜒䛣䜜䜙䛾3ศ䛾1䛾⳦䛿2ḟ௦ㅰ⏘≀䜢⏕⏘䛩䜛䛣 䛸䛜▱䜙䜜䛶䛔䜛(Whalley and Edwards 1995)䚹䛘䜀䚸Xylariaceae ⛉䛻ᒓ䛩䜛Xylaria longipes 䛜⏕⏘䛩䜛Xylaramide䛿Nematospora coryliཬ䜃Saccharomyces cerevisiae䛻ᑐ䛧䛶ᢠ⳦άᛶ 䜢♧䛩䛣䛸䛜ሗ࿌䛥䜜䛶䛔䜛(Schneider et al. 1996)䚹䛣䜜䜙䛾୰䛻䛿䜲䝛䛔䜒䛱䜢ྵ䜐᳜≀ཎ
⳦䛻ᑐ䛧䛶ᢚไຠᯝ䜢♧䛩≀㉁䜒ྵ䜎䜜䛶䛔䜛ྍ⬟ᛶ䛜䛒䜛䚹ᚋ䚸Annulohypoxylon sp.䛾ᇵ㣴 䜝ᾮ䛾ᢚไ≀㉁䛻䛴䛔䛶ㄪᰝ䛩䜛ᚲせ䛜䛒䜛䚹ᅇ䛾ᐇ㦂䛻䜘䜚㔝⏕䛝䛾䛣䛛䜙ศ㞳䛥䜜䛯⳦㢮 䛾ᇵ㣴䜝ᾮ୰䛻䛿䜲䝛䛔䜒䛱⳦䜢ᢚไ䛷䛝䜛」ᩘ䛾≀㉁䛜Ꮡᅾ䛧䛶䛔䜛䛣䛸䛜♧䛥䜜䛯䚹
21
➨
➨3❶ O821⳦ᰴࡢ᳜≀ཎ⳦ᑐࡍࡿᢚไຠᯝࡘ࠸࡚
๓❶࠾࠸࡚ࠊ᪥ᮏᅜෆ࡛᥇ྲྀࡋࡓ㔝⏕ࡁࡢࡇࡽศ㞳ࡉࢀࡓ⳦㢮ࡢᇵ㣴ࢁᾮࡀࢿ࠸
ࡶࡕ⳦ࡢឤᰁ⾜ືཬࡧᩬᙧᡂࢆᢚไࡍࡿࡇࢆ᫂ࡽࡋࡓࠋࡲࡓࠊ㜰ᗓෆ࡛᥇ྲྀࡋ ࡓ5✀㢮ࡢ㔝⏕ࡁࡢࡇࡽࡣࠊྜィ15⳦ᰴࡀศ㞳ࡉࢀ࡚࠸ࡿࠋࡇࢀࡽࡢศ㞳⳦(15⳦ᰴ)
ࢿ࠸ࡶࡕ⳦ࡢᑐᓖᇵ㣴࡛ࡣࠊ⏕⫱㜼Ṇᖏࡣほᐹࡉࢀ࡞ࡗࡓࠋ୍᪉ࠊศ㞳⳦ࡢᇵ㣴ࢁᾮ
ࡼࡿࢿ࠸ࡶࡕ⳦ࡢឤᰁ⾜ືࡢᢚไຠᯝヨ㦂࡛ࡣࠊࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆཬࡧ
╔ჾᙧᡂࢆᏳᐃⓗᢚไࡍࡿ O821 ⳦ᰴࡀ㑅ᢤࡉࢀࡓࠋᮏ❶࡛ࡣࠊO821 ⳦ᰴࡢྠᐃཬࡧ O821⳦ᰴࡢᇵ㣴ࢁᾮࢆ⏝࠸ࡓࢿ࠸ࡶࡕ⳦ࡢᢚไຠᯝࡘ࠸࡚㏙ࡿࠋ
➨1⠇ O821⳦ᰴࡢ≉ᚩཬࡧྠᐃ 1㸬ᮦᩱཬࡧ᪉ἲ
a)౪ヨ⳦
O821⳦ᰴࢆᐇ㦂⏝࠸ࡓࠋ⳦ࡣ➨2❶ࠊ➨2⠇ࠊ1a)࡛㏙ࡓ᪉ἲࡼࡾ‽ഛࡋࡓࠋ b)⳦ྀཬࡧ⬊Ꮚࡢほᐹ
PSA ᇵᆅୖ࡛ 7᪥㛫ᇵ㣴ࡋࡓO821 ⳦ᰴࡢ⳦ྀࡢᵝᏊࢆほᐹࡋࡓࠋගᏛ㢧ᚤ㙾ࢆ⏝࠸࡚
ᙧែⓗ࡞≉ᚩࢆほᐹࡋࡓࠋࡲࡓࠊO821⳦ᰴࡢ⳦ྀࢆࢢࣝࢱࣝࣝࢹࣄࢻᅄ㓟࢜ࢫ࣑࢘
࣒ࡼࡾ㔜ᅛᐃࡋࠊ㟁Ꮚ㢧ᚤ㙾(᪥❧㉸㧗ศゎ⬟㟁⏺ᨺฟᙧ㉮ᰝ㟁Ꮚ㢧ᚤ㙾S-4800ࠊᰴᘧ
♫᪥❧ࣁࢸࢡࣀࣟࢪ࣮ࢬ)ࢆ⏝࠸࡚ほᐹࡋࡓࠋ
c)DNAࡢᢳฟཬࡧITS㡿ᇦࡢࢩ࣮ࢡ࢚ࣥࢫゎᯒ
DNAᢳฟࡣSuzuki et al. (2006) ࡢ᪉ἲᚑࡗ࡚⾜ࡗࡓࠋ➨2❶ࠊ➨3⠇ࠊ1b)ྠᵝ࡞᪉
