JP2008061530A - Insect-pathogenic microorganism and method for controlling harmful organism by using the same - Google Patents
Insect-pathogenic microorganism and method for controlling harmful organism by using the same Download PDFInfo
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- JP2008061530A JP2008061530A JP2006240633A JP2006240633A JP2008061530A JP 2008061530 A JP2008061530 A JP 2008061530A JP 2006240633 A JP2006240633 A JP 2006240633A JP 2006240633 A JP2006240633 A JP 2006240633A JP 2008061530 A JP2008061530 A JP 2008061530A
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Abstract
Description
本発明は、アブラムシ類、コナジラミ類及びアザミウマ類に対して同時に高い病原性を有する昆虫病原性微生物、並びに新規バーティシリウム・レカニ株群に関する。 The present invention relates to an entomopathogenic microorganism having high pathogenicity simultaneously against aphids, whiteflies and thrips, and a novel Verticillium lecani strain group.
植物の病害虫の防除には、化学物質を用いた防除の開発が進展し、現在、化学殺虫剤や殺菌剤による防除がその主流を占めている。しかしながら、化学物質を用いた農薬は、その効果が優れている反面、人畜に対して毒性を有しているものや、自然環境に残留して、他の生態系に影響を及ぼすものがある。また化学物質を用いた農薬では、長期間の使用によって当該化学物質に対する抵抗性を獲得した病害虫が出現したり、本来天然に存在した天敵まで殺して逆に病害虫の発生する環境を創出してしまうことがある。 For the control of plant pests, the development of control using chemical substances has progressed, and currently control by chemical insecticides and fungicides is the mainstream. However, pesticides using chemical substances have excellent effects, but some are toxic to human livestock, while others remain in the natural environment and affect other ecosystems. In addition, with pesticides that use chemical substances, pests that have acquired resistance to the chemical substance will appear after long-term use, or even natural enemies that originally existed will be killed, creating an environment where pests will occur. Sometimes.
このような化学物質を用いた農薬に対して、他の生態系への影響を極力抑え、防除の目的とする病害虫のみを特異的に駆除する手段として、生物を用いる方法が研究されてきた。このような生物を用いた病害虫の防除方法の一つとして、微生物を用いた病害虫の防除方法がある。例えば、糸状菌のような微生物を用いて、害虫を防除する方法として、ボーベリア（Ｂｅａｕｖｅｒｉａ）属菌、メタリジウム（Ｍｅｔｈａｒｈｉｚｉｕｍ）属菌、ヒルステラ（Ｈｉｒｓｕｔｅｌｌａ）属菌、ノムラエ（Ｎｏｍｕｒａｅ）属菌、及びバーティシリウム（Ｖｅｒｔｉｃｉｌｌｉｕｍ）属菌、等の微生物を用いて、昆虫等の害虫を防除する方法が知られている。ボーベリア属菌やメタリジウム属菌は、鱗翅目、鞘翅目の害虫の防除に用いられ、ヒルステラ属菌は、サビダニ類の防除に用いられ、ノムラエ属菌は、鱗翅目の害虫の防除に用いられ、バーティシリウム属菌は、半翅目、総翅目及びハダニ類の害虫の防除に用いられている。 For agricultural chemicals using such chemical substances, methods that use organisms have been studied as a means to specifically control only the pests targeted for control while minimizing the impact on other ecosystems. As one method for controlling pests using such organisms, there is a method for controlling pests using microorganisms. For example, as a method for controlling pests using microorganisms such as filamentous fungi, the genus Beauveria, the genus Metalithium, the genus Hirstella, the genus Nomurae, and the vertici There is known a method for controlling pests such as insects using microorganisms such as bacteria belonging to the genus Verticillium. Boberia spp. And Metalidium spp. Are used to control Lepidoptera and Coleoptera pests, Hilstella spp. Are used to control Rust mites, and Nomurae spp. Are used to control Lepidoptera pests. Verticillium spp. Are used to control pests of the order Hemiptera, Panacea and Spider mites.
上記に用いられている菌株の中でバーティシリウム・レカニはネイトナー博士によって１８６１年にスリランカでコーヒー樹を加害するカイガラムシの一種Ｌｅｃａｎｉｕｍ ｃｏｆｆｅａｅの寄生菌として報告され、その後、多種類の昆虫やダニ類から分離されている。またこの菌株は、熱帯と温帯地域に広く分布することが知られている。我が国では、北沢らが１９８４年に、オンシツコナジラミＴｒｉａｌｅｕｒｏｄｅｓ ｖａｐｏｒａｒｉｏｒｕｍ、モモアカアブラムシＭｙｚｕｓ ｐｅｒｓｉｃａｅ、ワタアブラムシＡｐｈｉｓ ｇｏｓｓｙｐｉｉ、及びダイコンアブラムシＢｒｅｖｉｃｏｒｙｎｅ ｂｒａｓｓｉｃａｅから本菌を初めて分離し、菌そうの特徴や寄主昆虫などから３種類の菌株の存在が明らかになっている。 Among the strains used above, Verticillium lecani was reported by Dr. Neytner as a parasitic fungus of Lecanium coffeae, a kind of scale insect that injured coffee trees in Sri Lanka in 1861, and then many kinds of insects and mites Has been separated from. This strain is known to be widely distributed in the tropics and temperate regions. In Japan, Kitazawa et al. First isolated three species of the main fungus such as the white-spotted whitefly Trialeurodes vaporiarum, the peach aphid Myzus persicae, the cotton aphid Aphis gossypii, and the main fungus from the main species, the radish aphid Brevicory brasicae. The existence of the strain is revealed.
