JPH08191685A - Artificial culture method for vesicular arbuscular mycorrhizae - Google Patents

Abstract

PURPOSE: To artificially obtain a vesicular arbuscular mycorrhizae useful in the field of agriculture and horticulture without causing contamination with various germs by culturing a mycelium and a spore of VA mycorrhizae by using an extract of a root of a plant to be infected with VA mycorrhizae but not a live root of a plant. CONSTITUTION: A root of a crop belonging to the genus Paspalum Thunbergii of the family Gramineae (e.g. America Paspalum Thunbergii) to be infected with VA mycorrhizae is immersed in a water-soluble organic solvent (e.g. methanol) for 24 hours and extracted, the extracted solution is concentrated by an evaporator and purified by a flash chromatography to give an extract. Then, the extract is dissolved in a liquid containing 1.4% agar and sterilized by an autoclave at 120 deg.C for 15 minutes to give a medium for artificial culture of VA mycorrhizae. A mycelium and/or a spore of VA mycorrhizae is cultured by using the medium without using a live root of a plant to artificially and inexpensively culture VA mycorrhizae useful in the field of agriculture and horticulture.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for artificially culturing VA mycorrhizal fungi useful in fields such as agriculture and horticulture, without using living roots of plants.

[0002]

2. Description of the Related Art Vesicular Arbuscular Myc
orrhizal fungi) is known to improve the growth promotion and disease resistance of various plants by infecting various plants and coexisting with them (Makoto Ogawa, “VA mycorrhizal and its function, forest location”). , 30 (2), pp. 57-65, 1
1988; "Agriculture and Horticulture," Vol. 62, No. 8, 93.
0-937, 1987; "Plant Protection", Volume 42,
No. 5, pp. 259-266, 1988). This is V
This is because the mycelium of the mycorrhizal fungus infects the roots of the plant, and the absorption of phosphorus and trace elements in the soil is promoted through the mycelium.

However, the VA mycorrhizal fungus is said to be a plant absolute symbiotic bacterium, and it has been impossible to artificially culture the same symbiotic bacterium without a living plant like Rhizobia or Francia. For this reason, attempts have been made to cultivate VA mycorrhizal fungi all over the world, but it has not been possible to carry out artificial culture without living plants. If VA mycorrhizal fungi can be artificially cultured without using the roots of living plants, V
The A mycorrhizal fungus can be supplied at low cost.

As a method for culturing VA mycorrhizal fungi, a method in which a plant is grown on a medium such as a porous carrier and the roots of the plant are infected with VA mycorrhizal fungus to proliferate has been widely used. There is. On the other hand, attempts have been made to aseptically culture VA mycorrhizal fungi in a container (JP-A-3-
No. 83,322), and a technique of adding an oligosaccharide in order to assist the growth of mycelium at that time has also been proposed (Japanese Patent Laid-Open No. 4-320676). However, all of these techniques use living plant roots, and in any case, it was not possible to culture VA mycorrhizal fungi without using living plant roots.

VA mycorrhizal fungi naturally form spores and overwinter. These spores germinate and grow hyphae if conditions of water, temperature, and oxygen necessary for growth are adjusted without particular nutrition. The elongation of this mycelium is continuous with the spores, and extends while the nutrients from the spores are supplied. However, once the hyphae and spores are cut, the hyphal extension stops and loses the ability to infect the plant roots. Therefore, in order for hyphae to infect roots of plants, it is necessary that hyphae are continuous with spores or that roots of plants are once infected and nutrients are supplied from the plants.

By the way, plant tissues and root extracts are VA
There are several reports on promoting germination of mycorrhizal spores and elongation of germinated hyphae. For example, it has been reported that the addition of the cells of the kudzu group to the medium promoted the germination of Scutellospora heterogama spores and the elongation of hyphae (CIENC PRA
T, 14 (3), 1990, 308-316). Moreover, it was reported that the extract extracted from the root of carrot promoted the elongation of hyphae germinated from spores (New Phytol., 118 (2), 1991, 289-29.
4; Can. J. Bot., 68 (6), 1990, 1260-1264; Appl. Env
Iron. Microbiol., 55 (9), 1989, 2320-2325) and flavonols and flavones extracted from the roots of carrot promoted the growth of mycelium of Gigaspora margarita (J. Chem. Ecol. 19 (1
0), 1993, 2317-2327) and flavonoid compounds contained in plants, daidzein, myricetin, and quercetin, extend mycelia sprouted from spores of Glomus moseae and Glomus intraradicies. Report to let (J. Plant Phys
iol. 141 (1), 1993, 54-60).

