JP6030908B2 - Mycorrhizal culturing method, mycorrhizal spore formation promoting method and mycorrhizal spore formation promoting composition - Google Patents
Mycorrhizal culturing method, mycorrhizal spore formation promoting method and mycorrhizal spore formation promoting composition Download PDFInfo
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Description
本発明は、菌根菌の生長、特に胞子形成を著しく促進させる菌根菌胞子形成促進用物質による菌根菌の培養方法に関し、さらに菌根菌の胞子形成促進方法、およびその菌根菌胞子形成促進用組成物を提供する。 The present invention relates to a mycorrhizal fungus culture method using a mycorrhizal fungus sporulation-promoting substance that significantly promotes mycorrhizal growth, particularly spore formation, and further to a mycorrhizal fungus spore formation promoting method and mycorrhizal fungal spore A composition promoting composition is provided.
従来、この種の菌根菌の培養方法としては、宿主の根を用いた菌糸の培養や胞子生産方法が知られている。また、消毒した宿主の根を用いて、体外的なインビトロ（in vitro）において無菌状態下で菌根菌を培養することも知られている。すなわち、従来は、これら菌根菌の培養方法のように、絶対共生菌である菌根菌の培養においては、宿主の根なくして無菌状態下で純粋に菌根菌を培養し、胞子を得るまで増殖できないと考えられていた。 Conventionally, as a method for culturing this mycorrhizal fungus, a method for culturing mycelia using a host root and a method for producing spores are known. It is also known to cultivate mycorrhizal fungi under aseptic conditions in vitro in vitro using the sterilized host roots. That is, conventionally, in the culture of mycorrhizal fungi, which is an absolute symbiotic fungus, as in the method for culturing mycorrhizal fungi, the mycorrhizal fungi are cultured purely under aseptic conditions without the host roots to obtain spores. It was thought that it could not grow until.
ところが、近年、フラッシュクロマトグラフィで得たバヒアグラス根の２５質量％のメタノール溶出物を用い、菌根菌の中でも代表的なアーバスキュラー菌根（Arbuscular Mycorrhiza：ＡＭ）菌の純粋培養が成功している（例えば、特許文献１、並びに非特許文献１および２参照）。そして、この培養方法においては、赤色光から遠赤色光までの光の照射によって、菌根菌の菌糸生長が著しく促進することも知られている（例えば、特許文献２参照）。しかしながら、これらの培養方法は、バヒアグラスという宿主から得た根抽出物を用いた培養基においてのものであり、バヒアグラス根の採取時期の判断が極めて重要であるとともに、胞子の生産効率が余り良くなく、実用化が容易ではない（例えば、非特許文献３参照）。 However, in recent years, pure culturing of Arbuscular Mycorrhiza (AM) fungus, which is a typical mycorrhizal fungus, has been successful using 25 mass% methanol eluate of bahiagrass root obtained by flash chromatography ( For example, see Patent Document 1 and Non-Patent Documents 1 and 2). In this culture method, it is also known that mycelial growth of mycorrhizal fungi is significantly promoted by irradiation with light from red light to far red light (see, for example, Patent Document 2). However, these culturing methods are in a culture medium using a root extract obtained from a host called bahiagrass, and it is extremely important to determine the timing of collecting bahiagrass roots, and the production efficiency of spores is not so good. It is not easy to put into practical use (for example, see Non-Patent Document 3).
これに対し、本発明者らは、ＡＭ菌の生長に関与する物質の単離・同定によって、宿主の根や根抽出物を全く用いない人工の培養基内でも、ＡＭ菌母胞子から新胞子を生産でき、数世代も継代培養できる純粋培養技術を確立した（例えば、特許文献３、並びに非特許文献４および５参照）。この純粋培養用培地には、ＡＭ菌菌糸を生長促進させて、誘引する作用を持ち、胞子形成をも促進させるトリプトファンダイマー（Trp-Trp）、ロイシルプロリン（Leu-Pro）などのペプチドが含まれている。 In contrast, the present inventors have isolated and identified substances involved in the growth of AM fungi, so that new spores can be obtained from AM fungal spores even in an artificial culture medium that does not use any host roots or root extracts. A pure culture technique that can be produced and subcultured for several generations has been established (see, for example, Patent Document 3 and Non-Patent Documents 4 and 5). This pure culture medium contains peptides such as tryptophan dimer (Trp-Trp) and leucylproline (Leu-Pro) that have the effect of promoting and attracting the mycelium of AM mycelium and also promoting sporulation. It is.
一方、ＡＭ菌の純粋培養技術の実用化を図るためには、ＡＭ菌の生長に関与する物質のさらなる探索が必要である。その探索の過程で、菌根菌の胞子形成を著しく促進する新規の物質、すなわちアミンおよびアミン誘導体を発見し、菌根菌を純粋培養技術で安定的に生産できる技術を作り上げたと言える。 On the other hand, in order to put the pure culture technology of AM bacteria into practical use, it is necessary to further search for substances involved in the growth of AM bacteria. In the process of searching, we discovered a novel substance that significantly promotes mycorrhizal spore formation, that is, amines and amine derivatives, and created a technique that enables stable production of mycorrhizal fungi using pure culture techniques.