バーティシリウム・レカニは分生子表面の粘質物により、昆虫の体表面に付着し、そこで菌糸を伸長させ繁殖する。体表面に付着した分生子は発芽し発芽管が宿主の表皮、クチクラを貫通して、宿主の体腔内に侵入する。侵入後、円筒状に分断された短い菌糸が出芽あるいは***を繰り返しながら体液中で増殖し、各組織、器官に侵入して栄養分を奪取する。死の直前に血液及びリンパ系中に、いわゆる、“菌糸体”を形成する。７〜１０日経過した後、多数の菌糸体が形成されると、感染した幼虫は死亡し、組織の破壊が始まる。 Verticillium recani adheres to the body surface of insects due to the mucus on the conidia surface, where it expands and reproduces mycelia. The conidia attached to the surface of the body germinate, and the germ tube penetrates the epidermis and cuticle of the host and enters the body cavity of the host. After the invasion, the short mycelium divided into cylinders grows in the body fluid while repeating budding or division, and invades each tissue and organ to take up nutrients. The so-called “mycelium” forms in the blood and lymphatic system just before death. When a large number of mycelium are formed after 7 to 10 days, the infected larvae die and tissue destruction begins.
バーティシリウム・レカニは、湿度が高い場合、更に成長し、クチクラを突き破り、虫体の表面に菌糸を伸ばし、２次的に分生子を形成する。菌糸はあらゆる方向に伸び、これから分生子柄（長さ：１６〜２０μｍ）を出す。各分生子柄の上に分生子形成細胞であるフィアライドが輪生し、この上に粘質物に包まれた分生子が球状に塊まってできる。粘質物は水に触れると溶け、分生子は菌糸から離れ、飛散し、広がっていく。 Verticillium regani grows further when humidity is high, breaks through the cuticle, extends mycelia onto the surface of the worm body, and forms conidia secondarily. The mycelium extends in all directions, from which a conidia pattern (length: 16 to 20 μm) is produced. On each conidia pattern, a phialide, which is a conidia-forming cell, rotates, and conidia encapsulated in a sticky material are formed in a spherical shape. The mucilage melts when it touches water, and the conidia separates from the mycelium, scatters and spreads.
高濃度の分生子液を散布すると、２〜３日で虫体表面に分生子形成がみられるが、分生子は最初に感染虫の脚や触覚にみられる。虫が死亡した後でない限り、胸部、腹部には殆ど分生子の形成はみられない。生きている虫の表面に分生子が形成されるのがこの菌の特徴であり、他の昆虫病原性糸状菌類にはみられない。低濃度の分生子液では虫体内の組織の破壊は起こらない。非常に高濃度の分生子液を散布すると４８時間以内に死亡する。このとき虫体表面は大量の菌糸と分生子で覆われているが、菌は体内組織に侵入していない。この死因は大量の分生子による物理的な損傷やショック、呼吸器官の閉塞による酸素不足と考えられている。湿度が非常に高く、菌にとって非常に好都合な条件であれば、自然に２次感染が起こることもあるが、通常はバーティシリウム・レカニの作用は伝搬によるのではなく、昆虫が分生子に直接触れることによる。 When a high concentration of conidia is sprayed, conidia formation is observed on the surface of the worm body in 2 to 3 days, but the conidia is first seen on the legs and touch of the infecting insect. There is almost no conidia formation in the chest and abdomen unless the insects have died. A characteristic of this fungus is the formation of conidia on the surface of living insects, which is not found in other entomopathogenic fungi. At low concentrations of conidia, tissue destruction in the worm body does not occur. If a very high concentration of conidia is sprayed, it will die within 48 hours. At this time, the surface of the worm body is covered with a large amount of hyphae and conidia, but the bacterium does not enter the body tissue. The cause of death is thought to be oxygen deficiency due to physical damage and shock caused by a large number of conidia and obstruction of respiratory organs. If the humidity is very high and the conditions are very favorable for the fungus, secondary infection may occur spontaneously, but the action of Verticillium regani is not usually due to transmission, but the insect becomes a conidia. By touching directly.
感染は若い幼虫に起こりやすいが、齢の進んだ幼虫や蛹にも感染する。環境が適温、多湿であれば、成虫にも感染する。
感染した成虫は、正常な虫と殆ど変わらず活動し、ほぼ通常の速度で次世代を生産し、健全な幼虫を生む。このように虫体に侵入する前に虫体の表面で、腐生的に生育すること、及び感染虫の体内に菌糸体が大量に増えてもかなり活発に活動することは、本菌の作用が毒素によるものでないことを示すと考えられている。バーティシリウム・レカニには昆虫寄生性や分生子の形状等が異なる各種の系統があり、これらは多くの昆虫に寄生している。土壌中にも存在するが、これは菌が寄生した昆虫またはその死体が土壌に混入したものと考えられている。植物、哺乳動物中には存在しない。地理的には、熱帯および温帯に広く分布している。日本でも各種の系統があることが報告されている。アブラムシに寄生する系統は北沢や増田らによって報告されている（北沢１９８４、増田１９９０）。
Infection is likely to occur in young larvae but also in older larvae and pupae. If the environment is appropriate temperature and humidity, adults can also be infected.
Infected adults act almost the same as normal insects, produce the next generation at almost normal speed, and produce healthy larvae. The fact that the fungus grows on the surface of the worm body before entering the worm body and that it is quite active even if the mycelium increases in the body of the infecting worm, the action of this bacterium is It is thought to indicate that it is not due to a toxin. Verticillium lecani has various strains that differ in insect parasitism and conidia shape, and these parasites parasitize many insects. It is also present in the soil, but it is thought that the insects parasitized by fungi or their dead bodies were mixed in the soil. It does not exist in plants and mammals. Geographically, it is widely distributed in the tropics and temperate zones. It is reported that there are various strains in Japan. Lines parasitic on aphids have been reported by Kitazawa and Masuda et al. (Kitazawa 1984, Masuda 1990).
