JP3694739B2 - How to identify fungi - Google Patents

Description

【００６９】
VA 菌根菌胞子の増殖
供試VA菌根菌は、基本的に白クローバー、バヒアグラス、タマネギ等を宿主として増殖させた。滅菌処理した培土（赤玉土、白川砂、黒ボク土等を含む）に無菌播種し発芽した宿主植物苗を植え、あらかじめ表面殺菌したVA菌根菌胞子を苗の根の先端部分に置き、培土を載せた。グロスキャビネット内で45μM/m²s (3000lux以上）の光照射下、25℃で約3ヶ月栽培した。栽培終了時に、ポット内の培土を回収し、培土中の増殖したVA菌根菌胞子を、デカンテーション-ふるい分け法により収集した。得られた胞子は蒸留水に入れ超音波処理を数秒行い、胞子を洗浄した。
【００７０】
粗酵素液の調製
VA菌根菌胞子をPBSで洗浄し、エッペンドルフチューブのふたのウェル内に入れた。これに、タンパク質抽出バッファー(10mM Tris−HCl、10mM NaHCO₃、10mM MgCl₂・６H₂O、0.1mM EDTA-2Na 、10mM β−メルカプトエタノール、0.1％ Triton X-100、pH7に調整、使用前に4℃で保存。)を加え、氷冷しながら精密ピンセットで胞子を1個づつ潰した。なお、G. margaritaの場合、75〜100個の胞子をウェルにいれ、胞子15個に対し10μlの割合で抽出バッファーを添加した。抽出液はエッペンドルフチューブ内に回収し、4℃、11,700rpmで20分間遠心分離した。得られた上清を10μlづつエッペンドルフチューブに分注し、−70℃で凍結保存した。
【００７１】
タンパク質濃度の測定
粗酵素液中のタンパク質濃度はBradford法に基づいたBio-Rad プロテインアセイキットII（Cat. No. 500-0002）を用いて測定した。Tris-HCl (0.05M, pH8.5)を用いて粗酵素液を適宜希釈した。この希釈液800μlに染色液（Bio-Rad, Cat. No. 500-0006）200μlを添加し、よく混合した後、25℃で5分間インキュベートした。この試料の595nmにおける吸光度を測定し、牛血清アルブミン(BSA)を標準タンパク質として作成した検量線を基にタンパク質濃度を測定した。
【００７２】
電気泳動
泳動装置はミニプロティアン３セル(Bio-Rad, No.165-3302)を用いた。また、泳動用のゲルとして、7.5％ポリアクリルアミドゲル(Bio-RadレディーゲルJ, No.161-J311)、泳動バッファーとしてTris-Glycine バッファー(Bio-Rad、No.161-0734)を使用した。また、サンプルである粗酵素液は、サンプルバッファー(Bio-Rad native サンプルバッファー、No.161-0738)と1:1(w/w)の割合で良く混合した後、それぞれのウェルに20μｌずつ分注した。泳動は、4℃、200Vの条件下で、ブロモフェノールブルーのフロントマーカーが所定の位置に移動するまで行なった(泳動時間にして約1時間)。泳動終了後直ちに活性染色に供した。
【００７３】
各種タンパク質（酵素）の活性染色法
各種アイソザイムの活性染色は、下記の染色法を用いて25℃でインキュベートした。
【００７４】
(i)酸性フォスファターゼ（Acid phosphatase；ACP）
0.15M 酢酸-NaOHバッファー（pH5.0）50mlにFast blue RR塩50mg を溶解し、ろ過により不溶物を除いた。この溶液に、基質としてα-ナフチルリン酸・2Naを50mg溶解し、ゲルを染色した。染色液が濁ったら同量の染色液と交換した。
【００７５】
(ii)アルカリフォスファターゼ（Alkaline phosphatase；AKP）
0.05M Tris-NaOH-HClバッファー（pH8.7）50mlに、0.5M MgCl₂ 300μl、および0.25M MnCl₂ 600μlを加え、Fast blue RR 塩50mgを溶解した。不溶物をろ過により除去した後、α-ナフチルリン酸・2Na を50mgを溶解し、これを染色に使用した。染色液が濁った場合、同量の新しい染色液と交換した。
【００７６】
(iii)エステラーゼ（Esterase；EST）
0.05M Tris-HClバッファー（pH7.1）50mlにFast blue RR塩50mgを溶解し、ろ過により不溶物を除去した。染色直前に1%α-ナフチル酢酸および1％β-ナフチル酢酸（ともに50%アセトン溶液に溶解）をそれぞれ1.5ml 加えた。この染色液に泳動の終了したゲルを浸し、染色した。染色液に濁りが見られたら新しい染色液50mlと交換し、さらに染色を行った。
【００７７】
(iv)グルタミン酸−オキザロ酢酸トランスアミナーゼ（Glutamate-oxaloacetate transaminase；GOT）
0.2M Tris-HClバッファー（0.04%EDTAを含む、pH8.0）30mlに、L-アスパラギン酸100mg、10% 2-ケトグルタル酸溶液1ml、および0.33%ピリドキサルリン酸溶液1.5mlを加え、1M Tris溶液を用いてpHを8.0に調整した。この溶液に、泳動の終了したゲルを25℃30分間浸漬した。20mlのTris-HClバッファー（0.04％EDTAを含む）にFast blue BB塩 100mgを溶解し、不溶物をろ過した溶液を調製し、先の溶液に加え、染色を行った。
【００７８】
(v)グルコース-6-リン酸脱水素酵素（Glucose-6-Phosphate dehydrogenase；G6PD）
0.2M Tris-HClバッファー（0.04%EDTAを含む、pH8.0）50mlに、0.5% MgCl₂溶液 1ml、0.67% NADP溶液 1ml、0.5% MTT tetrazorium 1ml、および1.25% グルコース-６-リン酸溶液1mlを加えて良く攪拌した。使用直前に0.5% PMS（Phenazine
methosulphate）溶液200μlを加え、染色に使用した。
(vi)ヘキソキナーゼ（Hexokinase；HK）
【００７９】
0.2M Tris-HClバッファー（0.04%EDTAを含む、pH8.0） 50mlに、Glucose 0.5gを溶解し、さらにこれに0.5M MgCl₂溶液 1ml、10% ATP溶液 1ml、0.67% NADP溶液 1ml、1% NAD溶液 1mlを加え、良く攪拌した。使用直前にグルコース-6-リン酸脱水素酵素 17unit、0.5% MTT tetrazolium溶液 1ml、および0.5% PMS溶液 200μlを加えて、染色を行った。
【００８０】
(vii)ロイシンアミノペプチダーゼ（Leucine aminopeptidase；LAP）
0.2M Tris-Malateバッファー（pH5.5）50mlに、0.5%MgCl₂溶液 5mlおよびＬ-Leucyl-β-naphthylamide 20mgを加えた。あらかじめTris-Malateバッファー5mlにFast black K塩 30mgを溶解させておいた溶液を、使用直前に加え、染色した。
【００８１】
(viii)リンゴ酸脱水素酵素（Malate dehydrogenase；MDH）
0.05M Tris-HClバッファー（pH7.0）50mlに、1M DL-リンゴ酸溶液（0.98M炭酸ナトリウム溶液に溶解した）5ml、2.5%NAD溶液1ml、および1%NBT（Nitro Blue Tetrazolium）溶液1mlをそれぞれ加えて良く攪拌した。染色直前にこの溶液に0.5%PMS溶液 200μlを加え、染色を行った。
【００８２】
ザイモグラムの解析
ノギスを用いて各バンドの泳動距離と幅を測定して各バンドの位置を記録した。さらに、フロントマーカーであるBPBの移動距離から、各バンドのRf値を以下の数式により算出した。
Rf＝(泳動の始点から各バンドの中央までの距離)／(泳動の始点からフロントラインまでの距離)
【００８３】
バンドの濃さは目視により3段階で評価した。各泳動図は、染色後のゲルをライトボックス上に設置して撮影した写真を基に作成した。
【００８４】
アイソザイム分析の結果
G. margarita MAFF520054胞子の無細胞抽出液を用いて、８つの酵素系（EST、ACP、AKP、MDH、G6PD、GOT、HK、LAP）についてアイソザイム分析を実施した。アイソザイム分析から得られたザイモグラムを図１に示す。
【００８５】
その結果、ACP以外の酵素系で薄いバンドが得られ、識別はかろうじて可能であった。本実施例においては、1酵素の分析に約15個の胞子を使用したことになるが、この胞子数で回収できる酵素の絶対量は非常に少ないため、薄いバンドしか得られなかった。よりシャープなバンドを得るには、約20個〜数十個の胞子が必要であることが判明した。VA菌根菌の単一胞子レベルでのアイソザイム分析による識別には、単一胞子を宿主植物に接種しポット栽培等を行うことで、遺伝的に同一の胞子（クローン）を多数増殖させる作業をあらかじめ行い、このようにして得られた多数のクローンを用いて無細胞抽出液を調製し、アイソザイム分析に使用する必要があることが判明した。
【００８６】
核酸分析
次に、核酸分析を行なった。
既知の菌として、VA菌根菌、Gigaspora margarita MAFF520054株を用いた。まず、G．margarita MAFF520054株のITS領域および5.8SrDNAの塩基配列を決定し、既従の配列とともに分子系統樹を作成し、系統間で系統樹上で何らかの分布の特異性があるかどうかを検討した。図２は、Gigaspora 属の5.8S rDNA及びITS領域の塩基配列に基づく分子系統樹を示す。図２中、ｍは、G. margaritaを、ｒは、G. roseaを、１は、単一胞子クローンを、１０は、10胞子クローンを、C,A,Kは、それぞれG. margarita MAFF520054、 G. margarita Ni-A、 G. margarita MAFF520052を示す。
【００８７】
次に、G. margarita MAFF520054株のゲノムDNAに特異的な塩基配列を検出することを目的として、Scutellospora属で見いだされた反復配列を３’-末端に持つ市販のランダムプライマーを用いてRAPD解析すると共に、再現性の高いバンドの塩基配列を決定して数種の新規なプライマーを設計した。さらに、既往の文献に記載のある反復配列やミニサテライトなどのプライマーや同プライマーの塩基配列を基に新たに設計したプライマーを用いて、PCRを実施した。以上のプライマーより2つのプライマーを選び、PCRを行い、G. margarita MAFF520054株に特異的なバンドを検出するプライマーセットを検索した。具体的には、以下の通り行なった。
【００８８】
プライマー 639R の設計とプライマーセットＭ 13/639 Ｒについて
（1）Scutellospora の反復配列を3'-末端に持つ市販の12塩基ランダムプライマーを選定し、単一プライマーでRAPDを行った。このうち、比較的再現性の良いバンドを抽出し、それらの塩基配列を決定した。このうち、500bp以上の配列6本についてそれぞれの内部でセンス/アンチセンスプライマーを設計した（639F/R, 714F/R, 621F/R, 641F/R, 581F/R, 1014F/R）。621F/Rでは、 G.margarita MAFF520054株からは増幅産物は得られなかった。他のプライマーセットは、 G.margarita MAFF520054株特異的ではなかったが、PCR産物の高い再現性からG.margarita各系統のゲノム内にここで得られた配列が存在することが期待された。
【００８９】
（2）既往の文献にある各種プライマーのうち、M13ミニサテライトプライマーと(GACA)4プライマーは、G. margarita MAFF520054株から、総合的なRAPDパターンは変動するものの、比較的再現性の良いメジャーバンドを生じた。また、前者は既往の文献で既にG.margaritaに対して用いられた経緯がある。これらのプライマーを単独に用いても系統間の識別はできなかったが、G. margarita各系統のゲノム内にここで得られた配列が存在することが期待された。
（3）この時点までに、M13ミニサテライト、(GACA)4、ITS1, ITS4及び（1）で設計した10種のプライマーは、G. margaritaゲノムに存在する配列に対応することが期待されたので、これらを2つずつ組み合わせてPCRを行った。その結果、M13と639Rの組み合わせの時にのみ、235bpのG. margarita MAFF520054株に特異的なバンドが形成された(図３)。
【００９０】
実施例２
次に、核酸分析を、既知の菌類に特異的な塩基配列に一部又は全部が相補的な塩基配列を有するDNA断片をプローブとして、未知の菌類について相補的な塩基配列を検出して行なった。
【００９１】
実施例1で得られた235bpの特異的な塩基配列をもとにして、プローブ(230PBC)を設計した。
【００９２】
