JP4979551B2 - Mycorrhizal fungus culture method - Google Patents

Description

  The present invention relates to a mycorrhizal culture method for culturing mycelium and spores of mycorrhizal fungi.

  Conventionally, as this mycorrhizal fungi culture method, a mycelial culture or spore production method using a host root is known. It is also known to cultivate mycorrhizal fungi under aseptic conditions in vitro in vitro using disinfected host roots. In other words, conventionally, in the culture of mycorrhizal fungi, which are absolute symbiotic fungi, like these mycorrhizal culture methods, the mycorrhizal fungi are cultured purely under aseptic conditions without the roots of the host, and the spores are obtained. It was thought that it could not grow until it was obtained.

  However, in recent years, pure culture of Arbuscular Mycorrhiza (AM), a representative mycorrhizal fungus, has succeeded using 25% by mass methanol eluate of bahiagrass root obtained by flash chromatography. (See, for example, 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 successful 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 spore production efficiency is not so good. It is not easy to put into practical use (for example, see Non-Patent Document 2).

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, this mycorrhizal fungus can be used for 1) sustainable crop production with low inputs, 2) environmentally friendly, safe and secure food production, 3) environmental greening in dry and semi-arid areas, etc. It is attracting attention. However, since mycorrhizal fungi are absolutely symbiotic microorganisms, it was thought that pure culture of these fungi was not easy (see, for example, Non-Patent Document 3), which greatly limited basic and applied research on these fungi. It was.
Japanese Patent Laid-Open No. Hei 8-19185 JP 2003-274930 A Takaaki Ishii, 4 others, Horticultural Society, 1995, Vol. 64, separate volume 1, p. 190-191 Takaaki Ishii, 4 others, ICOM4 (The Fourth International Conference on Mycorrhizae), (Canada), 2003, p. 696 Declerck S., two others, In Vitro Culture of Mycorrhizae, (Germany), 2005, p. 1-388

  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.

  This invention is made | formed in view of such a point, and it aims at providing the mycorrhizal fungus culture method which can perform pure culture | cultivation of mycorrhizal fungi easily.

請 Motomeko 1 method mycorrhizal fungus culture described, plus a peptide as mycorrhizal growth-promoting substances represented by the following formula (1), mineral nutrients, vitamins, saccharides, phospholipids and nucleic acid material was added Mycorrhizal fungi are cultured in a culture medium that is a solid medium or a liquid medium.

  A mycorrhizal fungus culture method according to claim 2 is the mycorrhizal fungus culture method according to claim 1, wherein a peptide represented by the following formula (2) is added to the culture medium.

The mycorrhizal fungus culture method according to claim 3 is the mycorrhizal fungus culture method according to claim 1 or 2, wherein the mycorrhizal fungus is any one of arbuscular mycorrhizal fungi, ericoidal mycorrhizal fungi, and ectomycorrhizal fungi. Ru der thing is.

A mycorrhizal fungus culture method according to claim 4 is the mycorrhizal fungi culture method according to any one of claims 1 to 3 , wherein activated carbon fibers having activated carbon fibers are added to the culture medium.

The mycorrhizal fungi culture method according to claim 5 is the mycorrhizal fungus culture method according to any one of claims 1 to 4 , wherein the mycorrhizal fungi are cultured under light environment conditions of red light irradiation.

According to the mycorrhizal fungi culture method according to claim 1, pure mycorrhiza is obtained by culturing mycorrhizal fungi in a culture medium to which a peptide as a mycorrhizal mycelia growth promoting substance represented by formula (1) is added. Since the mycelium of the fungus can be grown and the spores of the mycorrhizal fungus can be produced, the pure culture of the mycorrhizal fungus can be facilitated.

In addition, the growth of mycorrhizal mycelium can be vigorous and the spore formation rate by using either a solid medium or a liquid medium containing inorganic nutrients, vitamins, sugars, phospholipids and nucleic acid substances. Can be improved.

  According to the mycorrhizal fungus culture method of claim 2, in addition to the effect of the mycorrhizal fungi culture method of claim 1, the mycorrhizal fungi are cultured in a culture medium to which the peptide represented by formula (2) is added. By doing so, the mycelium of mycorrhizal fungi grows more efficiently and can produce spores of the mycorrhizal fungi, so that the pure culture of the mycorrhizal fungi can be performed more efficiently.

According to the mycorrhizal fungus culture method of claim 3, in addition to the effects of the mycorrhizal fungus culture method of claim 1 or 2, any of arbuscular mycorrhizal fungi, ericoidal mycorrhizal fungi and ectomycorrhizal fungi even mycorrhizal fungi, Ru can efficiently grow the spores of these mycorrhizal fungi.