バーティシリウム・レカニの宿主域は、菌株によって非常に偏在しており、アブラムシ類に最もよく寄生する菌株、コナジラミ、アザミウマ類に最もよく寄生する菌株など株ごとに異なる。このため、現在、生物農薬として市販されているバーティシリウム・レカニを有効成分とする製品は、アブラムシ類用、コナジラミ類用で異なる菌株が用いられている。このため、トマト、ピーマンなど野菜類でのアブラムシ類用、コナジラミ類用などの防除には別々の製品を利用しなければならない。 The host range of Verticillium lecani is highly ubiquitous depending on the strain and varies from strain to strain, such as the strain most parasitic on aphids, the strain most frequently parasitic on whiteflies and thrips. For this reason, different strains are currently used for aphids and whiteflies for products containing Verticillium lecani, which are commercially available as biological pesticides, as active ingredients. For this reason, separate products must be used for controlling aphids and whiteflies in vegetables such as tomatoes and peppers.
菌類における細胞融合技術は、酵母、麹菌、キノコ等でＰＥＧ（ポリエチレングリコール）法を用いた研究が盛んに行われている。また生物防除資材における細胞融合の利用は，植物病原菌の生物防除用トリコデルマ・ハージアナムで実施されており，他の植物病原菌に対しての拮抗力が親株のそれよりも強い株が得られている（Ｐｅ’ｅｒ＆Ｃｈｅｔ １９９０）．昆虫寄生菌ではボーベリア・バッシアーナ×ボーベリア・スルフレスセンス（Ｃｏｕｔｗａｕｄｉｅｒ ｅｔ ａｌ １９９６）や、メタリジウム・アニソプリエ（Ｓｉｌｖｅｉｒａ＆Ａｚｅｖｅｄｏ １９８７）等の報告があり、いずれも病原性の改良を目的としている。これらの細胞融合株を検出する際の遺伝的なマーカーとしては抗生物質耐性や栄養要求性を用いている。なお、後述する通り、本試験で実施したバーティシリウム・レカニの細胞融合には、不完全菌類の遺伝的なグルーピング（栄養体親和性群）を決定するのに用いる硝酸体窒素利用能変異株（ｎｉｔ ｍｕｔａｎｔ）を用いた。
本発明の課題は、昆虫病原性糸状菌バーティシリウム・レカニにおいて、広い宿主域で病原性を示す微生物菌株を創出することである。また、本発明の更なる課題は、この微生物菌株を有効成分として、広い宿主域で病原性を示す微生物農薬を提供することにある。 An object of the present invention is to create a microbial strain exhibiting pathogenicity in a wide host range in the entomopathogenic filamentous fungus Verticillium regani. Moreover, the further subject of this invention is providing the microbial pesticide which shows pathogenicity in a wide host range by using this microbial strain as an active ingredient.
本発明者らは、上記課題を達成するために、葉面等の植物体表面での定着能力をもつ糸状菌バーティシリウム・レカニ Ｂ−２株（ＭＡＦＦ２３８４２９）（特許公開２００３−３３５６１２（参照により本明細書に組み込む））と、アブラムシ類用、コナジラミ類用、アザミウマ類用でそれぞれ高い活性を有するバーティシリウム・レカニＩＭＩ １７９１７２株、又はバーティシリウム・レカニＩＭＩ ２６８３１７とを融合させて、特定のＤＮＡバンドパターンを示す、アブラムシ類、コナジラミ類及びアザミウマ類に対して同時に高い病原性を有する菌株を創出し、本発明の完成に至った。本発明の菌株は、植物体表面定着能力を有し、且つ広い宿主域で病原性を示すため、ひとつの昆虫病原性糸状菌によって、アブラムシ類用、コナジラミ類用及びアザミウマ類など広範囲の害虫を防除できる微生物農薬を提供することができる。 In order to achieve the above-mentioned problems, the present inventors have established a filamentous fungus Verticillium regani B-2 strain (MAFF238429) (patent publication 2003-335612) (with reference). Incorporated herein)) and Verticillium regani IMI 179172 strain or Verticillium regani IMI 268317, which have high activity for aphids, whiteflies, and thrips, respectively, A strain having a high pathogenicity against aphids, whiteflies and thrips having the same DNA band pattern was simultaneously created, and the present invention was completed. Since the strain of the present invention has a plant surface fixing ability and exhibits pathogenicity in a wide host range, a wide range of pests such as for aphids, whiteflies, and thrips can be obtained by one entomopathogenic fungus. A microbial pesticide that can be controlled can be provided.
具体的には、本発明は、アブラムシ類、コナジラミ類及びアザミウマ類に対して同時に高い病原性を有し、図１に示すＤＮＡバンドパターンを示すバーティシリウム・レカニ株群又はＢ−２とマイコタールの融合株群でＤＮＡタイプはＢ−２型を示す菌株群或いはその変異株を含む有害生物防除剤からなる。 Specifically, the present invention relates to a group of Verticillium lecani strains or B-2 and mycotal which have high pathogenicity simultaneously against aphids, whiteflies and thrips and which show the DNA band pattern shown in FIG. The DNA type is composed of a pest control agent containing a strain group showing B-2 type or a mutant strain thereof.
