【０００１】
【産業上の利用分野】
アブラナ科作物はハクサイ、キャベツ等に代表される重要な野菜として大量に生産されている。生産効率の追求、産地の指定化等により同一作物を長年にわたって栽培する連作が日常的に行われてきた。
【０００２】
【従来の技術】
その結果連作による弊害として土壌病害、要素欠乏症などが大きな問題となっている。そこでアブラナ科作物の根こぶ病の防除にはＰＣＮＢ（ペンタクロルニトロベンゼン）、ネビジン等の化学農薬散布、アルカリ資材の投入による土壌ｐＨの調整、品種改良による抵抗性品種の作出等の対策が採られている。
【０００３】
【発明が解決しようとする課題】
しかしながら、化学農薬の使用は耐性菌の出現等で効果が不十分になってきた。更に残留毒性、散布時の吸入による障害などの問題が起こり、使用は限定されるようになっている。加えて消費者においても化学農薬に対する嫌悪感は増加する一方であり、化学農薬を使用した野菜もまた敬遠される様になってきた。また、土壌ｐＨの調整では充分な効果が得られず、抵抗性品種の作出も多大な時間を要するため幼苗価格が高価となるに加えて、舌ざわり、味もこれまでのものと微妙に異なる。
【０００４】
近年、微生物を用いる土壌病害防除方法が開発され、注目を集めバチルス・ズブチルスＳＣ−３を用いて植物病害を防除する方法（特開平５−５１３０５号公報）、バチルス・スフェリカスの芽胞含有培養物を用いた野菜類病害防除剤（特許第２５２６３６５号公報）が提案されている。本発明は後に詳述する特定の微生物を用いてアブラナ科根こぶ病を防除することを目的とするものである。
【０００５】
尚、本発明と前記特開平５−５１３０５号公報記載の菌とは、例えば分類学的性質に於いて食塩濃度での生育、Ｖ−Ｐ試験のｐＨが異なり、また病害防除に於いてはバチルス・ズブチルスＴ２はうどんこ病等の地上部病害に対しては効果がなく両者は全く別の菌株である。
【０００６】
【課題を解決するための手段】
即ち、本発明はバチルス・ズブチルスＴ２（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ Ｔ２）を含有してなるアブラナ科根こぶ病防除材に関し、またバチルス・ズブチルスＴ２を１０3ｃｆｕ／ｇ以上含有した培土からなるアブラナ科根こぶ病防除材に関し、更にまたバチルス・ズブチルスＴ２をアルギン酸のアルカリ金属塩とカルシウム化合物とで固定化した固定化菌を用いたアブラナ科根こぶ病防除材に関する。
【０００７】
【発明の実施の形態】
本発明に使用するバチルス・ズブチルスＴ２は兵庫県加古川市別府町の圃場から分離した微生物であり、菌株コード番号をＴ２と命名した。この本発明菌は平成１０年４月１４日付で生命研寄託番号「Ｎｏ ＦＥＲＭ Ｐ−１６７５９号」として寄託されている。表１に本発明バチルス・ズブチルスＴ２（以下本発明菌という）の分類学的性状を示す。
【０００８】
【表１】
【０００９】
本発明菌の培養は一般的細菌における通常の培養方法に従って行えば良く、固体培養あるいは振とう培養、ジャーファメンター培養等の液体培養のいずれでも利用することができる。
【００１０】
本発明の培養培地としては通常細菌に使用され培養培地で良く、具体的には糖質としてはグルコース、蔗糖、果糖、デンプン等、窒素源としては肉エキス、ペプトンの他に酵母エキス、大豆粉末、硝酸ナトリウム、硝酸アンモニウム、硫酸アンモニウム等を利用することができ、また生育促進効果を持つエキス類として麦芽エキス、ジャガイモ煎汁等も用いることができる。さらに金属塩の補給、ｐＨ或いは浸透圧調整に塩化ナトリウム、りん酸カリウム塩、マグネシウム塩等の無機塩類を加えても良い。培養温度は２０〜５０℃、好ましくは２５〜４０℃であれば良く、好気的培養であれば振とう、通気攪拌等の手段は問わない。ｐＨは５．５〜８．０好ましくは６．５〜７．５の範囲であれば良い。また固体培養を行った後殺菌水を添加・混合し菌懸濁液とすることもできる。
【００１１】
本発明の使用方法について云えば、本発明菌それ自体、あるいは培養液をそのまま使用することもできるが、一般的には固体あるいは液体担体と混合して使用することが望ましい。固体担体としてはカオリン、タルク、バーミキュライト、珪藻土、モンモリロナイト等の天然鉱物微粉末、合成微粉末シリカ、活性炭、炭酸カルシウム、酸性白土、酸化カルシウム等の人工鉱物微粉末、発泡ポリスチレン、発泡ポリエチレン、発泡ポリプロピレン、ポリウレタン等多孔性プラスチック等を使用することができる。また液体担体としては水、特に滅菌水が最も望ましい。