ἲࢆ⏝࠸࡚ศ㞳⳦O821⳦ᰴࡢྠᐃࢆ⾜ࡗࡓࠋ⣔⤫ᶞࡣSaitou and Nei (1987) ᇶ࡙࠸࡚స ᡂࡋࡓࠋ
2㸬⤖ᯝ
a)O821 ⳦ᰴࡢほᐹ
O821 ⳦ᰴࡢ⳦ྀཬࡧ⬊Ꮚࡢᙧែほᐹࢆ⾜ࡗࡓࠋPSAᇵᆅ࡛ᇵ㣴ࡋࡓO821⳦ᰴࡣᇵ㣴7
᪥┠௨㝆ࠊⓑⰍࡽⷧ㯤Ⰽࡢ⳦ྀ࡞ࡾࠊࡑࡢᚋࠊ⃰⥳Ⰽኚࡋࠊᇵ㣴28᪥┠ࡣࠊ⳦
ྀࡢⰍࡣ㯮Ⰽኚࡋ࡚࠸ࡓ(Fig. 3-1)ࠋ⬊Ꮚࡢᙧᡂࡀ☜ㄆࡉࢀ࡞ࡗࡓ(Fig. 3-1)ࠋ
b)O821 ⳦ᰴࡢࢩ࣮ࢡ࢚ࣥࢫゎᯒ
O821 ⳦ᰴࡢITS㡿ᇦࡢࢩ࣮ࢡ࢚ࣥࢫࡢᚋࠊBLAST᳨⣴GENETYXࢆ⏝࠸ࡓゎᯒࢆ
⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊO821⳦ᰴࡣBiscogniauxia sp. HHP54ᰴ㧗࠸┦ྠᛶ(98 %)ࢆ♧ࡋࡓ(Fig.
3-2)ࠋ
22
Fig. 3-1. Morphological characteristics of isolate O821 on PSA medium. A: Isolate O821 grown on PSA for 7 days. B: Isolate O821 grown on PSA for 28 days. C, D: Mycelia (Light microscopy).
E, F: Mycelia (Scanning electron microscopy).
10 μm 5 μm
23
Fig. 3-2. Phylogenetic tree based on ITS sequences of isolate O821. A bootstrap consensus neighbor-joining tree for the isolate O821 was created based on the Kimura 2-Parameter distance matrix (1000 replicates). Lopadostoma amoenum strain LA (KC774569) was used as the out-group. The scale bar represents 10% sequence dissimilarity.