具体的には、M13/639RによるPCR産物のDNA配列を決定し、DDBJ/EMBL/GenBankデータベース上でFASTAプログラムを用いて相同性検索を行った。この結果、比較的広い範囲で既知のDNA配列と相同性が低かった領域（45塩基）を選び出し、プローブ領域とした。この配列を人工的に合成し（BEX株式会社）、ECL 3'オリゴラベリングキット（アマシャムファルマシア株式会社）あるいはDIG オリゴヌクレオチドテーリングキット(ロシュダイアグノスティック）で標識してプローブとした(図４)。
【００９３】
G．margarita NAFF520054、G．margarita Ni-A、G．margarita NAFF520052、G．margarita INVAM及びG. margarita ネパール株の胞子から抽出したDNAを鋳型として、M13/639Rプライマーセットを用いたPCRと230PBCによるプロービングを行った。また、G. margarita MAFF520054株と同一のセラキンコン(G. margarita sp. CK、セントラル硝子社製）の胞子DNAについても、PCRとプロービングを実施した。その結果、G．margarita NAFF520054株及びセラキンコン（G. margarita sp. CK）胞子の235bpのバンドにハイブリダイズした(図５、６)。なお、 G．margarita NAFF520054は、セントラル硝子（株）の製造するVA菌根菌接種資材中に含まれる菌株であり、1992年に畜産草地試験場に分譲され、その後ポット培養法により継代培養したラボラトリーストレインである。 G．margarita NAFF520054と同資材中に含まれるG．margarita胞子は同一系統であるが、資材として緑化現場に導入されたものは便宜上G. margarita sp. CK株として、 G．margarita NAFF520054と区別して扱う。今回の実施例からも明らかなように、8年間個別に世代交代を繰り返したMAFF520054とCK株の両方について235bpのバンドに230PBCはハイブリダイズしたことから、実用上、この塩基配列は充分な安定性があることが判明した。
【００９４】
したがって、当該プローブを用いれば、 G．margarita NAFF520054株およびsp. CK株を特異的に検出することが可能であり、たとえPCR後の電気泳動により検出されるバンドが不鮮明な場合であっても、特異的に標的とする菌類を容易に識別することができることが分かる。
【００９５】
実施例３
次に、アイソザイム分析及び核酸分析を行なう前に、形態的特徴や組織化学的特徴によって選別した後に、菌類の識別を行なった。
【００９６】
形態的特徴
供試菌株として、実施例1と同様に表1に記載のものを用いた。まず、雲仙普賢岳火砕流跡地試験区にコロナイズしたVA菌根菌及び接種VA菌根菌の形態的特徴を比較した。
【００９７】
土着VA菌根菌胞子を採取するために、雲仙普賢岳中尾川流域の治山施工地において、試験区を設置した。試験区内に侵入し生育した植物を任意に10個体選び、根圏土壌に含まれるVA菌根菌胞子の同定、及び採取した植物根のVA菌根菌感染に関する分析を実施した。各VA菌根菌胞子を任意に選び、各胞子の形態的特徴を実体顕微鏡下で観察した。
【００９８】
採取土壌より検出されたVA菌根菌胞子を形態的特徴に基づき、タイプ分けしたところ、5つのタイプに分類された。これらのVA菌根菌は、火砕流跡地周囲から風による飛散や雨水の流入などにより侵入し、宿主植物に感染、増殖したと考えられる。検出された胞子は、AcaulosporaceaeおよびGlomaceaeのいずれかに分類され、その直径は200μm以下であった。
【００９９】
一般に、Gigasporaceaeに属する胞子は、GlomaceaeやAcaulosporaceaeの胞子に比べて大きく、その直径は300μm以上といわれている(図７)。図７は、各種VA菌根菌胞子の形態的特徴を示す。図７中、aはGigaspora margarita MAFF520054、bはGigaspora margarita MAFF520052、cはGigaspora margarita Ni-A、dはGlomus intraradices、eはAcaulospora longula MAFF520060を示す。
【０１００】
本研究で標的としているG. margarita MAFF520054及び同種のNAFF520052およびNi-Aの胞子の平均直径は、350μmを超えていた。胞子の色は、3株ともWhite−pale yallowであり、形態は球状であり、大きさを除いた形態的特徴に特に大きな違いはなかった(図７a-c)。
【０１０１】
このような形態的特徴による選別は、G. margarita MAFF520054株と明らかに異なる形態の菌を、検出対象から効率的に除外することができ、アイソザイム分析、核酸分析の回数をより少なくし、検出時間を短縮できることが判明した。
【０１０２】
組織化学的特徴
次に、組織化学的特徴についても、同様に調べた。供試菌株として、実施例1と同様に表1に記載のものを用いた。
【０１０３】
G. margaritaを含む代表的なGlomales目のVA菌根菌数種の胞子壁およびSubtending hyphaeの表層の糖鎖構成を糖鎖認識プローブを用いて定性的に分析し、加えて自家蛍光についても分析を行なった。以上の分析により、G. margarita MAFF520054株に特異的な組織化学的マーカーを検索するだけでなく、供試VA菌根菌の識別における有効な組織化学的特徴の有無についても併せて検証した。
【０１０４】
VA菌根菌の胞子壁およびSubtending hyphaeの表層の構成糖鎖の定性的分析には、6種の糖鎖認識プローブを使用し、蛍光顕微鏡（OLYMPUS BX-FLA）を用いて結合に伴う蛍光の発生を観察した。自家蛍光は、Ｕ励起（励起フィルター、BP330-385；ダイクロイックミラー、DM400；吸収フィルター、BA420）、IB励起（BP400-410、DM455、BA455）及びIG励起（BP460-490、DM505、BA515IF）のもとで観察した。
【０１０５】
図８は、Gigaspora margarita MAFF520054の胞子の自家蛍光を示す図である。図８中、aは透過光を、ｂはU励起、ｃはIB励起、ｄはIG励起を示す。また、Cは、胞子内容物を、Sは、subtending hyphaeを、Ｗは、胞子壁を示す。組織化学的特徴を利用した特異的検出の対象であるG. margarita MAFF520054株の胞子壁は、U,IB及びIG励起下においていずれも強い自家蛍光を示した(図８)。さらに、Subtending hyphae及び胞子内容物も同様に自家蛍光を示した。同様の現象は、他のG. margarita２株、G. rosea及び同科のScuttellospora cerradensisにおいても認められた(図９)。図９は、Gigasporaceaeの胞子の自家蛍光を示す。図９中、a−dについては、Gigaspora roseaMAFF520062の透過光(a)、U励起(b)、IB励起(c)、IG励起(d)を示す。e‐fについては、Gigaspora margarita MAFF520052の透過光(e)、U励起(f)を示す。g−hについては、Scutellospora cerradensisMAFF520056の透過光(g)、U励起(h)を示す。また、Ｃは胞子内容物を、Ｓはsubtending hyphaeを、Ｗは胞子壁を、それぞれ示す。
【０１０６】
これに対して、Glomaceae及びAcaulosporaceaeでは、胞子壁及びsubtending hyphaeに自家蛍光は認めれらたが、胞子内容物には認められなかった(図１０)。図１０は、Glomaceae 及びAcaulosporaceaeの胞子の自家蛍光を示す。図１０中、a−dについては、Glomus clarum-like MAFF520061の透過光(a)、U励起(b)、IB励起(c)、IG励起(d)を示す。e‐hについては、Glomus intradicesの透過光(e)、U励起(f)、IB励起(g)、IG励起(h)を示す。i−ｊについては、Glomus etunicatum MAFF520053の透過光(i)、U励起(j)を示す。ｋ−ｌについては、Acaulospora longula MAFF520060の透過光(i)、U励起(j)を示す。また、Ｃは胞子内容物を、Ｓはsubtending hyphaeを、Ｗは胞子壁を、それぞれ示す。
【０１０７】
この結果は、今回供試したGlomales目9株に限定されるが、胞子内容物の自家蛍光は標的VA菌根菌を含むGigasporaceaeとGlomaceaeおよびAcaulosporaceaeとを区別する有効な指標として利用できることを示唆した。
【０１０８】
一方、糖鎖認識プローブであるCWとFITC結合レクチンの胞子壁及びsubtending hyphaeに対する反応性であるが、CWは、G. margarita MAFF520054およびその他のGigasporaceae の胞子壁に対して結合しなかった。結果を図１１に示す。図１１は、G. margarita MAFF520054の胞子壁及びsubtending hyphaeに対するCW及びWGAの反応を示す図である。図１１中、aは、CWの反応（U励起）を示す。subtending hyphaeにCWが結合し、青色の蛍光を発した(矢印)。ｂは、ａと同じ視野における透過光での観察を示す。ｃは、ＷＧＡの反応（ＩＢ励起）を示す。subtending hyphae に対してＷＧＡが結合し、緑色の蛍光を発した(矢印)。一方、WGAは胞子壁表層には反応せず、自家蛍光を示すのみであった。さらに、他の4種のFITC結合レクチンもGigasporaceaeの胞子壁表層に対して反応性を示さなかった。一方、G. margarita MAFF520054、その他の2系統、およびG. rosea MAFF520062のsuntending hyphaeに対して、CWおよびWGAは結合した(図１１)。
【０１０９】
また、図１２は、Gigaspora rosea MAFF520062の胞子壁及びsubtending hyphaeに対するConA の反応を示す。図１２中、aは、Con Aの結合に伴う緑色蛍光を示す。Suntenging hyphaeの周縁部に緑色粒子(矢印)が付着していた(IB励起)。ｂは、ａと同じ視野における透過光での観察を示す。G. roseaのsubtending hyphaeでは、ConAの結合による粒子状の緑色蛍光が菌糸周縁部に認められた(図１２)。
【０１１０】
また、図１３はGlomaceaeの胞子壁及びsubtending hyphaeに対するCW及びFITC結合レクチンの反応を示す。ａ‐bは、Glomus clarum−like MAFF520061である。ａは、胞子壁及びsubtending hyphae(矢印)に対するCWの反応を示す。ｂは、胞子壁に対するWGAの反応を示す。胞子壁の第一層にWGAが結合し、緑色の蛍光を発していた(矢印)。ｃ−dは、Glomus intraradicesである。ｃは、胞子壁及びsubtending hyphae(矢印)に対するCWの反応を示す。菌糸周縁部に付着している顆粒状の物質(矢印)が緑色の蛍光を発していた。Glomaceae 3株では、CWは胞子壁およびSubtending hyphaeに対して結合した(図１３a、c)。また、WGAは、G. clarum-like MAFF520061の胞子壁にのみ結合した(図１３b)。各種FITCレクチンのsubtending hyphaeに対する反応性は、種によって異なっており、その結合は、菌糸同縁部に限定されていた(図１３d)。
【０１１１】
今回の分析では、使用した6種の糖鎖認識プローブにより、標的VA菌根菌G. margarita MAFF520054の特異的マーカーを見つけだすことはできなかった。しかし、CWやWGAの胞子壁に対する反応性は、供試菌の範囲に限定されるが、科のレベルでの区別化や特定の種の識別に利用可能であることが判明した。
【０１１２】
そして、このような菌類に特有の組織化学的特徴を利用することにより、菌類を大まかに選別することができる否かを確認した。
【０１１３】
具体的に、上述の組織化学的特徴による選別を行なった後に、実施例1と同様のアイソザイム分析及び核酸分析を行なった。その結果、明らかに異なる組織化学的特徴を有する菌類を、検出対象から効率的に除外することができた。
【０１１４】
組織化学的特徴による選別を行なえば、対象となる菌類を絞ることができ、アイソザイム分析、核酸分析の回数をより少なくでき、検出時間を短縮できることが判明した。
【０１１５】
実施例４
次に、実際に、雲仙普賢岳の緑化施工区に既に微生物資材として投与されているVA菌根菌の挙動を調べた。すなわち、施工区に生育している植物に投与したVA菌根菌が感染し定着しているかについて調べた。
【０１１６】
1997年の施工時に植物種子とともにG. margarita sp. CK株が導入された雲仙普賢岳水無川火砕流跡地において、2001年3月に植物体を採取し、根圏土壌からVA菌根菌胞子を抽出した。植物個体当たりの胞子数を計数するとともに、形態的特徴、組織化学的特徴および核酸分析によりG. margarita sp. CK株の選別と識別を試みた。