【００１２】
さて、本発明者らが固体担体と共に使用する別法として、最も望ましい使用形態として推奨する方法は、本発明菌を育苗培土と混合して使用する方法である。育苗培土としては、バーミキュライト、ピートモス、腐植土、山土、あるいは市販育苗培土をそのまま好ましくは通気性、通水性、保水性を考慮して混合使用することもできるが、更に望ましくは１００〜２００℃の範囲で０．５〜１時間焼成した焼成培土を使用することである。焼成することにより培土中の雑菌を死滅させ無菌化することにより、本発明菌を容易に増殖させることができる。また更に殖増を推進するために糖質等の炭素源、肉エキス、麦芽エキス等の生育促進剤を培土に添加混合しておくと良い。
【００１３】
このように調製した培土に本発明菌を混合し、無菌室で菌濃度が１０3ｃｆｕ／ｇ以上になるまで培養する。
培養は通常１５〜３０℃で１週間以上行えば良い。１０3ｃｆｕ／ｇ以上とする理由は、このような培土に播種して育苗したアブラナ科植物は根こぶ病発病率が極めて低いことによる。
【００１４】
次に更に望ましい育苗培土の製造方法について述べる。
先ず、本発明菌濃度が１０4ｃｆｕ／ｇ、好ましくは１０6ｃｆｕ／ｇ以上となるように本発明菌をアルギン酸のアルカリ金属塩水溶液に懸濁させる。
このときアルギン酸のアルカリ金属塩水溶液の濃度は、０．０１〜１重量％の範囲が良い。アルギン酸のアルカリ金属塩の使用割合は育苗培土に対しアルギン酸のアルカリ金属塩として０．０１〜１．０重量％である。この菌体懸濁液を焼成培土との混合により、アルギン酸のアルカリ金属塩は培土表面に存在する二価陽イオンとの置換によって、培土表面に水不溶性膜を形成する。菌体は水不溶性膜と共に培土表面に固定されて増殖する。
【００１５】
しかしながら、更に本発明菌の固定を安定化させるためにはアルギン酸のアルカリ金属塩添加混合後にカルシウム化合物を添加する。カルシウム化合物としては、炭酸カルシウム、塩化カルシウム、硫酸カルシウム、硝酸カルシウム等が好例として挙げられるが、とりわけ炭酸カルシウムが望ましい。カルシウム化合物の添加量は、使用するアルギン酸のアルカリ金属塩の量、本発明菌の量、育苗培土の物性等により異なるが大略育苗培土に対して０．０１〜１．０重量％である。アルギン酸のアルカリ金属塩とカルシウム化合物使用割合は重量で４：１〜１：１０の範囲が望ましい。
【００１６】
本発明に於いて本発明菌を育苗培土に固定化する理由は、灌水等による本発明菌の移動を防ぎ、本発明菌を効率よく利用するためである。尚、本発明菌とアルギン酸のアルカリ金属塩水溶液の混合液を土壌に施用した後、カルシウム化合物を加えて本発明菌を固定化しても良いし、土壌にカルシウム化合物を混合した後、
混合液を加えて本発明菌を固定化しても良い。
【００１７】
さて、以上詳しく述べたように本発明菌は、それ自体または本発明菌培養液自体あるいはこれらを水に懸濁させた懸濁液、更にまたこれらを固体担体と混合した混合物をそのまま土壌に混合することもできるし、散布することもできる。土壌に混合あるいは散布する場合の量としては、例えば使用濃度が１０5cell／ｍｌの場合100ｍｌ／ｍ2〜１０Ｌ／ｍ2、特に好ましくは１Ｌ／ｍ2〜５Ｌ／ｍ2である。
【００１８】
本発明菌濃度の調製は、水希釈により容易に行うことができる。土壌へ直接適用する場合、散布回数は植物の生育状況により１回〜５回行えば良い。また別法として本発明菌培養液に種子を浸漬して利用することもできる。かかる場合に於ける培養液中の菌濃度としては種子の種類により異なるが１０3ｃｆｕ／ｇ以上あれば良く、種子濃度としては１〜５０重量％が良い。浸漬時間は１〜２時間で充分である。浸漬温度は５〜２５℃が良い。
【００１９】
しかしながら、本発明菌がその効果を最も良く発揮するのは、先に記載した如く本発明菌を混合した培土または本発明菌を固定化した培土に播種し、育苗後定植する方法である。
【００２０】
