JP2008137946A - Controlling agent for biological control of soybean stem and root blight and controlling method - Google Patents
Controlling agent for biological control of soybean stem and root blight and controlling method Download PDFInfo
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本発明は植物病害、特にダイズ茎疫病の生物的防除のための防除剤及びそれを用いた植物病害の防除方法に関する。 The present invention relates to a control agent for biological control of plant diseases, particularly soybean stem blight, and a method for controlling plant diseases using the same.
ダイズは、古来よりわが国において生産される重要な作物の一つであるが、近年になり水田転換畑を利用した栽培が主体となったことから、連作や排水不良により茎疫病が発生・蔓延し大きな被害を生じている。 Soybean has been one of the important crops produced in Japan since ancient times, but in recent years it has mainly been cultivated using paddy field conversion fields. There is a lot of damage.
ダイズ茎疫病に対する登録農薬としては、オキサジル・銅水和剤があるが、耐性菌問題により本剤の製造は中止され、代替え農薬としてボルドー剤が新規登録となったものの効果が弱く現実的ではない。このため、本病防除は主に、耕種的な対策を中心に行っている。本病の発生は土壌水分が高い場所で多いことから、圃場の排水能を高めるための高畝のほか明渠の設置や発病株の抜き取りおよび田畑輪換などの栽培技術で対応しているものの、その効果は十分ではない。 Registered pesticides for soybean pesticidal diseases include oxazil and copper wettable powder, but the production of this drug was discontinued due to the problem of resistant bacteria, but the effect of the newly registered Bordeaux agent as an alternative pesticide was weak and impractical . For this reason, this disease control mainly focuses on cultivating measures. The occurrence of this disease is common in places with high soil moisture, and in addition to Takatsuki to increase drainage capacity in the field, it is supported by cultivation techniques such as the installation of alum and extraction of diseased strains and field rotation. The effect is not enough.
また、国民の環境に対する関心の高まりから低環境負荷型病害防除技術は今日的な農業技術として強く求められている。 In addition, low environmental impact disease control technology is strongly demanded as a modern agricultural technology due to the increasing public interest in the environment.
上記の通りダイズ茎疫病に対する生物的防除技術としては、有望な生物防除素材(BCA)が明らかにされておらず確立されていない。また、耕種的な防除技術だけでは効果が不十分である。登録促進によって農薬が使用できたとしても、低環境負荷型の病害防除技術として生物的防除技術開発への要望は大きい。 As described above, a promising biological control material (BCA) has not been clarified or established as a biological control technique for soybean stem blight. Moreover, the effect is not sufficient only by cultivating control technology. Even if pesticides can be used by promoting registration, there is a great demand for the development of biological control technology as a low environmental load disease control technology.
そこで本発明は、ダイズ茎疫病に対する低環境負荷型の防除技術として、微生物の有する拮抗作用を利用した生物的防除技術に利用できる有望な拮抗菌を土壌中から選抜することを目的とする。 Then, this invention aims at selecting the promising antagonistic bacteria which can be utilized for the biological control technique using the antagonism which microorganisms have as a low environmental load type control technique with respect to a soybean stem rot.
本発明者らは、微生物の有する拮抗作用を利用した生物的防除技術に利用できる有望な拮抗菌を土壌中から選抜することを試み、本発明を完成させるに至った。 The present inventors have attempted to select promising antagonistic bacteria that can be used in biological control technology using the antagonistic action of microorganisms from the soil, and have completed the present invention.
本発明は以下の発明を包含する。
(1)ダイズにおける茎疫病を防除する能力を有するトリコデルマ属に属する微生物を有効成分として含有する、ダイズにおける茎疫病の防除剤。
(2)トリコデルマ属に属する微生物がトリコデルマ・ヴィリデ (Trichoderma viride)又はトリコデルマ・ヴィレンス (Trichoderma virens)に属する、(1)に記載の防除剤。
(3)トリコデルマ属に属する微生物がトリコデルマ・ヴィリデ T-3菌株(受領番号 NITE AP-279)もしくはその変異株、トリコデルマ・ヴィレンス T04398菌株(受領番号 NITE AP-280)もしくはその変異株、トリコデルマ・ヴィレンス T04401菌株(受領番号 NITE AP-281)もしくはその変異株、又はトリコデルマ・ヴィレンス T04464菌株(受領番号 NITE AP-282)もしくはその変異株である、(2)に記載の防除剤。
(4)(1)〜(3)のいずれかに記載の防除剤をダイズに施用することを含む、ダイズにおける茎疫病の防除方法。
(5)トリコデルマ・ヴィリデ T-3菌株(受領番号 NITE AP-279)。
(6)トリコデルマ・ヴィレンス T04398菌株(受領番号 NITE AP-280)。
(7)トリコデルマ・ヴィレンス T04401菌株(受領番号 NITE AP-281)。
(8)トリコデルマ・ヴィレンス T04464菌株(受領番号 NITE AP-282)。
The present invention includes the following inventions.
(1) A pesticidal control agent in soybean, which contains, as an active ingredient, a microorganism belonging to the genus Trichoderma having the ability to control pesticidal disease in soybean.
(2) The control agent according to (1), wherein the microorganism belonging to the genus Trichoderma belongs to Trichoderma viride or Trichoderma virens.
(3) The microorganism belonging to the genus Trichoderma is Trichoderma viride T-3 strain (reception number NITE AP-279) or its mutant strain, Trichoderma virens T04398 strain (reception number NITE AP-280) or its mutant strain, Trichoderma virence The control agent according to (2), which is T04401 strain (reception number NITE AP-281) or a mutant thereof, or Trichoderma virens T04464 strain (reception number NITE AP-282) or a mutant thereof.
