56 Diseases severity (%)㻌erity 60㻌
100
㻌
sease 40㻌
20㻌
57
➨
➨5⠇ ⪃ᐹ
᳜≀ࡣ᳜≀ཎ⳦ࡽ⮬㌟ࢆᏲࡿࡓࡵࡢᵝࠎ࡞ࢩࢢࢼࣝ⤒㊰ࢆࡋࡓ㜵ᚚ࣓࢝ࢽࢬ࣒ࢆ
ᣢࡗ࡚࠸ࡿ(Tomiyama 1963)ࠋ㐣㓟Ỉ⣲ࡀࢩࢢࢼࣝศᏊࡋ࡚⣽⬊Ṛ㔜せ࡞ᙺࢆᯝࡓ ࡋ࡚࠸ࡿࡇࡣࡼࡃ▱ࡽࢀ࡚࠸ࡿ(Jabs 1999㸹Levine et al. 1994㸹Thordal-Christense et al. 1997㸹
Chamnongpol et al. 1998)ࠋᮏ◊✲࡛ࡣࠊࢿⴥO821 ⳦ᰴࡢᇵ㣴ࢁᾮ(100ಸ⃰⦰)ࢆฎ⌮ࡋ
࡚ࠊ12ࠊ24ཬࡧ48㛫ᚋࡢ㐣㓟Ỉ⣲ࡢ⏕ᡂࢆDABἲࡼࡾㄪᰝࡋࡓࠋࡑࡢ⤖ᯝࠊᇵ㣴
ࢁᾮࢆฎ⌮ࡋࡓࢿⴥ࡛ࡣ〓ⰍࡢDABᛂࡀほᐹࡉࢀࡓࠋࡇࡢࡇࡣࠊᇵ㣴ࢁᾮฎ⌮ࡼ
ࡾࢿⴥ㐣㓟Ỉ⣲ࡀ⏕ᡂࡉࢀࡿࡇࢆ♧ࡋࡓࠋ㐣㓟Ỉ⣲ࡢ⏕ᡂࡣ⣽⬊࡛ࡢ㐣ᩄឤ⣽
⬊Ṛࢆㄏᑟࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿࠋ୍᪉ࠊ༙ά≀ᐤ⏕⳦࡛࠶ࡿࢿ࠸ࡶࡕ⳦ࡀ⣽⬊
ධࡍࡿࡓࡵࡣࠊ୍ᐃᮇ㛫᳜≀⣽⬊ࡀ⏕ࡁ࡚࠸ࡿᚲせࡀ࠶ࡿࠋ࢝ࢱ࣮ࣛࢮࡀ㐣㓟Ỉ⣲ࡢᾘ ཤ࡛࠶ࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿࠋᅇࡢᐇ㦂࡛ࡣࠊO821⳦ᰴࡢᇵ㣴ࢁᾮ࢝ࢱ࣮ࣛࢮࡢྠ
ฎ⌮ࡼࡾࠊࢿ࠸ࡶࡕࡢᢚไຠᯝࡣపୗࡋࡓࠋࡇࡢࡇࡽࡶO821⳦ᰴࡢᇵ㣴ࢁᾮ ࡣ 1 ࡘࡢస⏝ࡋ࡚㐣㓟Ỉ⣲ࡢ⏕ᡂࢆಁࡋࠊ⣽⬊Ṛࡼࡾࢿ࠸ࡶࡕ⳦ࡢ⣽⬊ࡢ
ධࢆᢚไࡋ࡚࠸ࡿ⪃࠼ࡽࢀࡓࠋ
ࡲࡓࠊࢿO821⳦ᰴࡢᇵ㣴ࢁᾮࢆฎ⌮ࡋࠊ12ࠊ24ཬࡧ48㛫ᚋࡢPBZ1㑇ఏᏊࡢⓎ
⌧㔞ࢆㄪᰝࡋࡓࠋࡑࡢ⤖ᯝࠊO821 ⳦ᰴࡢᇵ㣴ࢁᾮฎ⌮ࡼࡾࢿⴥ࡛ࡢPBZ1㑇ఏᏊࡢⓎ
⌧ࡀቑຍࡋࡓࠋPBZ1㑇ఏᏊࡣ࣏ࣜ࢟ࢩࢤࢼ࣮ࢮࠊ࢟ࢳࢼ࣮ࢮ࡞ࡢᐖ㜵ᚚ㛵ࡋ࡚࠾
ࡾࠊ㌟ⓗ࡞ᢠᛶࢆάᛶࡉࡏ࡚ࠊࢿ࠸ࡶࡕᑐࡍࡿᢠᛶࢆㄏᑟࡍࡿࡇࡀ▱ࡽࢀ
࡚࠸ࡿࠋO821⳦ᰴࡢᇵ㣴ࢁᾮࢆฎ⌮ࡋࡓࢿ࡛ࡶྠᵝ㌟ⓗ࡞ᢠᛶࡀ㛵ࡋࡓᢠᛶ ࡀㄏᑟࡉࢀࡿྍ⬟ᛶࢆ♧ࡋࡓࠋKim et al. (2011) ࡣPBZ1ࢱࣥࣃࢡ㉁ࡢ⤌ࡳ࠼ࢿ࡛ࡣ⣽
⬊Ṛࡀㄏᑟࡉࢀࡿࡇࢆሗ࿌ࡋࡓࠋࡲࡓࠊࢿ⣽⬊㠀ぶᛶ⳦ᰴAcidovorax avenae ࢆ᥋
✀ࡍࡿࡇࡼࡗ࡚㐣㓟Ỉ⣲ࡢ⏕ᡂඹ PALࠊ Cht-1 ཬࡧ PBZ1 㑇ఏᏊࡢⓎ⌧ࢆㄏᑟ ࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ(Tanaka et al. 2003)ࠋࢿ࠸ࡶࡕ⳦ࡣேᕤᇵᆅ࡛ࡣ⏕⫱ࡍࡿࡶ
ࡢࡢࠊឤᰁࡀᡂࡾ❧ࡘࡓࡵࡣᐟ᳜≀ࡢ୍ⓗ࡞ඹ⏕㛵ಀࡀᚲせ࡛࠶ࡿࡇࡀ♧ࡉࢀ
࡚࠸ࡿ(Koga 1994; Suzuki 1965)ࠋࡇࢀࡽࡢ⤖ᯝࡽࠊO821⳦ᰴࡢᇵ㣴ࢁᾮࢆฎ⌮ࡋࡓࢿ
࡛ࡣࠊ㐣㓟Ỉ⣲ࡀ⏕ᡂࡉࢀࠊ㐣ᩄឤ⣽⬊Ṛࡢㄏᑟࡼࡾࠊඹ⏕㛵ಀࡀᡂ❧࡞ࡗࡓࡔࡅ
࡛࡞ࡃࠊPBZ1࡞ࡢࢿࡢᐖᢠᛶ㑇ఏᏊࡢⓎ⌧ࡀቑຍࡋࠊࢿ࠸ࡶࡕ⳦ࡢධࢆᢚ ไ࡛ࡁࡿ⪃࠼ࡽࢀࡓࠋ
ࢧࣜࢳࣝ㓟ࡣࢩࣟࢾࢼࢬࢼ࠾࠸࡚ Pseudomonas syringae pv. tomato ཬࡧ Peronospora
parasitica ᑐࡍࡿᢠᛶࡸ࢟ࣗ࢘ࣜ࠾࠸࡚ࡣⅣᑐࡍࡿ㌟ⓗ࡞ᢠᛶࢆㄏᑟࡍࡿ
ࡇࡀ▱ࡽࢀ࡚࠸ࡿࠋࡇࢀࡲ࡛ࠊࣉࣟ࣋ࢼࢰ࣮ࣝཬࡧࢳࢪࢽࣝࡀࢿࢿ࠸ࡶࡕ⳦
ᑐࡍࡿᢠᛶࢆㄏᑟࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ(Iwata 2001㸹Yasuda et al. 2004)ࠋࣉࣟ࣋ࢼࢰ
࣮ࣝࡣࠊ2ࡘࡢ␗࡞ࡿస⏝ࢆ㏻ࡋ࡚ᐖᢠᛶࢆㄏᑟࡍࡿࡇࡀ᥎ ࡉࢀࠊ1ࡘࡣ」ᩘࡢ㜵 ᚚ㛵㐃㑇ఏᏊࡢⓎ⌧ࢆㄏᑟࡍࡿస⏝ࠊࡶ࠺1ࡘࡣࣉࣟ࣋ࢼࢰ࣮ࣝࡽ௦ㅰࡉࢀ࡚⏕ࡌࡓBIT
ࡼࡿస⏝࡛࠶ࡿ(Midoh and Iwata 1997)ࠋࣉࣟ࣋ࢼࢰ࣮ࣝࡢฎ⌮࡛ࡣࠊ㐣㓟Ỉ⣲ࡀㄏᑟࡉ
ࢀࠊࢧࣜࢳࣝ㓟ࡢ✚ࢆಁࡋࠊࢧࣜࢳࣝ㓟ࢢࣝࢥࢩࢻࡀྜᡂࡉࢀࠊPBZ1㑇ఏᏊࡢⓎ⌧㔞ࡀ
58
ቑຍࡋࠊᢠᛶࡀㄏᑟࡉࢀࡿࠋࡋࡋࠊO821⳦ᰴࡢᇵ㣴ࢁᾮࡢฎ⌮ࡼࡾࢿⴥ୰࡛ࡢࢧ
ࣜࢳࣝ㓟ࡢቑຍࡀㄆࡵࡽࢀ࡞ࡗࡓࠋࡇࡢࡇࡣO821⳦ᰴࡢᇵ㣴ࢁᾮࡼࡿࢿ࡛ࡢᢠ ᛶㄏᑟࡣࢧࣜࢳࣝ㓟ࡀ㛵ಀࡋ࡚࠸࡞࠸ࡇࢆ♧ࡋࡓࠋࡇࢀࡲ࡛ࠊࢳࢪࢽࣝࡢฎ⌮࡛ࡣࠊ ࢧࣜࢳࣝ㓟ࢆࡉ࡞࠸ᢠᛶࡀㄏᑟࡉࢀࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ(Yasuda et al. 2006)ࠋO821
⳦ᰴࡢᇵ㣴ࢁᾮࡣࢳࢪࢽࣝྠᵝࢧࣜࢳࣝ㓟ࡢୗὶస⏝ࡋࠊࢿᢠᛶࢆㄏᑟࡋ
࡚࠸ࡿྍ⬟ᛶࡶ⪃࠼ࡽࢀࡓࠋᢠᛶㄏᑟ⤒㊰ࢆ᫂ࡽࡍࡿࡓࡵࠊࡢᢠᛶ㛵㐃㑇ఏᏊ
ࡘ࠸࡚ࡶㄪᰝࡍࡿᚲせࡀ࠶ࡿࠋ
59
➨
➨6❶ O821⳦ᰴࡢᇵ㣴ࢁᾮ⏕ᡂࡉࢀࡿᢚไ≀㉁ࡢศ㞳ཬࡧྠᐃ
๓❶࡛ࡣࠊO821⳦ᰴࡢᇵ㣴ࢁᾮࡀࢿ࠸ࡶࡕ⳦ࡢឤᰁ⾜ືࡸᇵᆅୖ࡛ࡢ⳦⣒⏕⫱ࢆᢚ ไࡍࡿࡇࢆ᫂ࡽࡋࡓࠋࡲࡓࠊࢿࡢO821 ⳦ᰴࡢᇵ㣴ࢁᾮࡢ๓ฎ⌮ࡼࡾࢿ࠸ࡶ
ࡕࡢᩬᙧᡂࡀᢚไࡉࢀࡓࠋࡉࡽࠊO821⳦ᰴࡢᇵ㣴ࢁᾮ୰ࢿ࠸ࡶࡕ⳦ࢆᢚไࡍ
ࡿ≀㉁ࡀᏑᅾࡋࠊࡑࡢ≀㉁ࡀ㓑㓟࢚ࢳࣝྍ⁐࡛ࠊ⇕Ᏻᐃ࡞≀㉁࡛࠶ࡿࡇࢆ᫂ࡽࡋࡓࠋ ᮏ❶࡛ࡣࠊO821⳦ᰴࡢᇵ㣴ࢁᾮ୰⏕⏘ࡉࢀࡿ≀㉁ࡢศ㞳࣭⢭〇ཬࡧྠᐃࡘ࠸࡚㏙ࡿࠋ
➨1⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮ⏕ᡂࡉࢀࡿᢚไ≀㉁ࡢศ㞳 1㸬ᮦᩱཬࡧ᪉ἲ
a)౪ヨ⳦ཬࡧᇵ㣴ࢁᾮࡢㄪ〇
౪ヨ⳦ࡋ࡚ࢿ࠸ࡶࡕ⳦ࠊO821⳦ᰴࡣ➨2❶ࠊ➨2⠇ࠊ1a)࡛㏙ࡓ᪉ἲࡼࡾ‽ഛ ࡋࡓࠋ
O821⳦ᰴࢆPSᾮయᇵᆅ(500 ml)ࡀධࡗࡓ1 L୕ゅࣇࣛࢫࢥ7 mmࢥࣝࢡ࣮࣮࡛࣎ࣛ⳦
ྀࢆࡃࡾᢤࡁࠊ25∦⛣᳜ࡋࡓࠋ⛣᳜ᚋࠊ14᪥㛫ࠊ130ᅇ㌿/ศ࡛┞ᇵ㣴ࡋࡓࠋ┞ᇵ㣴ᚋࠊ ᇵ㣴ᾮࢆࣇࣝࢱ࣮(No. 101 Advantec Toyo, Tokyo, Japan )࡛ࢁ㐣ࡋࠊᇵ㣴ࢁᾮࢆㄪ〇ࡋࡓࠋ b)࣒࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮ࡼࡿᇵ㣴ࢁᾮࡢศ㞳
㏫┦ࢡ࣐ࣟࢺࢢࣛࣇ࣮(10×45×3 cm) ࣒࢝ࣛணࡵࢩࣜ࢝ࢤࣝ(Silica Gel, ODS-Q3; Wako
Pure Chemical, Osaka, Japan)ࢆ10 cmሸࡋ࡚࠾ࡁࠊࡑࡢୖࢆ࢞ࣛࢫ࣮࡛࢘ࣝそࡗࡓࠋᇵ㣴ࢁ
ᾮ(400 ml)ࢆຍ࠼ࠊࢩࣜ࢝ࢤࣝ྾╔ࡉࡏࡓᚋࠊ80%࣓ࢱࣀ࣮ࣝ⁐ᾮ(100 ml)ࢆ⏝࠸࡚ࠊ⁐
ฟࡋࡓࠋྜィ4 Lࡢᇵ㣴ࢁᾮࢆ⏝࠸ࡓࠋ⁐ฟᾮࡣ࢚ࣂ࣏࣮ࣞࢱ࣮ࢆ⏝࠸࡚ࠊ45Υ࡛␃Ỉࡢ
ࡳ࡞ࡿࡲ࡛ῶᅽୗ࡛⃰⦰ࡋࡓࠋ40 mlࡢ࣓ࢱࣀ࣮ࣝᢳฟỈ⁐ᾮㄪ〇ࡋࠊ㓑㓟࢚ࢳࣝࢆ⏝
࠸࡚ᢳฟࡋࡓࠋྠ㔞ࡢ㓑㓟࢚ࢳࣝࢆຍ࠼࡚ศ㓄ᢳฟࡋࡓࠋࡇࡢᢳฟ᧯సࢆ3ᅇ⧞ࡾ㏉ࡋࡓࠋ ᚓࡽࢀࡓ㓑㓟࢚ࢳࣝྍ⁐㒊␃Ỉ(⣙ 45 ml)ࢆຍ࠼ࠊᆺࣇࣛࢫࢥࢆ⏝࠸࡚ࠊ࢚ࣂ࣏࣮ࣞ
ࢱ࣮ࡼࡾῶᅽୗ࡛⃰⦰ࡋࡓࠋ㓑㓟࢚ࢳࣝࢆ㝖ཤࡋࡓỈ⁐ᾮ(40 ml)ࢆㄪ〇ࡋࠊᇵ㣴ࢁᾮ㓑㓟
࢚ࢳࣝᢳฟᾮࡋ࡚ᐇ㦂⏝࠸ࡓࠋ
c)㧗㏿ᾮయࢡ࣐ࣟࢺࢢࣛࣇ࣮ࡼࡿᇵ㣴ࢁᾮࡢศ㞳
ᇵ㣴ࢁᾮ㓑㓟࢚ࢳࣝᢳฟᾮࢆ0.22 μmࣇࣝࢱ࣮(RephiLe Bioscience, Boston, USA)࡛ࢁ㐣 ࡋࠊ㧗㏿ᾮయࢡ࣐ࣟࢺࢢࣛࣇ࣮(HPLC㸸᪥❧࣏ࣥࣉ L-6000ᆺࠊHPLC࣒࢝ࣛሸ Wakosil-ϩ5C18 AR column size 4.6/250 mmࠊ⛣ືᒙ㸸80%࣓ࢱࣀ࣮ࣝࠊὶ㏿㸸1 ml/minࠊ᳨ฟἼ㛗㸸
204 nm)ࢆ⏝࠸࡚ࠊO821⳦ᰴࡢᇵ㣴ࢁᾮ୰ࡢᢚไ≀㉁ࡢศ㞳ࢆ⾜ࡗࡓࠋHPLCࡢࡑࢀࡒࢀࡢ
ࣆ࣮ࢡࢆᅇࡋࠊ࢚ࣂ࣏࣮ࣞࢱ࣮ࡼࡾῶᅽୗ࡛⃰⦰ࡋࠊᐇ㦂⏝࠸ࡓࠋ d)⏕≀᳨ᐃヨ㦂
➨2❶ࠊ➨2⠇ࠊ1b)ྠᵝࡢ᪉ἲࡼࡾࠊHPLC࡛ศ㞳ࡋࡓRT 4.26㏆ࡢࣆ࣮ࢡᢳฟᾮ
ࡼࡿࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆヨ㦂ࢆ⾜ࡗࡓࠋ
ࡲࡓࠊ➨4❶ࠊ➨3⠇ࠊ1d)ྠᵝࡢ᪉ἲࢆ⏝࠸࡚ࠊTLCୖ࡛ࡢᢚไάᛶࢆㄪᰝࡋࡓࠋୖ
60
グヨᩱ50 μlࢆⷧᒙࢡ࣐ࣟࢺࢢࣛࣇ࣮⏝ࣉ࣮ࣞࢺTLC (Silica gel 60, Merck KGaA, Darmstadt,
Germany)ࢫ࣏ࢵࢺࡋࠊࢺ࢚ࣝࣥ㸸㓑㓟࢚ࢳࣝ㸻1㸸1 (v/v) ࡢᒎ㛤⁐፹ࢆ⏝࠸࡚ᒎ㛤ࡉࡏࡓࠋ
ᒎ㛤ᚋࠊTLCࣉ࣮ࣞࢺPSAᇵᆅ(ࢡ࣒ࣟࣛࣇ࢙ࢽࢥ࣮ࣝ2 mg/100 ml)ᠱ⃮ࡉࡏࡓࢿ࠸
ࡶࡕ⳦ࡢ⬊Ꮚࢆᄇ㟝ࡋࡓࠋࣉ࣮ࣞࢺࡣ‵ᐊࡋࡓࣉࣛࢫࢳࢵࢡࢣ࣮ࢫෆ⣡ࡵࡓᚋࠊࣝ
࣑⨁࡛そ࠸ࠊ26Υࠊᬯ㯮᮲௳ୗ࡛ᇵ㣴ࡋࠊࣉ࣮ࣞࢺୖࡢ⏕⫱㜼Ṇᖏࡢ᭷↓ࢆㄪᰝࡋࡓࠋ
2㸬㸬⤖ᯝ
࣒࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮ཬࡧHPLCࢆ⏝࠸࡚ࠊO821⳦ᰴࡢᇵ㣴ࢁᾮᏑᅾࡋ࡚࠸ࡿᢚ ไ≀㉁ࡢศ㞳ࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊHPLCࡣ2ࡘࡢࡁ࡞ࣆ࣮ࢡࡀ᳨ฟࡉࢀࠊRT 4.26
㏆ࡢࣆ࣮ࢡࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆࢆᢚไࡍࡿࡇࡀ☜ㄆࡉࢀࡓࡓࡵࠊRT 4.26㏆ࡢ
ࣆ࣮ࢡࢆᅇࡋࠊᐇ㦂⏝࠸ࡓࠋᅇࡢᐇ㦂ࡣO821⳦ᰴࡢᇵ㣴ࢁᾮ㓑㓟࢚ࢳࣝᢳฟᾮཬ ࡧࡑࢀࢆHPLC࡛ศ㞳ࡋࠊ༢㞳ࡋࡓRT 4.26㏆ࡢࣆ࣮ࢡࢆ⏝࠸࡚ࠊヲ⣽ࢿ࠸ࡶࡕ⳦
ࡢ⬊ᏊⓎⱆཬࡧ╔ჾᙧᡂ࠼ࡿᙳ㡪ࢆㄪᰝࡋࡓࠋࡑࡢ⤖ᯝࠊO821⳦ᰴࡢᇵ㣴ࢁᾮ㓑㓟
࢚ࢳࣝᢳฟᾮཬࡧRT 4.26㏆ࡢࣆ࣮ࢡ࡛ࡣࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆཬࡧ╔ჾᙧᡂࡢᢚ ไຠᯝࡀ☜ㄆࡉࢀࡓࠋᑐ↷༊ࡋ࡚⏝࠸ࡓ␃Ỉฎ⌮༊࡛ࡣ⬊ᏊⓎⱆ⋡ཬࡧ╔ჾᙧᡂ⋡
ࡣࡑࢀࡒࢀࠊ100±0.0% ཬࡧ 99.9±0.5% ࡛࠶ࡗࡓ(Fig. 6-1AࠊB)ࠋ୍᪉ࠊO821⳦ᰴࡢᇵ㣴ࢁ
ᾮ㓑㓟࢚ࢳࣝᢳฟᾮRT 4.26㏆ࡢࣆ࣮ࢡ࡛ࡣࠊ⬊ᏊⓎⱆཬࡧ╔ჾᙧᡂࡣᢚไࡉ
ࢀࡓ(Fig. 6-1AࠊB)ࠋ
ࡲࡓࠊࡑࢀࡒࢀࡢᢳฟᾮ50 μlࢆⷧᒙࢡ࣐ࣟࢺࢢࣛࣇ࣮ᒎ㛤ࡉࡏࡓࠋᒎ㛤ᚋࠊࣉ࣮ࣞࢺ
ࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚࢆᄇ㟝ࡍࡿ7 ᪥ᚋRf ್0.5-0.6 ㏆⏕⫱㜼Ṇᖏࡀㄆࡵࡽࢀ
ࡓ(Fig. 6-2)ࠋ
61 100㻌
80㻌
60㻌
40㻌 20㻌 0㻌 8
6
4
2
Percentage of spore germination (%)㻌
b㻌 b㻌
a㻌
Percentage of appressorium formation (%)㻌gpp() 100㻌 80㻌 60㻌
40㻌 20㻌
0㻌
DW㻌 O821-CF㻌
b㻌 b㻌
a㻌
b RT 4.26 peak㻌
Fig. 6-1. Effect of the high-performance liquid chromatography peak (RT 4.26 peak) from O821-CF on infection behavior of Magnaporthe oryzae. Spores of M. oryzae were suspended in RT 4.26 peak and dropped on glass slides. Distilled water (DW) 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 per experiment were examined. Bars represent ± SD. Means followed by different letters are significantly different using the Tukey-Kramer test (p < 0.05).
