JP2004217637A - Insecticidal filamentous fungal preparation - Google Patents
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本発明は、殺虫性糸状菌製剤等に関するものである。 The present invention relates to an insecticidal filamentous fungus preparation and the like.
化学合成化合物を有効成分として使用しない害虫防除方法のひとつとして、殺虫性糸状菌の利用が注目されており、いくつかの殺虫性糸状菌を利用した殺虫性糸状菌製剤が知られている（例えば、特許文献１、特許文献２参照）。 As one of the pest control methods that do not use a chemically synthesized compound as an active ingredient, utilization of insecticidal filamentous fungi has attracted attention, and insecticidal filamentous fungal preparations utilizing some insecticidal filamentous fungi are known (for example, , Patent Documents 1 and 2).
殺虫性糸状菌製剤の有効成分である殺虫性糸状菌は、生きている菌体が害虫に付着、感染することによって殺虫活性を発現することから、殺虫性糸状菌製剤には、製剤中に含有される殺虫性糸状菌が生きた状態で保持されていること、即ち生存安定性が高いことが要求される。
しかしながら、殺虫性糸状菌は単独では生存安定性の高くないものがあり、このような殺虫性糸状菌を用いた殺虫性糸状菌製剤の場合必ずしも十分な生存安定性を有していないことが多かった。そのため、製剤中における殺虫性糸状菌の生存安定性を向上させた製剤が求められていた。
Insecticidal filamentous fungi, which is an active ingredient of an insecticidal filamentous fungal preparation, expresses insecticidal activity when living cells adhere to and infect pests. It is required that the insecticidal filamentous fungus be kept alive, that is, have high survival stability.
However, some insecticidal filamentous fungi alone do not have high survival stability.In many cases, insecticidal filamentous fungal preparations using such insecticidal filamentous fungi do not always have sufficient survival stability. Was. Therefore, there has been a demand for a preparation having improved survival stability of insecticidal filamentous fungi in the preparation.
本発明者らは、鋭意検討した結果、殺虫性糸状菌に特定の界面活性剤及び水に溶解するか若しくは水により糊化するデンプンを含有させることによって、製剤中における殺虫性糸状菌の生存安定性、さらには製剤の沈降性が向上することを見出し、本発明に至った。
即ち、本発明は、
１．ペーシロマイセス（Paecilomyces）属に属する殺虫性糸状菌、ポリカルボン酸型界面活性剤、及び、水に溶解するか若しくは水により糊化するデンプンを含有することを特徴とする殺虫性糸状菌製剤（以下、本発明製剤と記すこともある。）；
２．製剤の全重量に対して、ポリカルボン酸型界面活性剤を５重量％以上３０重量％以下、及び、水に溶解するか若しくは水により糊化するデンプンを２０重量％以上６５重量％未満含有することを特徴とする前項１記載の殺虫性糸状菌製剤；
３．殺虫性糸状菌が下記のいずれかの糸状菌であることを特徴とする前項１記載の殺虫性糸状菌製剤
（１）核の５．８ＳリボゾームＲＮＡをコードするＤＮＡが配列番号１で示される塩基配列を有し、かつ、核の２８ＳリボゾームＲＮＡをコードするＤＮＡが配列番号２で示される塩基配列を有する糸状菌
（２）ペーシロマイセス・テヌイペス（Paecilomyces tenuipes）に属する糸状菌
（３）独立行政法人産業技術総合研究所特許生物寄託センターに寄託番号ＦＥＲＭ ＢＰ−７８６１として寄託されているペーシロマイセス・テヌイペス（Paecilomyces tenuipes）Ｔ１菌株である糸状菌
４．ポリカルボン酸型界面活性剤が、(a)イソブチレン若しくはジイソブチレンと(b)マレイン酸との共重合物又はその塩からなるポリカルボン酸型界面活性剤であることを特徴とする前項１〜３のいずれか記載の殺虫性糸状菌製剤；
５．前項１〜３のいずれか記載の製剤を、害虫、害虫の生育場所又は害虫から保護するべき植物に施用することを特徴とする殺虫方法；
６．ポリカルボン酸型界面活性剤及び水に溶解するか若しくは水により糊化するデンプンと、ペーシロマイセス（Paecilomyces）属に属する殺虫性糸状菌とを混合する工程を有することを特徴とする殺虫性糸状菌製剤の製造方法；
等を提供するものである。
The present inventors have conducted intensive studies and found that the pesticidal filamentous fungi contained a specific surfactant and a starch which was dissolved in water or gelatinized with water, thereby stabilizing the survival of the insecticidal filamentous fungi in the preparation. The present inventors have found that the properties and the sedimentation properties of the preparations are improved, leading to the present invention.
That is, the present invention
1. Insecticidal filamentous fungi belonging to the genus Paecilomyces, a polycarboxylic acid type surfactant, and an insecticidal filamentous fungus preparation characterized by containing starch which is dissolved or gelatinized by water (hereinafter, referred to as " It may also be referred to as the preparation of the present invention.);
2. 5% to 30% by weight of a polycarboxylic acid type surfactant and 20% to less than 65% by weight of starch which is dissolved in water or gelatinized by water, based on the total weight of the preparation. The insecticidal filamentous fungal preparation according to the above item 1, which is characterized in that:
3. 2. The insecticidal filamentous fungus preparation according to item 1, wherein the insecticidal filamentous fungus is any of the following filamentous fungi: (1) a base encoding the DNA encoding the nuclear 5.8S ribosomal RNA; A filamentous fungus having a sequence and a DNA encoding the nuclear 28S ribosomal RNA having the nucleotide sequence of SEQ ID NO: 2; (2) a filamentous fungus belonging to Paecilomyces tenuipes; and (3) an industrial corporation. 3. Filamentous fungus, Paecilomyces tenuipes T1 strain, deposited at the National Institute of Advanced Industrial Science and Technology Patent Depositary under the deposit number FERM BP-7861. The above-mentioned items 1 to 3, wherein the polycarboxylic acid surfactant is a polycarboxylic acid surfactant comprising (a) isobutylene or diisobutylene and (b) a copolymer of maleic acid or a salt thereof. The insecticidal filamentous fungal preparation according to any of the above,
5. An insecticidal method, which comprises applying the preparation according to any one of the above items 1 to 3 to a pest, a place where the pest is grown or a plant to be protected from the pest;
6. Insecticidal filamentous fungal preparation, comprising a step of mixing a polycarboxylic acid type surfactant and starch which is dissolved in water or gelatinized by water, and a pesticidal filamentous fungus belonging to the genus Paecilomyces. Production method;
Etc. are provided.
本発明によれば、製剤中におけるペーシロマイセス属に属する殺虫性糸状菌の生存安定性、さらには製剤の沈降性を向上させることができ、当該方法により得られる殺虫性糸状菌の生存安定性及び製剤の沈降性に優れた殺虫性糸状菌製剤及び当該殺虫性糸状菌製剤を用いた殺虫方法等を提供することができる。 According to the present invention, it is possible to improve the survival stability of insecticidal filamentous fungi belonging to the genus Pessilomyces in a preparation, and further to improve the sedimentation of the preparation, the survival stability of the insecticidal filamentous fungus obtained by the method, and the preparation. The present invention can provide an insecticidal filamentous fungal preparation excellent in sedimentation, a method for killing insects using the insecticidal filamentous fungal preparation, and the like.
以下、詳細に本発明を説明する。
本発明製剤において用いられる殺虫性糸状菌は、ペーシロマイセス（Paecilomyces）属に属する糸状菌であり、例えば、ペーシロマイセス・テヌイペス（Paecilomyces tenuipes）に属する糸状菌、ペーシロマイセス・フモソロセウス（Paecilomyces fumosoroseus）に属する糸状菌、ペーシロマイセス・ファリノーサス（Paecilomyces farinosus）に属する糸状菌等を挙げることができ、具体的にはペーシロマイセス・テヌイペス（Paecilomyces tenuipes）Ｔ１菌株、ペーシロマイセス・テヌイペス ＡＴＣＣ４４８１８、ペーシロマイセス・フモソロセウス ＡＴＣＣ２０８７４、ペーシロマイセス・フモソロセウス ＡＴＣＣ４２１８８、ペーシロマイセス・フモソロセウス ＮＲＢＣ８５５５等を挙げることができる。
Hereinafter, the present invention will be described in detail.
The insecticidal filamentous fungus used in the preparation of the present invention is a filamentous fungus belonging to the genus Paecilomyces, for example, a filamentous fungus belonging to Paecilomyces tenuipes, a filamentous fungus belonging to Paecilomyces fumosoroseus, or a filamentous fungus belonging to Paecilomyces fumosoroseus. Filamentous fungi belonging to Paecilomyces farinosus and the like can be mentioned. Specifically, Paecilomyces tenuipes T1 strain, Paecilomyces tenuipes ATCC44818, Paecilomyces fumerotheus ATCC20874 ATCC20874 ATCC20874 ATCC20874 ATCC20874 ATCC20874 NRBC 8555 and the like.