(4) A method for controlling stem blight in soybean, comprising applying the control agent according to any one of (1) to (3) to soybean.
(5) Trichoderma viride T-3 strain (reception number NITE AP-279).
(6) Trichoderma virens strain T04398 (reception number NITE AP-280).
(7) Trichoderma virens strain T04401 (reception number NITE AP-281).
(8) Trichoderma virens strain T04464 (reception number NITE AP-282).
本発明により、ダイズにおける茎疫病を生物的に防除する手段が提供される。 The present invention provides a means for biologically controlling stem blight in soybean.
本発明による防除の対象となる茎疫病の原因となる微生物(ダイズ茎疫病菌)はPhytophthora sojaeである。 Phytophthora sojae is a microorganism (soybean stem rot) that causes stem rot that is subject to control according to the present invention.
病原菌は、ダイズのみを侵し、茎疫病を引き起こす。本発明の防除剤はダイズにおける茎疫病の防除に適する。 Pathogenic bacteria invade only soybeans and cause stem blight. The control agent of the present invention is suitable for controlling stem blight in soybean.
本発明に使用することができるトリコデルマ属に属する微生物としては、茎疫病を防除することができるものであれば特に限定されないが、典型的にはトリコデルマ・ヴィリデ (Trichoderma viride)又はトリコデルマ・ヴィレンス (Trichoderma virens)に属するものが挙げられる。なかでも独立行政法人製品評価技術基盤機構特許微生物寄託センターに寄託申請されたトリコデルマ・ヴィリデ T-3菌株(受領番号 NITE AP-279、受領日2006年11月14日)、トリコデルマ・ヴィレンス T04398菌株(受領番号 NITE AP-280、受領日2006年11月14日)、トリコデルマ・ヴィレンス T04401菌株(受領番号 NITE AP-281、受領日2006年11月14日)、又はトリコデルマ・ヴィレンス T04464菌株(受領番号 NITE AP-282、受領日2006年11月14日)が好ましい。 The microorganism belonging to the genus Trichoderma that can be used in the present invention is not particularly limited as long as it can control stem rot, but typically, Trichoderma viride or Trichoderma vilens (Trichoderma virens). Among them, Trichoderma viride T-3 strain (reception number NITE AP-279, date of receipt November 14, 2006), Trichoderma virens strain T04398 (deposited with the National Institute of Technology and Evaluation) Receipt number NITE AP-280, receipt date November 14, 2006), Trichoderma virens strain T04401 (reception number NITE AP-281, receipt date November 14, 2006), or Trichoderma virens strain T04464 (receipt number NITE) AP-282, date of receipt, November 14, 2006) is preferred.
本発明にはまた上記の特定の4種類の菌株の変異誘発処理された変異株を用いることもできる。変異誘発処理は任意の適当な変異原を用いて行なうことができる。ここで、「変異原」なる語は、その広義において、例えば変異原効果を有する薬剤のみならずUV照射のごとき変異原効果を有する処理をも含むものと理解すべきである。適当な変異原の例としてエチルメタンスルホネート、UV照射、N−メチル−N′−ニトロ−N−ニトロソグアニジン、ブロモウラシルのようなヌクレオチド塩基類似体及びアクリジン類が挙げられるが、他の任意の効果的な変異原もまた使用することができる。 In the present invention, mutant strains obtained by mutagenesis treatment of the above-mentioned four specific strains can also be used. The mutagenesis treatment can be performed using any appropriate mutagen. Here, the term “mutagen” should be understood in a broad sense to include not only a drug having a mutagenic effect but also a treatment having a mutagenic effect such as UV irradiation. Examples of suitable mutagens include ethyl methanesulfonate, UV irradiation, N-methyl-N'-nitro-N-nitrosoguanidine, nucleotide base analogs such as bromouracil, and acridines, but any other effect A typical mutagen can also be used.
本発明に使用されるトリコデルマ属菌は、往復振とう培養、ジャーファメンター培養、培養タンク培養等の液体培養(固形物含有液体の流動培養を含む)、棚段培養等の固体培養等の通常の培養法によって培養することができる。トリコデルマ属菌の培養のための培地は、該菌が効率的に繁殖し胞子形成し得るものであれば特に限定されない。かかる培地としては例えば、天然物を利用したものではオオムギ粒培地(六条カワムギ20g、水20g)やジャガイモ煎汁培地(PSA培地またはPDA培地:ジャガイモ200g、サッカロースまたはデキストロース15〜20g、寒天15〜20g)のほか、炭素源としてグルコース、シュークロース、デンプン、デキストリン、黒砂糖、フスマ、コメヌカなどの糖類を、窒素源として硫酸アンモニウム、塩化アンモニウム、硝酸アンモニウム等のアンモニウム塩、硝酸塩等の無機窒素源、または、酵母エキス、コーン・スティープ・リーカー、肉エキス、小麦胚芽、ポリペプトン、サトウキビ絞り粕、ビール粕、大豆粕、コメヌカ、魚粉等の有機窒素源を、無機塩としてリン酸一カリ、硫酸マグネシウム、硫酸マンガン、硫酸第一鉄等の、リン、カリウム、マンガン、マグネシウム、鉄等を含む塩類を、それぞれ含有する合成または天然の培地が挙げられる。 Trichoderma species used in the present invention are usually used for liquid culture (including fluid culture of solid-containing liquid) such as reciprocal shaking culture, jar fermenter culture, culture tank culture, and solid culture such as shelf culture. The culture method can be used. A culture medium for culturing Trichoderma is not particularly limited as long as the bacterium can efficiently propagate and sporulate. Examples of such a medium include a barley grain medium (6 g raw walnuts, 20 g water) and a potato broth medium (PSA medium or PDA medium: potato 200 g, saccharose or dextrose 15-20 g, agar 15-20 g). ), Sugars such as glucose, sucrose, starch, dextrin, brown sugar, bran and rice bran as a carbon source, ammonium salts such as ammonium sulfate, ammonium chloride and ammonium nitrate as nitrogen sources, inorganic nitrogen sources such as nitrates, or Organic nitrogen sources such as yeast extract, corn steep liaker, meat extract, wheat germ, polypeptone, sugarcane squeezed potato, beer lees, soybean meal, rice bran, fish meal, etc., inorganic potassium salt of potassium phosphate, magnesium sulfate, manganese sulfate , Ferrous sulfate Of phosphorus, potassium, manganese, magnesium, salts containing iron, and a medium of synthetic or natural, each containing.