A
B
62
FRONT㻌
ORIGIN㻌 Rf 0.5-0.6㻌
Fig. 6-2. Thin layer chromatography (TLC) bioautography of the ethyl acetate extract of culture filtrates of isolate O821 (CF) and the high-performance liquid chromatography peak (RT 4.26 peak). The O821-CF and RT 4.26 peak were spotted onto a TLC plate. After development, the TLC plate was sprayed with a concentrated spore suspension of Magnaporthe oryzae in the presence of potato sucrose agar medium.
Inoculated plate was kept in a moist chamber at 26°C for 7 days.
O821-CF㻌 RT 4.26 peak㻌
63
➨
➨2⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮ⏕ᡂࡉࢀࡿᢚไ≀㉁ࡢྠᐃ 1㸬ᮦᩱཬࡧ᪉ἲ
౪ヨ⳦ࡋ࡚ࢿ࠸ࡶࡕ⳦ཬࡧ O821⳦ᰴࢆ➨2 ❶ࠊ➨2⠇ࠊ1a)࡛㏙ࡓ᪉ἲࡼࡾ
‽ഛࡋࡓࠋ
O821⳦ᰴࡢᇵ㣴ࢁᾮཬࡧᢚไ≀㉁ࡣ➨6❶ࠊ➨1⠇ࠊ1࡛㏙ࡓ᪉ἲࡼࡾ‽ഛࡋࡓࠋ O821⳦ᰴࡢᇵ㣴ࢁᾮ㓑㓟࢚ࢳࣝᢳฟᾮࢆHPLCࡼࡾศ㞳ࡋࠊ༢㞳࣭⢭〇ࡋࡓࠋNMR⨨
(Agilent Unity INOVA700ཬࡧ500)ࢆ⏝࠸࡚1H NMRࠊ13C NMRࢆ ᐃࡋ࡚ࠊᵓ㐀ゎᯒࢆ⾜ࡗ ࡓࠋࡲࡓࠊGC/MS/MSゎᯒࡼࡿศᏊ㔞ࡢỴᐃFT-IRࡼࡿゎᯒࡶ⾜ࡗࡓࠋ
2㸬⤖ᯝ
HPLC ࡛ࡢศ㞳ࢆ⾜ࡗࡓ⤖ᯝࠊRT 4.26㏆ࡢࣆ࣮ࢡࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆཬࡧ
╔ჾᙧᡂࢆᢚไࡍࡿ≀㉁ࡀᏑᅾࡋ࡚࠸ࡓࠋࡑࡇ࡛ࠊHPLCࢆ⏝࠸࡚ศ㞳ࡋࡓRT 4.26㏆ࡢ
ࣆ࣮ࢡᏑᅾࡍࡿ≀㉁ࢆ㉁㔞ศᯒ᰾☢Ẽඹ㬆ศගἲ (NMR)ཬࡧ㉁㔞ศᯒἲ(GC/MS/MS)
ࡼࡾศᯒࡋࠊ⤖ᯝᇶ࡙࠸࡚ᵓ㐀ゎᯒࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊⅣ⣲ᩘཬࡧỈ⣲ᩘࡀ᥎ᐃ࡛ࡁࠊ
HH COSY HSQC HMBCࠊINADEQUATEḟඖࢫ࣌ࢡࢺࣝゎᯒࡼࡾࠊࡑࢀࡒࢀࡢඖ⣲ࡢ
ࡘ࡞ࡀࡾࡀ᫂ࡽ࡞ࡗࡓࠋࡲࡓࠊGC/MS/MSࡼࡾศᏊ㔞248࡛࠶ࡾࠊFT-IRࡼࡾỈ㓟 ᇶࡀ࠶ࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋ᭱⤊ⓗᮏ≀㉁ࡀ(3aS,4aR,8aS,9aR)-3a-hydroxy-8a-methyl-3,5-dimethylenedecahydronaphtho[2,3-b]furan-2(3H)-one࡛࠶ࡾࠊศᏊ㔞248ࡢ≀㉁࡛࠶ࡿࡇ
ࡀ᫂ࡽ࡞ࡗࡓ (Fig. 6-3)ࠋ
64
Fig. 6-3. The structure of inhibitory compound from culture filtrate of isolate O821.
65
➨
➨3⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮ⏕ᡂࡉࢀࡿᢚไ≀㉁ࡼࡿࢿ࠸ࡶࡕ⳦ࡢឤᰁ⾜ືࡢ ᢚไຠᯝ
1㸬ᮦᩱཬࡧ᪉ἲ
౪ヨ⳦ࡋ࡚ࢿ࠸ࡶࡕ⳦ཬࡧ O821⳦ᰴࡣ➨2 ❶ࠊ➨2⠇ࠊ1a)࡛㏙ࡓ᪉ἲࡼࡾ
‽ഛࡋ࡚⏝࠸ࡓࠋ
O821⳦ᰴࡢᇵ㣴ࢁᾮࡽ༢㞳࣭⢭〇ࡉࢀࡓᢚไ≀㉁ࡣ␃Ỉ࡛1ࠊ2ࠊ5ࠊ10ࠊ50ࠊ100ཬ
ࡧ200 ppmㄪ〇ࡋࡓࠋ➨2❶ࠊ➨2⠇ࠊ1b)ྠᵝࡢ᪉ἲ࡛ࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆヨ
㦂ࢆ⾜ࡗࡓࠋ
2. ⤖ᯝ
O821⳦ᰴࡢᇵ㣴ࢁᾮࡽ༢㞳࣭⢭〇ࡉࢀࡓᢚไ≀㉁ࢆ1-200 ppmㄪ〇ࡋ࡚ࠊࢿ࠸ࡶ
ࡕ⳦ࡢឤᰁ⾜ືࢆㄪᰝࡋࡓࠋࡑࡢ⤖ᯝࠊ␃Ỉࢆ⏝࠸ࡓᑐ↷༊ࡢ⬊ᏊⓎⱆ⋡ཬࡧ╔ჾᙧ ᡂ⋡ࡀࡑࢀࡒࢀ99.9 ± 0.5% ཬࡧ 99.8 ± 0.6%࡛࠶ࡗࡓ(Fig. 6-4 AࠊB)ࠋ୍᪉ࠊ1ཬࡧ2 ppm ฎ⌮༊࡛ࡣ⬊ᏊⓎⱆ⋡ࡀࡑࢀࡒࢀ71.2 ± 35.8% ཬࡧ20.2 ± 27.4%࡛࠶ࡗࡓ(Fig. 6-4 A)ࠋࡲࡓࠊ
╔ჾᙧᡂ⋡ࡣࠊࡑࢀࡒࢀ70.9 ± 35.8% ཬࡧ18.2 ± 27.5%࡛࠶ࡗࡓ(Fig. 6-4 B)ࠋࡋࡋࠊ5 ppm ௨ୖ࡛ࡣࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆ⋡ཬࡧ╔ჾᙧᡂ⋡ࡣⴭࡋࡃᢚไࡉࢀࠊ࡚ࡢฎ
⌮༊࡛0%࡞ࡾࠊࢿ࠸ࡶࡕ⳦ࡢ㧗࠸ឤᰁ⾜ືᢚไࡀㄆࡵࡽࢀࡓ(Fig. 6-4 AࠊB)ࠋ
66 ᴾ
DW ᴾ ᴾ 1ppm ᴾ ᴾ2ppm 5ppmᴾ
10ppmᴾ 50ppm ᴾ 100ppm 200ppmᴾ
Percentage of spore germination (%) Percentage of spore germination(%)100 80
60 40
20 0
c a b
d d d d d
Fig. 6-4. Effect of O821 inhibitory compound on infection behavior of Magnaporthe oryzae. Spores of M. oryzae were suspended in O821 inhibitory compound and dropped on glass slides. Distilled water (DW) 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 per experiment were examined. Bars represent ± SD.
Means followed by different letters are significantly different using the Tukey-Kramer test (p < 0.05).
A
B
Percentage of appressorium formation (%) Percentage of appressorium formation(%) 100 80
60 40 20
0 DW ᴾ 1ppm ᴾ2ppm 5ppmᴾ
i f ti b h i fM th S
10ppmᴾ 50ppm 100ppm c
a b
d d d d d
m 200ppm
67
➨
➨4⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮࡽศ㞳ࡉࢀࡓᢚไ≀㉁ࡼࡿࢿ࡛ࡢࢿ࠸ࡶࡕࡢⓎ
ᢚไຠᯝ
1㸬ᮦᩱཬࡧ᪉ἲ a)౪ヨ⳦ཬࡧ౪ヨ᳜≀
౪ヨ⳦ࡋ࡚⏝࠸ࡓࢿ࠸ࡶࡕ⳦ཬࡧO821 ⳦ᰴࡣ➨2❶ࠊ➨2 ⠇ࠊ1a)࡛㏙ࡓ᪉ἲ
ࡼࡾ‽ഛࡋࡓࠋ౪ヨ᳜≀ࡋ࡚ࢿ(ရ✀㸸ࢥࢩࣄ࢝ࣜ)ࢆᐇ㦂⏝࠸ࡓࠋࢿࡣ➨ 4 ❶ࠊ
➨1⠇ࠊ1a)࡛㏙ࡓ᪉ἲࡼࡾ‽ഛࡋࡓࠋ
b)O821⳦ᰴࡢᇵ㣴ࢁᾮࡽ༢㞳࣭⢭〇ࡉࢀࡓᢚไ≀㉁ࡢㄪ〇
O821⳦ᰴࡢᇵ㣴ࢁᾮࡽ༢㞳࣭⢭〇ࡉࢀࡓᢚไ≀㉁ࡣ➨6❶ࠊ➨3⠇ࠊ1࡛㏙ࡓ᪉ἲ
ࡼࡾㄪ〇ࡋࡓࠋ
c)ࢿࡢ᥋✀᪉ἲⓎᗘࡢㄪᰝ
ࢿࡢ᥋✀᪉ἲⓎᗘࡢㄪᰝࡣ➨ 4 ❶ࠊ➨ 1 ⠇࡛㏙ࡓ᪉ἲࡼࡾᐇ㦂ࢆ⾜ࡗࡓࠋ ᐇ㦂ࡣ1ฎ⌮༊࠶ࡓࡾࢩ࣮ࣕࣞ2ᯛ(20ಶయ)⏕⫱ࡉࡏࡓࢿࢆ⏝࠸ࡓࠋ1×105spores/ml ࡢࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊࢆO821 ⳦ᰴࡢᇵ㣴ࢁᾮࡽ༢㞳࣭⢭〇ࡉࢀࡓᢚไ≀㉁(5 ཬࡧ 10
ppm)ᠱ⃮ࡋ࡚ࠊ20ಶయࡢࢿ2 mlࢆᄇ㟝᥋✀ࡋࡓࠋᑐ↷༊ࡋ࡚␃Ỉࢿ࠸ࡶࡕ
⳦⬊Ꮚࢆᠱ⃮ࡋ࡚ࠊྠᵝࡢ᪉ἲࡼࡾᄇ㟝᥋✀ࡋࡓࠋฎ⌮ࡋࡓࢿࡣ‵ᐊࠊᬯ㯮ୗ 24
㛫ಖࡕࠊࡑࡢᚋࠊ࢞ࣛࢫᐊ࡛᱂ᇵࡋࡓࠋ᥋✀5᪥ᚋࠊ1༊࠶ࡓࡾ15ಶయࡢ➨2ⴥࡢ࠾
ࡅࡿⓎ≧ἣࢆㄪᰝࡋࠊ➨4❶ࠊ➨1⠇ࠊ1c)࡛㏙ࡓ᪉ἲࡼࡾⓎᗘࢆㄪᰝࡋࡓࠋ
2㸬⤖ᯝ
O821⳦ᰴࡢᇵ㣴ࢁᾮࡽ༢㞳࣭⢭〇ࡉࢀࡓᢚไ≀㉁ࢿ࠸ࡶࡕ⳦ࡢΰྜ᥋✀ࡼࡿ
Ⓨᗘࡢㄪᰝࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊᑐ↷༊ࡋ࡚⏝࠸ࡓ␃Ỉฎ⌮༊࡛ࡣࠊࢿ࠾ࡅࡿⓎ
ᗘࡣ98.6 ± 3.4%࡛࠶ࡗࡓ(Fig. 6-5 B)ࠋ୍᪉ࠊO821⳦ᰴࡢᇵ㣴ࢁᾮࡽ༢㞳࣭⢭〇ࡉࢀࡓ
ᢚไ≀㉁ࡢฎ⌮༊࡛ࡣࠊ5ཬࡧ10 ppm࡛Ⓨᗘࡣࡑࢀࡒࢀ22.2 ± 9.7% ཬࡧ9.7 ± 10.3%࡛
࠶ࡗࡓ(Fig. 6-5 B)ࠋᑐ↷༊ẚ㍑ࡋ࡚ࠊ5 ppmฎ⌮༊࡛ࡶⓎᗘࡣ⣙1/5௨ୗῶᑡࡋࠊ10
ppmฎ⌮༊࡛ࡣ1/10ῶᑡࡋࡓ(Fig. 6-5 AࠊB)ࠋ
68
Diseases severity (%)Diseases severity (%)
100 80 60 40 20
0 DW ᴾ 5 ppm ᴾ 10 ppm b
b a
Fig. 6-5. Effect of treatment with O821 inhibitory compound on blast lesion formation by Magnaporthe oryzae in rice leaves. Rice leaves were inoculated with O821 inhibitory compound or with distilled water (DW) mixed with a spore suspension of M. oryzae and maintained in a moist chamber for 24 h at 26°C in the dark. 5 days after inoculation, lesion formation (A) and the extent of disease severity (B) was measured. The experiments were independently repeated three times. A total of 15 rice plants per experiment were examined. Bars represent ± SD. Means followed by different letters are significantly different using the Tukey-Kramer test (p < 0.05).
A
B
69
➨
➨5⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮࡽศ㞳ࡉࢀࡓᢚไ≀㉁ࡢࢿࡢ๓ฎ⌮ࡼࡿࢿ࠸ࡶࡕ
ࡢⓎᢚไຠᯝ 1㸬ᮦᩱཬࡧ᪉ἲ a)౪ヨ⳦ཬࡧ౪ヨ᳜≀
౪ヨ⳦ࡋ࡚⏝࠸ࡓࢿ࠸ࡶࡕ⳦ཬࡧO821 ⳦ᰴࡣ➨2❶ࠊ➨2 ⠇ࠊ1a)࡛㏙ࡓ᪉ἲ
ࡼࡾ‽ഛࡋࡓࠋ౪ヨ᳜≀ࡋ࡚ࢿ(ရ✀㸸ࢥࢩࣄ࢝ࣜ)ࢆᐇ㦂⏝࠸ࡓࠋࢿࡣ➨ 4 ❶ࠊ
➨1⠇ࠊ1a)࡛㏙ࡓ᪉ἲࡼࡾ‽ഛࡋࡓࠋ
b)O821⳦ᰴࡢᇵ㣴ࢁᾮࡽ༢㞳࣭⢭〇ࡉࢀࡓᢚไ≀㉁ࡢㄪ〇
O821⳦ᰴࡢᇵ㣴ࢁᾮࡽ༢㞳࣭⢭〇ࡉࢀࡓᢚไ≀㉁ࡣ➨6❶ࠊ➨3⠇ࠊ1࡛㏙ࡓ᪉ἲ
ࡼࡾㄪ〇ࡋࡓࠋ
c)ࢿࡢ᥋✀᪉ἲཬࡧⓎᗘࡢㄪᰝ
ࢿࡢ᥋✀᪉ἲⓎᗘࡢㄪᰝࡣ➨ 4 ❶ࠊ➨ 2 ⠇࡛㏙ࡓ᪉ἲࡼࡾᐇ㦂ࢆ⾜ࡗࡓࠋ ᐇ㦂ࡣ1ฎ⌮༊ᙜࡓࡾ20ಶయࡢࢿࢆ⏝࠸ࡓࠋ100ཬࡧ200 ppmࡢO821⳦ᰴࡢᇵ㣴ࢁ
ᾮࡽ༢㞳࣭⢭〇ࡉࢀࡓᢚไ≀㉁ࢆ20ಶయࡢࢿ1 ml๓ฎ⌮ࡋࡓࠋ๓ฎ⌮24㛫ᚋ
ࢿ࠸ࡶࡕ⳦⬊Ꮚᠱ⃮ᾮ(1×105spores/ml)ࢆ20ಶయࡢࢿ2 mlᄇ㟝᥋✀ࡋࡓࠋ᥋✀ᚋࠊ
‵ᐊࠊᬯ㯮ୗ24㛫ಖࡕࠊࡑࡢᚋ࢞ࣛࢫᐊ࡛᱂ᇵࡋࡓࠋ᥋✀5᪥ᚋࠊ1༊࠶ࡓࡾ15ಶ యࡢ➨2ⴥࡢ࠾ࡅࡿⓎ≧ἣࢆㄪᰝࡋࠊ➨4❶ࠊ➨1⠇ࠊ1c)࡛㏙ࡓ᪉ἲࡼࡾⓎᗘࢆ
ㄪᰝࡋࡓࠋ
2㸬⤖ᯝ
O821⳦ᰴࡢᇵ㣴ࢁᾮࡽ༢㞳࣭⢭〇ࡉࢀࡓᢚไ≀㉁ࡢ24㛫๓ฎ⌮ࡼࡾࠊࢿ࠸ࡶࡕ
ࡢⓎᗘࡢㄪᰝࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊᑐ↷༊ࡋ࡚⏝࠸ࡓ␃Ỉฎ⌮༊࡛ࡣࠊࢿ࠾ࡅ
ࡿⓎᗘࡣ99.6 ± 0.9%࡛࠶ࡗࡓ(Fig. 6-6 B)ࠋ୍᪉ࠊO821⳦ᰴࡢᇵ㣴ࢁᾮࡽ༢㞳࣭⢭〇ࡉ
ࢀࡓᢚไ≀㉁ࡢฎ⌮༊࡛ࡣࠊ100ཬࡧ200 ppm࡛ࡢⓎᗘࡣࡑࢀࡒࢀ53.4 ± 1.9% ཬࡧ45.8
± 2.9%࡞ࡾࠊᑐ↷༊ẚ㍑ࡋ࡚ࠊ࠸ࡎࢀࡶⓎᗘࡀ⣙༙ศ⛬ᗘῶᑡࡋࡓ(Fig. 6-6 AࠊB)ࠋ
70 100
80 60 40 20 0
O821 inhibitory compound 8
6 4 Diseases severity (%) 2
DW ᴾ ᴾ ᴾ ᴾ100 ppmᴾ 200 ppmᴾ
Fig. 6-6. Effect of pretreatment with O821 inhibitory compound on blast lesion formation by Magnaporthe oryzae in rice leaves. Rice leaves were pretreated with O821 inhibitory compound (100 ppm or 200 ppm) or with distilled water (DW). After 24 h, rice leaves were inoculated with a spore suspension of M. oryzae and maintained in a moist chamber for 24 h at 26°C in the dark. 5 days after inoculation, lesion formation (A) and the extent of disease severity (B) was measured. The experiments were independently repeated three times. A total of 15 rice plants per experiment were examined. Bars represent ± SD. Means followed by different letters are significantly different using the Tukey-Kramer test (p < 0.05).