これらは、天然から分離してもよいし、菌株保存機関等から購入してもよい。
天然から分離する場合には、まず、体が硬化し、体からキノコ状のものが生えている死亡虫を野外から採取する。当該死亡虫に形成されている分生子を白金耳で触れ、SDY培地（組成：ペプトン 1％（Ｗ／Ｖ）、酵母エキス 1％（Ｗ／Ｖ）、ブドウ糖 2％（Ｗ／Ｖ）、寒天 1.5％（Ｗ／Ｖ））やＣｚａｐｅｋ培地（組成：NaNO3 0.3％（Ｗ／Ｖ）、K2HPO4 0.1％（Ｗ／Ｖ）、MgSO4・7H2O 0.05％（Ｗ／Ｖ）、KCl 0.05％（Ｗ／Ｖ）、FeSO4・7H2O 0.001％（Ｗ／Ｖ）、ショ糖 3％（Ｗ／Ｖ）、寒天 1.5％（Ｗ／Ｖ））等の固体培地に線を引くように擦りつける。２５℃で培養し、数日後に生えてきた菌の独立したコロニーを切り取り、新しいSDY培地やＣｚａｐｅｋ培地等の固体培地に移植し、さらに２５℃で培養する。生育してきた菌について、植物防疫特別増刊号Ｎｏ．２天敵微生物の研究手法（社団法人日本植物防疫協会発行）記載の方法等に従って、ペーシロマイセス属に属する糸状菌であるか同定を行い、ペーシロマイセス属に属する糸状菌を選抜すればよい。
つぎに、選抜されたペーシロマイセス属に属する糸状菌の殺虫活性の有無を確認する。選抜されたペーシロマイセス属に属する糸状菌をSDY培地やＣｚａｐｅｋ培地等の固体培地で２５℃で培養し、形成された分生子を１×１０８cfu／mlとなるように滅菌水に懸濁し、分離源となった死亡虫と同種の昆虫１０頭を懸濁液に３０秒間浸漬する。浸漬した昆虫を２５℃、湿度１００％の条件下で飼育し、接種後６日後に死亡虫が観察される菌株を、ペーシロマイセス属に属する殺虫性糸状菌として選抜することができる。
These may be isolated from nature or purchased from a strain preservation institution or the like.
In the case of separation from nature, first, a dead insect whose body has hardened and mushroom-like ones grow from the body is collected from the field. Touch the conidia formed on the dead insect with a platinum loop, and use an SDY medium (composition: peptone 1% (W / V), yeast extract 1% (W / V), glucose 2% (W / V), agar 1.5% (W / V) or Czapek medium (composition: NaNO 3 0.3% (W / V), K 2 HPO 4 0.1% (W / V), MgSO 4 .7H 2 O 0.05% (W / V), KCl 0.05% (W / V) , FeSO 4 · 7H 2 O 0.001% (W / V), sucrose 3% (W / V), draw a line on a solid medium 1.5% agar (W / V)), etc. Rub. After culturing at 25 ° C., independent colonies of bacteria that grew several days later are cut out, transplanted to a new solid medium such as SDY medium or Czapek medium, and further cultured at 25 ° C. About the fungus which grew, the special issue No. (2) According to the method described in the research method for natural enemy microorganisms (issued by the Japan Plant Protection Association), it is sufficient to identify whether the filamentous fungus belongs to the genus Pessilomyces and to select the filamentous fungus belonging to the genus Pesilomyces.
Next, the presence or absence of the insecticidal activity of the selected filamentous fungi belonging to the genus Pesilomyces is confirmed. The selected filamentous fungi belonging to the genus Paecilomyces are cultured at 25 ° C. in a solid medium such as SDY medium or Czapek medium, and the formed conidia are suspended in sterilized water to 1 × 10 8 cfu / ml and separated. 10 insects of the same species as the source dead insect are immersed in the suspension for 30 seconds. The immersed insects are bred under conditions of 25 ° C. and 100% humidity, and a strain in which dead insects are observed 6 days after the inoculation can be selected as insecticidal filamentous fungi belonging to the genus Pesiromyces.
尚、ペーシロマイセス・テヌイペス（Paecilomyces tenuipes）Ｔ１菌株は、独立行政法人産業技術総合研究所特許生物寄託センターに寄託され、ＦＥＲＭ ＢＰ−７８６１（旧FERM P18487）の寄託番号が付与されている。菌学的性状は次のとおり。
（１）生育速度（２５℃、７日間）
集落の直径：２５〜３０ｍｍ（２％マルトエキス寒天平板培地）、２５〜３０ｍｍ（オートミール寒天平板培地）
（２）集落表面の色調
白色（２％マルトエキス寒天平板培地）、白色（オートミール寒天平板培地）
（３）集落裏面の色調
白色（２％マルトエキス寒天平板培地）、白色〜明るい黄色（オートミール寒天平板培地）
（４）集落表面の組織
羊毛状〜綿毛状
（５）分生子柄
滑面、分岐して不規則な輪生となる。
（６）分生胞子
滑面、楕円形〜円筒形、連鎖する、約４μｍ×約２μｍ
（７）厚膜胞子
形成せず（２５℃、９日間）
（８）核の５．８ＳリボソームＲＮＡをコードするＤＮＡの塩基配列及び核の２８ＳリボゾームＲＮＡをコードするＤＮＡの塩基配列
核の５．８ＳリボゾームＲＮＡをコードするＤＮＡの塩基配列を配列番号１に、核の２８ＳリボゾームＲＮＡをコードするＤＮＡの塩基配列を配列番号２に示す。
The strain Paecilomyces tenuipes T1 has been deposited with the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, and has a deposit number of FERM BP-7861 (formerly FERM P18487). Mycological properties are as follows.
(1) Growth rate (25 ° C, 7 days)
Colony diameter: 25-30 mm (2% malt extract agar plate), 25-30 mm (oatmeal agar plate)
(2) Color tone of colony surface White (2% malt extract agar plate medium), white (oatmeal agar plate medium)
(3) Color tone of the back of the colony White (2% malt extract agar plate medium), white to bright yellow (oatmeal agar plate medium)
(4) Tissue-like fluffy to fluffy surface of colonies (5) Conidiophore smooth surface, diverging into irregular rings.
(6) Conidiospore smooth surface, oval to cylindrical, linked, about 4 μm × about 2 μm
(7) No thick spore formation (25 ° C, 9 days)
(8) The nucleotide sequence of the DNA encoding the nuclear 5.8S ribosomal RNA and the nucleotide sequence of the DNA encoding the nuclear 28S ribosomal RNA The nucleotide sequence of the DNA encoding the nuclear 5.8S ribosomal RNA is represented by SEQ ID NO: 1, SEQ ID NO: 2 shows the nucleotide sequence of the DNA encoding the nuclear 28S ribosomal RNA.
本発明製剤に用いられる殺虫性糸状菌は、液体培地又は固体培地を用いて培養することにより調製することができる。
当該菌の培養に用いられる液体培地又は固体培地は当該菌が増殖するものであれば特に限定されるものではなく、微生物培養に通常使用される炭素源、窒素源、有機塩及び無機塩等を適宜含む培地が用いられる。
液体培地は、通常水に炭素源、窒素源、有機塩、無機塩、ビタミン類等を適宜混合することにより調製できる。
液体培地に用いられる炭素源としては、例えば、グルコース、デキストリン、シュークロース等の糖類、グリセロール等の糖アルコール類、フマル酸、クエン酸、ピルビン酸等の有機酸、動植物油及び糖蜜等が挙げられる。培地に含まれる炭素源の量は、通常０．１〜２０％（ｗ／ｖ）である。
液体培地に用いられる窒素源としては、例えば、肉エキス、ペプトン、酵母エキス、麦芽エキス、大豆粉、コーン・スティープ・リカー（Corn Steep Liquor）、綿実粉、乾燥酵母、カザミノ酸等の天然有機窒素源、硝酸ナトリウム、塩化アンモニウム、硫酸ナトリウム、リン酸アンモニウム等の無機酸のアンモニウム塩や硝酸塩、フマル酸アンモニウム、クエン酸アンモニウム等の有機酸のアンモニウム塩、尿素及びアミノ酸類が挙げられる。培地に含まれる窒素源の量は、通常０．１〜３０％（ｗ／ｖ）である。
液体培地に用いられる有機塩や無機塩としては、例えば、カリウム、ナトリウム、マグネシウム、鉄、マンガン、コバルト、亜鉛等の塩化物、硫酸塩、酢酸塩、炭酸塩、リン酸塩が挙げられ、具体的には例えば、塩化ナトリウム、塩化カリウム、硫酸マグネシウム、硫酸第一鉄、硫酸マンガン、塩化コバルト、硫酸亜鉛、硫酸銅、酢酸ナトリウム、炭酸カルシウム、炭酸ナトリウム、リン酸一水素カリウム及びリン酸ニ水素カリウムが挙げられる。培地に含まれる無機塩や有機塩の量は、通常０．０００１〜５％（ｗ／ｖ）である。
ビタミン類としては、チアミン等が挙げられる。
固体培地としては、例えば、米類、麦類等の主穀類、トウモロコシ、栗、稗、コーリャン、蕎麦等の雑穀類、オガ粉、バガス、籾殻、フスマ、莢、藁、コ−ンコブ、綿実粕、オカラ、寒天、ゼラチン等を挙げることができる。また、これらの２種以上を混合して使用することもでき、さらに、前記液体培地に使用される炭素源、窒素源、有機塩、無機塩、ビタミン等を適宜混合したものが挙げられる。
The insecticidal filamentous fungi used in the preparation of the present invention can be prepared by culturing using a liquid medium or a solid medium.