Furthermore, it was reported that the clover root extract promoted spore germination and hyphal elongation (Symbiosi
s, 7 (3), 1989, 243-256; Appl. Environ. Microbiol.,
53 (8), 1987, 1928-1933) and citrus root extract promote spore germination and hyphal elongation (Mycolog).
ia, 74 (5), 1982, 831-835).

However, these reports are all reports that spore germination and hyphal elongation are promoted, and are not techniques for growing VA mycorrhizal fungi themselves. That is, even if the growth of hyphae is promoted, the growth of hyphae eventually stops until the roots of the plant are infected, leading to death.

As described above, attempts have been made to independently culture VA mycorrhizal fungi around the world, but it has not been possible to artificially culture VA mycorrhizal fungi without living plants (plant roots). Is the reality. If VA mycorrhizal fungi can be artificially cultured without using plant roots, VA mycorrhizal fungi can be supplied at low cost. If the VA mycorrhizal fungus can be cultivated alone without using live hairy roots, etc., the fungus body not contaminated with miscellaneous bacteria can be easily obtained. This is
In addition to broadening the industrial applicability of VA mycorrhizal fungi, it is also possible to study the physiological properties of VA mycorrhizal fungi that could only be investigated through symbiosis with plants. is there.

[0010]

DISCLOSURE OF THE INVENTION The present invention provides a method for artificially culturing VA mycorrhizal fungi useful in fields such as agriculture and horticultural industry without using living roots of plants. That is the purpose. The present invention is the world's first V
It is intended to provide a method for artificially culturing A mycorrhizal fungus alone.

[0011]

That is, the present invention provides a VA
A VA mycorrhizal fungus characterized by culturing mycelium and / or spores of a VA mycorrhizal fungus without using a live root of the plant, using an extract of a root of a plant which can be infected by the mycorrhizal fungus An artificial culture method is provided.

Here, there are various plants which can be infected with VA mycorrhizal fungi. Specifically, Iridaceae, Gramineae, Pinus family, Ginkgo family, Cucurbitaceae, Oxaceae family, Cicaceae family, Asteraceae family, Asteraceae family, Ranunculaceae family, Moraceae family,
Sesameaceae, sakura, primrose, taro, perilla, sycamore, cedar, seriaceae, polygonaceae, communis family, solanaceae, caryophyllaceae, rose family, cypress family, amaranthaceae family, floss family, vine family , One of the plants selected from the family of Persimmonaceae, Leguminosae, Rutaceae, Palmaceae, Yamaceae, Liliaceae, Rybaceae and Gentianaceae. In the present invention, plants of the Gramineae family are preferred among these.

[0013] The plants belonging to the family Gramineae include the genus Astragalus,
Acne, Merikenkarkaya, Maize, Juzudama, Ushinosippei, Ayasi, Platypus, Megalucaya, Meatgrass, Ogarkaya, Cobnagus, Pleurotus spp., Sorghum, Itataga, Unnuke, Ashivoso , Genus Vase, Genus Abrasski, Genus Miscanthus, Genus Chigaya, Genus Abrasski,
The genus Karimataga, Genus Tsukiige, Genus Chigusa, Genus velvet, Genus Musca, Genus Nycium, Narcobier, Genus Astragalus, Genus Citrus, Millet, Genus Numerigus, Genus Corydalis, Genus Fern, Uxiba, Todashiba,
Genus Sheba, genus Shiba, genus Rhizoma, genus Rhizophora, genus Rhizophora, genus Rhododendron, Trichogusa, genus Azegaya, genus Galleria, genus Oshiba, genus Suzumegaya, genus Urhagusa, Numagaya, genus Tatsunogi, Tachibiki, dactylum Reed genus, genus genus, genus genus, genus Tsukushigaya, genus Sayanukagusa, genus Hogarigaya, genus Komegaya, genus Rhododendron, genus Nagahusa, genus Rhododendron, genus Neuroptera, genus Aspergillus, Saturniidae, Scutellaria, serrata Cypress,
Barley genus, Erythrium genus, Dactyla genus, Kobo genus,
The genus Hurghaya, the genus Kusayoshi, the genus Ibukinukabo, the genus Honegaya, the genus Shiragegaya, the genus Komesski, the genus Minoro, the genus Cyperaceae, the genus Miyamayamahi, the genus Oedum, the genus Odinosaur, the genus Nogariya, the genus Minatome, the genus Fagali,
Examples include plants belonging to the genus Nucabo, genus Corydalis and spp.