According to the mycorrhizal fungi culture method according to claim 4 , in addition to the effects of the mycorrhizal fungi culture method according to any one of claims 1 to 3 , by adding activated carbon fiber to the culture medium, the growth of mycorrhiza mycelium And since the branching can be promoted, the spore formation rate of this mycorrhizal fungus can be further improved.

According to the mycorrhizal fungi culture method according to claim 5 , in addition to the effects of the mycorrhizal fungi culture method according to any one of claims 1 to 4 , by culturing mycorrhizal fungi under light environment conditions of red light irradiation Since the growth of the mycelium of this mycorrhizal fungus becomes more vigorous, the spore formation rate of this mycorrhizal fungus can be further improved.

  Next, an embodiment of the mycorrhizal fungus culture method of the present invention will be described.

  First, this mycelium culture method uses an artificial culture medium that does not use any host roots or root extract that can be infected with mycorrhizal fungi, and adds activated carbon fiber to this culture medium or adjusts the light environment conditions. This is a pure culture technique of mycorrhizal fungi that can purely grow mycelium and spores of mycorrhizal fungi. As this mycorrhizal fungus, any one of arbuscular mycorrhizal fungi (VA mycorrhizal fungi), ericoidal mycorrhizal fungi and ectomycorrhizal fungi that are representative of this type of mycorrhizal fungi is used. However, even mycorrhizal fungi other than these arbuscular mycorrhizal fungi, ericoidal mycorrhizal fungi and ectomycorrhizal fungi can be used.

  In addition, the mycorrhizal fungus has a pure culture technique that includes a special chemical substance, a mycorrhizal fungi growth-promoting substance, and mycorrhizal fungi nutrients such as inorganic nutrients, vitamins, sugars, phospholipids and nucleic acid substances. An artificial culture medium such as solid medium or liquid medium supplemented with Here, the mycorrhizal growth-promoting substance includes an AM fungal growth-promoting substance that is a tryptophan dimer (Trp-Trp) represented by the following formula (1) and an active component represented by the following formula (2). A low molecular weight peptide such as leucylproline (Leu-Pro) is used.

  Here, the tryptophan dimer represented by the above formula (1) has a function of promoting hyphal growth of AM fungi and is a signal substance that attracts the mycelium. In addition, leucylproline represented by the above formula (2) has an effect of promoting mycelial growth of AM fungi, and has an effect as an adjuvant for tryptophan dimer.

  Next, activated carbon fiber (Activated Carbon Fiber: ACF) is obtained by activating normal carbon fiber and added to a solid medium or liquid medium to which a mycorrhizal growth promoting substance is added. Or the growth and branching of mycorrhizal mycelium cultivated in a liquid medium are promoted, and the spore formation rate of this mycorrhizal fungus is improved.

Further, the light environment conditions adjusted during the culture period of the mycorrhizal fungi include dark or red light irradiation. And as red light irradiation, the light irradiation (5 microEm ^<-2 > S ^{< -1} >) using a red light emitting diode (redLED) is preferable. Here, this mycorrhizal fungus grows mycelium even under dark conditions to form spores, but under red light irradiation conditions, the mycelium grows more vigorously and the spore formation rate increases.

  Next, a method for extracting tryptophan dimer and leucylproline used in the mycorrhizal fungus culture method of the above embodiment will be described.

  First, bahiagrass roots or exudates of the roots are used as raw materials, and methanol eluate is filtered from the bahiagrass roots or exudates of the roots.

  Thereafter, the methanol eluate obtained by this filtration is concentrated under reduced pressure, and fractionated using a flash chromatography apparatus, and a 25 mass% methanol elution fraction containing a peptide that promotes the growth of mycorrhizal fungi. Get. Here, flash chromatography is a kind of column chromatography, and is a method of separating a sample under a pressurized condition such as compressed air.

  Subsequently, the 25 mass% methanol elution fraction is purified by an isoelectric point floating device called a lotofer to obtain a purified solution. Here, the isoelectric point movement is a method of separating and purifying a target purified solution using the difference in isoelectric point. Further, the purified portion purified by isoelectric point migration is separated by separating the active portion by high performance liquid chromatography, and the portion where the peptide is detected by the ninhydrin reaction solution is isolated.

  After this, the isolated part is identified with an amino acid sequencer which is a device for determining the amino acid sequence (sequence) of proteins and peptides, or a nuclear magnetic resonance (NMR) device. . As a result, it can be confirmed that the peptides contained in this part are tryptophan dimer and leucylproline. It should be noted that peptides other than tryptophan dimer and leucylproline contained in this part are not easy to identify because they are trace amounts.