The liquid medium or solid medium used for culturing the bacterium is not particularly limited as long as the bacterium grows.A carbon source, a nitrogen source, an organic salt, an inorganic salt, and the like usually used for culturing a microorganism are used. A culture medium containing an appropriate medium is used.
The liquid medium can be usually prepared by appropriately mixing water with a carbon source, a nitrogen source, organic salts, inorganic salts, vitamins and the like.
Examples of the carbon source used in the liquid medium include sugars such as glucose, dextrin and sucrose, sugar alcohols such as glycerol, organic acids such as fumaric acid, citric acid and pyruvic acid, animal and vegetable oils and molasses. . The amount of the carbon source contained in the medium is usually 0.1 to 20% (w / v).
Examples of the nitrogen source used in the liquid medium include natural organic substances such as meat extract, peptone, yeast extract, malt extract, soybean powder, corn steep liquor, cottonseed powder, dried yeast, casamino acid and the like. Examples include nitrogen sources, ammonium salts of inorganic acids such as sodium nitrate, ammonium chloride, sodium sulfate and ammonium phosphate, nitrates, ammonium salts of organic acids such as ammonium fumarate and ammonium citrate, urea and amino acids. The amount of the nitrogen source contained in the medium is usually 0.1 to 30% (w / v).
Examples of the organic and inorganic salts used in the liquid medium include potassium, sodium, magnesium, iron, manganese, cobalt, zinc, and other chlorides, sulfates, acetates, carbonates, and phosphates. Specifically, for example, sodium chloride, potassium chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, cobalt chloride, zinc sulfate, copper sulfate, sodium acetate, calcium carbonate, sodium carbonate, potassium monohydrogen phosphate and dihydrogen phosphate Potassium. The amount of the inorganic salt or the organic salt contained in the medium is usually 0.0001 to 5% (w / v).
Examples of the vitamins include thiamine.
Examples of the solid medium include, for example, main grains such as rice and wheat, cereals such as corn, chestnut, leeches, kolyan, and buckwheat, sawdust, bagasse, rice husk, bran, pods, straw, corn cob, and cottonseed. Grains, okara, agar, gelatin and the like can be mentioned. In addition, two or more of these can be used as a mixture, and examples further include those obtained by appropriately mixing a carbon source, a nitrogen source, organic salts, inorganic salts, vitamins, and the like used in the liquid medium.
殺虫性糸状菌の培養に用いられる培地の具体的例としては、液体培地として、２％マルトエキス液体培地、オートミール液体培地、ポテトデキストロース液体培地、サブロー液体培地及びＬ−ｂｒｏｔｈ液体培地等が挙げられ、固体培地として、米、大麦、フスマ、寒天培地（２％マルトエキス寒天培地、オートミール寒天培地、ポテトデキストロース寒天培地、サブロー寒天培地及びＬ−ｂｒｏｔｈ寒天培地等）等が挙げられる。 Specific examples of the culture medium used for cultivating the insecticidal filamentous fungi include a liquid culture medium such as a 2% malt extract liquid culture medium, an oatmeal liquid culture medium, a potato dextrose liquid culture medium, a Sabouraud liquid culture medium, and an L-broth liquid culture medium. Examples of the solid medium include rice, barley, bran, and agar medium (2% malt extract agar medium, oatmeal agar medium, potato dextrose agar medium, Sabouraud agar medium, L-broth agar medium, and the like).
当該菌の培養は、微生物の培養に通常使用される方法を用いて行うことができる。
即ち、液体培地を用いて培養する方法としては、例えば、試験管振盪式培養、往復式振盪培養、ジャーファーメンター培養及びタンク培養が挙げられ、固体培地を用いて培養する方法としては、例えば、静置培養が挙げられ、必要に応じ切り返しを加えてもよい。
培養温度は、当該菌が生育可能な範囲で適宜変更することができるが、通常１５℃〜３５℃の範囲であり、培地のｐＨは通常約５〜７の範囲である。培養時間は培養条件により異なるが、通常約１日間〜約２ヶ月間の範囲である。
当該菌は、当該菌を培養した培養液を遠心分離する方法、当該菌を培養した固体培地上に蒸留水等を加えて表面から菌体をかきとる方法や、固体培地を乾燥させ粉砕した後、篩により分画する方法等により得ることができる。
The cultivation of the bacterium can be performed using a method usually used for culturing a microorganism.
That is, as a method of culturing using a liquid medium, for example, a test tube shaking culture, a reciprocal shaking culture, a jar fermenter culture and a tank culture can be mentioned. As a method of culturing using a solid medium, for example, Static culture may be mentioned, and if necessary, cutback may be added.
The culture temperature can be appropriately changed within a range in which the bacterium can grow, but is usually in the range of 15 ° C to 35 ° C, and the pH of the medium is usually in the range of about 5 to 7. The culturing time varies depending on the culturing conditions, but usually ranges from about 1 day to about 2 months.
The bacterium may be obtained by centrifuging a culture solution obtained by culturing the bacterium, by removing distilled cells or the like from the surface by adding distilled water or the like to a solid medium on which the bacterium is cultured, or by drying and grinding the solid medium. And a method of fractionating through a sieve.
本発明製剤に含まれる殺虫性糸状菌の配合量は、施用した場合に必要な効力が得られるように調製する限り特に限定されないが、本発明製剤１ｇあたり当該菌を通常１０3〜１０15 ＣＦＵ（ＣＦＵ：コロニー形成単位）含有させればよい。 The compounding amount of the insecticidal filamentous fungi contained in the preparation of the present invention is not particularly limited as long as it is adjusted so as to obtain the necessary efficacy when applied, and the bacterium is usually contained in an amount of 10 3 to 10 15 CFU per gram of the preparation of the present invention. (CFU: colony forming unit).
本発明製剤において用いられるポリカルボン酸型界面活性剤とは、高分子中にカルボキシル基を有する化合物又はその塩を指し、通常、繰り返し単位中に平均0.2残基以上、好ましくは平均1残基以上のカルボキシル基を有する高分子化合物又はその塩を意味する。ポリカルボン酸型界面活性剤としては、例えば、アクリル酸とマレイン酸との共重合物、(a)イソブチレン若しくはジイソブチレンと(b)マレイン酸との共重合物、アクリル酸とイタコン酸との共重合物、メタアクリル酸とイタコン酸との共重合物、マレイン酸とスチレンとの共重合物、マレイン酸とスチレンスルホン酸との共重合物等、或いはこれらの塩が挙げられ、好ましくは、(a)イソブチレン若しくはジイソブチレンと(b)マレイン酸との共重合物、或いはその塩を挙げることができる。ポリカルボン酸型界面活性剤としては、具体的には例えば、パナカヤクＣＰ（日本化薬製）、ＡＱＵＰＥＣ ＨＶ−５０１、ＡＱＵＰＥＣ ＨＶ−５０４、ＡＱＵＰＥＣ ＨＶ−５０５（以上、住友精化製）、ジュリマーＡＣ−１０ＮＰ（日本純薬製）、Ｓ−ＳＭＡ３０００、Ｓ−ＳＭＡ１０００、Ｓ−ＳＭＡ１４４０Ｈ（以上、ＡＲＣＯ ＣＨＥＭＩＣＡＬ製）、ポリスターＯＭＰ、ポリスターＯＭＡ、ポリスターＳＭＸ、ポリスターＳＭ−１０１５、ポリスターＡ−１０６０（以上、日本油脂製）、ソカランＣＰ−5、ソカランＣＰ−７、ソカランＣＰ−９、ソカランＣＰ−10（以上、ＢＡＳＦ製）、ＧＥＲＯＰＯＮ Ｔ／３６、ＧＥＲＯＰＯＮ ＴＡ／７２、ＧＥＲＯＰＯＮ ＳＣ／２１３（以上、ローディア日華製）、デモールＥＰ（花王製）、トキサノンＧＲ−３０、トキサノンＧＲ−３１Ａ（以上、三洋化成製）等が挙げられ、好ましくは、ＧＥＲＯＰＯＮ Ｔ／３６、ＧＥＲＯＰＯＮ ＳＣ／２１３、デモールＥＰ等を挙げることができる。
本発明製剤に含まれるポリカルボン酸型界面活性剤の配合量は、本発明製剤の全重量に対して、通常０．５重量％以上５０重量％以下、好ましくは１重量％以上３０重量％以下、より好ましくは５重量％以上３０重量％以下である。
本発明製剤において用いられる水に溶解するか若しくは水により糊化するデンプンとしては、例えば、α化デンプン、デキストリン、アミロース及びアミロペクチンの分解物、酸化デンプン及び酸処理デンプン等の加工デンプンを挙げることができ、具体的には例えば、マツノリン５００、マツノリンＭ、マツノリンＭ−２２（以上、松谷化学工業製）、アミコールＮｏ．１、アミコールＷ、キプロガムＦ−５００、ペトロサイズJ（以上、日澱化学製）等が挙げられる。
The polycarboxylic acid-type surfactant used in the preparation of the present invention refers to a compound having a carboxyl group in a polymer or a salt thereof, and is usually 0.2 residues or more in a repeating unit, preferably 1 residue or more. Or a salt thereof having a carboxyl group. Examples of the polycarboxylic acid type surfactant include a copolymer of acrylic acid and maleic acid, a copolymer of (a) isobutylene or diisobutylene and (b) maleic acid, and a copolymer of acrylic acid and itaconic acid. Polymers, copolymers of methacrylic acid and itaconic acid, copolymers of maleic acid and styrene, copolymers of maleic acid and styrene sulfonic acid, and the like, or salts thereof, preferably, ( Examples thereof include a) a copolymer of isobutylene or diisobutylene and (b) maleic acid, or a salt thereof. Specific examples of the polycarboxylic acid surfactant include, for example, Panakayak CP (manufactured by Nippon Kayaku), AQUAPEC HV-501, AQUPEC HV-504, AQUAPEC HV-505 (all manufactured by Sumitomo Seika), Julimer AC -10NP (manufactured by Nippon Junyaku), S-SMA3000, S-SMA1000, S-SMA1440H (manufactured by ARCO CHEMICAL), POLYSTAR OMP, POLYSTAR OMA, POLYSTAR SMX, POLYSTAR SM-1015, POLYSTAR A-1060 (nominated, Japan Fats and oils), Sokaran CP-5, Sokalan CP-7, Sokalan CP-9, Sokalan CP-10 (all made by BASF), GEROPON T / 36, GEROPON TA / 72, GEROPON SC / 213 (all, Rhodia Nichika) Made), Demol EP (made by Kao), Examples include Toxanone GR-30 and Toxanone GR-31A (all manufactured by Sanyo Chemical Industries, Ltd.), and preferably, GEROPON T / 36, GEROPON SC / 213, Demol EP, and the like.