【０１１７】
緑化施工区から採取したウィーピングラブグラス10個体のうち2個体の根圏土壌から、形態的特徴および組織化学的特徴に基づきGigaspora様と思われる胞子が選別された（表2、図１４）。図１４中、AはGigaspora様胞子を、Ｂは同胞子の直径の頻度分布を、ＣはGigaspora様以外の胞子を、Dは同胞子の直径の頻度分布を示す。図14のＡにおいて、Gifasporaceaeに特徴的なbulbous suspensor (矢印で示す)が認められる。そのうち1個体のWLGから24個のGigaspora様胞子が採取され、そのうち10胞子をPCRとプロービングに供した。DNAが得られた8胞子に関しては、230PBCとハイブリする235bpのバンドが得られた（図１５）。図１５の上段泳動図において、矢印はITS領域増幅産物のバンドを示す。C及びfの胞子では、同バンドは検出されなかった(＊印で示す)。残り８胞子については、バンドの出現によりDNAの存在が示された。図１５の下段泳動図は、２３０PBCを用いたサザンハイブリダイゼ―ションの結果を示す。DNAが得られた８胞子について、２３０PBCとハイブリダイズする２３５bpのバンド(矢印)が得られた。
【０１１８】
【表２】

【０１１９】
以上の核酸分析等の解析により、緑化現場の土壌より採取したVA菌根菌と接種菌との同一性を確認することができた。
よって、前記分析手法により、特定系統のVA菌根菌を迅速に同定できることが確かめられ、緑化現場での接種VA菌根菌の土壌中での挙動を把握できることが判明した。
【０１２０】
よって、定期的に土着菌と接種菌とを比較することにより、種々の菌が混在する場合であっても、特定の菌をスクリーニングすることが可能であることが分かり、接種菌の土壌中での挙動を把握することができることが判明した。
【０１２１】
【発明の効果】
本発明の菌類の識別法によれば、同種内での異系統間の菌類を識別できるという有利な効果を奏する。
【０１２２】
また、本発明の菌類の識別法によれば、アイソザイム分析及び核酸分析によって、より精度よく菌類の同一性を判定することができ、ひいては、土壌に接種した場合の菌類の挙動を定期的に把握することもできるという有利な効果を奏する。
【０１２３】
【配列表】
＜１１０＞山口大学長
＜１２０＞菌類の識別方法
＜１３０＞Ｕ２００１Ｐ０６３
＜１６０＞２
＜２１０＞１
＜２１１＞235
＜２１２＞核酸
＜２１３＞Gigaspora margarita C株
＜４００＞１
gagggtggcg gttcttttat gacaacaata acaaaaagac caagaaagat ttataactcc 60
gaaagtggtt tctctcaaat aactaccgca atacttacat tctattcaat agtaaaagga 120
gggcggagta taacgacttc tacttacaac atctccatca tcatctaacc atttatcgca 180
tttttcgcag tagcgagtgt attgccaagt tgtggggtca tcttggcatt aggta 235
＜２１０＞２
＜２１１＞45
＜２１２＞核酸
＜２１３＞Gigaspora margarita MAFF520054
＜４００＞２
ctaaccattt atcgcatttt tcgcagtagc gagtgtattg ccaag 45
【図面の簡単な説明】
【図１】 種々の酵素を用いたアイソザイム分析から得られたザイモグラムを示す図である。
【図２】 Gigaspora 属の5.8SrDNA及びITS領域の塩基配列に基づく分子系統樹を示す図である。
【図３】 M13/639Rプライマーによる各種胞子のPCR産物の比較を示す図である。
【図４】 G. margarita MAFF520054株を識別するためのプローブの一例を示す図である。
【図５】 プローブと、各菌類由来のPCR産物とのハイブリダイゼーションを示す図である。
【図６】 プローブと、各菌類由来のPCR産物とのハイブリダイゼーションを示す図である。
【図７】 各種VA菌根菌胞子の形態的特徴を示す図である。
【図８】 G. margarita MAFF520054の胞子の自家蛍光を示す図である。
【図９】 Gigasporaceaeの各種胞子の自家蛍光を示す図である。
【図１０】 Glomaceae 及びAcaulosporaceaeの胞子の自家蛍光を示す図である。
【図１１】 G. margarita MAFF520054の胞子壁およびsubtending hyphaeに対するCW及びWGAの反応を示す図である。
【図１２】 Gigaspora rosea MAFF520062の胞子壁及びsubtending hyphaeに対するCon Aの反応を示す図である。
【図１３】 Glomaceaeの胞子壁およびsubtending hyphaeに対するCW及びFITC結合レクチンの反応を示す図である。
【図１４】 VA菌根菌を導入した緑化施工区より採取されたVA菌根菌胞子の特徴を示す図である。
【図１５】 プローブと、緑化施工区の土壌より採取されたVA菌根菌胞子由来のPCR産物とのハイブリダイゼーションを示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for identifying fungi, and more particularly to a method for identifying fungi by performing isozyme analysis and nucleic acid analysis.
[0002]
[Prior art]
Conventionally, methods based on morphological features such as hyphae, spores, and breeding organs are known as fungal identification methods. Specifically, the method based on morphological features such as spores is a comparison between the size of fungi, such as conidia, spores, pupa fruit, pupa, pupa spore, zygospore, basidiospore. This is a method for identification and classification.
[0003]
[Problems to be solved by the invention]
However, the method based on the above morphological characteristics has a problem that it is impossible to identify a specific strain at the hyphal and mycorrhiza level in the same species. That is, according to the method based on the morphological features described above, the classification is based only on morphological criteria such as spore size, mycelial features, and spore patterns, and narrows down to the level of the species of the same genus. It is necessary to examine the morphological characteristics in more detail, such as the fine structure, color, etc. Such classification based on detailed morphological features such as fine structure, color, etc. of spores, mycelia, breeding organs, etc. is necessary for those other than researchers who are skilled and who have experience in observing and classifying many different spores. Has the problem of being very difficult.
[0004]
In recent years, the regeneration of forests and greening techniques that make use of mycorrhizal symbiosis have attracted attention, and the introduction of inoculum during the introduction of trees in eroded and degraded areas has been discussed. For example, infection of mycorrhizal fungi with planted trees and plants not only increases the growth of the host plant accompanying the absorption of nutrients, but also improves the survival rate after transplanting, resistance to water stress, etc. As a result, early recovery of vegetation is expected.
[0005]
However, soil microbiota including mycorrhizal fungi are poor in oligotrophic soil environments such as eroded and degraded areas. When fungi are introduced into such a soil environment, it has been difficult to analyze in detail how they coexist with indigenous bacteria or compete and inhabit, using only the method based on the morphological features described above. Therefore, in addition to the morphological features described above, it has been desired to develop an identification method for knowing the details of fungal species and species identification and evaluation of their diversity.
[0006]
Then, this invention is providing the identification method of the fungi which enables identification of the specific strain | stump | stock at a mycelium and mycorrhiza level.
[0007]
[Means for Solving the Problems]
In order to achieve the above-mentioned object, the inventors have intensively studied a method for identifying specific strains of fungi from histochemical, biochemical, and molecular biological viewpoints. I came to find.
[0008]
The method for identifying a fungus of the present invention is characterized in that a known fungus and an unknown fungus are subjected to isozyme analysis and nucleic acid analysis, and the fungi are identified by comparing both fungi.
