Method for producing japanese black pine seedlings having rhizopogon roseolus mycorrhiza

Abstract

To realize a method for producing Japanese black pine seedlings having Rhizopogon roseolus mycorrhiza, which is excellent in a seedling raising rate.SOLUTION: A method for producing Japanese black pine seedlings having Rhizopogon roseolus mycorrhiza comprises: a seed soaking and sterilization step of subjecting Japanese black pine seeds to running-water soaking in water for a predetermined period and then immersing the seeds in an aqueous solution containing hydrogen peroxide; a sowing step of sowing the seeds in an inorganic culture medium that is held in contact with water in a seedling-raising container that retains water, the inorganic culture medium having a soil pH of 4 or more and 7 or less; a contact step of bringing roots of seedlings germinated from the seeds into contact with at least one of Rhizopogon roseolus spores and hyphae; and a cultivation step of cultivating the seedlings in the inorganic culture medium for 3 months or more after germination.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for producing black pine seedlings having mycorrhizae. [Background technology]

[0002] The fruiting bodies of the Japanese yew tree are in demand as a luxury food ingredient. The Japanese yew tree forms fruiting bodies in symbiosis with Pinus thunbergii and other pine trees. Furthermore, the growth of Pinus thunbergii trees is promoted by the symbiosis of mycorrhizal fungi, such as those of the Japanese yew tree, with the roots. For this reason, methods are being investigated to artificially create a symbiotic relationship between the Japanese yew tree and the Japanese black pine, both from the perspective of cultivating the fruiting bodies of the Japanese yew tree and growing Pinus thunbergii trees.

[0003] For example, Patent Documents 1 and 2 disclose a method of inoculating seedlings of Japanese black pine with spores or mycelia of Camphora camphora. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2003-52243 [Patent Document 2] Japanese Patent Application Laid-Open No. 2012-80811 Summary of the Invention [Problem to be solved by the invention]

[0005] However, in the methods described in Patent Documents 1 and 2, seedlings of Japanese black pine inoculated with spores or mycelia of Camphor tree sometimes germinate poorly or grow poorly due to mold and pest infestation, leaving room for improvement.

[0006] One aspect of the present invention aims to realize a method for producing black pine seedlings having rhizosaccharomycorrhizae, which has an excellent seedling growth rate. [Means for solving the problem]

[0007] In order to solve the above-mentioned problems, one embodiment of the present invention provides a method for producing black pine seedlings with Perilla mycorrhizae, which comprises the following steps: a soaking and sterilizing step of soaking Black pine seeds in running water for a predetermined period of time and then soaking them in an aqueous solution containing hydrogen peroxide; a sowing step of sowing the seeds in mineral culture soil with a soil pH of 4 to 7 as measured in accordance with JIS Z8802:2011, the soil being held in a seedling container that holds water so that at least a portion of the soil is in contact with the water; a contacting step of contacting the roots of seedlings germinated from the seeds with at least one of Perilla spores and mycelium; and a cultivation step of cultivating the seedlings in the mineral culture soil for at least three months after germination. [Effects of the Invention]

[0008] According to one aspect of the present invention, a method for producing black pine seedlings having rhizosaccharomycorrhizae with an excellent seedling growth rate can be realized. [Brief explanation of the drawings]

[0009] [Figure 1] 1 is a schematic diagram showing a series of steps in a production method according to an embodiment of the present invention. [Figure 2] 1 is a cross-sectional view schematically showing an example of a seedling raising container used in a production method according to one embodiment of the present invention. [Figure 3] FIG. 10 is a perspective view schematically showing another example of a seedling raising container used in a production method according to one embodiment of the present invention. [Figure 4] FIG. 10 is a graph showing the atmospheric temperature and humidity on each day from the day the seeds were sown in Reference Example 4. [Figure 5] FIG. 10 is a graph showing the atmospheric temperature and humidity on each day from the day the seeds were sown in Reference Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, one embodiment of the present invention will be described in detail.

[0011] [Summary of the Invention] The method for producing black pine seedlings with Peronosporium mycorrhizae according to one embodiment of the present invention improves the germination rate of black pine seeds and the raising rate of black pine seedlings compared to conventional methods. As a result, the success of raising black pine seedlings with Peronosporium mycorrhizae can be achieved with a much higher probability than conventional methods.

[0012] In this specification, the method for producing black pine seedlings having rhizosaccharomycorrhizae according to one embodiment of the present invention may be simply referred to as the "production method." The seedling growth rate of black pine seedlings refers to the probability that a black pine seedling will take root in the culture soil, become established in the culture soil, and grow to a state where it can continue to grow.