The compounding amount of the polycarboxylic acid surfactant contained in the preparation of the present invention is usually 0.5% by weight or more and 50% by weight or less, preferably 1% by weight or more and 30% by weight or less based on the total weight of the present preparation. And more preferably 5% by weight or more and 30% by weight or less.
Examples of the starch to be dissolved in water or gelatinized by water used in the preparation of the present invention include, for example, pregelatinized starch, dextrin, degraded products of amylose and amylopectin, modified starch such as oxidized starch and acid-treated starch. Specifically, for example, Matsunoline 500, Matsunoline M, Matsunoline M-22 (all manufactured by Matsutani Chemical Industry Co., Ltd.), AMICOL No. 1, Amicol W, Kipro Gum F-500, Petrosize J (all manufactured by Nissei Chemical), and the like.
本発明製剤中の水に溶解するか若しくは水により糊化するデンプンの配合量は、本発明製剤の全重量に対して、通常０．５重量％以上９０重量％以下、好ましくは２０重量％以上６５重量％未満である。 The amount of starch dissolved or gelatinized in water in the preparation of the present invention is usually 0.5% by weight or more and 90% by weight or less, preferably 20% by weight or more based on the total weight of the present preparation. Less than 65% by weight.
本発明製剤は、上記構成成分のほかに、さらに必要に応じて他構成成分若しくは残部として、本発明で用いられる殺虫性糸状菌の殺虫活性を喪失させない範囲において、通常農薬に使用される副資材、例えば、固体担体、液体担体、液性調整剤（ｐＨ調整剤等）、拡展剤、展着剤、湿潤剤、安定化剤（防腐剤、乾燥剤、凍結防止剤、固結防止剤、抗酸化剤、紫外線吸収剤）、ドリフト防止剤等を添加することができる。
これらの副資材を添加する場合、その添加量は合計で、本発明製剤の全重量に対して、通常０．１重量％以上９０重量％以下、好ましくは０．５重量％以上７５重量％以下である。
The preparation of the present invention, in addition to the above-mentioned constituents, may further contain, as necessary, other constituents or the remainder, as long as the insecticidal activity of the insecticidal filamentous fungi used in the present invention is not lost. For example, solid carriers, liquid carriers, liquid property adjusting agents (pH adjusting agents, etc.), spreading agents, spreading agents, wetting agents, stabilizers (preservatives, drying agents, antifreezing agents, anti-caking agents, (An antioxidant, an ultraviolet absorber), a drift inhibitor and the like.
When these auxiliary materials are added, the total amount thereof is generally 0.1% by weight or more and 90% by weight or less, preferably 0.5% by weight or more and 75% by weight or less based on the total weight of the preparation of the present invention. It is.
本発明製剤の剤型としては、粉剤、水和剤等の粉末製剤、粒剤、顆粒水和剤、錠剤等の造粒型製剤を挙げることができる。
本発明製剤の製造方法には、通常の農薬製剤の製造方法を適用することができる。本発明製剤が粉末製剤の場合には、例えば、前述の方法により得られた殺虫性糸状菌と、ポリカルボン酸型界面活性剤及び水に溶解するか若しくは水により糊化するデンプンとを、さらに必要に応じて他構成成分若しくは残部として、副資材を、混合することにより製造することができる。混合の際には、乳鉢・乳棒、薬さじ等を用いて混合することもできるし、例えば、リボンミキサー、ナウタミキサー等の混合機を用いて混合することもできる。
本発明製剤が造粒型製剤の場合には、例えば、乾式造粒法、湿式押し出し造粒法、噴霧乾燥法、流動層造粒法等の通常の造粒方法で製造することができる。
Examples of the dosage form of the preparation of the present invention include powder preparations such as powders and wettable powders, and granulated preparations such as granules, wettable powders and tablets.
As the method for producing the preparation of the present invention, a general method for producing a pesticide preparation can be applied. When the formulation of the present invention is a powder formulation, for example, the insecticidal filamentous fungus obtained by the method described above, and a polycarboxylic acid surfactant and starch dissolved or gelatinized with water, If necessary, it can be produced by mixing an auxiliary component or another component as a balance. At the time of mixing, mixing can be performed using a mortar / pestle, a spoonful of medicine, or the like, or for example, using a mixer such as a ribbon mixer or a Nauta mixer.
When the preparation of the present invention is a granulated preparation, it can be produced by a usual granulation method such as a dry granulation method, a wet extrusion granulation method, a spray drying method, and a fluidized bed granulation method.
本発明製剤が殺虫性効力を有する害虫としては、例えば、以下の害虫が挙げられる。
鱗翅目害虫：ニカメイガ（Chilo suppressalis）、コブノメイガ（Cnaphalocrocis medinalis）等のメイガ類、ヨトウガ（Mamestra brasicae）、オオタバコガ（Helicoverpa armigera）、タマナギンウワバ（Autographa nigrisigna）等のヤガ類、モンシロチョウ（Pieris rapae）等のシロチョウ類、コナガ（Plutella xylostella）等のスガ類、ドクガ（Euproctis taiwana）、マイマイガ（Lymantria dispar）、モンシロドクガ（Euproctis similis）等のドクガ類、ヒメクロイラガ（Scopelodes contracus）等のイラガ類、マツカレハ（Dendrolimus spectabilis）等のカレハガ類
半翅目害虫：ワタアブラムシ（Aphis gossypii）、モモアカアブラムシ（Myzus persicae）、ニセダイコンアブラムシ（Lipaphis pserudobrassicae）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）、シルバーリーフコナジラミ（Bemisia argentifolli）等のコナジラミ類等
双翅目害虫：イエバエ（Musca domestica）等のイエバエ類、アカイエカ（Culex pipiens pallens）等のイエカ類等
アザミウマ目害虫：ミナミキイロアザミウマ（Thrips palmi）、ミカンキイロアザミウマ（Frankliniella occidentalis）等
シロアリ目害虫：ヤマトシロアリ（Reticulitermes speratus）、イエシロアリ（Coptotermes formosanus）等。
Examples of the pests in which the preparation of the present invention has insecticidal efficacy include the following pests.
Lepidopteran pests: Japanese platymes such as Chilo suppressalis and Cnaphalocrocis medinalis; Motestra brasicae; Helicoverpa armigera; Autographa nigrisigna; Species, Suga such as Plutella xylostella, etc., Dokuga (Euproctis taiwana), Gypsy moth (Lymantria dispar), Dokuga such as Monprodulga (Euproctis similis), Iragus such as Scopelodes contracus, etc., and Dendros specilis (Dendrolim) Hemiptera pests: Aphids such as cotton aphids (Aphis gossypii), peach aphids (Myzus persicae), and aphids (Lipaphis pserudobrassicae), etc .; -Whiteflies, such as whiteflies (Bemisia argentifolli), etc. Diptera: Houseflies such as housefly (Musca domestica), houseflies such as Culex pipiens pallens, etc. Thripidae: Thrips palmi, Thrips palmi Thrips (Frankliniella occidentalis), etc.