24
➨
➨2⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮࡼࡿࢿ࠸ࡶࡕ⳦ࡢᢚไຠᯝ (⇕ฎ⌮) 1㸬ᮦᩱཬࡧ᪉ἲ
a)౪ヨ⳦ཬࡧᇵ㣴᮲௳
ࢿ࠸ࡶࡕ⳦ཬࡧO821⳦ᰴࡣ➨2❶ࠊ➨2⠇ࠊ1a)࡛㏙ࡓ᪉ἲࡼࡾ‽ഛࡋࡓࠋ b)ᇵ㣴ࢁᾮࡢㄪ〇
O821⳦ᰴࡣணࡵPSAᇵᆅ࡛ᇵ㣴ᚋࠊ7 mmࢥࣝࢡ࣮࣮࡛࣎ࣛ⳦ྀࢆࡃࡾᢤࡁࠊPSᾮయᇵ ᆅ(20 ml)1∦ࡎࡘ⛣᳜ࡋࡓࠋ⛣᳜ᚋࠊ7᪥㛫ࠊ120ᅇ㌿/ศ࡛┞ᇵ㣴ࡋࡓࠋ┞ᇵ㣴ᚋࠊ
ᇵ㣴ᾮ20 mlྠ㔞ࡢ㓑㓟࢚ࢳࣝࢆຍ࠼࡚ศ㓄ᢳฟࡋࡓࠋࡇࡢᢳฟ᧯సࢆ1ヨᩱࡘࡁ2ᅇ
⧞ࡾ㏉ࡋࡓࠋᚓࡽࢀࡓ㓑㓟࢚ࢳࣝྍ⁐㒊(⣙40 ml)⁛⳦Ỉࢆຍ࠼ࠊᆺࣇࣛࢫࢥࢆ⏝࠸࡚ࠊ
࢚ࣂ࣏࣮ࣞࢱ࣮(NA-1 SGSࠊࢬ࣡ࣥᰴᘧ♫)ࡼࡾῶᅽୗ࡛⃰⦰ࡋࡓࠋ㓑㓟࢚ࢳࣝࢆ㝖ཤ ࡋࡓỈ⁐ᾮࢆㄪ〇ࡋࠊᵝࠎ࡞⃰ᗘ࡛ᇵ㣴ࢁᾮ㓑㓟࢚ࢳࣝྍ⁐㒊Ỉ⁐ᾮ(௨ୗ㸸ᇵ㣴ࢁᾮ)ࡋ
࡚ᐇ㦂⏝࠸ࡓࠋỈ⁐ᾮ20 mlㄪ〇ࡋࡓࡶࡢࢆཎᾮ(1ಸ)ࡋࡓࠋᑐ↷༊ࡋ࡚ࠊPSᾮయ ᇵᆅࢆྠᵝࡢ᪉ἲ࡛ㄪ〇ࡋࡓࡶࡢࢆ⏝࠸ࡓࠋ
c)⇕ฎ⌮
ୖ㏙ࡢ᪉ἲ࡛ㄪ〇ࡋࡓᇵ㣴ࢁᾮ(ཎᾮ㸸1ಸ)ࢆ࣮࢜ࢺࢡ࣮ࣞࣈ(121Υࠊ20ศ㛫)ࡋࠊ⇕ฎ⌮
ࡋࡓᇵ㣴ࢁᾮࡋ࡚ᐇ㦂⏝࠸ࡓࠋ d)⏕≀᳨ᐃἲ
ྛᇵ㣴ࢁᾮࡣ➨2❶ࠊ➨2⠇ࠊ1b)࡛㏙ࡓ᪉ἲࡼࡾࢿ࠸ࡶࡕ⳦ᑐࡍࡿᢚไຠᯝ
ࢆㄪᰝࡋࡓࠋ
2㸬⤖ᯝ
O821 ⳦ᰴࡢᇵ㣴ࢁᾮ୰ྵࡲࢀࡿࢿ࠸ࡶࡕ⳦ࡢឤᰁ⾜ືࢆᢚไࡍࡿ≀㉁ࡢ⇕Ᏻᐃ
ᛶࢆㄪᰝࡋࡓࠋࡑࡢ⤖ᯝࠊᑐ↷༊ࡋ࡚⏝࠸ࡓ PS ᾮయᇵᆅࡢ↓ฎ⌮༊ཬࡧ⇕ฎ⌮༊࡛ࡣࠊ
⬊ᏊⓎⱆ⋡ࡣ 98.7±1.7%ཬࡧ 99.8±0.7%࡛࠶ࡗࡓࡢᑐࡋ࡚ࠊO821 ⳦ᰴࡢᇵ㣴ࢁᾮࡢ↓ฎ
⌮༊ཬࡧ⇕ฎ⌮༊࡛ࡣࠊ⬊ᏊⓎⱆ⋡1.4±1.8%ࡣཬࡧ1.3±1.5%࡛࠶ࡗࡓ(Fig. 3-3 A)ࠋࡲࡓࠊ
╔ჾᙧᡂ⋡ࡣPSᾮయᇵᆅࡢ↓ฎ⌮༊ཬࡧ⇕ฎ⌮༊ࠊO821 ⳦ᰴࡢᇵ㣴ࢁᾮࡢ↓ฎ⌮༊ཬࡧ
⇕ฎ⌮༊࡛ࡑࢀࡒࢀ97.9±1.6%ࠊ99.1±1.2%ࠊ0.2±0.6%ཬࡧ0.0±0.0%(Fig. 3-3 B)࡞ࡾࠊO821
⳦ᰴࡢᇵ㣴ࢁᾮ୰ྵࡲࢀࡿࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆཬࡧ╔ჾᙧᡂࢆᢚไࡍࡿ≀㉁ࡣࠊ
⇕Ᏻᐃ࡛࠶ࡗࡓࠋ
25
Fig. 3-3. Inhibition by heat-treated ethyl acetate extract of culture filtrate of isolate O821 (O821- CF) on the infection behaviors of Magnaporthe oryzae. O821-CF was treated at 121Υ, 20 minutes.