また本発明に使用されるトリコデルマ属菌は、フスマ、ムギワラ、コメヌカ、イナワラ、トウモロコシガラ、オートミール、ムギ粒、大豆粕、コーンミール、食品残渣等の有機物粉末に菌体を接種し、適当な条件、例えば24〜26℃暗黒条件下で培養することもできる。またこれらの有機物粉末にベントナイト、珪藻土、タルク類、パーライト、バーミキュライト等の無機物を適宜添加したものもまた培地として使用することができる。こうして得られた培養物又はそれを適宜乾燥させたものはそれ自体が植物病害の防除資材として好適に使用できるため好ましい。トリコデルマ属菌は上記有機物粉末を培地として用いた場合に優れた胞子形成能を示すことから、かかる防除資材は大量生産が容易である。 Trichoderma bacterium used in the present invention is inoculated with organic powder such as bran, wheat straw, rice bran, rice bran, corn straw, oatmeal, wheat grain, soybean cake, corn meal, food residue, etc. For example, it can also be cultured under dark conditions at 24-26 ° C. Moreover, what added inorganic substances, such as bentonite, diatomaceous earth, talc, pearlite, vermiculite suitably, to these organic substance powder can also be used as a culture medium. A culture thus obtained or a product obtained by appropriately drying it is preferable because it can be suitably used as a plant disease control material. Since Trichoderma spp. Exhibit excellent sporulation ability when the organic powder is used as a medium, such a control material is easily mass-produced.
本発明の防除剤としては、上記トリコデルマ属菌の培養物をそのまま使用することができる。培養物中には、分生胞子、子のう胞子、厚膜胞子等の各種胞子(本明細書ではこれらを総称して単に「胞子」と称することがある)が含まれていることが好ましいが、菌糸体又はその断片が更に含まれていてもよい。かかる培養物は適宜乾燥して使用することもできる。 As the control agent of the present invention, the above Trichoderma spp. Culture can be used as it is. The culture preferably contains various spores such as conidia spores, ascospores and thick film spores (in the present specification, these may be collectively referred to simply as “spores”). The mycelium or a fragment thereof may be further contained. Such a culture can be appropriately dried before use.
本発明の防除剤としてはまた、上記培養物から、形成された胞子を常法により分離または高濃度化したもの又はその乾燥物を使用することができる。この場合もまた菌糸体又はその断片が含まれていてもよい。分離または高濃度化の方法としては、ろ過、遠心分離等の方法によりペレット状又は懸濁液状の組成物を得る方法のほか、例えば次の方法が挙げられる。すなわち、固形物含有液体の流動培養では、菌糸体を先ず生育させ、液体をろ過により除去し、残固形物ごと乾燥させ、胞子形成を促し、十分に胞子形成させたのちに、解砕し篩分けする方法や、この工程の胞子形成後に、界面活性剤を含む水にて処理し、胞子懸濁液をろ過により回収し乾燥して胞子を得る方法も可能である。固体培養では、固体表面に胞子形成後に培地ごと篩分けしたり、もしくは培地を乾燥後、解砕し篩分けすることより胞子を回収できる。 As the control agent of the present invention, it is also possible to use a product obtained by separating or increasing the concentration of spores formed from the above culture by a conventional method or a dried product thereof. Again, mycelium or fragments thereof may be included. As a method for separation or concentration enhancement, for example, the following method may be mentioned in addition to a method of obtaining a pellet-like or suspension-like composition by a method such as filtration or centrifugation. That is, in fluid culture of a solid-containing liquid, the mycelium is first grown, the liquid is removed by filtration, the whole remaining solid is dried, the spore formation is promoted, and the spore is formed sufficiently. It is also possible to divide the spore, or after spore formation in this step, to treat with water containing a surfactant, collect the spore suspension by filtration and dry it to obtain spores. In solid culture, spores can be collected by sieving together with the medium after spore formation on the solid surface, or by pulverizing and sieving the medium after drying.
本発明の防除剤としてはまた、上記トリコデルマ属菌を液体培養(固形物含有液体の流動培養を含む)により培養した場合の培養液を活性炭粉末、珪藻土、タルク等の多孔吸着体に吸着させ乾燥させたものを使用することもできる。 In addition, as the control agent of the present invention, the Trichoderma spp. Can be adsorbed on a porous adsorbent such as activated carbon powder, diatomaceous earth, or talc when dried by liquid culture (including fluid culture of solid-containing liquid) and dried. You can also use the
なお上記のいずれにおいても乾燥方法は通常の方法でよく、例えば自然乾燥や低温での通風乾燥の他、凍結乾燥、減圧乾燥でよい。これらの乾燥物は乾燥後さらにボールミル等の粉砕手段で適宜粉砕してもよい。 In any of the above, the drying method may be a normal method, for example, natural drying or ventilation drying at a low temperature, as well as freeze drying and drying under reduced pressure. These dried products may be further pulverized appropriately by a pulverizing means such as a ball mill after drying.
