JP2013180995A - Carbonic acid ester extract from angleworm fecal soil and method for production thereof - Google Patents
Carbonic acid ester extract from angleworm fecal soil and method for production thereof Download PDFInfo
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本発明はミミズ糞土のカルボン酸エステル抽出物およびその製造方法に関する。 The present invention relates to a carboxylic acid ester extract of earthworm manure and a method for producing the same.
植物病原糸状菌は植物に深刻な病気を引き起こすため、農業生産の安定の観点から当該菌による病害を防除することが重要な課題となっている。このため植物病原糸状菌による病害を防除する種々の化合物が提案されている。例えば特許文献１には、工業的に合成された複素環を有する化合物が開示されている。 Since phytopathogenic fungi cause serious diseases in plants, it is important to control diseases caused by the fungi from the viewpoint of stable agricultural production. For this reason, various compounds that control diseases caused by phytopathogenic fungi have been proposed. For example, Patent Document 1 discloses an industrially synthesized compound having a heterocyclic ring.
消費者は工業的に合成された農薬を用いない安全かつ安心な食物を求めている。従って、植物病原糸状菌による病害を防除する化合物または組成物においても、工業的に合成されたものではなく、生体材料由来のものへのニーズが高まりつつある。しかしながら、生体材料由来でありながら植物病原糸状菌によってもたらされる病害に対して優れた防除活性を有する化合物または組成物は未だ得られていない。 Consumers are demanding safe and secure food that does not use industrially synthesized pesticides. Accordingly, there is an increasing need for compounds or compositions that control diseases caused by phytopathogenic fungi that are not industrially synthesized but derived from biomaterials. However, a compound or composition having excellent control activity against diseases caused by phytopathogenic fungi while being derived from biomaterials has not yet been obtained.
かかる事情を鑑み、本発明は、植物病原糸状菌による病害を防除する生体材料由来の化合物または組成物を提供することを課題とする。 In view of such circumstances, an object of the present invention is to provide a biomaterial-derived compound or composition that controls diseases caused by phytopathogenic fungi.
発明者らは、ミミズ糞土のカルボン酸エステル抽出物およびその中に含まれる特定の化合物が、植物病原糸状菌による病害を防除する活性を有することを見出し、本発明を完成させた。 The inventors have found that the carboxylic acid ester extract of earthworm feces and a specific compound contained therein have an activity of controlling diseases caused by phytopathogenic fungi, and have completed the present invention.
すなわち、前記課題は以下の本発明により解決される。
［１］（Ａ）ミミズ糞土を水抽出工程に供して、第一水相を得る工程、
（Ｂ）前記第一水相を、一般式（Ｉ）で表されるカルボン酸エステル以外の有機溶媒抽出工程に供して、有機相と第二水相とを得る工程、
（Ｃ）前記第二水相を一般式（Ｉ）：Ｒ−ＣＯＯＲ’（Ｒは炭素数１〜８のアルキル基、Ｒ’は炭素数１〜４のアルキル基である）
で表されるカルボン酸エステル抽出工程に供してカルボン酸エステル相と第三水相とを得る工程、および
（Ｄ）前記カルボン酸エステル相からカルボン酸エステルを除去してカルボン酸エステル抽出物を得る工程、
を含む、ミミズ糞土のカルボン酸エステル抽出物の製造方法。
［２］前記［１］に記載の方法で得たミミズ糞土のカルボン酸エステル抽出物。
［３］前記［１］または［２］に記載の方法で得たミミズ糞土のカルボン酸エステル抽出物を含む、植物病原糸状菌による植物病害を防除するための組成物。
［４］後述の一般式（ＩＩ）または（ＩＩＩ）で表される化合物を含む、植物病原糸状菌による植物病害を防除するための組成物。
That is, the said subject is solved by the following this invention.
[1] (A) A step of subjecting earthworm feces to a water extraction step to obtain a first aqueous phase;
(B) A step of subjecting the first aqueous phase to an organic solvent extraction step other than the carboxylic acid ester represented by the general formula (I) to obtain an organic phase and a second aqueous phase;
(C) The second aqueous phase is represented by the general formula (I): R-COOR ′ (R is an alkyl group having 1 to 8 carbon atoms, R ′ is an alkyl group having 1 to 4 carbon atoms)
And (D) removing the carboxylic acid ester from the carboxylic acid ester phase to obtain a carboxylic acid ester extract. Process,
A method for producing a carboxylic acid ester extract of earthworm manure.
[2] A carboxylic acid ester extract of earthworm manure obtained by the method described in [1] above.
[3] A composition for controlling plant diseases caused by phytopathogenic fungi, comprising a carboxylic acid ester extract of earthworm feces obtained by the method described in [1] or [2].
[4] A composition for controlling plant diseases caused by phytopathogenic fungi, comprising a compound represented by the following general formula (II) or (III).
本発明により、植物病原糸状菌による病害を防除する生体材料由来の化合物または組成物が提供できる。 According to the present invention, a biomaterial-derived compound or composition that can control diseases caused by phytopathogenic fungi can be provided.
１．ミミズ糞土のカルボン酸エステル抽出物の製造方法
本発明の製造方法は、（Ａ）ミミズ糞土を水抽出工程に供して、第一水相を得る工程、（Ｂ）前記第一水相を、後述する一般式（Ｉ）で表されるカルボン酸エステル以外の有機溶媒抽出工程に供して、有機相と第二水相とを得る工程、（Ｃ）前記第二水相を一般式（Ｉ）で表されるカルボン酸エステル抽出工程に供してカルボン酸エステル相と第三水相とを得る工程、および（Ｄ）前記カルボン酸エステル相からカルボン酸エステルを除去してカルボン酸エステル抽出物を得る工程、を含む。図１に本発明の製造方法の概要を示す。以下、必要に応じて符号を参照し本発明を説明する。本発明において「〜」はその両端の値を含む。
1. Production method of carboxylic acid ester extract of earthworm excreta The production method of the present invention includes (A) a step of subjecting earthworm excrement to a water extraction step to obtain a first aqueous phase, and (B) the first aqueous phase described later. A step of obtaining an organic phase and a second aqueous phase by subjecting to an organic solvent extraction step other than the carboxylic acid ester represented by the general formula (I), and (C) the second aqueous phase represented by the general formula (I) A step of obtaining a carboxylic acid ester phase and a third aqueous phase by subjecting to the represented carboxylic acid ester extraction step, and (D) a step of removing the carboxylic acid ester from the carboxylic acid ester phase to obtain a carboxylic acid ester extract ,including. FIG. 1 shows an outline of the production method of the present invention. Hereinafter, the present invention will be described with reference to the reference numerals as necessary. In the present invention, “to” includes values at both ends thereof.
１−１．Ａ工程
本工程では、ミミズ糞土１を水抽出工程に供して第一水相Ａ１および固相Ａ２を得る。ミミズ糞土とは、ミミズの***物を含む土である。ミミズ糞土については後で詳しく説明する。
1-1. Step A In this step, the earthworm dung 1 is subjected to a water extraction step to obtain a first aqueous phase A1 and a solid phase A2. Earthworm dung is soil that contains excrement of earthworms. Earthworm dung will be explained in detail later.