本発明の対象とする植物はアブラナ科植物であるが、具体的には、キャベツ、チンゲンサイ、蕪、小松菜、ブロッコリー、水菜、コールラビ（蕪甘藍）、高菜、山東菜、壬生菜、からし菜、タアサイ、カリフラワー、体菜等であるがこれらに限定されるものではない。以下に実施例を記し、本発明を更に詳述する。
【００２１】
【実施例】
根こぶ病汚染土の調製
根こぶ病に感染したハクサイ肥大根に等量の水を加え磨砕し、２重のガ−ゼで濾す。さらにろ液を2500rpmで5分間遠心分離する。遠心分離後上澄液を捨て、沈殿を少量の殺菌水に懸濁する。この操作を３回繰り返した。こうして調製した休眠胞子懸濁液の休眠胞子数を顕微鏡で計数した。これを休眠胞子数が1.1×106／ｇの乾土になるように加古川沖積土壌に添加・混合し根こぶ病汚染土とした。この根こぶ病汚染土を用いて本発明の根こぶ病防除効果について対照および農薬等と比較を行った。なお以下の実施例でハクサイ等の栽培で「病土」と記載している場合すべてこの根こぶ病汚染土に定植することを示す。
【００２２】
（実施例１）
バ−ミキュライト２容に赤玉土と多木園芸培土（多木物産株式会社製品）を８：１で混合したもの１容を混合した培土22.5kgを150℃で1時間加熱殺菌した後、室温まで放冷した。肉エキス5g／ｌ、ペプトン10g／ｌ、塩化ナトリウム5g／ｌを含有する液体培地で30℃、40時間振盪培養した本発明菌培養液4Lとアルギン酸ナトリウムを１重量％含有した殺菌水3.5Lとを混合し本発明菌−アルギン酸ナトリウム混合液とした。これを上記の培土に加え混合（培土に対しアルギン酸ナトリウムとして0.12重量％）後、炭酸カルシウム１重量％を加えて良く混合し、これを4Lずつビニ−ル袋詰め後脱気し、25℃で1週間熟成し、本発明防除材１、（菌数６．７×１０7ｃｆｕ／ｇ）とした。またパ−ライト１容に真砂土（六甲山系花崗岩風化土）４容、ピ−トモス１容の割合で混合した培土7kgに本発明菌−アルギン酸ナトリウム混合液2.33Ｌを添加し、ついで炭酸カルシウムを１重量％加えた。更に本発明防除資材１と同様に袋詰め・脱気・熟成を行い本発明菌濃度の異なる本発明防除材２（２．４×１０3ｃｆｕ／ｇ）を作製した。また本発明防除材２に用いた培土を使用して本発明菌濃度の異なる本発明防除材３（５．２×１０4ｃｆｕ／ｇ）、本発明防除材４（８．１×１０5ｃｆｕ／ｇ）を作製した。
【００２３】
本発明防除材１〜４を各々128穴セルトレイに充填し、ハクサイ（品種「無双」：タキイ種苗(株)製）を播種した。温室で21日間育苗後病土に20株ずつ定植した。対照区、農薬区の育苗は本発明菌添加前の本発明防除材２の培土を用いた。農薬区はネビジン粉剤30Kg／10a相当量を植穴施用した。施肥等栽培管理は慣行に準じた。定植28日目に感染発病の程度を調査し、その結果を表２に示した。調査の結果発病株率が対照区100％、農薬区30％、本発明防除材１区は5％、本発明防除材２区は３０％、本発明防除材３区は２５％、本発明防除材４区は２０％であった。防除価は農薬区70に対し本発明防除材１〜４区はそれぞれ９５、７０、７５、８０であった。本発明防除材区は根こぶ病汚染土において化学農薬より安定した防除効果を示したことから、ハクサイ根こぶ病防除に有効な微生物資材として評価できる。
【００２４】
【表２】
＊１） 発病株率：発病株数を栽培本数で除した値の百分率
＊２） 防除価：対照区の発病株率から本発明の発病株率を差し引いた値を対照区の発病株率で除した値に１００を乗じた値
【００２５】
（実施例２）
本発明菌培養液（菌数4.2×108cfu／ｇ）を水で菌数5.1×106cfu／ｇに希釈した。これを植穴（直径４ｃｍ）当たり100ml直接病土に施用し、直ちにハクサイの苗（「無双」：21日間育苗）を20株ずつ定植した（直接区）。本発明菌培養液（菌数4.2×108cfu／ｇ）40mlとアルギン酸ナトリウムを１重量％の濃度に溶解した殺菌水35mlとを混合し本発明菌−アルギン酸ナトリウム混合液とした。これを水で希釈し、菌数5.1×106cfu／ｇの希釈液とした。炭酸カルシウム5gを植穴施用した部分へ本発明菌−アルギン酸ナトリウム混合液希釈液100mlを施用し、固定化を行なった。30分以上経過後ハクサイの苗（「無双」：21日間育苗）を20株ずつ定植した（土壌固定化菌体区）。上記の本発明菌−アルギン酸ナトリウム混合液75mlを２重量％の塩化カルシウム溶液200mlに滴下し拮抗菌の固定化を行った。塩化カルシウム溶液中で30分以上固定化を行った後1mm目のステンレス網で固定化菌体を濾過捕集した。
