JP2009296977A - Method for producing japanese red pine young seedling infected with mycorrhizal fungus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing infected seedlings in large volume by an easy way in a short period by using a natural infection route of mycorrhizal fungi to Japanese red pine (Pinus densiflora) young seedlings and forming mycorrhizae in an oligotrophic medium. <P>SOLUTION: This method for producing the Japanese red pine young seedlings infected with mycorrhizal fungi includes: (1) a process of sowing Japanese red pine seeds on an oligotrophic medium which can support the root of Japanese red pine young seedlings, has water retention, and contains nutrient as little as possible; (2) a process of inoculating mycorrhizal fungi to the vicinity of the root of Japanese red pine young seedlings after at least sprouting from the Japanese red pine seeds; (3) a process of covering the first and second roots of Japanese red pine young seedlings when hyphae of the inoculated mycorrhizal fungi extend, and (4) a process of transplanting and acclimating the Japanese red pine young seedlings infected with mycorrhizal fungi. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing a red pine seedling infected with mycorrhizal fungi by aseptically sowing seeds of red pine in a sterilized container, inoculating and infecting mycorrhizal fungi such as matsutake, kotake, and chichitake. .

Conventionally, many researchers have engaged in artificial cultivation of Matsutake, which belongs to mycorrhizal fungi, but there are no successful examples yet. The first reason for making this kind of artificial cultivation difficult is that matsutake mycelia produce mycorrhiza at the roots of Pinus densiflora and use the sugars that are the photosynthetic products of Pinus densiflora directly through the mycorrhiza. Second, the mechanism of the symbiotic relationship between matsutake and red pine has not been fully elucidated, and the third is that the growth of matsutake fungi is extremely slow.

By the way, the production of matsutake tends to drastically decrease nationwide, and the cause of this is that red pine forests have been neglected due to the reduction in the production of charcoal due to the fuel revolution that began in the 1960s. It is thought that factors such as acid pollution, air pollution, and global warming overlapped, making the soil physicochemical properties and soil microflora unsuitable for the environment where matsutake fungi inhabit.

On the other hand, several cases have been reported in which pine mushroom-infected seedlings were produced by a mixed culture using a red pine aseptic seedling and mycorrhizal mycelium under aseptic conditions.
(1) Mycorrhiza was synthesized by mixing and cultivating matsutake mycelium and red pine seedlings in a plant culture flask using vermiculite immersed in a liquid medium as a medium (Non-patent Document 1).
(2) The matsutake mycelium colony was aseptically crushed, and the roots of Pinus densiflora were aseptically immersed in a culture solution containing the obtained mycelium (Patent Document 1).
(3) Sterile seedlings of trees were transplanted to an agar medium in a test tube, inoculated with ginger mycelia, and transplanted into sterilized sea sand placed in a large beaker to synthesize mycorrhiza (Non-Patent Document 2).
(4) The pulverized mycelium of matsutake fungus that had been cultured in liquid was cultured in a mixed medium of vermiculite and sphagnum moss, and aseptic seedlings of Pinus densiflora were transplanted to synthesize mycorrhizae. (Patent Document 3)

However, in any of the above-mentioned culture methods, after raising red pine seedlings and cultivating mycorrhizal fungi in separate containers, transplant the sterile red pine seedlings to a medium filled with cultured mycelia or artificially transfer the cultured mycelium to red pine seedlings. It is a method to synthesize mycorrhiza by directly attaching, and it is inefficient due to special culture container and complicated operation, and symbiotic relationship is maintained under the risk of contamination and harsh natural environment after acclimatization It is unclear whether mycorrhiza colonizes and grows as white, and it is difficult to say that it is an infectious seedling production method that can withstand practical use.
Japanese Patent No. 3263730 (rapid artificial synthesis of matsutake mycorrhiza). JP-A-2004-129653 (Method for forming pine seedlings infected with matsutake by co-culture of matsutake fungi and sterile germinated seedlings). (Shinya Eto (1990) Raising matsutake fungus seedlings by mycorrhiza synthesis, Hiroshima Prefectural Forest Research Institute, 24, 1-6). 2004 Katsushi Tamada Miyagi Forestry Experiment Station Business Report 37 No. 10-11

  Accordingly, the present invention has been made in view of the above circumstances, and is a natural infection route to the red pine root of mycorrhizal fungi that expands the mycelium extension area in the root rhizosphere of the host by seeking a nutrient source in thin land The present invention has been completed by finding a method for mass-producing infected seedlings in a short period of time by forming mycorrhiza under certain conditions such as an oligotrophic medium, etc.