  As described above, according to the above-described embodiment, artificial culture media each containing tryptophan dimer and leucylproline, which are special chemical substances that do not use any host roots or root extracts that can be infected by mycorrhizal fungi. The mycorrhizal fungi can be vigorously grown vigorously by culturing the mycorrhizal fungi. Therefore, the mycorrhizal fungus spores can be efficiently propagated without using plant roots or root extracts at all, and thus the mycorrhizal fungi spores can be efficiently produced in large quantities. Pure culture of spores can be easily performed.

At this time, by adding inorganic nutrients, vitamins, saccharides, phospholipids and nucleic acid substances to the culture medium to obtain a solid or liquid medium for the peptide, the growth of mycorrhizal fungi in this culture medium can be improved. The

  Furthermore, the growth and branching of mycorrhizal mycelium can be further promoted by adding activated carbon fibers to the culture medium. Also, by adjusting the light environment conditions by irradiating with red light during the culture period of this mycorrhizal fungus, the growth of mycelium of this mycorrhizal fungus becomes more vigorous and the production of spores of this mycorrhizal fungus is further promoted Therefore, this spore production can be further stabilized. In addition, when arbuscular mycorrhizal fungi are used as the mycorrhizal fungi, the effect of pure culture can be obtained particularly remarkably.

  First, as Example 1, new AM mycospore production by a pure culture technique using the mycorrhizal fungus culture method of the present invention will be described.

  For the experiment in Example 1, a gellite (1.5% by mass) solid medium in which 10 ppm each of tryptophan dimer and leucylproline was added to the basic culture solution (Base Media: BM) shown in Table 1 was used. .

In addition to this medium alone, when ACF is added and used, when light is irradiated with redLED (5 μEm ⁻² S ⁻¹ ), and when these ACF and redLED are used in combination, the respective effects are also confirmed. did. Here, the culture temperature of AM fungi as mycorrhizal fungi was set to 25 ° C. using this medium. Moreover, as this AM bacterium, mainly Gigaspora albida, Glomus etunicatum and Glomus caledonium were used. The surface of these AM spore was thoroughly sterilized with a disinfectant. A potato dextrose agar medium (PDA) widely used for culturing filamentous fungi was used for comparison. And after starting culture | cultivation of AM fungi using these culture media, the mycelial growth of these AM fungi and newly formed spores were observed under red light.

  As a result, when mycorrhizal fungi were cultured under dark conditions on potato dextrose agar medium (PDA) (PDA + dark ward), no mycelial growth or spore formation could be confirmed in any mycorrhizal fungi. In addition, when mycorrhizal fungi were cultured under dark conditions on the basic culture solution (BM) shown in Table 1 (BM + dark ward), mycelial growth of any mycorrhizal fungi was good, but spore formation was completely I could not confirm.

  On the other hand, when mycorrhizal fungi were cultured under dark conditions on a medium obtained by adding tryptophan dimer and leucylproline to the basic culture solution shown in Table 1 (BM + peptide + dark ward), any mycorrhiza The mycelial growth of fungi was promoted, and sporulation of these mycorrhizal fungi was confirmed. This tendency was promoted by the addition of ACF to the medium and red light irradiation by redLED.

  In particular, in the case where mycorrhizal fungi are cultured under the condition of adding red tryptophan dimer and leucylproline to the basic culture solution shown in Table 1 and irradiating red light with redLED on a medium containing ACF (BM + peptide + ACF + redLED), As shown in Table 2, it was confirmed that mycorrhizal growth was remarkably promoted by any mycorrhizal fungus and the formation of new spores was further increased.

  Further, in this (BM + peptide + ACF + redLED) treatment group, as shown in FIG. 1, several new spores were obtained from one mother spore even after 2 weeks of culture. Here, Δ marks in FIG. 1 are spores of AM fungi (Gromus caledonium) newly formed from hyphae sprouted from mother spores.

  Furthermore, in this (BM + peptide + ACF + redLED) treatment group, as shown in FIG. 2, newly formed AM fungal spores became almost the same size as mother spores after one month of culture. The Δ mark in FIG. 2 is a spore of an AM bacterium (Gromus caledonium) that is newly formed from the mother spore and has almost the same size as this mother spore.

The growth of the hyphae of the AM fungus is suppressed by light irradiation of 10 μEm ⁻² S ⁻¹ or more using a blue light emitting diode (blueLED).

  Next, as Example 2, the infectivity of AM fungal spores produced by the mycorrhizal fungus culture method in Example 1 will be described.

  In the experiment in Example 2, a cultivated Bahiagrass seedling was transplanted and used in a No. 2 plastic pot containing vermiculite. Then, one month after planting the spore of Glomus caledonium in the pure culture in Example 1, from the gellite medium containing the newly formed spore (diameter 50 μm to 80 μm), Then, the gellite slices having a diameter of about 1 cm were cut out and inoculated with the newly formed spores and mycelia of AM fungi contained in the gellite slices in contact with the roots of bahiagrass.