ここで、菌根菌は、植物の根に共生し、宿主の植物から光合成産物を得る見返りとして、植物の養水分を促進させ、生長を旺盛にさせるとともに、病害虫や環境ストレスに対する抵抗性を植物に付与する働きを有している。このため、菌根菌の利用としては、（１）低投入で持続可能な作物生産、（２）環境に優しく、安心・安全な食料生産、（３）乾燥地・半乾燥地における環境緑化、等に着目されている。 Here, mycorrhizal fungi are symbiotic to the roots of plants, and in return for obtaining photosynthetic products from the host plant, they promote plant nourishment, vigorous growth, and resistance to pests and environmental stress. It has a function to give to. Therefore, the use of mycorrhizal fungi includes (1) sustainable crop production with low input, (2) environmentally friendly, safe and safe food production, (3) environmental greening in dry and semi-arid areas, Etc.
以上のように、菌根菌は絶対共生微生物であることから、この菌根菌の純粋培養が容易ではないと考えられていたという問題を有している。 As described above, since mycorrhizal fungi are absolute symbiotic microorganisms, there is a problem that pure culture of mycorrhizal fungi has been considered not easy.
本発明は、このような点に鑑みなされたもので、新規の菌根菌胞子形成促進用物質を見出し、菌根菌の純粋培養が容易にできる菌根菌の培養方法に加え、新規の菌根菌胞子形成促進用物質を用いた菌根菌の胞子形成促進方法、および菌根菌胞子形成促進用組成物を提供することを目的とする。 The present invention has been made in view of these points, and has found a novel mycorrhizal spore formation-promoting substance, and in addition to a mycorrhizal fungus culture method capable of easily purifying mycorrhizal fungi, a novel fungus sporulation method of promoting mycorrhizal using Nekin sporulation promoting substance, and an object of the invention to provide a mycorrhizal sporulation promoting composition.
請求項１記載の菌根菌の培養方法は、第１アミンによって構成されるジアミン類、ジメチルアミン、ジエタノールアミン、ピペラジン、およびクロラミンＴの少なくともいずれかを含む培養基で菌根菌を培養し、前記菌根菌の胞子形成を促進させるものである。 The method for cultivating mycorrhizal fungi according to claim 1 comprises culturing mycorrhizal fungi in a culture medium containing at least one of diamines composed of primary amines, dimethylamine, diethanolamine, piperazine, and chloramine T , It promotes sporulation of root fungi .
請求項２記載の菌根菌の培養方法は、請求項１記載の菌根菌の培養方法において、前記培養基にぺプチドを加えるとともに、無機養分、ビタミン類、糖類、リン脂質および核酸物質を適宜加えた固形培地または液体培地で菌根菌を培養するものである。 The method for culturing mycorrhizal fungi according to claim 2 is the method for culturing mycorrhizal fungi according to claim 1, wherein a peptide is added to the culture medium and inorganic nutrients, vitamins, sugars, phospholipids and nucleic acid substances are appropriately added. Mycorrhizal fungi are cultured in the added solid medium or liquid medium .
請求項３記載の菌根菌の培養方法は、請求項１または２記載の菌根菌の培養方法において、前記菌根菌は、アーバスキュラー菌根菌、エリコイド菌根菌、および外生菌根菌のいずれかであるものである。 The method for culturing mycorrhizal fungi according to claim 3 is the method for culturing mycorrhizal fungi according to claim 1 or 2, wherein the mycorrhizal fungi are arbuscular mycorrhizal fungi, ericoidal mycorrhizal fungi, and ectomycorrhizal fungi. It is one of fungi.
請求項４記載の菌根菌の培養方法は、請求項１ないし３いずれかに記載の菌根菌の培養方法において、前記第１アミンによって構成されるジアミン類は、エチレンジアミン、１，２−プロパンジアミン、プトレッシン、１，４−オクタンジアミンのいずれかであるものである。 The method for culturing mycorrhizal fungi according to claim 4 is the method for culturing mycorrhizal fungi according to any one of claims 1 to 3, wherein the diamine constituted by the first amine is ethylenediamine or 1,2-propane. One of diamine, putrescine, and 1,4-octanediamine .
請求項５記載の菌根菌の培養方法は、請求項１ないし４いずれかに記載の菌根菌の培養方法において、前記クロラミンＴは、８０００ｐｐｍから１４０００ｐｐｍの濃度の溶液とするものである。 The method for culturing mycorrhizal fungi according to claim 5 is the method for culturing mycorrhizal fungi according to any one of claims 1 to 4, wherein the chloramine T is a solution having a concentration of 8000 ppm to 14000 ppm .