Although any of these Gramineae plants may be used, in the present invention, the genus Maize (corn), the sorghum genus (Sorghum etc.), the genus Suski (Suki, Caryas etc.), the genus Chigaya (eg Chigaya), the barnyard grass Genus (such as barnyard grass), genus Aedes genus (such as Aedes aegypti, etc.), genus Crabgrass (such as crabgrass and rice clover), genus Millet (such as brackish millet), genus Echinochloas (such as foxtail millet, foxtail moss), genus Shiva
Preferred are plants belonging to the genus Astragalus, etc., the genus Duckweed (such as Duckweed), the genus Oatus (such as oats), and the genus Occidentalis (Setogaya, Apocynaceae, etc.). More preferably, plants belonging to the genus Prunus chinensis (Prunus chinensis, Prunus chinensis, Kikususumenohie, Sawasumenohie, Shimasumenohie,
Tachisumenohie, Nagabusumaenohie, Aedes aegypti and the like), and especially American Astragalus (Bahiagrass) and Astragalus japonicus are preferred. When using these plants, it is more preferable to select a plant from a growth stage to a growth end stage in which a substance (active ingredient in the extract) that grows VA mycorrhizal fungi is more accumulated. Moreover, as these plants, not only one kind but also two or more kinds can be used in combination.

The method of the present invention is characterized by culturing VA mycorrhizal fungi without using live roots of plants, using the root extract of plants which can be infected with VA mycorrhizal fungi as described above. It is what The root extract of a plant that can be infected with VA mycorrhizal fungus is extracted from the root of a plant that can be infected with VA mycorrhizal fungus as described above, and its components are not always clear. It is eluted with a water-soluble organic solvent from the root of a plant that can be infected with root fungi. Examples of the water-soluble organic solvent include one selected from methanol, ethanol, propanol, i-propanol, butanol, i-butanol, t-butanol, acetone, methyl ethyl ketone, and a mixture thereof, particularly methanol. Optimal.

In the present invention, it is more preferable to use a water-soluble organic solvent as described above, which contains water, that is, a water-containing water-soluble organic solvent. The water content of such a water-containing water-soluble organic solvent varies depending on the type of the water-soluble organic solvent used and is difficult to determine uniquely, but it is usually about 5 to 95% by weight. When a water-free water-soluble organic solvent is used as the extraction solvent here, the extraction rate becomes low, and if the water content is too high, the extraction rate will decrease and impurities may increase. is there.

The pH of the extraction solvent is not particularly limited, but if the pH is extremely low or high after extraction, the pH of the growth medium of VA mycorrhizal fungus is changed, and therefore the pH after extraction is 4 to 4. It is preferable to use one having a range of 9. The extraction temperature may be a normal extraction temperature range, and is in the range of -20 ° C to 100 ° C. The extract thus obtained is stored in an autoclave at 120 ° C.
It is a stable substance that does not decompose when heated to sterilized medium. Further, the extraction time depends on the type of solvent, water content, and temperature, but at a high temperature it takes a few minutes, and at a low temperature it can take from 1 hour to a few days.

The extract used in the method of the present invention is thus obtained, and the water-soluble organic solvent is removed as it is or by evaporating to dryness, and preferably further using an autoclave or the like. Sterilized and VA
It can be used as an artificial medium (or a part of artificial medium) for culturing mycelia or spores of mycorrhizal fungi. In the present invention, the water content of the water-soluble organic solvent-water elution fraction obtained by fractionating such a solvent extract is 5%.
% Or more of water-soluble water-soluble organic solvent
Can be used as an artificial medium for culturing hyphae and spores of VA mycorrhizal fungi. In this case, purification by a method such as ion exchange resin, activated carbon, silica gel chromatography, reverse phase silica gel chromatography, molecular sieve, organic solvent fractionation, etc. is more effective than purification of the solvent extract as it is without impurities. It is more preferable because it reduces the amount of substances and inhibitors. When a component (eluate) that elutes in a water-containing water-soluble organic solvent having a water content of less than 5%, for example, a 100% methanol eluate is used, the growth of hyphae of VA mycorrhizal fungi is not sufficient. In the fractionation, the solvent extract may be concentrated using an evaporator or the like, and the obtained concentrate may be fractionated using a flash chromatograph or the like. The water-soluble organic solvent is removed by evaporating and drying the component (eluate) eluted in the water-containing water-soluble organic solvent having a water content of 5% or more thus obtained, and preferably further using an autoclave or the like. After sterilization, it may be used as an artificial medium for VA mycorrhizal culture. In addition,
In order to form a medium, components of Murashige-Skoog medium (MS medium) or agar may be added to the above extract or eluate, if necessary.

