JP7071386B2 - 線虫防除のための材料および方法 - Google Patents
線虫防除のための材料および方法 Download PDFInfo
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- JP7071386B2 JP7071386B2 JP2019547247A JP2019547247A JP7071386B2 JP 7071386 B2 JP7071386 B2 JP 7071386B2 JP 2019547247 A JP2019547247 A JP 2019547247A JP 2019547247 A JP2019547247 A JP 2019547247A JP 7071386 B2 JP7071386 B2 JP 7071386B2
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Description
本出願は、2016年11月16日に提出された米国仮出願第62/422,918号の恩典を主張し、それは参照によりその全体として本明細書に組み入れられる。
収量を高めるため、ならびに作物を病原菌、害虫、および病害から守るために、農場経営者は、合成化学物質および化学肥料の使用に大きく依存している;しかしながら、過度に用いられたまたは不適正に適用された場合、これらの物質は地表水に流出し得、地下水に浸出し得、および空気中に蒸発し得る。空気および水の汚染源として、これらの物質はますます精査され、それらの責任ある使用が生態学的および商業的な責務になっている。適正に用いられた場合でさえ、ある特定の化学肥料および殺害虫剤の過度の依存および長期にわたる使用は、土壌生態系を有害に変更し得、ストレス耐性を低下させ得、害虫抵抗性を増加させ得、植物および動物の成長および生命力を妨げ得る。
本発明は、線虫を防除するための組成物および方法を提供する。加えて、本発明は、線虫による作物への損傷を防止し、ゆえに収量増加をもたらすための方法および組成物を提供する。有利なことに、本発明に従った殺害虫剤は、微生物および微生物培養の副産物などの非毒性物質を利用する。
[本発明1001]
線虫を防除するための方法であって、該方法は、微小生物が生物界面活性剤を産生する条件下で微小生物を培養することによって微生物に基づく殺線虫組成物を調製する工程を含み、該殺線虫組成物は、微小生物および/または微小生物が培養される培養液を含み、かつ該方法は、線虫および/またはそれらの環境に該殺線虫組成物を適用する工程をさらに含む、該方法。
[本発明1002]
生物界面活性剤が糖脂質である、本発明1001の方法。
[本発明1003]
糖脂質がソホロ脂質(SLP)である、本発明1002の方法。
[本発明1004]
糖脂質がマンノシルエリスリトール脂質(MEL)である、本発明1002の方法。
[本発明1005]
微小生物が、スターメレラ(Starmerella)、ピキア(Pichia)、およびシュードザイマ(Pseudozyma)より選択される真菌である、本発明1001の方法。
[本発明1006]
真菌がスターメレラ・ボンビコーラ(Starmerella bombicola)である、本発明1005の方法。
[本発明1007]
真菌がシュードザイマ・アフィディス(Pseudozyma aphidis)である、本発明1005の方法。
[本発明1008]
真菌がピキア・アノマラ(Pichia anomala)(ウィッカーハモマイセス・アノマルス(Wickerhamomyces anomalus))である、本発明1005の方法。
[本発明1009]
培養液が生物界面活性剤を含む、本発明1001の方法。
[本発明1010]
培養液を処理して生物界面活性剤を細胞塊から分離し、それに続いて生物界面活性剤を線虫および/またはそれらの環境に適用する工程をさらに含む、本発明1009の方法。
[本発明1011]
最初に生物界面活性剤を細胞塊から分離することなく、培養液を線虫および/またはそれらの環境に適用する、本発明1009の方法。
[本発明1012]