b b a
A
B
71
➨
➨6⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮࡽศ㞳ࡉࢀࡓᢚไ≀㉁ࡼࡿࢿ࡛ࡢ㐣㓟Ỉ⣲ࡢ⏕ᡂ 1㸬ᮦᩱཬࡧ᪉ἲ
a)౪ヨ᳜≀
ࢿ(ရ✀㸸ࢥࢩࣄ࢝ࣜ)ࢆᐇ㦂⏝࠸ࡓࠋࢿࡣ➨4❶ࠊ➨1⠇ࠊ1a)࡛㏙ࡓ᪉ἲࡼࡾ
⏕⫱ࡉࡏ࡚ᐇ㦂⏝࠸ࡓࠋ
b)O821⳦ᰴࡢᇵ㣴ࢁᾮࡽ༢㞳࣭⢭〇ࡉࢀࡓᢚไ≀㉁ࡢㄪ〇ཬࡧࢿⴥࡢฎ⌮ἲ
O821⳦ᰴࡢᇵ㣴ࢁᾮࡽ༢㞳࣭⢭〇ࡉࢀࡓᢚไ≀㉁ࡣ➨6❶ࠊ➨3⠇ࠊ1࡛㏙ࡓ᪉ἲ
ࡼࡾㄪ〇ࡋࡓࠋ
ࢿࡣࢩ࣮ࣕࣞࡽ᰿ࢆษ᩿ࡋ࡞࠸ࡼ࠺ᢤࡁྲྀࡾࠊ‵ࡽࡏࡓ࢟ࢵࢳ࣮ࣥ࣌ࣃ࣮ࢆᩜ࠸
ࡓࣉࣛࢫࢳࢵࢡࢣ࣮ࢫ7ಶయࡎࡘ୪ࡓࠋୖグ࡛ㄪ〇ࡋࡓ100ಸ⃰⦰ࡢO821⳦ᰴࡢᇵ㣴
ࢁᾮࠊ100 ppmཬࡧ350 ppmㄪ〇ࡋࡓO821⳦ᰴࡢᇵ㣴ࢁᾮࡽ༢㞳࣭⢭〇ࡉࢀࡓᢚไ≀
㉁ࢆ➨2ⴥ1ⴥᙜࡓࡾ5 μlࡎࡘ 3-5ᡤୗࡋࡓࠋᑐ↷༊ࡋ࡚␃Ỉࢆ⏝࠸ࡓࠋฎ⌮
ᚋࠊࢿࡣ26Υ࡛12 㛫ࡢග࿘ᮇ࡛ᇵ㣴ࡋࡓࠋᇵ㣴24㛫ᚋ㐣㓟Ỉ⣲ࡢ⏕ᡂࡢㄪᰝ
ࢆ⾜ࡗࡓࠋ
c)㐣㓟Ỉ⣲ࡢ⤌⧊Ꮫⓗ᳨ฟ
Thordal-Christense et al. (1997)ࡢ᪉ἲࢆ⏝࠸࡚ࠊࢿ⤌⧊ࡢ㐣㓟Ỉ⣲ࡢ⏕ᡂࢆ⤌⧊Ꮫⓗ
ㄪᰝࡋࡓࠋᐇ㦂ࡣ➨5❶ࠊ➨1⠇ࠊ1c)࡛㏙ࡓ᪉ἲࢆ⏝࠸࡚ࠊ⾜ࡗࡓࠋ
2㸬⤖ᯝ
O821 ⳦ᰴࡢᇵ㣴ࢁᾮ(100ಸ)ࠊ100 ppmཬࡧ350 ppmࡢO821⳦ᰴࡢᇵ㣴ࢁᾮࡽ༢㞳࣭
⢭〇ࡉࢀࡓᢚไ≀㉁ࢆฎ⌮ࡋ࡚ࠊ24㛫ᚋࡢ㐣㓟Ỉ⣲ࡢ⏕ᡂࢆDABἲࡼࡾㄪᰝࡋࡓࠋ ࡑࡢ⤖ᯝࠊO821 ⳦ᰴࡢᇵ㣴ࢁᾮࢆฎ⌮ࡋࡓࢿⴥ࡛ࡣ㐣㓟Ỉ⣲ࡢ⏕ᡂࢆ♧ࡍ〓ⰍࡢDAB
ᛂࡀほᐹࡉࢀࡓ(Fig. 6-7)ࠋ୍᪉ࠊᑐ↷༊ࡋ࡚⏝࠸ࡓ␃Ỉฎ⌮ࡢࢿⴥ࡛ࡣ㐣㓟Ỉ⣲
ࡢ⏕ᡂࢆ♧ࡍ〓Ⰽࡢᛂࡣほᐹࡉࢀ࡞ࡗࡓ(Fig. 6-7)ࠋ100 ppmࠊ350 ppmཬࡧ100ಸ⃰⦰
ࡋࡓO821 ⳦ᰴࡢᇵ㣴ࢁᾮࡽ༢㞳࣭⢭〇ࡉࢀࡓᢚไ≀㉁ࡢฎ⌮༊࡛ࡣྠᵝ〓Ⰽࡢ DAB
ᛂࡀほᐹࡉࢀࡓ(Fig. 6-7)ࠋࢫࢥࣝࣅࣥ㓟ࢆྵ᭷ࡋࡓ༊࡛ࡣࠊ࠸ࡎࢀࡶ〓Ⰽࡢᛂࡣほᐹ ࡉࢀ࡞ࡗࡓ(Fig. 6-7)ࠋ
72
DW O821-CFᴾ 100 ppm 350 ppmpp pp ᴾ㻌 O821 inhibitory compound㻌 DAB+AAᴾ㻌
DABᴾ㻌
Fig. 6-7. Histochemical detection of H2O2 generation using 3,3'-diaminobenzidine (DAB) in rice leaves treated with the ethyl acetate extract of culture filtrate of isolate O821 (O821-CF), O821 inhibitory compound (100 ppm or 350 ppm) or distilled water (DW). Rice leaves were treated with O821-CF, O821 inhibitory compound, or DW and maintained at room temperature for 24 h. Treated rice leaves were immersed in DAB solution in the presence or absence of ascorbic acid (AA) for 8 h.
73
➨
➨7⠇ O821⳦ᰴࡢᇵ㣴ࢁᾮࡽศ㞳ࡉࢀࡓᢚไ≀㉁ࡼࡿࢿ࡛ࡢPBZ1㑇ఏᏊࡢⓎ⌧
1㸬ᮦᩱཬࡧ᪉ἲ a)౪ヨ᳜≀
ࢿ(ရ✀㸸ࢥࢩࣄ࢝ࣜ)ࢆᐇ㦂⏝࠸ࡓࠋࢿࡣ➨4❶ࠊ➨1⠇ࠊ1a)࡛㏙ࡓ᪉ἲࡼࡾ
⏕⫱ࡉࡏ࡚ᐇ㦂⏝࠸ࡓࠋ
b)O821⳦ᰴࡢᇵ㣴ࢁᾮࡽศ㞳ࡉࢀࡓᢚไ≀㉁ࡢㄪ〇ཬࡧࢿⴥࡢฎ⌮ἲ
O821⳦ᰴࡢᇵ㣴ࢁᾮࡽ༢㞳࣭⢭〇ࡉࢀࡓᢚไ≀㉁ࡣ➨6❶ࠊ➨3⠇ࠊ1࡛㏙ࡓ᪉ἲ
ࡼࡾㄪ〇ࡋࡓࠋࢿࡢฎ⌮ࡣ➨6 ❶ࠊ➨6⠇ࠊ1b)࡛㏙ࡓ᪉ἲࡼࡾ⾜ࡗࡓࠋ
c)ࢿࡢⴥࡽTotal RNAࡢᢳฟࠊcDNAྜᡂཬࡧRT-PCR
ࢿࡢⴥࡽTotal RNAࡢᢳฟࠊcDNAྜᡂࡣ➨5❶ࠊ➨2⠇ࠊ1c)࡛㏙ࡓ᪉ἲࢆ⏝࠸
࡚ࠊ⾜ࡗࡓࠋ
MiliQ (14.9 μl)ࠊ10×ࣂࢵࣇ(2.0 μl)ࠊ2.5mM dNTPs (1.6 μl)ࠊࣉ࣐࣮ࣛ1 (0.2 μl)ࠊࣉࣛ
࣐࣮2 (0.2 μl)ࠊTaq (0.1 μl)ࠊࢧࣥࣉࣝDNA⁐ᾮ(2 μl)ࢆΰྜࡋࠊࢧ࣮࣐ࣝࢧࢡ࣮ࣛࢆ⏝࠸
࡚ࠊRT-PCRࢆ⾜ࡗࡓࠋRT-PCRࡢ᮲௳ࡣ95Υ࡛3ศ㛫ࡢ⇕ኚᛶᚋࠊ98Υ࡛10⛊㛫ࠊ50Υ
࡛30⛊㛫ࠊ72Υ࡛1ศ㛫ࢆ30ࢧࢡࣝ⧞ࡾ㏉ࡋࠊ᭱ᚋ72Υ࡛10ศ㛫ࡋࡓࠋ㑇ఏᏊ
ࡢⓎ⌧ࡣ1%࣮࢞ࣟࢫࢤ࡛ࣝ㟁ẼὋືࡋ࡚ࠊࣂࣥࢻࡢ㐪࠸࡛☜ㄆࡋࡓࠋ
2㸬⤖ᯝ
O821⳦ᰴࡢᇵ㣴ࢁᾮ(100ಸ)ཬࡧ༢㞳࣭⢭〇ࡉࢀࡓᢚไ≀㉁(100 ppmཬࡧ350 ppm)ࢆ
ࢿⴥฎ⌮ࡋࠊ24 㛫ᚋࡢࢿⴥ࡛ࡢᐖᢠᛶㄏᑟ㛵㐃㑇ఏᏊ࡛࠶ࡿ PBZ1 㑇ఏᏊࡢⓎ
⌧㔞ࢆRT-PCRࡼࡾㄪᰝࡋࡓࠋࡑࡢ⤖ᯝࠊO821⳦ᰴࡢᇵ㣴ࢁᾮཬࡧ༢㞳࣭⢭〇ࡉࢀࡓᢚ
ไ≀㉁(100 ppmཬࡧ350 ppm)ࢆฎ⌮ࡋࡓࢿⴥ࡛ࡢPBZ1㑇ఏᏊࡢⓎ⌧ࡣ␃Ỉࢆฎ⌮ࡋ ࡓࢿⴥ࡛ࡢPBZ1㑇ఏᏊࡢⓎ⌧㔞ẚ㍑ࡋ࡚ࠊ㧗ࡗࡓ(Fig. 6-8)ࠋ
74
O821 inhibitory compound㻌 PBZ1ᴾ㻌
Actinᴾ 㻌
DW O821-CF 100 ppm 350 ppmᴾ 㻌
Fig. 6-8. Effect of O821 inhibitory compound on PBZ1 expression by RT-PCR in rice leaves.
Rice leaves were treated with O821-CF, O821 inhibitory compound (100 ppm or 350 ppm) or distilled water (DW). RNA samples were extracted from rice leaves treated for 24 h. The experiments were independently repeated three times.
75
➨
➨8⠇ ⪃ᐹ
࣒࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮ཬࡧHPLCࢆ⏝࠸࡚ࠊO821⳦ᰴࡢᇵ㣴ࢁᾮᏑᅾࡋ࡚࠸ࡿᢚ ไ≀㉁ࡢศ㞳ࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊHPLC࡛ศྲྀࡋࡓRT 4.26㏆ࡢࣆ࣮ࢡࢿ࠸ࡶࡕ
⳦ࡢ⬊ᏊⓎⱆཬࡧ╔ჾᙧᡂࢆᢚไࡍࡿ≀㉁ࡀᏑᅾࡋ࡚࠸ࡓࠋࡲࡓࠊศྲྀࡋࡓ≀㉁ࢆ TLC
ࡼࡾศ㞳ࡋࡓ⤖ᯝࠊRf್0.5-0.6㏆ࢿ࠸ࡶࡕ⳦ࡢ⏕⫱㜼ṆᖏࡀㄆࡵࡽࢀࡓࠋTLC ࡢᒎ㛤⁐፹ࡣࠊࢺ࢚ࣝࣥ㸸㓑㓟࢚ࢳࣝ(1:1)ࢆ⏝࠸ࡓࡇࡽࠊẚ㍑ⓗỈᛶࡢ≀㉁࡛࠶ࡿ
ࡇࡀ⪃࠼ࡽࢀࡓࠋࡲࡓࠊ254 nm࡛ࡢ྾ࡀࢇㄆࡵࡽࢀ࡞ࡗࡓࡇࡽ࣋ࣥࢮࣥ
⎔ࢆᣢࡗ࡚࠸࡞࠸ྍ⬟ᛶࡶ♧၀ࡉࢀࡓࠋ୍᪉ࠊ➨4❶࡛ࡣࠊTLCࢆ⏝࠸ࡓᐇ㦂ࡼࡾࠊྠᵝ ࡢRf್࢟ࣗ࢘ࣜ〓ᩬ⳦ཬࡧⅣ⳦ࡢ⏕⫱㜼ṆࡀほᐹࡉࢀࡓࠋࡇࡢࡇࡣO821⳦ᰴ
ࡢᇵ㣴ࢁᾮ୰ྵࡲࢀࡿẚ㍑ⓗᢠ⳦ࢫ࣌ࢡࢺࣝࡀᗈࡃࠊ༢୍ࡢ≀㉁ࡀࢿ࠸ࡶࡕ⳦ࠊ࢟ࣗ
࢘ࣜ〓ᩬ⳦ཬࡧⅣ⳦ࢆᢚไࡋࡓ⪃࠼ࡽࢀࡓࠋ
๓❶࡛ࡣࠊO821⳦ᰴࡢᇵ㣴ࢁᾮࢿ࠸ࡶࡕ⳦ࡢឤᰁ⾜ືࢆᢚไࡍࡿ≀㉁ཬࡧࢿ
ᢠᛶࢆㄏᑟࡍࡿ≀㉁ࡀᏑᅾࡋ࡚࠸ࡿࡇࢆ᫂ࡽࡋࡓࠋᅇࠊO821⳦ᰴࡢᇵ㣴ࢁᾮ୰
⏕ ⏘ ࡉ ࢀ ࡓ ᢚ ไ ≀ ㉁ ࡀ (3aS,4aR,8aS,9aR)-3a-hydroxy-8a-methyl-3,5-dimethylenedecahydronaphtho[2,3-b]furan-2(3H)-one࡛࠶ࡿࡇࢆ᫂ࡽࡋࡓࠋᮏ≀㉁ࡣࡇࢀ
ࡲ࡛ࠊሗ࿌ࡉࢀ࡚࠸࡞࠸᪂つࡢ≀㉁࡛࠶ࡿࡇࡀ᥎ ࡉࢀࡓࠋᮏ≀㉁㢮ఝࡋ࡚࠸ࡿ≀㉁
ࡋ࡚ࠊ࢜ࢢ࣐ࣝᒓࡢ᳜≀(Inula helenium L., Inula japonica)ࡀ⏕⏘ࡍࡿࣛࣥࢺࣛࢡࢺࣥཬ ࡧࢯࣛࣥࢺࣛࢡࢺࣥࡀ࠶ࡿࠋࡇࢀࡽࡢ≀㉁ࡣᢠ⅖ᛶࠊᢠ┿⳦ᛶཬࡧᢠࡀࢇάᛶ࡞
ࡢ⸆⌮Ꮫⓗ࡞άᛶࡀሗ࿌ࡉࢀ࡚࠸ࡿ(Picman and Schneider 1993㸹Khan et al. 2012㸹Kataria and
Chahal 2013㸹Rasul et al. 2013)ࠋࣛࣥࢺࣛࢡࢺࣥཬࡧࢯࣛࣥࢺࣛࢡࢺࣥࡣ⣙100ᖺ
๓ᵓ㐀ࡀỴᐃࡉࢀࠊ10 ppm ࡢ⃰ᗘࡽᢠ⳦άᛶࢆ♧ࡍࡇࡀ▱ࡽࢀ࡚࠸ࡿ(Picman 1983)ࠋ
ᅇࠊO821⳦ᰴࡢᇵ㣴ࢁᾮ୰⏕⏘ࡉࢀࡓᢚไ≀㉁ࡣ5 ppm௨ୖࡢ⃰ᗘࡽࢿ࠸ࡶࡕ
⳦ࢆᢚไࡋࡓࠋᮏ≀㉁ࡣࢯࣛࣥࢺࣛࢡࢺࣥࡢᵓ㐀ࡢ1ࡘࡢỈ⣲(㸫H)ࡀࣄࢻࣟ࢟ࢩᇶ (㸫OH)⨨ࡉࢀࡓࡶࡢ࡛࠶ࡿࠋࣄࢻࣟ࢟ࢩᇶ(㸫OH)⨨ࡉࢀࡿࡇࡼࡾᮏ≀㉁ࡣ
ࢯࣛࣥࢺࣛࢡࢺࣥࡼࡾ㟁㞳ࡋࡸࡍࡃࠊᛂᛶࡀ㧗࠸ྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋࡲࡓࠊࢭࢫ࢟
ࢸ࣭ࣝ࣌ࣥࣛࢡࢺࣥࡢ⏕≀άᛶࡣࣛࢡࢺࣥ⎔ࡢ㔜⤖ྜࡀ㔜せ࡛࠶ࡾࠊࢯࣛࣥࢺࣛࢡ ࢺࣥࡢC4ࡢ⨨⎔እ㔜⤖ྜࡢᏑᅾࡼࡾࠊࢯࣛࣥࢺࣛࢡࢺࣥࡣࣛࣥࢺࣛࢡࢺ
ࣥࡼࡾᢠ⳦άᛶࡀ㧗࠸ࡇࡀ▱ࡽࢀ࡚࠸ࡿ(Kataria and Chahal 2013㸹Klochkov et al. 2011)ࠋᮏ
≀㉁ࡀࢯࣛࣥࢺࣛࢡࢺࣥྠᵝC4ࡢ⨨⎔እ㔜⤖ྜࡀ࠶ࡿࡇࡼࡾ㧗࠸ᢠ
⳦άᛶࡀ࠶ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ
ࡇࢀࡲ࡛ࡢࢿ࠸ࡶࡕ⳦ᑐࡍࡿ⸆ࡋ࡚ࠊ㣗⏝࢟ࣀࢥࡽⓎぢࡉࢀࡓኳ↛⏕⌮ά ᛶ≀㉁࡛࠶ࡿࢫࢺࣟࣅࣝࣜࣥAཬࡧBࡀ▱ࡽࢀ࡚࠾ࡾࠊ10 ppm௨ୖࡽ⣽⳦ཬࡧ⣒≧⳦
ᑐࡍࡿᢚไࢆ♧ࡍ(Anke et al. 1977)ࠋࡲࡓࠊᐇ㝿ࣈࣛࢩࣥ(᭷ຠᡂศ㸸ࣇࢧࣛࢻ)ࡣ 150 ppmࠊ࢝ࢫ࣑ࣥ(࢝ࢫ࣐࢞ࢩࣥ)ࡣ20 ppmࠊࣄࣀࢨࣥ(࢚ࢹࣇ࢙ࣥ࣍ࢫ)ࡣ300 ppmࠊ࣋ࣥ
࣮ࣞࢺ(࣋ࣀ࣑ࣝ)ࡣ 500 ppmཬࡧࢡ࣑ ࣅ࣮࣒ࢰࣝ(ࢺࣜࢩࢡࣛࢰ࣮ࣝ)ࡣ200 ppmࡢ⃰
ᗘ࡛ࢿ࠸ࡶࡕࡢ㎰⸆ࡋ࡚ࢃࢀ࡚࠸ࡿࠋO821⳦ᰴࡢᇵ㣴ࢁᾮࡽ༢㞳࣭⢭〇ࡉࢀࡓ
76
ᢚไ≀㉁ࡣ 5 ppm ௨ୖ࡛ࠊࢿయୖ࡛ࢿ࠸ࡶࡕࡢⓎࢆᢚไࡍࡿࡇࡽࠊⓏ㘓ࡉࢀ
࡚࠸ࡿ㎰⸆ྠ➼࡞ຠᯝࢆ♧ࡍࡇࡀ࡛ࡁࡿ⪃࠼ࡽࢀࡓࠋO821⳦ᰴࡢᇵ㣴ࢁᾮࡽ༢㞳࣭
⢭〇ࡉࢀࡓᢚไ≀㉁ࡀࢿ࠸ࡶࡕࡢ㜵㝖⏝࡛ࡁࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ
୍᪉ࠊO821⳦ᰴࡢᇵ㣴ࢁᾮࡽ༢㞳࣭⢭〇ࡉࢀࡓᢚไ≀㉁ࡢฎ⌮࡛ࡣࠊ㐣㓟Ỉ⣲ࡢ⏕
ᡂࢆㄪᰝࡍࡿ〓ⰍࡢᛂࡀほᐹࡉࢀࡓࠋJabs (1999)ࡣ㐣㓟Ỉ⣲ࡀࢩࢢࢼࣝศᏊࡋ࡚⣽
⬊Ṛ㔜せ࡞ᙺࢆᯝࡓࡋ࡚࠸ࡿࡇࢆሗ࿌ࡋ࡚࠸ࡿࠋࡉࡽࠊO821⳦ᰴࡢᇵ㣴ࢁᾮࡽ
ศ㞳ࡋࠊ༢㞳࣭⢭〇ࡉࢀࡓᢚไ≀㉁ࡣࢿⴥ࡛ࡢPBZ1㑇ఏᏊࡢⓎ⌧ࢆቑຍࡉࡏࡓࡇࡽࠊ
ᢠᛶࡶㄏᑟࡋ࡚࠸ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋ୍᪉ࠊO821⳦ᰴࡢᇵ㣴ࢁᾮࡽศ㞳ࡋࠊ༢㞳࣭
⢭〇ࡉࢀࡓᢚไ≀㉁ࡢฎ⌮༊࡛ࡣ➨4❶ࠊ➨2⠇࡛O821⳦ᰴࡢᇵ㣴ࢁᾮࡢ๓ฎ⌮༊ࡢᢚไ
ຠᯝẚ㍑ࡋ࡚Ⓨᗘࡀ㧗࠸ࡇࡽࠊࢿⴥୖ࡛ᇵ㣴ࢁᾮ୰ᮏ≀㉁┦ຠᯝࢆ♧ࡍ
≀㉁ࡀᏑᅾࡋࠊࡑࡢ≀㉁ࡀᢠᛶㄏᑟ㛵ࢃࡿ⿵㊊ⓗ࡞ᙺࢆᯝࡓࡍྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋ ࡇࢀࡽࡢ⤖ᯝࡽࠊO821⳦ᰴࡢᇵ㣴ࢁᾮ୰⏕⏘ࡉࢀࡓᢚไ≀㉁ࡣࢿ࠸ࡶࡕ⳦ᑐࡍ
ࡿ┤᥋ⓗ࡞స⏝ࡔࡅ࡛࡞ࡃࠊࢿ࡛ࡢ㐣㓟Ỉ⣲ࡢ⏕ᡂࢆㄏᑟࡋࠊࢿࡢᐖᢠᛶ㑇ఏᏊ ࡢⓎ⌧ࡶቑຍࡉࡏࡿస⏝ࢆᣢࡗ࡚࠸ࡿྍ⬟ᛶࡀ♧ࡉࢀࡓࠋ
77
➨
➨7❶ H921⳦ᰴࡢᇵ㣴ࢁᾮࡀࢿ࠸ࡶࡕ⳦ཬࡰࡍᙳ㡪