Spores of M. oryzae were dropped on glass slides in the presence of heat-pretreated O821-CF and kept in a moist chamber at 26Υ. As a control, potato sucrose broth (PSB) was used. After 24 h of incubation in a moist chamber, the spore germination (A) and appressorium formation (B) were observed by light microscopy. The rates of spore germination, and appressorium formation per germinating spore were calculated. Experiments were repeated three times and a total of 300 spores were examined per experiment. Bars represent ± SD. Means followed by different letters indicate significant difference (Tukey-Kramer test, p < 0.05).
Percentage of spore germination (%)
PSB No treatment
O821-CF 121Υ No treatment 121Υ
)100
tion
80
germ 60
of sp 40
centa
20 0
a a
b b
Percentage of appressorium formation (%) ) 100 80
60
40 20
0 No treatment atment 121Υ 12 No treatment PSB
121Υ eatment 12
O821-CF
b a
c c
A
B
26
➨
➨3⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮࡼࡿࢿ࠸ࡶࡕ⳦ࡢᢚไຠᯝ (ᇵ㣴᮲௳) 1㸬ᮦᩱཬࡧ᪉ἲ
a)౪ヨ⳦ཬࡧᇵ㣴᮲௳
ࢿ࠸ࡶࡕ⳦ཬࡧO821⳦ᰴࡣ➨2❶ࠊ➨2⠇ࠊ1a)࡛㏙ࡓ᪉ἲࡼࡾ‽ഛࡋࡓࠋ b)ᇵ㣴ࢁᾮࡢㄪ〇
O821 ⳦ᰴࡣPSᾮయᇵᆅ࡛1-7᪥㛫ᇵ㣴ࡋࡓࠋᇵ㣴ᚋࠊᇵ㣴ࢁᾮࡢㄪ〇ࡣ➨3 ❶ࠊ➨ 2
⠇ࠊ1b)࡛㏙ࡓ᪉ἲࡼࡾ‽ഛࡋࡓࠋ c)⏕≀᳨ᐃἲ
ྛᇵ㣴ࢁᾮࡣ➨2❶ࠊ➨2⠇ࠊ1b)࡛㏙ࡓ᪉ἲࡼࡾࢿ࠸ࡶࡕ⳦ᑐࡍࡿᢚไຠᯝ
ࢆㄪᰝࡋࡓࠋ
2㸬⤖ᯝ
PSᾮయᇵᆅ࡛1-7᪥㛫ᇵ㣴ࡋࡓO821⳦ᰴࡽㄪ〇ࡋࡓᇵ㣴ࢁᾮ(ཎᾮ㸸1ಸ)ࢆ⏝࠸࡚ࠊ ࡑࢀࡒࢀࡢᇵ㣴ࢁᾮࡢࢿ࠸ࡶࡕ⳦ࡢឤᰁ⾜ືᑐࡍࡿຠᯝࢆㄪᰝࡋࡓࠋࡑࡢ⤖ᯝࠊᑐ↷
༊ࡋ࡚⏝࠸ࡓPSᾮయᇵᆅ࡛ࡣࠊ⬊ᏊⓎⱆ⋡ࡣ99.1±1.4%࡛࠶ࡗࡓࡢᑐࡋ࡚ࠊO821⳦ᰴ
ࢆ1-7᪥㛫ᇵ㣴ᚋㄪ〇ࡋࡓᇵ㣴ࢁᾮ࡛ࡣࠊࡑࢀࡒࢀ98.4±2.6%ࠊ98.4±2.6%ࠊ99.4±0.9%ࠊ 35.0±47.3%ࠊ30.9±44.3%ࠊ0.8±1.0%ཬࡧ0.3±0.8%࡛࠶ࡗࡓ(Fig. 3-4 A)ࠋࡲࡓࠊ╔ჾᙧᡂ⋡
ࡣPSᾮయᇵᆅ࡛ࡣ98.9±1.4%࡞ࡾࠊO821⳦ᰴࢆ1-7᪥㛫ᇵ㣴ᚋㄪ〇ࡋࡓᇵ㣴ࢁᾮ࡛ࡣ ࡑࢀࡒࢀ98.2±2.6%ࠊ97.4±3.0%ࠊ99.1±1.0%ࠊ33.6±48.1%ࠊ25.1±38.1%ࠊ0.0±0.0% ཬࡧ0.0±0.0%
࡛࠶ࡗࡓ(Fig. 3-4 B)ࠋO821⳦ᰴࡢᇵ㣴ࢁᾮࡼࡿࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆཬࡧ╔ჾᙧ ᡂࡢᢚไࡣᇵ㣴4᪥௨㝆ࡽほᐹࡉࢀࠊ6᪥௨㝆ࡣ㧗࠸ᢚไຠᯝࡀほᐹࡉࢀࡓ(Fig. 3-4 Aࠊ B)ࠋ
27 100
80 60 40 20 0
100 80 60 40 20 0
Fig. 3-4. Time course of inhibitory activity of the ethyl acetate extract of culture filtrates of isolate O821 (O821-CF) on spore germination and appressorium formation of Magnaporthe oryzae. Spores of M. oryzae were suspended in O821-CF for different time periods and dropped on glass slides.