  One month after the inoculation, bahiagrass roots were collected and the mycorrhizal formation of the AM fungus inoculated into the bahiagrass was confirmed by Phillips, JM and Hayman, DS, Transactions of the British Mycological. Society (Transactions of the British Mycological Society), (UK), 1970, 55, p. Stained by the method described in 158-161.

  After this, the mycorrhiza formation of the stained AM fungus was examined by Ishii, T. and Kadoya, K., Journal of the Japanese Society for Horticultural Science. ), 1994, 63, p. It confirmed with the method of 529-535.

  Furthermore, this experiment was performed inside a glass house whose side and ceiling surfaces were covered with glass, and in order to prevent mixing of other spores, the pots transplanted with bahiagrass were isolated. During this experiment, water was sufficiently irrigated and fertilized with 10 ml of a Hogland solution in which the phosphorous concentration was reduced to ½ after 2 weeks and 3 weeks after transplantation, respectively.

  As a result, as shown in FIG. 3, a large number of mycelia can be confirmed around the roots of bahiagrass inoculated with gellite sections containing spores of AM fungus (Gromus caledonium) produced in the pure culture of Example 1, Numerous formations of rods and spores were confirmed inside the bahiagrass root.

  Next, as Example 3, subculture of AM fungal spores produced by the mycorrhizal fungus culture method in Example 1 will be described.

In the experiment of Example 3, a moderately sized ACF was placed in a liquid medium obtained by adding a nucleic acid substance to the culture medium used in Example 1, and newly formed Glomus caledonium spores (diameter). About 80 μm). Thereafter, the liquid medium was shaken at 90 rpm under irradiation of redLED (5 μEm ⁻² S ⁻¹ ), and observed with a CCD camera after 3 weeks.

  As a result, as shown in FIG. 4, on the ACF added to the liquid medium, hyphae extended from newly formed spores can be confirmed, and the third generation of Glomus caledonium (Glomus caledonium) is observed at the tip of the hyphae. Spores were formed. The size of the spore was about 60 μm and yellowish white.

  As a result, certain peptides can be added to an artificial culture medium supplemented with inorganic nutrients, vitamins, sugars, phospholipids, and nucleic acid substances without using host roots or root extracts that can be infected by mycorrhizal fungi. By adding tryptophan dimer and leucylproline, the mycelium of the mycorrhizal fungi can be grown to produce pure mycelium fungi in large quantities.

  Furthermore, the addition of activated carbon fiber to the artificial culture medium of this mycorrhizal fungus and the irradiation of red light during the culture period of this mycorrhizal fungus further improve the mycelial mycelium growth and spore formation. Very effective above. In addition, the mycorrhizal spore produced by this pure culture matures in a short period of one month and has the ability to infect plants, and from newly formed second generation spores. Can be subcultured.

  Therefore, the establishment of pure culture technology for this mycorrhizal fungus not only provides a clue to elucidating the symbiotic mechanism between mycorrhizal fungi and plants, which has been limited due to the difficulty of culturing, but also the spores of this mycorrhizal fungus. It has an important meaning that it can be mass-produced purely and stably.

It is a CCD camera image photograph which shows the spore of arbuscular mycorrhizal fungi cultured using the mycorrhizal fungi culture method of the present invention, (a) shows 2 weeks after culture, and (b) shows 1 month after culture. Indicates. It is an optical micrograph which shows the spore of the arbuscular mycorrhizal fungus cultured from the mother spore by the same mycorrhizal fungi culture method. It is an optical microscope photograph which shows mycorrhizal formation in bahiagrass by the spore of Glomus caledonium (Glomus caledonium) cultured by the same mycorrhizal fungi culture method. It is a CCD camera image photograph which shows the spore of the 3rd generation glomus caledonium cultured on activated carbon fiber by the same mycorrhizal fungus culture method.

Claims (5)

In a culture medium which is a solid medium or liquid medium to which a peptide as a mycorrhizal growth-promoting substance represented by the following formula (1) is added and inorganic nutrients, vitamins, sugars, phospholipids and nucleic acid substances are added , bacteria Nekin culture how to comprises culturing.
The peptide represented by following formula (2) is added to a culture medium. The mycorrhizal fungi culture method of Claim 1 characterized by the above-mentioned.
The mycorrhizal fungus is any one of arbuscular mycorrhizal fungi, ericoidal mycorrhizal fungi, and ectomycorrhizal fungi. The mycorrhizal fungi culture method according to claim 1 or 2. 4. The method of cultivating mycorrhizal fungi according to any one of claims 1 to 3 , wherein activated carbon fibers having activated carbon fibers are added to the culture medium. The mycorrhizal fungus culture method according to any one of claims 1 to 4 , wherein the mycorrhizal fungi are cultured under light environment conditions of red light irradiation.