᪥ᮏᅜෆ࡛ࠕ㔝⏕ࡁࡢࡇࠖࢆ᥇ྲྀࡋ࡚ࠊࠕࡁࡢࡇᐤ⏕ཪࡣඹ⏕ࡋ࡚࠸ࡿ⳦㢮ࠖࢆศ㞳ࡋࠊ ࡑࢀࡽࡢᇵ㣴ࢁᾮࢆ⏝࠸࡚ࠊࢿ࠸ࡶࡕ⳦ᑐࡍࡿᢚไຠᯝࢆㄪᰝࡋ࡚ࡁࡓࠋࡇࢀࡲ࡛
㜰ᗓෆ࡛᥇ྲྀࡋࡓࡁࡢࡇࡽศ㞳ࡉࢀࡓO821⳦ᰴࡢᢚไຠᯝࡘ࠸࡚㏙ࡓࠋᮏ❶࡛ࡣࠊ ᗈᓥ┴ෆ࡛᥇ྲྀࡋࡓࡁࡢࡇࡽศ㞳ࡉࢀࡓ H921 ⳦ᰴࡢᇵ㣴ࢁᾮࢆ⏝࠸ࡓࢿ࠸ࡶࡕ⳦
ࡢᢚไࡘ࠸࡚ヲࡋࡃ㏙ࡿࠋ
➨1⠇ H921⳦ᰴࡢᇵ㣴ࢁᾮࡀࢿ࠸ࡶࡕ⳦ࡢឤᰁ⾜ື࠼ࡿᙳ㡪 1㸬ᮦᩱཬࡧ᪉ἲ
a)౪ヨ⳦ཬࡧᇵ㣴᮲௳
ࢿ࠸ࡶࡕ⳦ཬࡧH921⳦ᰴࡣ➨2❶ࠊ➨2⠇ࠊ1a)࡛㏙ࡓ᪉ἲࡼࡾ‽ഛࡋࡓࠋ b)ᇵ㣴ࢁᾮࡢㄪ〇
ᇵ㣴ࢁᾮࡢㄪ〇ࡣ➨3❶ࠊ➨2⠇ࠊ1b)࡛㏙ࡓ᪉ἲࡼࡾ‽ഛࡋࡓࠋ c)⇕ฎ⌮
ୖ㏙ࡢ᪉ἲ࡛ㄪ〇ࡋࡓᇵ㣴ࢁᾮࢆ Disposable Culture Tubes(13×100 mm) (Thermo Fisher Scientific Waltham, Massachusetts, USA)ຍ࠼࡚ࠊ࣑࡛ࣝ࣍ࣝࢆࡋ࡚ࠊ࣮࢜ࢺࢡ࣮ࣞࣈ
ࢆ⏝࠸࡚ࠊ⇕ฎ⌮ࡋࡓࠋ⇕ฎ⌮ࡣ105Υཬࡧ121Υ࡛20ศ㛫ࡋࠊᑐ↷༊ࡋ࡚⇕ฎ⌮࡞ࡋ
ࢆ⏝࠸ࡓࠋ d)⏕≀᳨ᐃἲ
➨2❶ࠊ➨2⠇ࠊ1b)ྠᵝࡢ᪉ἲ࡛ࢿ࠸ࡶࡕ⳦⬊Ꮚᠱ⃮ᾮࢆㄪ〇ࡋࠊࢿ࠸ࡶࡕ
⳦ࡢ⬊ᏊⓎⱆཬࡧ╔ჾᙧᡂࢆほᐹࡋࡓࠋ
2㸬⤖ᯝ
H921 ⳦ᰴࡢᇵ㣴ࢁᾮ(2ಸ⃰⦰)ࢆ105Υཪࡣ121Υ࡛⇕ฎ⌮ࢆ⾜ࡗࡓ㝿ࡢࢿ࠸ࡶࡕ⳦
ࡢ⬊ᏊⓎⱆཬࡧ╔ჾᙧᡂࡢᙳ㡪ࢆㄪᰝࡋࡓࠋࡑࡢ⤖ᯝࠊPS ᾮయᇵᆅࢆ⏝࠸ࡓᑐ↷༊࡛
ࡣࠊ↓ฎ⌮༊ࠊ105Υཬࡧ121Υ⇕ฎ⌮༊ࡢ⬊ᏊⓎⱆ⋡ࡣࡑࢀࡒࢀ94.7 ± 6.2%ࠊ98.1 ± 2.3%
ཬࡧ 98.2 ± 2.0%࡛࠶ࡗࡓ(Fig. 7-1 A)ࠋ୍᪉ࠊH921 ⳦ᰴࡢᇵ㣴ࢁᾮ⇕ฎ⌮࡞ࡋࠊ105Υཬࡧ 121Υ⇕ฎ⌮༊࡛ࡣࠊ⬊ᏊⓎⱆ⋡ࡣࡑࢀࡒࢀ6.0 ± 4.7%ࠊ4.8 ± 3.8% ཬࡧ4.2 ± 2.9%࡞ࡾࠊ 㧗࠸⬊ᏊⓎⱆࡢᢚไࡀほᐹࡉࢀࡓ(Fig. 7-1 A)ࠋH921⳦ᰴࡢᇵ㣴ࢁᾮࡢ⇕ฎ⌮ (105Υཬࡧ 121Υ) ࡣࠊ⬊ᏊⓎⱆࡢᢚไᙳ㡪ࢆ࠼࡞ࡗࡓࠋࡲࡓࠊ╔ჾᙧᡂ⋡ࡶ⬊ᏊⓎⱆ⋡ྠ
ᵝᑐ↷༊ࡣࠊࡑࢀࡒࢀ79.6 ±25.1%ࠊ91.1 ± 15.6% ཬࡧ97.8 ± 2.6%࡛࠶ࡗࡓࡢᑐࡋࠊH921
⳦ᰴࡢᇵ㣴ࢁᾮ࡛ࡣ⇕ฎ⌮ࡢ᭷↓㛵ࢃࡽࡎࠊࡑࢀࡒࢀࡢฎ⌮༊ࡢ╔ჾᙧᡂ⋡ࡀ 0.3 ± 0.8%ࠊ0.1 ± 0.5% ཬࡧ0.1 ± 0.5%࡞ࡾࠊ╔ჾᙧᡂࡢᢚไࡀほᐹࡉࢀࡓ(Fig. 7-1 B)ࠋ
78 100㻌
80㻌 60㻌 40㻌 20㻌 0㻌 100
80 60 40 20 Percentage of spore germination (%)㻌Percentage of appressorium formation (%) Percentage of appressorium formation (%%) 0
100 80 60 40 20 0
a
b A
b b
B a 㻌 ab
No treatment 105°C 㻌 121°C No treatment 105°C 121°C
c c c
PSB H921-CF
Fig. 7-1. Inhibition by heat-treated the ethyl acetate extract of culture filtrates of isolate H921 (H921-CF) on the infection behaviors of Magnaporthe oryzae. H921-CF was treated at 105Υ or 121Υ, 20 minutes. Spores of M. oryzae were dropped on glass slides in the presence of heat-pretreated H921-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 are significantly different using the Tukey-Kramer test (p < 0.05).
a a
b
79
➨
➨2⠇ H921⳦ᰴࡢᇵ㣴ࢁᾮࡼࡿࢿ࠸ࡶࡕ⳦ࡢᢚไຠᯝ 1㸬ᮦᩱཬࡧ᪉ἲ
a)౪ヨ⳦ཬࡧᇵ㣴᮲௳
ࢿ࠸ࡶࡕ⳦ཬࡧH921⳦ᰴࡣ➨2❶ࠊ➨2⠇ࠊ1a)࡛㏙ࡓ᪉ἲࡼࡾ‽ഛࡋࡓࠋ b)ᇵ㣴ࢁᾮࡢㄪ〇
H921 ⳦ᰴ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ࡋࡓH921⳦ᰴࡽㄪ〇ࡋࡓᇵ㣴ࢁᾮ(2ಸ⃰⦰)ࢆ⏝࠸࡚ࠊ
ࢿ࠸ࡶࡕ⳦ࡢឤᰁ⾜ືᑐࡍࡿຠᯝࢆㄪᰝࡋࡓࠋࡑࡢ⤖ᯝࠊPSᾮయᇵᆅࢆ⏝࠸ࡓᑐ↷༊
࡛ࡣࠊ⬊ᏊⓎⱆ⋡ཬࡧ╔ჾᙧᡂ⋡ࡣ 99.4±1.1%ཬࡧ 98.0±2.8%࡛࠶ࡗࡓ(Fig. 7-2 AࠊB)ࠋ H921⳦ᰴࢆ1᪥ᇵ㣴ࡋࡓᇵ㣴ࢁᾮࡢ⬊ᏊⓎⱆ⋡ࡣ99.3 ± 1.3% (Fig. 7-2 A). ࡇࢀᑐࡋ࡚ࠊ H921⳦ᰴࢆ3ࠊ5ཬࡧ7᪥㛫ᇵ㣴ᚋㄪ〇ࡋࡓᇵ㣴ࢁᾮࡢ⬊ᏊⓎⱆ⋡ࡣࡑࢀࡒࢀ1.7 ± 2.7%ࠊ 6.5 ± 4.8%ཬࡧ6.0 ± 4.7%࡛࠶ࡗࡓ(Fig. 7-2 A)ࠋࡲࡓࠊH921⳦ᰴࢆ1᪥㛫ᇵ㣴ᚋㄪ〇ࡋࡓ ᇵ㣴ࢁᾮࡢ╔ჾᙧᡂ⋡ࡣ98.9 ± 1.7%࡛࠶ࡗࡓ(Fig. 7-2 B)ࠋ୍᪉ࠊH921⳦ᰴࢆ3ࠊ5ཬࡧ7
᪥㛫ᇵ㣴ᚋㄪ〇ࡋࡓᇵ㣴ࢁᾮ࡛ࡣࡑࢀࡒࢀ0.5 ± 1.5%, 4.8 ± 9.4%ཬࡧ0.3 ± 0.8%࡛࠶ࡗࡓ
(Fig. 7-2 B)ࠋH921 ⳦ᰴࡢᇵ㣴ࢁᾮ࡛ࡣᇵ㣴3᪥௨㝆ᢚไຠᯝࢆ♧ࡍᢚไ≀㉁ࡀᏑᅾࡍࡿ
ࡇࡀ♧ࡉࢀࡓࠋ
80 b
b b
A a
B
a
b
b
b a
a
Fig. 7-2. Time course of inhibitory activity of the ethyl acetate extract of culture filtrates of isolate H921 (H921-CF) on spore germination and appressorium formation of Magnaporthe oryzae. Spores of M. oryzae were suspended in H921-CF for different time periods and dropped on glass slides.
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 are significantly different using the Tukey-Kramer test (p < 0.05).
Percentage of spore germination (%) Percentage of appressorium formation (%) Percentage of appressorium formation 100
80 60 40 20 0 Percentage of spore germination(%)n (%)
0 100 100
80 60 40 20 0
Incubation period of H921 (days) PSB 1 3 5 7
81
➨
➨3⠇ H921⳦ᰴࡢྠᐃ 1㸬ᮦᩱཬࡧ᪉ἲ
࣭౪ヨ⳦
H921⳦ᰴࡣPSAᇵᆅࢆ⣙20 mlὶࡋࡓࣉࣛࢫࢵࢳࢡࢩ࣮ࣕࣞ(┤ᚄ7 cm)⛣᳜ࡋࠊᬯ㯮
ୗࠊ26Υ࡛⣙14᪥㛫ᇵ㣴ࡋࡓࡶࡢࢆᐇ㦂⏝࠸ࡓࠋ
࣭⳦ྀཬࡧ⬊Ꮚࡢほᐹ
PSA ᇵᆅୖ࡛ 7᪥㛫ᇵ㣴ࡋࡓH921 ⳦ᰴࡢ⳦ྀࡢᵝᏊࢆほᐹࡋࡓࠋගᏛ㢧ᚤ㙾ࢆ⏝࠸࡚
ᙧែⓗ࡞≉ᚩࢆほᐹࡋࡓࠋࡲࡓࠊH921⳦ᰴࡢ⳦ྀࡣࢢࣝࢱࣝࣝࢹࣄࢻᅄ㓟࢜ࢫ࣑࢘
࣒ࡼࡿ㔜ᅛᐃࢆࡋࠊ㟁Ꮚ㢧ᚤ㙾(᪥❧㉸㧗ศゎ⬟㟁⏺ᨺฟᙧ㉮ᰝ㟁Ꮚ㢧ᚤ㙾S-4800ࠊᰴ
ᘧ♫᪥❧ࣁࢸࢡࣀࣟࢪ࣮ࢬ)ࢆ⏝࠸࡚ほᐹࡋࡓࠋ
࣭DNAࡢᢳฟཬࡧITS㡿ᇦࡢࢩ࣮ࢡ࢚ࣥࢫゎᯒ
DNAᢳฟࡣSuzuki et al. (2006) ࡢ᪉ἲᚑࡗ࡚⾜ࡗࡓࠋ➨2❶ࠊ➨3⠇ࠊ1b)ྠᵝ࡞᪉
ἲࢆ⏝࠸࡚H921 ⳦ᰴࡢྠᐃࢆ⾜ࡗࡓࠋ⣔⤫ᶞࡣSaitou and Nei (1987) ᇶ࡙࠸࡚సᡂࡋࡓࠋ
2㸬⤖ᯝ
࣭H921 ⳦ᰴࡢ⳦ྀཬࡧ⬊Ꮚࡢほᐹ
H921 ⳦ᰴࡢ⳦ྀཬࡧ⬊Ꮚࡢᙧែほᐹࢆ⾜ࡗࡓࠋ⳦ྀࡣᇵ㣴ึᮇ(⛣᳜4᪥┠㡭)ࡣⓑⰍ
࡛࠶ࡗࡓࡀࠊḟ➨ⓑⰍࡽ⥳Ⰽኚࡋࡓ(Fig. 7-3 AࠊB)ࠋ⬊Ꮚࡣ⳦ྀࡀ⥳ⰍኚⰍࡋࡓ 㡭ࡽᙧᡂࡉࢀጞࡵࡓࠋගᏛ㢧ᚤ㙾(Fig. 7-3 CࠊD)ཬࡧ㟁Ꮚ㢧ᚤ㙾(Fig. 7-3 EࠊF)ࡼࡗ࡚⌫
≧ࡢ⬊Ꮚࡀほᐹࡉࢀࡓࠋࡇࢀࡽࡢ≉ᚩࡣࠊTrichodermaᒓ⳦ࡢ⳦ྀࡢᙧែཬࡧ⬊Ꮚྠᵝ࡛
࠶ࡗࡓ(Fig. 7-3)ࠋ
࣭H921 ⳦ᰴࡢITS㡿ᇦࡢࢩ࣮ࢡ࢚ࣥࢫゎᯒ
H921 ⳦ᰴࡢITS㡿ᇦࡢࢩ࣮ࢡ࢚ࣥࢫࡢᚋࠊBLAST᳨⣴GENETYXࢆ⏝࠸ࡓゎᯒࢆ
⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊH921 ⳦ᰴࡣTrichoderma koningiopsis㧗࠸┦ྠᛶ(100 %)ࢆ♧ࡋࡓ(Fig.
7-4)ࠋ
82
Fig. 7-3. Morphological characteristics of isolate H921 on PSA medium. A: Colony of isolate H921 grown on PSA for 4 days. B: Colony of isolate H921 grown on PSA for 7 days. C, D: Spore (Light microscopy). E, F: Spore (Scanning electron microscopy).
83 Trichoderma viride㻌(NBRC30546)㻌
H921㻌
Trichoderma koningiopsis㻌(FR670342) 㻌
Trichoderma ovalisporum (NBRC101778) 㻌
Trichoderma gamsii (KM491886) 㻌
Trichoderma atroviride (NBRC30543)㻌
Trichoderma aeroaquaticum (NBRC108034)㻌
Trichoderma asperellum (NBRC101777) 㻌
Trichoderma yunnanense (NBRC108643) 㻌
Trichoderma hamatum (NBRC31932) 㻌
Trichoderma piluliferum (NBRC104992) 㻌
Trichoderma brevicompactum (NBRC107663) 㻌
Trichoderma longibrachiatum (NBRC5720) 㻌
Trichoderma rossicum (NBRC9065) 㻌
Trichoderma crissum (NBRC100846) 㻌
Trichoderma virens (NBRC6355)㻌
Trichoderma harzianum (NBRC9066)
㻌
Trichoderma spirale (NBRC30544)㻌
0.01
㻌
Fig. 7-4. Phylogenetic tree based on ITS sequences of strain H921. A bootstrap consensus Neighbor-Joining tree for the Trichoderma strain H921 was created based on the Kimura 2-Parameter distance matrix (1,000 replicates). The Trichoderma viride (NBRC30546) was used as the out-group. The scale bar represents 1% sequence dissimilarity.