[0009]
Further, in a preferred embodiment of the method for identifying a fungus of the present invention, the nucleic acid analysis determines a specific base sequence for DNA of a known fungus by RAPD (Random Amplified polymorphic DNA) analysis of the known fungus, and the unknown The present invention is characterized in that the presence or absence of the specific base sequence is investigated for the fungus.
[0010]
Further, in a preferred embodiment of the method for identifying fungi of the present invention, a base sequence specific to DNA of a known fungus is determined, and a base sequence is determined for a reproducible band obtained by RAPD analysis of a known fungus. A primer that hybridizes to a genomic DNA of a known fungus is designed based on this base sequence, and this primer is used.
[0011]
Further, in a preferred embodiment of the method for identifying a fungus of the present invention, the determination of a base sequence specific to the DNA of a known fungus is carried out by performing PCR using a combination of several primers of the above design and known primers. It is characterized in that it is carried out by detecting a base sequence constituting a band specific to a known bacterium having good reproducibility.
[0012]
Further, in a preferred embodiment of the method for identifying a fungus of the present invention, the nucleic acid analysis is performed by using a DNA fragment having a base sequence partially or entirely complementary to the specific base sequence as a probe and complementary to the probe. The nucleotide sequence isUnknownIt is the analysis of whether it exists in the fungi of this.
[0013]
Further, in a preferred embodiment of the method for identifying a fungus of the present invention, the investigation of the presence or absence of the specific base sequence for the unknown fungus is performed by PCR using a combination of several kinds of primers of the design and known primers. Thus, the detection is performed by detecting a base sequence constituting a band specific to an unknown fungus having good reproducibility.
[0014]
In a preferred embodiment of the method for identifying a fungus of the present invention, the complementary base sequence is analyzed by hybridization to a base sequence constituting a band specific to the probe and the unknown fungus. And
[0015]
In a preferred embodiment of the method for identifying a fungus of the present invention, it is characterized in that it is identified whether or not the fungus belongs to the same species.
[0016]
In a preferred embodiment of the method for identifying a fungus of the present invention, it is characterized in that it is identified whether or not the fungus belongs to the same strain.
[0017]
In a preferred embodiment of the method for identifying a fungus of the present invention, the fungus is a fungus derived from a mycorrhizal fungus.
[0018]
In a preferred embodiment of the method for identifying a fungus of the present invention, the mycorrhizal fungus is a VA mycorrhizal fungus.
[0019]
Further, in a preferred embodiment of the method for identifying a fungus of the present invention, the known fungus is a VA mycorrhizal fungus, and PCR based on a combination of the primers is a base sequence represented by base numbers 1-235 of SEQ ID NO: 1 in the sequence listing It is characterized by amplifying a partial DNA.
[0020]
In a preferred embodiment of the method for identifying a fungus of the present invention, the known fungus is a VA mycorrhizal fungus, and the probe consists of a base sequence represented by base numbers 1-45 of SEQ ID NO: 2 in the sequence listing. Features.
[0021]
Further, in a preferred embodiment of the method for identifying a fungus of the present invention, the isozyme is acid phosphatase (ACP), alkaline phosphatase (AKP), esterase (EST), glutamate-oxaloacetate transaminase (GOT), hexokinase (HK), Leucine aminopeptidase (LAP), malate dehydrogenase (MDH), alanine aminopeptidase (AAP), aspartate aminotransferase (AAT), aconitase (ACO), alcohol dehydrogenase (ADH), amylase (AMY) Diaphorase (DIA), fumarase (FM), glutamate dehydrogenase (GDH), glycerate dehydrogenase (G2D), glucokinase (GK), glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase ( GR), Ngonate enzyme pH 8.0 (ME8), malate enzyme pH 7.0 (ME7), menadione reductase (MNR), 6-phosphogluconate dehydrogenase (6PGD), phosphoglucose isomerase (PGI), phosphoglucomutase (PGM) ), Peroxidase (POD), shikimate dehydrogenase (SKD), sorbitol dehydrogenase (SODH), triosephosphate isomerase (TPI), and tetrazolium oxidase (TZO). It is characterized by being.
[0022]
In a preferred embodiment of the method for identifying a fungus of the present invention, the isozyme analysis and the nucleic acid analysis are performed after selecting the fungus based on morphological characteristics.
[0023]
Further, in a preferred embodiment of the method for identifying a fungus of the present invention, the morphological characteristics are zygospore, gamete, zoospore, zoospore, dormant spore, thick film, gamete, spore. Spore spores, conidia, spore spore, spore stalk, pseudoroot, microspore sac, hyphae, pseudozygous spore, pupa, pupa, spore spore, basidiospore, basidiode, conidia Features such as at least one size and / or form selected from the group consisting of a handle, a conidial bundle, a conidial fruit and fruit, a clamp formation, a septum, and a mycorrhiza.
[0024]
In a preferred embodiment of the method for identifying a fungus according to the present invention, the isozyme analysis and the nucleic acid analysis are performed after selecting the fungus based on histochemical characteristics.
[0025]
In a preferred embodiment of the method for identifying a fungus of the present invention, the histochemical characteristic is autofluorescence and / or the presence of a sugar chain.
[0026]
DETAILED DESCRIPTION OF THE INVENTION
In the fungus identification method of the present invention, the target fungus is not particularly limited. Fungi are located in independent worlds that are fundamentally different from the 4th world of monella, protists, plants and animals. Examples of the fungi that are the subject of the present invention include Aspergillus fungi, zygomycetes, basidiomycetes, ascomycetes, and incomplete fungi. Among various fungi, for example, zygomycetes, ascomycetous fungi, basidiomycetes, etc., there are those called mycorrhizal fungi.
[0027]
The mycorrhiza is a complex of the higher plant root and the mycelium in the soil in contact with each other to form a symbiotic relationship. In general, fungi obtain organic matter from the roots of plants, while plant roots receive nutrient salts, water, and vitamins that are insufficient from fungi. Mycorrhizal fungi can mainly include endomycorrhizal fungi and ectomycorrhizal fungi. Endogenous mycorrhizal fungi are those in which fungi enter a plant root cell to form a symbiotic relationship. The ectomycorrhizal fungi are those that form a mycelium layer around the fine plant roots, and the hyphae also enter the interstices of the skin layer of the fine roots but do not enter the plant cells. In the present invention, it can be widely applied to fungi including mycorrhizal fungi such as endomycorrhizal fungi and ectomycorrhizal fungi. In particular, from the viewpoint of supplying phosphorus to a host plant growing in an oligotrophic environment, examples of mycorrhizal fungi include VA mycorrhizal fungi. VA mycorrhizal fungi are bacteria belonging to the zygomycetes. VA mycorrhizal fungus is a typical fungus that is administered as a microbial material for greening of wasteland that has been depleted of nutrients, and if it becomes possible to grasp the behavior of such fungi, Can contribute.
[0028]
In the method for identifying fungi of the present invention, it is possible to distinguish even if the species of one fungus to be compared and the other fungus are the same species or different species. can do. This is because, as will be described later, it is possible to improve the discrimination accuracy between different strains in the fungal species by combining isozyme analysis and nucleic acid analysis.
[0029]
The fungal identification method of the present invention utilizes isozyme analysis and nucleic acid analysis.
First, the principle of isozyme analysis will be described. The isozyme method is a method for identifying fungal strains by utilizing the difference in behavior of each isozyme due to electrophoresis or the like. The isozyme is a group of enzymes in which chemically different protein molecules catalyze the same chemical reaction.
[0030]
The isozyme method will be specifically described. For example, when there is a biological reaction of A → B and the enzyme that advances this reaction is C, there are several types of enzymes called C, each having a different molecular weight. have. Since the enzyme is a protein, by using a technique called electrophoresis, the individual enzymes constituting C can be separated based on the difference in the molecular size of the protein and the charged state. Electrophoresis usually uses polyacrylamide gel or agarose gel as a carrier. The degree of movement of the isozyme in electrophoresis depends on the molecular weight of the isozyme and the charged state, and proteins can be fractionated based on this feature.
[0031]
When these electrophoresed carriers are attached to a solution containing a specific substrate and a staining agent to proceed with the reaction, a portion where an enzyme that catalyzes the specific substrate is stained and detected as a band. Each such band is an enzyme with the same catalytic activity but different molecular weight. This band pattern may be the same or different between genera, species and strains. It is the isozyme method that discriminates based on such pattern differences.
[0032]
The isozyme procedure includes (1) preparation of cell-free extract of fungal tissue, (2) measurement of protein (enzyme) concentration in cell-free extract, (3) electrophoresis, and (4) various proteins. (Enzyme) activity staining, (5) Zymogram analysis.
[0033]
Preparation of cell-free extract of fungal tissue
First, it begins with collecting fungal extracts. The fungus used as a sample may be freeze-dried. Dilute fungal sample in appropriate buffer and homogenize. After homogenization, it is centrifuged at 0 to 25 ° C., preferably 0 to 10 ° C. and 8000 to 15000 g for 5 to 30 minutes, and the resulting supernatant can be used as a crude extract. Centrifugation conditions can be appropriately changed depending on the type of fungi and the like, and are not limited to the above range.
[0034]
Protein in the extract ( enzyme ) Concentration measurement
The protein concentration can be measured by a conventional method, for example, UV method, Biuret method, Lowry method, BCA method, Bradford method and the like.
[0035]
Activity staining of various proteins
Examples of staining methods include acid phosphatase (hereinafter referred to as ACP), alkaline phosphatase (hereinafter referred to as AKP), esterase (hereinafter referred to as EST), glutamate-oxaloacetate transaminase (hereinafter referred to as GOT), and hexokinase. (Hereinafter referred to as HK), leucine aminopeptidase (hereinafter referred to as LAP), malate dehydrogenase (hereinafter referred to as MDH), alanine aminopeptidase (hereinafter referred to as AAP), aspartate aminotransferase ( Hereafter referred to as AAT), aconitase (hereinafter referred to as ACO), alcohol dehydrogenase (hereinafter referred to as ADH), amylase (hereinafter referred to as AMY), diaphorase (hereinafter referred to as DIA), fumarase (hereinafter referred to as AIA). FM), glutamate dehydrogenation Element (hereinafter referred to as GDH), glycerate dehydrogenase (hereinafter referred to as G2D), glucokinase (hereinafter referred to as GK), glucose-6-phosphate dehydrogenase (hereinafter referred to as G6PD), glutathione. Reductase (hereinafter referred to as GR), malate enzyme pH 8.0 (hereinafter referred to as ME8), malate enzyme pH 7.0 (hereinafter referred to as ME7), menadione reductase (hereinafter referred to as MNR), 6-phospho Gluconic acid dehydrogenase (hereinafter referred to as 6PGD), phosphoglucose isomerase (hereinafter referred to as PGI), phosphoglucomutase (hereinafter referred to as PGM), peroxidase (hereinafter referred to as POD), shikimate dehydrogenase (Hereinafter referred to as SKD), sorbitol dehydrogenase (hereinafter referred to as SODH), triose phosphate isomerase (hereinafter referred to as TPI). And tetrazolium oxidase is a method that utilizes dye to (hereinafter, referred to. TZO) enzymes, such as, the reaction product resulting substrate by the enzyme and a substrate for the enzyme.