（１）抽出操作および条件等
抽出は、ビーカー等の容器にミミズ糞土と水を装入して撹拌することで行なえる。抽出時の水の温度は室温〜１００℃としてよいが、抽出を効率よく行なう観点から、７０℃以上が好ましく、８０℃以上がより好ましい。同様に、水の温度の上限に関しては、１００℃以下が好ましく、９０℃以下がより好ましい。抽出時間は適宜調整してよいが、７０〜１００℃で抽出を行なう場合、０．５〜５時間が好ましく、１〜３時間がより好ましい。
(1) Extraction operation and conditions, etc. Extraction can be carried out by charging earthworm feces and water into a container such as a beaker and stirring. Although the temperature of the water at the time of extraction may be room temperature to 100 ° C, it is preferably 70 ° C or higher, more preferably 80 ° C or higher, from the viewpoint of efficiently performing extraction. Similarly, regarding the upper limit of the temperature of water, 100 degrees C or less is preferable and 90 degrees C or less is more preferable. Although extraction time may be adjusted suitably, when extracting at 70-100 degreeC, 0.5-5 hours are preferable and 1-3 hours are more preferable.
抽出を効率よく行なう観点から、ミミズ糞土１００質量部に対して、水の量は２００〜１０００質量部が好ましく、３００〜５００質量部がより好ましい。
抽出が完了した後の容器内容物は、ろ過等により、ミミズ糞土の残渣である固相Ａ２と第一水相Ａ１とに分離される。第一水相Ａ１から水を除去すると第一水抽出物を単離できる。この場合、水を蒸発させて除去することが好ましい。第一水抽出物は、通常、固体または液体である。
From the viewpoint of efficiently performing the extraction, the amount of water is preferably 200 to 1000 parts by mass, and more preferably 300 to 500 parts by mass with respect to 100 parts by mass of earthworm dung.
The contents of the container after the extraction is completed are separated into a solid phase A2 and a first aqueous phase A1 that are residues of earthworm dung by filtration or the like. The first water extract can be isolated by removing water from the first aqueous phase A1. In this case, it is preferable to remove the water by evaporating. The first water extract is usually solid or liquid.
第一水相Ａ１またはこれから単離された第一水抽出物を保存する場合は、成分の劣化等を防ぐために、−２０℃以下程度の低温にて保存することが好ましい。
（２）ミミズ糞土
ミミズ糞土とはミミズの***物を含む土である。ミミズとは環形動物門貧毛綱に属する動物である。本発明においては、入手容易性等の観点から、フトミミズ科、ツリミミズ科に分類されるミミズ由来のミミズ糞土が好ましく、中でもシマミミズ由来のミミズ糞土が好ましい。
In the case of storing the first aqueous phase A1 or the first water extract isolated therefrom, it is preferable to store it at a low temperature of about −20 ° C. or lower in order to prevent deterioration of the components.
(2) Earthworm droppings Earthworm droppings are soil containing earthworm excrement. An earthworm is an animal belonging to the annelid oleander. In the present invention, from the viewpoint of availability, earthworm-derived earthworm feces classified into the earthworm family and the earthworm family are preferable, and earthworm earth-derived earthworm earth is particularly preferable.
ｉ）ミミズ糞土の構造
本発明で用いるミミズ糞土は、団粒構造を有することが好ましい。図２はミミズ糞土の団粒構造を模式的に示す。図２中、１２は団粒であり団粒１２が複数個凝集することにより凝集体１０および団粒間隙間１６が形成される。団粒１２とは、糞土の微粒子が糊状の有機物を介して結合されてできた微小な団粒内隙間１４を有する粒である。糊状の有機物とは、有機物が微生物等により分解されて生成された粘着性の物質である。
i) Structure of earthworm dung The earthworm dung used in the present invention preferably has a aggregate structure. FIG. 2 schematically shows the aggregate structure of earthworm dung. In FIG. 2, reference numeral 12 denotes aggregates, and agglomerates 10 and intergranular gaps 16 are formed by aggregating a plurality of aggregates 12. The aggregate 12 is a grain having a minute intra-aggregate gap 14 formed by combining fine particles of manure via a paste-like organic substance. The pasty organic substance is an adhesive substance produced by decomposing organic substances by microorganisms or the like.
凝集体１０は、団粒内隙間１４および団粒間隙間１６に水を保持できる。また、団粒１２は水には溶解しないので、この保持された水は効率よく排水される。従って、このような団粒構造を有するミミズ糞土を用いると、本工程において効率よく第一水抽出物を得ることができる。この凝集体の直径は約０．１〜０．２ｍｍが好ましい。本発明で用いるミミズ糞土は前記凝集体がさらに凝集して構成された粒子であってもよい。本発明で用いるミミズ糞土粒子の平均粒子径は０．１〜０．６ｍｍが好ましい。このような平均粒子径を有するミミズ糞土粒子は飛散しにくいので取り扱い性に優れる。 Aggregate 10 can retain water in inter-granular gap 14 and inter-granular gap 16. Further, since the aggregate 12 is not dissolved in water, the retained water is drained efficiently. Therefore, if earthworm dung having such a aggregate structure is used, the first water extract can be efficiently obtained in this step. The diameter of the aggregate is preferably about 0.1 to 0.2 mm. The earthworm feces used in the present invention may be particles formed by further agglomerating the aggregates. The average particle diameter of the earthworm excrement particles used in the present invention is preferably 0.1 to 0.6 mm. The earthworm excrement particles having such an average particle diameter are excellent in handleability because they are not easily scattered.
ｉｉ）ミミズ糞土の産生方法
ミミズ糞土は、餌をミミズに摂食させて産生することが好ましい。餌としては特に限定されないが、例えば、木材のチップ、食物の残渣、キノコ類の菌床粕等が挙げられる。中でも、キノコ類の菌床粕を含む餌が好ましく、シイタケの菌床粕を含む餌がより好ましい。菌床粕とは、キノコ類の栽培に用いた菌床の廃棄物である。菌床とは、キノコ栽培における培地であって、オガクズ等の木質基材に栄養源を混ぜた培地である。シイタケの菌床粕を含む餌を摂食したミミズにより産生されたミミズ糞土を用いると、植物病原糸状菌による病害を防除する活性がより向上する。
ii) Method for producing earthworm feces Earthworm feces are preferably produced by feeding food to earthworms. Although it does not specifically limit as bait, For example, the chip | tip of a wood, the residue of food, the fungus bed of a mushroom, etc. are mentioned. Among these, a feed containing fungus mushrooms is preferable, and a food containing mushroom fungi is more preferable. The fungus bed is waste of the fungus bed used for the cultivation of mushrooms. The fungus bed is a medium for mushroom cultivation, which is a medium in which a nutrient source is mixed with a woody base material such as sawdust. The activity of controlling diseases caused by phytopathogenic fungi is further improved by using earthworm feces produced by earthworms that have eaten a diet containing shiitake fungi.
ミミズへの給餌量は、ミミズ１００質量部あたり５０〜１００質量部とすることが好ましい。給餌量が５０質量部未満であると、ミミズ糞土の生産性が十分でないことがある。給餌量が１００質量部を超えると、コストが増加することがある。この餌を与え、土壌でミミズを飼育することにより、ミミズが食餌、***して、ミミズ糞土が産生される。 The amount of feeding to the earthworm is preferably 50 to 100 parts by mass per 100 parts by mass of the earthworm. When the amount of feeding is less than 50 parts by mass, the productivity of earthworm feces may not be sufficient. When the amount of feeding exceeds 100 parts by mass, the cost may increase. By giving this food and raising earthworms in the soil, the earthworms feed and excrete, and earthworm dung is produced.