84
➨
➨4⠇ H921⳦ᰴࡢᇵ㣴ࢁᾮࡀࢿ࡛ࡢࢿ࠸ࡶࡕࡢⓎ࠼ࡿᙳ㡪 1㸬ᮦᩱཬࡧ᪉ἲ
a)౪ヨ⳦ཬࡧ౪ヨ᳜≀
ࢿ࠸ࡶࡕ⳦ཬࡧH921⳦ᰴࡣ➨2❶ࠊ➨2⠇ࠊ1a)ྠᵝࡢ᪉ἲ࡛‽ഛࡋࡓࠋ࣒࢜࢜
ࢠ(ရ✀:࣡ࢭࢻࣜᅄ᮲)ࡣ➨2❶ࠊ➨2⠇ࠊ1c)ྠᵝࡢ᪉ἲ࡛⏕⫱ࡉࡏࠊᐇ㦂⏝࠸ࡓࠋ ΰྜ᥋✀ࡢᐇ㦂⏝࠸ࡿࢿ(ရ✀㸸ࢥࢩࣄ࢝ࣜ)ࡣ➨4❶ࠊ➨1⠇ࠊ1a)ྠᵝࡢ᪉ἲ࡛‽
ഛࡋࡓࠋ
ࢿ(ရ✀㸸ࢥࢩࣄ࢝ࣜ)ࢆࢿⴥ㠧ࡢH921⳦ᰴࡢᇵ㣴ࢁᾮࡢ๓ฎ⌮ࡢᐇ㦂⏝࠸ࡓࠋ ணࡵࠊⓎⱆࡉࡏࡓ✀Ꮚࡣࢢ࣮ࣜࣥࢯࣝ (ฟ㞼ࢢ࣮࢚࣏ࣜࣥࢵࢡᰴᘧ♫)ࢆຍ࠼ࡓࢩ࣮ࢻ
ࣜࣥࢢࢣ࣮ࢫࠊ1ࢣ࣮ࢫ⣙20⢏ࡎࡘ✀ࡋࡓࠋそᅵࡣࢢ࣮ࣜࣥࢯࣝそᅵ(ฟ㞼ࢢࣜ
࣮࢚࣏ࣥࢵࢡᰴᘧ♫)ࢆ⏝࠸ࡓࠋ✀ᚋࠊ࢞ࣛࢫᐊෆ࡛1᭶㛫(5ⴥᮇࡲ࡛)⏕⫱ࡉࡏࡓࡶ
ࡢࢆᐇ㦂⏝࠸ࡓࠋ
࣭H921 ⳦ᰴࡢᇵ㣴ࢁᾮཬࡧࢿ࠸ࡶࡕ⳦⬊Ꮚᠱ⃮ᾮࡢㄪ〇
H921 ⳦ᰴࡢᇵ㣴ࢁᾮࡣ➨3❶ࠊ➨2⠇ࠊ1b)ྠᵝࡢ᪉ἲ࡛ㄪ〇ࡋࠊ2ಸᕼ㔘ࠊཎᾮ(1ಸ)ࠊ
2ಸཬࡧ5ಸ⃰⦰ࡋࡓᇵ㣴ࢁᾮࢆᐇ㦂⏝࠸ࡓࠋࢿ࠸ࡶࡕ⳦⬊Ꮚᠱ⃮ᾮࡣ➨2❶ࠊ➨2
⠇ࠊ1a)ྠᵝࡢ᪉ἲ࡛‽ഛࡋࡓࠋ
b) ࣒࢜࢜ࢠࡢH921⳦ᰴࡢᇵ㣴ࢁᾮࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚࡢΰྜ᥋✀ࡀࢿ࠸ࡶࡕ
⳦ࡢឤᰁ࠼ࡿᙳ㡪
2ⴥᮇࡢ࣒࢜࢜ࢠࢆࢩ࣮ࢻࣜࣥࢢࢣ࣮ࢫࡽ᰿ࢆษ᩿ࡋ࡞࠸ࡼ࠺ᢤࡁྲྀࡾࠊᅵተࢆ㝖 ཤࡋࡓࠋࡑࡢᚋࠊ‵ࡽࡏࡓ࢟ࢵࢳ࣮ࣥ࣌ࣃ࣮ࢆᘬ࠸ࡓࣉࣛࢫࢳࢵࢡࢣ࣮ࢫ7ಶయࡎࡘ୪
ࡓࠋᇵ㣴ࢁᾮ(2ಸ⃰⦰)ࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚ(1×105 spores/ml)ࢆᠱ⃮ࡋࠊ 5 mlࡎࡘ࢜
࣒࢜ࢠᄇ㟝᥋✀ࡋࡓࠋᑐ↷༊ࡋ࡚↓᥋✀ࡢPSᾮయᇵᆅࡢ㓑㓟࢚ࢳࣝᢳฟỈ⁐ᾮࢆ2 ಸ⃰⦰ࡋࡓᾮࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚࢆᠱ⃮ࡉࡏࡓࡶࡢࢆᄇ㟝᥋✀ࡋࡓࠋࣉࣛࢫࢳࢵࢡࢣ
࣮ࢫࡣࡩࡓࢆ㛢ࡵࠊࢭࣟࣁࣥࢸ࣮ࣉ࡛ᐦ㛢ࡋࠊேᕤẼ㇟ჾෆ(LIB-302(H), IWAKI)ධࢀࠊ ᬯ㯮ୗ࡛24㛫ᇵ㣴ᚋࠊ12㛫ග᮲௳ୗಖᣢࡋࡓࠋ᥋✀3᪥ᚋࠊ࠸ࡶࡕᩬࡢᙧᡂࢆほ ᐹࡋࡓࠋ
c) ࢿࡢ H921 ⳦ᰴࡢᇵ㣴ࢁᾮࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚࡢΰྜ᥋✀ࡀࢿ࠸ࡶࡕ
⳦ࡢឤᰁ࠼ࡿᙳ㡪
3ⴥᮇࡢࢿࢆ1ฎ⌮༊࠶ࡓࡾ2ࢩ࣮ࣕࣞ(20ಶయ)⏝࠸ࡓࠋࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊࢆH921
⳦ᰴࡢ2ಸᕼ㔘ࠊ1ಸࠊ2ಸཬࡧ5ಸ⃰⦰ࡋࡓᇵ㣴ࢁᾮΰྜࡋࠊ20ಶయࡢࢿ2 mlࡎ ࡘᄇ㟝᥋✀ࡋࡓࠋᑐ↷༊ࡋ࡚ࠊ↓᥋✀ࡢPSᾮయᇵᆅࡢ㓑㓟࢚ࢳࣝᢳฟỈ⁐ᾮ5ಸ⃰⦰ࡋ ࡓᾮࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚࢆᠱ⃮ࡋࡓࡶࡢࢆᐇ㦂⏝࠸ࡓࠋฎ⌮ࡋࡓࢿࡣ‵ᐊෆࠊᬯ㯮
ୗ࡛24㛫ಖᣢࡋࠊ⮬↛᮲௳࡛᱂ᇵࡋࡓࠋ᥋✀5᪥ᚋࠊⓎ≧ἣࢆ➨4 ❶ࠊ➨1⠇ࠊ1c)
ྠᵝ࡞᪉ἲࡼࡗ࡚ㄪᰝࡋࠊⓎᗘࢆồࡵࡓࠋ
d) ࢿⴥ㠧ࡢH921⳦ᰴࡢᇵ㣴ࢁᾮࡢ๓ฎ⌮ࡀࢿ࠸ࡶࡕ⳦ࡢឤᰁ⾜ື࠼ࡿᙳ㡪
࣭ࢿⴥ㠧ࡢῶᅽᾐ㏱ฎ⌮ཬࡧ᥋✀
85
H921⳦ᰴࡢᇵ㣴ࢁᾮ(2ಸ⃰⦰)ࢆヨ㦂⟶(24×130 mm)7 mlຍ࠼ࠊࢿⴥ㠧ࢆྛฎ⌮༊࠶
ࡓࡾ5ࠊ6 ᮏࡎࡘษ᩿ࡋࠊⴥ㠧ᇵ㣴ࢁᾮࢆᾐₕࡉࡏࡓࠋᑐ↷༊ࡋ࡚↓᥋✀ࡢPSᾮయᇵ ᆅࢆ㓑㓟࢚ࢳ࡛ࣝᢳฟࡋࡓỈ⁐ᾮ(2ಸ⃰⦰)ࢆ⏝࠸ࠊྠᵝࡢ᪉ἲ࡛ฎ⌮ࡋࡓࠋⴥ㠧ࡣῶᅽᾐ
㏱ฎ⌮ࢆ10ศ㛫ࠊ3ᅇ⧞ࡾ㏉ࡋ࡚ࠊⴥ㠧ෆྛฎ⌮ᾮࢆᾐ㏱ࡉࡏࡓࠋࡑࡢᚋࠊⴥ㠧ࢆྲྀࡾ
ฟࡋࠊ␃Ỉ࡛ᑀὙίࡋࠊฎ⌮ᾮࢆ㝖ཤࡋ࡚‵ᗘࢆಖࡓࡏࡓࣉࣛࢫࢳࢵࢡࢣ࣮ࢫ୪
ࡓࠋὀᑕჾࢆ⏝࠸ࠊࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚᠱ⃮ᾮࢆⴥ㠧ෆ᥋✀ࡋࠊᬯ㯮ୗࠊ26ΥࡢேᕤẼ
㇟ჾෆ(LIB-302(H), IWAKI)࡛48㛫ᇵ㣴ࡋࡓࠋ
࣭ⴥ㠧ษ∦ࡢస〇ཬࡧࢿ࠸ࡶࡕ⳦ឤᰁ⾜ືࡢほᐹ
48 㛫ᇵ㣴ᚋࠊ࣑࢝ࢯࣜࢆ⏝࠸࡚ⴥ㠧㠃⾲⓶ษ∦ࢆస〇ࡋࠊࣛࢡࢺࣇ࢙ࣀ࣮ࣝࣝࢥ
࣮ࣝᾐࡋࠊගᏛ㢧ᚤ㙾ୗ࡛⣽⬊ෆࡢධ⳦⣒ࢆほᐹࡋࡓࠋධ⳦⣒ᩘࡣࠊ⬊Ꮚ50ಶ୰ࡢ
╔ჾᙧᡂࡋࡓ⬊Ꮚ୰ࡢධ⳦⣒ࢆᙧᡂࡋ࡚࠸ࡿ⬊Ꮚᩘࢆィᩘࡋࠊධ⳦⣒ᙧᡂ⋡ࢆ⟬ฟ ࡋࡓࠋ3ᅇ⧞ࡾ㏉ࡋࠊྜィ650⬊Ꮚࢆほᐹࡋࡓࠋ
2㸬㸬⤖ᯝ
a) ࣒࢜࢜ࢠࡢH921⳦ᰴࡢᇵ㣴ࢁᾮࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚࡢΰྜ᥋✀ࡀࢿ࠸ࡶࡕ
⳦ࡢឤᰁཬࡰࡍᙳ㡪
࣒࢜࢜ࢠH921⳦ᰴᇵ㣴ࢁᾮࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚࢆΰྜࡋࠊᄇ㟝᥋✀ࢆ⾜࠸ࠊ࠸
ࡶࡕᩬࡢᙧᡂࢆほᐹࡋࡓࠋࡑࡢ⤖ᯝࠊᑐ↷༊࡛ࡣࠊ࣒࢜࢜ࢠࡢⴥࡀ㯤Ⰽኚࡋࠊ⅊Ⰽ ࡢ࠸ࡶࡕᩬࡀయᙧᡂࡉࢀ࡚࠸ࡓࠋ୍᪉ࠊH921⳦ᰴࡢᇵ㣴ࢁᾮฎ⌮༊࡛ࡣࠊ࡞≧
ែࡢ࣒࢜࢜ࢠࡢⴥࡀほᐹࡉࢀࠊ࠸ࡶࡕᩬࡢᙧᡂࡣᑐ↷༊ẚ㍑ࡋ࡚ῶᑡࡋ࡚࠸ࡓ(Fig. 7-5)ࠋ
b) ࢿࡢH921 ⳦ᰴࡢᇵ㣴ࢁᾮࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚࡢΰྜ᥋✀ࡀࢿ࠸ࡶࡕ⳦
ࡢឤᰁཬࡰࡍᙳ㡪
H921 ⳦ᰴࡢᇵ㣴ࢁᾮࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚࢆΰྜࡋࠊࢿᄇ㟝᥋✀ࡋࠊⓎᗘࢆㄪ
ᰝࡋࡓࠋᑐ↷༊ࡢ5ಸ⃰⦰ࡋࡓPSᾮయᇵᆅฎ⌮༊࡛ࡣࠊⓎᗘࡣ99.5 ± 0.8%࡛࠶ࡗࡓ(Fig.
7-6 B)ࠋ୍᪉ࠊH921 ⳦ᰴࡢᇵ㣴ࢁᾮࡢฎ⌮༊࡛ࡣ2ಸᕼ㔘ࠊ1ಸࠊ2ಸཬࡧ5ಸ⃰⦰ࡋࡓᾮ
ࡣࡑࢀࡒࢀ31.9 ± 18.9%ࠊ16.7 ± 12.7%ࠊ7.6 ± 12.6%ཬࡧ3.3 ± 7.4%࡛࠶ࡗࡓ(Fig. 7-6 B)ࠋᑐ
↷༊ẚ㍑ࡋ࡚ࠊH921⳦ᰴࡢᇵ㣴ࢁᾮࡢΰྜ᥋✀࡛ࡣ2ಸᕼ㔘࡛ࡶⓎᗘࡀ1/3௨ୗῶ ᑡࡋࡓ(Fig. 7-6 AࠊB)ࠋ
c) ࢿⴥ㠧ࡢH921⳦ᰴࡢᇵ㣴ࢁᾮࡢ๓ฎ⌮ࡀࢿ࠸ࡶࡕ⳦ࡢឤᰁ⾜ືཬࡰࡍᙳ㡪 H921⳦ᰴࡢᇵ㣴ࢁᾮࢆࢿⴥ㠧ῶᅽᾐ㏱ฎ⌮ࡋࠊࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚᠱ⃮ᾮࢆ᥋✀
ࡋ࡚ࢿ࠸ࡶࡕ⳦ࡢࢿ⣽⬊ෆࡢධࢆㄪᰝࡋࡓࠋࡑࡢ⤖ᯝࠊPS ᾮయᇵᆅࢆ⏝࠸ࡓᑐ
↷༊࡛ࡣධ⳦⣒ᙧᡂ⋡ࡣ98.6 ± 1.7%࡛࠶ࡗࡓࡢᑐࡋ࡚ࠊH921⳦ᰴࡢᇵ㣴ࢁᾮࡢฎ⌮༊
࡛ࡣࠊ8.0 ± 9.7%࡛࠶ࡗࡓ(Fig. 7-7)ࠋࢿ࠸ࡶࡕ⳦ࡢධ⳦⣒ᙧᡂ⋡ࡀ1/10௨ୗῶᑡࡋࠊ
ࢿ࠸ࡶࡕ⳦ࡢࢿ⣽⬊ෆࡢධࡢᢚไࡀほᐹࡉࢀࡓ(Fig. 7-7)
86
Fig. 7-5. Inhibition of Magnaporthe oryzae blast lesion formation on barley leaves by the ethyl acetate extract of culture filtrate of isolate H921 (H921-CF). Barley leaves were inoculated with M.
oryzae in the presence of H921-CF and kept in a moist chamber for 24 h. 3 days after inoculation, blast lesion formation was observed.
PSB ᴾ ᴾ ᴾ ᴾ ᴾ ᴾH921-CF㻌
87 a
b
bc
c
c
Disease severity (%)㻌
A㻌
B㻌
PSB-5 㻌 H921-1/2 㻌 㻌 H921-1 H921-2 H921-5 㻌
Disease severity (%)
100㻌 80㻌 60㻌 40㻌 20㻌 0㻌
PSB-5 H921-1/2 H921-1 㻌H921-2 㻌 H921-5
Fig. 7-6. Inhibition of blast lesion formation on rice leaves by the ethyl acetate extract of the culture filtrates of isolate H921 (H921-CF). Rice plants were inoculated with Magnaporthe oryzae in the presence of H921-CF isolate and kept in a moist chamber for 24 h. 5 days after inoculation, lesion formation (A) and disease severity (B) was measured. Data are representative of the mean values of the results from three separate experiments with 15 rice plants in each treatment per experiment. Bars represent ± SD. Means followed by different letters are significantly different using the Tukey-Kramer test (p < 0.05).
88
PSB㻌 H921-CF㻌
100㻌 80㻌
60㻌
40㻌
20㻌 0㻌
PSB㻌 H921-CFᴾ ᴾ ᴾ ᴾ ᴾ㻌
10
8
6
4
Infection hypha formation rate (%)㻌 2
Fig. 7-7. Effect of pretreatment with the ethyl acetate extract of the culture filtrates of isolate H921 (H921-CF) on infection behavior of Magnaporthe oryzae in the rice leaf sheaths. Rice leaf sheaths were pretreated with H921-CF, inoculated with M. oryzae and kept in a moist chamber at 26°C in the dark. 48 h after inoculation percentage of infection was determined. The rates of infection hypha formation per appressorium were calculated. The data are the mean the results of three experiments.
A total 650 infection bites were examined. Bars represent ± SD. Asterisks indicate significant differences compared with the control (t-test, p< 0.05).
* Infection hypha㻌
89
➨
➨5⠇ H921⳦ᰴࡢᇵ㣴ࢁᾮࡽࡢࢿ࠸ࡶࡕ⳦ࡢᢚไ≀㉁ࡢศ㞳 1㸬ᮦᩱཬࡧ᪉ἲ
a)౪ヨ⳦ཬࡧᇵ㣴ࢁᾮࡢㄪ〇
౪ヨ⳦ࡋ࡚ࢿ࠸ࡶࡕ⳦ཬࡧH921⳦ᰴࡣ➨2 ❶ࠊ➨2⠇ࠊ1a)࡛㏙ࡓ᪉ἲࡼࡾ
‽ഛࡋࡓࠋ
ࢿࡈࡲⴥᯤ⳦(Bipolaris oryzae)ࡣ➨3❶ࠊ➨5⠇ࠊ1a)࡛㏙ࡓ᪉ἲࡼࡾ‽ഛࡋ࡚⏝
࠸ࡓࠋ
ᇵ㣴ࢁᾮࡣ➨3❶ࠊ➨2⠇ࠊ1b)࡛㏙ࡓ᪉ἲࡼࡾ‽ഛࡋࡓࠋ b)ⷧᒙࢡ࣐ࣟࢺࢢࣛࣇ࣮ࡼࡿᇵ㣴ࢁᾮࡢศ㞳
100 ಸ⃰⦰ࡋࡓ H921 ⳦ᰴࡢᇵ㣴ࢁᾮ(50 μl)ࢆⷧᒙࢡ࣐ࣟࢺࢢࣛࣇ࣮(TLC)⏝ࣉ࣮ࣞࢺ (Silica gel 60, Merck KGaA, Darmstadt, Germany)ࢫ࣏ࢵࢺࡋࠊࢺ࢚ࣝࣥ㸸㓑㓟࢚ࢳࣝ㸻1㸸1
(v/v) ࡢᒎ㛤⁐፹ࢆ⏝࠸࡚ࠊᒎ㛤ࡉࡏࡓࠋ
d)⏕≀᳨ᐃヨ㦂
ᒎ㛤ᚋࠊTLCࣉ࣮ࣞࢺPDAᇵᆅ(39 g/L)ᠱ⃮ࡉࡏࡓB. oryzae⬊Ꮚࢆᄇ㟝ࡋࡓࠋྠᵝ
TLCࣉ࣮ࣞࢺPSAᇵᆅ(ࢡ࣒ࣟࣛࣇ࢙ࢽࢥ࣮ࣝ2 mg/100 ml)ᠱ⃮ࡉࡏࡓM. oryzae⬊ Ꮚࢆᄇ㟝ࡋࡓࠋࣉ࣮ࣞࢺࡣ‵ᐊࡋࡓࣉࣛࢫࢳࢵࢡࢣ࣮ࢫෆ⣡ࡵࡓᚋࠊ࣑ࣝ⨁࡛そ࠸ࠊ 26Υᬯ㯮᮲௳ୗ࡛ᇵ㣴ࡋࠊࣉ࣮ࣞࢺୖࡢ⏕⫱㜼Ṇᖏࡢ᭷↓ࢆㄪᰝࡋࡓࠋ
2㸬⤖ᯝ
H921⳦ᰴࡢᇵ㣴ࢁᾮࢆⷧᒙࢡ࣐ࣟࢺࢢࣛࣇ࣮ᒎ㛤ࡉࡏࡓࠋࢿ࠸ࡶࡕ⳦ཬࡧࢿࡈ
ࡲⴥᯤ⳦ᑐࡍࡿᢚไ≀㉁ࢆ᳨ฟࡋࡓࠋࡑࡢ⤖ᯝࠊࢿ࠸ࡶࡕ⳦࡛ࡣࠊTLCࡢRf್0ࠊ
0.13ࠊ0.19ࠊ0.25ཬࡧ0.56㏆⏕⫱㜼Ṇᖏࡀ☜ㄆࡉࢀࡓ(Fig. 7-8)ࠋࡉࡽࠊࢿࡈࡲⴥᯤ
⳦࡛ࡶྠᵝࡢ⏕⫱㜼Ṇᖏࡀほᐹࡉࢀࡓ(Fig. 7-8)ࠋ
90
Fig. 7-8. Thin layer chromatography (TLC) bioautography of the ethyl acetate extract of the culture filtrates of isolate H921 (H921-CF). The H921-CF was spotted onto a TLC plate. After development, the TLC plate was sprayed with a concentrated spore suspension of Magnaporthe oryzae (left) and Bipolaris oryzae (right) in the presence of potato sucrose agar medium or potato dextrose agar medium. Inoculated plate was kept in a moist chamber at 26°C for 1 (B. oryzae) or 7 days (M. oryzae).