Potato sucrose Broth (PSB) was used as a control. After 24 h of incubation in a moist chamber, the spore germination (A) and appressorium formation (B) were observed by light microscopy. The rates of spore germination, and appressorium formation per germinating spore were calculated.
Experiments were repeated three times and a total of 300 spores were examined per experiment.
Bars represent ± SD. Means followed by different letters indicate significant difference (Tukey- Kramer test, p < 0.05).
Percentage of spore germination (%)
PSB 1 2 3 4 5 6 7 a
c b
b
Incubation period of O821 (days)
Percentage of appressorium formation (%)
A
B
a a a
c
PSB 1 2 3 4 5 6 7 Incubation period of O821 (days)
a a a a
b
b
c c
28
➨
➨4⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮࡼࡿࢿ࠸ࡶࡕ⳦ࡢᢚไຠᯝ (⳦⣒⏕⫱) 1. ᮦᩱཬࡧ᪉ἲ
a)౪ヨ⳦ཬࡧᇵ㣴᮲௳
ࢿ࠸ࡶࡕ⳦ཬࡧO821⳦ᰴࡣ➨2❶ࠊ➨2⠇ࠊ1a)࡛㏙ࡓ᪉ἲࡼࡾ‽ഛࡋࡓࠋ b)ᇵ㣴ࢁᾮࡢㄪ〇
ᇵ㣴ࢁᾮࡢㄪ〇ࡣ➨3 ❶ࠊ➨2⠇ࠊ1b)࡛㏙ࡓ᪉ἲࡼࡾ‽ഛࡋࡓࠋ c)⳦⣒⏕⫱ᢚไヨ㦂
ணࡵࠊ⡿ࡠᇵᆅ࡛ᇵ㣴ࡋࡓࢿ࠸ࡶࡕ⳦ࡢ⳦ྀ⁛⳦Ỉ2 mlࢆὀࡂࠊࣛࣂ࣮ࢫࣃࢳ
࡛ࣗࣛࡁࡗࡓࠋPSAᇵᆅࢆ⣙20 mlὶࡋࡓࣉࣛࢫࢵࢳࢡࢩ࣮ࣕࣞ(┤ᚄ7 cm)20 μlࡢ
⳦⣒ᠱ⃮ᾮࢆῧຍࡋࠊࢭࣝࢫࣉࣞࢵࢲ࣮(Heathrow Scientific ® LLC. Delta™Disposable Cell Spreader )ࢆ⏝࠸࡚ࠊࢩ࣮ࣕࣞሬᕸࡋࡓࠋࡑࡢᚋࠊᢠ⏕≀㉁᳨ᐃ⏝࣮࣌ࣃ࣮ࢹࢫࢡ(┤ᚄ 8 mm)(Advantec ®. Filter paper )ཎᾮ(1ಸ)ࠊ2ಸࠊ5ಸࠊ10ಸཬࡧ20ಸ⃰⦰ࡋࡓO821⳦ᰴ
ࡢᇵ㣴ࢁᾮࢆฎ⌮ࡋࠊୖグࡢࢿ࠸ࡶࡕ⳦ࡢ⳦⣒ࡀሬᕸࡉࢀࡓࢩ࣮ࣕࣞࡢ୰ᚰ⨨࠸ࡓ
(Palaniyandi et al. 2011)ࠋᑐ↷༊ࡋ࡚20ಸ⃰⦰ࡋࡓPSᾮయᇵᆅࡢ㓑㓟࢚ࢳࣝྍ⁐㒊Ỉ⁐
ᾮࢆ⏝࠸ࡓࠋᇵ㣴ࡣࠊᬯ㯮ୗࠊ26Υ࡛⾜࠸ࠊᇵ㣴5᪥ཬࡧ28᪥ᚋࠊࢿ࠸ࡶࡕ⳦⳦⣒
ࡢ⏕⫱㜼Ṇᖏࡢ┤ᚄࢆ ᐃࡋࠊᖹᆒࢆ⟬ฟࡋࡓࠋ1ࡘࡢฎ⌮༊ࡘࡁ6ᯛࡢࢩ࣮ࣕࣞࢆ⏝࠸
࡚ࠊᐇ㦂ࡣ3ᅇ⧞ࡾ㏉ࡋࡓࠋ
2㸬⤖ᯝ
ᢠ⏕≀㉁᳨ᐃ⏝࣮࣌ࣃ࣮ࢹࢫࢡࢆ⏝࠸࡚ࠊO821⳦ᰴࡢᇵ㣴ࢁᾮࡀࢿ࠸ࡶࡕ⳦ࡢ⳦