本発明の新規なアブラムシ類、コナジラミ類及びアザミウマ類に対して同時に高い病原性を有し、図１に示すＤＮＡバンドパターンを示すバーティシリウム・レカニ株群は、（独）製品評価技術基盤機構・特許微生物寄託センターに受託番号（バーティシリウム・レカニ２ａＦ１ ＦＥＲＭ ＡＰ−２０９８０； バーティシリウム・レカニ２ａＦ４ ＦＥＲＭ ＡＰ−２０９８１；バーティシリウム・レカニ２ａＦ２６ ＦＥＲＭ ＡＰ−２０９８２；バーティシリウム・レカニ２ａＦ４３ ＦＥＲＭ ＡＰ−２０９８３）として寄託されている。バーティシリウム・レカニ株群は、一般糸状菌の生育可能な培地を用い、通常の発酵手法を用いて培養が可能である。培地としては、麦芽エキス寒天培地（麦芽エキス３０．０ｇ 大豆ペプトン３．０ｇ 寒天１５．０ｇ、ｐＨ５．６）などが挙げられ、至適生育湿度８０％以上、至適生育温度２０〜２５℃で培養できる。 The novel aphids, whiteflies and thrips of the present invention, which are simultaneously highly pathogenic and have the DNA band pattern shown in FIG.・ Registration number (Verticillium regani 2aF1 FERM AP-20980; Verticillium regani 2aF4 FERM AP-20981; Verticillium regani 2aF26 FERM AP-20982; Verticillium regani 2aF43 FERMAP FERMAP FERMAP -20983). The Verticillium lecani strain group can be cultured using a normal fermentation technique using a medium capable of growing general filamentous fungi. Examples of the medium include malt extract agar medium (malt extract 30.0 g soybean peptone 3.0 g agar 15.0 g, pH 5.6) and the like, at an optimum growth humidity of 80% or more and at an optimum growth temperature of 20 to 25 ° C. Can be cultured.
炭素源としては、グルコース、フルクトース、サッカロース、マルトース、糖蜜、可溶性デンプン、コーンスターチなどが利用できる。窒素源としては、塩化アンモニウム、硫酸アンモニウム、尿素、酵母エキス、ペプトン、大豆粉、カゼインなどが利用できる。さらに、その他の無機塩類、ビタミンなどとして、ＮａＨ２ＰＯ４、Ｋ２ＨＰＯ４、ＭｎＳＯ４、ＦｅＳＯ４、ＭｇＳＯ４，ＮａＣｌ，糖蜜、酵母エキス、エビオス（ビタミン剤）などを添加することが好ましい。ｐＨは６〜８が好ましく、培養温度は２０〜２５℃が好ましく、培養時間は２４〜１２０時間が好ましい。培養方法は、通気撹拌培養等の好気的条件によるものが好ましい。 As the carbon source, glucose, fructose, saccharose, maltose, molasses, soluble starch, corn starch and the like can be used. As the nitrogen source, ammonium chloride, ammonium sulfate, urea, yeast extract, peptone, soybean flour, casein and the like can be used. Further, as other inorganic salts and vitamins, it is preferable to add NaH 2 PO 4 , K 2 HPO 4 , MnSO 4 , FeSO 4 , MgSO 4 , NaCl, molasses, yeast extract, shrimp (vitamin) and the like. The pH is preferably 6-8, the culture temperature is preferably 20-25 ° C., and the culture time is preferably 24-120 hours. The culture method is preferably based on aerobic conditions such as aeration and agitation culture.
培養後、培養液から昆虫病原性糸状菌を分離する場合、通常の遠心分離法、濾過法等が利用できる。また、上記の新規なアブラムシ類、コナジラミ類及びアザミウマ類に対して同時に高い病原性を有し、図１に示すＤＮＡバンドパターンを示すバーティシリウム・レカニ株群を元菌株として自然または誘発突然変異により、上記菌株と同様の特性を有する変異株を得て、本発明による有害生物防除剤の有効成分として用いることができる。これらの変異株を調整する方法としては、従来知られている慣用の方法、例えば元菌株を紫外線照射あるいはＮ−メチル−Ｎ’−ニトロ−Ｎ−ニトロソグアニジン（ＮＴＧ）等の薬剤による人工突然変異処理を施して、スキムミルク等を含む寒天培地に広げ、生育してくる菌株の中からコロニーのまわりに形成されるクリアゾーンがより大きいコロニーを選抜し、昆虫病原性の高い優れた菌株を選別する方法を用いることができる。 When the entomopathogenic filamentous fungi are separated from the culture solution after culturing, ordinary centrifugation, filtration, etc. can be used. In addition, the above-mentioned novel aphids, whiteflies and thrips are simultaneously highly pathogenic and naturally or induced mutations using the Verticillium lecani strain group having the DNA band pattern shown in FIG. Thus, a mutant having the same characteristics as the above strain can be obtained and used as an active ingredient of the pest control agent according to the present invention. As a method for preparing these mutant strains, conventionally known methods, for example, the original strain is irradiated with ultraviolet rays or artificial mutations by drugs such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) are used. Apply to treatment and spread on agar medium containing skim milk etc., select colonies with larger clear zone around colonies from growing strains, and select excellent strains with high entomopathogenicity The method can be used.