Termite pests: Yamato termites (Reticulitermes speratus), house termites (Coptotermes formosanus) and the like.
本発明製剤は、通常、害虫、害虫の生息場所又は害虫から保護すべき植物等に施用することにより使用される。害虫から保護すべき植物に施用する場合、通常、当該植物の茎葉等に対して、本発明製剤のうち水和剤、顆粒水和剤又は錠剤を水で希釈した後、当該希釈液を散布処理することにより使用することがよい。
本発明製剤を害虫、害虫の生息場所又は害虫から保護すべき植物等に施用する際、その施用量は、通常１０００ｍ2あたり本発明製剤で用いられる殺虫性糸状菌株の菌体の量として、１０5〜１０19ＣＦＵ、好ましくは１０7〜１０17ＣＦＵである。水和剤、顆粒水和剤等は通常、前記菌体の量として、その濃度が１０3〜１０12ＣＦＵ／ｍｌとなるように水で希釈して使用すればよく、粉剤、粒剤等は通常、そのままで使用すればよい。
The preparation of the present invention is usually used by applying it to pests, habitats of pests or plants to be protected from pests. When applied to a plant to be protected from insect pests, usually, a water-dispersible powder, a water-dispersible granule or a tablet of the preparation of the present invention is diluted with water to the foliage of the plant, and then the diluted solution is sprayed. It is better to use it.
The present formulation pests when applied to the plants to be protected from habitat or pest pests, the application amount thereof is, as the amount of cells of insecticidal filamentous strain to be used in the normal 1000 m 2 per the present invention formulation, 10 5 to 10 19 CFU, preferably 10 7 ~10 17 CFU. The wettable powder, the wettable powder for granules and the like may be usually used by diluting with water so that the concentration of the cells is 10 3 to 10 12 CFU / ml. Normally, it can be used as is.
以下、実施例及び試験例を挙げて本発明を具体的に説明するが、本発明はこれらに限定されるものではない。 Hereinafter, the present invention will be specifically described with reference to Examples and Test Examples, but the present invention is not limited thereto.
実施例１ （ペーシロマイセス属に属する殺虫性糸状菌の単離）
体が硬化し、体からキノコ状のものが生えている死亡虫を野外から採取する。当該死亡虫に形成されている分生子を白金耳で触れ、ＳＤＹ培地に線を引くように擦りつける。２５℃で培養し、数日後に生えてきた菌の独立したコロニーを切り取り、新しいＳＤＹ培地に移植し、さらに２５℃で培養する。
得られた菌株のうち、以下のア）〜ク）に記載された性質をもつ菌株を、ペーシロマイセス属に属する糸状菌として選抜する。
ア）栄養菌糸が隔壁を持つ
イ）有性生殖が認められない。
ウ)分生子は分生子殻と呼ばれる壺上の器官の中に作られるのではなく、外生する。
エ）分生子はフィアライド頂端にフィアロ型に形成され、乾燥し連鎖状をなす。
オ）分生子柄は先端に小のうを持たない。
カ）フィアライドは分生子柄束上に柵状に配列されない。
キ）分生子の連鎖は束をなさない。
ク）フィアライドは頸部が明瞭、不規則あるいは緩く輪生である。
選抜されたペーシロマイセス属に属する糸状菌をSDY培地で２５℃で培養し、形成された分生子を１×１０８ＣＦＵ／ｍｌとなるように滅菌水に懸濁し、分離源となった死亡虫と同種の昆虫１０頭を懸濁液に３０秒間浸漬する。浸漬した昆虫を２５℃、湿度１００％の条件下で飼育し、接種後６日後に死亡虫が観察されるものを、ペーシロマイセス属に属する殺虫性糸状菌として選抜する。
Example 1 (Isolation of insecticidal filamentous fungi belonging to the genus Pesilomyces)
A dead insect whose body has hardened and mushrooms grow from the body is collected from the field. The conidia formed on the dead insect are touched with a platinum loop and rubbed so as to draw a line on the SDY medium. After culturing at 25 ° C., independent colonies of bacteria that grew several days later are cut out, transplanted to a new SDY medium, and further cultured at 25 ° C.
Among the obtained strains, strains having the properties described in (a) to (c) below are selected as filamentous fungi belonging to the genus Pesiromyces.
A) Vegetative hyphae have septum a) No sexual reproduction is observed.
C) Conidia are exogenous, not formed in an organ on the jar called conidial shell.
D) Conidia are formed in the form of phialo at the apex of phialide, and are dried to form a chain.
E) The conidium has no sac at the tip.
F) Fialides are not arranged in a fence on conidiophores.
G) Conidia chains do not form a bundle.
F) Fialide has a distinct neck, irregular or loose ring.
The selected filamentous fungi belonging to the genus Paecilomyces are cultured in an SDY medium at 25 ° C., and the formed conidia are suspended in sterilized water so as to have a concentration of 1 × 10 8 CFU / ml. 10 insects of the same kind are immersed in the suspension for 30 seconds. The immersed insects are bred under conditions of 25 ° C. and 100% humidity, and those in which dead insects are observed 6 days after the inoculation are selected as insecticidal filamentous fungi belonging to the genus Pesilomyces.
実施例２ （殺虫性糸状菌の調製）
５００ｍｌ容フラスコに入れた１００ｍｌのポテトデキストロース培地（Ｄｉｆｃｏ Ｌａｂｏｒａｔｏｒｉｅｓ製）に予めポテトデキストロース寒天培地（Ｄｉｆｃｏ Ｌａｂｏｒａｔｏｒｉｅｓ製）で培養されたペーシロマイセス・テヌイペス（Paecilomyces tenuipes）Ｔ１菌株の菌体を接種し、２５℃で３日間振とう培養することにより、培養液を得た。滅菌水１６０ｍｌが添加された滅菌済みフスマ８０ｇに、前記培養液２０ｍｌを接種し、２５℃、湿度９０％の条件下で、光（２０００〜３０００ルクス）を間欠照射（明条件：連続１４時間／日、暗条件：連続１０時間／日）しながら１４日間培養した。培養後、菌体（分生子を多く含む）が形成されたフスマを乾燥させ、乾燥後のフスマ及び直径２０ｍｍの瑪瑙ボール５個を日本工業規格標準ふるい（ＪＩＳ Ｚ ８８０１：６０メッシュのふるいを使用）に入れ、これを日本工業規格標準ふるい（ＪＩＳ Ｚ ８８０１：１００、２００メッシュのふるいを使用）と重ねて、自動ふるい振とう機（ＦＲＩＴＳＣＨ社）で１０分間振とうすることにより、２００メッシュ以下の画分に前記菌株の菌体粉末２．０ｇを得た。
Example 2 (Preparation of insecticidal filamentous fungi)
A 100-ml potato dextrose medium (manufactured by Difco Laboratories) in a 500-ml flask was inoculated with Paecilomyces tenuipes T1 strain 25 ° C. cells previously cultured on a potato dextrose agar medium (manufactured by Difco Laboratories) at 25 ° C. A culture solution was obtained by shaking culture for 3 days. 20 ml of the above culture solution was inoculated into 80 g of sterilized bran to which 160 ml of sterilized water was added, and intermittently irradiated with light (2000 to 3000 lux) at 25 ° C. and 90% humidity (bright conditions: continuous 14 hours / Day, dark conditions: continuous 10 hours / day) for 14 days. After the cultivation, the bran on which the cells (including a large amount of conidia) are formed is dried, and the dried bran and five agate balls having a diameter of 20 mm are sifted using a Japanese Industrial Standard Standard sieve (JIS Z 8801: a 60 mesh sieve). ), And put it on a Japanese Industrial Standards sieve (JIS Z 8801: 100, using a 200-mesh sieve) and shake it for 10 minutes with an automatic sieve shaker (FRITSCH) to obtain 200 mesh or less. In this fraction, 2.0 g of bacterial cell powder of the above strain was obtained.
実施例３ （本発明製剤の調製）
プラスチックカップにＧＥＲＯＰＯＮ Ｔ／３６（ローディア日華製）１８．４重量％、マツノリン５００（松谷化学工業製）３７．６重量％、カープレックス＃８０（塩野義製薬製）８．８重量％、勝光山ＳＰクレー（勝光山鉱業所製）１２．８重量％及びプロピレングリコール（関東化学製）２．４重量％を入れ、薬さじでよく混和した。これに実施例２で得られた菌体粉末２０．０重量％を加え、さらに薬さじで混和することにより、本発明製剤１を得た。
Example 3 (Preparation of the preparation of the present invention)
18.4% by weight of GEROPON T / 36 (manufactured by Rhodia Nika), 37.6% by weight of Matsunoline 500 (manufactured by Matsutani Chemical Industry), 8.8% by weight of Carplex # 80 (manufactured by Shionogi & Co., Ltd.) in a plastic cup 12.8% by weight of Mitsuyama SP clay (manufactured by Katsumitsu Mining Co., Ltd.) and 2.4% by weight of propylene glycol (manufactured by Kanto Chemical Co., Ltd.) were added and mixed well with a medicine spoon. 20.0% by weight of the bacterial cell powder obtained in Example 2 was added thereto, and the mixture was further mixed with a spoonful to obtain Preparation 1 of the present invention.