In order to achieve the above object, the method for producing a pine seedling infected with mycorrhizal fungus according to claim 1 comprises: (1) a poorly nutrient-free plant that can support the roots of red pine seedling and has water retention. Seeding red pine seeds in a nutrient medium; (2) inoculating mycorrhizal fungi near the root of the red pine seedling after at least germination of the red pine seeds; and (3) And a step of covering the primary and secondary roots of the red pine seedling by extending the mycelium, and (4) a step of transplanting and acclimatizing the red pine seedling infected with the mycorrhizal fungus.
The method for producing a mycorrhizal fungus-infected red pine seedling according to claim 2 is such that the oligotrophic medium is a gel-like substance, and the method for producing a mycorrhizal-infected red pine seedling is according to claim 3 wherein the gel-like substance is It is a gellan gum.
The method for producing a mycorrhizal-infected red pine seedling according to claim 4 is characterized in that the mycorrhizal fungus is any one of matsutake fungus, mushroom fungus, and tititake fungus.
The method for producing a mycorrhizal-infected red pine seedling according to claim 5 is characterized in that the mycorrhizal fungus is a fungus subcultured in a medium containing a malt extract, a yeast extract and peptone.
The method for producing mycorrhizal fungi inoculated in the vicinity of the root of a mycorrhizal-infected red pine seedling according to claim 6, wherein the mycorrhizal fungus is a medium obtained by mixing cereal seeds containing starch and porous fine particles. It is characterized by being cultured.

When mycorrhizal fungi such as matsutake fungus are inoculated in the vicinity of the roots of the red pine seedlings after germination from at least red pine seeds, the mycorrhizal fungi descend to the primary and secondary roots of the germinated red pine seedlings.
In that process, the medium is an oligotrophic medium that can support the roots of Japanese red pine seedlings and has water retention, and contains no nutrients as much as possible. Since it has the characteristics that it can withstand, it expands the mycelia by seeking the nutrient source, extending the hyphae, passing through the primary roots and secondary roots to the roots that store sugars and other nutrients by photosynthesis of red pine seedlings. To form mycorrhiza.
At this time, if the oligotrophic medium is a gel-like substance (eg gellan gum), it can support the roots of red pine seedlings and have water retention, germinate in a highly transparent state, and primary roots. And secondary root elongation and mycorrhizal fungal infection can be visually observed.
At this time, if the mycorrhizal fungi such as matsutake fungus, mushroom fungus, and tititake fungus are cultured in a medium containing malt extract, yeast extract and peptone, vigorous hyphae with vigorous growth Thus, it has a property capable of withstanding poor nutrition, and the infection rate of mycorrhizal fungi is increased, so that more infected seedlings can be obtained.
At the same time, when mycorrhizal fungi are cultured in a medium that is mixed with grain seeds containing starch such as barley and porous fine particles such as pumice, zeolite, and Otani stone, sufficient oxygen is supplied to mycorrhizal fungi Therefore, it becomes a vigorous and vigorous inoculum, and has properties that can withstand poor nutrition.
As a result, the present invention does not require special culture devices, tools, or complicated operations, and can reliably mass-produce pine seedlings infected with mycorrhizal fungi such as matsutake fungi.

  The present invention inoculates seedlings of germinated red pine seeds with vigorous and vigorous matsutake seedlings to obtain matsutake fungus-infected seedlings in a short period of time using the natural infection route of mycorrhizal fungi Then, the specific embodiment will be described based on the order of the production process and based on the drawings and tables.

The first series in FIG. 1 will be described.
First, the first series in FIG. 1 will be described. In order to grow red pine seedlings from red pine seeds, an oligotrophic medium that can support the roots of red pine seedlings and has water retention as much as possible and does not contain nutrients. For example, it is set as the process which prepares and solidifies a gel-like culture medium and gellan gum with high transparency.
The oligotrophic medium here refers to a medium that can support the roots of Japanese red pine seedlings and has water retention, but itself contains no nutrients as much as possible, and adds new nutrients other than the medium substrate. And a nutrient-free medium that itself promotes the growth of inorganic and organic plants as much as possible. For example, gel-like raw gellan gum with high transparency can be mentioned. However, the oligotrophic medium is not limited to gellan gum.
Here, attention was paid to the above-mentioned oligotrophic medium. The seeds of Pinus densiflora germinate by the nutrients of their own endosperm, and then the roots grow and the foliage develops. In a medium containing nutrients, seed germination rate and root growth are not good. Therefore, we focused on the natural infection route of mycorrhizal fungi by expanding mycelium by seeking nutrients in thin land and expanding the hyphal elongation area. A gel medium with high transparency that can be formed was used.
Specifically, a heat-resistant container was prepared, 1000 ml of ion-exchanged water was added, 15 g of gellan gum was added while stirring little by little, and it was confirmed that the solution was dissolved by heating and completely dissolved. Dispense into tubes. Thereafter, the mouth of the test tube is covered with aluminum foil or a silicon stopper, autoclaved at 1.2 atm. 120 ° C. for 15 minutes, and cooled to prepare an oligotrophic, translucent gel and elementary gellan gum medium.