請求項６記載の菌根菌の胞子形成促進方法は、菌根植物の根に菌根菌を感染させて第１アミンによって構成されるジアミン類、ジメチルアミン、ジエタノールアミン、ピペラジンの少なくともいずれかを生成させた前記根、または第１アミンによって構成されるジアミン類、ジメチルアミン、ジエタノールアミン、ピペラジン、およびクロラミンＴの少なくともいずれかを、他の菌根植物の根に触れさせることによって、前記他の菌根植物の根における菌根菌の胞子形成を促進させるものである。 Sporulation method for promoting mycorrhizal fungi according to claim 6, wherein the generation, diamines composed of primary amines by infecting Mycorrhizal the roots of mycorrhizal plants, dimethylamine, diethanolamine, at least one of piperazine By contacting the roots of other mycorrhizal plants with the roots of other mycorrhizal plants, the roots of other mycorrhizal plants, or at least one of diamines composed of primary amines, dimethylamine, diethanolamine, piperazine, and chloramine T are used. It promotes sporulation of mycorrhizal fungi in plant roots .
請求項７記載の菌根菌胞子形成促進用組成物は、第１アミンによって構成されるジアミン類、ジメチルアミン、ジエタノールアミン、ピペラジン、およびクロラミンＴの少なくともいずれかを含むものである。 The composition for promoting mycorrhizal spore formation according to claim 7 includes at least one of diamines composed of primary amines , dimethylamine , diethanolamine, piperazine , and chloramine T.
請求項１記載の菌根菌の培養方法によれば、菌根菌胞子形成促進用物質である第１アミンによって構成されるジアミン類、ジメチルアミン、ジエタノールアミン、ピペラジン、およびクロラミンＴの少なくともいずれかを含む培養基で菌根菌を培養することにより、純粋に菌根菌の胞子を生産できるから、菌根菌の純粋培養をさらに容易にできる。 According to the method for culturing mycorrhizal fungi according to claim 1 , at least one of diamines, dimethylamine, diethanolamine, piperazine, and chloramine T constituted by a primary amine that is a substance for promoting mycorrhizal spore formation is obtained. By cultivating mycorrhizal fungi with the culture medium containing them, it is possible to produce pure mycorrhizal spores, so that pure culture of mycorrhizal fungi can be further facilitated.
請求項２記載の菌根菌の培養方法によれば、請求項１記載の菌根菌の培養方法において、例えばトリプトファンダイマーおよびロイシルプロリン等のぺプチドを加えた培養基で菌根菌を培養することにより、菌根菌の菌糸生長を回復でき、菌根菌の胞子を生産できるから、菌根菌の純粋培養をより効率良くできる。また、無機養分、ビタミン類、糖類、リン脂質および核酸物質を適宜加えた固形培地または液体培地のいずれかの培地基とすることにより、菌根菌の胞子形成率を向上できる。 According to the method for culturing mycorrhizal fungi according to claim 2, in the method for culturing mycorrhizal fungi according to claim 1, for example, the mycorrhizal fungi are cultured in a culture medium to which peptides such as tryptophan dimer and leucylproline are added. As a result, mycelial growth of mycorrhizal fungi can be recovered and spores of mycorrhizal fungi can be produced, so that pure culture of mycorrhizal fungi can be performed more efficiently. Moreover, the spore formation rate of mycorrhizal fungi can be improved by using a medium group of either a solid medium or a liquid medium to which inorganic nutrients, vitamins, saccharides, phospholipids and nucleic acid substances are appropriately added.
請求項３記載の菌根菌の培養方法によれば、請求項１または２記載の菌根菌の培養方法の効果に加え、アーバスキュラー菌根菌、エリコイド菌根菌および外生菌根菌のいずれの菌根菌であっても、これら菌根菌の胞子を効率良く増殖することができる。 According to the method for culturing mycorrhizal fungi according to claim 3, in addition to the effect of the method for culturing mycorrhizal fungi according to claim 1 or 2, arbuscular mycorrhizal fungi, ericoidal mycorrhizal fungi and ectomycorrhizal fungi Any mycorrhizal fungus can efficiently proliferate spores of these mycorrhizal fungi.
請求項４記載の菌根菌の培養方法によれば、請求項１ないし３いずれかに記載の菌根菌の培養方法において、第１アミンによって構成されるジアミン類を、エチレンジアミン、１，２−プロパンジアミン、プトレッシン、１，４−オクタンジアミンのいずれかとすることにより、菌根菌の胞子形成率を向上できる。 According to the method for culturing mycorrhizal fungi according to claim 4, in the method for culturing mycorrhizal fungi according to any one of claims 1 to 3, the diamine constituted by the first amine is ethylenediamine, 1,2- The spore formation rate of mycorrhizal fungi can be improved by using any one of propanediamine, putrescine, and 1,4-octanediamine .
請求項５記載の菌根菌の培養方法によれば、請求項１ないし４いずれか記載の菌根菌の培養方法において、クロラミンＴを８０００ｐｐｍから１４０００ｐｐｍの濃度の溶液として用いることにより、菌根菌の胞子形成率をより向上できる。 According to the method for culturing mycorrhizal fungi according to claim 5, in the method for culturing mycorrhizal fungi according to any one of claims 1 to 4, by using chloramine T as a solution having a concentration of 8000 ppm to 14000 ppm, The spore formation rate can be further improved.