A medium comprising the extract obtained as described above, a medium comprising the eluate, a liquid medium in which a carbon source such as inorganic salts, sucrose, oligosaccharides or the like is added to the medium, and further agar. VA using a solid medium containing
The mycelia and spores of mycorrhizal fungi may be cultured. The culture conditions may be ordinary conditions, the temperature is 10 to 45 ° C., and the culture may be performed under aerobic conditions. The artificial culture method may be carried out by suspending the solvent extract or the specific eluate as described above in distilled water, sterilizing, and aseptically inoculating the mycelium or spores of VA mycorrhizal fungus and culturing, Alternatively, a solvent extract or a specific eluate as described above may be added to a medium such as MS medium, for example, MS3 medium, and after sterilization treatment, hyphae and spores of VA mycorrhizal fungi may be inoculated and cultured. For the above-mentioned culture, various nutrient solutions usually used for culture can be used, and the concentration thereof is not particularly limited and may be within the range usually used.

Here, VA mycorrhizal fungus is a kind of zygomycete existing in soil, and it is known that its hyphae form mycorrhizal roots of various plant roots and both coexist. It is known that this VA mycorrhizal fungus has a function of coexisting with a plant, promoting the growth of a plant infected with it, and improving disease resistance. V which can be artificially cultured by the method of the present invention
There are various types of mycorrhizal fungi, for example, genus Gigaspora, genus Glomus, genus Acaulospora, and entropospora (E
ntrophospora), Sclerocysti
Examples include microorganisms belonging to the genus s) and the genus Scutellospora.

Specific examples of these VA mycorrhizal fungi include, for example, those belonging to the genus Gigaspora:
Gigaspora ramisporop
hora), Gigaspora albida
), Gigaspora margarita
), Gigaspora gigante
a), Gigaspora rosea
, Gigaspora heterogama
) And the like. In addition, Gigaspora ramisporophora is a VA mycorrhizal preservation facility INVAM at the University of Virginia in the United States.
It is registered and stored as JA101 in. Further, in Japan, the contract was refused at the Institute of Biotechnology, Institute of Biotechnology, AIST.

[0023] Examples of the genera belonging to the genus Glomus include Glomus moss.
eae), Glomus fascuratorum
ciculatum), Glomus caledonium
edonium), Glomus Intraradices
intraradicies), Glomus Manihotis (Glomu
s manihotis), etc. In addition, Scutellospora gregaria
), Acaulospora laev
is), Entrofossora frequens (Entro
phospora infrequens), Sclerocystis dussii, and the like.

Among the VA mycorrhizal fungi that can be artificially cultured by the method of the present invention, among these, Gigaspora
Microorganisms belonging to the genus), the genus Glomus are preferred.

According to the method of the present invention, by using the root extract of a plant which can be infected with VA mycorrhizal fungi, such VA mycorrhizal fungi can be obtained without using a living plant (plant root). The VA mycorrhizal fungus can be artificially cultivated only by using mycelia and / or spores. The VA mycorrhizal fungi to be used may be only hyphae or spores alone, or may be a mixture of hyphae and spores. Hairy roots can also be added, but this is not essential. As a method for collecting VA mycorrhizal spores, a method of collecting from the natural world using a sieve (Tatsuhiko Suzuki, VA mycorrhizal problems 5, agriculture and horticulture,
62, No. 3, p28-33, 1987) and a method by centrifugation (Japanese Patent Laid-Open No. 63-309178). In addition, a nutrient thin film culture method (Japanese Patent Application Laid-Open No. 55-1
18390) or a method using organ-cultured roots (Japanese Patent Publication No. 62-49037), and the like
There is also a method to grow mycorrhizal fungus A and form spores,
There is no particular limitation on the collection method. On the other hand, when the VA mycorrhizal inoculum is used, its origin is not particularly limited, and a commercially available product can be used. When using mycelia or spores collected under the condition that various bacteria are mixed as an inoculum, it is desirable to sterilize the surface with chloramine T or the like before use. Furthermore, in order to prevent other VA mycorrhizal fungi from mixing, it is preferable to inoculate monospores as the spores.