Rf 0.56㻌
ORIGIN㻌 FRONT㻌
Rf 0.25㻌
Magnaporthe oryzae㻌
Bipolaris oryzae㻌 Rf 0.25
Rf 0.19㻌 Rf 0.19 Rf 0.13㻌
91
➨
➨6⠇ ⪃ᐹ
H921⳦ᰴࡢᇵ㣴ࢁᾮ୰ࡣࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚࡢឤᰁ⾜ືࢆᢚไࡍࡿ≀㉁ࡀᏑᅾࡋࡓ ࡇࡽࠊࡇࡢᢚไ≀㉁ࡢ⇕ฎ⌮ࡀࠊࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆཬࡧ╔ჾᙧᡂࡢᢚไຠ
ᯝᙳ㡪ࢆ࠼ࡿࢆㄪᰝࡋࡓࠋࡑࡢ⤖ᯝࠊH921⳦ᰴࡢᇵ㣴ࢁᾮ୰Ꮡᅾࡍࡿᢚไ≀㉁ࡣ
105Υཬࡧ121Υࡢ⇕ฎ⌮ࡼࡗ࡚ࡶᢚไຠᯝࡣపୗࡋ࡞ࡗࡓࠋࡇࢀࡽࡢ⤖ᯝࡽࠊH921
⳦ᰴࡢᇵ㣴ࢁᾮ୰ࡢᢚไ≀㉁ࡀࢱࣥࣃࢡ㉁ࡢࡼ࠺࡞⇕Ᏻᐃ࡞≀㉁࡛ࡣ࡞࠸ࡇࡀ⪃࠼
ࡽࢀࡓࠋ
ᮏ◊✲࡛ࡣࠊPSᾮయᇵᆅࢆ⏝࠸࡚ࠊ1-7᪥㛫ᇵ㣴ࡋࡓH921⳦ᰴࡽㄪ〇ࡋࡓᇵ㣴ࢁᾮࡢ
ࢿ࠸ࡶࡕ⳦ᑐࡍࡿᢚไຠᯝࢆ⤒ⓗㄪᰝࡋࡓࠋH921⳦ᰴࡢᇵ㣴ࢁᾮࡣᇵ㣴ᚋ3᪥
┠௨㝆ࡽࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆཬࡧ╔ჾᙧᡂࡢᢚไࡀほᐹࡉࢀࡓࠋH921⳦ᰴࡢᇵ 㣴ࢁᾮ୰ከࡃࡢ௦ㅰ⏘≀ࡀ⏕⏘ࡉࢀ࡚࠸ࡿ⪃࠼ࡽࢀࠊᑡ࡞ࡃࡶ 1 ࡘ௨ୖࡢ≀㉁ࡀ
ࢿ࠸ࡶࡕ⳦ࡢឤᰁ⾜ືࢆᢚไࡋ࡚࠸ࡿ⪃࠼ࡽࢀࡿࠋᇵ㣴 7 ᪥ᚋࡣ⳦⣒ࡣ༑ศ⏕⫱
ࡋ࡚࠸ࡿࡇࡽ⳦⣒⏕⫱ᚋከࡃࡢᢚไ≀㉁ࡀ⏕⏘ࡉࢀ࡚࠸ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋ
H921 ⳦ᰴࡢ⳦ྀཬࡧ⬊Ꮚࡢᙧែⓗ࡞ほᐹࢆ⾜ࡗࡓࡇࢁࠊTrichoderma ᒓ⳦㢮ఝⅬࡀ
ከࡃᏑᅾࡋࡓࠋࡲࡓࠊH921 ⳦ᰴࡢ ITS 㡿ᇦࡢࢩ࣮ࢡ࢚ࣥࢫゎᯒࡢ⤖ᯝࠊTrichoderma koningiopsisࡢ┦ྠᛶࡀ᭱ࡶ㧗ࡗࡓࠋࡇࢀࡽࡢࡇࡽࠊH921 ⳦ᰴࡣTrichodermaᒓ⳦
࡛࠶ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋࡇࢀࡲ࡛ࠊTrichoderma koningiopsisࡀ᱂ᇵ࢟ࣀࢥឤᰁࡍࡿ
ࡀሗ࿌ࡉࢀ࡚࠸ࡿ(ዟ⏣࣭༑ᔒ 2010)ࠋ୍᪉࡛ࠊTrichodermaᒓ⳦ࡀከࡃࡢ᳜≀ᐖࡢ㜵 㝖⏝ࡉࢀࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿࠋTrichoderma virens ࡣ⏕⏘ࡋࡓᢠ⳦௦ㅰ⏘≀ࡼࡗ࡚
࢝ࣛࣥᩬⅬࡀ㜵㝖࡛ࡁࡿࡇࡀሗ࿌ࡉࢀࡓ(Intana et al. 2005)ࠋࡲࡓࠊTrichoderma harzianum ࡽศἪࡉࢀࡓࣅࣜࢪ࢜ࣇࣥࢪࣥ A ࡣ Fusarium moniliformeࠊ Phytophthora infestans ཬࡧSclerotinia sclerotiorumࢆᢚไࡋࡓ(El-Hasan et al. 2009)ࠋ
H921⳦ᰴࡢᇵ㣴ࢁᾮࢆ⏝࠸࡚ࠊࢿ࠸ࡶࡕ⳦ࡢΰྜ᥋✀ࢆ⾜ࡗࡓ⤖ᯝࠊ࣒࢜࢜ࢠⴥ
࡛ࡣ᳜≀ẘᛶࢆ♧ࡍࡇ࡞ࡃᩬᙧᡂ㐪࠸ࡀㄆࡵࡽࢀࡓࠋࡇࡢ⤖ᯝࡽࠊH921 ⳦ᰴࡢᇵ 㣴ࢁᾮ୰ᢚไ≀㉁ࡀᏑᅾࡋࠊࡑࡢᢚไ≀㉁ࡣࢿ࠸ࡶࡕ⳦ࡢឤᰁ⾜ືᙳ㡪ࢆ࠼ࡿ
ࡇࡼࡾࠊᩬᙧᡂࢆᢚไࡋ࡚࠸ࡿࡇࡀ♧၀ࡉࢀࡓࠋࡉࡽࠊH921⳦ᰴࡢᇵ㣴ࢁᾮࡢ ฎ⌮ࡼࡾࠊࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚࡢධ⳦⣒ᙧᡂࡀᢚไࡉࢀ࡚࠸ࡓࠋࢫࣛࢻ࢞ࣛࢫୖ࡛
ࡢ⬊ᏊⓎⱆヨ㦂࡛ࠊH921⳦ᰴࡢᇵ㣴ࢁᾮࢿ࠸ࡶࡕ⳦⬊Ꮚࢆΰྜࡋࡓ㝿ࠊⓎⱆཬࡧ
╔ჾᙧᡂࢆ࠸ࡎࢀࡶᢚไࡋ࡚࠸ࡓࠋࡑࡋ࡚ࠊH921⳦ᰴࡢᇵ㣴ࢁᾮࢆࢿⴥ㠧ῶᅽᾐ㏱ฎ
⌮ࡋࠊࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚᠱ⃮ᾮࢆ᥋✀ࡋ࡚ࢿ࠸ࡶࡕ⳦ࡢࢿ⣽⬊ෆࡢධࢆㄪ ᰝࡋࡓࠋH921⳦ᰴࡢᇵ㣴ࢁᾮࢆࢿ⣽⬊ෆῶᅽᾐ㏱ฎ⌮ࡋࡓࡓࡵࠊࢿ࠸ࡶࡕ⳦ࡀ
ࢿ⣽⬊ෆධࡋࡓ㝿ࡶᢚไຠᯝࢆ♧ࡋࡓ⪃࠼ࡽࢀࡿࠋࡇࢀࡽࡢ⤖ᯝࡽࠊH921⳦ᰴ
ࡢᇵ㣴ࢁᾮࡣࠊࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚࡢឤᰁ⾜ືࡢᵝࠎ࡞ẁ㝵ࢆᢚไࡍࡿࡇࡀ⪃࠼ࡽࢀ
ࡓࠋ
ࡇࢀࡲ࡛ࡶࠊTrichodermaᒓ⳦ࡀ⏕⏘ࡋࡓ≀㉁ࢆ⏝࠸ࡓ᳜≀ᐖ㜵㝖ࡢሗ࿌ࡣከ࠸(John et
al. 2010)ࠋᮏ◊✲࡛ࡣH921⳦ᰴࡢᇵ㣴ࢁᾮ୰Ꮡᅾࡍࡿᢚไ≀㉁ࢆTLCࡼࡾศ㞳ࡋࠊ2
92
ࡘ௨ୖࡢ⏕⫱㜼ṆᖏࢆほᐹࡋࡓࠋࡲࡓࠊH921⳦ᰴࡢᇵ㣴ࢁᾮࡣࢿ࠸ࡶࡕ⳦ࡔࡅ࡛࡞ࡃࠊ Colletotrichum orbiculareࡸ Corynespora cassiicola ࡞ࡢ⬊ᏊⓎⱆࢆᢚไࡋࡓ(Nguyen et al.
2012 b)ࠋࡇࢀࡽࡢ⤖ᯝࡽࠊH921⳦ᰴࡢᇵ㣴ࢁᾮ୰⏕⏘ࡉࢀࡿ≀㉁ࡣᗈ⠊ᅖ࡛ຠᯝࢆ♧
ࡋࠊ᳜≀ᐖࡢ㜵㝖⏝࡛ࡁࡿྍ⬟ᛶࢆ♧ࡋࡓࠋࡇࢀࡲ࡛ࠊTrichoderma harzianumࡢᇵ 㣴ࢁᾮ⬊Ꮚࢆ⏝࠸ࡓ࣐࣓㢮ࡉࡧࠊࣛࣥࣈ㸫ࢱࣥ〓ᩬ⣽⳦ࠊⅣࠊ㍈⭉ࢀࡢ㜵㝖ࠊ Trichoderma atrovirideࡢᇵ㣴ࢁᾮࢆ⏝࠸ࡓࢪࣕ࢞ࣔ࠾ࡅࡿRhizoctonia solani AG3ࡢ⳦⣒
⏕㛗ࡢᢚไࠊⓎᣦᩘࡢపୗࡀሗ࿌ࡉࢀ࡚࠸ࡿ(Burmeister and Hau 2009㸹Sivakumar et al.
2000㸹Lahlali and Hijri 2010)ࠋࡲࡓࠊTrichodermaᒓ⳦ࡢᇵ㣴ࢁᾮᏑᅾࡍࡿⓎᛶ≀㉁ࡀࢥ
࣒ࢠ❧ࡕᯤࢀ⳦ࡢ⳦⣒⏕㛗ࢆᢚไࡍࡿሗ࿌ࡶ࠶ࡿ(Zafari et al. 2008)ࠋ㎰⸆ࡋ࡚Ⓩ㘓ࡉࢀ
ࡓ Trichoderma ᒓ⳦ࡢ⳦ᰴࡀᏑᅾࡍࡿࡇࡶ▱ࡽࢀ࡚࠸ࡿ(ᑠᕝ 1998)ࠋᮏ◊✲ࡼࡾࠊ
Trichoderma ᒓ⳦࡛࠶ࡿH921 ⳦ᰴࡢᇵ㣴ࢁᾮࡶࠊ᳜≀ᐖࡢ㜵㝖⏝࡛ࡁࡿྍ⬟ᛶࡀ♧
၀ࡉࢀࡓࠋ
93
➨
➨8❶ ⥲ྜ⪃ᐹ
ࢿ࠸ࡶࡕࡣ⣒≧⳦ࡢᏊᄞ⳦㢮ᒓࡍࡿMagnaporthe oryzae (Hebert) Barr(ୡ௦ࠊ
Pyricularia oryzae Cavara)ࡼࡗ࡚ᘬࡁ㉳ࡇࡉࢀࡿࢿࡢ㔜せᐖࡢ 1 ࡘ࡛࠶ࡿࠋᮏࡢ㜵
㝖ࡣᢠᛶရ✀ࡢ⏝ࡸᏛྜᡂ㎰⸆ࡢ⏝ࡀ୍⯡ⓗ࡛࠶ࡿࡀࠊ᪂つࡢཎᛶ࣮ࣞࢫࡢฟ
⌧ࡸ⸆⪏ᛶ⳦ࡢฟ⌧ࡀၥ㢟࡞ࡗ࡚࠸ࡿࠋࡑࡇ࡛ࠊ㏆ᖺࠊᚤ⏕≀ࢆ⏝ࡋࡓ⏕≀㜵㝖㛵 ࡍࡿ◊✲ࡸᚤ⏕≀ࡀ⏕⏘ࡍࡿᢚไ≀㉁ࢆ⏝ࡋࡓ㜵㝖㛵ࡍࡿ◊✲ࡀ┒ࢇ⾜ࢃࢀ࡚࠸ࡿࠋ
୍᪉ࠊ㔝⏕ࡁࡢࡇࡣᚤ⏕≀ࡢ1ࡘ࡛࠶ࡿ⳦㢮࡛ࠊ᪥ᮏከࡃ⏕ᜥࡋ࡚࠸ࡿࡀࠊከࡃࡢࡶࡢ ࡀᮍྠᐃ࡛࠶ࡾࠊᮍ▱ࡢ᭷⏝࡞⳦ࡀᏑᅾࡍࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋࡑࡇ࡛ࠊᮏ◊✲࡛ࡣࠊ᪥
ᮏᅜෆ࡛᥇ྲྀࡋࡓ㔝⏕ࡁࡢࡇࡽࠕࡁࡢࡇᐤ⏕ཪࡣඹ⏕ࡋ࡚࠸ࡿ⳦㢮ࠖࢆศ㞳ࡋࠊศ㞳⳦
ࡢᇵ㣴ࢁᾮࢆ⏝࠸࡚ࠊձࢿ࠸ࡶࡕ⳦➼ࢆᢚไࡍࡿศ㞳⳦ࡢ㑅ᢤ᳜≀ୖ࡛ࡢᢚไຠᯝࠊ ղᢚไࡋࡓ⳦ࡢྠᐃཬࡧճᢚไ≀㉁ࡢ᥈⣴࣭ྠᐃࢆ⾜࠸ࠊ㜵㝖ࡢ⏝ࡘ࠸࡚⪃ᐹࡋࡓࠋ
ᅇࠊᓥ᰿ࠊ㫽ྲྀࠊᗈᓥࠊ㜰ࠊ⚟ཬࡧ⇃ᮏ┴ෆ࡛᥇ྲྀࡋࡓ46ಶయࡢࡁࡢࡇࡽࠊPSA ᇵᆅࢆ⏝࠸ࡓ⤌⧊ᇵ㣴ࡼࡾ105⳦ᰴࡀศ㞳ࡉࢀࡓࠋࡇࢀࡽࡢศ㞳⳦ࡣࠊࡁࡢࡇᐤ⏕ཪ ࡣඹ⏕ࡋ࡚࠸ࡓ⳦㢮ࡀྵࡲࢀ࡚࠸ࡿ⪃࠼ࡽࢀࡿࠋࡑࡇ࡛ࠊࢿ࠸ࡶࡕ⳦ࡢឤᰁ⾜ືࢆᣦ ᶆࠊPSᾮయᇵᆅࢆ⏝࠸࡚ㄪ〇ࡋࡓศ㞳⳦ࡢᇵ㣴ࢁᾮࡽᢚไຠᯝࢆ♧ࡍศ㞳⳦ࢆࢫࢡࣜ
࣮ࢽࣥࢢࡋࡓࠋࡑࡢ⤖ᯝࠊࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆཬࡧ╔ჾᙧᡂࢆᢚไࡍࡿ20⳦ᰴࡀ ࢫࢡ࣮ࣜࢽࣥࢢࡉࢀࡓࠋ⇕ฎ⌮ࡼࡾࡇࢀࡽࡢศ㞳⳦ࡢᇵ㣴ࢁᾮࡣࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚ
Ⓨⱆཬࡧ╔ჾᙧᡂඹᢚไࡋ࡞࠸ࢢ࣮ࣝࣉ1ࠊࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆཬࡧ╔ჾᙧ ᡂඹᢚไࡍࡿࢢ࣮ࣝࣉ2ࠊ⬊ᏊⓎⱆࢆᢚไ࡛ࡁ࡞࠸ࡀࠊ╔ჾᙧᡂࢆᢚไࡍࡿࢢ࣮ࣝࣉ
3ࡢ3ࡘࡢࢢ࣮ࣝࣉศ㢮ࡉࢀࡓࠋࡇࢀࡽࡢࡇࡽࢢ࣮ࣝࣉ1ࡣ⇕Ᏻᐃ࡞ᢚไ≀㉁ࠊࢢ
࣮ࣝࣉ2ࡣ⇕Ᏻᐃ࡞ᢚไ≀㉁ࠊࢢ࣮ࣝࣉ3ࡣẚ㍑ⓗ⇕Ᏻᐃࠊࡶࡋࡃࡣࠊ⇕Ᏻᐃ࡞ᢚไ≀
㉁⇕Ᏻᐃ࡞ᢚไ≀㉁ࡢ୧᪉ࢆ⏕⏘ࡍࡿศ㞳⳦࡛࠶ࡿࡇࡀ⪃࠼ࡽࢀࡓࠋࡇࢀࡲ࡛ࠊከࡃࡢ
᳜≀ᐖࡢ㜵㝖ຠᯝⓗ࡞⇕Ᏻᐃ࡞≀㉁ࡋ࡚ࡣࠊ᳜≀ཎ⳦ࡢ⣽⬊ቨཬࡧ⣽⬊⭷ࢲ
࣓࣮ࢪࢆ࠼ࡿ࢟ࢳࢼ࣮ࢮཬࡧβ-1, 3ࢢࣝ࢝ࢼ࣮ࢮࡀ▱ࡽࢀ࡚࠾ࡾࠊࡑࢀࡽࢆ⏕⏘ࡍࡿᚤ⏕
≀ࡘ࠸࡚ࡢሗ࿌ࡀ࠶ࡿ (Lorito et al. 1994㸹Shoresh and Harman 2010㸹Sakdapetsiri et al. 2016)ࠋ
ࡲࡓࠊBacillus subtilis NSRS 89-24ࡢᇵ㣴ࢁᾮ୰ࡣࠊࢿ࠸ࡶࡕ⳦ཬࡧ⣠ᯤ⳦ࢆᢚไࡍ
ࡿ⇕Ᏻᐃ࡞≀㉁ࡶ⇕Ᏻᐃ࡞ β-1, 3 ࢢࣝ࢝ࢼ࣮ࢮࡶྵࡲࢀ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ
(Leelasuphakul et al. 2006)ࠋ㔝⏕ࡁࡢࡇࡣᢠ㓟≀㉁ཬࡧᢚไ≀㉁ࢆ⏕⏘ࡋࠊHypholoma
fasciculare ཬࡧCantharellus cibarius ࡢ࣓ࢱࣀ࣮ࣝᢳฟ≀ࡀBacillus cereusࠊ Bacillus subtilis ཬࡧStaphylococcus aureusࢆⴭࡋࡃᢚไࡍࡿࡇࡀሗ࿌ࡉࢀࡓ(Barros et al. 2007㸹Barros et al.