これまでに説明したトリコデルマ属菌の培養物、分離もしくは高濃度化された胞子、培養液の吸着物又はそれらの乾燥物(以下、「培養物等」という)は、単独で又は適宜組み合わせて本発明の防除剤として使用することができる。培養物等は、各種土壌や麦粒、木材片、サトウキビ絞り粕、ビール粕、大豆粕、コメヌカ、魚粉、各種堆肥、食品残渣等の有機物と組み合わせた堆肥状資材の形態で使用することもできる。培養物等はまた、他の任意成分と組み合わせて通常の微生物製剤と同様の形態(例えば粉剤、水和剤、乳剤、液剤、フロアブル剤、塗布剤等の形態)に製剤化することもできる。水和剤の形態が特に好ましい。水和剤化することにより植物の播種時や生育時での土壌灌注や散布による施用が可能になり施設内だけでなく露地での広範な用途に対応可能となるからである。これらの資材や製剤中において組み合わせて使用される任意成分としては例えば固体担体、補助剤が挙げられる。固体担体としては例えばベントナイト、珪藻土、タルク類、パーライト、バーミキュライト、カルボキシメチルセルロースナトリウム、ビール粕、サトウキビ絞り粕、オカラ、フスマ、キチン、コメヌカ、小麦粉等が挙げられ、補助剤としては例えばゼラチン、アラビアガム、糖類、ジェランガム等の固着剤や増粘剤が挙げられる。 The cultures of Trichoderma genus described so far, separated or highly concentrated spores, adsorbents of culture solution or dried products thereof (hereinafter referred to as “cultures etc.”) may be used alone or in appropriate combination. It can be used as a control agent of the invention. Cultures etc. can also be used in the form of compost-like materials combined with organic matter such as various soils, wheat grains, wood pieces, sugarcane squeezed rice cake, beer rice cake, soybean meal, rice bran, fish meal, various composts, food residues, etc. . Cultures and the like can also be formulated in the same form as normal microbial preparations (for example, powders, wettable powders, emulsions, solutions, flowables, coatings, etc.) in combination with other optional components. The wettable form is particularly preferred. This is because the use of a wettable powder enables application by soil irrigation and spraying at the time of sowing and growth of plants, and it can be used for a wide range of uses not only in the facility but also in the open ground. Examples of optional components used in combination in these materials and preparations include solid carriers and adjuvants. Examples of the solid carrier include bentonite, diatomaceous earth, talc, perlite, vermiculite, sodium carboxymethylcellulose, beer lees, sugar cane pomace, okara, bran, chitin, rice bran, flour, etc., and auxiliary agents include, for example, gelatin, gum arabic , Sugars, gellan gum and other sticking agents and thickeners.
本発明の防除剤の植物への施用方法は、植物病害の発生状況、施用対象である植物の種類、トリコデルマ属菌の剤形などの諸条件に応じて適宜選択され、例えば、地上部散布、施設内施用、土壌混和施用、土壌灌注施用、表面処理(種子粉衣処理、種子塗布処理)等の各処理により行われ得る。より具体的な施用方法としては、各種形状のトリコデルマ属菌含有防除剤を植物の種子に粉衣・塗布する処理、植物の育苗培土や栽培土壌に灌注する処理、植物の育苗培土や栽培土壌に混和する処理、植物の茎葉に散布する処理、および、植物の付傷部に接触させる処理が挙げられる。これらの処理は単独または複数が組み合わされて行われてよい。また施用時期は播種時、生育時等いずれの時期であってもよい。丹波黒大豆のような苗移植栽培を行なうダイズでは、育苗培土に本発明の防除剤を混和するか、トリコデルマ属菌を繁殖させた育苗土(すなわち育苗土資材)を用いて育苗を行なうことにより、出芽直後の幼苗を茎疫病菌の感染から保護することができ、初期生育の健全化が図れる。 The method of applying the control agent of the present invention to a plant is appropriately selected according to various conditions such as the occurrence of plant diseases, the type of plant to be applied, and the dosage form of Trichoderma sp. It can be performed by each treatment such as in-facility application, soil admix application, soil irrigation application, surface treatment (seed dressing treatment, seed application treatment). More specific application methods include treatment of applying various forms of Trichoderma spp. Control agent on plant seeds, irrigation of plant seedling cultivation soil and cultivation soil, plant seedling cultivation soil and cultivation soil The process which mixes, the process sprayed on the foliage of a plant, and the process made to contact the wound part of a plant are mentioned. These processes may be performed singly or in combination. The application time may be any time such as sowing or growing. In soybeans such as Tamba black soybeans, seedling transplantation cultivation is carried out by adding the control agent of the present invention to the seedling culture soil or by raising seedlings using the seedling soil (that is, the seedling material) on which Trichoderma spp. The seedlings immediately after emergence can be protected from the infection of the stem rot fungus, and the initial growth can be improved.
更にまた、上記トリコデルマ属菌の植物への施用に際しては、必要に応じて通常使用される他の有効成分、例えば殺虫剤、殺線虫剤、殺ダニ剤、除草剤、殺真菌剤、殺細菌剤、抗ウイルス剤、肥料、土壌改良剤等を混合施用するか、または、混合せずに交互施用もしくは同時施用することも可能である。 Furthermore, when applying the above Trichoderma spp. To plants, other active ingredients usually used as necessary, such as insecticides, nematicides, acaricides, herbicides, fungicides, bactericides An agent, an antiviral agent, a fertilizer, a soil improver, etc. can be mixed and applied, or can be applied alternately or simultaneously without mixing.