本発明での新規なアブラムシ類、コナジラミ類及びアザミウマ類に対して同時に高い病原性を有し、図１に示すＤＮＡバンドパターンを示すバーティシリウム・レカニ株群を有効成分とした有害生物防除剤を作成する場合、一般農薬と同様に水和剤、粒剤、粉剤、フロアブル剤などの任意な剤型として作成することができる。これらは、それぞれの剤型にふさわしい担体、例えば、ロウ石、タルク、カオリン、炭酸カルシウム、ベントナイト、珪石粉、石灰石粉末、酸性白土、珪藻土類粉末、石膏、軽石粉末、貝殻類粉末、雲母粉末、及びコロイド性含水珪酸ソーダなどの鉱物質粉末、並びに／或いは水、緩衝液などの水溶液と混合して用いられ、好ましくは、アルキルベンゼンスルホネート、及びアルキルスルホネート等の固着剤、ポリオキシエチレン（ＰＯＥ）アルキルエーテル、ＰＯＥアルキルフェニルエーテル、ＰＯＥジアルキルフェニルエーテル、ＰＯＥアルキルアミン、及びジアルキルスルホサクシネート等の湿潤剤、アルキルサルフェート、ＰＯＥアルキルエーテルサルフェート、ＰＯＥアルキルフェニルエーテルサルフェート、ＰＯＥベンジル化（あるいはサルチル化）フェニル（またはフェニルフェニル）エーテルサルフェート、パラフィン（アルカン）スルホネート、アルファオレフィンスルホネート（ＡＯＳ）、アルキルベンゼンスルホネート、モノまたはジアルキルナフタレンスルホネート、ナフタレンスルホネート・ホルマリン縮合物、アルキルジフェニルエーテルジスルホネート、リグニンスルホネート、ＰＯＥアルキルエーテルスルホコハク酸ハーフエステル、及びＰＯＥベンジル（あるいはスチリル化）フェニル（またはフェニルフェニル）エーテルフォスフェート等の分散剤、並びに／或いはパラオキシ安息香酸誘導体、サリチルアニライド、１，２−ベンズイソチアゾリン−３−オン、テトラフタロニトリル（ＴＰＮ）、２−ニトロブロモ等の防黴剤を添加して用いられる。 A novel pest control agent comprising the Verticillium lecani strain group which has high pathogenicity at the same time for the novel aphids, whiteflies and thrips in the present invention and which exhibits the DNA band pattern shown in FIG. In the case of preparing a pesticide, it can be prepared as an arbitrary dosage form such as a wettable powder, a granule, a powder, a flowable agent and the like in the same manner as general agricultural chemicals. These are carriers suitable for each dosage form, such as wax, talc, kaolin, calcium carbonate, bentonite, quartzite powder, limestone powder, acid clay, diatomaceous earth powder, gypsum, pumice powder, shellfish powder, mica powder, And / or a mineral powder such as colloidal hydrous sodium silicate and / or mixed with an aqueous solution such as water or a buffer, and preferably a sticking agent such as alkylbenzene sulfonate and alkyl sulfonate, polyoxyethylene (POE) alkyl Wetting agents such as ether, POE alkyl phenyl ether, POE dialkyl phenyl ether, POE alkyl amine, and dialkyl sulfosuccinate, alkyl sulfate, POE alkyl ether sulfate, POE alkyl phenyl ether sulfate, POE benzylation ( Or salylated) phenyl (or phenylphenyl) ether sulfate, paraffin (alkane) sulfonate, alpha olefin sulfonate (AOS), alkyl benzene sulfonate, mono- or dialkyl naphthalene sulfonate, naphthalene sulfonate-formalin condensate, alkyl diphenyl ether disulfonate, lignin sulfonate Dispersants such as POE alkyl ether sulfosuccinic acid half ester and POE benzyl (or styryl) phenyl (or phenylphenyl) ether phosphate, and / or paraoxybenzoic acid derivatives, salicylanilide, 1,2-benzisothiazoline- Used by adding antifungal agents such as 3-one, tetraphthalonitrile (TPN), 2-nitrobromo It is.
また、本発明の、新規なアブラムシ類、コナジラミ類及びアザミウマ類に対して同時に高い病原性を有し、図１に示すＤＮＡバンドパターンを示すバーティシリウム・レカニ株群を有効成分とする有害生物防除剤を作成する場合、本発明の菌株を単一の有効成分とするのではなく、他の有害生物に有効な除草剤、各種殺虫剤、殺菌剤、植物生長調節剤または効果を助長させる共力剤、誘引剤さらには他の効用を目的とする植物栄養剤、肥料等を混合することも可能である。本発明のバーティシリウム・レカニ株群を有効成分とする有害生物防除剤では、バーティシリウム・レカニ２ａＦ４３株（寄託番号：ＦＥＲＭ ＡＰ−２０９８３）、バーティシリウム・レカニ２ａＦ４株（受託番号：ＦＥＲＭ ＡＰ−２０９８１）、バーティシリウム・レカニ２ａＦ２６株（受託番号：ＦＥＲＭ ＡＰ−２０９８２）及び／又はバーティシリウム・レカニ２ａＦ１株（受託番号：ＦＥＲＭ ＡＰ−２０９８０）の分生胞子粉末（１ｘ１０６／ｇ、好ましくは１ｘ１０６／ｇ〜１ｘ１０８／ｇ）を、全成分中、１０〜９９％、好ましくは４０〜９０％含有することが適当であるが、対象有害生物、栽培作物、使用方法、使用時期等に応じて、有効成分含有量は調整される。
ご確認下さい。
In addition, the pests of the present invention that have high pathogenicity against the novel aphids, whiteflies and thrips at the same time, and whose active ingredient is the Verticillium lecani strain group having the DNA band pattern shown in FIG. When preparing a control agent, the strain of the present invention is not a single active ingredient, but a herbicide effective for other pests, various insecticides, fungicides, plant growth regulators or co-promoting effects. It is also possible to mix a power agent, an attractant, and other plant nutrients and fertilizers for other purposes. In the pest control agent comprising the Verticillium lecani strain group of the present invention as an active ingredient, Verticillium lecani 2aF43 strain (deposit number: FERM AP-20983), Verticillium lecani 2aF4 strain (deposit number: FERM) AP-20981), Verticillium regani 2aF26 strain (Accession number: FERM AP-20982) and / or Verticillium regani 2aF1 strain (Accession number: FERM AP-20980) conidial spores (1 × 10 6 / g) , Preferably 1 × 10 6 / g to 1 × 10 8 / g) is 10 to 99%, preferably 40 to 90%, of the total components, but the target pests, cultivated crops, methods of use and uses The active ingredient content is adjusted according to the time and the like.
Please check it.