According to the method of the present invention, a living root of a plant is obtained by culturing mycelium and / or spores of a VA mycorrhizal fungus using an extract of the root of a plant which can be infected with VA mycorrhizal fungus. It became possible for the first time to artificially culture VA mycorrhizal fungi without using.

[0026]

EXAMPLES Next, the present invention will be described in detail with reference to Examples. Example 1 Bahiagrass roots collected during the growth period from July to September were washed with distilled water, and then extracted and purified as follows. That is, this Bahiagrass root was placed in 80% methanol, soaked all day and night, and then filtered with Toyo filter paper No. 5C. After extracting the VA mycorrhizal-promoting substance and the like in this manner, this extract (filtrate) was concentrated using an evaporator. The resulting concentrate is chromatorex ODS DM1020T
Purified using a flash chromatograph (manufactured by Fuji Silysia Chemical Ltd.) equipped with (diameter 20 mm, length 25 cm), 5 kinds of methanol eluents (0% methanol eluate, 10% methanol eluate, 25% methanol eluate) , 50% methanol eluate, 100% methanol eluate) were separated.
These eluates (equivalent to 0.4 to 6 g root) were evaporated to dryness,
10 ml of liquid (distilled water) containing 1.4% agar was added and dissolved, and then sterilized by autoclave at 120 ° C. for 15 minutes to obtain a medium for VA mycorrhizal artificial culture.

This medium was aseptically transferred to a petri dish (7 cm in diameter), and VA mycorrhizal fungi [Gigaspora ramisporophora] sterilized on the petri dish were placed on a clean bench. VA mycorrhizal fungus conservation institution INVA
It has been registered and stored in M as JA101. In addition, this bacterium was refused to be entrusted by the Institute of Biotechnology, Institute of Biotechnology, AIST. ], And cultured in the dark at 30 ° C. In addition, disinfection of VA mycorrhizal spores was carried out by adding an antibiotic (5% chloramine T
Drop 0.04% streptomycin) and
It was carried out by a method of treating for a minute. Further, as a control group, the culture was performed in the same manner for the group containing only agar as the medium.

Three weeks after the start of culture, the elongation of mycelia was measured by an image processing method using a stereoscopic microscope equipped with a CCD camera and a personal computer. First result
It is shown in the table and FIG. In Table 1, the column of VA mycorrhizal mycelium length shows average value ± standard error. Also,
FIG. 1 is a drawing showing the growth state of Bahiagrass root extract and VA mycorrhizal mycelium in each section measured by an image processing method using a stereoscopic microscope equipped with a CCD camera and a personal computer. In FIG. 1, a is a control section, b is a 0% methanol elution section, c is a 10% methanol elution section,
d shows a 25% methanol elution group, e shows a 50% methanol elution section, and f shows a 100% methanol elution section.

[0029]

[Table 1]

From Table 1 and FIG. 1, it can be seen that the elongation of the mycelium of VA mycorrhizal fungi is significantly promoted in the 25% methanol elution group as compared with the control group. Also, 100%
It can be seen that the mycelium of VA mycorrhizal fungus does not extend in the methanol elution zone of.

Example 2 8 from Bahiagrass roots obtained in Example 1 above
A 25% methanol eluate obtained by flash chromatography of a 0% methanol extract (hereinafter referred to as a 25% methanol eluate of a Bahiagrass root extract) is widely used for culturing filamentous fungi, because of its hyphal elongation action. It was examined using potato dextrose (PDA) medium. That is, a 25% methanol eluate of a Bahiagrass root extract obtained by the same method as in Example 1 was evaporated to dryness, and a liquid medium (distilled water) containing 1.4% agar was added and dispersed and dissolved. Then, it was sterilized by autoclave at 121 ° C. for 15 minutes and transferred to a petri dish to prepare a plate medium for VA mycorrhizal artificial culture.