本発明の防除剤の植物への施用量は、植物病害の発生状況、施用対象である植物の種類、トリコデルマ属菌の剤形などの諸条件に応じて適宜決定される。例えば、ふすま・バーミキュライト培地培養物では10a当たり250kg〜約2tを全面土壌混和することで高い茎疫病防除効果が生まれる。 The application amount of the control agent of the present invention to a plant is appropriately determined according to various conditions such as the occurrence of plant diseases, the type of plant to be applied, and the dosage form of Trichoderma. For example, in the bran / vermiculite medium culture, a high effect of controlling pesticidal diseases can be obtained by mixing the whole soil with 250 kg to about 2 t per 10a.
以下本発明を実施例に基づいて説明するが本発明はこれらの実施例によっては限定されない。 Hereinafter, the present invention will be described based on examples, but the present invention is not limited to these examples.
参考例1: 温室内ポット選抜
(茎疫病菌)
茎疫病菌としてPhytophthora soja S-2 (兵庫農技センター分離株)を用いた。本茎疫病菌を、ふすま・バーミキュライト培地で25℃暗黒下20日間培養した。ふすま・バーミキュライト培地は、コムギふすま:バーミキュライト:水を重量比で1:1:3の割合で混合後,容器に入れ加圧滅菌して得られたものを用いた。
(候補菌株)
候補菌株として各地の土壌(圃場など)から分離した220菌株を使用した。各候補菌株を、ふすま・バーミキュライト培地で25℃暗黒下10日間培養した。
(植物)
試験植物としてダイズ品種タチナガハ(茨城県経済連購入種子)を用いた。
(土壌)
上記ダイズを栽培するための土壌として殺菌クロボク土(高圧滅菌消毒)を用いた。
(試験方法)
殺菌土壌200mlに、培養した候補トリコデルマ菌及び茎疫病菌の培養物を各2gづつ同時に混和した。混和後の土壌をプラスチック製ポット(280ml)に入れ、ダイズ種子をポット当たり5粒播種した。播種後ガラス温室(25℃,70%Rh)において保持した。試験は1処理につき6ポットを用い、3反復で行った。
Reference example 1: Selection of pots in a greenhouse (stalk fungus)
Phytophthora soja S-2 (Hyogo Agricultural Technology Center isolate) was used as a stem rot fungus. This stem blight fungus was cultured in a dark bran and vermiculite medium at 25 ° C. for 20 days. The bran / vermiculite medium used was obtained by mixing wheat bran: vermiculite: water in a weight ratio of 1: 1: 3, and then placing in a container and autoclaving.
(Candidate strain)
220 strains isolated from various soils (such as fields) were used as candidate strains. Each candidate strain was cultured on a bran / vermiculite medium in the dark at 25 ° C. for 10 days.
(plant)
Soybean variety Tachinagaha (Ibaraki Prefectural Economical Purchasing Seeds) was used as a test plant.
(soil)
Sterilized Kuroboku soil (high-pressure sterilization) was used as the soil for cultivating the soybean.
(Test method)
2 g of each cultured culture of candidate Trichoderma fungus and pesticidal fungus were mixed simultaneously with 200 ml of sterilized soil. The mixed soil was placed in a plastic pot (280 ml), and 5 soybean seeds were sown per pot. After sowing, it was kept in a glass greenhouse (25 ° C, 70% Rh). The test was performed in triplicate using 6 pots per treatment.
播種20日後にダイズを引き抜き、下記式に従って発病度を求めた。
発病度={Σ(程度別発病茎数×指数)/(調査茎数×4)}×100
Soybean was extracted 20 days after sowing, and the disease severity was determined according to the following formula.
Disease severity = {Σ (number of diseased stems by index × index) / (number of surveyed stems × 4)} × 100
上記式中、「指数」とは発病程度別階級指数を意味する。同指数は図1に示す指標に沿って評価した。具体的な評価基準は以下の通りである。0:発病無し(健全),1:地際茎部に病斑をわずかに認める(萎凋無し),2:地際部に病斑が認められ,葉の一部に萎凋が生じる。3:地際部に病斑を生じ葉全体に萎凋を生じる。4:枯死。 In the above formula, “index” means a class index according to the severity of disease. The index was evaluated according to the index shown in FIG. Specific evaluation criteria are as follows. 0: No disease (healthy), 1: Slight lesions are observed on the ground stem (no wilt), 2: Spots are observed on the ground, and some leaves are wilted. 3: Causes lesions on the border and causes wilt throughout the leaves. 4: Death.
発病度に基づく防除価は下記式に従って求めた。
発病度に基づく防除価=100×(無処理区の発病度−処理区の発病度)/(無処理区の発病度)
The control value based on the severity was determined according to the following formula.
Control value based on disease severity = 100 × (morbidity of untreated area−morbidity of treated area) / (morbidity of untreated area)
(結果)
候補となる220菌株を20菌株ずつに分けて試験を行った。そのうち一回の実験結果を表1に示す。
(result)
220 candidate strains were divided into 20 strains and tested. Table 1 shows the results of one experiment.
本実験から、T04398菌株、T04401菌株、T04464菌株、T-3菌株等の9菌株が茎疫病に対して防除効果があることが示された。 From this experiment, it was shown that 9 strains, such as T04398 strain, T04401 strain, T04464 strain, T-3 strain, etc. have the control effect with respect to stem rot.
参考例2: 野外設置コンクリート枠ポット条件下での選抜
(ポット)
野外設置コンクリート製枠ポット(50cm×50cm×50cm)を使用した。1処理あたり3ポット供試した。土壌は、クロルピクリン剤(30L/10a)により土壌消毒したクロボク土を用いた。
Reference example 2: Selection under the condition of outdoor installation concrete frame pot (pot)
A field-installed concrete frame pot (50 cm x 50 cm x 50 cm) was used. Three pots were tested per treatment. As the soil, Kuroboku soil sterilized with chloropicrin (30L / 10a) was used.