本発明の方法で防除し得る害虫としてはアブラムシ類、コナジラミ類、アザミウマ類では以下の害虫が挙げられる。すなわち、具体的にはモモアカアブラムシ（Ｍｙｚｕｓ ｐｅｒｓｉｃａｅ）、ワタアブラムシ（Ａｐｈｉｓ ｇｏｓｓｙｐｉｉ）、ダイコンアブラムシ（Ｂｒｅｖｉｃｏｒｙｎｅ ｂｒａｓｓｉｃａｅ）、ジャガイモヒゲナガアブラムシ（Ａｕｌａｃｏｒｔｈｕｍ ｓｏｌａｎｉ）、ニセダイコンアブラムシ（Ｌｉｐａｐｈｉｓ ｅｌｉｓｉｍｉ）、チューリップヒゲナガアブラムシ（Ｍａｃｒｏｓｉｐｈｕｍ ｅｕｐｈｏｒｂｉａｅ）、及びネギアブラムシ（Ｎｅｏｔｏｘｏｐｔｅｒａ ｆｏｒｍｏｓａｎａ）等のアブラムシ類、オンシツコナジラミ（Ｔｒｉａｌｅｕｒｏｄｅｓ ｖａｐｏｒａｒｉｏｒｕｍ）、タバココナジラミ（Ｂｅｍｉｓｉａ ｔａｂａｃｉ）、及びミカンコナジラミ（Ｄｉａｌｅｕｒｏｄｅｓ ｃｉｔｒｉ）等のコナジラミ類、ヒラズハナアザミウマ（Ｆｒａｎｋｌｉｎｉｅｌｌａ ｉｎｔｏｎｓａ）、ミカンキイロアザミウマ（Ｆｒａｎｋｌｉｎｉｅｌｌａ ｏｃｃｉｄｅｎｔａｌｉｓ）、ミナミキイロアアミウマ（Ｔｈｒｉｐｓ ｐａｌｍｉ）、及びチャノキイロアザミウマ（Ｓｃｉｒｔｏｔｈｒｉｐｓ ｄｏｒｓａｌｉｓ）等のアザミウマ類を挙げることができるが、これらに限定されるものではなく、その他の半翅目昆虫、鞘翅目昆虫、鱗翅目昆虫、等翅目、例えばツマグロヨコバイ、ヒメトビウンカ、カクシュコガネムシ、イエバエ、ゴキブリなどにも適用可能である。 Pests that can be controlled by the method of the present invention include the following pests for aphids, whiteflies, and thrips. Specifically, the peach aphid (Myzus persicae), cotton aphid (Aphis gossypii), radish aphid (Brevicoryne brasicae), potato aphid aphid (Aulacorthum aphid) ), And aphids such as Neoxoptera formosa, Trialeurodes vaporiarum, Bemisia tabaci, and citrus whitefly (Dialeuridae) These include, but are not limited to, Miridia thrips (Franklinella intonsa), Citrus thrips (Franklinella occidentalis), Thrips palmi, etc. The present invention is not limited to other insects, and is also applicable to other Hemiptera insects, Coleoptera insects, Lepidoptera insects, etc., for example, the leafhopper leafhopper, tiger beetle, hornbill beetle, house fly, cockroach and the like.
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.
実施例１．
現在市場に流通しているバータレック水和剤（バーティシリウム・レカニＩＭＩ １７９１７２株）、マイコタール水和剤（バーティシリウム・レカニＩＭＩ ２６８３１７）に加え、本大学の温室より分離され、植物の葉面における定着能力の高さが知られている系統であるバーティシリウム・レカニＢ−２株（ＭＡＦＦ２３８４２９）（特許公開２００３−３３５６１２）を用いてプロトプラスト融合を行った。その際、融合によって生じたヘテロカリオンを視覚化するための遺伝子マーカーとしてｎｉｔ（硝酸体窒素利用能）変異株（Ｃｏｒｒｅｌｌ，ｅｔ ａｌ．１９８７）を用いた。それぞれの系統をＰＤＢ（ジャガイモ煎汁液体培地）で発芽胞子を得た後、ノボザイム１８８でプロトプラスト化した。遺伝的マーカーの異なるｎｉｔ変異体の組み合わせでＰＥＧ法によりプロトプラスト融合を実施した。その結果バータレックとマイコタール、バータレックとＢ−２、マイコタールとＢ−２の組み合わせからそれぞれ１２６（ＡａＦ１〜１０６、ＢｂＦ１〜２０）系統、４（２ＢＦ１〜４）系統、４４（２ａＦ１〜４４）系統の融合株が作出された。２ａＦ１，４，２６，４３（マイコタール×Ｂ−２）の４株ともＲＡＰＤ，ＥＲＩＣ−ＰＣＲではＢ−２型に偏る。２ａＦ１では親株には存在しない２７０ｂｐと４２０ｂｐのバンド（アンプリコン；ともに赤矢印）が存在し、Ｂ−２の７５０ｂｐのアンプリコン（黄色矢印）が欠失している。２ａＦ４では親株Ｂ−２の７５０ｂｐのアンプリコン（黄色矢印）が欠失している。２ａＦ２６では７５０ｂｐのアンプリコン（黄色矢印）が欠失しているが、Ｂ−２に存在する２００ｂｐのアンプリコン（白点線）が確認できる。２ａＦ４３では他の融合株にはないＭｙｃｏｔａｌの１２５０ｂｐ、１３５０ｂｐのアンプリコン（黄色点線）が確認でき、親株にはない新規の３００ｂｐのアンプリコン（赤矢印）が存在する（図）。
ＲＡＰＤ，ＥＲＩＣ−ＰＣＲの解析方法の概略は、以下のとおりである。
融合株よりＤＮＡを抽出し、１０ｎｇの鋳型ＤＮＡを含んだ１．０μｌ のＴＥ ｂｕｆｆｅｒ、．５μｌ ｏｆ １０×ｂｕｆｆｅｒ（１００ｍＭ Ｔｒｉｓ−ＨＣｌ，ｐＨ８．３，５００ｍＭ ＫＣｌ，１５ｍＭ ＭｇＣｌ２ ａｎｄ ０．０１％ ｇｅｌａｔｉｎ），０．５μｌのｄＮＴＰ（１０ｍＭ ｄＡＴＰ，１０ｍＭ ｄＣＴＰ，１０ｍＭ ＧＴＰ，１０ｍＭ ＴＴＰ），０．２５ｕｎｉｔｓのＴａｑポリメラーゼ（ＳＩＧＭＡ ＧＥＮＯＳＹＳ ＪＡＰＡＮ，Ｉｎｃ．，Ｊａｐａｎ）とプライマー（ＡＡＧ ＴＡＡ ＧＴＧ ＡＣＴ ＧＧＧ ＧＴＧ ＡＧＣ Ｇ）を０．５ｍＭを含んだ２５μｌの反応液をバイオラッド社のｉＣｙｃｌｅｒにより反応させた。反応サイクルは９４℃（５分）、｛９４℃（３０秒）５５℃（３０秒）７２℃（１分）｝を４０サイクル、最後の伸長サイクルは７２℃５分間実施した。反応産物を５μｌをアガロースゲルにアプライし、電気泳動後にエチジウムブロマイドで染色し、ＵＶ照射下で撮影した。
Example 1.