根瘤線虫(サツマイモネコブセンチュウ(Meloidogyne incognital))、刺毛線虫(sting nematode)(ベロノライムス・ロンギカウダツス(Belonolaimus longicaudatus))、大豆シスト線虫(ダイズシストセンチュウ(Heterodera glycines))、病変線虫(プラチレンクス(Pratylenchus)種)、ダガー線虫(dagger nematode)(キシフィネマ(Xiphinema)種)、および柑橘類線虫(ミカンネセンチュウ(Tylenchulus semipenetrans))より選択される線虫を防除するために用いられる、本発明1001の方法。
[本発明1013]
植物の線虫害虫を防除するために用いられる、本発明1001の方法。
[本発明1014]
植物が、トマト、大豆、コーン、柑橘類、および芝草より選択される、本発明1013の方法。
[本発明1015]
前記組成物が種子処理として適用される、本発明1013の方法。
[本発明1016]
前記組成物が土壌に適用される、本発明1001の方法。
[本発明1017]
線虫誘引物を適用する工程をさらに含む、本発明1001の方法。
[本発明1018]
生物界面活性剤および生物界面活性剤を産生する微小生物を含む、線虫を防除するための組成物。
[本発明1019]
生物界面活性剤が糖脂質である、本発明1018の組成物。
[本発明1020]
糖脂質がSLPである、本発明1019の組成物。
[本発明1021]
糖脂質がMELである、本発明1019の組成物。
[本発明1022]
線虫誘引物質をさらに含む、本発明1019の組成物。
[本発明1023]
線虫誘引物質がカノコソウ(Valerian)の根抽出物を含む、本発明1022の組成物。
[本発明1024]
マンノシルエリスリトール脂質(MEL)を線虫および/または線虫の環境に適用する工程を含む、線虫を防除するための方法。
[本発明1025]
線虫にSLPを適用する工程をさらに含む、本発明1024の方法。
[本発明1026]
線虫誘引物質をさらに含む、本発明1025の方法。
[本発明1027]
根瘤線虫(サツマイモネコブセンチュウ)、刺毛線虫(ベロノライムス・ロンギカウダツス)、大豆シスト線虫(ダイズシストセンチュウ)、病変線虫(プラチレンクス種)、ダガー線虫(キシフィネマ種)、および柑橘類線虫(ミカンネセンチュウ)より選択される線虫を防除するために用いられる、本発明1025の方法。
本発明は、線虫を防除するための材料および方法を提供する。本発明はまた、線虫を防除するための殺害虫剤として用いられ得る組成物を提供する。本発明は、そのような組成物を調製するための方法をさらに提供する。加えて、本発明は、線虫による作物の損傷を防止し、ゆえに収量増加をもたらすための方法および組成物を提供する。
本明細書において使用するとき、生物界面活性剤または生物界面活性剤産生微小生物によってもたらされる活性に関連して用いられる「防除」という用語は、害虫を殺傷する、不能にする、もしくは動きを止める、または別様に害虫が実質的に害を引き起こし得なくすることにまで及ぶ。
微生物生物界面活性剤は、細菌、真菌、および酵母などの多様な微小生物によって産生される化合物である。
生物界面活性剤は、多くの微小生物において生じる二次代謝産物の重要なクラスを形成する。生物界面活性剤は生分解性であり、再生可能な基板上で選択生物を用いて容易かつ安価に産生され得る。生物界面活性剤を産生するほとんどの生物は、成長培地における炭化水素供給源(例えば、油、糖類、グリセロール等)の存在に応答して生物界面活性剤を産生する。他の培地構成要素も、生物界面活性剤産生に大いに影響を及ぼし得る。例えば、シュードモナス・エルギノーサ細菌によるラムノ脂質の産生は、窒素の供給源としてアンモニウムよりもむしろニトレートが用いられる場合に増加し得る。鉄、マグネシウム、ナトリウム、およびカリウムの濃度;炭素:リン比;ならびに撹拌も、ラムノ脂質産生に大きな影響を及ぼし得る。
本発明は、微小生物の培養、ならびに生物界面活性剤および/または微生物増殖の他の副産物の産生のための方法を提供する。これらの培養過程には、液内培養/発酵、表面培養、固体状発酵(SSF)、およびそれらの組み合わせが含まれるが、それらに限定されるわけではない。