(茎疫病菌)
茎疫病菌としてPhytopthora soja S-2 (兵庫農技センター分離株)を用いた。本茎疫病菌を、ふすま・バーミキュライト培地で25℃暗黒下40日間培養した。このように培養した茎疫病菌培養物500gを、細かくほぐした後各ポット土壌の表層(0-10cm間)に混和して接種した。
(Pesticide)
Phytopthora soja S-2 (Hyogo Agricultural Technology Center isolate) was used as a stem rot fungus. The stem rot fungus was cultured in a bran / vermiculite medium at 25 ° C. in the dark for 40 days. 500 g of the cultivated pesticidal fungus thus cultured was finely loosened and mixed with the surface layer (between 0-10 cm) of each pot soil and inoculated.
(試験方法)
温室内ポット試験で選抜した9菌株をふすま・バーミキュライト培地で10日間培養した培養物500gを、各ポットの土壌表層(0-10cm間)に混和し、ダイズ(品種タチナガハ)を1箇所 3粒、ポットあたり15箇所播種した。
(Test method)
500 g of 10 strains cultured in bran / vermiculite medium for 9 days selected in the greenhouse pot test were mixed in the soil surface of each pot (between 0-10 cm), and 3 soybeans (variety Tachinagaha) in one place, 15 seeds were sown per pot.
播種20日後に出芽阻害率(出芽前発病率)を調べると共に、収穫時に発病程度を調べ、下記式に従って発病度を求めた。
発病度={Σ(程度別発病茎数×指数)/(調査茎数×4)}×100
20 days after sowing, the germination inhibition rate (pre-emergence disease incidence rate) was examined, the degree of disease occurrence was examined at the time of harvest, and the disease incidence was determined according to the following formula.
Disease severity = {Σ (number of diseased stems by index × index) / (number of surveyed stems × 4)} × 100
上記式中、「指数」とは発病程度別階級指数を意味する。同指数の評価基準は以下の通りである。0:発病無し(健全),1:地際茎部に病斑をわずかに認める(萎凋無し)、2:株全体の1/3以下に枯れ・萎凋を生じる。3:株全体の1/3〜2/3の枯れ・萎凋を生じる。4:株全体の2/3以上の枯れ・萎凋を生じる。
発病度に基づく防除価=100×(無処理区の発病度−処理区の発病度)/(無処理区の発病度)
In the above formula, “index” means a class index according to the severity of disease. The evaluation criteria for the index are as follows. 0: No disease (healthy) 1: Slight lesions are observed on the ground stem (no wilt), 2: Withering or wilt occurs in 1/3 or less of the entire strain. 3:
Control value based on disease severity = 100 × (morbidity of untreated area−morbidity of treated area) / (morbidity of untreated area)
(結果)
結果を図2に示す。図2において星印のついたT04398菌株、T04401菌株、T04464菌株及びT-3菌株が特に高い防除価を示した。
(result)
The result is shown in figure 2. In FIG. 2, the T04398, T04401, T04464 and T-3 strains marked with an asterisk showed particularly high control values.
参考例3: 4菌株の同定
(顕微鏡による観察)
T-3菌株は、2%マルツ培地上における分生子柄の分岐様式およびフィアライドの着生様式はともに規則的でフィアライドは分岐に単生する。分生胞子は濃緑色球形〜亜球形を呈し、表面には小突起を呈しラフである。大きさは3.0〜4.5×2.8〜3.8μmである。
Reference Example 3: Identification of 4 strains (observation with a microscope)
In the T-3 strain, both the conidial pattern branching pattern and the phialide settlement pattern on a 2% Marz medium are regular, and the phialide grows monotonically. The conidia are dark green spheres to subspheres, and are rough with small protrusions on the surface. The size is 3.0 to 4.5 × 2.8 to 3.8 μm.
一方、T04398菌株、T04401菌株、T04464菌株は2%マルツ培地上において箒状の分生子柄を形成しその最先端のフィアライド上に分生胞子塊を形成する。分生子柄は無色直立し、単純または分岐する。数個のセプタ(隔膜)を有する。分生胞子は濃緑色球形、亜球形〜倒卵形を呈し、大きさは3.3〜5.8×3.0〜4.0μmである。 On the other hand, T04398 strain, T04401 strain, and T04464 strain form rod-like conidia on a 2% Marz medium and form a conidial spore mass on the most advanced phialide. The conidia pattern is colorless and upright, simple or branched. It has several septa. The conidia are dark green spheres, sub-spheres to fallen eggs, and have a size of 3.3 to 5.8 × 3.0 to 4.0 μm.
(培養性質)
T-3菌株は、PSA培地において10℃〜30℃の範囲で生育し、25℃で最適生育温度(菌糸生育速度219.8mm/日)を有し、10℃での菌糸生育速度は3.9 mm/日であった。また30℃では4.4 mm/日の菌糸生育速度を示した。PSA培地25℃培養2日後の菌叢は白く平滑に伸長し、気中菌糸は少なかった。菌糸細胞には平均3.0個の核を有した。緑色の分生胞子を良好に形成した。
(Culture properties)
The T-3 strain grows in the range of 10 ° C to 30 ° C in the PSA medium, has an optimum growth temperature (mycelium growth rate 219.8 mm / day) at 25 ° C, and the mycelial growth rate at 10 ° C is 3.9 mm / It was a day. At 30 ° C., the mycelium growth rate was 4.4 mm / day. The bacterial flora after 2 days of culture at 25 ° C. in PSA medium expanded smoothly and white, and there were few aerial hyphae. Mycelium cells had an average of 3.0 nuclei. Green conidia were formed well.