[0036]
That is, in this staining method, the presence or absence of a product generated by an enzyme and a substrate is used to know the band position in electrophoresis of a specific enzyme. Even if a product is produced by the reaction between the enzyme and the substrate, the position of the product coincides with the position of the enzyme because it is immobilized by the gel. Since the position of the product is dyed by the dye, the position of the product, that is, the band pattern of the enzyme can be known.
[0037]
Analysis of zymogram
The position of a specific enzyme separated by electrophoresis is revealed by a band pattern by the staining method described above. The analysis of the zymogram can be performed by calculating the Rf value ((distance from the starting point of electrophoresis to the center of each band) / (distance from the starting point of electrophoresis to the front line)) of each band.
[0038]
Nucleic acid analysis
Next, nucleic acid analysis will be described. Nucleic acid analysis, like isozyme analysis, is an effective means for grasping species specificity. This is because a specific base sequence exists in the same manner as a specific isozyme exists depending on individual species.
[0039]
In finding a base sequence peculiar to fungi, for example, RFLP, PCR, RAPD method and the like can be used.
[0040]
From the viewpoint that various PCR amplification products can be obtained, it is preferable to detect the base sequence peculiar to fungi by the RAPD method. The RAPD method differs from normal PCR in that the DNA reaction polymorphisms (hereinafter referred to as RAPD) of various PCR amplification products from multiple sites obtained by arbitrarily changing the PCR reaction conditions, the base sequence and length of the synthetic primer, etc. )). DNA polymorphism can be grasped by a band pattern obtained by subjecting the obtained PCR amplification product to electrophoresis.
[0041]
Specifically, a part of the base sequence of known fungi is determined by RAPD (Random Amplified polymorphic DNA) analysis of known fungi, and various primers are designed. By carrying out PCR by combining these primers and known primers, etc., the base sequence specific to the DNA of the known bacterium is determined, and the presence or absence of the specific base sequence is investigated for the unknown fungus. This makes it possible to know whether a known fungus and an unknown fungus are of the same type, and whether they belong to the same strain.
[0042]
In order to obtain a specific nucleotide sequence using the RAPD method, it is necessary to randomly amplify fungal genomic DNA using various primers and search for a stable band when electrophoresis is performed. it can. The accuracy can be improved by confirming the reproducibility of a stable band. That is, when a stable band is obtained, a base sequence can be determined for the obtained band, and a specific sequence can be found by the fungus.
[0043]
Furthermore, by designing various primers based on the base sequence of a stable band and performing PCR using a combination of these primers and known primers, a more stable band can be found.
[0044]
Furthermore, if a specific base sequence can be specified,ConcernedIt is also possible to identify fungi using a probe having a base sequence partially or entirely complementary to a specific base sequence. That is, the nucleic acid of an unknown fungus is examined for the presence of a specific base sequence specific to the known fungus. If it has a specific base sequence, the same type or strain of the known fungus is used. It is possible to determine that there is a high possibility of belonging. On the other hand, if it does not have a specific sequence, it can be judged that it is highly likely that it belongs to the known fungus or belongs to a different strain.
[0045]
By using the probe, even if the band derived from the nucleic acid of the fungus to be examined is unclear, the fungi can be identified by hybridization with the nucleic acid of the probe and the fungal to be examined. it can.
[0046]
The fungal identification method of the present invention can be directly identified by the above isozyme analysis and nucleic acid analysis. However, a screening method based on morphological and / or histochemical characteristics as described below is introduced. By doing so, the efficiency of sorting can be improved.
[0047]
The selection method based on morphological features is a method based on morphological features such as hyphae, spores, and breeding organs. Specifically, methods based on morphological features such as spores include zygospores, gametes, zoospores, zoospores, dormant spores, thick membranes, gametes, spore spores, Spores, spore spore, spore stalk, pseudoroot, microspore spore, hyphae, pseudozygous spore, pupa fruit, pupa, pupa, spore spore, basidiospore, basidiode, conidia spore, conidia This is a method for identifying and classifying fungi by comparing at least one size and / or form selected from the group consisting of stalk bundles, conidia, clump formation, septum, mycorrhiza. By roughly selecting in advance according to such morphological features, it can be advantageous in terms of time and cost.
[0048]
Further explanation according to the type of fungus is as follows.
When the fungus is a fungus, these fungi have zygospores, zoospores, gametes, zoosporangia, dormant spores, thick membranes, etc., so these spores, presence or absence of organs, form, And / or identification based on size and the like.
[0049]
When the fungus is a zygomycete, the fungus is a zygospore, a gamete, a spore spore, a spore spore, a spore stalk, a temporary root, a conidia, a microspore, a hypha, a pseudozygote Can be identified based on the presence, form, and / or size of these spores and organs.
[0050]
When the fungus is a baby larva, the fungus has a baby spore, a baby spore, a conidia, a mycelium, a baby fruit, etc., so these spores, presence or absence of an organ, form, and / or Identification is possible based on size and the like.
[0051]
When the fungus is a basidiomycete, the fungus has a basidiomycete, basidiomycetes, conidia, conidia, basidiomycetes, clamps, etc., so these spores, the presence or absence, form and / or size of organs It is possible to identify based on the like.
[0052]
When the fungus is an incomplete fungus, the fungus has a conidia, a conidial pattern, a conidial pattern bundle, a conidia fruit, etc., so that these spores, the presence or absence of an organ, form, and / or size, etc. Can be identified based on
[0053]
In addition, the fungi and zygomycetes do not have a partition wall, and the ascomycetous fungi, basidiomycetes, and incomplete fungi have a partition wall, so that the fungi can be identified by the presence or absence of the partition wall. Needless to say, the present invention is not limited to the above description, and a conventional identification method based on morphological features can also be incorporated and used here.
[0054]
Examples of the screening method based on histochemical characteristics include a method based on autofluorescence and qualitative analysis of saccharides. This method based on histochemical characteristics utilizes the properties of mycelia, mycelium, spores, and spore contents, so that, for example, even if each part of the collected spores is missing and sorting based on morphological characteristics is not possible There is an advantage that can be done.
[0055]
First, a method using autofluorescence will be described. Autofluorescence refers to a phenomenon in which when a substance is irradiated with specific excitation light, a component of the irradiated part of the substance emits fluorescence of a specific wavelength. The fungus identification method using autofluorescence is a method of irradiating a fungus with specific excitation light and discriminating the type, strain, etc. of the fungus using the difference in fluorescence emitted.
[0056]
Specifically, a part of the fungal tissue is placed on a slide glass and is referred to as phosphate buffered saline (0.12 M NaCl, 0.0027 M KCl, 10 mM phosphate buffer, pH 7.5, hereinafter PBS). This can be done by observing with a fluorescence microscope (Nikon, OPTIPHOTO). The set of excitation light and filter can be the following set, but is not particularly limited, and can be appropriately changed depending on conditions such as the type and nature of the fungus, the model of the microscope, and the like.
[0057]
For example, 1) UV excitation: Excitation filter EX330-380, Dichroic mirror DM400, Absorption filter BA420, 2) V excitation: EX380-425, DM430, BA450, 3) B2 excitation: EX450-490, DM510, BA520, etc. be able to.
[0058]
If there is autofluorescence, the sample will color from blue-white to blue (UV), green to blue-green (V), yellow to ocher to brown (B2), so it is possible to distinguish fungi using the difference in color development. it can.
[0059]
In addition, in the present invention, in addition to the above-described isozyme analysis and nucleic acid analysis, qualitative analysis of saccharides can be performed, and fungal discrimination efficiency can be further increased.
[0060]
The method based on the qualitative analysis of saccharides is a method utilizing the confirmation of the presence or absence of saccharides. In this method, the presence of a specific sugar chain is confirmed by the presence or absence of binding using a substance (sugar chain recognition probe) that specifically binds to a specific sugar chain that constitutes the cell wall surface layer of fungal hyphae or plant tissue. be able to.
[0061]
As the sugar chain recognition probe, one or more fluorescent dyes, one or more lectins and the like can be used. Examples of the former fluorescent dye include Calcofluor white M2R and Aniline blue. Preferable examples include Calcofluor white M2R and Aniline blue.
[0062]
Calcofluor white M2R and Aniline blue specifically bind to β-1,4-glucan and β-1,3-glucan, respectively. When a mycelium treated with a Calcofluor white M2R solution or a root chip with mycorrhizae is observed with a fluorescence microscope under UV excitation (for example, EX330-380, DM400, BA420), the binding portion shows blue fluorescence. On the other hand, when the sample treated with the Aniline blue solution is observed under a V excitation (EX380-425, DM430, BA450) using a fluorescence microscope, the binding portion shows green fluorescence. The presence of the specific sugar chain can be confirmed by these fluorescent emissions.
[0063]
The latter lectin is a protein that recognizes a specific sugar chain structure, and various types of lectins have been isolated and purified from animals, plants, and microorganisms, and their sugar-binding specificity has been revealed. When a fluorescent dye called Fluorescein isothiocyanate (FITC) bound to a lectin that recognizes a specific sugar chain is added to the sample to be analyzed, the lectin binds to the portion of the sample surface where the specific sugar chain exists. When this sample is observed under a fluorescence microscope (B2 excitation: EX450-490, DM510, BA520), the lectin-bound portion shows green fluorescence derived from FITC, indicating that a specific sugar chain exists in that portion. . Utilizing such properties, the presence of sugar chains can be confirmed.
[0064]
For example, Con A, Concanavalin A (from Canavalia ensiformis); WGA, wheat germ agglutinin (from Triticum vulgaris); LTA, lotus agglutinin (from Tetragonolobus purpureas); SBA, soy bean agglutinin (from Glycine max); PNA, You can use peanut agglutinin (from Arachis hypogaea). These lectins specifically bind to the following sugar chains. That is, Con A is a sugar chain such as methyl-α-D-mannnopyranoside, D-mannose, WGA is a sugar chain such as N-acetylneuraminic acid, N, N'-diacetylchitobiose, LTA is L-fucose, etc. SBA binds to sugar chains such as N-acetyl-D-galactosamine and D-galactose, and PNA binds to sugar chains such as D-galactose and α-lactose.