In addition to the Bata Lec wettable powder (Verticillium regani IMI 179172 strain) and Mycotal wettable powder (Verticillium regani IMI 268317) currently on the market, they have been separated from the greenhouse of the University, and leaves of plants Protoplast fusion was performed using Verticillium lecani B-2 strain (MAFF238429) (patent publication 2003-335612), which is a line known for its high fixing ability. At that time, a nit (nitrate nitrogen utilization ability) mutant (Correll, et al. 1987) was used as a genetic marker for visualizing heterokaryons produced by fusion. After germinating spores of each strain with PDB (potato broth liquid medium), protoplasts were made with Novozyme 188. Protoplast fusion was performed by the PEG method with a combination of nit mutants with different genetic markers. As a result, fusion of 126 (AaF1 to 106, BbF1 to 20), 4 (2BF1 to 4), 44 (2aF1 to 44) from the combination of Batalek and Mycotal, Batalek and B-2, and Mycotal and B-2, respectively. A stock was created. All 4a strains of 2aF1,4,26,43 (mycotal x B-2) are biased to B-2 in RAPD and ERIC-PCR. In 2aF1, there are 270 bp and 420 bp bands (amplicon; both red arrows) that are not present in the parent strain, and the B-2 750 bp amplicon (yellow arrow) is missing. In 2aF4, the 750 bp amplicon (yellow arrow) of the parent strain B-2 is deleted. In 2aF26, a 750 bp amplicon (yellow arrow) is deleted, but a 200 bp amplicon (white dotted line) existing in B-2 can be confirmed. In 2aF43, a Mycotal 1250 bp and 1350 bp amplicon (yellow dotted line) not found in other fusion strains can be confirmed, and there is a new 300 bp amplicon (red arrow) not found in the parent strain (Fig.).
The outline of the analysis method of RAPD and ERIC-PCR is as follows.
DNA was extracted from the fused strain and 1.0 μl of TE buffer,. 5 μl of 10 × buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl 2 and 0.01% gelatin), 0.5 μl dNTP (10 mM dATP, 10 mM dCTP, 10 mM GTP, 10 mM TTP), 0. 25 μl of a reaction solution containing 0.5 mM of 25 units of Taq polymerase (SIGMA GENOSYS JAPAN, Inc., Japan) and a primer (AAG TAA GTG ACT GGG GTG AGC G) was reacted with iCycler of Bio-Rad. The reaction cycle was 94 ° C. (5 minutes), {94 ° C. (30 seconds) 55 ° C. (30 seconds) 72 ° C. (1 minute)}, and the last extension cycle was 72 ° C. for 5 minutes. 5 μl of the reaction product was applied to an agarose gel, stained with ethidium bromide after electrophoresis, and photographed under UV irradiation.
実施例２．培養方法
ＰＤＡ（ジャガイモ煎汁寒天培地）上で２５℃、暗黒下で３週間培養し、コロニーを筆もしくはコンラージ棒で分生胞子をかきとり、遠沈法（３０００ｒｐｍ、５分）により分生胞子を蒸留水で洗浄した。その後、トーマの血球計算板により１×１０６になるよう光学顕微鏡下で調整した。
Example 2 Cultivation method Culture on PDA (potato broth agar) at 25 ° C in the dark for 3 weeks, colonies are scraped off with a brush or a congeal rod, and conidia are clarified by centrifugation (3000 rpm, 5 minutes). Washed with distilled water. Then, it adjusted under the optical microscope so that it might become 1 * 10 < 6 > by the hemocytometer of Toma.
実施例３．ワタアブラムシに対する殺虫活性
２ｃｍ２のキュウリのリーフディスクにそれぞれ約５０頭ずつワタアブラムシの若虫を放飼し、１×１０６分生胞子／ｍｌに調整した胞子懸濁液を約０．６５ｍｌスプレー散布した。３日後に菌糸に覆われている、若しくは体表から菌糸が生えている死亡個体を計数し死亡率を算出した（１菌株につき、５反復）。結果は表に示した。
Example 3 Insecticidal activity against cotton aphids About 50 Aphid nymphs are released on 2 cm 2 cucumber leaf discs each and sprayed with about 0.65 ml of spore suspension adjusted to 1 × 10 6 conidia / ml did. After 3 days, the number of dead individuals covered with mycelia or growing mycelium from the body surface was counted and the mortality was calculated (5 repetitions per strain). The results are shown in the table.