On the other hand, potato leachate powder in 1 liter 4.
Melt 0g, glucose 20.0g, agar 15.0g,
Sterilization was performed at 121 ° C for 15 minutes, and 10 ml of each was transferred to a petri dish to prepare a plate medium. Glucose only, 1 /
A medium reduced to 4, 1/20 was similarly prepared.

On these media, disinfected VA mycorrhizal fungi [Gigaspora ramisporopho
)) spores were placed and cultured in the dark at 30 ° C. for 2 weeks, and mycelial elongation was measured in the same manner as in Example 1. The disinfection of VA mycorrhizal spores was performed in the same manner as in Example 1. Further, as a control group, a group containing only agar as a medium was similarly cultured and measured. The results are shown in Table 2. As a result, mycelial elongation hardly progressed in the PDA medium, and remarkable mycelial elongation occurred in the medium containing the 25% methanol eluate of the Pahiagrass root extract (the present invention group). In Table 2, the column of VA mycorrhizal mycelium length shows average value ± standard error.

[0034]

[Table 2]

Example 3 (Pure culture experiment using VA mycorrhizal mycelium) Pure culture was performed using VA mycorrhizal mycelium. That is, VA mycorrhizal fungus [Gigaspora ramisporophora (Gigaspora ramisporophora) that was previously grown in a medium containing only agar.
)] On a clean bench, the whole agar was punched out with a 7 mm diameter cork borer and collected (preparation of agar disc). On the other hand, a solid substance obtained by evaporating a 25% methanol eluate of a Bahiagrass root extract (corresponding to 0.4 g root) obtained by the same method as in Example 1 to dryness was added to a liquid medium containing 1.4% agar (distillation). It was suspended in 10 ml of water), sterilized at 121 ° C. for 15 minutes, and then transferred to a petri dish to prepare a plate medium for VA mycorrhizal artificial culture. The agar disc containing only the mycelium obtained as described above was placed on the plate medium and cultured in the dark at 30 ° C. Further, as a control group, the culture was performed in the same manner for the group containing only agar as the medium.

60 days after the start of the culture, the spread of hyphae from the agar disc was examined under a stereoscopic microscope. The results are shown in Table 3. The length of the culture flora (R) of VA mycorrhizal fungi in Table 3 was measured as shown in FIG. 2, and the length was shown as an average value ± standard error. As a result, no elongation of hyphae from the agar disc was observed in the control group, but in the group to which the 25% methanol eluate (liquid) of the Bahiagrass root extract was added, as shown in FIG. The hyphal elongation was remarkable. Note that FIG.
The part indicated by the arrow in the middle is an agar disc containing VA mycorrhizal mycelia.

[0037]

[Table 3]

Example 4 (Pure Culture Experiment Using Mycelia of VA Mycorrhizal Fungus) It was investigated that the mycelia that had been completely artificially cultured could infect plants and form mycorrhiza in the roots. A 25% methanol eluate of the Bahiagrass root extract obtained by the same method as in Example 1 (equivalent to 0.8 g of root) was evaporated to dryness to give a solid product.
20 ml of liquid (distilled water) containing 4% agar was added and dissolved, and this liquid was placed in a 200 ml glass bottle and
Sterilization was performed in an autoclave at 1 ° C for 15 minutes. On the other hand, VA mycorrhizal fungus [Gigaspora ramisporophora (Gigaspora ramisporophora) that was previously grown in a medium containing only agar
)] On a clean bench, the whole agar was punched out with a 7 mm diameter cork borer and collected (preparation of agar disc). After the agar medium solidified on the bottom of the glass bottle, an agar disk containing only the collected hyphae was placed in the center of the agar medium. Prepare two such glass bottles, one of which is 2 mm from the agar disc.
Disinfected leek seeds were placed at a remote location, and disinfected bahiagrass seeds were placed on the other side. The mouth of this glass bottle was closed with a cotton plug, and after culturing under a light condition of 26 ° C. for 1 month, the formation state of VA mycorrhizal fungi in each plant root was stained and observed. As a result, as shown in FIG. 4 (Bahiagrass root) and FIG. 5 (Allium root), hyphae invaded roots, dendritic bodies were observed, and VA mycorrhizal fungi propagated from hyphae infected roots of plants. , It was confirmed that a symbiotic relationship was established.