⣒⏕⫱࠼ࡿᙳ㡪ࢆㄪᰝࡋࡓࠋࡑࡢ⤖ᯝࠊᇵ㣴5᪥┠ࡢ⏕⫱㜼Ṇᖏࡢ┤ᚄࡣᑐ↷༊ࠊ1ಸࠊ 2ಸࠊ5ಸࠊ10ಸཬࡧ20ಸ⃰⦰ࡋࡓO821⳦ᰴࡢᇵ㣴ࢁᾮ࡛ࡑࢀࡒࢀ0.0±0.0 mmࠊ0.0±0.0 mmࠊ0.0±0.0 mmࠊ4.6±6.7 mmࠊ14.9±2.8 mmཬࡧ20.8±3.2 mm࡛࠶ࡗࡓ(Fig. 3-5)ࠋࡲࡓࠊᇵ 㣴28᪥┠࡛ࡢ⏕⫱㜼Ṇᖏࡢ┤ᚄࡣࡑࢀࡒࢀ0.0±0.0 mmࠊ0.0±0.0 mmࠊ0.0±0.0 mmࠊ2.9±4.4 mmࠊ11.6±2.1 mmཬࡧ15.6±2.1 mm࡛࠶ࡗࡓ(Fig. 3-6)ࠋᇵ㣴5᪥┠࡛ࡢ⏕⫱㜼Ṇᖏࡣ5ಸ⃰
⦰௨ୖ࡛ほᐹࡉࢀࠊ⃰ᗘ౫Ꮡⓗᢚไࡣ㧗ࡃ࡞ࡗ࡚࠸ࡓࠋࡲࡓࠊࡑࡢᢚไࡣᇵ㣴28᪥┠࡛
ࡶᣢ⥆ࡋ࡚࠸ࡓࠋ
29
Growth inhibition zone (mm)
O821-20
mm)
25
one 20
bitio 15
10 5 0 30
PSB-20 0 ‒ O821-1 1 ‒ O821-2 O821-5‒ O821-10 a a a
b
c
d O821-20
PSB-20 ‒ O821-1 ‒ O821-2
O821-5 O821-10
Fig. 3-5. Effect of the ethyl acetate extract of culture filtrates of isolate O821 (O821-CF) on mycelial growth of Magnaporthe oryzae on the PSA plate. A mycelial suspension of M. oryzae was spread onto PSA medium. After that, a paper disk immersed in 1-, 2-, 5-, 10- or 20-fold- concentrated O821-CF was transferred onto PSA medium. After 5 days of incubation at 26Υ, growth inhibition zone was measured. Bars represent ± SD. Means followed by different letters are significantly different using the Tukey-Kramer test (p < 0.05).
30
Fig. 3-6. Effect of the ethyl acetate extract of culture filtrates of isolate O821 (O821-CF) on mycelial growth of Magnaporthe oryzae on the PSA plate. A mycelial suspension of M. oryzae was spread onto PSA medium. After that, a paper disk immersed in 1-, 2-, 5-, 10- or 20-fold- concentrated O821-CF was transferred onto PSA medium. After 28 days of incubation at 26Υ, growth inhibition zone was measured. Bars represent ± SD. Means followed by different letters are significantly different using the Tukey-Kramer test (p < 0.05).