[0065]
By conducting a qualitative analysis of these sugar chains in advance, it is possible to select to some extent when detecting the target fungus, and the detection efficiency can be improved.
[0066]
【Example】
Here, although one Example of this invention is described, this invention is limited to the following Example and is not interpreted. Moreover, it cannot be overemphasized that it can change suitably, without deviating from the summary of this invention.
[0067]
Example 1
Strain
In this example, a specific strain was detected using VA mycorrhizal spores as fungi. Specifically, the VA mycorrhizal fungus G. margarita MAFF520054 was used as a known fungus. Furthermore, other VA mycorrhizal fungi listed in Table 1 were used as test fungi.
[0068]
[Table 1]

[0069]
VA Mycorrhizal Spore Growth
The test VA mycorrhizal fungi were basically grown using white clover, bahiagrass, onion, etc. as the host. Sterile seedlings (including Akadama soil, Shirakawa sand, Kuroboku soil, etc.) are sown and germinated host plant seedlings are planted, and VA mycorrhizal spore sterilized in advance is placed on the tip of the seedling roots. I put. 45 μM / m in the gross cabinet²Grown for about 3 months at 25 ° C. under light irradiation of 3000 s or more. At the end of the cultivation, the soil in the pot was collected, and the VA mycorrhizal spores grown in the soil were collected by the decantation-sieving method. The obtained spores were placed in distilled water and subjected to ultrasonic treatment for several seconds to wash the spores.
[0070]
Preparation of crude enzyme solution
The VA mycorrhizal spore was washed with PBS and placed in the lid well of an Eppendorf tube. To this, protein extraction buffer (10 mM Tris-HCl, 10 mM NaHCO_Three, 10 mM MgCl₂・ 6H₂O, 0.1 mM EDTA-2Na, 10 mM β-mercaptoethanol, 0.1% Triton X-100, adjusted to pH 7, stored at 4 ° C before use. ) And crush each spore one by one with precision tweezers while cooling with ice. In the case of G. margarita, 75 to 100 spores were placed in a well, and extraction buffer was added at a ratio of 10 μl to 15 spores. The extract was collected in an Eppendorf tube and centrifuged at 4 ° C. and 11,700 rpm for 20 minutes. The obtained supernatant was dispensed in 10 μl aliquots into Eppendorf tubes and stored frozen at −70 ° C.
[0071]
Protein concentration measurement
The protein concentration in the crude enzyme solution was measured using Bio-Rad Protein Assay Kit II (Cat. No. 500-0002) based on the Bradford method. The crude enzyme solution was appropriately diluted with Tris-HCl (0.05M, pH 8.5). 200 μl of staining solution (Bio-Rad, Cat. No. 500-0006) was added to 800 μl of this diluted solution, mixed well, and then incubated at 25 ° C. for 5 minutes. The absorbance of this sample at 595 nm was measured, and the protein concentration was measured based on a calibration curve prepared using bovine serum albumin (BSA) as a standard protein.
[0072]
Electrophoresis
The electrophoresis apparatus used was a mini-PROTEAN 3 cell (Bio-Rad, No. 165-3302). In addition, 7.5% polyacrylamide gel (Bio-Rad ready gel J, No. 161-J311) was used as the gel for electrophoresis, and Tris-Glycine buffer (Bio-Rad, No. 161-0734) was used as the electrophoresis buffer. The sample crude enzyme solution was mixed well with the sample buffer (Bio-Rad native sample buffer, No.161-0738) at a ratio of 1: 1 (w / w), and then 20 μl was dispensed into each well. Noted. Electrophoresis was performed under conditions of 4 ° C. and 200 V until the bromophenol blue front marker moved to a predetermined position (electrophoresis time was about 1 hour). Immediately after completion of electrophoresis, it was subjected to activity staining.
[0073]
Activity staining method of various proteins (enzymes)
Activity staining of various isozymes was incubated at 25 ° C. using the following staining method.
[0074]
(i) Acid phosphatase (ACP)
50 mg of Fast blue RR salt was dissolved in 50 ml of 0.15 M acetic acid-NaOH buffer (pH 5.0), and insoluble matters were removed by filtration. In this solution, 50 mg of α-naphthyl phosphate · 2Na as a substrate was dissolved to stain the gel. When the staining solution became cloudy, it was replaced with the same amount of staining solution.
[0075]
(ii) Alkaline phosphatase (AKP)
0.05M Tris-NaOH-HCl buffer (pH8.7) 50ml, 0.5M MgCl₂ 300 μl, and 0.25M MnCl₂600 μl was added to dissolve 50 mg of Fast blue RR salt. After insoluble matter was removed by filtration, 50 mg of α-naphthyl phosphate · 2Na was dissolved and used for staining. When the staining solution became cloudy, it was replaced with the same amount of new staining solution.
[0076]
(iii) Esterase (EST)
50 mg of Fast blue RR salt was dissolved in 50 ml of 0.05 M Tris-HCl buffer (pH 7.1), and insoluble matters were removed by filtration. Immediately before staining, 1.5 ml of 1% α-naphthylacetic acid and 1% β-naphthylacetic acid (both dissolved in 50% acetone solution) were added. The gel after electrophoresis was immersed in this staining solution and stained. When turbidity was observed in the staining solution, it was replaced with a new staining solution 50 ml, and further staining was performed.
[0077]
(iv) Glutamate-oxaloacetate transaminase (GOT)
To 30 ml of 0.2M Tris-HCl buffer (containing 0.04% EDTA, pH 8.0), add 100 mg of L-aspartic acid, 1 ml of 10% 2-ketoglutaric acid solution, and 1.5 ml of 0.33% pyridoxal phosphate solution, and add 1M Tris solution. Was used to adjust the pH to 8.0. The gel after the electrophoresis was immersed in this solution at 25 ° C. for 30 minutes. A solution obtained by dissolving 100 mg of Fast blue BB salt in 20 ml of Tris-HCl buffer (containing 0.04% EDTA) and filtering insoluble matter was prepared, added to the previous solution, and stained.
[0078]
(v) Glucose-6-Phosphate dehydrogenase (G6PD)
0.2M Tris-HCl buffer (containing 0.04% EDTA, pH 8.0) in 50ml, 0.5% MgCl₂1 ml of solution, 1 ml of 0.67% NADP solution, 1 ml of 0.5% MTT tetrazorium, and 1 ml of 1.25% glucose-6-phosphate solution were added and stirred well. Just before use, 0.5% PMS (Phenazine
200 μl of methosulphate solution was added and used for staining.
(vi) Hexokinase (HK)
[0079]
Glucose 0.5g is dissolved in 50ml of 0.2M Tris-HCl buffer (containing 0.04% EDTA, pH 8.0), and 0.5M MgCl₂1 ml of solution, 1 ml of 10% ATP solution, 1 ml of 0.67% NADP solution and 1 ml of 1% NAD solution were added and stirred well. Immediately before use, 17 units of glucose-6-phosphate dehydrogenase, 1 ml of 0.5% MTT tetrazolium solution, and 200 μl of 0.5% PMS solution were added for staining.
[0080]
(vii) Leucine aminopeptidase (LAP)
0.2M Tris-Malate buffer (pH5.5) 50ml, 0.5% MgCl₂5 ml of the solution and 20 mg of L-Leucyl-β-naphthylamide were added. A solution prepared by dissolving 30 mg of Fast black K salt in 5 ml of Tris-Malate buffer in advance was added immediately before use and stained.
[0081]
(viii) Malate dehydrogenase (MDH)
50 ml of 0.05M Tris-HCl buffer (pH 7.0), 5 ml of 1M DL-malic acid solution (dissolved in 0.98M sodium carbonate solution), 1 ml of 2.5% NAD solution, and 1 ml of 1% NBT (Nitro Blue Tetrazolium) solution Each was added and stirred well. Immediately before staining, 200 μl of 0.5% PMS solution was added to this solution for staining.
[0082]
Analysis of zymogram
The position of each band was recorded by measuring the migration distance and width of each band using calipers. Furthermore, the Rf value of each band was calculated from the movement distance of BPB, which is the front marker, by the following formula.
Rf = (Distance from the starting point of electrophoresis to the center of each band) / (Distance from the starting point of electrophoresis to the front line)
[0083]
The density of the band was visually evaluated in three stages. Each electrophoretic diagram was prepared based on a photograph taken by placing the stained gel on a light box.
[0084]
Results of isozyme analysis
Isozyme analysis was performed on eight enzyme systems (EST, ACP, AKP, MDH, G6PD, GOT, HK, LAP) using a cell-free extract of G. margarita MAFF520054 spores. The zymogram obtained from the isozyme analysis is shown in FIG.
[0085]
As a result, a thin band was obtained with an enzyme system other than ACP, and discrimination was barely possible. In this example, about 15 spores were used for the analysis of one enzyme, but since the absolute amount of enzyme that can be recovered with this number of spores was very small, only a thin band was obtained. It was found that about 20 to several tens of spores are required to obtain a sharper band. To identify VA mycorrhizal fungi by isozyme analysis at the level of a single spore, inoculate the host plant with a single spore and perform pot cultivation, etc., to grow a large number of genetically identical spores (clone). It was carried out in advance, and it was found that it was necessary to prepare a cell-free extract using a large number of clones obtained in this way and to use it for isozyme analysis.
[0086]
Nucleic acid analysis
Next, nucleic acid analysis was performed.
VA mycorrhizal fungi and Gigaspora margarita MAFF520054 strain were used as known bacteria. First, G. The base sequence of margarita MAFF520054 strain ITS region and 5.8S rDNA was determined, a molecular phylogenetic tree was created together with the existing sequences, and it was examined whether there was any distribution specificity in the phylogenetic tree between the lines. FIG. 2 shows a molecular phylogenetic tree based on the 5.8S rDNA of the genus Gigaspora and the base sequence of the ITS region. In FIG. 2, m is G. margarita, r is G. rosea, 1 is a single spore clone, 10 is a 10 spore clone, C, A, and K are G. margarita MAFF520054, G. margarita Ni-A, G. margarita MAFF520052 are shown.
[0087]
Next, for the purpose of detecting a base sequence specific to the genomic DNA of G. margarita MAFF520054 strain, RAPD analysis is performed using a commercially available random primer having a repetitive sequence found in the genus Scutellospora at the 3′-end. In addition, several new primers were designed by determining the base sequence of the band with high reproducibility. Furthermore, PCR was performed using a primer such as a repetitive sequence or minisatellite described in the previous literature, or a primer newly designed based on the base sequence of the primer. Two primers were selected from the above primers, PCR was performed, and a primer set for detecting a band specific to the G. margarita MAFF520054 strain was searched. Specifically, it was performed as follows.