本発明の微生物に基づく製品の1つは、微小生物および/または微小生物によって産生された微生物代謝産物(生物界面活性剤など)および/または任意の残留栄養素を含有する単なる発酵培養液である。発酵の産物は、抽出、単離、または精製なしで直接用いられ得る。所望の場合には、抽出、単離、および/または精製は、本明細書および/または文献に記載される標準的な方法または技法を用いて容易に達成され得る。
さらに別の局面において、本発明は、微生物に基づく製品を適用するための方法、システム、および装置を含む。
本発明の好ましいが非限定的な態様において、防除される線虫は以下から選定される。
(1)稲(例えば、サツマイモネコブセンチュウ、ジャワネコブセンチュウ、またはイネネコブセンチュウ(M. graminicola))、大豆(例えば、サツマイモネコブセンチュウまたはアレナリアネコブセンチュウ)、綿(例えば、サツマイモネコブセンチュウ)、じゃがいも(例えば、コロンビアネコブセンチュウ(M. chitwoodi)またはキタネコブセンチュウ)、トマト(例えば、コロンビアネコブセンチュウ)、タバコ(例えば、サツマイモネコブセンチュウ、ジャワネコブセンチュウ、またはアレナリアネコブセンチュウ)、およびコーン(例えば、サツマイモネコブセンチュウ)における根瘤線虫(ネコブセンチュウ種);稲(例えば、イネシストセンチュウ(H. oryzae))、大豆(例えば、ダイズシストセンチュウ(H. glycines))、およびコーン(例えば、トウモロコシシストセンチュウ(H. zeae))におけるシスト線虫(ヘテロデラ種);じゃがいも(例えば、ジャガイモシロシストセンチュウまたはジャガイモシストセンチュウ(G. rostochiensis))におけるシスト線虫(グロボデラ種);綿(例えば、R.レニフォルミス(reniformis))におけるニセフクロ(Reniform)線虫(ロチレンチュラス(Rotylenchulus)種);バナナ(例えば、ミナミネグサレセンチュウまたはP.グーデイ)における根病変線虫(プラチレンクス種);バナナ(例えば、バナナネモグリセンチュウ(R. similis))における穴掘り線虫(ラドフォラス(Radopholus)種);ならびに稲根線虫(ヒルシュマニエラ(Hirschmaniella)種、例えばイネネモグリセンチュウ(H. oryzae))などの稲に損傷を与える他の線虫などであるがそれらに限定されない、植物病原性線虫である線虫;
(2)蟯虫症を引き起こす蟯虫(pinworm)であるヒトギョウチュウ(Enterobius vermicularis);回虫症を引き起こす大腸回虫であるヒトカイチュウ(Ascaris lumbridoides);十二指腸虫症を引き起こす2つのタイプの鉤虫であるネカトール(Necator)およびアンシロストマ(Ancylostoma);鞭虫症を引き起こす鞭虫であるベンチュウ(Trichuris trichiura);糞線虫症を引き起こすストロンギロイデス・ステルコラリス(Strongyloides stercoralis);ならびに旋毛虫症(trichinosis)を引き起こすトリキネラ・スピラリス(Trichonella spirae);リンパ系フィラリア症として知られる蠕虫感染症およびその肉眼的兆候である象皮症と関連した糸状線虫であるマレー糸状虫(Brugia malayi)およびバンクロフト糸状虫(Wuchereria bancrofti)、ならびに河川盲目症を引き起こす回旋糸状虫(Onchocerca volvulus)などであるがそれらに限定されない、ヒトに蔓延し得る線虫;
(3)イヌ(鉤虫、例えばイヌコウチュウ(Ancylostoma caninum)またはウンシナリア・ステノセファラ(Uncinaria stenocephala)、回虫類(Ascarids)、例えばイヌカイチュウ(Toxocara canis)もしくはトキサスカリス・レオニナ(Toxascaris leonina)、または鞭虫、例えばイヌベンチュウ(Trichuris vulpis))、ネコ(鉤虫、例えばアンシロストマ・ツバエホルメ(Ancylostoma tubaeforme)、回虫類、例えばネコカイチュウ(Toxocara cati))、魚類(ニシン蠕虫もしくはタラ蠕虫、例えばアニサキス(Anisakid)、または条虫、例えば裂頭条虫(Diphyllobothrium))、ヒツジ(ハリガネムシ、例えば捻転胃虫(Haemonchus contortus))、およびウシ(消化管蠕虫、例えばオステルターグ胃虫(Ostertagia ostertagi)、クーペリア・オンコホラ(Cooperia oncophora))などであるがそれらに限定されない、動物に蔓延し得る線虫;