ｉｉｉ）ミミズ糞土の成分
本発明で用いるミミズ糞土は、炭素、窒素、リン、カリウム、カルシウム、マグネシウム、およびナトリウムを含むことが好ましい。各成分の濃度は以下のとおりであることが好ましい。これらの濃度は独立行政法人農業環境技術研究所が定める肥料分析法に基づいて求められるが、以下にその測定方法の概要も合わせて示す。
iii) Components of Earthworm Feces Earthworm Feces used in the present invention preferably contain carbon, nitrogen, phosphorus, potassium, calcium, magnesium, and sodium. The concentration of each component is preferably as follows. These concentrations are determined based on the fertilizer analysis method established by the National Institute for Agro-Environmental Sciences. The outline of the measurement method is also shown below.
全炭素濃度：２５０〜４００ｍｇ／ｇ。全炭素濃度は、ミミズ糞土に含まれる炭素の総量であり、ミミズ糞土を燃焼させて発生する二酸化炭素を定量して求められる。
全窒素濃度：１５〜３０ｍｇ／ｇ。全窒素濃度は、ミミズ糞土に含まれる窒素の総量であり、ミミズ糞土を燃焼させて発生する酸化窒素を定量することにより求められる。
Total carbon concentration: 250-400 mg / g. The total carbon concentration is the total amount of carbon contained in earthworm feces, and is determined by quantifying carbon dioxide generated by burning earthworm feces.
Total nitrogen concentration: 15-30 mg / g. The total nitrogen concentration is the total amount of nitrogen contained in earthworm feces, and is determined by quantifying the nitric oxide generated by burning earthworm feces.
アンモニア態窒素濃度：１５〜３０ｍｇ／ｋｇ。アンモニア態窒素は、アンモニウム塩の形態で存在する窒素である。この濃度は、ミミズ糞土を２Ｍの塩化カリウム水溶液で抽出し、抽出物をアルカリ性にしてアンモニウム塩をアンモニアにし、水蒸気蒸留してアンモニアを分留して定量して求められる。 Ammonia nitrogen concentration: 15-30 mg / kg. Ammonia nitrogen is nitrogen present in the form of an ammonium salt. This concentration is obtained by extracting earthworm feces with a 2M aqueous potassium chloride solution, making the extract alkaline, converting the ammonium salt to ammonia, steam distillation, fractionating the ammonia, and determining the concentration.
硝酸態窒素濃度：７００〜１０００ｍｇ／ｋｇ。硝酸態窒素は、硝酸イオンのように酸化窒素の形で存在する窒素である。この濃度は、１）ミミズ糞土を２Ｍの塩化カリウム水溶液で抽出し、抽出液にデバルタ合金等の還元性の金属を装入して、亜硝酸イオン、硝酸イオンをアンモニウムイオンに還元し、２）この液をさらにアルカリ性にして、アンモニウムイオンをアンモニアにし、アンモニアを水蒸気蒸留して定量し、３）この値から、別に求めたアンモニア態窒素濃度を差し引くことで求められる。 Nitrate nitrogen concentration: 700-1000 mg / kg. Nitrate nitrogen is nitrogen that exists in the form of nitric oxide like nitrate ions. This concentration is as follows: 1) Extract earthworm feces with 2M aqueous potassium chloride solution, and charge the extract with a reducing metal such as debarta alloy to reduce nitrite and nitrate ions to ammonium ions. 2) This solution is further made alkaline, ammonium ions are converted into ammonia, ammonia is quantified by steam distillation, and 3) it is obtained by subtracting the ammonia nitrogen concentration obtained separately from this value.
全リン濃度：５〜２０ｍｇ／ｇ。全リン濃度は、ミミズ糞土に含まれるリンの総量であり、ミミズ糞土を乾式で灰化した後、バナドモリブデンイエロー溶液を添加して吸光光度分析して求められる。 Total phosphorus concentration: 5-20 mg / g. The total phosphorus concentration is the total amount of phosphorus contained in earthworm feces, and is determined by spectrophotometric analysis after ashing earthworm feces and adding a vanad molybdenum yellow solution.
全カリウム濃度：５〜２０ｍｇ／ｇ
全カルシウム濃度：３０〜６０ｍｇ／ｇ
全マグネシウム濃度：５〜２０ｍｇ／ｇ
全ナトリウム濃度：０．５〜２ｍｇ／ｇ
全カリウム濃度等は、ミミズ糞土に含まれるカリウム等の総量であり、ミミズ糞土を乾式で灰化した後、フレーム原子吸光分析して求められる。
Total potassium concentration: 5-20 mg / g
Total calcium concentration: 30-60 mg / g
Total magnesium concentration: 5-20 mg / g
Total sodium concentration: 0.5-2 mg / g
The total potassium concentration and the like are the total amount of potassium and the like contained in earthworm feces, and are determined by flame atomic absorption analysis after ashing earthworm feces in a dry manner.
本発明で用いるミミズ糞土は、上記成分の他にミミズの消化器官に由来する酵素等の生態由来物質を含むことが好ましい。
また、本発明で用いるミミズ糞土の抽出水は中性〜弱アルカリ性であることが好ましく、ｐＨが７〜８であることがより好ましい。ミミズ糞土の前記抽出水の電気伝導度は、０．５〜２ｄＳ／ｍが好ましい。前記ｐＨと電気伝導度が上記範囲にあるミミズ糞土からは、栄養素が抽出されやすい。ｐＨと電気伝導度は、独立行政法人農業環境技術研究所が定める肥料分析法に準じて、１質量部のミミズ糞土を１０質量部の水で抽出して得た抽出水から求められる。
The earthworm feces used in the present invention preferably contains an ecologically derived substance such as an enzyme derived from an earthworm digestive organ in addition to the above components.
Moreover, it is preferable that the extraction water of earthworm dung used by this invention is neutral-weakly alkaline, and it is more preferable that pH is 7-8. The electric conductivity of the extracted water of earthworm dung is preferably 0.5 to 2 dS / m. Nutrients are easily extracted from earthworm feces having pH and electrical conductivity in the above ranges. The pH and electrical conductivity are determined from the extracted water obtained by extracting 1 part by weight of earthworm dung with 10 parts by weight of water in accordance with the fertilizer analysis method established by the National Institute for Agricultural Environment.
１−２．Ｂ工程
本工程では、前記第一水相Ａ１を、一般式（Ｉ）で表されるカルボン酸エステル以外の有機溶媒抽出工程に供して第二水相Ｂ１と有機相Ｂ２とを得る。
1-2. Step B In this step, the first aqueous phase A1 is subjected to an organic solvent extraction step other than the carboxylic acid ester represented by the general formula (I) to obtain a second aqueous phase B1 and an organic phase B2.
