JPS6033444B2 - How mycorrhiza forms - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for forming mycorrhizae, and in particular provides a method for forming mycorrhizae by artificial means, which is the basis for the artificial cultivation or propagation of edible mycorrhizal fungi such as matsutake and honshimeji. It is.

The main methods that have been attempted in the past to artificially propagate Matsutake, which is a representative mycorrhizal fungus, have been to naturally infect the roots of Matsutake with spores by scraping fallen leaves, raking the ground, and digging up existing Matsutake mountains. There are methods to promote this, methods such as laying down the umbrella, spore inoculation method by spraying or irrigation of spore liquid, mycorrhizal transplantation method, cultured mycelium inoculation method, etc.

Among them, many attempts have been made to inoculate cultured mycelia, which is considered to be the best method due to the advantage that the inoculum source can be easily obtained at any time, but no complete success story has yet been heard. The methods of inoculating cultured mycelia that have been tried so far include: [1' A method of burying a mass of cultured mycelium, including a beach, in the ground, and guiding and contacting the roots of matt.

The main method is to excavate the roots of the mat, inoculate the new roots with mycelial masses from which the medium has been washed off, and then backfill.
The co-suitability of these methods is quite large (number 1
The two points are that the inoculation source is hyphae containing a medium or hyphae (estimated to be 9 to several 100 times), and that the inoculum source is buried in the soil immediately after inoculation. For this reason, in both cases where the inoculum source contains a medium or when the inoculum source does not include camellia land, the remains of excess dead mycelium remain in the soil due to the limited amount of nutrients supplied from the inoculated roots. The soil bacteria that assimilate these substances begin to proliferate at an early stage. In this case, since the target fungi are aerobic, they often suffocate in the buried soil, inhibiting their growth, or dying. Contrary to the common sense in the case of inoculation with plant pathogens, which is easy to establish, settlement is not achieved. In order to prevent the proliferation of these soil bacteria (mainly filamentous fungi), an excellent attempt was made to use a selective fungicide that suppressed the growth of miscellaneous fungi without inhibiting the growth of mycorrhizal fungi (Tokusei Sho 50).
-157152), but this method has drawbacks in terms of the cost of the drug and the labor involved in applying it.

Furthermore, even if a high rate of settlement is achieved, a large amount of inoculum is required, and this is because the growth rate of mycorrhizal fungi in the medium is extremely slow when inoculating a large number of inocula. Problems also arise from above.・The purpose of the present invention is to reliably form mycorrhizae at a high rate by an extremely simple method, mainly by artificial inoculation of cultured hyphae, which has traditionally had an extremely low success rate and was thought to be practically impossible. It's about achieving.

That is, the present invention provides a mycorrhizal fungus nutrient source consisting of a host tree and soil attached to its roots as a tower, and sufficiently isolates the new roots of the host tree that will become the mycorrhizal fungus inoculation area from the soil. to form a predetermined free space adjacent to the soil and configured to a size that can exclude harmful bacteria, and in this state, the line of the host tree is allowed to grow so that new roots extend to the free space. None, this new root was inoculated with a specified mycorrhizal fungus, and the free space was kept in a high humidity and well-ventilated state to achieve initial colonization of the inoculated mycorrhizal fungus. The present invention relates to a method for forming mycorrhizae characterized by the following.

In order to overcome the drawbacks of the conventional method described above, the inventor conducted various studies that took into consideration the mechanism of mycorrhizal propagation in natural conditions, the characteristics of the growing point of the plant, etc., and developed the method without using any chemicals. We devised a method to reliably form mycorrhizae using a small amount of inoculum and simple operations.