実施例４ オンシツコナジラミに対する殺虫活性
オンシツコナジラミ成虫に自由に産卵させたインゲンの葉面を採取し、５０頭程度の幼虫のいるリーフディスクに１×１０６分生胞子／ｍｌに調整した胞子懸濁液を約０．６５ｍｌスプレー散布した。３日後に菌糸に覆われている、又は体表から菌糸が生えている死亡個体を計数し死亡率を算出した（１菌株につき、５反復）。結果は表に示した。
Example 4 Insecticidal activity against whitefly whitefly Leaf surfaces of green beans that were freely spawned by adult whitefly were collected, and a spore suspension adjusted to 1 × 10 6 conidia spores / ml was placed on a leaf disk containing about 50 larvae. About 0.65 ml spray was applied. After 3 days, the number of dead individuals covered with mycelia or growing mycelia from the body surface was counted and the mortality was calculated (5 repetitions per strain). The results are shown in the table.
実施例５．ミナミキイロアザミウマに対する殺虫活性
インゲンリーフディスクにミナミキイロアザミウマ３令幼虫を１０頭接種し、１×１０６分生胞子／ｍｌに調整した胞子懸濁液を約０．６５ｍｌスプレー散布した。３日後に菌糸に覆われている、若しくは体表から菌糸が生えている死亡個体を計数し死亡率を算出した（１菌株につき、５反復）。結果は表に示した。
実施例６．
実施例２で示した分生胞子懸濁液を乾燥させ、分生胞子粉末を得た。これを用い、分生胞子粉末４５部、ポリオキシエチレンアルキルアリールエーテル硫酸エステル１０部、ジアルキルスルホサクシネート５部、リグニンスルホン酸ナトリウム１０部、カオリン３０部を秤量し、サンプルミルにて２０秒間混合し１×１０６分生胞子／ｇの水和剤を得た。本剤を５℃の冷蔵条件下で保存し、有効成分である分生胞子の生存率を（生死の判断基準は希釈平板法で行った）調査した。
The conidia spore suspension shown in Example 2 was dried to obtain conidia spores powder. Using this, 45 parts of conidial spore powder, 10 parts of polyoxyethylene alkylaryl ether sulfate, 5 parts of dialkyl sulfosuccinate, 10 parts of sodium lignin sulfonate and 30 parts of kaolin are weighed and mixed in a sample mill for 20 seconds. 1 × 10 6 conidia / g wettable powder was obtained. This agent was stored under refrigerated conditions at 5 ° C., and the survival rate of the conidia as an active ingredient was examined (life and death criteria were determined by the dilution plate method).
本発明により、アブラムシ類、コナジラミ類及びアザミウマ類に対して同時に高い病原性を有し、図１に示すＤＮＡバンドパターンを示すバーティシリウム・レカニ株群を見出し、これらの菌株を有効成分として含有することを特徴とする有害生物防除剤を製剤化することで、その病原性を示す宿主域が広い微生物農薬を供給できた。特に本発明の有害生物防除剤は、野菜類の大害虫であるアブラムシ類、コナジラミ類、アザミウマ類ではなどの害虫に対し、従来の化学合成殺虫剤や微生物農薬等に比べ、効果、価格面でより有効なものとなる。 According to the present invention, a group of Verticillium lecani strains having high pathogenicity against aphids, whiteflies and thrips at the same time and having the DNA band pattern shown in FIG. 1 is found, and these strains are contained as active ingredients. By formulating a pest control agent characterized in that it was possible to supply a microbial pesticide with a wide host range showing its pathogenicity. In particular, the pest control agent of the present invention is effective against the harmful insects such as aphids, whiteflies, and thrips, which are large pests of vegetables, in terms of effectiveness and price compared to conventional chemical synthetic insecticides and microbial pesticides. It becomes more effective.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2010018830A1 (en) * | 2008-08-11 | 2010-02-18 | 石原産業株式会社 | Lecanicillium muscarium strain v-5, pest extermination method using the same, and microorganism pesticide comprising the same |
| RU2598251C1 (en) * | 2015-08-25 | 2016-09-20 | Федеральное государственное бюджетное научное учреждение "Всероссийский научно-исследовательский институт защиты растений" | Lecanicillium muscarium FUNGUS STRAIN HAVING INSECTO-ACARICIDAL AND ANTIBIOTIC ACTIVITY FOR FIGHTING AGAINST SUCKING PESTS, FUNGAL AND BACTERIAL DISEASES |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2010018830A1 (en) * | 2008-08-11 | 2010-02-18 | 石原産業株式会社 | Lecanicillium muscarium strain v-5, pest extermination method using the same, and microorganism pesticide comprising the same |
| CN102171327B (en) * | 2008-08-11 | 2013-01-02 | 石原产业株式会社 | Lecanicillium muscarium strain V-5, pest extermination method using the same, and microorganism pesticide comprising the same |
| US8535932B2 (en) | 2008-08-11 | 2013-09-17 | Ishihara Sangyo Kaisha, Ltd | Lecanicillium muscarium strain V-5, pest extermination method using the same, and microorganism pesticide comprising the same |
| JP5515098B2 (en) * | 2008-08-11 | 2014-06-11 | 石原産業株式会社 | Recanicillium mass potassium V-5 strain, pest control method using the strain, and microbial pesticide containing the strain |
| RU2598251C1 (en) * | 2015-08-25 | 2016-09-20 | Федеральное государственное бюджетное научное учреждение "Всероссийский научно-исследовательский институт защиты растений" | Lecanicillium muscarium FUNGUS STRAIN HAVING INSECTO-ACARICIDAL AND ANTIBIOTIC ACTIVITY FOR FIGHTING AGAINST SUCKING PESTS, FUNGAL AND BACTERIAL DISEASES |
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