ここで用いる有機溶媒は、一般式（Ｉ）で表されるカルボン酸エステル以外の、水と分離可能な有機溶媒であれば特に限定されない。しかしながら沸点が高いと溶媒を除去することが困難になる場合があるので、沸点が１００℃以下の有機溶媒が好ましく、沸点が５０℃以下の有機溶媒がより好ましい。このような有機溶媒としては、ハロゲン含有炭化水素が挙げられ、中でも入手容易であることから炭素数が２〜５のハロゲン含有炭化水素が好ましく、炭素数が２〜３のハロゲン含有炭化水素がより好ましく、炭素数が２のハロゲン含有炭化水素がさらに好ましい。入手が容易であることから、塩化メチレンまたはクロロホルム等の塩素含有炭化水素が特に好ましい。 The organic solvent used here is not particularly limited as long as it is an organic solvent separable from water other than the carboxylic acid ester represented by the general formula (I). However, since it may be difficult to remove the solvent when the boiling point is high, an organic solvent having a boiling point of 100 ° C. or lower is preferable, and an organic solvent having a boiling point of 50 ° C. or lower is more preferable. Examples of such an organic solvent include halogen-containing hydrocarbons. Among them, halogen-containing hydrocarbons having 2 to 5 carbon atoms are preferable because they are easily available, and halogen-containing hydrocarbons having 2 to 3 carbon atoms are more preferable. A halogen-containing hydrocarbon having 2 carbon atoms is more preferable. Chlorine-containing hydrocarbons such as methylene chloride or chloroform are particularly preferred because they are readily available.
抽出操作には、分液漏斗等、公知の手段を用いることができる。使用する前記有機溶媒の量は、１００質量部の第一水相Ａ１に対して、５０〜２００質量部が好ましい。抽出操作は複数回（例えば２〜５回）実施してもよい。抽出における温度は、使用する有機溶媒により適宜選択してよいが、作業性を考慮すると２０〜３０℃程度が好ましい。 For the extraction operation, known means such as a separatory funnel can be used. The amount of the organic solvent to be used is preferably 50 to 200 parts by mass with respect to 100 parts by mass of the first aqueous phase A1. The extraction operation may be performed a plurality of times (for example, 2 to 5 times). The temperature in the extraction may be appropriately selected depending on the organic solvent to be used, but is preferably about 20 to 30 ° C. in consideration of workability.
第二水相Ｂ１および有機相Ｂ２から、それぞれ水および有機溶媒を除去すると、第二水抽出物および有機溶媒抽出物を単離できる。この場合、水および有機溶媒を蒸発させて除去することが好ましい。第二水抽出物および有機溶媒抽出物は、通常、液体か固体である。これらの相および抽出物を保存する場合は、成分の劣化を防ぐため、−２０℃以下程度の低温にて保存することが好ましい。 When the water and the organic solvent are removed from the second aqueous phase B1 and the organic phase B2, respectively, the second aqueous extract and the organic solvent extract can be isolated. In this case, it is preferable to remove water and the organic solvent by evaporation. The second water extract and the organic solvent extract are usually liquid or solid. When storing these phases and extracts, it is preferable to store them at a low temperature of about −20 ° C. or lower in order to prevent deterioration of the components.
１−３．Ｃ工程
本工程では、第二水相Ｂ１を特定のカルボン酸エステル抽出工程に供して、カルボン酸エステル相Ｃ１および第三水相Ｃ２を得る。本工程で用いるカルボン酸エステルは室温で液体であり、その構造は一般式（Ｉ）で表される。
1-3. Step C In this step, the second aqueous phase B1 is subjected to a specific carboxylic acid ester extraction step to obtain a carboxylic acid ester phase C1 and a third aqueous phase C2. The carboxylic acid ester used in this step is liquid at room temperature, and its structure is represented by the general formula (I).
Ｒ−ＣＯＯＲ’ （Ｉ）
Ｒは炭素数１〜８のアルキル基であり、Ｒ’は炭素数１〜４のアルキル基である。本発明においてアルキル基は、１級アルキル基、２級アルキル基、および３級アルキル基を含む。
R-COOR '(I)
R is an alkyl group having 1 to 8 carbon atoms, and R ′ is an alkyl group having 1 to 4 carbon atoms. In the present invention, the alkyl group includes a primary alkyl group, a secondary alkyl group, and a tertiary alkyl group.
次工程において、カルボン酸エステル相Ｃ１からカルボン酸エステルを除去することにより、カルボン酸エステル抽出物Ｄ１が単離できる。この際、カルボン酸エステルは公知の方法で除去できるが、蒸発させて除去することが好ましい。従って、Ｃ工程で用いるカルボン酸の沸点はあまり高くないことが好ましい。よって式（Ｉ）におけるＲのアルキル基の炭素数は１〜６が好ましく、１〜４がより好ましく、１〜３がさらに好ましく、２〜３が特に好ましい。同様にＲ’のアルキル基の炭素数は１〜３が好ましく、２〜３がより好ましい。好ましいカルボン酸エステルの具体例としては、酢酸エチル、酢酸ブチル、酢酸プロピルなどの酢酸のエステルや、酪酸ブチル、プロピオン酸メチル等が挙げられる。 In the next step, the carboxylic acid ester extract D1 can be isolated by removing the carboxylic acid ester from the carboxylic acid ester phase C1. At this time, the carboxylic acid ester can be removed by a known method, but is preferably removed by evaporation. Therefore, it is preferable that the boiling point of the carboxylic acid used in Step C is not so high. Therefore, 1-6 are preferable, as for carbon number of the alkyl group of R in Formula (I), 1-4 are more preferable, 1-3 are more preferable, and 2-3 are especially preferable. Similarly, the carbon number of the alkyl group of R ′ is preferably 1 to 3, and more preferably 2 to 3. Specific examples of preferable carboxylic acid esters include esters of acetic acid such as ethyl acetate, butyl acetate, and propyl acetate, butyl butyrate, methyl propionate, and the like.
抽出操作は、分液漏斗等、公知の手段を用いて行なうことが好ましい。使用するカルボン酸エステルの量は、１００質量部の第二水相Ｂ１に対して、５０〜２００質量部が好ましい。抽出操作は複数回（例えば２〜５回）実施してもよい。抽出における温度は、使用する有機溶媒により適宜選択してよいが、作業性を考慮すると２０〜３０℃程度が好ましい。 The extraction operation is preferably performed using a known means such as a separatory funnel. The amount of the carboxylic acid ester to be used is preferably 50 to 200 parts by mass with respect to 100 parts by mass of the second aqueous phase B1. The extraction operation may be performed a plurality of times (for example, 2 to 5 times). The temperature in the extraction may be appropriately selected depending on the organic solvent to be used, but is preferably about 20 to 30 ° C. in consideration of workability.
１−４．Ｄ工程
本工程ではカルボン酸エステル相Ｃ１からカルボン酸エステルを除去して、カルボン酸エステル抽出物Ｄ１を得る。前述のとおり、カルボン酸エステルを蒸発させて除去することが好ましい。具体的には、風乾、減圧乾燥、蒸留、エバポレーターを用いる乾燥等によりカルボン酸エステルをカルボン酸エステル相Ｃ１から除去できる。カルボン酸エステル相Ｃ１を加熱する場合は、温度が高すぎると成分が劣化する恐れがあるので、３０〜７０℃に加熱することが好ましい。本工程で得られるカルボン酸エステル抽出物Ｄ１は、通常、固体または液体である。
1-4. Step D In this step, the carboxylic acid ester is removed from the carboxylic acid ester phase C1 to obtain a carboxylic acid ester extract D1. As described above, it is preferable to remove the carboxylic acid ester by evaporation. Specifically, the carboxylic acid ester can be removed from the carboxylic acid ester phase C1 by air drying, drying under reduced pressure, distillation, drying using an evaporator, or the like. When heating the carboxylic acid ester phase C1, if the temperature is too high, the components may be deteriorated, so it is preferable to heat to 30 to 70 ° C. The carboxylic acid ester extract D1 obtained in this step is usually solid or liquid.