【００２６】
この固定化菌体2mlを植え穴に添加しハクサイの苗（「無双」：21日間育苗）を20株ずつ定植した（固定化菌体区）。対照区は植穴に水100mlを施用した後ハクサイの苗（無双：21日間育苗）を20株ずつ定植した。定植28日目に感染発病の程度を調査し、表３に示した。調査の結果発病株率は対照区100％、直接区は25％、土壌固定化菌体区、固定化菌体区は15％であった。防除価は直接区75、土壌固定化菌体区、固定化菌体区は85であった。本発明はいずれも対照より低い発病株率で、防除効果が認められた。土壌処理の方法でもハクサイ根こぶ病防除に有効な微生物資材として評価できる。
【００２７】
【表３】
【００２８】
（実施例３）
本発明菌培養液（菌数4.2×108cfu／ｇ）とアルギン酸ナトリウムを１重量％の濃度に溶解した殺菌水とを混合し本発明菌−アルギン酸ナトリウム混合液（5.5×１０7ｃｆｕ／ｇ）とした。この混合液100mlにハクサイ（無双）の種子100粒を室温で1時間浸漬した。この種子を取り出し、２重量％の塩化カルシウム溶液200mlに滴下し本発明菌の固定化を行った（固定化菌体区）。また本発明菌培養液希釈液100ml（５．５×１０7ｃｆｕ／ｇ）にハクサイ（無双）の種子100粒を室温で1時間浸漬した（菌体区）。対照として水100mlにハクサイ（無双）の種子100粒を室温で1時間浸漬した（対照区）。以上3種類の処理を行ったハクサイ（無双）の種子を市販野菜用培土に50粒播種し、21日間育苗した。この苗20株ずつを病土に定植し、28日間栽培した。28日後に各処理区の感染発病株率を調べ、表４に示した。調査の結果発病株率が対照区100％、固定化菌体区は15％、菌体区は25％であった。防除価は固定化菌体区85、菌体区75であった。本発明防除材は種子処理でもハクサイ根こぶ病防除に有効な微生物資材として評価できる。
【００２９】
【表４】
【００３０】
（実施例４）
実施例１の本発明防除材１を128穴セルトレイに充填し、これにキャベツ（松波：石井種苗）、ブロッコリ−（ハイツ：タキイ種苗）を播種した。同様に200穴セルトレイに充填後チンゲンサイ（青帝：タキイ種苗）、蕪（金町小かぶ：サカタの種）、晩生小松菜（小林種苗）を播種した。チンゲンサイ、蕪、小松菜は育苗14日目に病土に20株ずつ定植した。キャベツ、ブロッコリ−は育苗21日目に病土に20株ずつ定植した。チンゲンサイ、蕪、小松菜は定植後21日目に、キャベツ、ブロッコリ−は定植後28日目に感染の程度を調査した。また対照区はバーミキュライト２容に赤玉土と多木園芸培土（多木物産株式会社製品）を８：１で混合したもの１容を混合した培土を、育苗培土として使用し各作物を本発明と同期間育苗後、定植し感染の程度を調査した。その結果を表５に示した。いずれの作物においても本発明のほうが対照より低い発病株率で根こぶ病防除効果が認められた。
【００３１】
【表５】
以上から明らかなように、本発明のアブラナ科根こぶ病防除材はハクサイを始め各種のアブラナ科作物の根こぶ病の防除に有効であることが判る。
【００３２】
【発明の効果】
本発明のアブラナ科根こぶ病防除材は化学農薬のような環境上の問題を引き起こすことがなく優れた防除効果を発揮する。[0001]
[Industrial applications]
Brassicaceae crops are produced in large quantities as important vegetables such as Chinese cabbage and cabbage. Continuous crop cultivation for cultivating the same crop for many years has been performed on a daily basis due to the pursuit of production efficiency and the designation of a production area.