実施例４〜５ （本発明製剤の調製）
ＧＥＲＯＰＯＮ Ｔ／３６に代えて、ＧＥＲＯＰＯＮ ＳＣ２１３（ローディア日華製）（本発明製剤２）又はデモールＥＰ（花王製）（本発明製剤３）を用いること以外は実施例３と同様にして調製することにより、本発明製剤２及び本発明製剤３を得た。
Examples 4 to 5 (Preparation of the preparation of the present invention)
Prepared in the same manner as in Example 3 except that GEROPON SC213 (manufactured by Rhodia Nika) (formulation of the present invention 2) or Demol EP (manufactured by Kao) (formulation of the present invention 3) is used instead of GEROPON T / 36. As a result, Formulations 2 and 3 of the present invention were obtained.
実施例６（殺虫性糸状菌の生存安定性）
本発明製剤１〜３をそれぞれ１００ｍｇ計り取り、これを１０ｍｌの滅菌水に懸濁した。この懸濁液を適当な濃度に希釈して、得られた希釈液をポテトデキストロース寒天培地に１００μｌ滴下し塗り広げ、２５℃で２日間培養した。培養後、生育したコロニー数を計測することにより、当該製剤中の生菌数を求めた。一方、本発明製剤１〜３をそれぞれねじ口の付いたガラス瓶に入れ密栓した後、２５℃暗所で１５日間保存した。保存された本発明製剤１〜３をそれぞれ１００ｍｇ計り取り、これを１０ｍｌの滅菌水に懸濁した。この懸濁液を適当な濃度に希釈して得られた希釈液をポテトデキストロース寒天培地に１００μｌ滴下し塗り広げ、２５℃で２日間培養した。培養後、生育したコロニー数を計測することにより、保存後の当該製剤中の生菌数を求めた。このようにして求めた保存の前後における当該製剤中の生菌数の比を保存１５日後の生存率として算出した。
Example 6 (survival stability of insecticidal filamentous fungi)
100 mg of each of Preparations 1 to 3 of the present invention were weighed and suspended in 10 ml of sterilized water. This suspension was diluted to an appropriate concentration, and the resulting diluted solution was dropped on a potato dextrose agar medium (100 μl), spread, and cultured at 25 ° C. for 2 days. After the culture, the number of viable colonies in the preparation was determined by counting the number of grown colonies. On the other hand, each of Formulations 1 to 3 of the present invention was placed in a glass bottle provided with a screw hole, sealed, and then stored in a dark place at 25 ° C. for 15 days. 100 mg of each of the stored preparations 1 to 3 of the present invention was weighed, and suspended in 10 ml of sterilized water. This suspension was diluted to an appropriate concentration, and a diluted solution obtained was dropped on a potato dextrose agar medium (100 μl), spread, and cultured at 25 ° C. for 2 days. After culturing, the number of viable colonies in the preparation after storage was determined by counting the number of colonies that grew. The ratio of the number of viable bacteria in the preparation before and after storage thus determined was calculated as the survival rate 15 days after storage.
本発明製剤１〜３における保存１５日後の生存率を以下に示す。 The survival rates after 15 days of storage in Formulations 1 to 3 of the present invention are shown below.
実施例７ （殺虫性糸状菌の生存安定性）
本発明製剤１を１００ｍｇ計り取り、これを１０ｍｌの滅菌水に懸濁した。この懸濁液を適当な濃度に希釈して、得られた希釈液をポテトデキストロース寒天培地に１００μｌ滴下し塗り広げ、２５℃で２日間培養した。培養後、生育したコロニー数を計測することにより、当該製剤中の生菌数を求めた。一方、本発明製剤１をねじ口の付いたガラス瓶に入れ密栓した後、２５℃暗所で２８日間保存した。保存された本発明製剤１を１００ｍｇ計り取り、これを１０ｍｌの滅菌水に懸濁した。この懸濁液を適当な濃度に希釈して得られた希釈液をポテトデキストロース寒天培地に１００μｌ滴下し塗り広げ、２５℃で２日間培養した。培養後、生育したコロニー数を計測することにより、保存後の当該製剤中の生菌数を求めた。このようにして求めた保存の前後における当該製剤中の生菌数の比を保存２８日後の生存率として算出した。
Example 7 (Survival stability of insecticidal filamentous fungi)
100 mg of Formulation 1 of the present invention was weighed and suspended in 10 ml of sterilized water. This suspension was diluted to an appropriate concentration, and the resulting diluted solution was dropped on a potato dextrose agar medium (100 μl), spread, and cultured at 25 ° C. for 2 days. After the culture, the number of viable colonies in the preparation was determined by counting the number of grown colonies. On the other hand, Formulation 1 of the present invention was placed in a glass bottle with a screw hole, sealed, and stored in a dark place at 25 ° C. for 28 days. 100 mg of the stored preparation 1 of the present invention was weighed and suspended in 10 ml of sterilized water. This suspension was diluted to an appropriate concentration, and a diluted solution obtained was dropped on a potato dextrose agar medium (100 μl), spread, and cultured at 25 ° C. for 2 days. After culturing, the number of viable colonies in the preparation after storage was determined by counting the number of colonies that grew. The ratio of the number of viable bacteria in the preparation before and after storage thus determined was calculated as the survival rate 28 days after storage.
本発明製剤１における保存２８日後の生存率を以下に示す。 The survival rate of the formulation 1 of the present invention 28 days after storage is shown below.
比較例１ （殺虫性糸状菌の生存安定性）
ＧＥＲＯＰＯＮ Ｔ／３６に代えて、リグニンスルホン酸ナトリウム（東京化成工業製）（比較製剤１）又はソルポール５０６０（東邦化学工業製）（比較製剤２）を用いること以外は実施例３と同様にして調製することにより、比較製剤１及び比較製剤２を得た。
比較製剤１及び比較製剤２をそれぞれ１００ｍｇ計り取り、これを１０ｍｌの滅菌水に懸濁した。この懸濁液を適当な濃度に希釈して、得られた希釈液をポテトデキストロース寒天培地に１００μｌ滴下し塗り広げ、２５℃で２日間培養した。培養後、生育したコロニー数を計測することにより、当該製剤中の生菌数を求めた。一方、比較製剤１及び比較製剤２をそれぞれねじ口の付いたガラス瓶に入れ密栓した後、２５℃暗所で１５日間保存した。保存された比較製剤１及び比較製剤２をそれぞれ１００ｍｇ計り取り、これを１０ｍｌの滅菌水に懸濁した。この懸濁液を適当な濃度に希釈して得られた希釈液をポテトデキストロース寒天培地に１００μｌ滴下し塗り広げ、２５℃で２日間培養した。培養後、生育したコロニー数を計測することにより、保存後の当該製剤中の生菌数を求めた。このようにして求めた保存の前後における当該製剤中の生菌数の比を保存１５日後の生存率として算出した。
Comparative Example 1 (viability stability of insecticidal filamentous fungi)
Prepared in the same manner as in Example 3 except that instead of GEROPON T / 36, sodium ligninsulfonate (manufactured by Tokyo Chemical Industry) (comparative preparation 1) or Solpol 5060 (manufactured by Toho Chemical Industry) (comparative preparation 2) is used. As a result, Comparative Formulation 1 and Comparative Formulation 2 were obtained.
100 mg of each of Comparative Formulation 1 and Comparative Formulation 2 was weighed and suspended in 10 ml of sterilized water. This suspension was diluted to an appropriate concentration, and the resulting diluted solution was dropped on a potato dextrose agar medium (100 μl), spread, and cultured at 25 ° C. for 2 days. After the culture, the number of viable colonies in the preparation was determined by counting the number of grown colonies. On the other hand, Comparative Formulation 1 and Comparative Formulation 2 were respectively sealed in glass bottles with screw holes, and then stored in a dark place at 25 ° C. for 15 days. 100 mg of each of the stored comparative preparations 1 and 2 was weighed out and suspended in 10 ml of sterilized water. This suspension was diluted to an appropriate concentration, and 100 μl of the diluted solution obtained was dropped on a potato dextrose agar medium, spread, and cultured at 25 ° C. for 2 days. After culturing, the number of viable colonies in the preparation after storage was determined by counting the number of colonies that grew. The ratio of the number of viable bacteria in the preparation before and after storage thus determined was calculated as the survival rate 15 days after storage.
比較製剤１及び比較製剤２における保存１５日後の生存率を以下に示す。また、実施例６で算出した本発明製剤１における保存１５日後の生存率も併せて示す。 The survival rates after 15 days of storage in Comparative Preparation 1 and Comparative Preparation 2 are shown below. In addition, the survival rate of the preparation 1 of the present invention calculated in Example 6 after 15 days of storage is also shown.