２．カルボン酸エステル抽出物
（１）特性
カルボン酸エステル抽出物Ｄ１は、植物病原糸状菌による病害を防除する活性を有する。糸状菌とは糸状の菌糸で生活する真菌や細菌であり、植物病原糸状菌とは、植物に寄生して生育し、感染して発病させる糸状菌である。植物病原糸状菌の胞子は、宿主（イネ等）に接着すると発芽管を伸ばし、その先端に形成される付着器と呼ばれる器官を経由して宿主に侵入することが知られている。従って、植物病原糸状菌による病害を防除する活性は、付着器形成および発芽の状態を観察することで評価できる。
2. Carboxylic ester extract (1) Characteristics The carboxylic ester extract D1 has an activity of controlling diseases caused by phytopathogenic fungi. Filamentous fungi are fungi and bacteria that live in filamentous mycelia, and phytopathogenic filamentous fungi are filamentous fungi that grow parasitically on plants and infect and cause disease. It is known that spores of phytopathogenic fungi extend the germ tube when adhering to a host (such as rice) and invade the host via an organ called an appendage formed at the tip of the germ tube. Therefore, the activity of controlling diseases caused by phytopathogenic fungi can be evaluated by observing the state of attachment and germination.
カルボン酸エステル抽出物Ｄ１が前記活性を有する機構は、限定されないが次のように考えられる。カルボン酸エステル抽出物Ｄ１を、さらにクロロホルムとメタノールを溶媒とするクロマトグラフィーで分画すると、特定の画分において顕著な活性が見られる。当該画分の解析から、前記活性を有する化合物は式（ＩＩ）で表されるアルキルジ脂肪酸、または式（ＩＩＩ）で表されるジフタル酸のエステルであると考えられる。
ＨＯＯＣ−Ｒ２−ＣＯＯＨ （ＩＩ）
Ｒ２は炭素数１０〜２０のアルキル基であり、好ましくは炭素数１１〜１３のアルキル基である。
The mechanism by which the carboxylic ester extract D1 has the activity is not limited, but is considered as follows. When the carboxylic acid ester extract D1 is further fractionated by chromatography using chloroform and methanol as solvents, remarkable activity is observed in a specific fraction. From the analysis of the fraction, the compound having the activity is considered to be an alkyldifatty acid represented by the formula (II) or an ester of diphthalic acid represented by the formula (III).
HOOC-R 2 -COOH (II)
R 2 is an alkyl group having 10 to 20 carbon atoms, preferably an alkyl group having 11 to 13 carbon atoms.
Ｒ３は独立に炭素数３〜１０のアルキル基であり、好ましくは炭素数４〜７のアルキル基である。「独立に」とは、２つのＲ３が同一または異なってよいことを意味するが、２つのＲ３は同一であることが好ましい。 R 3 is independently an alkyl group having 3 to 10 carbon atoms, preferably an alkyl group having 4 to 7 carbon atoms. “Independently” means that two R 3 s may be the same or different, but preferably the two R 3 s are the same.
Ｒ４は独立に炭素数１〜４のアルキル基であり、好ましくは炭素数１〜２のアルキル基である。ｎは独立に０〜４の整数である。２つのＲ４およびｎは同一または異なってよいが、同一であることが好ましい。
これらの化合物は、植物病原糸状菌の呼吸を阻害する作用を有すると考えられる。
R 4 is independently an alkyl group having 1 to 4 carbon atoms, preferably an alkyl group having 1 to 2 carbon atoms. n is an integer of 0-4 independently. Two R 4 and n may be the same or different, but are preferably the same.
These compounds are considered to have an action of inhibiting respiration of phytopathogenic fungi.
（２）用途
カルボン酸エステル抽出物Ｄ１、および前記式（ＩＩ）または（ＩＩＩ）で表される化合物は前記の活性を有するので、植物病原糸状菌による植物病害を防除する用途に好適である。例えば、カルボン酸エステル抽出物Ｄ１または前記化合物を水または有機溶剤に溶解して薬剤組成物として使用できる。
(2) Use Since the carboxylate extract D1 and the compound represented by the formula (II) or (III) have the activity described above, they are suitable for use in controlling plant diseases caused by phytopathogenic fungi. For example, the carboxylic acid ester extract D1 or the compound can be dissolved in water or an organic solvent and used as a pharmaceutical composition.
あるいは、カルボン酸エステル抽出物Ｄ１または前記化合物を、公知の方法で製剤化して農園芸用組成物として使用できる。製剤の形態としては、粒剤、乳剤、水性液剤、油剤、マイクロカプセル剤等が挙げられる。 Alternatively, the carboxylic acid ester extract D1 or the compound can be formulated as a composition for agriculture and horticulture by a known method. Examples of the form of the preparation include granules, emulsions, aqueous liquids, oils, microcapsules and the like.
［実施例１］酢酸エチル抽出物の調製
以下、図１を参照しながら実施例１を説明する。
Ａ工程
ミミズ糞土（商品名：みみず太郎１００、株式会社豊徳製）１５０ｇおよび蒸留水６００ｍＬをビーカーに装入した。ビーカーを外部から加熱して内容物の温度を９０℃とした。この温度を保ちながら、２時間、内部を撹拌して水抽出を行なった。次いで、内容物の温度を４０℃に冷却し、遠心分離機（株式会社日立製作所製）を用いて、ビーカー内容物を分離しミミズ糞土を沈殿させた。回転速度は３０００ｒｐｍとし１５分間分離を行なった。この時のビーカー内の水の温度は４℃であった。セライト（関東化学株式会社製）を用いて上清液を濾過し、第一水相Ａ１と、固相Ａ２を得た。
[Example 1] Preparation of ethyl acetate extract Example 1 will be described below with reference to FIG.
Step A A beaker was charged with 150 g of earthworm dung (trade name: Mizutaro 100, manufactured by Toyokuto Co., Ltd.) and 600 mL of distilled water. The beaker was heated from the outside, and the temperature of the content was 90 degreeC. While maintaining this temperature, the inside was stirred for 2 hours for water extraction. Next, the temperature of the contents was cooled to 40 ° C., and the contents of the beaker were separated using a centrifuge (manufactured by Hitachi, Ltd.) to precipitate earthworm feces. The rotation speed was 3000 rpm and separation was performed for 15 minutes. The temperature of the water in the beaker at this time was 4 ° C. The supernatant was filtered using Celite (manufactured by Kanto Chemical Co., Inc.) to obtain a first aqueous phase A1 and a solid phase A2.