O821-20 PSB-20 ‒ O821-1 ‒ O821-2
O821-5 O821-10
O821-20 PSB-20 ‒ O821-1 ‒ O821-2 O821-5‒ O821-10
Growth inhibition zone (mm) zone
20
hibiti15
wthi
10
G 5
0 a a a
b
c
d
mm)
25 30
31
➨
➨5⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮࡼࡿ᳜≀ཎ⳦ࡢᢚไຠᯝ 1㸬ᮦᩱཬࡧ᪉ἲ
a)౪ヨ⳦ཬࡧᇵ㣴᮲௳
O821⳦ᰴࡣ➨2❶ࠊ➨2⠇ࠊ1a)࡛㏙ࡓ᪉ἲࡼࡾ‽ഛࡋࡓࠋ
ࢿࡈࡲⴥᯤ⳦(Bipolaris oryzae)ࠊ࣐ࣥࢦ࣮Ⅳ⳦(Colletotrichum gloeosporioides)ཬࡧ
࢘ࣜ㢮Ⅳ⳦(Colletotrichum orbiculare)ࡣ⣙20 ml ࡎࡘࣉࣛࢫࢳࢵࢡࢩ࣮ࣕࣞ(┤ᚄ 7 cm)
ศὀࡋ࡚ᅛࡵࡓ⡿ࡠᐮኳᇵᆅࢆ⏝࠸࡚ࠊ26Υࡢᬯ㯮ୗ࡛7-10᪥㛫ᇵ㣴ࡋࠊ⬊Ꮚᙧᡂࡉ ࡏࡓࠋ
࢟ࣗ࢘ࣜ〓ᩬ⳦(Corynespora cassiicora)ཬࡧࢼࢩ㯮ᩬ⳦(Alternaria alternate Japanese
pear pathotype)ࡣ⣙20 ml ࡎࡘࣉࣛࢫࢳࢵࢡࢩ࣮ࣕࣞ(┤ᚄ7 cm)ศὀࡋ࡚ᅛࡵࡓ⡿ࡠᐮ
ኳᇵᆅࢆ⏝࠸࡚ࠊ26Υࡢᬯ㯮ୗ࡛7-10᪥㛫ᇵ㣴ࡋࡓᚋࠊ⳦⣒ࢆ㝖ཤࡋࠊBLB↷ᑕୗ࡛2᪥ 㛫ᇵ㣴ࡋࠊ⬊Ꮚᙧᡂࡉࡏࡓࠋ
ࢳࢪࢡᰴᯤ⳦(Ceratocysts fimbriata)ࠊ࣍࢘ࣞࣥࢯ࢘ⴎ⳦(Fusarium oxysporum f. sp.
spinaciae)ࠊ࢝ࣈⴎ㯤⳦(Fusarium oxysporum f. sp. conglutinans) ཬࡧࢩࣕࢡࣖࢡ᰿㯮ᩬ⳦
(Cylindrocarpon destructans )ࡣPSAᇵᆅࢆ⏝࠸࡚ࠊ26Υࡢᬯ㯮ୗ࡛7-10᪥㛫ᇵ㣴ࡋࠊ⬊Ꮚᙧ ᡂࡉࡏࡓࠋ
⅊Ⰽࡧ⳦(Botrytis cinerea)ࡣPSAᇵᆅࢆ⏝࠸࡚ࠊ26ΥࡢBLB↷ᑕୗ࡛7-10᪥㛫ᇵ㣴 ࡋࠊ⬊Ꮚᙧᡂࡉࡏࡓࠋ
⬊Ꮚᙧᡂᚋࠊࢩ࣮ࣕࣞ⁛⳦Ỉࢆὀࡂࠊࣛࣂ࣮ࢫࣃࢳࣗࣛࢆ⏝࠸࡚ࠊ⬊Ꮚࢆᅇࡋࠊ⃰ᗘ (1×105spores/ml)ࢆㄪ〇ࡋ࡚ࠊᐇ㦂⏝࠸ࡓࠋ
b)ᇵ㣴ࢁᾮࡢㄪ〇
ᇵ㣴ࢁᾮࡢㄪ〇ࡣ➨3 ❶ࠊ➨2⠇ࠊ1b)࡛㏙ࡓ᪉ἲࡼࡾ‽ഛࡋࡓࠋཎᾮ(1ಸ)ࠊ2ಸࠊ 5ಸཬࡧ10ಸ⃰⦰ࡋࡓᇵ㣴ࢁᾮࢆᐇ㦂⏝࠸ࡓࠋ
c)⏕≀᳨ᐃἲ
ྛᇵ㣴ࢁᾮࡣ➨2❶ࠊ➨2⠇ࠊ1b)࡛㏙ࡓ᪉ἲࡼࡾࡑࢀࡒࢀࡢ᳜≀ཎ⳦ᑐࡍࡿᢚ ไຠᯝࢆㄪᰝࡋࡓࠋ
2㸬⤖ᯝ
O821⳦ᰴࡢᇵ㣴ࢁᾮࡀࢿ࠸ࡶࡕ⳦௨እࡢ᳜≀ཎ⳦ᑐࡍࡿᢚไຠᯝࢆㄪᰝࡋࡓࠋ ࡑࡢ⤖ᯝࠊᑐ↷༊࡛࠶ࡿ10 ಸ⃰⦰ࡋࡓPSᾮయᇵᆅࡢฎ⌮༊࡛ࡣࠊ⏝࠸ࡓ᳜≀ཎ⳦ࡢⓎ
ⱆ⋡ࡣ࠸ࡎࢀࡶ99%௨ୖ࡛࠶ࡗࡓࡢᑐࡋ࡚ࠊO821⳦ᰴࡢᇵ㣴ࢁᾮࡢฎ⌮༊࡛ࡣࠊ1ಸࠊ 2ಸࠊ5ಸཬࡧ10ಸ⃰⦰ࡋࡓᇵ㣴ࢁᾮࡢ⬊ᏊⓎⱆ⋡ࡣ⅊Ⰽࡧ⳦࡛ࡣࡑࢀࡒࢀ52.9±36.0%ࠊ 26.8±38.6%ࠊ0.1±0.5%ཬࡧ 0.0±0.0%࡛࠶ࡗࡓ(Table 3-1)ࠋࢼࢩ㯮ᩬ⳦࡛ࡣ 85.3±11.4%ࠊ 80.3±15.2%ࠊ2.9±3.2%ཬࡧ0.1±0.5%࡛࠶ࡗࡓࠋࢿࡈࡲⴥᯤ⳦࡛ࡣ28.0±28.0%ࠊ27.7±32.1%ࠊ 0.1±0.5%ཬࡧ0.2±0.6%࡛࠶ࡗࡓࠋ࢘ࣜ㢮Ⅳ⳦࡛ࡣ4.9±11.4%ࠊ0.9±1.7%ࠊ0.2±0.6%ཬࡧ 0.0±0.0%࡛࠶ࡗࡓࠋ࢟ࣗ࢘ࣜ〓ᩬ⳦࡛ࡣ27.1±22.0%ࠊ2.0±2.8%ࠊ1.1±1.6%ཬࡧ0.4±0.9%࡛