(4)食材、種子、木材、塗料、プラスチック、衣類等を攻撃する線虫など、基材または材料への不要な損傷を引き起こす線虫。そのような線虫の例には、ネコブセンチュウ種(例えば、サツマイモネコブセンチュウ、ジャワネコブセンチュウ、アレナリアネコブセンチュウ、イネネコブセンチュウ、コロンビアネコブセンチュウ、またはキタネコブセンチュウ);ヘテロデラ種(例えば、イネシストセンチュウ、ダイズシストセンチュウ、トウモロコシシストセンチュウ、またはテンサイシストセンチュウ);グロボデラ種(例えば、ジャガイモシロシストセンチュウまたはジャガイモシストセンチュウ);クキセンチュウ種(例えば、ナミクキセンチュウ(D. dipsaci)、イモグサレセンチュウ(D. destructor)、またはイネクキセンチュウ(D. angustus));ベロノライムス(Belonolaimus)種;ロチレンチュラス種(例えば、R.レニフォルミス);プラチレンクス種(例えば、ミナミネグサレセンチュウ、P.グーデイ、またはモロコシネグサレセンチュウ);ラドフォラス種(例えば、バナナネモグリセンチュウ);ヒルシュマニエラ種(例えば、イネネモグリセンチュウ);アフェレンコイデス(Aphelenchoides)種(例えば、イネシンガレセンチュウ(A. besseyi));クリコネモイデス(Criconemoides)種;ロンギドラス(Longidorus)種;ヘリコチレンチュス(Helicotylenchus)種;ホプロライムス(Hoplolaimus)種;キシフィネマ(Xiphinema)種;パラトリコドラス(Paratrichodorus)種(例えば、ヒメユリハリセンチュウ(P. minor));チレンコリンクス(Tylenchorhynchus)種が含まれるが、それらに限定されるわけではない;
(5)ウイルス伝播線虫(例えば、プルヌスえそ輪点ウイルスを伝播するロンギドラス・マクロソマ(Longidorus macrosoma)、タバコ輪点ウイルスを伝播するキシフィネマ・アメリカナム(Xiphinema americanum)、エンドウ早期褐変ウイルス(pea early browning virus)を伝播するパラトリコドラス・テレス(Paratrichadorus teres)、またはタバコラットルウイルスを伝播するトリコドラス・シミリス(Trichodorus similis))。
本発明の製品および方法の適用により利益を受け得る植物には、列にして植える作物(例えば、コーン、大豆、ソルガム、落花生、じゃがいも等)、農作物(例えば、アルファルファ、小麦、穀物等)、樹木作物(例えば、クルミ、アーモンド、ピーカン、ヘーゼルナッツ、ピスタチオ等)、柑橘作物(例えば、オレンジ、レモン、グレープフルーツ等)、果実作物(例えば、リンゴ、ナシ等)、芝生作物、観賞作物(例えば、花、つる植物等)、野菜(例えば、トマト、ニンジン等)、つる作物(例えば、ブドウ、イチゴ、ブルーベリー、ブラックベリー等)、森林地(例えば、マツ、トウヒ、ユーカリ、ポプラ等)、管理された牧草地(放牧動物を支持するために用いられる植物の任意の混合)が含まれる。
水濾過、温度制御ユニット、および搭載された送風機を備えたPLCによって運転される、可搬式のエアリフト型の完全に密閉されたリアクターを用いる。リアクターは、SLP産生のためにスターメレラ・ボンビコーラを増殖させる場合、90Lの作業体積を有する。
このリアクターは、空気スパージャー(spurge)およびインペラーを備えた加圧滅菌可能な被覆ガラス容器である。それは、溶存酸素、pH、温度、および気泡プローブ(prob)が配備されており;それは、カラータッチスクリーンインターフェース、内蔵式ポンプ、ガスフロー制御装置、およびpH/DO気泡/レベル制御装置を備えた統合制御ステーションを有する。リアクターの作業体積は10リットルである。