Next, as shown in FIG. 1, red pine seeds are sown in a test tube containing the sterilized gellan gum to grow sterile red pine seedlings.
FIG. 1 shows a state in which a single red pine seed 3 is sown in a test tube 1 on the surface of a gel-like raw gellan gum medium 2 having poor nutrition and high transparency. The seed coat of the Pinus densiflora seed 3 is strongly adhered and contaminated with various fungi such as fungi and bacteria, sterilized with ethyl alcohol, sterilized with a chlorine-based disinfectant such as sodium hypochlorite, and rinsed several times with sterilized water. Sow. After that, when cultivated in a culture room at 21 to 25 ° C. for 12 hours, the red pine seeds germinate in two weeks due to the nutrients of the endosperm that they own. In FIGS. The seed germinates and roots due to the nutrients of the endosperm, and then the hypocotyl extends and the foliage develops, and a sterile red pine seedling 4 grows.

Next, based on the 2nd series | sequence shown in FIG. 1, the production process of the matsutake inoculum which consists of a matsutake hypha collection, subculture, and a barley is demonstrated in order.
First, as shown in FIG. 1, matsutake mycelia was collected. For example, matsutake mycelia separated from fold tissue of Hiroshima-grown matsutake mushrooms in 1/2 Hamada medium were transplanted to HY medium for 1-2 times. The subcultured and grown mycelia are used as an inoculation source for matsutake inoculum. Table 1 shows the composition of the HY medium and the content per liter of the medium.

Next, as shown in the second series of FIG. 1, the matsutake mycelia cultured and proliferated in the HY medium are propagated to the barley medium.
Barley is a medium containing 20% pumice or zeolite or Otani stone fine particles mixed with a commercial pressed barley overnight, and 300 ml in a heat-resistant glass container with a large 450 ml upper mouth. After filling and steaming for 60 minutes, sterilization was performed in an autoclave under the conditions of 1.2 atm, 120 ° C. and 60 minutes. The upper mouth portion of the heat-resistant glass container is covered with a cap with a hole of φ5 mm, and a filter is pressure-bonded to the hole to be used for breathing matsutake mycelia. Furthermore, for fine granules of pumice, zeolite, or Otani stone, the vigorous matsutake mycelia can be supplied by providing sufficient oxygen without causing the medium to tighten during growth of the matsutake mycelia by giving the barley medium softness and aeration. Serving to grow.
As shown in FIG. 4, the matsutake mycelium slant 5 subcultured for 40 days in a HY medium is cut into a size of 1 to 2 cm square, and the heat resistant glass is desirably removed in the barley 7 medium. By embedding in the side surface of the container 6 and culturing for two months, the matsutake fungus bred in the so-called barley that grows vigorous and infectious matsutake from the matsutake mycelia is grown.
When pine bamboo hyphae that grows white and covers the surface of barley 7 on the heat resistant glass container 6 side and upper surface side of the barley 7, the white hyphae part is carefully scraped off with tweezers. Transplanted into barley medium of composition.