一方、T04398菌株、T04401菌株、T04464菌株は、PSA培地において10℃〜35℃の範囲で生育し、30℃で最適生育温度(菌糸生育速度219.8mm/日)を有し、10℃では菌叢の生育はほとんど認められなかった。30℃では20.8〜31.4 mm/日の菌糸生育速度を示した。また、35℃では9.4〜14.6 mm/日の菌糸生育速度を示した。PSA培地25℃培養2日後の培地は淡黄色から褐色に着色。菌叢は白く平滑に伸長し、気中菌糸は少なかった。菌糸細胞には平均2.0〜2.6個の核を有した。また、緑色の分生胞子を形成し分生胞子形成は良好であった。 On the other hand, the strains T04398, T04401, and T04464 grow on PSA medium in the range of 10 ° C to 35 ° C and have an optimum growth temperature (mycelium growth rate 219.8 mm / day) at 30 ° C. Little growth was observed. At 30 ° C., the mycelial growth rate was 20.8 to 31.4 mm / day. In addition, the mycelial growth rate was 9.4 to 14.6 mm / day at 35 ° C. The medium after 2 days of PSA culture at 25 ° C is colored from pale yellow to brown. The flora was white and extended smoothly, and there were few aerial hyphae. Mycelium cells had an average of 2.0-2.6 nuclei. In addition, green conidia were formed and conidia formation was good.
以上の性質から、T-3菌株は新規株と特定された。本株はリファイの分類(Rifai1969)に従いトリコデルマ・ヴィリデ(Trichoderma viride)と同定し、トリコデルマ・ヴィリデT-3菌株として独立行政法人製品評価技術基盤機構特許微生物寄託センターに寄託申請された(受領番号 NITE AP-279)。同様に、T04398菌株、T04401菌株、T04464菌株も新規株と特定された。これら菌株は、Bisstの分類(Bisset1991)に従いトリコデルマ・ヴィレンス(Trichoderma virens)と同定し、トリコデルマ・ヴィレンスT04398菌株(受領番号 NITE AP-280)、T04401菌株(受領番号 NITE AP-281)、T04464菌株(受領番号 NITE AP-282)として独立行政法人製品評価技術基盤機構特許微生物寄託センターに寄託申請された。 From the above properties, the T-3 strain was identified as a new strain. This strain was identified as Trichoderma viride according to the Riffai classification (Rifai 1969), and was filed as an application for deposit with the National Institute of Technology and Evaluation of the National Institute of Technology and Evaluation as the Trichoderma viride T-3 strain (reception number NITE). AP-279). Similarly, T04398 strain, T04401 strain, and T04464 strain were also identified as new strains. These strains were identified as Trichoderma virens according to the Bisst classification (Bisset 1991), and Trichoderma virens strain T04398 (reception number NITE AP-280), T04401 strain (reception number NITE AP-281), T04464 strain ( Application No. NITE AP-282) was filed with the National Institute for Product Evaluation Technology Patent Microorganism Depositary.
実施例1: 圃場試験
参考例で選抜された4種のトリコデルマ属菌を用いて圃場試験を行った。
(場所)
試験は兵庫県篠山市内の農家圃場で行った。この圃場は茎疫病自然発生圃場であり、水田転換畑である。
(試験区)
1区の大きさは1.5m×5.4mであり、1区あたり1条12株のダイズを用いた。
(供試トリコデルマ属菌)
T04398菌株、T04401菌株、T04464菌株及びT-3菌株の4菌株を試験した。
Example 1: A field test was carried out using four species of the genus Trichoderma selected in the field test reference example.
(place)
The test was conducted at a farmer's field in Sasayama City, Hyogo Prefecture. This field is a field where stalk disease occurs naturally and is a paddy field conversion field.
(Test area)
The size of 1 ward is 1.5m x 5.4m, and 1 shoot and 12 soybeans per ward were used.
(Test Trichoderma spp.)
Four strains T04398, T04401, T04464 and T-3 were tested.
これらの菌株はいずれもふすま・バーミキュライト培地で25℃暗黒下30日間培養したものを用いた。培養された菌株を小型管理機で土壌表面(0-10cm間)に250kg/10aの割合で混和した。 All of these strains were cultured in a bran / vermiculite medium at 25 ° C. in the dark for 30 days. The cultured strain was mixed at a rate of 250 kg / 10a on the soil surface (between 0-10 cm) with a small control machine.
(ダイズ品種)
ダイズ品種として丹波黒大豆を使用した。
ダイズは畝幅150cm、株間45cm間隔で1箇所当たり3粒を圃場に直まきした。播種15日後に1本立ちとした。
(方法)
平成17年6月8日に、トリコデルマ属菌を上記手順で混和した土壌にダイズを播種した。
(Soybean variety)
Tamba black soybean was used as a soybean variety.
Soybeans were directly planted on the field at 3 centimeters per paddy at 150 cm wide and 45 cm apart. One stand was established 15 days after sowing.
(Method)
On June 8, 2005, soybean was sown in soil mixed with Trichoderma sp.
茎疫病の発生が認められ出した平成17年8月25日、9月2日、及び9月29日の3回、発病調査を行った。 The disease was investigated three times on August 25, 2005, September 2, and September 29, when the outbreak of stem plague was observed.
播種15日後に出芽阻害率(出芽前発病率)を調べると共に、経時的に発病程度を調べ、下記式に従って発病度を求めた。
発病度={Σ(程度別発病茎数×指数)/(調査茎数×4)}×100
15 days after sowing, the budding inhibition rate (pre-emergence morbidity rate) was examined, and the morbidity was examined over time, and the morbidity was determined according to the following formula.