[0002]
[Prior art]
As a result, soil diseases and element deficiency have become serious problems as adverse effects of continuous cropping. Therefore, in order to control the clubroot disease of cruciferous crops, measures such as spraying chemical pesticides such as PCNB (pentachloronitrobenzene) and nevidin, adjusting the soil pH by adding alkaline materials, and creating resistant varieties by improving the variety are taken. ing.
[0003]
[Problems to be solved by the invention]
However, the use of chemical pesticides has become insufficiently effective due to the emergence of resistant bacteria. In addition, problems such as residual toxicity and obstacles due to inhalation at the time of spraying occur, and the use thereof has been limited. In addition, consumer aversion to chemical pesticides has been increasing, and vegetables using chemical pesticides have also been shunned. In addition, adjustment of soil pH does not provide a sufficient effect, and the production of resistant varieties requires a lot of time, so that seedling prices are expensive, and tongues and tastes are slightly different from those in the past.
[0004]
In recent years, a method for controlling soil diseases using microorganisms has been developed, and has attracted attention. A method for controlling plant diseases using Bacillus subtilis SC-3 (Japanese Patent Application Laid-Open No. 5-51305), a method for spore-containing culture of Bacillus sphaericus, A vegetable disease controlling agent used (Japanese Patent No. 2526365) has been proposed. An object of the present invention is to control cruciferous clubroot using a specific microorganism described in detail below.
[0005]
The present invention differs from the bacterium described in JP-A-5-51305 in, for example, the growth at a salt concentration in terms of taxonomic properties, the pH in a VP test, and the bacillus in controlling a disease. -Subtilis T2 has no effect on aboveground diseases such as powdery mildew and both are completely different strains.
[0006]
[Means for Solving the Problems]
That is, the present invention relates to a brassica root-knot control material containing Bacillus subtilis T2, and a brassica root-knot disease comprising Bacillus subtilis T2 in an amount of 10 3 cfu / g or more. The present invention further relates to a material for controlling root-knot disease of Brassicaceae using immobilized bacteria obtained by immobilizing Bacillus subtilis T2 with an alkali metal salt of alginic acid and a calcium compound.
[0007]
BEST MODE FOR CARRYING OUT THE INVENTION
Bacillus subtilis T2 used in the present invention is a microorganism isolated from a field in Beppu town, Kakogawa city, Hyogo prefecture, and its strain code number is designated as T2. This bacterium of the present invention was deposited on April 14, 1998 under the deposit number of No. FERM P-16759. Table 1 shows the taxonomic properties of the Bacillus subtilis T2 of the present invention (hereinafter referred to as the present fungus).
[0008]
[Table 1]
[0009]
The cultivation of the bacterium of the present invention may be performed according to a general culturing method for general bacteria, and any of solid culturing, shaking culturing, and liquid culturing such as jar fermenter culturing can be used.
[0010]
The culture medium of the present invention may be a culture medium which is usually used for bacteria and may be a culture medium. Specifically, glucose, sucrose, fructose, starch, etc., as carbohydrates, meat extract, peptone as a nitrogen source, yeast extract, soybean powder, etc. , Sodium nitrate, ammonium nitrate, ammonium sulfate, etc., and malt extract, potato decoction, etc. can also be used as extracts having a growth promoting effect. Further, inorganic salts such as sodium chloride, potassium phosphate, magnesium salt and the like may be added for replenishment of metal salts, adjustment of pH or osmotic pressure. The culturing temperature may be 20 to 50 ° C, preferably 25 to 40 ° C. For aerobic culturing, any means such as shaking and aeration and stirring can be used. The pH may be in the range of 5.5 to 8.0, preferably 6.5 to 7.5. After solid culture, sterile water may be added and mixed to form a bacterial suspension.
[0011]
Regarding the method of use of the present invention, the bacterium of the present invention itself or a culture solution can be used as it is, but it is generally preferable to use it by mixing it with a solid or liquid carrier. As a solid carrier, natural mineral fine powder such as kaolin, talc, vermiculite, diatomaceous earth, montmorillonite, synthetic fine powder silica, activated carbon, calcium carbonate, acid clay, artificial mineral fine powder such as calcium oxide, expanded polystyrene, expanded polyethylene, expanded polypropylene And a porous plastic such as polyurethane. Water, particularly sterile water, is most desirable as the liquid carrier.
[0012]
Now, as another method of using the present invention together with a solid carrier, a method recommended as the most desirable use form is a method of using the microorganism of the present invention in combination with a seedling culture medium. As the seedling cultivation soil, vermiculite, peat moss, humus, mountain soil, or commercially available seedling cultivation soil can be used as it is, preferably in consideration of breathability, water permeability, water retention, and more preferably 100 to 200 ° C. In the range of 0.5 to 1 hour. By calcination to kill and sterilize various bacteria in the soil, the bacteria of the present invention can be easily proliferated. Further, in order to further promote the growth, it is preferable to add and mix a carbon source such as a saccharide and a growth promoter such as a meat extract and a malt extract to the soil.