Ｂ工程
前記水相Ａ１約６００ｍＬに塩化メチレン（関東化学株式会社製）３００ｍＬを加え、分液漏斗を用いて水相と有機相とに分液した。得られた水相に、再度塩化メチレン３００ｍＬを加えて抽出を行なった。さらに同様の操作を繰り返し、塩化メチレンによる抽出を合計で３回行った。このようにして第二水相Ｂ１を得た。さらに３回の抽出で得た塩化メチレン相を合わせて有機相Ｂ２を得た。
Step B 300 mL of methylene chloride (manufactured by Kanto Chemical Co., Inc.) was added to about 600 mL of the aqueous phase A1, and the mixture was separated into an aqueous phase and an organic phase using a separatory funnel. The obtained aqueous phase was extracted again by adding 300 mL of methylene chloride. Further, the same operation was repeated, and extraction with methylene chloride was performed three times in total. A second aqueous phase B1 was thus obtained. Further, the methylene chloride phases obtained by the extraction three times were combined to obtain an organic phase B2.
Ｃ工程
６００ｍＬの第二水相Ｂ１に酢酸エチル（関東化学株式会社製）３００ｍＬを加え、分液漏斗を用いてカルボン酸エステル相Ｃ１と第三水相Ｃ２とに分液した。
Step C 300 mL of ethyl acetate (manufactured by Kanto Chemical Co., Inc.) was added to 600 mL of the second aqueous phase B1, and the solution was separated into a carboxylic acid ester phase C1 and a third aqueous phase C2 using a separatory funnel.
Ｄ工程
カルボン酸エステル相Ｃ１をシャーレに流し込み、室温（２０℃）にて酢酸エチルを蒸発させて除去し、カルボン酸エステル抽出物（酢酸エチル抽出物）Ｄ１を単離した。当該酢酸エチル抽出物Ｄ１をＤＭＳＯに溶解し、１０ｍｇ／ｍＬの濃度の酢酸エチル抽出物溶液を得た。
Step D Carboxylic acid ester phase C1 was poured into a petri dish, and ethyl acetate was removed by evaporation at room temperature (20 ° C.) to isolate carboxylic acid ester extract (ethyl acetate extract) D1. The ethyl acetate extract D1 was dissolved in DMSO to obtain an ethyl acetate extract solution having a concentration of 10 mg / mL.
［実施例２］酢酸エチル抽出物の活性
（１）オートミール寒天培地（５％［ｗ／ｖ］粉末オートミール（オートミール（The Quaker Oats Company社製）をコーヒーミル（ＭＫ−６１Ｍ−Ｇ、パナソニック株式会社製）で粉末にしたもの）、０．５％［ｗ／ｖ］のスクロース（ナカライテスク株式会社製）、１．５％［ｗ／ｖ］寒天（和光純薬工業株式会社製）、およびＹＧ（０．５％Yeast Extract、２．０％glucose（関東化学株式会社製））寒天培地を、滅菌済みプラスチック培養シャーレ（滅菌シャーレ（深型）ＣＳＰＤ９０−２０、関東化学株式会社製）に流し込み平板培地を準備した。
[Example 2] Activity of ethyl acetate extract (1) Oatmeal agar medium (5% [w / v] powdered oatmeal (Oatmeal (manufactured by The Quaker Oats Company)) was used in a coffee mill (MK-61MG, Panasonic Corporation). Made into a powder), 0.5% [w / v] sucrose (manufactured by Nacalai Tesque), 1.5% [w / v] agar (made by Wako Pure Chemical Industries, Ltd.), and YG (0.5% Yeast Extract, 2.0% glucose (manufactured by Kanto Chemical Co., Inc.)) Agar medium was poured into a sterilized plastic culture petri dish (sterile petri dish (deep type) CSPD90-20, manufactured by Kanto Chemical Co., Ltd.) A medium was prepared.
（２）イネいもち病菌（Magnaporthe oryzae）菌株として、東京理科大学理工学部鎌倉研究室で維持している日本産イネ病原菌株であるＰ２株を準備した。前記平板培地を用い、２８℃にて当該菌株を７日間静置培養した。 (2) As a rice blast fungus (Magnaporthe oryzae) strain, a P2 strain, a Japanese rice pathogen strain maintained in the Kamakura Laboratory, Tokyo University of Science was prepared. The strain was statically cultured at 28 ° C. for 7 days using the plate medium.
これにより得た菌叢から、滅菌筆および滅菌水で気中菌糸を除去した後、ＢＬＢランプ（ＦＬ２０Ｓ・ＢＬＢ、株式会社東芝製）照射下でさらに３日間培養することにより胞子形成を誘導した。この際、培養用シャーレにパラフィルム（PECHINEY PASTIC PACKAGING社製）を巻いた後、ハサミにより通気口を作成したシャーレを用いた。 After removing the aerial hyphae from the thus obtained microflora with a sterilized brush and sterilized water, spore formation was induced by further culturing under irradiation of a BLB lamp (FL20S / BLB, manufactured by Toshiba Corporation) for 3 days. At this time, a parafilm (manufactured by PECHINEY PASTIC PACKAGING) was wound around the petri dish for culturing, and then a petri dish with an air vent formed by scissors was used.
形成された胞子を滅菌筆で滅菌蒸留水中に懸濁した。その濃度を、血球計算盤（THOMA、HIRSCHMANN（登録商標）Laborgerate製）を用いて胞子数が、３×１０４ｃｅｌｌｓ／ｍＬになるように調整した。次に当該胞子懸濁液に、実施例１で調製した１０ｍｇ／ｍＬの濃度の酢酸エチル抽出物溶液を添加し、胞子懸濁液中の酢酸エチル抽出物濃度を１００μｇ／ｍＬとした。 The formed spores were suspended in sterile distilled water with a sterile brush. The concentration was adjusted using a hemocytometer (THOMA, manufactured by HIRSCHMANN (registered trademark) Laborgerate) so that the number of spores was 3 × 10 4 cells / mL. Next, the ethyl acetate extract solution having a concentration of 10 mg / mL prepared in Example 1 was added to the spore suspension to adjust the concentration of the ethyl acetate extract in the spore suspension to 100 μg / mL.
（３）２５×７５ｍｍのポリカーボネート板を準備し、滅菌水中で振盪して洗浄した後乾燥した。当該ポリカーボネート板上に、（２）で得た胞子懸濁液を２０μｌずつ３ヶ所に滴下した。次いで水で湿らせた紙ウエス（キムタオル、日本製紙クレシア株式会社製）を敷いたプラスチックケースを準備し、その中にポリカーボネート板を装入して蓋をした。胞子懸濁液滴下後、６時間、１２時間、２４時間、３６時間後に顕微鏡観察を行ない、付着器形成率を調査した。付着器形成率は以下のようにして算出した。
発芽率＝発芽している胞子数／全胞子数
付着器形成率＝発芽管の先端に付着器を形成している胞子数／発芽している胞子数
この実験を、独立して３回行い、これらの結果を平均した。
(3) A 25 × 75 mm polycarbonate plate was prepared, washed by shaking in sterilized water, and then dried. On the polycarbonate plate, 20 μl of the spore suspension obtained in (2) was dropped at three locations. Next, a plastic case laid with a paper cloth (Kim towel, manufactured by Nippon Paper Crecia Co., Ltd.) moistened with water was prepared, and a polycarbonate plate was inserted therein and covered. Microscopic observation was performed 6 hours, 12 hours, 24 hours, and 36 hours after dropping of the spore suspension, and the rate of formation of the attachment device was investigated. The depositor formation rate was calculated as follows.