請求項６記載の菌根菌の胞子形成促進方法によれば、菌根植物の根では菌根菌の成長をつかさどる第１アミンによって構成されるジアミン類、ジメチルアミン、ジエタノールアミン、ピペラジンの少なくともいずれが生成されるので、これらを生成させた根や、第１アミンによって構成されるジアミン類、ジメチルアミン、ジエタノールアミン、ピペラジン、およびクロラミンＴの少なくともいずれかを、他の菌根植物の根に触れさせることによって、他の菌根植物の根における菌根菌の胞子形成を促進させることにより、農業場面で活用することができる。 According to the method for promoting spore formation of mycorrhizal fungi according to claim 6 , at least any one of diamines, dimethylamine, diethanolamine, and piperazine constituted by primary amines that control the growth of mycorrhizal fungi in the roots of mycorrhizal plants. Since they are produced, the roots that produced them, and the diamines composed of primary amines, dimethylamine, diethanolamine, piperazine, and chloramine T are brought into contact with the roots of other mycorrhizal plants. By promoting spore formation of mycorrhizal fungi in the roots of other mycorrhizal plants, it can be utilized in agricultural situations.
請求項７記載の菌根菌胞子形成促進用組成物によれば、第１アミンによって構成されるジアミン類、ジメチルアミン、ジエタノールアミン、ピペラジン、およびクロラミンＴの少なくともいずれかを含むため、この菌根菌胞子形成促進用組成物にて菌根菌を培養することにより、純粋に菌根菌の胞子を生産でき、菌根菌の純粋培養をさらに容易にできる。 According to the composition for promoting mycorrhizal spore formation according to claim 7 , since it contains at least one of diamines composed of primary amines , dimethylamine , diethanolamine, piperazine , and chloramine T, the mycorrhizal fungi By culturing mycorrhizal fungi with the composition for promoting spore formation , spores of mycorrhizal fungi can be produced purely, and pure culture of mycorrhizal fungi can be further facilitated.
次に、本発明の菌根菌の培養方法の一実施形態について説明する。 Next, an embodiment of the method for culturing mycorrhizal fungi of the present invention will be described.
まず、本発明に係る菌糸菌の培養方法は、菌根菌が感染し得る宿主の根や根抽出物を全く用いない人工の培養基を用い、この培養基に活性炭素繊維を添加したり、光環境条件を調整したりすることによって、菌根菌の菌糸および胞子を純粋に増殖できる菌根菌の純粋培養技術である。なお、この菌根菌としては、この種の菌根菌を代表とするアーバスキュラー菌根（ＡＭ菌）（ＶＡ菌根菌）、エリコイド菌根菌および外生菌根菌のいずれかが用いられる。ただし、これらＡＭ菌、エリコイド菌根菌および外生菌根菌以外の菌根菌であっても用いることができる。 First, the mycelial fungus culture method according to the present invention uses an artificial culture medium that does not use any host root or root extract that can be infected with mycorrhizal fungi, and adds activated carbon fiber to the culture medium, This is a mycorrhizal fungus pure culture technique that can grow mycorrhizal fungi and spores purely by adjusting the conditions. As this mycorrhizal fungus, any one of arbuscular mycorrhizal (AM fungus) (VA mycorrhizal fungus), ericoidal mycorrhizal fungus, and ectomycorrhizal fungus that are representative of this mycorrhizal fungus is used. . However, mycorrhizal fungi other than these AM fungi, ericoidal mycorrhizal fungi and ectomycorrhizal fungi can also be used.
また、この菌根菌の純粋培養技術には、新たに発見した菌根菌生長調整用物質としての菌根菌生長促進物質であるアミンとともに、これまでに見出されていた菌根菌生長促進物質であるペプチドや、菌根菌の栄養素、例えば無機養分、ビタミン類、糖類、リン脂質および核酸物質等を適宜添加した固形培地または液体培地のような人工の培養基を用いる。また、菌根菌生長促進物質としては、エチレンジアミン、１、２−プロパンジアミンおよびジメチルアミン等のアミンや、クロラミンＴ等のアミン誘導体が用いられ、これらアミンおよびアミン誘導体の少なくともいずれかを含む菌根菌生長調整用物質が、菌根菌の培養基として用いられる。 In addition, this mycorrhizal fungus cultivation technology includes the newly discovered mycorrhizal fungi growth-promoting substance, an amine that is a mycorrhizal fungus growth-promoting substance, and the mycorrhizal fungi growth promotion that has been found so far. An artificial culture medium such as a solid medium or a liquid medium to which peptides, which are substances, and mycorrhizal fungi, such as inorganic nutrients, vitamins, sugars, phospholipids, and nucleic acid substances are appropriately added is used. As the mycorrhizal growth promoting substance, amines such as ethylenediamine, 1,2-propanediamine and dimethylamine, and amine derivatives such as chloramine T are used, and mycorrhiza containing at least one of these amines and amine derivatives. A substance for adjusting mycorrhizal growth is used as a culture medium for mycorrhizal fungi.