比較例３（製剤の沈降性）
マツノリン５００（松谷化学工業製）に代えて、馬鈴薯デンプン（和光純薬工業製）（比較製剤３）を用いること以外は実施例３と同様にして調製することにより、比較製剤３を得た。
本発明製剤１及び比較製剤３を２００ｍｇ計り取り、これを２５０ｍｌ容のメスシリンダーに入った２５０ｍｌの３度硬水に懸濁した。懸濁した直後の懸濁液の中間層から５ｍｌの懸濁液を採取し、濁度ＯＤ６６０を測定した。一方、懸濁後１０分間静置した後、最下層の１０ｍｌを残して上層の懸濁液を取り除き、沈殿物を最下層の１０ｍｌ懸濁液に懸濁して回収し、該回収液の濁度ＯＤ６６０を測定した。
Comparative Example 3 (Precipitation of formulation)
Comparative preparation 3 was obtained in the same manner as in Example 3, except that potato starch (manufactured by Wako Pure Chemical Industries) (comparative preparation 3) was used instead of Matsunoline 500 (manufactured by Matsutani Chemical Industry).
200 mg of Formulation 1 of the present invention and Comparative Formulation 3 were weighed out and suspended in 250 ml of 3rd hard water in a 250 ml measuring cylinder. 5 ml of the suspension was collected from the intermediate layer of the suspension immediately after the suspension, and the turbidity OD660 was measured. On the other hand, after the suspension was allowed to stand for 10 minutes, the upper layer suspension was removed except for the lowermost 10 ml, and the precipitate was suspended and recovered in the lowermost 10 ml suspension. OD660 was measured.
本発明製剤１及び比較製剤３における懸濁直後の採取液、静置１０分間後の回収液の濁度ＯＤ６６０を以下に示す。 The turbidity OD660 of the collected liquid immediately after suspension and the recovered liquid after standing for 10 minutes in the present preparation 1 and comparative preparation 3 are shown below.
実施例８ （本発明製剤の殺虫試験）
本発明製剤１を水に１０００倍（W/V）に希釈して試験液とした。シルバーリーフコナジラミの幼虫が寄生しているトマト幼苗（播種から３〜４週間経過したもの）を準備し、寄生しているシルバーリーフコナジラミの幼虫の数を計測した。当該トマト幼苗に前述の試験液１０ｍｌを噴霧し、２５℃、湿度１００％の条件下で光（２０００〜３０００ルクス）を間欠照射（明条件：連続１４時間／日、暗条件：連続１０時間／日）しながら、当該試験液が噴霧されたトマト幼苗を６日間栽培した。栽培後、トマト幼苗に寄生しているシルバーリーフコナジラミの幼虫の数を計測することにより、当該試験液の噴霧の前後の幼虫数から死虫率を算出した。その結果、死虫率は９５．０％であった。
Example 8 (Insecticidal test of the preparation of the present invention)
Formulation 1 of the present invention was diluted 1000 times (W / V) in water to prepare a test solution. Tomato seedlings (three to four weeks after sowing) infested with silver leaf whitefly larvae were prepared, and the number of parasitic silver leaf whitefly larvae was counted. The above-mentioned test solution (10 ml) is sprayed on the tomato seedlings, and intermittently irradiated with light (2000 to 3000 lux) at 25 ° C. and 100% humidity (bright condition: continuous 14 hours / day, dark condition: continuous 10 hours / day) On the other hand, tomato seedlings sprayed with the test solution were cultivated for 6 days. After the cultivation, the number of larvae of silver leaf whitefly parasitic on the tomato seedlings was counted, and the mortality was calculated from the number of larvae before and after spraying the test solution. As a result, the mortality was 95.0%.
実施例９ （殺虫性糸状菌の調製）
５００ｍｌ容フラスコに入れた１００ｍｌのポテトデキストロース培地（Ｄｉｆｃｏ Ｌａｂｏｒａｔｏｒｉｅｓ製）に予めポテトデキストロース寒天培地（Ｄｉｆｃｏ Ｌａｂｏｒａｔｏｒｉｅｓ製）で培養されたペーシロマイセス・フモソロセウス（Paecilomyces fumosoroseus ）ＮＲＢＣ８５５５株の菌体を接種し、２５℃で３日間振とう培養することにより、培養液を得た。滅菌水１６０ｍｌが添加された滅菌済みフスマ８０ｇに、前記培養液２０ｍｌを接種し、２５℃、湿度９０％の条件下で、光（２０００〜３０００ルクス）を間欠照射（明条件：連続１４時間／日、暗条件：連続１０時間／日）しながら１４日間培養した。培養後、菌体（分生子を多く含む）が形成されたフスマを乾燥させ、乾燥後のフスマ及び直径２０ｍｍの瑪瑙ボール５個を日本工業規格標準ふるい（ＪＩＳ Ｚ ８８０１：６０メッシュのふるいを使用）に入れ、これを日本工業規格標準ふるい（ＪＩＳ Ｚ ８８０１：１００、２００メッシュのふるいを使用）と重ねて、自動ふるい振とう機（ＦＲＩＴＳＣＨ社）で１０分間振とうすることにより、２００メッシュ以下の画分に前記菌株の菌体粉末０．６ｇを得た。
Example 9 (Preparation of insecticidal filamentous fungi)
A 100 ml potato dextrose medium (manufactured by Difco Laboratories) in a 500 ml flask was previously inoculated with Paecilomyces fumosoroseus strain NRBC8555 inoculated with Paecilomyces fumosoroseus strain 25 ° C, which had been cultured on a potato dextrose agar medium (manufactured by Difco Laboratories). A culture solution was obtained by shaking culture for 3 days. 20 ml of the above culture solution was inoculated into 80 g of sterilized bran to which 160 ml of sterilized water was added, and intermittently irradiated with light (2000 to 3000 lux) at 25 ° C. and 90% humidity (bright conditions: continuous 14 hours / Day, dark conditions: continuous 10 hours / day) for 14 days. After the cultivation, the bran on which the cells (including a large amount of conidia) are formed is dried, and the dried bran and five agate balls having a diameter of 20 mm are sifted using a Japanese Industrial Standard Standard sieve (JIS Z 8801: a 60 mesh sieve). ), And put it on a Japanese Industrial Standards sieve (JIS Z 8801: 100, using a 200-mesh sieve) and shake it for 10 minutes with an automatic sieve shaker (FRITSCH) to obtain 200 mesh or less. In this fraction, 0.6 g of bacterial cell powder of the above strain was obtained.
実施例１０ （殺虫性糸状菌の調製）
予めポテトデキストロース寒天培地（Ｄｉｆｃｏ Ｌａｂｏｒａｔｏｒｉｅｓ製）で培養されたペーシロマイセス・テヌイペス（Paecilomyces tenuipes）ＡＴＣＣ４４８１８菌株の菌体を、直径９０ｍｍのシャーレのポテトデキストロース寒天培地（Ｄｉｆｃｏ Ｌａｂｏｒａｔｏｒｉｅｓ製）２０枚に接種し、２５℃で１ヶ月培養した。培養後、ペーシロマイセス・テヌイペス（Paecilomyces tenuipes）ＡＴＣＣ４４８１８菌株の菌体をスパチュラで掻き取り、掻き取った菌体及び直径２０ｍｍの瑪瑙ボール５個を日本工業規格標準ふるい（ＪＩＳ Ｚ ８８０１：６０メッシュのふるいを使用）に入れ、これを日本工業規格標準ふるい（ＪＩＳ Ｚ ８８０１：１００、２００メッシュのふるいを使用）と重ねて、自動ふるい振とう機（ＦＲＩＴＳＣＨ社）で１０分間振とうすることにより、２００メッシュ以下の画分に前記菌株の菌体粉末０．４ｇを得た。
Example 10 (Preparation of insecticidal filamentous fungi)
Cells of Paecilomyces tenuipes ATCC 44818 strain previously cultured on a potato dextrose agar medium (manufactured by Difco Laboratories) were inoculated on a potato dextrose agar medium (Difco Laboratories, 25 ° C., at 25 ° C.) on a potato dextrose agar medium having a diameter of 90 mm. For one month. After the culturing, the cells of Paecilomyces tenuipes ATCC 44818 strain were scraped off with a spatula, and the scraped cells and 5 agate balls having a diameter of 20 mm were sieved with a Japanese Industrial Standard sieve (JIS Z 8801: 60 mesh sieve). Use), and the resultant is superimposed on a Japanese Industrial Standard Standard sieve (JIS Z 8801: 100, using a 200-mesh sieve), and shaken with an automatic sieve shaker (FRITSCH) for 10 minutes to obtain 200 mesh. 0.4 g of bacterial cell powder of the above strain was obtained in the following fractions.