2008)ࠋࡉࡽࠊ㔝⏕ࡢ㣗⏝ࡁࡢࡇ࡛࠶ࡿLycoperdon perlatumࠊPleurotus pulmonariusࠊClavaria vermiculrisཬࡧMarasmius oreadesࡢ࣓ࢱࣀ࣮ࣝᢳฟ≀ࡀEscherichia coliཬࡧBacillus subtilis
ࢆᢚไࡍࡿሗ࿌ࡶ࠶ࡿ(Ramesh and Pattar 2010)ࠋᮏ◊✲ࡢ⤖ᯝࡶྠᵝ㔝⏕ࡁࡢࡇࡽศ㞳 ࡉࢀࡓ⳦ࡀࢿ࠸ࡶࡕ⳦ࢆᢚไࡍࡿ≀㉁ࢆ⏕⏘ࡋࠊ⇕Ᏻᐃ࡞≀㉁ࡶ⇕Ᏻᐃ࡞≀㉁ࡶ
Ꮡᅾࡍࡿࡇࢆ♧ࡋࡓࠋࡉࡽࠊAnnulohypoxylon sp. (S9)䚸Nigrospora sp. (S5)ཬ䜃 Penicillium
94
sp. (O1 ཬ䜃 O3)䛿䚸࣒࢜࢜ࢠࡢᇵ㣴ࢁᾮࡢ๓ฎ⌮ࡼࡾࢿ࠸ࡶࡕࡢᩬᙧᡂࢆᢚไ
ࡋࡓࠋPenicillium spp.䛜⏕⏘䛩䜛Mycophenolic acid䚸Patulin䚸3-omethylfunicone䛸 Decumbenone A 䛿᳜≀ཎ⳦䛾ឤᰁ䜢ᢚไ䛩䜛ᢚไ≀㉁䛸䛧䛶▱䜙䜜䛶䛔䜛(Fujii et al. 2002; Nicoletti and Stefano 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䛩䜛(Kim et al. 2001)䚹୍᪉䚸Nigrospora oryzae (Berk. & Br.) Petch 䛾ᇵ㣴䜝ᾮ䛻⏕⏘䛥䜜䜛Nigrosporin Aཬ䜃B䛿 ᳜≀ẘ⣲䛷䛒䜛(Tanaka et al. 1997)䚹䛧䛛䛧䚸ᮏ
◊✲䛷䛿Nigrospora sp.䛾ᇵ㣴䜝ᾮ䛿䜸䜸䝮䜼䛻ᑐ䛧䛶䚸ẘᛶ䜢♧䛥䛪䚸Phomalactone 䛸䜒␗䛺䜛
ᛶ㉁䛾≀㉁䛷䛒䛳䛯䚹䛣䜜䜙䛾⤖ᯝ䛛䜙䚸᪂䛯䛺ᢚไ≀㉁䜢⏕⏘䛧䛶䛔䜛ྍ⬟ᛶ䜒♧၀䛥䜜䛯䚹䜎 䛯䚸Annulohypoxylon sp.䛻䛴䛔䛶䛿䚸䛣䜜䜎䛷䛻ᇵ㣴䜝ᾮ䛻䜘䜛᳜≀ཎ⳦䛾㜵㝖䛻㛵䛩䜛ሗ࿌
䛜䛺䛔䚹Annulohypoxylon sp.䛿Xylariaceae ⛉䛻ᒓ䛧䚸ෆ⏕⳦䛸䛧䛶▱䜙䜜䛶䛔䜛䚹Xylariaceae 䛿 36௨ୖ䛾ᒓ䛜ྵ䜎䜜䛶䛔䜛䛝䛺⛉䛷䛒䜚䚸Whalley and Edwards (1995)䛻䜘䜚䚸Xylariaceae䛾ከ 䛟䛾2ḟ௦ㅰ⏘䛜ሗ࿌䛥䜜䛶䛔䜛䚹ࡇࢀࡽࡢ୰ࡣࠊࢿ࠸ࡶࡕ⳦ࢆྵࡴ᳜≀ཎ⳦ᑐࡋ
࡚ࡶᢚไຠᯝࢆ♧ࡍ≀㉁ࡀྵࡲࢀ࡚࠸ࡿྍ⬟ᛶࡀ࠶ࡿࠋᚋࠊAnnulohypoxylon sp.ࡢ⏕⏘ࡍ
ࡿᢚไ≀㉁ࡘ࠸࡚ࡶㄪᰝࡍࡿࡇࡼࡾࠊࢿ࠸ࡶࡕࡢ㜵㝖⏝࡛ࡁࡿ᪂ࡓ࡞ᢚไ
≀㉁ࡀ᥈⣴࡛ࡁࡿ⪃࠼ࡽࢀࡿࠋࡇࢀࡽࡢ⤖ᯝࡼࡾࠊ㔝⏕ࡁࡢࡇࡽศ㞳ࡉࢀࡓ⳦㢮ࡀᏛ
ⓗ㜵㝖ࡸ⏕≀ⓗ㜵㝖ࡢ㛤Ⓨ⏝࡛ࡁࡿྍ⬟ᛶࢆ♧ࡋࡓࠋ
ᮏ◊✲࡛ࡣᇵ㣴ࢁᾮࢆ⏝࠸࡚ᢚไຠᯝࢆ♧ࡍ⳦ࡢ㑅ᢤࢆ⾜ࡗࡓࠋᚤ⏕≀ࡢᇵ㣴ࢁᾮࢆ⏝
࠸ࡓ᳜≀ཎ⳦ࡢ㜵㝖ࡘ࠸࡚ࡣ᳜≀ཎ⳦ࢆ┤᥋ⓗᢚไࡍࡿຠᯝࢆ♧ࡍࡶࡢࡔࡅ࡛࡞
ࡃࠊ᳜≀㌟ⓗ࡞ᢠᛶࢆㄏᑟࡍࡿࡇࡼࡾ㜵㝖ࢆヨࡳࡓ◊✲ࡶᏑᅾࡍࡿ(బ➉ࡽ
2003㸹ᑠᓥࡽ 2006㸹ㅖぢ㔛ࡽ 2005㸹Kock and Löffler 2009㸹Li et al. 2011㸹Lai et al. 2012㸹
Zhang et al. 2013)ࠋᮏ◊✲࡛ศ㞳ࡉࢀࡓO821⳦ᰴࡢᇵ㣴ࢁᾮࡣࢿ࠸ࡶࡕ⳦ࡔࡅ࡛࡞ࡃࠊ
ᆅୖᐖཬࡧᅵተᐖࢆᘬࡁ㉳ࡇࡍ」ᩘࡢ᳜≀ཎ⳦ᑐࡍࡿឤᰁ⾜ືᢚไຠᯝࢆ♧ࡋࡓࠋ ࡑࡇ࡛ࠊࢿࢿ࠸ࡶࡕ⳦ࠊ࢟ࣗ࢘ࣜ࢟ࣗ࢘ࣜ〓ᩬ⳦ཬࡧⅣ⳦ࡢ⤌ࡳྜࢃࡏ࡛ࠊ O821⳦ᰴࡢᇵ㣴ࢁᾮࡢຠᯝࢆㄪᰝࡋࡓࠋࡑࡢ⤖ᯝࠊ࠸ࡎࢀࡢ᳜≀ཎ⳦ࡢ⤌ࡳྜࢃࡏ
࠾࠸࡚ࡶࠊΰྜ᥋✀ࡼࡾࠊ᳜≀య᳜≀ẘᛶࢆ♧ࡍࡇ࡞ࡃࠊⓎࢆᢚไࡋࡓࠋO821⳦
ᰴࡢᇵ㣴ࢁᾮྵࡲࢀࡿᢚไ≀㉁ࡣ㓑㓟࢚ࢳࣝྍ⁐࡞⇕Ᏻᐃ࡞≀㉁࡛࠶ࡗࡓࠋࡑࡇ࡛ࠊⷧ
ᒙࢡ࣐ࣟࢺࢢࣛࣇ࣮(TLC)ࢆ⏝࠸࡚ࠊࢺ࢚ࣝࣥ㸸㓑㓟࢚ࢳࣝ=1㸸1 (v/v) ࡢᒎ㛤⁐፹࡛ศ㞳 ᚋࡑࢀࡒࢀࡢ⳦ࢆᄇ㟝ࡍࡿࡇ࡛ࠊ⏕⫱㜼Ṇᖏࢆ☜ㄆࡋࡓࠋࡑࡢ⤖ᯝࠊO821⳦ᰴࡢᇵ㣴
ࢁᾮྵࡲࢀࡿᢚไຠᯝࢆ♧ࡍ୰ᚰⓗ࡞≀㉁ࡣ 1 ࡘ࡛࠶ࡾࠊࡑࡢ≀㉁ࡀẚ㍑ⓗỈᛶ࡛࠶
ࡾࠊ㓑㓟࢚ࢳࣝྍ⁐࡞⇕Ᏻᐃ࡞≀㉁࡛࠶ࡿྍ⬟ᛶࡀ♧ࡉࢀࡓࠋ
୍᪉ࠊO821⳦ᰴࡢᇵ㣴ࢁᾮࡣ᳜≀యࡢ๓ฎ⌮࡛ࡶࢿ࠸ࡶࡕࠊ࢟ࣗ࢘ࣜ〓ᩬཬࡧ
Ⅳࢆᢚไࡋࡓࠋࡇࢀࡲ࡛ࠊᚤ⏕≀ࡢᇵ㣴ࢁᾮ୰ࡣ㌟ⓗ࡞ᢠᛶࢆㄏᑟࡍࡿ≀㉁ࡶྵ
ࡲࢀ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ (Hossain et al. 2007; Kojima et al. 2013; Shimizu et al. 2013;
Spence et al. 2014)ࠋࡑࡇ࡛ࠊࢿࢆ⏝࠸࡚ࠊࢿయୖ࡛ࡢᢠᛶࡢྍ⬟ᛶࢆㄪᰝࡋࡓࠋࡑࡢ
⤖ᯝࠊO821⳦ᰴࡢᇵ㣴ࢁᾮࢆฎ⌮ࡋࡓࢿ࡛ࡣࠊ㐣㓟Ỉ⣲ࡢ⏕ᡂࡸᢠᛶㄏᑟ㛵㐃㑇ఏ Ꮚࡢ1ࡘ࡛࠶ࡿPBZ1㑇ఏᏊࡢⓎ⌧㔞ࡀቑຍࡋࡓࠋࡉࡽࠊ㐣㓟Ỉ⣲ࡢᾘཤ࡛࠶ࡿ࢝ࢱ
95
࣮ࣛࢮࢆ⏝࠸ࡓሙྜࡣࠊO821⳦ᰴࡢᇵ㣴ࢁᾮࡼࡿᢚไຠᯝࡣⴭࡋࡃపୗࡋࡓࠋ㐣㓟
Ỉ⣲ࡀࢩࢢࢼࣝศᏊࡋ࡚⣽⬊Ṛ㔜せ࡞ᙺࢆᯝࡓࡋ࡚࠸ࡿࡇࡣࡼࡃ▱ࡽࢀ࡚࠸ࡿ
(Jabs 1999㸹Levine et al. 1994㸹Thordal-Christense et al. 1997㸹Chamnongpol et al. 1998)ࠋࡲࡓࠊ
ࢿ⣽⬊㠀ぶᛶ⳦ᰴAcidovorax avenae ࢆ᥋✀ࡍࡿࡇࡼࡗ࡚㐣㓟Ỉ⣲ࡢ⏕ᡂඹ
PALࠊCht-1ཬࡧ PBZ1㑇ఏᏊࡢⓎ⌧ࢆㄏᑟࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ (Tanaka et al. 2003)ࠋ ࡉࡽࠊKim et al. (2011) ࡣPBZ1ࢱࣥࣃࢡ㉁ࡢ⤌ࡳ࠼ࢿ࡛ࡣ⣽⬊Ṛࡀㄏᑟࡉࢀࡿࡇ
ࢆሗ࿌ࡋࡓࠋ୍᪉࡛ࠊ㌟ⓗ࡞ᢠᛶࡢㄏᑟࡣࢧࣜࢳࣝ㓟⤒㊰ࡀ㛵ࡋ࡚࠸ࡿࡇࡀ▱ࡽ
ࢀ࡚࠸ࡿࠋࡋࡋࠊᮏ◊✲࠾࠸࡚ࠊO821⳦ᰴࡢᇵ㣴ࢁᾮࡢฎ⌮ࡋࡓࢿ࡛ࠊࢧࣜࢳࣝ㓟 ࡢ✚㔞ࡢቑຍࡀ☜ㄆ࡛ࡁ࡞ࡗࡓࠋ㏆ᖺࠊࢧࣜࢳࣝ㓟ࡢୗὶస⏝ࡍࡿ≀㉁ࡋ࡚ࠊࢳࢪ
ࢽࣝࡸࢩ࣋ࣥࢰࣛࣝ S ࣓ࢳࣝࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡲࡓࠊࢧࣜࢳࣝ㓟ࢆࡉ࡞࠸ᢠᛶ ㄏᑟࡢሗ࿌ࡶ࠶ࡿ(Yoshioka et al. 2001㸹Nakashita et al. 2002㸹Lin et al. 2008)ࠋࡇࢀࡽࡢ⤖ᯝ
ࡽࠊO821⳦ᰴࡢᇵ㣴ࢁᾮࢆฎ⌮ࡋࡓࢿ࡛ࡣࠊᢚไ≀㉁ࡢཎ⳦ᑐࡍࡿ┤᥋ⓗ࡞ຠᯝ
ࡸάᛶ㓟⣲✀࡛࠶ࡿ㐣㓟Ỉ⣲ࡀ⏕ᡂࡋࠊPBZ1࡞ࡢࢿࡢᐖᢠᛶ㑇ఏᏊⓎ⌧ㄏᑟࡀ
㉳ࡇࡾࠊ༙ά≀ᐤ⏕⳦࡛࠶ࡿࢿ࠸ࡶࡕ⳦ࢿࡢึᮇඹ⏕㛵ಀࡀ㜼ᐖࡉࢀࠊධࢆ㜼Ṇ
࡛ࡁࡿ⪃࠼ࡽࢀࡓࠋ
ITS㡿ᇦࡢࢩ࣮ࢡ࢚ࣥࢫゎᯒࡢ⤖ᯝࡽࠊO821⳦ᰴࡀBiscogniauxiaᒓ࡛࠶ࡿࡇࡀ᥎ᐃ ࡉࢀࡓࠋO821⳦ᰴࡣAlbizia sp.ࡽศ㞳ࡉࢀࡓBiscogniauxia sp. HHP54㧗࠸┦ྠᛶࢆ♧ࡋ ࡓࠋࡋࡋࠊBiscogniauxia sp. HHP54㛵ࡍࡿヲ⣽࡞ሗࡀ࡞࠸ࠋBiscogniauxiaᒓ⳦ࡣࠊ࢚
ࣥࢻࣇࢺࡋ࡚ศ㞳ࡉࢀࡓࡇࡀከࡃࠊୡ⏺࡛57✀௨ୖࡀⓎぢࡉࢀ࡚࠸ࡿࠋᏊᐇయࡢ ᙧᡂࡶሗ࿌ࡉࢀ࡚࠸ࡿࡀ O821 ⳦ᰴ࡛ࡣᏊᐇయࡢᙧᡂࡀ☜ㄆ࡛ࡁ࡚࠸࡞࠸ࠋࡇࢀࡲ࡛ࠊ Biscogniauxia mediterraneaࡸBiscogniauxia nummulariaࡀᮌᮦ⭉ᮙ⳦ࡋ࡚ሗ࿌ࡉࢀ࡚࠾ࡾࠊ
᳜ ≀ẘᛶ ࢆ♧ࡍ ≀㉁ ࡋ ࡚ࠊBiscopyranࠊ Phenylacetic acidࠊ 5-methylmellein ཬ ࡧ 6-methoxymelleinࡀྠᐃࡉࢀ࡚࠸ࡿ (Evidente et al. 2005㸹Luchi et al. 2006㸹Cheng et al. 2011)ࠋ O821⳦ᰴࡢ✀࡛ࣞ࣋ࣝࡢྠᐃࡣ⮳ࡗ࡚࠾ࡽࡎࠊᏊᐇయᙧᡂࠊᐟ⠊ᅖࠊ⏕⏘ࡉࢀࡿ≀㉁
ࢆヲ⣽᫂ࡽࡍࡿࡇࡼࡾࠊO821⳦ᰴࡢヲࡋ࠸ྠᐃࡀ⾜࠼ࡿ⪃࠼ࡽࢀࡿࠋ ᮏ◊✲࡛ࡣࠊBiscogniauxiaᒓ࡛࠶ࡿO821⳦ᰴࡀᇵ㣴ࢁᾮ୰⏕⏘ࡍࡿ≀㉁ࡢ༢㞳࣭⢭〇 ཬࡧྠᐃࡶ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊNMRࠊGC/MS/MSཬࡧFT-IRࡼࡿゎᯒࡽศᏊ㔞248ࡢ (3aS,4aR,8aS,9aR)-3a-hydroxy-8a-methyl-3,5-dimethylenedecahydronaphtho[2,3-b]furan-2(3H)-one
࡛࠶ࡿࡇࡀ᫂ࡽ࡞ࡾࠊ᪂つࡢ≀㉁࡛࠶ࡗࡓࠋᮏ≀㉁᭱ࡶ㢮ఝࡋ࡚࠸ࡿ≀㉁ࡋ࡚ࠊ
࢜ࢢ࣐ࣝᒓࡢ᳜≀(Inula helenium L., Inula japonica)ࡀ⏕⏘ࡍࡿࣛࣥࢺࣛࢡࢺࣥཬࡧࢯ
ࣛࣥࢺࣛࢡࢺࣥࡀ▱ࡽࢀ࡚࠸ࡿࠋࡇࢀࡽࡢ≀㉁ࡣᢠ⅖ᛶࠊᢠ┿⳦ᛶཬࡧᢠࡀࢇάᛶ࡞
⸆⌮Ꮫⓗ࡞άᛶࡀሗ࿌ࡉࢀ࡚࠸ࡿ(Picman and Schneider 1993㸹Khan et al. 2012㸹Kataria and Chahal 2013㸹Rasul et al. 2013)ࠋᮏ≀㉁ࡣࢯࣛࣥࢺࣛࢡࢺࣥࡢᵓ㐀ࡢ1ࡘࡢỈ⣲(㸫H) ࡀࣄࢻࣟ࢟ࢩᇶ(㸫OH)⨨ࡉࢀࡓᵓ㐀࡞ࡗ࡚࠸ࡿࠋࣄࢻࣟ࢟ࢩᇶ(㸫OH)⨨ࡉࢀࡿ
ࡇࡼࡾᮏ≀㉁ࡣࢯࣛࣥࢺࣛࢡࢺࣥࡼࡾ㟁㞳ࡋࡸࡍࡃࠊᛂᛶࡀ㧗࠸ྍ⬟ᛶࡀ⪃
࠼ ࡽ ࢀ ࡓ ࠋ ༢ 㞳 ࡋ ࡓ
(3aS,4aR,8aS,9aR)-3a-hydroxy-8a-methyl-3,5-96
dimethylenedecahydronaphtho[2,3-b]furan-2(3H)-one ࡣࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚࡢឤᰁ⾜ືࢆᢚ ไࡍࡿඹࢿ࡛ࡢⓎᢚไຠᯝࡸࢿ࡛ࡢ㐣㓟Ỉ⣲ࡢ⏕ᡂ PBZ1 㑇ఏᏊࡢⓎ⌧ࡶ
ቑ ຍ ࡉ ࡏ ࡓ ࠋ
(3aS,4aR,8aS,9aR)-3a-hydroxy-8a-methyl-3,5-dimethylenedecahydronaphtho[2,3-b]furan-2(3H)-one ࡢ᳜≀ཎ⳦ᑐࡍࡿస⏝ᶵᵓ᳜≀ࡢᢠᛶㄏᑟࡘ࠸࡚ࡣヲ⣽
᳨ウࡍࡿᚲせࡀ࠶ࡿࠋ
ᮏ◊✲࡛ࡣࠊࡑࡢᗈᓥ┴ෆ࡛᥇ྲྀࡋࡓ㔝⏕ࡁࡢࡇࡽศ㞳ࡉࢀࡓTrichodermaᒓ࡛࠶
ࡿH921⳦ᰴࡢᇵ㣴ࢁᾮࡢࢿ࠸ࡶࡕ⳦ࡢᢚไຠᯝࡶㄪᰝࡋࠊࢿࡸ࣒࢜࢜ࢠ࡛ࡢᢚไຠ
ᯝࡇࢀࡽ㛵ࡍࡿ⪃࠼ࡽࢀࡿ⇕Ᏻᐃ࡞」ᩘࡢᢚไ≀㉁ࡢ⏕⏘ࢆ☜ㄆࡋࡓࠋࡇࢀࡽࡢ
≀㉁ࡘ࠸࡚ࡶྠᐃཬࡧస⏝ᶵᵓࢆ᫂ࡽࡍࡿࡇ࡛ࠊ㜵㝖⏝ྍ⬟࡞≀㉁࡞ࡿ
⪃࠼ࡽࢀࡿࠋ
ᮏ◊✲ࡼࡾࠊ㔝⏕ࡁࡢࡇࡣࠕࡁࡢࡇᐤ⏕ཪࡣඹ⏕ࡋ࡚࠸ࡿ⳦㢮ࠖ⪃࠼ࡽࢀࡿ⳦㢮 ࡀከࡃᏑᅾࡋࠊࡑࢀࡽࡢᇵ㣴ࢁᾮ୰ࡣࠊᐖ㜵㝖⏝࡛ࡁࡿྍ⬟ᛶࡀ࠶ࡿ᪂つ≀㉁ࡶ⏕
⏘ࡉࢀ࡚࠸ࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋ㔝⏕ࡁࡢࡇࡽศ㞳ࡉࢀࡓ⳦㢮ࡀᏛⓗ㜵㝖ࡸ⏕≀
ⓗ㜵㝖ࡢ㛤Ⓨ⧅ࡀࡿྍ⬟ᛶࢆ♧ࡋࡓࠋ
97 せ
᳜≀ᐖࡢ㜵㝖ࡣࠊᢠᛶရ✀ࡸᏛྜᡂ㎰⸆ࡢ⏝ࡀ୍⯡ⓗ࡛࠶ࡿࡀࠊ㧗ᗘᢠᛶရ
✀ࡢ⨯ࡸ㐣ᗘ࡞㎰⸆⏝ࡼࡿ⸆⪏ᛶ⳦ࡢฟ⌧ࡀၥ㢟࡞ࡗ࡚࠸ࡿࠋ㏆ᖺࠊᚤ⏕≀ࢆ
⏝࠸ࡓ᳜≀ᐖࡢ㜵㝖㛵ࡍࡿ◊✲ࡀ┒ࢇ⾜ࢃࢀ࡚࠸ࡿࠋ㔝⏕ࡁࡢࡇᐤ⏕࣭ඹ⏕ࡍࡿ⳦
㢮ࡣࠊᮍࡔᮍྠᐃࡢࡶࡢࡀከࡃࠊᐖ㜵㝖⏝ྍ⬟࡞ᮍ▱ࡢ᭷⏝࡞⳦ࡀᏑᅾࡍࡿ⪃
࠼ࡽࢀࡿࠋᮏ◊✲ࡣ㔝⏕ࡁࡢࡇࡽศ㞳ࡋࡓ⳦㢮ࡼࡿ᳜≀ཎ⳦ࡢᢚไࡘ࠸࡚ㄪᰝࡋ ࡓࠋᚓࡽࢀࡓ⤖ᯝࡣ௨ୗࡢࡼ࠺せ⣙ࡉࢀࡿࠋ
1. 㔝⏕ࡁࡢࡇࡽࡢ⳦ศ㞳ࢫࢡ࣮ࣜࢽࣥࢢ
᪥ᮏᅜෆ࡛᥇ྲྀࡋࡓ46✀㢮ࡢ㔝⏕ࡁࡢࡇࡽࠊ105⳦ᰴࡀศ㞳ࡉࢀࡓࠋࡇࢀࡽࡢศ㞳⳦
ࡽࢿ࠸ࡶࡕ⳦ࡢឤᰁ⾜ືࢆᣦᶆࡋ࡚ᢚไຠᯝࢆ♧ࡍศ㞳⳦ࢆࢫࢡ࣮ࣜࢽࣥࢢࡋࡓࠋࡑ ࡢ⤖ᯝࠊ90ศ㞳⳦ࡢ࠺ࡕ20⳦ᰴࡢᇵ㣴ࢁᾮࡀࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆཬࡧ╔ჾᙧᡂࢆ
ᢚไࡋࡓࠋࡇࢀࡽࡢศ㞳⳦ࡢᇵ㣴ࢁᾮࡢ⇕ฎ⌮ࢆ⾜ࡗࡓ⤖ᯝࠊࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆཬ ࡧ╔ჾᙧᡂᢚไࡋ࡞࠸ࢢ࣮ࣝࣉࠊࢿ࠸ࡶࡕ⳦ࡢឤᰁ⾜ືࢆᢚไࡍࡿࢢ࣮ࣝࣉࠊ⬊Ꮚ
Ⓨⱆࢆᢚไࡋ࡞࠸ࡀࠊ╔ჾᙧᡂࢆᢚไࡍࡿࢢ࣮ࣝࣉࡢ3ࡘࡢࢢ࣮ࣝࣉศ㢮ࡉࢀࡓࠋࡲࡓࠊ
࣒࢜࢜ࢠⴥୖ࡛ࠊO1ࠊO3ࠊS5ཬࡧ S9ศ㞳⳦ࡢᇵ㣴ࢁᾮࡢ๓ฎ⌮ࡍࡿࡇࡼࡾࢿ࠸ࡶࡕ
ࡢᩬᙧᡂࢆᢚไࡋࡓࠋITS㡿ᇦࡢゎᯒࡼࡾࠊࡇࢀࡽࡢ⳦ࡣAnnulohypoxylon sp. (S9)ࠊ Nigrospora sp. (S5) ཬࡧ Penicillium sp. (O1 ཬࡧO3) 㧗࠸┦ྠᛶࢆ♧ࡋࡓࠋࡇࢀࡽࡢ⤖
ᯝࡽࠊ㔝⏕ࡁࡢࡇࡽศ㞳ࡉࢀࡓ⳦㢮ࡀࢿ࠸ࡶࡕࡢ㜵㝖⏝࡛ࡁࡿྍ⬟ᛶࡀ♧ࡉࢀ
ࡓࠋ
2. O821 ⳦ᰴࡢᇵ㣴ࢁᾮࡢ᳜≀ཎ⳦ᑐࡍࡿᢚไຠᯝࡘ࠸࡚
᭱ࡶࢿ࠸ࡶࡕ⳦ࡢឤᰁᢚไຠᯝࡢ㧗࠸ᇵ㣴ࢁᾮࡋ࡚ࠊO821 ⳦ᰴὀ┠ࡋ࡚◊✲ࢆ
㐍ࡵࡓࠋITS㡿ᇦࡢࢩ࣮ࢡ࢚ࣥࢫゎᯒࡼࡾࠊᮏ⳦ᰴࡣBiscogniauxiaᒓ⳦࡛࠶ࡿࡇࡀ᥎ᐃ ࡉࢀࡓࠋ⇕ฎ⌮(121Υࠊ20ศ㛫)ࡋࡓO821 ⳦ᰴࡢᇵ㣴ࢁᾮ(O821-CF)ࡣ↓ฎ⌮ࡢO821-CF
ྠᵝࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆཬࡧ╔ჾᙧᡂࢆᢚไࡋࡓࠋO821-CFࡣ⃰ᗘ౫Ꮡⓗࢿ
࠸ࡶࡕ⳦ࡢ⳦⣒⏕⫱ࢆᢚไࡋࠊࢿ࠸ࡶࡕ௨እࡢ」ᩘࡢᆅୖᐖཬࡧᅵተᐖࡢ᳜≀
ཎ⳦ࡢឤᰁ⾜ືࢆ⃰ᗘ౫ᏑⓗᢚไࡋࡓࠋࡲࡓࠊO821 ⳦ᰴࢆPSᾮయᇵᆅ࡛1-7᪥㛫ᇵ㣴ࡋࠊ O821⳦ᰴࡢᇵ㣴ࢁᾮࡢࢿ࠸ࡶࡕ⳦ᑐࡍࡿᢚไຠᯝࢆㄪᰝࡋࡓ⤖ᯝࠊᇵ㣴4᪥┠ࡽO8 21⳦ᰴࡢᇵ㣴ࢁᾮࡣࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆཬࡧ╔ჾᙧᡂࢆᢚไࡋࡓࠋࡇࢀࡽࡢࡇ
ࡽࠊO821-CF୰ࡣᢚไ≀㉁ࡀᏑᅾࡋࠊࡑࡢ≀㉁ࡀ⇕Ᏻᐃ࡞≀㉁࡛࠶ࡿࡇࡀ♧၀ࡉࢀࡓࠋ
ࢿ࠸ࡶࡕ⳦ࡢΰྜ᥋✀࡛ࡣࠊO821-CFࡣ࠸ࡶࡕࡢᩬᙧᡂࢆᢚไࡋࠊ๓ฎ⌮࠾
࠸࡚ࡶⓎࡀᢚไࡉࢀࡓࠋࡉࡽࠊO821-CFࡣ࢟ࣗ࢘ࣜయୖ࡛ࡶ࢟ࣗ࢘ࣜࡢ㔜せ࡞ᐖ࡛࠶
ࡿ〓ᩬཬࡧⅣࡢᩬᙧᡂࡶᢚไຠᯝࢆ♧ࡋࡓࠋࡇࢀࡽࡢ⤖ᯝࡽࠊO821-CF୰
ࢿ࠸ࡶࡕ⳦ࡔࡅ࡛࡞ࡃࠊ࢟ࣗ࢘ࣜࡢ㔜せ࡞ཎ⳦࡛࠶ࡿ〓ᩬ⳦ཬࡧⅣ⳦ࢆᢚไࡍ
98
ࡿ≀㉁ࡶ⏕⏘ࡉࢀࡿࡇࡀ⪃࠼ࡽࢀࡓࠋ
3. O821⳦ᰴࡢᇵ㣴ࢁᾮࢿࡢᢠᛶㄏᑟ
O821-CFࢆฎ⌮ࡋࡓࢿⴥ࡛ࡣࠊ㐣㓟Ỉ⣲ࡢ⏕ᡂࢆ♧ࡍ〓Ⰽࡢ3,3'-diaminobenzidineᛂ ࡀほᐹࡉࢀࡓࠋྠᵝࢿࡢᐖᢠᛶㄏᑟ㛵㐃㑇ఏᏊࡋ࡚▱ࡽࢀ࡚࠸ࡿPBZ1ࡘ࠸࡚
ࡶࠊᑐ↷༊ẚ㍑ࡋ࡚ࠊⓎ⌧㔞ࡀቑຍࡋ࡚࠸ࡓࠋ࢝ࢱ࣮ࣛࢮࡢῧຍࡼࡾᇵ㣴ࢁᾮࡢᢚไά ᛶࡀⴭࡋࡃᢚไࡉࢀࠊ㐣㓟Ỉ⣲ࡀO821-CFࡢᢚไᶵᵓ࠾࠸࡚ࠊ㔜せ࡞ᙺࢆᯝࡓࡋ࡚࠸
ࡿࡇࡀ♧၀ࡉࢀࡓࠋ୍᪉ࠊࢧࣜࢳࣝ㓟ࡀᢠᛶㄏᑟ㔜せ࡛࠶ࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿࡀࠊ ࢧࣜࢳࣝ㓟ࡢ✚ࡣቑຍࡋ࡞ࡗࡓࠋࡇࢀࡽࡢࡇࡽO821-CF୰ࡣࢧࣜࢳࣝ㓟ࢆࡉࡎࠊ
ᢠᛶࢆㄏᑟࡍࡿ≀㉁ࡀᏑᅾࡍࡿࡇࡀ♧ࡉࢀࡓࠋ
4. O821ࡢᇵ㣴ࢁᾮ⏕⏘ࡉࢀࡿᢚไ≀㉁ࡢศ㞳ཬࡧྠᐃ
࣒࢝ࣛཬࡧHPLCࢆ⏝࠸࡚ࠊO821-CF୰⏕⏘ࡉࢀࡿᢚไ≀㉁ࡢศ㞳ཬࡧࠊNMRࠊGC/MS/