32
࠶ࡗࡓࠋ࣐ࣥࢦ࣮Ⅳ⳦࡛ࡣ0.0±0.0%ࠊ0.0±0.0%ࠊ0.0±0.0%ཬࡧ0.0±0.0%࡛࠶ࡗࡓࠋࢳ ࢪࢡᰴᯤ⳦࡛ࡣ0.6±1.5%ࠊ0.0±0.0%ࠊ0.0±0.0%ཬࡧ0.1±0.5%࡛࠶ࡗࡓࠋ࢝ࣈⴎ㯤⳦࡛ࡣ 4.7±7.3%ࠊ0.7±1.2%ࠊ0.7±1.4%ཬࡧ0.1±0.5%࡛࠶ࡗࡓࠋࢩࣕࢡࣖࢡ᰿㯮ᩬ⳦࡛ࡣ5.0±13.6%ࠊ
0.6±2.4%ࠊ0.0±0.0%ཬࡧ 0.0±0.0%࡛࠶ࡗࡓࠋ࣍࢘ࣞࣥࢯ࢘ⴎ⳦࡛ࡣ 22.8±33.0%ࠊ
25.3±39.9%ࠊ1.8±2.8%ཬࡧ0.6±1.1%࡛࠶ࡗࡓࠋ
33 Botrytis cinerea 㻌
Alternaria alternate Japanese pear pathotype㻌
Bipolaris oryzae㻌
Colletotrichum orbiculare 㻌 Corynespora cassiicora㻌 Colletotrichum gloeosporioides㻌 Ceratocysts fimbriata
㻌
Fusarium oxysporum f.sp. conglutinans 㻌 Cylindrocarpon destructans 㻌
Fusarium oxysporum f.sp.spinaciae㻌
Table 3-1. Effect of the ethyl acetate extract of the culture filtrates of isolate O821 on infection behaviors of plant pathogens.㻌
Phytopathogens Treatment
㻌
PSB-10 O821-1 O821-2 O821-5 O821-10㻌
s
99.8±0.6 a 52.9±36.0 b 26.8±38.6 c 0.1±0.5 d 㻌 99.7±0.8 a 85.3±11.4 b 80.3±15.2 b 2.9±3.2 c 㻌
99.3±1.2 a 28.0±28.0 b 27.7±32.1 b 0.1±0.5 c 㻌 99.4±1.1 a 4.9±11.4 b 0.9±1.7 bc 0.2±0.6 bc 99.8±0.6 a 27.1±22.0 b 2.0±2.8 c 1.1±1.6 c 㻌 99.2±1.6 a 0.0±0.0 b 0.0±0.0 b 0.0±0.0 b 㻌 99.7±1.4 a 0.6±1.5 b 0.0±0.0 b 0.0±0.0 b
㻌
100.0±0.0 a 4.7±7.3 b 0.7±1.2 c 0.7±1.4 c 㻌 99.8±0.9 a 5.0±13.6 b 0.6±2.4 b 0.0±0.0 b 㻌 99.6±1.3 a 22.8±33.0 b 25.3±39.9 bc 㻌
The pecentage of spore germination (%)
Means followed by different letters are significantly different using the Tukey-Kramer test (p < 0.05).㻌 c
0.0±0.0 d㻌 0.1±0.5 c㻌
0.2±0.6 c㻌 0.0±0.0 c㻌 0.4±0.9 c㻌 0.0±0.0 b㻌 0.1±0.5 b
㻌
0.1±0.5 c㻌 0.0±0.0 b㻌 0.6±1.1 d㻌 c 1.8±2.8 cd 0.60