水濾過、温度制御ユニット、および十分な通気のための送風機を備えたPLCによって運転される、可動エアリフトリアクターを用いる。過程は、バッチ培養過程として行われ得る。リアクターは、SLP産生のためにウィッカーハモマイセスまたはピキアを増殖させる場合、400Lの作業体積を有する。
2つのタンクを同時に混合するのを助ける中心エアリフトによって作動される、2つのタンクに分割された可搬式リアクターを用いる。リアクターは、細胞産生のためにウィッカーハモマイセスおよび/またはピキアを増殖させる場合、600Lの作業体積を有する。
タンク間での質量交換のための2本のループを伴う2つの四角いタンクに分割された可搬式リアクターを用いる。リアクターは、細胞産生のためにウィッカーハモマイセスおよび/またはピキアを増殖させる場合、2000Lの作業体積を有する。
これは、空気スパージャーおよびRushtonインペラーを備えた加圧蒸気滅菌可能な被覆ガラス容器である。それは、DO、pH、温度、および気泡プローブが配備されている。それは、カラータッチスクリーンインターフェース、内蔵式ポンプ、ガスフロー制御装置、およびpH/DO気泡/レベル制御装置を備えた統合制御ステーションを有する。リアクターの作業体積は10リットルである。
スターメレラ・ボンビコーラを、上記のように増殖させた。発酵培養の終わりに、培養液は落ち着き、SLPのほとんどは暗褐色層として分離する(約500gm/Lの濃度)。この褐色層は、主としてSLP、および一部の細胞も含有する。層を回収し、pHをpH7.0に調整する。次いで、その溶液を水で希釈して、所望の濃度を達成する。
14日目:1%肥料(オスモコート(osmocote))を有する砂/ターフェス(turface)(1:1)を調製し、トマトの2週間苗を移植する。
28日目:4週齢の植物に土壌灌注によって微生物を接種する。各10cm×10cm正方形の鉢に対して、土壌を湿らせるのに80~100mlの培養物で十分であった。
32日目:微生物接種の4日後、各植物にRKN卵(1個の鉢あたり4000個の卵)を適用する。
76日目:卵接種の6週間後、植物を収穫する。
Nimitz(登録商標)は、20年を超える期間のあいだに開発された第一の新たな化学的殺線虫剤である。Nimitz(登録商標)は、適用直後に不可逆的でかつ迅速な殺線虫活性を引き起こす。接触の1時間以内に、線虫は摂食を止め、急速に麻痺する。24~48時間以内に、有機リン酸およびカルバメート殺線虫剤を用いて見られる一時的な静線虫(動きを止める)活性ではなく、害虫死亡が起こる。
根瘤線虫(サツマイモネコブセンチュウ)の死亡率(すなわち、どれぐらいの線虫が殺傷されたか)および運動性の欠如(すなわち、どれぐらいの線虫が死んだように状態が悪いか)を、様々な濃度のSLP(SD10)またはMEL(SD12)の処理の3日後に判定し、Nimitz(登録商標)の処理および未処理対照と比較した(図1および2)。死亡率パーセントを、死滅した線虫の数/検査前の線虫の総カウントの数によって算出した。非運動性線虫パーセントを、非運動性線虫の数/生きた線虫の数によって算出した。ゆえに、運動性は、殺傷されなかったが非運動性であると判定されるそれらの線虫のパーセンテージとして算出される。
刺毛線虫(ベロノライムス・ロンギカウダツス)の死亡率および運動性低下率を、様々な濃度のSLPまたはMELの処理の3日後に判定し、Nimitz(登録商標)の処理および未処理対照と比較した(図1および2)。
病変線虫(プラチレンクス種)の死亡率および運動性低下率を、様々な濃度のSLPまたはMELの処理の3日後に判定し、Nimitz(登録商標)の処理および未処理対照と比較した(図1および2)。
線虫が感染した土壌に直接適用されるSLP処理(SD10)およびMEL処理(SD12)を、単独でまたは一緒に2種類の割合で検査し、生きたサツマイモ根瘤線虫集団を減少させることに関してNimitz(登録商標)および未処理対照と比較した。処理後の3つの異なる時点でカウント数を取った。