Disease severity = {Σ (number of diseased stems by index × index) / (number of surveyed stems × 4)} × 100
上記式中、「指数」とは発病程度別階級指数を意味する。同指数の評価基準は以下の通りである。0:発病無し(健全)、1:地際茎部に病斑をわずかに認める(萎凋無し)、2:株全体の1/3以下に枯れ・萎凋を生じる。3:株全体の1/3〜2/3の枯れ・萎凋を生じる。4:株全体の2/3以上の枯れ・萎凋を生じる。
In the above formula, “index” means a class index according to the severity of disease. The evaluation criteria for the index are as follows. 0: No disease (healthy) 1: Slight lesions are observed on the stem of the ground (no wilt), 2: Withering / wilt occurs in 1/3 or less of the entire strain. 3:
また、発病度に基づく防除価を参考例1及び2と同様に算出した。
平成17年9月29日の最終調査の結果を図3に示す。図3において無処理区の発病率は16.9%、発病度は12.7であった。4菌株とも高い茎疫病防除効果を有することが明らかとなった。
Moreover, the control value based on the disease severity was calculated in the same manner as in Reference Examples 1 and 2.
The results of the final survey on September 29, 2005 are shown in Figure 3. In FIG. 3, the disease rate in the untreated area was 16.9%, and the disease severity was 12.7. All four strains were found to have a high control effect on stem rot.
実施例2: トリコデルマ資材の育苗培土処理による茎疫病防除効果
試験は野外設置コンクリート製枠ポット(50cm×50cm×50cm)を使用した。1処理あたり3ポット供試した。土壌は、クロルピクリン剤(30L/10a)により土壌消毒したクロボク土を用いた。
Example 2: A field-installed concrete frame pot (50 cm x 50 cm x 50 cm) was used in the test for controlling the stalk disease effect of Trichoderma material by raising seedlings . Three pots were tested per treatment. As the soil, Kuroboku soil sterilized with chloropicrin (30L / 10a) was used.
(茎疫病菌)
ダイズ茎疫病菌はS-2菌株(兵庫農技センター分離株)を使い、平成17年6月20日接種した。茎疫病菌は、ふすま・バーミキュライト培地で25℃暗黒下で40日間培養し、1ポット(50cm×50cm)当たり500gの培養したダイズ茎疫病菌S-2菌株の培養物を土壌表層土壌(10cm間)に混和した。
(Pesticide)
The soybean stem fungus was inoculated on June 20, 2005 using the S-2 strain (Hyogo Agricultural Technology Center isolate). Stem fungus is cultured in bran / vermiculite medium at 25 ° C in the dark for 40 days, and 500g per 1 pot (50cm x 50cm) of cultured soybean stem fungus S-2 is cultured on soil surface soil (between 10cm ).
(トリコデルマ属菌)
トリコデルマ属菌:T04464、T04401、T04398、T-3の4菌株。T04464、T04401およびT04398菌株は資材化(出光興産試作)させ、播種時に0.5〜4.0/紙筒の割合で育苗培土に混和した。一方、T-3菌株では胞子形成がT04464、T04401およびT04398菌株と比べると悪いため、資材の代わりにふすま培養物を使い、資材と同量を使用した。
(Trichoderma spp.)
Trichoderma spp .: 4 strains T04464, T04401, T04398, T-3. T04464, T04401 and T04398 strains were made into materials (Idemitsu Kosan Prototype) and mixed with the seedling culture soil at a rate of 0.5 to 4.0 / paper tube at the time of sowing. On the other hand, since the sporulation of T-3 strain was worse than that of T04464, T04401 and T04398 strains, bran culture was used instead of the material, and the same amount as the material was used.
(耕種概要)
平成17年6月17日ダイズ(品種タチナガハ)を紙筒に各1粒播種後、10日間温室で育苗し、6月27日(月)コンクリート枠にそれぞれ9株移植した。調査は10月28日に発病程度を調べ、下記式に従って発病度を求めた。
発病度={Σ(程度別発病茎数×指数)/(調査茎数×4)}×100
(Cultivation overview)
June 17, 2005 Soybean (variety Tachinagaha) was sown in a paper tube, seeded in a greenhouse for 10 days, and on June 27 (Monday), 9 strains were transplanted into a concrete frame. The survey was conducted on October 28 to determine the severity of the disease, and the severity was determined according to the following formula.
Disease severity = {Σ (number of diseased stems by index × index) / (number of surveyed stems × 4)} × 100
上記式中、「指数」とは発病程度別階級指数を意味する。同指数の評価基準は以下の通りである。0:発病無し(健全)、1:地際茎部に病斑をわずかに認める(萎凋無し)、2:株全体の1/3以下に枯れ・萎凋を生じる。3:株全体の1/3〜2/3の枯れ・萎凋を生じる。4:株全体の2/3以上の枯れ・萎凋を生じる。
In the above formula, “index” means a class index according to the severity of disease. The evaluation criteria for the index are as follows. 0: No disease (healthy) 1: Slight lesions are observed on the stem of the ground (no wilt), 2: Withering / wilt occurs in 1/3 or less of the entire strain. 3:
また、発病度に基づく防除価を参考例1及び2と同様に算出した。
結果を表2に示す。4菌株いずれも高い防除価を示した。T04464株については防除価が特に高く有用な菌株であると言える。
Moreover, the control value based on the disease severity was calculated in the same manner as in Reference Examples 1 and 2.
The results are shown in Table 2. All four strains showed high control values. The T04464 strain is a useful strain with a particularly high control value.
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