Example 5 Roots of soybean, chickweed, soybean, soybean, communis, oxalis, perilla, purslane, corn and sorghum collected from fields were washed with tap water to remove soil. After removing excess water from each root with filter paper, 5 g was weighed and placed in a separating funnel having a volume of 1 liter. 200 ml of a mixed solvent of 15 parts by weight of water, 45 parts by weight of methanol and 40 parts by weight of ethanol was added to this separating funnel, and the mixture was sealed and shaken for 24 hours for extraction. Then, the mixture was placed in a 1 liter eggplant-shaped flask while being filtered with a paper to prevent solids from entering, and evaporated to dryness. Then, 100 ml of n-hexane was added and the mixture was left for 24 hours, after which the supernatant hexane was gently discarded to remove the lipid fraction. Further, 100 ml of ethyl acetate was added and the mixture was allowed to stand for 1 hour, and then the supernatant ethyl acetate was discarded.
Next, the eggplant-shaped flask was sucked with an evaporator to completely remove the solvent. After removing the solvent, 100 ml of distilled water was added to disperse the solid matter in water. After filtering this solution with a paper jar, the filtrate was transferred to an Erlenmeyer flask and
g of agar was added and sterilized at 121 ° C. for 15 minutes. Then, this medium was dispensed into 8 petri dishes to prepare a plate medium.

On the other hand, as a VA mycorrhizal fungus, 10 g of spores of Glomus sp. R-10 (ATCC 74311) were used.
The suspension was suspended in 1 sterilized water, several drops of 5% chloramine T and 0.04% streptomycin were dropped, and the mixture was treated for 15 minutes. Then, the spores were washed with sterilized water, spread on 10 sheets of 1.4% water agar medium, and cultured under dark conditions at 30 ° C. for 20 days. After 20 days, the hyphae that had grown on four petri dishes that were not contaminated with miscellaneous bacteria were punched out with a 7 mm cork borer. Using this spore-free hypha, artificial culture of Glomus sp. R-10 was carried out. That is, five petri dishes containing the extracts of roots of various plants prepared above were prepared, and the mycelium punched by a cork borer was placed in the center of the petri dish, and dark culture was carried out at 25 ° C. for 40 days. Then, the average value of 5 plates was calculated | required about the length of the extended hypha. The results are shown in Table 4.

[0041]

[Table 4]

[0042]

According to the present invention, it became possible to artificially culture VA mycorrhizal fungi without living plants (plant roots) for the first time in the world. Therefore, according to the present invention, VA mycorrhizal fungi can be supplied at low cost, and fungal cells free from contamination by miscellaneous bacteria can be easily obtained. This is VA
In addition to expanding the possibilities of industrial use of mycorrhizal fungi, it is also possible to investigate the physiological properties of VA mycorrhizal fungi that could only be investigated through symbiosis with plants, which is of great academic importance. .
Therefore, the present invention can be effectively used in fields such as agriculture and horticulture.

The various aspects of the present invention are as follows. (1). A VA mycorrhizal fungus characterized by culturing mycelium and / or spores of a VA mycorrhizal fungus without using a live root of the plant, using an extract of a root of a plant which can be infected with the VA mycorrhizal fungus Artificial culture method.

(2). Plants that can be infected with VA mycorrhizal fungus
Iridaceae, Gramineae, Pinus, Ginkgo, Cucurbitaceae,
Oxalis family, vine family, syringaceae family, chrysanthemum family, buttercup family, mulberry family, sesameaceae family, cherry family, primrose family, taro family, perilla family, cyperaceae family, cedar family,
Seriaceae, Polygonaceae, Astragalus, Solanaceae, Caryophyllaceae, Rosaceae, Cypress, Amaranthaceae, Furoaceae, Vineaceae, Pleurotus, Legumes, Rutaceae, Palm, Yamanoimo, Lily, Rybaceae And the method according to (1) above, which is a kind of plant selected from the Gentianaceae.

(3). Plants that can be infected with VA mycorrhizal fungus
The method according to (1) above, which is a gramineous crop.

(4). The method according to (3) above, wherein the gramineous plant crop is a plant belonging to the genus Astragalus.

(5). The method according to (4) above, wherein the Gramineae plant belonging to the genus Astragalus is Bahiagrass.