Furthermore, as shown in the third series of FIG. 1, the inoculation of the matsutake fungus to the red pine seedling and the mycorrhizal infection process will be described.
The matsutake fungus propagated on the barley medium is inoculated in the vicinity of the root of the red pine seedling to form a mycorrhiza formation process for infecting the red pine seedling.
That is, as shown in FIG. 5, when the primary root 8 of the red pine seedling 4 is sufficiently elongated and the secondary root 9 is generated, one matsutake seedling 10 is inoculated near the root of the sterile seedling. Eventually, as shown in FIG. 5 and FIG. 6, the matsutake mycelium 11 derived from the matsutake inoculum 10 responds to the growth of the red pine seedling 4 within about 2 weeks of inoculation, in response to the root metabolites that exude to the nutrient medium, Along with the primary root 8 and the secondary root 9 of the red pine seedling 4, it penetrates into the internal tissue of the root, and sufficient mycorrhizal mycorrhizal fungi such as matsutake fungi are formed at the root of the red pine seedling. The situation where the matsutake mycelium 11 descends along the root is enlarged and shown by the dotted line in FIG. Subsequently, the matsutake hyphae 11 continue to descend to the primary roots 8 and secondary roots 9 of the red pine seedling 4, finally covering the surface of the primary roots 8 and secondary roots 9 of the red pine seedling 4, Enters the inside and forms mycorrhiza. When mycorrhiza is formed, generation of capillary roots is suppressed. FIG. 7 is a schematic photograph showing a state in which mycorrhizal formation has been performed in a culture vessel in a mature state.
In other words, red pine seeds are originally pine seeds that seek nutrient sources and extend mycelia to expand the mycelial growth area. When seeds grow and extend primary and secondary roots, mycorrhizal fungi such as matsutake fungi sown near the roots seek nutrient sources such as sugars created by photosynthesis of red pine seedlings, or in an oligotrophic medium By detecting the exudation of metabolites associated with the vital activity of the root tissue, which stores adjacent photosynthesis product starch particles, into the oligotrophic medium, the area of infection is expanded to the surface of the root tissue, thereby Sufficient mycorrhizal mycorrhizal fungi such as matsutake fungi are formed at the root.
This attempt is due to the failure of conventional matsutake fungus-infected seedlings to be produced. Mycorrhizal fungi such as matsutake fungi are originally symbiotic with red pine in poorly cultivated and thin land, and the pine fungus is filled with rich mycelia. When transplanting seedlings or attaching cultured mycelia directly to Pinus densiflora seedlings, mycorrhizal fungi receive an abundance of nutrients in the medium, seeking hygiene sources on thin land, extending mycelia, and extending mycelia This is based on the fact that the mycorrhizal fungi that reduce the mycorrhizal fungi, which increase the size of the mycorrhiza, and as a result the area of infection cannot be expanded to the surface of the root tissue, but the mycorrhiza once formed is supposed to degenerate and disappear after acclimatization.
After this mycorrhiza formation, matsutake fungi become symbiotic with primary and secondary roots of Pinus densiflora seedlings. In other words, matsutake fungus seeds the sugars of the photosynthesis product of red pine seedlings, and red pine seedlings sow moisture and nutrients from matsutake fungi from the medium or soil where their roots do not reach, while increasing resistance to drought and nutrient deficiency, Form a coexistence cycle.

At this time, the mycorrhizal fungi such as the above-mentioned matsutake fungus, mushroom fungus, and mushroom fungus are those that have been subcultured in a culture solution containing a malt extract, yeast extract and peptone, and further in a HY medium. It becomes a vigorous and vigorous inoculum, has a property that can withstand poor nutrition, and becomes an inoculum exhibiting a high infection rate of mycorrhizal fungi.
In addition, when mycorrhizal fungi are cultured in a medium in which grain seeds containing starch such as barley are mixed with porous fine particles such as pumice, zeolite, and Otani stone, mycorrhizal fungi need aeration. Therefore, it becomes a vigorous hypha similar to the above, and has a high vitality that can withstand poor nutrition.

Once the infected seedlings have grown to a size that can be adapted, they are transplanted and acclimatized in a pot of mixed soil of pumice and red soil.
Acclimatized Pinus densiflora seedlings, when planted in artificial rocks built with crushed rocks containing weathered rocks, will form and expand with the growth of the seedlings several years later. Creation is possible.

  An example of the present invention was manufactured based on the above embodiment. The implementation status will be described below. 1000 ml of ion-exchanged water was prepared in a heat-resistant container, and 15 g of gellan gum was added and dissolved little by little with stirring to prepare a gellan gum solution. 10 ml of the gellan gum solution was dispensed into a test tube, and the mouth was covered with aluminum foil and sterilized at 1.2 atm and 120 ° C. for 15 minutes to prepare a gel-free medium gellan gum with no nutrition and high transparency. . The test tube containing the sterilized gellan gum is pre-sterilized with ethyl alcohol, then sown pine seeds sterilized with a chlorine-based disinfectant such as sodium hypochlorite and cultivated in a culture room at 25 ° C. for 12 hours. Germination was performed in 2 weeks, and the primary roots were elongated to grow sterile red pine seedlings. The collected matsutake mycelia were transplanted to a HY medium containing nutrients and subcultured to grow matsutake mycelia. Then, 20% Oyaishi fine granules by volume ratio were added to the absorbed barley, mixed, filled in a heat-resistant glass container with a large volume of 450 ml in the upper mouth, and steamed for 60 minutes, and 1.2. A so-called barley medium was obtained by sterilization in an autoclave under atmospheric pressure and 120 ° C. for 60 minutes. The matsutake mycelium slant that has been subcultured for 40 days in the HY medium is cut to a size of 1 to 2 cm square, embedded in the side of the heat-resistant glass container in the barley medium, and cultured in February. Breeding and breeding high matsutake inoculum. One matsutake seedling was inoculated in the vicinity of the root of a sterile seedling in which the primary roots of red pine seedlings were elongated and secondary roots were generated. Two weeks after the inoculation, the matsutake inoculum begins to descend along with the primary roots of the red pine seedlings, finally reaches the secondary roots of the red pine seedlings, and finally enters the cell space from the root epidermis. The mycorrhiza was formed.