MSཬࡧFT-IRࡼࡿྠᐃࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊᮏ≀㉁ࡀศᏊ㔞248ࡢ (3aS,4aR,8aS,9aR)-3a- hydroxy-8a-methyl-3,5-dimethylenedecahydronaphtho[2,3-b]furan-2(3H)-one࡛ࠊ᪂つ࡞≀㉁࡛࠶
ࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋᒎ㛤⁐፹ࡢࢺ࢚ࣝࣥ㸸㓑㓟࢚ࢳࣝ=1㸸1 (v/v)ࢆ⏝࠸ࡓሙྜࠊ TLC ࡢRf ್0.5-0.6㏆ࡑࡢ≀㉁ࡀ☜ㄆ࡛ࡁࠊ5 ppmࡽࢿ࠸ࡶࡕ⳦ࡢ⬊ᏊⓎⱆཬࡧ
ᩬᙧᡂࢆᢚไࡍࡿࡇࢆ᫂ࡽ࡞ࡗࡓࠋࡲࡓࠊᮏ≀㉁ࢆ๓ฎ⌮ࡋࡓࢿ࠾࠸࡚ࡶࢿ
࠸ࡶࡕࡢᩬᙧᡂࢆᢚไࡍࡿࡇࡀ♧ࡉࢀࡓࠋᮏ≀㉁ࢆฎ⌮ࡋࡓࢿ࡛ࡣࠊ㐣㓟Ỉ⣲ࡢ
⏕ᡂཬࡧPBZ1㑇ఏᏊࡢⓎ⌧㔞ࡢቑຍࡀ☜ㄆࡉࢀࠊཎ⳦ᑐࡍࡿ┤᥋ⓗ࡞ᢚไຠᯝࡔࡅ࡛
࡞ࡃࠊᮏ≀㉁ࡣࢿᢠᛶࡶㄏᑟ࡛ࡁࡿྍ⬟ᛶࡀ♧ࡉࢀࡓࠋ
5. H921⳦ᰴࡢᇵ㣴ࢁᾮࡀࢿ࠸ࡶࡕ⳦ཬࡰࡍᙳ㡪
H921⳦ᰴࡢᇵ㣴ࢁᾮ(H921-CF)୰ࡣࢿ࠸ࡶࡕ⳦ࡢ⬊Ꮚࡢឤᰁ⾜ືࢆᢚไࡍࡿ≀㉁
ࡀᏑᅾࡋࠊࡇࡢᢚไ≀㉁ࡣ㓑㓟࢚ࢳࣝྍ⁐࡛⇕Ᏻᐃ࡛࠶ࡗࡓࠋࡲࡓࠊH921-CFࡣ࣒࢜࢜ࢠ ཬࡧࢿୖ࡛ࡶࢿ࠸ࡶࡕࡢᩬᙧᡂࢆᢚไࡋࡓࠋITS㡿ᇦࡢࢩ࣮ࢡ࢚ࣥࢫゎᯒࡼࡾࠊ
ᮏ⳦ᰴࡀTrichodermaᒓ࡛࠶ࡿࡇࡀ♧၀ࡉࢀࡓࠋᒎ㛤⁐፹ࡢࢺ࢚ࣝࣥ㸸㓑㓟࢚ࢳࣝ=1㸸1 (v
/v)ࢆ⏝࠸ࡓሙྜࠊTLCୖ࡛ࡣࠊRf ್ 0ࠊ0.13ࠊ0.19ࠊ0.25ཬࡧ0.56ࡢ⨨ࢿ࠸ࡶࡕ
⳦ཬࡧࢿࡈࡲⴥᯤ⳦ࡢ⏕⫱㜼ṆᖏࡀㄆࡵࡽࢀࡓࠋࡇࢀࡽࡢࡇࡽH921-CF୰ࢿ࠸
ࡶࡕ⳦ᑐࡋ࡚ᢚไάᛶࢆ♧ࡍ」ᩘࡢ≀㉁ࡀᏑᅾࡍࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ
ᮏ◊✲ࡢ⤖ᯝࡽࠊ㔝⏕ࡁࡢࡇ࠾࠸࡚ࡣࠊࠕࡁࡢࡇᐤ⏕࣭ඹ⏕ࡍࡿ⳦㢮ࠖࡀከࡃᏑᅾ ࡋࠊࡑࢀࡽࡢ⳦㢮ࡀ⏕⏘ࡍࡿ≀㉁ࡀ᳜≀ཎ⳦ࡢ㜵㝖⏝࡛ࡁࡿྍ⬟ᛶࡀ♧ࡉࢀࡓࠋ
99 Summary
Resistant cultivars and chemical fungicides are important in plant disease management. However, the durability of genetic resistance in improved rice cultivars is often short-lived in the field and excess using of chemical fungicides is concerned. Recently, studies that use microorganisms and microbial metabolites to control disease have been remarked. In this study, fungal strains isolated from the fruiting body of wild mushrooms were evaluated for inhibitory activity against plant pathogens such as Magnaporthe oryzae causes rice blast disease.
1. Isolation of fungi from wild mushrooms and screening
Fungal isolates (n = 105) were obtained from 46 samples of wild mushrooms. Infection behavior of M. oryzae was assessed in the presence of culture filtrates from 90 fungal isolates, of which 20 fungal isolates inhibited spore germination. Heat-treated culture filtrates of these isolates were classified into 3 groups according to biological activity. Blast lesion formation by M. oryzae was significantly inhibited by pretreatment with culture filtrates from 4 fungal isolates. Internal transcribed spacer (ITS) region sequence analysis indicated these isolates shared similarities with species of the genera Annulohypoxylon, Nigrospora, and Penicillium. This result suggested that symbiotic and parasitic fungi from wild mushrooms produce inhibitory compounds for control of plant diseases such as the rice blast disease.
2. Inhibitory activity of culture filtrate from isolate O821 on plant pathogens
The ethyl acetate extract of culture filtrate O821 (O821-CF) significantly inhibited spore germination and appressorium formation of M. oryzae. ITS region sequence analysis indicated that this isolate shared similarities with fungal species of the genus Biscogniauxia. The inhibitory activity of O821-CF was heat-stable. To investigate the effect of incubation period on the inhibitory compound production by isolate O821, O821-CF was prepared at different incubation periods. The presence of inhibitory compounds in O821-CF increased over time of culture of isolate O821. 4-days-incubated O821-CF inhibited spore germination and appressorium formation of M. oryzae. Additionally, infection behaviors of air-borne and soil-borne plant pathogens were inhibited by O821-CF. Blast lesion formation was significantly suppressed in the presence of O821-CF. Also, blast lesion formation by M. oryzae was significantly inhibited in rice plant subjected to 24 h pretreatment with O821-CF.
Furthermore, Corynespora leaf spot and anthracnose in cucumber leaves inoculated with Corynespora cassiicola and Colletotrichum orbiculare spores were suppressed by O821-CF. These results suggested that Biscogniauxia sp. O821 produce a broad spectrum inhibitory compound in culture filtrate.
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3. Induction of resistance in rice by culture filtrates of isolate O821
We demonstrate that O821-CF treatment elicited the production of hydrogen peroxide (H2O2), and increased transcription of the PBZ1 gene in rice leaves. A time course analysis of H2O2 production indicated an increase with time after O821-CF treatment. The expression of the PBZ1 gene similarly peaked at 24 h after O821-CF treatment. Inhibitory activity of O821-CF on blast lesion formation was decreased by treatment with catalase. It showed that H2O2 production play important role in inhibitory mechanism of O821-CF. However, no accumulation of Salicylic acid (SA) was observed at 24 h after O821-CF treatment. These results suggested that SA-independent resistance to rice blast is induced by O821-CF.
4. Purification and identification of inhibitory compound produced by isolate O821
Inhibitory compound was purified by using column chromatography and high performance liquid chromatography. Partial chemical structure determined by NMR combined with data concerning GC/MS/MS and FT-IR. In the result, chemical analysis identified the structures of new compound as (3aS,4aR,8aS,9aR)-3a-hydroxy-8a-methyl-3,5-dimethylenedecahydronaphtho[2,3-b]furan-2(3H)-one (Molecular weight: 248). This new compound was detected at Rf 0.5-0.6 on the thin layer chromatography (TLC). Also, this new compound concentration greater than 5 ppm significantly inhibited spore germination, and blast lesion formation of M. oryzae. Furthermore, blast lesion formation was inhibited even in the new compound-pretreated rice leaves. H2O2 generation was observed in rice leaves pretreated with new compound, and PBZ1 gene was significantly expressed in the leaves. These result suggested that this compound has strong inhibitory effect against M. oryzae, and has the potential to induce resistance in rice against M. oryzae.
5. Effect of culture filtrate from isolate H921 on rice blast
Spore germination and appressorium formation of M. oryzae spores were completely suppressed by an ethyl acetate extract of the culture filtrate of isolate H921 (H921-CF). Heat treatment of H921-CF did not alter its inhibitory effect on M. oryzae spore germination compared to non-heat-treated H921-CF. Blast lesion formation inhibition by H921-CF was dose-dependent in the rice and barley plant.
ITS region sequence analysis indicated these isolates shared similarities with species of the genera Trichoderma. In the H921-CF the growth inhibition zone of M. oryzae and Bipolaris oryzae were observed at Rf 0, 0.13, 0.19, 0.25 and 0.56 on TLC. These results suggests that H921-CF contains some inhibitory compounds that could be promising candidates for control of rice blast disease.
In conclusion, studies of symbiotic and parasitic fungi from fresh fruiting body of wild mushrooms may yield potential control agents for plant diseases such as the rice blast disease.