Germination rate = number of germinated spores / total spore number attachment rate = number of spores forming attachment at the tip of germ tube / number of germinating spores This experiment was performed three times independently. These results were averaged.
［比較例１］
実施例２の（２）において、胞子懸濁液に酢酸エチル抽出物溶液を添加しなかった以外は、実施例２と同様に発芽率および付着器形成率を評価した。
[Comparative Example 1]
In Example 2 (2), the germination rate and the attachment rate were evaluated in the same manner as in Example 2 except that the ethyl acetate extract solution was not added to the spore suspension.
［比較例２］
実施例２の（２）において、酢酸エチル抽出物溶液の代わりにＤＭＳＯを胞子懸濁液に添加した以外は、実施例２と同様に発芽率および付着器形成率を評価した。この際、胞子懸濁液中、ＤＭＳＯの濃度が１質量％となるようにした。
[Comparative Example 2]
In Example 2 (2), the germination rate and the attachment rate were evaluated in the same manner as in Example 2 except that DMSO was added to the spore suspension instead of the ethyl acetate extract solution. At this time, the concentration of DMSO in the spore suspension was adjusted to 1% by mass.
［比較例３］塩化メチレン抽出物の活性
実施例１の工程Ｂで得た有機相Ｂ２（塩化メチレン相）を風乾して塩化メチレンを除去し、有機溶媒抽出物（塩化メチレン抽出物）を得た。次に当該抽出物をＤＭＳＯに溶解し、１０ｍｇ／ｍＬの濃度の塩化メチレン抽出物溶液を得た。
[Comparative Example 3] Activity of methylene chloride extract The organic phase B2 (methylene chloride phase) obtained in Step B of Example 1 was air-dried to remove methylene chloride to obtain an organic solvent extract (methylene chloride extract). It was. Next, the extract was dissolved in DMSO to obtain a methylene chloride extract solution having a concentration of 10 mg / mL.
実施例２の（２）において、酢酸エチル抽出物溶液の代わりに塩化メチレン抽出物溶液を胞子懸濁液に添加した以外は、実施例２と同様に発芽率および付着器形成率を評価した。 In Example 2 (2), the germination rate and the attachment device formation rate were evaluated in the same manner as in Example 2 except that the methylene chloride extract solution was added to the spore suspension instead of the ethyl acetate extract solution.
［比較例４］水抽出物の活性
実施例１の工程Ｃで得た第三水相Ｃ３を減圧下（１０ｍｍＨｇ）５０℃で乾燥して水を除去し第三水抽出物を単離した。当該抽出物を、ＤＭＳＯに溶解した。不溶の固形分をろ過して除去し、第三水抽出物のＤＭＳＯ溶液を得た。このとき、ＤＭＳＯに溶解している第三水抽出物の成分（以下「ＤＭＳＯ可溶成分」という）の濃度が、溶液中１０ｍｇ／ｍＬとなるようにした。
[Comparative Example 4] Activity of water extract The third aqueous phase C3 obtained in Step C of Example 1 was dried at 50 ° C under reduced pressure (10 mmHg) to remove water, and the third water extract was isolated. The extract was dissolved in DMSO. The insoluble solid was removed by filtration to obtain a DMSO solution of the third water extract. At this time, the concentration of the component of the third water extract dissolved in DMSO (hereinafter referred to as “DMSO soluble component”) was adjusted to 10 mg / mL in the solution.
実施例２の（２）において、このように調製した第三水抽出物のＤＭＳＯ溶液を、酢酸エチル抽出物溶液の代わりに胞子懸濁液に添加した以外は、実施例２と同様に発芽率および付着器形成率を評価した。この際、胞子懸濁液中の前記ＤＭＳＯ可溶成分の濃度を１００μｇ／ｍＬとした。 In Example 2 (2), the germination rate was the same as in Example 2 except that the DMSO solution of the third water extract thus prepared was added to the spore suspension instead of the ethyl acetate extract solution. And the applicator formation rate was evaluated. At this time, the concentration of the DMSO-soluble component in the spore suspension was set to 100 μg / mL.
［比較例５］水抽出物の活性
比較例４においてろ過により除去した固形分を水に溶解して、固形分濃度が１０ｍｇ／ｍＬの水溶液を得た。
[Comparative Example 5] Activity of water extract The solid content removed by filtration in Comparative Example 4 was dissolved in water to obtain an aqueous solution having a solid content concentration of 10 mg / mL.
実施例２の（２）において、酢酸エチル抽出物溶液の代わりに当該水溶液を胞子懸濁液に添加した以外は、実施例２と同様に発芽率および付着器形成率を評価した。この際、胞子懸濁液中の固形分濃度を１００μｇ／ｍＬとした。 In Example 2 (2), the germination rate and the attachment rate were evaluated in the same manner as in Example 2 except that the aqueous solution was added to the spore suspension instead of the ethyl acetate extract solution. At this time, the solid content concentration in the spore suspension was set to 100 μg / mL.
実施例２および比較例１〜５の結果を図３に示す。図３の縦軸は、比較例２における発芽率および付着器形成率を１とした相対値である。図３から、実施例１の酢酸エチル抽出物が発芽率および付着器形成率を著しく低減させることが分かる。 The results of Example 2 and Comparative Examples 1 to 5 are shown in FIG. The vertical axis in FIG. 3 is a relative value with the germination rate and the applicator formation rate in Comparative Example 2 as 1. From FIG. 3, it can be seen that the ethyl acetate extract of Example 1 significantly reduces the germination rate and attachment rate.
［実施例３］酢酸エチル抽出物の分画
実施例１で得た酢酸エチル抽出物を、下記の溶媒を用いたフラッシュカラムクロマトグラフィー（Silica Gel 60N、関東化学株式会社製）により分画した。
[Example 3] Fractionation of ethyl acetate extract The ethyl acetate extract obtained in Example 1 was fractionated by flash column chromatography (Silica Gel 60N, manufactured by Kanto Chemical Co., Inc.) using the following solvent.
各画分について実施例２と同様にして発芽率および付着器形成率を評価した。結果を図４に示す。図４から、画分３および４に極めて優れた活性が確認された。 Each fraction was evaluated in the same manner as in Example 2 for the germination rate and the attachment rate. The results are shown in FIG. From FIG. 4, extremely excellent activity was confirmed in fractions 3 and 4.
［実施例４］酢酸エチル抽出物の分画
実施例３で得た画分３について、下記の溶媒を用いたフラッシュカラムクロマトグラフィーにより分画した。
[Example 4] Fractionation of ethyl acetate extract Fraction 3 obtained in Example 3 was fractionated by flash column chromatography using the following solvent.
各画分を２０℃で乾燥して溶媒を除去し、固体または液体の成分を得た後、当該成分をＤＭＳＯに溶解し溶液とした。当該溶液を胞子懸濁液に添加し、実施例２と同様にして発芽率および付着器形成率を評価した。ただし、胞子懸濁液における固体または液体の成分濃度を１０μｇ／ｍＬとした。結果を図５に示す。図５から、画分３−２に極めて優れた活性が確認された。 Each fraction was dried at 20 ° C. to remove the solvent to obtain a solid or liquid component, and then the component was dissolved in DMSO to obtain a solution. The solution was added to the spore suspension, and the germination rate and the attachment rate were evaluated in the same manner as in Example 2. However, the solid or liquid component concentration in the spore suspension was set to 10 μg / mL. The results are shown in FIG. From FIG. 5, extremely excellent activity was confirmed in fraction 3-2.