実施例１１〜１２（本発明製剤の調製）
プラスチックカップにＧＥＲＯＰＯＮ Ｔ／３６（ローディア日華製）１８．４重量％、マツノリン５００（松谷化学工業製）３７．６重量％、カープレックス＃８０（塩野義製薬製）８．８重量％、勝光山ＳＰクレー（勝光山鉱業所製）１２．８重量％及びプロピレングリコール（関東化学製）２．４重量％を入れ、薬さじでよく混和した。これに実施例９又は実施例１０で得られた菌体粉末２０．０重量％を加え、さらに薬さじで混和することにより、本発明製剤４又は本発明製剤５を得た。
Examples 11 to 12 (Preparation of the preparation of the present invention)
18.4% by weight of GEROPON T / 36 (manufactured by Rhodia Nika), 37.6% by weight of Matsunoline 500 (manufactured by Matsutani Chemical Industry), 8.8% by weight of Carplex # 80 (manufactured by Shionogi & Co., Ltd.) in a plastic cup 12.8% by weight of Mitsuyama SP clay (manufactured by Katsumitsu Mining Co., Ltd.) and 2.4% by weight of propylene glycol (manufactured by Kanto Chemical Co., Ltd.) were added and mixed well with a medicine spoon. 20.0% by weight of the bacterial cell powder obtained in Example 9 or Example 10 was added thereto, and the mixture was further mixed with a spoonful to obtain Formulation 4 or Formulation 5 of the present invention.
実施例１２（殺虫性糸状菌の生存安定性）
本発明製剤４及び本発明製剤５をそれぞれ１０ｍｇ計り取り、これを１０ｍｌの０．１％のＬ−７７（日本ユニカー製）および０．１％のＫＦ−６３０（信越化学工業製）を添加した滅菌水に懸濁した。この懸濁液を適当な濃度に希釈して、得られた希釈液をポテトデキストロース寒天培地に１００μｌ滴下し塗り広げ、２５℃で２日間培養した。培養後、生育したコロニー数を計測することにより、当該製剤中の生菌数を求めた。一方、本発明製剤４又は本発明製剤５をそれぞれねじ口の付いたガラス瓶に入れ密栓した後、２５℃暗所で１５日間保存した。保存された本発明製剤４又は本発明製剤５をそれぞれ１０ｍｇ計り取り、これを１０ｍｌの０．１％のＬ−７７（日本ユニカー製）および０．１％のＫＦ−６３０（信越化学工業製）を添加した滅菌水に懸濁した。この懸濁液を適当な濃度に希釈して得られた希釈液をポテトデキストロース寒天培地に１００μｌ滴下し塗り広げ、２５℃で２日間培養した。培養後、生育したコロニー数を計測することにより、保存後の当該製剤中の生菌数を求めた。このようにして求めた保存の前後における当該製剤中の生菌数の比を保存15日後の生存率として算出した。
Example 12 (viability stability of insecticidal filamentous fungi)
10 mg of each of Formulation 4 of the present invention and Formulation 5 of the present invention were weighed, and 10 ml of 0.1% L-77 (manufactured by Nippon Unicar) and 0.1% of KF-630 (manufactured by Shin-Etsu Chemical Co., Ltd.) were added. Suspended in sterile water. This suspension was diluted to an appropriate concentration, and the resulting diluted solution was dropped on a potato dextrose agar medium (100 μl), spread, and cultured at 25 ° C. for 2 days. After the culture, the number of viable colonies in the preparation was determined by counting the number of grown colonies. On the other hand, each of Formulation 4 of the present invention and Formulation 5 of the present invention was placed in a glass bottle with a screw hole, sealed, and then stored in a dark place at 25 ° C for 15 days. 10 mg of each of the stored Preparation 4 of the present invention or Preparation 5 of the present invention was weighed, and 10 ml of 0.1% L-77 (manufactured by Nippon Unicar) and 0.1% of KF-630 (manufactured by Shin-Etsu Chemical Co., Ltd.) were weighed. Was suspended in sterile water to which was added. This suspension was diluted to an appropriate concentration, and a diluted solution obtained was dropped on a potato dextrose agar medium (100 μl), spread, and cultured at 25 ° C. for 2 days. After culturing, the number of viable colonies in the preparation after storage was determined by counting the number of colonies that grew. The ratio of the number of viable bacteria in the preparation before and after storage thus determined was calculated as the survival rate 15 days after storage.
本発明製剤４又は本発明製剤５における保存15日後の生存率を以下に示す。 The survival rates after 15 days of storage in Formulation 4 of the present invention or Formulation 5 of the present invention are shown below.
本発明によれば、製剤中におけるペーシロマイセス属に属する殺虫性糸状菌の生存安定性、さらには製剤の沈降性を向上させることができ、当該方法により得られる殺虫性糸状菌の生存安定性及び製剤の沈降性に優れた殺虫性糸状菌製剤及び当該殺虫性糸状菌製剤を用いた殺虫方法等を提供することができる。 According to the present invention, it is possible to improve the survival stability of insecticidal filamentous fungi belonging to the genus Pessilomyces in a preparation, and further to improve the sedimentation of the preparation, the survival stability of the insecticidal filamentous fungus obtained by the method, and the preparation. The present invention can provide an insecticidal filamentous fungal preparation excellent in sedimentation, a method for killing insects using the insecticidal filamentous fungal preparation, and the like.
Claims (6)
（１）核の５．８ＳリボゾームＲＮＡをコードするＤＮＡが配列番号１で示される塩基配列を有し、かつ、核の２８ＳリボゾームＲＮＡをコードするＤＮＡが配列番号２で示される塩基配列を有する糸状菌。
（２）ペーシロマイセス・テヌイペス（Paecilomyces tenuipes）に属する糸状菌。
（３）独立行政法人産業技術総合研究所特許生物寄託センターに寄託番号ＦＥＲＭ ＢＰ−７８６１として寄託されているペーシロマイセス・テヌイペス（Paecilomyces tenuipes）Ｔ１菌株である糸状菌。 The insecticidal filamentous fungal preparation according to claim 1, wherein the insecticidal filamentous fungus is any of the following filamentous fungi.
(1) A filament having a DNA encoding the nuclear 5.8S ribosomal RNA having the nucleotide sequence represented by SEQ ID NO: 1 and a DNA encoding the nuclear 28S ribosomal RNA having the nucleotide sequence represented by SEQ ID NO: 2 Fungus.
(2) Filamentous fungi belonging to Paecilomyces tenuipes.
(3) A filamentous fungus which is a Paecilomyces tenuipes T1 strain deposited at the National Institute of Advanced Industrial Science and Technology under the Patent Organism Depositary under the deposit number FERM BP-7861.
Insecticidal filamentous fungal preparation, comprising a step of mixing a polycarboxylic acid type surfactant and starch which is dissolved in water or gelatinized by water, and a pesticidal filamentous fungus belonging to the genus Paecilomyces. Manufacturing method.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006141392A (en) * | 2004-10-21 | 2006-06-08 | Sumitomo Chemical Co Ltd | Method for producing conidium of microorganism belonging to paecilomyces |
| JP2006232775A (en) * | 2005-02-28 | 2006-09-07 | Sumitomo Chemical Co Ltd | Method for controlling noxious organism |
| JP2012518027A (en) * | 2009-02-19 | 2012-08-09 | ユーリア カサーレ ソシエテ アノニム | Granules containing filamentous fungi and method for preparing the same |
| US8771676B2 (en) * | 2006-06-23 | 2014-07-08 | Sumitomo Chemical Company, Limited | Agrochemically active microbial formulation |
| JPWO2017146147A1 (en) * | 2016-02-23 | 2018-12-13 | 三井化学アグロ株式会社 | Water-floating granule composition |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2003095834A (en) * | 2001-09-26 | 2003-04-03 | Sumitomo Chem Co Ltd | Insect pathogenic filamentous fungus having wide host range |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2003095834A (en) * | 2001-09-26 | 2003-04-03 | Sumitomo Chem Co Ltd | Insect pathogenic filamentous fungus having wide host range |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006141392A (en) * | 2004-10-21 | 2006-06-08 | Sumitomo Chemical Co Ltd | Method for producing conidium of microorganism belonging to paecilomyces |
| JP4715243B2 (en) * | 2004-10-21 | 2011-07-06 | 住友化学株式会社 | Method for producing conidia of microorganisms belonging to the genus Paecilomyces |
| JP2006232775A (en) * | 2005-02-28 | 2006-09-07 | Sumitomo Chemical Co Ltd | Method for controlling noxious organism |
| JP4692023B2 (en) * | 2005-02-28 | 2011-06-01 | 住友化学株式会社 | Pest control method |
| US8771676B2 (en) * | 2006-06-23 | 2014-07-08 | Sumitomo Chemical Company, Limited | Agrochemically active microbial formulation |
| JP2012518027A (en) * | 2009-02-19 | 2012-08-09 | ユーリア カサーレ ソシエテ アノニム | Granules containing filamentous fungi and method for preparing the same |
| JPWO2017146147A1 (en) * | 2016-02-23 | 2018-12-13 | 三井化学アグロ株式会社 | Water-floating granule composition |
| JP7283902B2 (en) | 2016-02-23 | 2023-05-30 | 三井化学クロップ＆ライフソリューション株式会社 | Water floating granule composition |
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