ここで、エチレンジアミンおよび１、２−プロパンジアミン等のアミンは、菌根菌の胞子形成を促進させる作用を有するとともに、菌根菌が感染した根で生成される物質である。また、クロラミンＴは、消毒剤などに用いられる物質であるが、菌根菌の胞子形成を誘発させる作用を有しているので、アミンおよびペプチドを加えた培養基で菌根菌を培養する前の胞子表面に生息する雑菌の消毒に効果的であるとともに、胞子形成を刺激させる有効な胞子処理剤である。 Here, amines such as ethylenediamine and 1,2-propanediamine are substances produced in roots infected with mycorrhizal fungi while having the effect of promoting sporulation of mycorrhizal fungi. Chloramine T is a substance used for disinfectants and the like, but has the effect of inducing sporulation of mycorrhizal fungi, so that before the mycorrhizal fungi are cultured in a culture medium to which amine and peptide are added. It is an effective spore treatment agent that is effective for disinfection of germs living on the spore surface and stimulates sporulation.
まず、実施例１として、本発明のアミンを用いた菌根菌の純粋培養技術による新しいＡＭ菌胞子生産について説明する。 First, as Example 1, the production of new AM fungal spores by the pure culture technique of mycorrhizal fungi using the amine of the present invention will be described.
石井および堀井（石井孝昭・堀井幸江、園芸学研究、２００７年、第６巻別冊２、ｐ．１３６）が開発した表１に示す基本培養液を含むゲルライト（１．５質量％）固形培地をオートクレーブした後、孔径０．２μｍの滅菌フィルタでろ過した図１に示すアミン類を１０μＭ、３０μＭおよび１００μＭの濃度になるように加え、直径５ｃｍのシャーレに入れた。
その後、この培地に表面殺菌を施したグロムス・クララム（Glomus clarum）ＩＫ９７胞子を置床し、２６℃の赤色発光ダイオード（ＬＥＤ）による光照射下(１０μｍｏｌ／ｍ２・ｓｅｃ)で培養した。殺菌方法は、胞子を消毒剤（chloramine T：７０００ｐｐｍ、streptomycin：５６ｐｐｍ、chloramphenicol：２１ｐｐｍ、およびTween ８０：数滴）に投入し、超音波洗浄機で数秒間処理した後、１２分間静置して殺菌した。さらに、培養２か月後、菌糸生長および新しく形成された胞子数を調査した。なお、菌糸長は、スタイラスペン付のタブレット（WACOM Tablet UD−0608II）と自作のプログラムソフトウェア（石井孝昭、日本農業教育学会誌 １９９３年、第２４巻、ｐ．２５−３６）を用いて計測した。 Thereafter, Glomus clarum IK97 spores subjected to surface sterilization were placed on this medium and cultured under light irradiation (10 μmol / m 2 · sec) with a 26 ° C. red light emitting diode (LED). The sterilization method is as follows. Spores are put into a disinfectant (chloramine T: 7000 ppm, streptomycin: 56 ppm, chloramphenicol: 21 ppm, and Tween 80: several drops), treated with an ultrasonic cleaner for several seconds, and then allowed to stand for 12 minutes. Sterilized. Furthermore, after 2 months of culture, mycelial growth and the number of newly formed spores were investigated. The mycelial length was measured using a tablet with a stylus pen (WACOM Tablet UD-0608II) and a self-made program software (Takaaki Ishii, Journal of the Japanese Agricultural Education Society, 1993, Vol. 24, p. 25-36). .
この結果、図１に示すように、供試したアミン類は、菌根菌の菌糸生長促進効果を示さず、むしろ生長を抑制するものが多かった。しかしながら、それらの中で、第一アミンおよび第二アミンに含まれるアミン類は、胞子形成を誘発させる作用を有していた。特に、図２に示すように、エチレンジアミン（ＥＤＡ）、１，２−プロパンジアミン（ＰＤＡ）およびジメチルアミンの効果は著しく大きいものであった。なお、これら図１および図２中の垂線は、標準誤差（ｎ＝８）を表している。 As a result, as shown in FIG. 1, the tested amines did not show the mycelial growth promotion effect of mycorrhizal fungi, but rather suppressed the growth. However, among them, the amines contained in the primary amine and the secondary amine have an action of inducing sporulation. In particular, as shown in FIG. 2, the effects of ethylenediamine (EDA), 1,2-propanediamine (PDA) and dimethylamine were remarkably large. 1 and 2 represent standard errors (n = 8).
次いで、胞子形成促進効果が大きなアミン、すなわちＥＤＡと、これまでに発見したペプチド（Ｔｒｐ−ＴｒｐおよびＬｕｅ−Ｐｒｏ、各１０ｐｐｍ）との併用処理が菌糸生長および胞子形成に及ぼす影響について、ゲルライト固形培地上で検討した。また、この研究成果を基に、最適な濃度下での液体培養の調査も行った。殺菌方法および培養方法については、上記実施例１と同様とした。 Next, the influence of the combined treatment of an amine having a large sporulation-promoting effect, that is, EDA, and the peptides discovered so far (Trp-Trp and Lue-Pro, 10 ppm each) on mycelial growth and sporulation will be described in Gelrite solid medium. Considered above. In addition, based on the results of this research, we also investigated liquid culture under optimal concentration. The sterilization method and culture method were the same as in Example 1 above.