The growth points of vigorously growing plants generally have an extremely high growth (elongation) rate, and are susceptible to the growth (elongation) of viruses and pathogens.
(long) speed, the small area at the tip is kept virus-free or sterile, and there have already been several attempts to cultivate virus-free strains by culturing the shoot apex, which is the growing point of the above-ground part. It has been put into practical use for garden plants, crops, etc. The root tip, which is the point of growth underground, grows in direct contact with soil, which is inhabited by a large number of bacteria, so it is usually contaminated with these bacteria. However, as in the present invention, it is constructed in a size that can eliminate harmful bacteria by sufficiently isolating it from contaminated soil and preventing it from touching it, and it can grow in a predetermined free space adjacent to the soil. If allowed to grow, its growth rate will exceed that of the various microorganisms that originally existed in the rhizosphere, and, like the top of a shoot above ground, a certain area near the tip will always be kept sterile, and a specific point will be kept sterile. If you think about it, it will remain sterile or semi-sterile to the extent necessary for mycorrhizal fungi to colonize it for a certain period of time. If the root tip in such a state is inoculated with daughter fungi, the desired colonization of mycorrhizal fungi can be achieved reliably. After settling, it produces antibacterial substances and is able to continue growing while eliminating bacteria. In addition, considering the situation in which fungal plate fungi colonize new roots near the front of "shiro" (a collection of mycorrhizae), which progresses with a 100% colonization rate in natural conditions, it seems that individual new roots actually The amount of hyphae directly involved in settlement is extremely small. Therefore, if we move away from the conventional method of inoculating a large number of unspecified new roots all at once and inoculating each new root one by one, the amount of inoculum hyphae required will be extremely small. Only a small amount is required, and the amount of organic matter that remains after the initial settlement and helps the growth of bacteria can be reduced to a negligible level. Furthermore, the ventilation near the front of the "shiro" is extremely good, and considering that a large area inside the "shiro" is highly impermeable, it is thought that it will hardly ever become wet even during rain. However, as in the present invention, new roots that are constructed in a size that can be sufficiently isolated from the soil and eliminate harmful bacteria and that have grown to extend in a predetermined free space adjacent to the soil can also be grown under almost the same conditions. , which is extremely advantageous for the colonization of fungal plaques. From the above, the advantages of the method according to the present invention can be summarized as follows.

01 Sterilization or semi-sterilization (removal of germs) of the inoculated area can be made possible by a simple method that utilizes the characteristics of the growing point of the plant, without relying on the conventional use of chemicals.

'2} We have changed the traditional method of burying the inoculated area in the soil immediately after inoculation, which caused bacterial contamination of the inoculated area and poor ventilation, and now we are cleaning the inoculated area from the time of inoculation until the seedlings have established. Maintaining a large and well-ventilated condition, the settlement rate is almost 100%.
can be dramatically improved.

'3} Control of miscellaneous fungi that inhibit the growth of mycorrhizae after inoculation has been made possible by implementing the simple principle of not introducing assimilated materials of miscellaneous fungi, instead of using conventional chemicals. thing.

In addition, in the method according to the present invention, the temperature of the space that should be kept at a high temperature is 90% RH (relative humidity) or higher).
, up to around 100% RH is good, and preferably about 95% RH or higher.

If the humidity is less than 90% RH, root growth will be insufficient.
Furthermore, if too much rinsing water is used, especially when the inoculum contains a culture medium, the inoculum is often contaminated with various bacteria through the flowing water.
In addition, the temperature during inoculation and settlement of mycorrhizal fungi is 12 to 28 oo.
(Temperature conditions that favor the growth of bacteria)
Although a temperature of 25 qo is desirable, there is no problem in temporarily leaving the above temperature range within a range of about 5 to 31°C (temperature conditions in which bacteria can grow). Furthermore, after initial settlement, it is desirable to remove excess inoculum from the inoculation site, which may inhibit the growth of mycorrhizas, depending on the case. Next, an example in which the present invention is applied to inoculation of Matsutake fungi will be described with reference to the drawings.
It will be understood that the following embodiments can be further modified based on the technical idea of the present invention.

Example 1 As shown in FIG. 1, a bottomless plastic box 3 whose inner surface is lined with water-absorbing cloth, such as vinylon nonwoven fabric 1, and has observation holes and working holes 2, is placed in a shallow blood box 5 filled with water 4. When placed inside the box 3, the inside of the box 3 is maintained at approximately saturated humidity.

When cultivating a clay pot 8 in which red pine 7 (3rd year old) was planted using chert mountain sand 6 as a culture medium was placed on top of this box and cultivation continued, new roots 9 of red pine 7 came out from the holes in the bottom of the pot. Then, it grows into the plastic box 3, but since the inside of the box 3 is kept at high humidity, it continues to grow without being hindered.
Then, fill the small test tube (5 volumes out of 15) with agar medium (2% glucose, 0.5% yeast, 1.5% agar, pH
4. Add 2) and plant Matsutake fungi on the agar surface to 24 qo.
Colony 10 that grew after 3 to 4 weeks of cultivation was left as is)
Each beach area was inoculated by contacting the new root 9 mentioned above. In this case, the new diameter was ~1.2 and the willow temperature was 25~5°C. Matsutake fungus colonization was determined by the browning of the white new roots near the cuttings, and bacterial contamination was determined by the occurrence of bacterial colonies on the agar base or hyphae surface. The results showed that the number of days until settlement was 4 to 8 days (average 6 days), and that settlement and contamination progressed almost simultaneously.