[0013]
The bacterium of the present invention is mixed with the culture medium thus prepared, and cultured in a sterile room until the bacterium concentration becomes 10 3 cfu / g or more.
The culture may be usually performed at 15 to 30 ° C. for one week or more. The reason for setting it to be 10 3 cfu / g or more is that the brassicaceae plant sown on such a soil and raised for seedling has an extremely low root-knot disease incidence.
[0014]
Next, a more desirable method for producing a seedling culture is described.
First, the bacterium of the present invention is suspended in an aqueous solution of an alkali metal salt of alginic acid so that the concentration of the bacterium of the present invention is 10 4 cfu / g, preferably 10 6 cfu / g or more.
At this time, the concentration of the aqueous alkali metal salt of alginic acid is preferably in the range of 0.01 to 1% by weight. The use ratio of the alkali metal salt of alginic acid is 0.01 to 1.0% by weight as the alkali metal salt of alginic acid based on the seedling culture soil. The suspension of the bacterial cells is mixed with the baked soil, and the alkali metal salt of alginic acid is replaced with a divalent cation present on the surface of the soil to form a water-insoluble film on the surface of the soil. The cells grow on the soil surface together with the water-insoluble membrane.
[0015]
However, in order to further stabilize the fixation of the bacterium of the present invention, a calcium compound is added after the addition and mixing of the alkali metal salt of alginic acid. Preferred examples of the calcium compound include calcium carbonate, calcium chloride, calcium sulfate, and calcium nitrate, and calcium carbonate is particularly desirable. The amount of the calcium compound to be added varies depending on the amount of the alkali metal salt of alginic acid used, the amount of the bacterium of the present invention, the physical properties of the seedling cultivation soil, and the like, but is generally 0.01 to 1.0% by weight based on the seedling cultivation soil. The use ratio of the alkali metal salt of alginic acid and the calcium compound is preferably in the range of 4: 1 to 1:10 by weight.
[0016]
The reason why the fungus of the present invention is immobilized on the seedling cultivation soil in the present invention is to prevent the fungus of the present invention from moving due to irrigation and the like, and to efficiently utilize the fungus of the present invention. Incidentally, after applying a mixed solution of the present bacterium and an aqueous solution of an alkali metal salt of alginic acid to soil, the bacterium of the present invention may be immobilized by adding a calcium compound, or after mixing the calcium compound into the soil,
The microorganism of the present invention may be immobilized by adding a mixed solution.
[0017]
As described in detail above, the bacterium of the present invention is the bacterium itself or the cultivation solution of the bacterium of the present invention itself or a suspension obtained by suspending them in water, and further, a mixture of these and a solid carrier is directly mixed with soil. You can do it or you can spray it. The amount in the case of mixed or sprayed on the soil, for example, when using concentration of 10 5 cell / ml 100ml / m 2 ~10L / m 2, particularly preferably 1L / m 2 ~5L / m 2 .
[0018]
The concentration of the bacterium of the present invention can be easily adjusted by dilution with water. When applied directly to the soil, the frequency of application may be 1 to 5 times depending on the growth condition of the plant. Alternatively, seeds can be used by immersing the seeds in the culture solution of the present invention. In such a case, the concentration of the bacteria in the culture solution varies depending on the type of seed, but may be at least 10 3 cfu / g, and the seed concentration is preferably 1 to 50% by weight. An immersion time of 1-2 hours is sufficient. The immersion temperature is preferably 5 to 25 ° C.
[0019]
However, the best effect of the fungus of the present invention is achieved by a method of sowing seeds in a soil mixed with the fungus of the present invention or a soil immobilized with the fungus of the present invention as described above, followed by planting after raising seedlings.
[0020]
The plants targeted by the present invention are cruciferous plants, and specifically, cabbage, bok choy, turnip, komatsuna, broccoli, mizuna, kohlrabi (bamboo indigo), takana, Shandong rape, Mibu rape, mustard rape, taasai , Cauliflower, body vegetables, etc., but are not limited thereto. Hereinafter, the present invention will be described in more detail with reference to Examples.