(6). The method according to (1) above, wherein the extract is extracted from a root of a plant that can be infected with VA mycorrhizal fungus with a water-soluble organic solvent.

(7). Plants that can be infected with VA mycorrhizal fungus
The method according to (6) above, which is a gramineous crop.

(8). The water-soluble organic solvent is methanol,
The method according to (6) above, which is one selected from ethanol, propanol, i-propanol, butanol, i-butanol, t-butanol, acetone, methyl ethyl ketone and mixtures thereof.

(9). Plants that can be infected with VA mycorrhizal fungus
It is a grass crop and the water-soluble organic solvent is one selected from methanol, ethanol, propanol, i-propanol, butanol, i-butanol, t-butanol, acetone, methyl ethyl ketone and mixtures thereof. The method according to (6) above.

(10). Among the extracts extracted with a water-soluble organic solvent from a root of a plant that can be infected with VA mycorrhizal fungus, a component that is eluted in a water-soluble organic solvent having a water content of 5% or more is used. (1) The method described.

[Brief description of drawings]

FIG. 1 is a drawing showing the growth state of Bahiagrass root extract and VA mycorrhizal mycelium in each section measured in Example 1.

FIG. 2 is an explanatory diagram showing measurement points of the length of a culture microflora (R) of VA mycorrhizal fungi examined in Example 3.

FIG. 3 is a micrograph (150 times) showing a culture flora of VA mycorrhizal fungi in Example 3.

FIG. 4 shows V of Bahiagrass root in Example 4.
It is a micrograph (150 times) which shows the formation state of A mycorrhizal fungus.

FIG. 5 is a photomicrograph (150 ×) showing the state of formation of VA mycorrhizal fungi of Allium root in Example 4.

[Description of sign]

 a Control section b 0% methanol elution section c 10% methanol elution section d 25% methanol elution section e 50% methanol elution section f 100% methanol elution section R VA Measurement location of mycorrhizal culture flora

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[Procedure amendment]

[Submission date] May 17, 1995

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[Procedure 3]

[Document name to be corrected] Drawing

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Claims (10)

[Claims] 1. A method of culturing mycelium and / or spores of a VA mycorrhizal fungus without using a live root of the plant, using an extract of a root of a plant which can be infected with the VA mycorrhizal fungus. A method for artificially culturing VA mycorrhizal fungi. 2. Plants that can be infected with VA mycorrhizal fungi include Iridaceae, Gramineae, Pinus, Ginkgo, Cucurbitaceae, Oxalisaceae, Cetaceae, Ranunculaceae, Asteraceae, Ranunculaceae,
Moraceae, sesameaceae, sakura, primrose, taro, perilla, sycamore, cedar, seriaceae, polygonaceae, communis family, solanaceae, caryophyllaceae, rose family, cypress family, amaranthaceae, flossaceae The method according to claim 1, which is a kind of plant selected from the group consisting of Vineaceae, Pleurotus, Leguminosae, Leguminaceae, Rutaceae, Palmaceae, Yamaceae, Liliaceae, Rybaceae and Gentianaceae. 3. The method according to claim 1, wherein the plant that can be infected with VA mycorrhizal fungus is a gramineous crop. 4. The method according to claim 3, wherein the Gramineae crop is a plant belonging to the genus Astragalus. 5. A gramineous crop belonging to the genus Astragalus,
The method according to claim 4, which is bahiagrass. 6. The method according to claim 1, wherein the extract is extracted from a root of a plant which can be infected with VA mycorrhizal fungus with a water-soluble organic solvent. 7. The method according to claim 6, wherein the plant which can be infected with VA mycorrhizal fungus is a gramineous crop. 8. The water-soluble organic solvent is methanol, ethanol, propanol, i-propanol, butanol,
The method according to claim 6, which is one selected from i-butanol, t-butanol, acetone, methyl ethyl ketone, and a mixture thereof. 9. A plant which can be infected with VA mycorrhizal fungus is a gramineous plant, and the water-soluble organic solvent is methanol, ethanol, propanol, i-propanol, butanol, i-butanol, t-butanol, The method according to claim 6, which is one selected from acetone, methyl ethyl ketone, and a mixture thereof. 10. Use of a component that is eluted from a root of a plant that can be infected with VA mycorrhizal fungus with a water-soluble organic solvent to a water-soluble organic solvent having a water content of 5% or more. The method of claim 1 characterized.