この結果、純粋培養下における菌根菌（Glomus clarum）の菌糸生長および胞子形成に及ぼすＥＤＡおよびＰＤＡの効果をまとめた表２に示すように、基本培養液のみの区においては菌糸生長が旺盛であったが、胞子形成は全くみられなかった。しかしながら、ペプチド（Ｔｒｐ−Ｔｒｐ，１０ｐｐｍ ＋ Ｌｕｅ−Ｐｒｏ，１０ｐｐｍ）添加区においては、既報（石井孝昭・堀井幸江、園芸学研究、２００７年、６巻別冊２、ｐ１３６）と同様に、菌糸生長が良好で胞子形成も観察された。一方、ＥＤＡ添加区では、１００μＭおよび２５０μＭのいずれにおいても菌糸生長が阻害され、特に２５０μＭ区では顕著であった。
ところが、図３に示すように、いずれのＥＤＡ添加区においても母胞子１個から新しい胞子が数個形成された。ＥＤＡとペプチドとの併用添加区では、ＥＤＡによる菌糸生長阻害効果が軽減され、胞子形成が促進される傾向が確認できた。なお、図３において、ＭＳは母胞子を示し、ＥＤＡの濃度が１００μＭとされている。 However, as shown in FIG. 3, several new spores were formed from one mother spore in any EDA-added section. In the combined addition of EDA and peptide, the mycelial growth inhibitory effect by EDA was reduced and the tendency to promote sporulation was confirmed. In FIG. 3, MS indicates a mother spore, and the concentration of EDA is 100 μM.
特に、図４（ａ）に示すように、ＥＤＡ２５０μＭ＋ペプチド添加区では、菌糸生長および胞子形成ともに良好であった。さらに、ＥＤＡとペプチドとの併用処理における胞子形成促進効果を液体培養下で検討したところ、図４（ｂ）に示すように、培養３か月後の解体時には１個の母胞子から多数の新胞子が形成されていた。なお、図４（ａ）において、ＭＳは母胞子を示し、Ｓは新胞子を示している。 In particular, as shown in FIG. 4A, in the EDA 250 μM + peptide addition group, both mycelial growth and spore formation were good. Furthermore, when the sporulation promotion effect in the combined treatment of EDA and peptide was examined in liquid culture, as shown in FIG. 4 (b), a large number of new spores were obtained from one mother spore at the time of disassembly after 3 months of culture. Spores were formed. In FIG. 4A, MS indicates mother spores, and S indicates new spores.
以上のように、胞子形成にアミンが関与することが明らかであることから、アミン誘導体であり消毒剤でもあるクロラミンＴを用いて、胞子表面の消毒とともに、胞子形成に及ぼす影響について調査した。方法としては、７０００ｐｐｍ、８０００ｐｐｍおよび１４０００ｐｐｍのクロラミンＴ溶液に３０分間浸漬し胞子を消毒した後、素寒天培地に置床した。その後、２６℃の赤色発光ダイオードによる光照射下(１０μｍｏｌ／ｍ２・ｓｅｃ)で培養した。この成果を踏まえて、クロラミンＴ８０００ｐｐｍ溶液内に、胞子を１時間、４時間および８時間浸漬させた後、素寒天培地に置床し培養を行った。培養条件は上記実施例１と同様とした。 As described above, since it is clear that amines are involved in spore formation, chloramine T, which is an amine derivative and a disinfectant, was used to investigate the effects on spore formation as well as spore surface disinfection. As a method, the spore was sterilized by immersing in 7000 ppm, 8000 ppm and 14000 ppm of chloramine T solution for 30 minutes, and then placed on an elementary agar medium. Then, it culture | cultivated under the light irradiation (10 micromol / m < 2 > * sec) by a 26 degreeC red light emitting diode. Based on this result, the spores were immersed in a chloramine T8000 ppm solution for 1 hour, 4 hours, and 8 hours, and then placed on an elementary agar medium and cultured. The culture conditions were the same as in Example 1 above.
この結果、菌根菌（Glomus clarum）胞子へのクロラミンＴ処理の効果をまとめた表３に示すように、一般に消毒に用いられている濃度、７０００ｐｐｍでは胞子形成はみられなかったものの、８０００ｐｐｍでは母胞子１個から新しい胞子が６個程度形成された。しかしながら、１４０００ｐｐｍになると胞子形成は阻害される傾向が確認できた。さらに、菌根菌胞子をクロラミンＴ８０００ｐｐｍ溶液内に１時間、４時間および８時間浸漬させたところ、いずれの区でも胞子形成が確認できたが、特に、４時間浸漬区では、図５に示すように、多数の新しい胞子が観察された。なお、図５中のＭＳは母胞子を示している。
無加温のガラスハウス内で育成させたＡＭ菌感染および非感染のバヒアグラス根内からアミン類を抽出して分析した。具体的に、このアミン類の抽出および分析方法は、図６に示すように、根内のアミン類を４ｍＭメタンスルホン酸で抽出し、ろ過した後、Shodex IC-YS-50カラムを装備した電気伝導度検出器付き高速液体クロマトグラフ（ＨＰＬＣ）で分析した。このＨＰＬＣの分析条件は、次のとおりである。 Amines were extracted and analyzed from the roots of infected and non-infected bahiagrass grown in an unheated glass house. Specifically, as shown in FIG. 6, the amines are extracted and analyzed by extracting the amines in the roots with 4 mM methanesulfonic acid, filtering, and then applying an electricity equipped with a Shodex IC-YS-50 column. The analysis was performed by a high performance liquid chromatograph (HPLC) equipped with a conductivity detector. The analysis conditions of this HPLC are as follows.