[0021]
【Example】
Preparation of clubroot contaminated soil An equal amount of water is added to the Chinese cabbage radish infected with clubroot, ground and filtered with a double gauze. Further, the filtrate is centrifuged at 2500 rpm for 5 minutes. After centrifugation, the supernatant is discarded, and the precipitate is suspended in a small amount of sterile water. This operation was repeated three times. The number of dormant spores of the dormant spore suspension thus prepared was counted under a microscope. This was added to and mixed with the alluvial soil of Kakogawa so that the number of dormant spores was 1.1 × 10 6 / g dry soil, and it was used as a clubroot contaminated soil. Using this clubroot contaminated soil, the control effect of the clubroot of the present invention was compared with that of a control, a pesticide and the like. In the following examples, when the term "pathological soil" is described in cultivation of Chinese cabbage and the like, it means that all the plants are planted on the clubroot contaminated soil.
[0022]
(Example 1)
A mixture of 2 parts of vermiculite and 8 parts of Akadama clay and Taki horticultural cultivation soil (manufactured by Taki Bussan Co., Ltd.) A mixture of 1 volume and 22.5 kg of cultivation soil is sterilized by heating at 150 ° C. for 1 hour and then to room temperature. Allowed to cool. 4 L of the bacterial culture of the present invention cultured in a liquid medium containing 5 g / l of meat extract, 10 g / l of peptone and 5 g / l of sodium chloride at 30 ° C. for 40 hours and 3.5 L of sterilized water containing 1% by weight of sodium alginate To obtain a mixed solution of the present invention-sodium alginate. This is added to the above-mentioned cultivation soil and mixed (0.12% by weight as sodium alginate with respect to the cultivation soil), and then 1% by weight of calcium carbonate is added and mixed well. After aging for one week, it was designated as the controlling material 1 of the present invention (number of bacteria: 6.7 × 10 7 cfu / g). Also, 2.33 L of the fungus-sodium alginate mixed solution of the present invention was added to 7 kg of cultivated soil mixed with 1 volume of pearlite and 4 volumes of masago soil (weathered weathered granite of Rokko Mountains) and 1 volume of peat moss, and then calcium carbonate. 1% by weight was added. Further, bagging, degassing, and aging were performed in the same manner as the control material 1 of the present invention to prepare a control material 2 of the present invention (2.4 × 10 3 cfu / g) having a different concentration of the bacterium of the present invention. In addition, using the cultivation soil used for the present control material 2, the present control material 3 (5.2 × 10 4 cfu / g) and the present control material 4 (8.1 × 10 5 cfu) having different concentrations of the present bacterium. / G).
[0023]
Each of the pest control materials 1 to 4 of the present invention was filled in a 128-well cell tray, and Chinese cabbage (variety “Musou”: manufactured by Takii Seed Co., Ltd.) was sown. After 21 days of raising seedlings in the greenhouse, 20 plants were planted on the diseased soil. Seedlings of the control material 2 of the present invention before addition of the fungus of the present invention were used for raising seedlings in the control plot and the agrochemical plot. In the pesticide plot, 30 kg / 10a equivalent amount of nevidin powder was planted. Cultivation management such as fertilization was in accordance with customary practices. On the 28th day of planting, the extent of infection was investigated, and the results are shown in Table 2. As a result of the investigation, the disease-causing strain rate was 100% in the control plot, 30% in the pesticide plot, 5% in the plot of the control material of the present invention, 5% in the plot of the control composition of the present invention, 30% in the plot of the control composition of the present invention, 25% in the plot of the control composition of the present invention, and 25% in the control plot of the present invention In the four sections, the ratio was 20%. The control value was 95, 70, 75, and 80 in the control materials of the present invention 1 to 4 in the pesticide section 70, respectively. Since the control material section of the present invention showed a more stable control effect than the chemical pesticide on the clubroot contaminated soil, it can be evaluated as an effective microbial material for controlling Chinese cabbage clubroot.
[0024]
[Table 2]
* 1) Diseased strain rate: Percentage of the value obtained by dividing the number of diseased strains by the number of cultivated trees * 2) Control value: The value obtained by subtracting the diseased strain rate of the present invention from the diseased strain rate in the control plot is divided by the diseased strain rate in the control plot. A value obtained by multiplying the calculated value by 100
(Example 2)
The bacterial culture of the present invention (4.2 × 10 8 cfu / g) was diluted with water to 5.1 × 10 6 cfu / g. This was applied directly to the diseased soil in an amount of 100 ml per planting hole (4 cm in diameter), and 20 Chinese cabbage seedlings ("Musou": seedlings raised for 21 days) were immediately planted by 20 plants (direct plot). 40 ml of the bacterial culture of the present invention (4.2 × 10 8 cfu / g of bacteria) and 35 ml of sterilized water in which sodium alginate was dissolved at a concentration of 1% by weight were mixed to obtain a bacterial-sodium alginate mixed solution of the present invention. This was diluted with water to obtain a diluent having a bacterial count of 5.1 × 10 6 cfu / g. 100 ml of a diluted solution of the bacterium of the present invention-sodium alginate was applied to the portion where 5 g of calcium carbonate had been planted, and immobilization was performed. After a lapse of 30 minutes or more, 20 Chinese cabbage seedlings ("Musou": seedlings raised for 21 days) were planted by 20 strains (soil-immobilized bacterial cell section). 75 ml of the above-mentioned mixed solution of the bacterium of the present invention and sodium alginate was dropped into 200 ml of a 2% by weight calcium chloride solution to immobilize the antibacterial agent. After immobilization in a calcium chloride solution for 30 minutes or more, the immobilized cells were collected by filtration with a 1 mm stainless steel mesh.