In some cases, settlement was delayed compared to the contamination, but when the Matsutake colonies used for inoculation were removed after settlement and the subsequent growth was observed, browning and blackening of the roots progressed in all cases, and there was no significant difference between them. It was not recognized. Example 2 As shown in FIG. 2, a non-woven fabric 11 that is breathable and water-permeable and does not allow roots to pass through is laid on the bottom of a clay pot 8, and agricultural polyurethane foam 12 (with three holes 15) is placed on top of it. 15
Place a PVC tube 1 with a thickness of 5 mm in the same D as the hole.
3 is embedded to about half the thickness of the urethane foam 12 and fixed, and a lid 14 is placed on the upper end.

Red pine 7 (3
If you continue to plant the hole 15 as shown in the figure,
White new roots 9 emerge on the nonwoven fabric 11 inside. For this new root, the hyphae cultured on the same agar medium as in Example 1 were cut into small pieces and the size of the hyphae was approximately 1 to 9 to 10 females, or dense hyphae and sparse plexiform hyphae were cut into small pieces. An inoculum consisting of a small piece of the complex was inoculated after washing the medium with sterile water. We also slightly considered the inoculation temperature. The number of days required for settlement is shown in the table below. At 25:00, which is considered to be the optimum temperature, no effect of the amount of inoculation was observed, and settlement occurred extremely quickly.

In addition, in the case of this type of inoculation, no contamination occurred, but evidence of feeding damage by mites, springtails, etc. was observed, and the inoculated bacteria disappeared in 1 to 2 weeks. However, no effect on settlement was observed. Also, since it was a collection, there was no need to remove excess seed sources. Example 3 As shown in Fig. 3, Chromat seedlings 17 (5th grade), which were planted in the tower ±6 in the clay pot 8 and cultivated in the usual manner for about 6 months, and whose root ball had hardened, were pulled out from the pot 8. A silk stand 18 is placed inside the pot 8 to create a gap 21 on the inner surface of the pot 8, and a wet vinylon nonwoven fabric 20 is placed around the pot 8 to prevent it from drying out. The new roots 19 that had been placed on the stand 16 and had grown into the gap 21 were inoculated in the same manner as in Example 2.

As a result, good adhesion was obtained. Example 4 As shown in FIG. 4, red pine 7 (3rd year) was planted in a pot 8 together with a plastic mesh tube 22 with a lid 23, and the tube 2
New roots 9 that appeared within 2 were inoculated.

This inoculation could be easily performed by removing the lid 23. As a result, good adhesion was obtained. Example 5 As shown in FIG. 5, a film or non-woven fabric 25 that does not allow roots to pass through is laid down, a mound, a ridge, etc. is made in the open ground, and two red pines 7 are planted.

A V-shaped groove 26 is provided at an appropriate position between them, and one wall of the groove is made of silk 27 and the other is made of board 28. In the case of the net 27, the new roots 9 that had passed through and came out into the groove 26 were inoculated, and in the case of the plate 28, the plate was removed at an appropriate time and the exposed new roots 9 were inoculated. This inoculation could be easily performed by removing the lid 29. As a result, good establishment of the new roots 9 on both sides was obtained.

[Brief explanation of the drawing]

The drawings show an example in which the present invention is applied to the inoculation of Matsutake fungi, and FIG. 1 is a cross-sectional view showing the state of inoculation and settlement according to the first example, and FIG. FIG. 3 is a sectional view showing the state of inoculation and settlement according to the third embodiment, and FIG. 4 is a cross-sectional view showing the state of inoculation and settlement according to the fourth embodiment. FIG. 5 is a sectional view showing the state at the time of inoculation and settlement according to the fifth embodiment. In addition, in the symbols used in the drawings, 3...
・Plastic box, 4...Water, 6...
Cultivator, 7... Red pine, 9... New root, 10
...Matsutake fungus colony, 12...Polyurethane foam, 13...PVC pipe, 17...
Chromat, 18...Net stand, 19...New root, 22...Plastic mesh tube, 27...Silk,
28...It is a board. Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

Claims (1)

[Claims] 1. Prepare a nutrient source for mycorrhizal fungi consisting of a host tree and a pot of soil attached to the roots of the host tree, and thoroughly isolate the new roots of the host tree that will become the inoculated area of mycorrhizal fungi from the pot to eliminate harmful bacteria. A predetermined free space is formed adjacent to the soil pot and is configured to a desired size, and in this state, the roots of the host tree are allowed to grow so that new roots extend to the free space. The mycorrhizal fungus is inoculated with a predetermined mycorrhizal fungus, and the free space is kept in a high humidity and well-ventilated state to achieve initial settlement of the inoculated mycorrhizal fungus. How to form.