The infection of Pinus densiflora roots with the above matsutake mycelia was performed through gellan gum medium with high transparency, and visual observation was possible with the naked eye. The fact of infection is that the occurrence of capillary roots was suppressed by infection, the presence of harhinnets in the cell space was observed by observation of the root tissue, and the mycorrhiza specific to matsutake that can be made into the roots after acclimatization The presence was observed (see FIG. 8), and was confirmed by the base sequence of the hyphal DNA attached to the surface of the root, and the like.

  The present invention can reliably produce mycorrhizal fungus-infected seedlings in a short period of time and does not cost, and can mass-produce matsutake fungus-infected seedlings. Furthermore, it can be applied not only to matsutake but also to mycorrhizal fungi such as chichitake and koitake. In addition, it is effective for the large-scale construction of matsutake mushrooms using matsutake fungus-infected seedlings. Since mycorrhizal-infected seedlings increase resistance to the environment and pests, environmental conservation is achieved through planned planting of mycorrhizal fungus-infected seedlings And contribute to the tourism industry.

It is the schematic which shows the flow of the process from sowing of a red pine seed to acclimatization of a red pine seedling. It is the schematic which shows the state as which the red pine seed was aseptically sown in the gel-like culture medium with high transparency. It is the schematic which shows the grown red pine seedling in a test tube. It is the schematic which shows the state which the culture matsutake hypha transplanted in the barley culture medium extended to the surrounding barley, and became a seed fungus. It is the schematic which shows the state which the matsutake mycelium extended from the placed barley inoculum descend | falls to the primary root and secondary root of a red pine seedling, and covered the surface. Fig. 6 is a schematic view further enlarging a part of Fig. 5. It is a photograph figure showing the infection state of the matsutake fungus to the primary root and secondary root of a red pine aseptic seedling in a culture container. It is a photograph figure showing the mycorrhiza peculiar to the matsutake formed in the red pine acclimatized seedling.

Explanation of symbols

1 Test tube 2 Gellan gum medium (poor nutrient medium)
3 Japanese red pine seeds 4 Japanese red pine seedlings 5 Matsutake mycelium slant 6 Heat-resistant glass container 7 Barley 8 Primary root 9 Secondary root 10 Matsutake inoculum 11 Matsutake hyphae

Claims (6)

(1) sowing red pine seeds in an oligotrophic medium capable of supporting roots of red pine seedlings and having water retention and containing as little nutrient as possible;
(2) a step of inoculating a mycorrhizal fungus near the root of the red pine seedling after germination from at least the red pine seed;
(3) a step of extending the mycelium of the inoculated mycorrhizal fungus to cover primary and secondary roots of red pine seedlings;
(4) transplanting and acclimatizing red pine seedlings infected with the mycorrhizal fungi;
A method for producing red pine seedlings infected with mycorrhizal fungi, comprising: The method for producing red pine seedlings infected with mycorrhizal fungi according to claim 1, wherein the oligotrophic medium is a gel substance. The method for producing a mycorrhizal-infected red pine seedling according to claim 2, wherein the gel-like substance of the oligotrophic medium is gellan gum. The mycorrhizal fungus is any one of matsutake fungus, mushroom fungus, and tititake fungus. The method for producing a mycorrhizal fungus-infected red pine seedling according to any one of claims 1 to 3. The mycorrhizal fungus is a fungus that has been subcultured in a medium containing a malt extract, a yeast extract and peptone, 5. The method for producing a mycorrhizal-infected red pine seedling according to any one of claims 1 to 4. The mycorrhizal fungus is a fungus cultivated in a medium in which starch seed-containing cereal seeds and porous fine particles are mixed, production of mycorrhizal-infected red pine seedlings according to any one of claims 1 to 5 Method.