[0026]
2 ml of the immobilized cells were added to the planting hole, and 20 Chinese cabbage seedlings ("Musou": raised for 21 days) were planted by 20 strains (immobilized cell section). In the control plot, 100 ml of water was applied to the planting hole, and then 20 Chinese cabbage seedlings (musou: raising seedlings for 21 days) were planted at a time. On the 28th day of planting, the extent of infection was investigated and the results are shown in Table 3. As a result of the investigation, the disease rate was 100% in the control plot, 25% in the direct plot, and 15% in the soil-fixed bacterial plot and the immobilized bacterial plot. The control value was 75 in the direct plot, 85 in the soil immobilized bacterial plot, and 85 in the immobilized bacterial plot. In all of the present invention, the controlling effect was recognized at a lower disease rate than the control. It can be evaluated as a microbial material that is effective for the control of Chinese cabbage clubroot by the soil treatment method.
[0027]
[Table 3]
[0028]
(Example 3)
A bacterial culture of the present invention (4.2 × 10 8 cfu / g of bacteria) was mixed with sterilized water in which sodium alginate was dissolved to a concentration of 1% by weight, and a mixed liquid of the bacterial of the present invention and sodium alginate (5.5 × 10 7 cfu / g) was mixed. ). 100 seeds of Chinese cabbage (musou) were immersed in 100 ml of this mixed solution at room temperature for 1 hour. The seeds were taken out and dropped into 200 ml of a 2% by weight calcium chloride solution to immobilize the fungus of the present invention (immobilized bacterial body section). Further, 100 seeds of Chinese cabbage (Musou) were immersed in 100 ml (5.5 × 10 7 cfu / g) of the diluent of the bacterial culture of the present invention at room temperature for 1 hour (cell section). As a control, 100 Chinese cabbage seeds were immersed in 100 ml of water at room temperature for 1 hour (control). Fifty seeds of Chinese cabbage (Musou) that had been subjected to the above three treatments were sown on commercial vegetable soil, and raised for 21 days. 20 seedlings of each seedling were planted on diseased soil and cultivated for 28 days. Twenty-eight days later, the percentage of infected strains in each treatment group was examined. As a result of the investigation, the disease-causing strain rate was 100% in the control plot, 15% in the immobilized bacterial plot, and 25% in the bacterial plot. The control value was 85 for the immobilized cells and 75 for the cells. The control material of the present invention can be evaluated as a microbial material that is effective for the control of Chinese cabbage clubroot even in seed treatment.
[0029]
[Table 4]
[0030]
(Example 4)
A 128-well cell tray was filled with the control material 1 of the present invention of Example 1, and cabbage (Matsunami: Ishii seedling) and broccoli (Heights: Takii seedling) were sown. Similarly, after filling into a 200-well cell tray, bok choy (Seishin: Takii seedlings), turnip (Kanamachi small turnip: Sakata seeds), and late Komatsuna (Kobayashi seedlings) were sown. Chingensai, turnip and komatsuna were planted on the diseased soil on the 14th day of the nursery, 20 plants each. On the 21st day of raising seedlings, 20 plants of cabbage and broccoli were planted on the diseased soil. Chingensai, turnip, and komatsuna were examined for the degree of infection on the 21st day after planting, and cabbage and broccoli were examined on the 28th day after planting. In the control plot, 2 volumes of vermiculite were mixed with Akadama clay and Taki horticultural cultivation soil (product of Taki Bussan Co., Ltd.) at a ratio of 8: 1. After raising the seedlings for the same period, they were planted and examined for the degree of infection. Table 5 shows the results. In all of the crops, the present invention was found to have an effect of controlling clubroot at a lower disease rate than the control.
[0031]
[Table 5]
As is clear from the above, it is understood that the material for controlling clubroot of cruciferous plants of the present invention is effective for controlling clubroot of various cruciferous crops including Chinese cabbage.
[0032]
【The invention's effect】
INDUSTRIAL APPLICABILITY The material for controlling root-knot disease of cruciferous plants of the present invention exhibits an excellent control effect without causing environmental problems such as chemical pesticides.
