JPH07110182B2 - Mycorrhizal formation method of mycorrhizal fungi - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention is directed to plants (host plants) pine, oak, which are host plants of mycorrhizal fungi, which are parasitic of active mycorrhizal fungi such as matsutake mushrooms and honshimeji mushrooms. The present invention relates to a method of artificially cultivating mycorrhizal mushrooms by infecting roots of Kunugi and the like.

(Prior art) Matsutake mushrooms, honshimeji, etc. attach themselves to the roots of the specific host plants to form mycorrhizas and grow sufficiently (form white).
Although it is an active parasitic fungus that forms fruit bodies after growth, since the hyphae of Matsutake mushrooms, honshimeji, etc. grow very slowly, when artificially inoculating the filamentous fungi before the hyphae attach to the roots of host plants, It has been difficult to produce white for inhibiting the growth of mycelium and the formation of mycorrhiza to form fruiting bodies by propagating various bacteria such as bacteria. Therefore, as a method for artificially forming mycorrhizas such as Matsutake mushrooms and Hon-shimeji mushrooms, the method of embedding cultured mycelium of artificially-cultivated Matsutake mushrooms and Hon-shimeji mushrooms mainly in the ground of the host plant forest is mainly for the propagation of various bacteria. In order to prevent this, the root of the host plant has been subjected to aseptic treatment, or it has been replaced with sterilized soil.

(Problems to be solved by the invention) However, ordinary soil contains abundant amounts of organic matter and microorganisms that feed it, and in cultures of mycelium such as matsutake mushrooms, honshimeji, etc. The culture medium is also included, which serves as a nutrient source for other microorganisms as well as mycelia such as matsutake mushrooms and honshimeji mushrooms, and thus inhibits the growth of hyphae such as matsutake mushrooms and honshimeji mushrooms. In addition, even if the roots and soil of the host plant are sterilized, as described above, there are numerous germs in the soil and a large amount of organic substances that are nutrients for them, so that the germs may propagate. It is considered expensive. For this reason, it has been considered difficult to artificially cultivate soil not only in forests but also indoors.

(Means for Solving Problems) According to the present invention, when the cultured mycelium of mycorrhizal fungi is inoculated into the root of a host plant of the mycorrhizal fungus, the cultured mycelium of the mycorrhizal fungus is wrapped with a gel. The gist is a mycorrhizal formation method of mycorrhizal fungi characterized by burying.

This will be described in detail below.

The gel embedding cultured mycelium of mycorrhizal fungus, which is a feature of the present invention, is a natural polymer such as agar, alginic acid, konjak, carrageenan, gellan, pullulan, guar gum, locust bean gum, xanthan gum, pectin, starch and the like. Alternatively, synthetic polymers such as polyacrylamide and polyvinyl alcohol can be used, and these may be used alone or in combination of two or more. These polymers are made into a gel with water, a buffer solution, a salt solution or the like according to a conventional method. These gels are used for the purpose of mycorrhizal formation, maintaining the gel state and isolating from germs until pure cultured mycorrhizal fungus survives and invades contact with the roots of sterile host plants. Therefore, it is not necessarily limited to the following conditions, but it is preferable to satisfy the following conditions.

(1) For six months to one and a half years, it is difficult to be decomposed by soil bacteria.

To compensate for this, an antibacterial agent such as chlorhexidine gluconate, siabendazole, benomyl, streptomycin, etc., which is not harmful to mycorrhizal fungi, may be added to the gel as appropriate.

(2) The gel must have sufficient strength for easy handling during inoculation and stability for soil pressure in soil.

(3) It does not adversely affect mycorrhizal fungi such as high temperature during gel preparation.

(4) After the gel is made, do not include substances or ions in the gel at concentrations that would be harmful to mycorrhizal fungi.

(5) The gel is soft enough to allow the roots of the host plant to penetrate.

(6) The polymer is stable against sterilization by heating, radiation, etc. before gel formation.

The polymer is not limited to the natural and synthetic polymers as long as the above conditions are satisfied.

Generally, a concentration of 0.5-2.5%, preferably 1.0-2.0% can be used.

As the mycorrhizal fungus, various ones such as matsutake mushroom, honshimeji bacterium, kotake mushroom, matsutake bacterium, amitake bacterium and the like can be used. Particularly useful mycorrhizal fungi of edible mushrooms include matsutake fungi and honshimeji fungi. These mycorrhizal fungi
What has been cultivated by liquid culture or solid culture under each suitable condition can be used, but in order to proliferate mycelia well, for example, the cultured mycelium is dispersed using a homogenizer or the like, and vermiculite or the like is further added. You can use a mixture. With these polymers, a gel layer is formed around the mycelium. As a forming method, for example, if a test tube thicker than the cultured mycelium is used, a gel layer having an appropriate thickness can be formed. All of the above operations should be performed under sterile conditions to prevent contamination by miscellaneous bacteria.