[0088]
Primer 639R Design and primer set M 13/639 About R
(1) A commercially available 12-base random primer having a Scutellospora repeat sequence at the 3′-end was selected, and RAPD was performed with a single primer. Among these, relatively reproducible bands were extracted and their base sequences were determined. Among these, sense / antisense primers were designed in each of 6 sequences of 500 bp or more (639F / R, 714F / R, 621F / R, 641F / R, 581F / R, 1014F / R). With 621F / R, no amplification product was obtained from the G. margarita MAFF520054 strain. The other primer sets were not specific to the G. margarita MAFF520054 strain, but due to the high reproducibility of the PCR products, the sequences obtained here were expected to be present in the genome of each G. margarita strain.
[0089]
(2) Among the various primers in the existing literature, M13 minisatellite primer and (GACA) 4 primer are major bands with relatively good reproducibility, although the overall RAPD pattern varies from G. margarita MAFF520054. Produced. The former has already been used for G. margarita in previous literature. Even when these primers were used alone, it was not possible to discriminate between strains, but it was expected that the sequences obtained here were present in the genome of each strain of G. margarita.
(3) Up to this point, the 10 primers designed in M13 minisatellite, (GACA) 4, ITS1, ITS4 and (1) were expected to correspond to sequences present in the G. margarita genome. These were combined two by two for PCR. As a result, a band specific to the 235 bp G. margarita MAFF520054 strain was formed only when M13 and 639R were combined (FIG. 3).
[0090]
Example 2
Next, nucleic acid analysis was performed by detecting a complementary base sequence for an unknown fungus using a DNA fragment partially or entirely complementary to the base sequence specific to a known fungus as a probe. .
[0091]
Based on the specific base sequence of 235 bp obtained in Example 1, a probe (230PBC) was designed.
[0092]
Specifically, the DNA sequence of the PCR product by M13 / 639R was determined, and homology search was performed on the DDBJ / EMBL / GenBank database using the FASTA program. As a result, a region (45 bases) having a low homology with a known DNA sequence was selected over a relatively wide range and used as a probe region. This sequence was artificially synthesized (BEX Co., Ltd.) and labeled with an ECL 3 ′ oligo labeling kit (Amersham Pharmacia Co., Ltd.) or DIG oligonucleotide tailing kit (Roche Diagnostics) (FIG. 4).
[0093]
G. margarita NAFF520054, G.M. margarita Ni-A, G. margarita NAFF520052, G.M. Using DNA extracted from spores of margarita INVAM and G. margarita Nepal as a template, PCR using M13 / 639R primer set and probing with 230PBC were performed. PCR and probing were also performed on spore DNA of the same Serakinkon (G. margarita sp. CK, Central Glass Co., Ltd.) as the G. margarita MAFF520054 strain. As a result, G. It hybridized with a 235 bp band of margarita NAFF520054 strain and Serakinkon (G. margarita sp. CK) spores (FIGS. 5 and 6). G. margarita NAFF520054 is a strain contained in the VA mycorrhizal fungus inoculum produced by Central Glass Co., Ltd., and is a laboratory strain that was transferred to a livestock pasture test site in 1992 and then subcultured by pot culture. G. G. contained in the same material as margarita NAFF520054. Although margarita spores are the same strain, G. margarita sp. CK strain was introduced as a material in the planting site for convenience. Treated separately from margarita NAFF520054. As is clear from this example, 230PBC hybridized to a 235 bp band for both MAFF520054 and CK strains that had been subjected to repeated generation changes for 8 years, so this nucleotide sequence was sufficiently stable for practical use. Turned out to be.
[0094]
Therefore, if the probe is used, The margarita NAFF520054 and sp. CK strains can be specifically detected, and even when the band detected by electrophoresis after PCR is unclear, the specific target fungi can be easily detected. It can be seen that it can be identified.
[0095]
Example 3
Next, before performing isozyme analysis and nucleic acid analysis, fungi were identified after sorting by morphological characteristics and histochemical characteristics.
[0096]
Morphological features
As the test strains, those described in Table 1 were used as in Example 1. First, we compared the morphological characteristics of VA mycorrhizal fungi and inoculated VA mycorrhizal fungi colonized in the Unzen Fugendake pyroclastic flow site test area.
[0097]
In order to collect indigenous VA mycorrhizal spores, a test zone was set up in a mountainous area in the Nakao River basin of Unzen Fugendake. Arbitrary 10 plants that invaded and grown in the test area were selected, and VA mycorrhizal spores contained in the rhizosphere soil were identified, and analysis of the collected plant roots for VA mycorrhizal fungal infection was performed. Each VA mycorrhizal spore was arbitrarily selected, and the morphological characteristics of each spore were observed under a stereomicroscope.
[0098]
When VA mycorrhizal spores detected from the collected soil were classified based on morphological characteristics, they were classified into five types. It is considered that these VA mycorrhizal fungi invaded and propagated to the host plant by intrusion from the surroundings of the pyroclastic flow site due to wind scattering or rainwater inflow. The detected spore was classified into either Acaulosporaceae or Glomaceae, and its diameter was 200 μm or less.
[0099]
In general, spores belonging to Gigasporaceaae are larger than those of Glomaceae and Acaulosporaceae, and the diameter is said to be 300 μm or more (FIG. 7). FIG. 7 shows the morphological characteristics of various VA mycorrhizal spore. In FIG. 7, a represents Gigaspora margarita MAFF520054, b represents Gigaspora margarita MAFF520052, c represents Gigaspora margarita Ni-A, d represents Glomus intraradices, and e represents Acaulospora longula MAFF520060.
[0100]
The average diameter of the spores of G. margarita MAFF520054 and the same kind of NAFF520052 and Ni-A targeted in this study was over 350 μm. The color of the spores was White-pale yallow in all three strains, the morphology was spherical, and there was no significant difference in the morphological characteristics except for the size (FIGS. 7a-c).
[0101]
Such selection by morphological characteristics can efficiently exclude bacteria of a form clearly different from the G. margarita MAFF520054 strain from the detection target, reduce the number of isozyme analysis and nucleic acid analysis, and detect the detection time. It was found that can be shortened.
[0102]
Histochemical characteristics
Next, the histochemical characteristics were examined in the same manner. As the test strains, those described in Table 1 were used as in Example 1.
[0103]
Qualitative analysis of the spore walls of several VA mycorrhizal fungi, including G. margarita, and the surface sugar chain composition of Subtending hyphae using a sugar chain recognition probe, as well as autofluorescence Was done. Based on the above analysis, not only was a histochemical marker specific to the G. margarita MAFF520054 strain searched, but the presence or absence of effective histochemical characteristics in the identification of the test VA mycorrhizal fungi was also verified.
[0104]
For the qualitative analysis of the spore walls of VA mycorrhizal fungi and the surface layer of Subtending hyphae, six types of glycan recognition probes were used, and fluorescence associated with binding was measured using a fluorescence microscope (OLYMPUS BX-FLA). The occurrence was observed. Autofluorescence is the same for U excitation (excitation filter, BP330-385; dichroic mirror, DM400; absorption filter, BA420), IB excitation (BP400-410, DM455, BA455) and IG excitation (BP460-490, DM505, BA515IF). And observed.
[0105]
FIG. 8 is a diagram showing the autofluorescence of spores of Gigaspora margarita MAFF520054. In FIG. 8, a indicates transmitted light, b indicates U excitation, c indicates IB excitation, and d indicates IG excitation. C represents the spore content, S represents the subtending hyphae, and W represents the spore wall. The spore wall of the G. margarita MAFF520054 strain, which is the target of specific detection utilizing histochemical characteristics, showed strong autofluorescence under U, IB and IG excitation (FIG. 8). Furthermore, Subtending hyphae and spore contents also showed autofluorescence. Similar phenomena were observed in other G. margarita 2 strains, G. rosea, and Scuttellospora cerradensis of the same family (FIG. 9). FIG. 9 shows autofluorescence of Gigasporaceae spores. In FIG. 9, a to d indicate transmitted light (a), U excitation (b), IB excitation (c), and IG excitation (d) of Gigaspora rosea MAFF520062. For ef, transmission light (e) and U excitation (f) of Gigaspora margarita MAFF520052 are shown. About g-h, the transmitted light (g) and U excitation (h) of Scutellospora cerradensis MAFF520056 are shown. C represents the spore contents, S represents the subtending hyphae, and W represents the spore wall.
[0106]
In contrast, in Glomaceae and Acaulosporaceae, autofluorescence was observed in the spore wall and subtending hyphae, but not in the spore contents (FIG. 10). FIG. 10 shows the autofluorescence of the spores of Glomaceae and Acaulosporaceae. In FIG. 10, a to d indicate the transmitted light (a), U excitation (b), IB excitation (c), and IG excitation (d) of Glomus clarum-like MAFF520061. For e-h, Glomus intradices transmitted light (e), U excitation (f), IB excitation (g), and IG excitation (h) are shown. For ij, the transmitted light (i) and U excitation (j) of Glomus etunicatum MAFF520053 are shown. For kl, the transmitted light (i) and U excitation (j) of Acaulospora longula MAFF520060 are shown. C represents the spore contents, S represents the subtending hyphae, and W represents the spore wall.
[0107]
This result is limited to the 9 Glomales strains tested this time, but the autofluorescence of the spore contents suggested that it can be used as an effective indicator to distinguish between Gigasparaceae containing the target VA mycorrhizal fungus and Glomaceae and Acaulosporaceae .
[0108]
On the other hand, CW, which is a sugar chain recognition probe, was reactive to the spore wall and subtending hyphae of FITC-binding lectin, but CW did not bind to the spore wall of G. margarita MAFF520054 and other Gigasporacaeae. The results are shown in FIG. FIG. 11 is a diagram showing the reaction of CW and WGA to the spore wall and subtending hyphae of G. margarita MAFF520054. In FIG. 11, a represents the CW reaction (U excitation). CW bound to subtending hyphae and emitted blue fluorescence (arrow). b shows observation with transmitted light in the same field of view as a. c shows WGA reaction (IB excitation). WGA bound to subtending hyphae and emitted green fluorescence (arrow). On the other hand, WGA did not react with the surface layer of the spore wall and only showed autofluorescence. In addition, the other four FITC-binding lectins were not reactive against the spore wall surface of Gigaspolaceae. On the other hand, CW and WGA bound to G. margarita MAFF520054, the other two lines, and sundending hyphae of G. rosea MAFF520062 (FIG. 11).
[0109]
FIG. 12 shows the reaction of ConA to the spore wall and subtending hyphae of Gigaspora rosea MAFF520062. In FIG. 12, a indicates green fluorescence associated with the binding of Con A. Green particles (arrows) were attached to the periphery of Suntenging hyphae (IB excitation). b shows observation with transmitted light in the same field of view as a. In G. rosea subtending hyphae, particulate green fluorescence due to the binding of ConA was observed at the periphery of the mycelium (FIG. 12).