カラム： Shodex IC-YS-50、４．６ｍｍＩＤ × １２５ｍｍ
溶離液：４ｍＭメタンスルホン酸
流速：１０ｍｌ／ｍｉｎ
カラム温度：４０℃
検出器：Hitachi L3720 Electric Conductivity Detector
ポンプ：Hitachi Pump L-2130
Column: Shodex IC-YS-50, 4.6 mm ID x 125 mm
Eluent: 4 mM methanesulfonic acid Flow rate: 10 ml / min
Column temperature: 40 ° C
Detector: Hitachi L3720 Electric Conductivity Detector
Pump: Hitachi Pump L-2130
この結果、非菌根のバヒアグラス根ではアミン類は検出されなかった。しかしながら、グロムス・ファシキュラータム（G. fasciculatum）、グロムス・クララム（G. clarum）およびギガスポラ・マルガリータ（Gi. margarita）のＡＭ菌が感染した根では、図７に示すように、ジエタノールアミン、ジメチルアミン、トリエチルアミン、ＰＤＡ、ＥＤＡ等のアミン類が検出された。なお、図７において、１はＮａ、２はＮＨ４、３はジエタノールアミン、４はＫ、５はジメチルアミン、６はトリエチルアミン、７はＭｇ、８はＣａ、９はＰＤＡ、１０はＥＤＡを示している。特に、ＥＤＡは、いずれの菌根菌感染根でも検出されたが、ＰＤＡは、グロムス・クララム感染根でのみ分析された。また、菌根菌が感染したバヒアグラスにおける根内のＥＤＡおよびＰＤＡの濃度をまとめた表４に示すように、それらの含量は、胞子形成促進に効果のある濃度で検出された。
以上の結果、胞子形成に効果のあるペプチド以外に、アミン、特に第一アミンおよび第二アミンが菌根菌の胞子形成を著しく促進させる重要な働きを持っていることや、これらの物質は菌根菌が感染した植物において生産されることが見出された。特に、菌根植物において生産されたアミンの中で、ＥＤＡ、ＰＤＡなどはキレート物質であるため、菌根菌の感染によって生成されたＥＤＡ、ＰＤＡなどのアミンが菌根植物の養分吸収に密接に関与していることは非常に興味深く、これらのアミンを土壌などに施用して植物における菌根形成や養分吸収の促進に効果を得ることができるものと考えられる。 As a result, in addition to peptides that are effective for sporulation, amines, especially primary amines and secondary amines, have an important role in significantly promoting mycorrhizal sporulation, and these substances are It has been found that root fungi are produced in infected plants. In particular, among the amines produced in mycorrhizal plants, EDA, PDA, etc. are chelating substances, so amines such as EDA, PDA, etc. produced by mycorrhizal fungal infection are closely related to nutrient uptake of mycorrhizal plants. It is very interesting to be involved, and it is considered that these amines can be applied to soil and the like to obtain an effect in promoting mycorrhiza formation and nutrient absorption in plants.
ＡＭ菌の純粋培養において、ＥＤＡのようなアミンとペプチドとの併用添加は、それぞれの単独添加よりも胞子形成を高めた。この傾向は液体培養下でも確認でき、この純粋培養技術を用いて、菌根菌の胞子を純粋に安定的に大量生産できるという重要な意味を有している。また実用化に向け、ＥＤＡとペプチドとを同時に添加して実験を行ったところ、ＥＤＡ等のアミンは、菌糸生長をわずかに阻害する傾向が認められたので、ＥＤＡは、菌糸生長が旺盛になったときに添加することが良いと思われる。すなわち、本発明に係る培養方法は、菌根菌が感染し得る宿主の根や根抽出物を用いずに、菌根菌の生長を旺盛にして大量に胞子を増殖できるので、さらに菌根菌の純粋培養技術を安定化できる。また、アミンは菌根菌感染植物の根で生成されるので、アミンあるいはその誘導体を農業場面で活用できる。 In the pure culture of AM bacteria, the combined addition of an amine such as EDA and a peptide increased sporulation compared to the individual addition. This tendency can be confirmed even under liquid culture, and it has an important meaning that the mycorrhizal spore can be mass-produced purely and stably by using this pure culture technique. In addition, when EDA and a peptide were added simultaneously for practical use, an amine such as EDA tended to slightly inhibit mycelial growth. Therefore, EDA has a strong hyphal growth. It seems to be good to add when. That is, the culturing method according to the present invention can proliferate mycorrhizal fungi and grow spores in large quantities without using host roots or root extracts that can be infected with mycorrhizal fungi. Can stabilize the pure culture technology. In addition, since amines are produced in the roots of mycorrhizal fungi-infected plants, amines or their derivatives can be used in agricultural situations.