［実施例５］酢酸エチル抽出物の分画
実施例３で得た画分４について、実施例４と同じ溶媒を用いたフラッシュカラムクロマトグラフィーにより分画し、画分４−１、画分４−２、および画分４−３を得た。
[Example 5] Fractionation of ethyl acetate extract Fraction 4 obtained in Example 3 was fractionated by flash column chromatography using the same solvent as in Example 4, and fractions 4-1 and 4 were collected. -2 and fractions 4-3 were obtained.
各画分について実施例４と同様にして発芽率および付着器形成率を評価した。結果を図６に示す。図６から、画分４−２に極めて優れた活性が確認された。
実施例４および５で得た画分３−２および４−２を混合し、ＬＣ／ＭＳ（Bruker Daltonics社製）およびＮＭＲ（Bruker Biospin社製）を用いて、活性物質の同定を行なった。その結果、以下の化合物を同定した。
Each fraction was evaluated in the same manner as in Example 4 for the germination rate and the attachment rate. The results are shown in FIG. From FIG. 6, extremely excellent activity was confirmed in fraction 4-2.
Fractions 3-2 and 4-2 obtained in Examples 4 and 5 were mixed and the active substance was identified using LC / MS (Bruker Daltonics) and NMR (Bruker Biospin). As a result, the following compounds were identified.
［実施例６］ウリ炭そ病菌（Colletotricum orbiculare）に対する活性
実施例４および５で得た画分３−２および４−２を混合し、以下の溶媒を用いたフラッシュカラムクロマトグラフィーにより分画した。
[Example 6] Activity against Colletotricum orbiculare Fractions 3-2 and 4-2 obtained in Examples 4 and 5 were mixed and fractionated by flash column chromatography using the following solvents. .
ウリ炭そ病菌として東京理科大学理工学部鎌倉研究室で維持している野生株を準備した。イネいもち病菌の代わりにウリ炭そ病菌を用い、かつ酢酸エチル抽出物の代わりに画分ａ〜ｅを用いて、実施例２と同様にして発芽率および付着器形成率を評価した。ただし、胞子懸濁液における固体または油状成分濃度は１０μｇ／ｍＬとした。結果を図７に示す。この結果から、酢酸エチル抽出物は、ウリ炭そ病菌による病害を防除する活性が高いことが明らかである。 We prepared a wild strain maintained at the Kamakura Laboratory, Faculty of Science and Technology, Tokyo University of Science as a cucumber anthracnose fungus. The germination rate and the attachment rate were evaluated in the same manner as in Example 2 using cucumber anthracnose fungus instead of rice blast fungus and fractions a to e instead of ethyl acetate extract. However, the solid or oily component concentration in the spore suspension was 10 μg / mL. The results are shown in FIG. From this result, it is clear that the ethyl acetate extract has a high activity of controlling diseases caused by cucurbit anthracnose fungi.
［実施例７］イチゴ炭そ病菌（Glomerella cingulata）に対する活性
イチゴ炭そ病菌として東京理科大学理工学部鎌倉研究室で維持している野生株を準備した。ウリ炭そ病菌の代わりにイチゴ炭そ病菌を用いた以外は、実施例６と同様にして付着器形成率を評価した。結果を図８に示す。この結果から、酢酸エチル抽出物は、イチゴ炭そ病菌による病害を防除する活性が高いことが明らかである。
[Example 7] Activity against strawberry anthracnose fungus (Glomerella cingulata) A wild strain maintained at Kamakura Laboratory, Tokyo University of Science was prepared as a strawberry anthracnose fungus. The attachment rate was evaluated in the same manner as in Example 6 except that strawberry anthracnose was used instead of cucumber anthracnose. The results are shown in FIG. From this result, it is clear that the ethyl acetate extract has high activity for controlling diseases caused by strawberry anthracnose fungi.
［参考例１］
実施例５で単離したＨＤＤＡがイネいもち病菌の呼吸に与える影響を検討した。具体的には、実施例１の（２）において、酢酸エチル抽出物溶液の代わりにＨＤＤＡを胞子懸濁液に添加し、溶存酸素計（Model SWC-301DO、株式会社三商製）の酸素電極を挿入した密閉容器内で撹拌培養することによって、イネいもち病菌の酸素消費量を測定した。この際、胞子懸濁液中のＨＤＤＡの濃度を５００μｇ／ｍＬとした。
[Reference Example 1]
The effect of HDDA isolated in Example 5 on the respiration of rice blast fungus was examined. Specifically, in Example 1 (2), HDDA was added to the spore suspension instead of the ethyl acetate extract solution, and the dissolved oxygen meter (Model SWC-301DO, manufactured by Sansho Co., Ltd.) oxygen electrode The oxygen consumption of rice blast fungus was measured by stirring and culturing in an airtight container in which was inserted. At this time, the concentration of HDDA in the spore suspension was 500 μg / mL.
比較のため、実施例１の（２）において、酢酸エチル抽出物溶液の代わりにＤＭＳＯを胞子懸濁液に添加した場合においての酸素消費量を測定した。胞子懸濁液中のＤＭＳＯの濃度は１質量％とした。結果を図９に示す。この結果から、ＨＤＤＡはイネいもち病菌の呼吸を阻害することが示唆された。 For comparison, in Example 1 (2), the oxygen consumption was measured when DMSO was added to the spore suspension instead of the ethyl acetate extract solution. The concentration of DMSO in the spore suspension was 1% by mass. The results are shown in FIG. From these results, it was suggested that HDDA inhibits the respiration of rice blast fungus.
１ ミミズ糞土
１０ 団粒の凝集体
１２ 団粒
１４ 団粒内隙間
１６ 団粒間隙間
Ａ１ 第一水相
Ａ２ 固相
Ｂ１ 第二水相
Ｂ２ 有機相
Ｃ１ カルボン酸エステル相
Ｃ２ 第三水相
Ｄ１ カルボン酸エステル抽出物
DESCRIPTION OF SYMBOLS 1 Earthworm dung 10 Aggregate of aggregates 12 Aggregate 14 Intergranular gap 16 Intergranular gap A1 First aqueous phase A2 Solid phase B1 Second aqueous phase B2 Organic phase C1 Carboxylic ester phase C2 Third aqueous phase D1 Carvone Acid ester extract
Claims (8)
（Ｂ）前記第一水相を、一般式（Ｉ）で表されるカルボン酸エステル以外の有機溶媒抽出工程に供して、有機相と第二水相とを得る工程、
（Ｃ）前記第二水相を一般式（Ｉ）：
（Ｄ）前記カルボン酸エステル相からカルボン酸エステルを除去してカルボン酸エステル抽出物を得る工程、
を含む、ミミズ糞土のカルボン酸エステル抽出物の製造方法。 (A) A step of subjecting earthworm feces to a water extraction step to obtain a first aqueous phase;
(B) A step of subjecting the first aqueous phase to an organic solvent extraction step other than the carboxylic acid ester represented by the general formula (I) to obtain an organic phase and a second aqueous phase;
(C) The second aqueous phase is represented by the general formula (I):
A method for producing a carboxylic acid ester extract of earthworm manure.
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