[0110]
FIG. 13 shows the reaction of CW and FITC-binding lectins on the spore wall and subtending hyphae of Glomaceae. ab is Glomus clarum-like MAFF520061. a shows the reaction of CW to the spore wall and subtending hyphae (arrow). b shows the response of WGA to the spore wall. WGA was bound to the first layer of the spore wall and emitted green fluorescence (arrow). cd is Glomus intraradices. c shows the reaction of CW to the spore wall and subtending hyphae (arrow). The granular substance (arrow) adhering to the periphery of the mycelium emitted green fluorescence. In Glomaceae strain 3, CW bound to the spore wall and Subtending hyphae (FIGS. 13a, c). WGA bound only to the spore wall of G. clarum-like MAFF520061 (FIG. 13b). The reactivity of various FITC lectins to subtending hyphae was different depending on the species, and the binding was limited to the mycelia. (FIG. 13d).
[0111]
In this analysis, it was not possible to find a specific marker of the target VA mycorrhizal fungus G. margarita MAFF520054 by using the six sugar chain recognition probes used. However, the reactivity of CW and WGA to the spore wall was limited to the range of the test bacteria, but it was found that it can be used for differentiation at the family level and identification of specific species.
[0112]
And it was confirmed whether fungi could be roughly selected by utilizing histochemical characteristics peculiar to such fungi.
[0113]
Specifically, after performing the selection based on the above-mentioned histochemical characteristics, the same isozyme analysis and nucleic acid analysis as in Example 1 were performed. As a result, fungi having clearly different histochemical characteristics could be efficiently excluded from detection targets.
[0114]
It was found that selection by histochemical characteristics can narrow down target fungi, reduce the number of isozyme analysis and nucleic acid analysis, and shorten detection time.
[0115]
Example 4
Next, we investigated the behavior of VA mycorrhizal fungi already administered as microbial materials in the greening area of Unzen Fugendake. That is, it was investigated whether VA mycorrhizal fungi administered to plants growing in the construction area were infected and established.
[0116]
At the site of Unzen Fugendake Mizunagawa pyroclastic flow where G. margarita sp. CK strain was introduced together with plant seeds during construction in 1997, plants were collected in March 2001 to extract VA mycorrhizal spore from rhizosphere soil did. In addition to counting the number of spores per plant, we attempted to select and identify G. margarita sp. CK strains by morphological, histochemical and nucleic acid analysis.
[0117]
Spores that seemed to be Gigaspora-like were selected from the rhizosphere soil of 2 individuals out of 10 weeping lovegrass individuals collected from the planting area (Table 2, FIG. 14). In FIG. 14, A represents a Gigaspora-like spore, B represents a frequency distribution of the diameter of the sib, C represents a spore other than the Gigaspora-like, and D represents a frequency distribution of the diameter of the spore. In FIG. 14A, a bulbous suspensor (indicated by an arrow) characteristic of Gifasporaceae is observed. 24 Gigaspora-like spores were collected from one WLG, and 10 spores were subjected to PCR and probing. Regarding the 8 spores from which DNA was obtained, a 235 bp band hybridizing with 230 PBC was obtained (FIG. 15). In the upper electrophoresis diagram of FIG. 15, the arrow indicates the band of the ITS region amplification product. The same band was not detected in C and f spores (indicated by *). For the remaining 8 spores, the presence of a band indicated the presence of DNA. The lower electrophoretic diagram of FIG. 15 shows the result of Southern hybridization using 230PBC. About 8 spores from which DNA was obtained, a 235 bp band (arrow) hybridizing with 230 PBC was obtained.
[0118]
[Table 2]

[0119]
By the above analysis such as nucleic acid analysis, the identity of the VA mycorrhizal fungus collected from the soil at the greening site and the inoculum was confirmed.
Therefore, it was confirmed that the VA mycorrhizal fungi of a specific strain could be quickly identified by the above-described analysis method, and it was found that the behavior of the inoculated VA mycorrhizal fungi in the soil at the greening site could be grasped.
[0120]
Therefore, by periodically comparing indigenous bacteria and inoculum, it can be seen that even if various bacteria are mixed, it is possible to screen for specific bacteria. It was found that the behavior of can be grasped.
[0121]
【The invention's effect】
According to the fungus identification method of the present invention, there is an advantageous effect that fungi between different strains within the same species can be identified.
[0122]
In addition, according to the fungal identification method of the present invention, the identity of the fungus can be determined with higher accuracy by isozyme analysis and nucleic acid analysis, and as a result, the behavior of the fungus when inoculated on the soil is periodically grasped. There is an advantageous effect that it can also be performed.
[0123]
[Sequence Listing]
<110> University of Yamaguchi
<120> Method for identifying fungi
<130> U2001P063
<160> 2
<210> 1
<211> 235
<212> Nucleic acid
<213> Gigaspora margarita C strain
<400> 1
gagggtggcg gttcttttat gacaacaata acaaaaagac caagaaagat ttataactcc 60
gaaagtggtt tctctcaaat aactaccgca atacttacat tctattcaat agtaaaagga 120
gggcggagta taacgacttc tacttacaac atctccatca tcatctaacc atttatcgca 180
tttttcgcag tagcgagtgt attgccaagt tgtggggtca tcttggcatt aggta 235
<210> 2
<211> 45
<212> Nucleic acid
<213> Gigaspora margaritaMAFF520054
<400> 2
ctaaccattt atcgcatttt tcgcagtagc gagtgtattg ccaag 45
[Brief description of the drawings]
FIG. 1 is a diagram showing zymograms obtained from isozyme analysis using various enzymes.
FIG. 2 is a diagram showing a molecular phylogenetic tree based on the 5.8S rDNA of the genus Gigaspora and the base sequence of the ITS region.
FIG. 3 shows comparison of PCR products of various spores using M13 / 639R primer.
FIG. 4 is a diagram showing an example of a probe for identifying G. margarita MAFF520054 strain.
FIG. 5 is a diagram showing hybridization between a probe and a PCR product derived from each fungus.
FIG. 6 is a diagram showing hybridization between a probe and a PCR product derived from each fungus.
FIG. 7 is a diagram showing morphological characteristics of various VA mycorrhizal mycosis spores.
FIG. 8 shows the autofluorescence of spores of G. margarita MAFF520054.
FIG. 9 shows autofluorescence of various spores of Gigasporaceae.
FIG. 10 shows autofluorescence of spores of Glomaceae and Acaulosporaceae.
FIG. 11 shows the reaction of CW and WGA to the spore wall and subtending hyphae of G. margarita MAFF520054.
FIG. 12 is a diagram showing the reaction of Con A on the spore wall and subtending hyphae of Gigaspora rosea MAFF520062.
FIG. 13 shows the reaction of CW and FITC-binding lectins on spore walls and subtending hyphae of Glomaceae.
FIG. 14 is a diagram showing the characteristics of VA mycorrhizal spores collected from a planting construction zone into which VA mycorrhizal fungi have been introduced.
FIG. 15 is a diagram showing hybridization between a probe and a PCR product derived from VA mycorrhizal spore collected from soil in a greening section.

Claims (12)

VA mycorrhizal fungus and unknown fungus, isozyme analysis, and nucleic acid analysis to identify the fungus by comparing both fungi ,
A method for identifying a fungus, wherein the nucleic acid analysis is an investigation of the presence or absence of a base sequence represented by base numbers 1-235 of SEQ ID NO: 1 in the sequence listing for the unknown fungus. In the nucleic acid analysis, a DNA fragment having a base sequence partially or entirely complementary to the base sequence represented by base numbers 1-235 of SEQ ID NO: 1 in the sequence listing is used as a probe, and the base sequence complementary to the probe is 2. The method for identifying a fungus according to claim 1, wherein the method is an analysis of whether or not it is present in an unknown fungus. For the unknown fungus, investigation of the presence or absence of the base sequence represented by base numbers 1-235 of SEQ ID NO: 1 in the sequence listing was performed. Based on the base sequence, a primer that hybridizes to the genomic DNA of VA mycorrhizal fungi was designed. And detecting a base sequence constituting a band specific to an unknown fungus with good reproducibility by performing PCR with a combination of several of the primers and known primers. 3. The method according to item 1 or 2 . Analysis of the nucleotide sequence complementary, identification method according to claim 2 or 3, wherein performing the hybridization to the nucleotide sequence constituting the specific bands on the unknown fungus and the probe. The method according to any one of claims 2 to 4, wherein the probe comprises a base sequence represented by base numbers 1-45 of SEQ ID NO: 2 in the sequence listing. 6. The method according to any one of claims 1 to 5 , wherein the fungus is identified as belonging to the same species. The method according to any one of claims 1 to 6 , wherein the fungus is identified as belonging to the same strain. Isozymes are acid phosphatase (ACP), alkaline phosphatase (AKP), esterase (EST), glutamate-oxaloacetate transaminase (GOT), hexokinase (HK), leucine aminopeptidase (LAP), malate dehydrogenase (MDH) ), Alanine aminopeptidase (AAP), aspartate aminotransferase (AAT), aconitase (ACO), alcohol dehydrogenase (ADH), amylase (AMY), diaphorase (DIA), fumarase (FM), glutamate dehydrogenase (GDH), glycerate dehydrogenase (G2D), glucokinase (GK), glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase (GR), malate enzyme pH 8.0 (ME8), malate Enzyme pH 7.0 (ME7), Menage Onreductase (MNR), 6-phosphogluconate dehydrogenase (6PGD), phosphoglucose isomerase (PGI), phosphoglucomutase (PGM), peroxidase (POD), shikimate dehydrogenase (SKD), sorbitol dehydrogenase enzyme (SODH), triose phosphate isomerase (TPI), and tetrazolium oxidase according to any one of claims 1 to paragraph 7, characterized in that at least one member selected from the group consisting of (TZO) Method. The method according to any one of claims 1 to 8 , wherein the isozyme analysis and nucleic acid analysis are performed after selecting fungi based on morphological characteristics. Morphological features are zygospore, gamete, zoospore, zoosporangia, dormant spore, thick membrane, gamete, spore, spore, conidia, spore, spore, Temporary root, microspore, hyphae, pseudozygous spore, pupa, pupa, pupa, spore, basidiospore, basidiode, conidia, conidia stalk, conidia, clamp formation, septum The method according to claim 9 , wherein the method is characterized by at least one size and / or form selected from the group consisting of mycorrhiza. The method according to any one of claims 1 to 10 , wherein the isozyme analysis and the nucleic acid analysis are performed after selecting fungi based on histochemical characteristics. The method according to claim 11 , wherein the histochemical characteristic is autofluorescence and / or the presence of a sugar chain.

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