It should be noted that the embedding state is not limited to the state in which all the mycelium in culture is covered with gel, as long as it is capable of isolating miscellaneous bacteria, and the part in which the mycelium is embedded is combined with other miscellaneous bacteria isolation means. If it can be embedded to the extent that the roots of the host plant can penetrate,
It may be partial.

In addition, examples of host plants infected with mycorrhizal fungi include plants of the pine family such as red pine for matsutake fungi, and oak, kunugi, red pine for honshimeji fungi. When inoculating the roots of these host plants, it can also be applied to artificial cultivation in pots or the like, but in order to obtain fruiting bodies, artificial cultivation in forest land is preferable.

(Function) It is known that when the tip of a fine root of a tree in a sterile state comes into contact with a bacterium in pure culture, the root is infected and a mycorrhiza is formed within a relatively short time. In addition, the root growth point in the natural state is even said to be virus-free. Therefore, if pure mycelium is embedded in a gel such as agar, and if the roots of aseptic trees grow while penetrating the gel and encounter mycorrhizal fungi, the infection state is easily established and mycorrhiza is formed. To be done.

(Examples) Hereinafter, the present invention will be described in more detail with reference to Examples.
In the examples, "parts" means "parts by weight".

Example 1 Yeast extract 0.15 parts Soyton (manufactured by Difco) 0.15 parts Glucose 2.0 parts Distilled water 100 parts Cultured matsutake fungi in a medium consisting of the above components of pH 5 for about 4 weeks, then dispersed with a homogenizer and dried with horticultural vermiculite. Mix 3 parts with 10 parts heat-sterilized, and add 3 parts of the above medium containing 2 parts of agar to the mixture. This is the inner diameter with a plug made of foam silicone rubber
Fill a 3 cm test tube and incubate at 23 ° C for 2-4 months.

After the hyphae have proliferated well, they are taken out from the test tube and heat-sterilized at 120 ° C for 15 minutes. Into a test tube with an inner diameter of 4 cm containing 60 ml of a 2% agar solution, quickly take out the mycelium when the agar solution temperature becomes 40 ° C or less. Add and cool with ice water.

In this way, the mycelium embedded in agar with a thickness of 5 to 8 mm was placed 10 at a distance of 2 to 5 m from the root of a pine tree 25 to 40 years old.
Approximately 15 cm dig and inoculate. Pine roots start to grow 4-5
If you inoculate on the moon, you can see that the new roots of the pine invade the agar in about two months. Moreover, mycorrhizal formation is observed after 1 month.

Example 2 Carrageenan 0.7 part Locust bean gum 0.3 part Potassium chloride 0.24 part 60 ml of a solution consisting of 100 parts distilled water was sterilized by heating at 120 ° C for 15 minutes, and an inner diameter of 4 cm.
When the temperature reaches about 40 ° C., the mycelium cultured as in Example 1 is quickly added and cooled with ice water. When the mycelium embedded in the gel was taken out of the test tube and inoculated in the same manner as in Example 1, similar results were obtained.

Example 3 Rhomethoxypectin 2 parts Siabendazole 0.1 part Streptomycin 0.2 part Distilled water 100 parts After adjusting the pH to 5, a solution was sterilized by heating at 120 ° C. for 5 minutes and then sterilized separately at 40 ° C. or lower. Equally mix 0.5% potassium chloride solution, 60 ml of it quickly with an inner diameter of 4 cm
Put in the test tube. When the same procedure as in Example 4 was performed below, similar results were obtained.

Example 1 The same results were obtained when the same procedure as in Example 1 was carried out except that the medium of pH 5 and the matsutake bacterium were changed to the medium of pH 6 and the honshimeji bacterium.

Example 5 Acrylamide 8 parts Methylenebisacrylamide 2 parts TEMED (N, N, N ', N'-tetramethylethylenediamine)
0.15 parts Riboflavin 0.0005 parts Distilled water 90 parts The mycelium cultured as in Example 1 was suspended in a test tube containing the above aqueous solution with a wire or the like, polymerized at 10 cm under a 20 W fluorescent lamp, and embedded in acrylamide. The obtained mycelium was obtained. When cells were inoculated in the same manner as in Example 1, similar results were obtained.

Comparative Example When the mycelium of matsutake fungus cultured as in Example 1 was not embedded in a gel and inoculated in the same manner as in Example 1, the matsutake fungus was almost dead after 2 months.

(Effect) In the present invention, the mycelium of mycorrhizal fungi is embedded in a gel so that it is isolated from miscellaneous bacteria.
During that time, new roots of the host plant can be aseptically invaded into the gel layer to allow infection with hyphae and form mycorrhizas.

Claims (1)

[Claims] 1. When the cultured mycelium of mycorrhizal fungus is inoculated into the root of a host plant of the mycorrhizal fungus, the cultured mycelium of mycorrhizal fungus is embedded in a gel. Mycorrhizal formation method of root fungi.