一方、ＡＭ菌の増殖方法としては、遺伝子組み換えの毛状根を宿主とした技術がある（Becard, G. & Fortin, J. A. 1988年. New Phytol. 108: 211−218; St-Arnaud, M. C., Hamel, C., Vimard, B., Caron, M. & Fortin, J. A. 1996年. Mycol. Res. 100: 328-332）。この技術を用いることにより、インビトロでの安全なＡＭ菌の生産が可能であるように思われる。しかしながら、ＡＭ菌は宿主から細胞核を奪うので、毛状根を用いた培養では、変異遺伝子によるＡＭ菌への影響やその変異遺伝子の自然環境への流出や拡大が懸念される。それゆえ、ＡＭ菌の増殖および保存を安心かつ安全に行うためには、本発明を用いてさらなる改良が加えられた菌根菌の純粋培養技術を用い、世界のＡＭ菌の増殖や保存を進めていかなければならない。 On the other hand, as a method for growing AM bacteria, there is a technique using a genetically modified hairy root as a host (Becard, G. & Fortin, JA 1988. New Phytol. 108: 211-218; St-Arnaud, MC, Hamel, C., Vimard, B., Caron, M. & Fortin, JA 1996. Mycol. Res. 100: 328-332). By using this technology, it seems possible to produce safe AM bacteria in vitro. However, since AM bacteria deprive cell nuclei from the host, there are concerns about the influence of mutant genes on AM bacteria and the outflow and expansion of the mutant genes into the natural environment in the culture using hairy roots. Therefore, in order to perform the growth and storage of AM bacteria safely and safely, using the mycorrhizal fungus pure culture technology that has been further improved by using the present invention, the growth and storage of AM bacteria in the world are promoted. I have to go.
Claims (7)
ことを特徴とする菌根菌の培養方法。 A mycorrhizal fungus is cultured in a culture medium containing at least one of diamines composed of primary amines, dimethylamine, diethanolamine, piperazine, and chloramine T, and the sporulation of the mycorrhizal fungi is promoted. Mycorrhizal culture method.
前記培養基にぺプチドを加えるとともに、無機養分、ビタミン類、糖類、リン脂質および核酸物質を適宜加えた固形培地または液体培地で菌根菌を培養する
ことを特徴とする菌根菌の培養方法。 In the method for cultivating mycorrhizal fungi according to claim 1,
A method for cultivating mycorrhizal fungi, comprising culturing mycorrhizal fungi in a solid medium or liquid medium to which a peptide is added to the culture medium and inorganic nutrients, vitamins, sugars, phospholipids, and nucleic acid substances are appropriately added .
前記菌根菌は、アーバスキュラー菌根菌、エリコイド菌根菌、および外生菌根菌のいずれかである
ことを特徴とする菌根菌の培養方法。 The method for culturing mycorrhizal fungi according to claim 1 or 2,
The mycorrhizal fungus is any one of arbuscular mycorrhizal fungi, ericoidal mycorrhizal fungi, and ectomycorrhizal fungi.
前記第１アミンによって構成されるジアミン類は、エチレンジアミン、１，２−プロパンジアミン、プトレッシン、１，４−オクタンジアミンのいずれかである
ことを特徴とする菌根菌の培養方法。 In the method for cultivating mycorrhizal fungi according to any one of claims 1 to 3,
The method for cultivating mycorrhizal fungi, wherein the diamine constituted by the primary amine is any one of ethylenediamine, 1,2-propanediamine, putrescine, and 1,4-octanediamine .
前記クロラミンＴは、８０００ｐｐｍから１４０００ｐｐｍの濃度の溶液とする
ことを特徴とする菌根菌の培養方法。 In the method for cultivating mycorrhizal fungi according to any one of claims 1 to 4,
A method for culturing mycorrhizal fungi, wherein the chloramine T is a solution having a concentration of 8000 ppm to 14000 ppm .
ことを特徴とする菌根菌の胞子形成促進方法。 Diamines composed of primary amines by infecting Mycorrhizal the roots of mycorrhizal plants, dimethylamine, diethanolamine, diamines made by the root or first amine was generated at least one piperazine Promoting spore formation of mycorrhizal fungi in the roots of the other mycorrhizal plants by contacting at least one of dimethylamine, diethanolamine, piperazine, and chloramine T with the roots of the other mycorrhizal plants. A method for promoting spore formation of mycorrhizal fungi characterized by the above.
ことを特徴とする菌根菌胞子形成促進用組成物。 A composition for promoting mycorrhizal spore formation , comprising at least one of diamines composed of primary amines , dimethylamine , diethanolamine, piperazine , and chloramine T.
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