SD10およびSD12産物に関するジャーテスト結果は、サツマイモ根瘤線虫に対する活性に関して正の用量応答を示唆する。低い割合で一緒に適用されたSD10およびSD12は、より高い割合における単独でのいずれかの産物ほど有効でなかった。Nimitz(登録商標)は、より優れた防除を提供した。
米国フロリダ州ソノトサッサに位置する屋外小区画を用いて、Nimitz(登録商標)および未処理対照と比較した、キュウリ植物に対するMEL線虫処理を調査した。それぞれが8つの植物からなる6つの異なる処理群を調査した。SD12(0.2% ai/v)処理を受ける群に関して、処理を、土壌に1回、2回、または3回適用した。収穫時に、線虫カウント数およびこぶ形成(galling)率の両方を収集した。植物活力、収量、および植物の重量も測定した。
こぶ形成(図7および表6(下記))および処理後の線虫カウント数(図8および表7(下記))において割合応答が観察された。SD12は商業規格に合致する能力を示した。SD12処理とNimitz(登録商標)処理との間に有意な格差はなかったが、SD12処理と未処理チェックとの間に有意な差が観察された。収穫時の植物活力、収量、および重量が、下記の表8に報告されている。
LSD(P=.05)=0.88;SD=0.86;CV=69.39;バートレットのX2=11.754;P(バートレットのX2)=0.019*;反復F=3.712;反復プローブ(Prob)(F)=0.0061;処理F=11.133;処理プローブ(F)=0.0001。
LSD(P=.05)=17.33;SD=16.3;CV=63.88;バートレットのX2=17.263;P(バートレットのX2)=0.002*;反復F=2.774;反復プローブ(F)=0.0261;処理F=10.44;処理プローブ(F)=0.0001。
誘引材料でスパイクされた、湖の微細な砂質土壌を含有する4個の11.6インチ(in.)×7.6インチの密閉チャンバーにおいて、サツマイモ根瘤線虫のカウント数および蔓延パーセンテージを取った。あらかじめ作られている購入したカノコソウの根抽出物と、水、植物性グリセリン、および20%穀物アルコールとを混ぜ合わせて、誘引物を産生した。
結果は、図10および表9(下記)に要約されている。総集団のパーセンテージとして、誘引物エリアまたは未処理エリアへの移動は、接種エリアと比べて有意に異なった。24時間および48時間のサンプリング事象の時点で、チャンバーの未処理エリアよりも誘引物ゾーンにおいて、14%を上回って、より多くのカウントされた線虫が存在した;しかしながら、線虫が接種された中心部ゾーンが、全体のほとんどの線虫を保持した。
Claims (4)
- 植物の線虫害虫を防除するための方法であって、該方法は、シュードザイマ・アフィディス(Pseudozyma aphidis)微小生物が発酵培養液においてマンノシルエリスリトール脂質(MEL)生物界面活性剤を産生する条件下でシュードザイマ・アフィディス微小生物を発酵培養液において培養することによって微小生物に基づく殺線虫組成物を調製する工程、
該培養液を処理して該生物界面活性剤を細胞塊から分離し、それに続いて該生物界面活性剤を水と混合する工程、および、該生物界面活性剤を線虫に、土壌に、および/または種子処理として適用する工程をさらに含む、該方法。 - 根瘤線虫(サツマイモネコブセンチュウ(Meloidogyne incognita))、刺毛線虫(sting nematode)(ベロノライムス・ロンギカウダツス(Belonolaimus longicaudatus))、大豆シスト線虫(ダイズシストセンチュウ(Heterodera glycines))、病変線虫(プラチレンクス(Pratylenchus)種)、ダガー線虫(dagger nematode)(キシフィネマ(Xiphinema)種)、および柑橘類線虫(ミカンネセンチュウ(Tylenchulus semipenetrans))より選択される線虫を防除するために用いられる、請求項1に記載の方法。
- 植物が、トマト、大豆、コーン、柑橘類、および芝草より選択される、請求項1に記載の方法。
- 線虫、土壌および/または種子にソホロ脂質を適用する工程をさらに含む、請求項1に記載の方法。
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