[0013] In one embodiment of the present invention, the Japanese black pine is a plant of the pine genus known by the scientific name "Pinus thunbergii." The shōro is a species of fungus of the shōro genus known by the scientific name "Rhizopogon roseolus." The shōro is known as an edible mushroom whose fruiting body is a luxury food ingredient, and is a species useful in the food industry.

[0014] It is known that the Japanese sedge is a type of ectomycorrhizal fungus that lives in symbiosis with Japanese black pine by attaching itself to the roots of the tree and forming ectomycorrhizae. In this specification, when the term "mycorrhizae" is simply used, it refers to ectomycorrhizae unless otherwise specified. The Japanese sedge that lives in symbiosis with Japanese black pine produces edible fruiting bodies during its life cycle. The growth of the Japanese black pine is promoted by the Japanese sedge growing symbiotically with the roots. In this way, the symbiosis between the Japanese sedge and the Japanese black pine not only allows for the production of Japanese sedge fruiting bodies, but also contributes to promoting greening by promoting the growth of the Japanese black pine.

[0015] However, a method for cultivating symbiotically Pinus thunbergii and Pinus thunbergii on an industrial scale has not been realized so far. The reasons for this include the difficulty of stabilizing both the germination rate and seedling growth rate of Pinus thunbergii, and the difficulty of efficiently forming Pinus thunbergii mycorrhizae on Pinus thunbergii and growing mycorrhizae.

[0016] This production method can improve the germination rate of black pine and reduce the possibility of inhibiting the growth of black pine seedlings due to the infestation of pests, mold, etc. Furthermore, it can quickly form mycorrhizae in black pine, further improving the rate of black pine seedling growth.

[0017] By promoting the production of black pine seedlings with pine mycorrhizae using this production method, it is possible to promote the cultivation of pine fruiting bodies as well as greening using black pine. Such effects will also contribute to the achievement of the United Nations' Sustainable Development Goals (SDGs), such as Goal 2.4 "Ensure sustainable food production systems" and Goal 15.2 "Substantially increase afforestation and reforestation globally."

[0018] [This production method] The details of this production method will be described below with reference to Figures 1 to 3. Figure 1 is a schematic diagram showing the flow of this production method. Figure 2 is a cross-sectional view showing a typical example of a seedling raising container used in this production method. The cross-section shown in Figure 2 is a vertical cross-section of the seedling raising container including a water passage hole formed in the bottom surface. Figure 3 is a perspective view showing a typical example of another seedling raising container used in this production method.

[0019] For the sake of convenience, the "up-down direction" in this specification refers to the vertical direction when the seedling container is placed horizontally, but this does not limit the orientation of the seedling container. For example, if the seedling container is placed on a surface that is inclined from the horizontal plane, the "up-down direction" may deviate from the vertical direction due to the inclination.

[0020] As shown in Figure 1, this production method includes a soaking and sterilization step, a sowing step, a contacting step, and a cultivation step. The production method may further include a transplanting step. Each step will be described below.

[0021] (seeding / sterilization process) The seed soaking and sterilization process involves soaking black pine seeds in running water for a specified period of time and then soaking them in an aqueous solution containing hydrogen peroxide. Black pine seeds may contain eggs of pests such as mites or fungi other than pine larvae, which can cause pest and mold infestations after germination. If pests or fungi appear after germination, they can cause root rot or leaf withering in seedlings, inhibiting growth and significantly reducing seedling growth rates. Therefore, black pine seeds are sterilized by soaking them in an aqueous solution containing hydrogen peroxide. Note that hereafter in this specification, the term "seeds" refers simply to black pine seeds.

[0022] Sterilization here refers to, for example, killing bacteria or weakening bacteria present in the seeds to the point where they cannot grow. Sterilization is preferably carried out as a sterilization treatment that almost completely kills bacteria, but is not limited to this, and it is sufficient if at least some of the bacteria or bacteria can be weakened. This can improve the rate of seedling growth when seedlings germinate from the seeds.

[0023] The water for soaking the seeds in running water is preferably tap water or well water, and the predetermined period for soaking the seeds in running water is preferably 3 days or more and 7 days or less.

[0024] "Soaking seeds in an aqueous solution containing hydrogen peroxide" means that at least a portion of the seeds is immersed in the aqueous solution. From the viewpoint of sterilization efficiency, it is preferable that the entire seeds are immersed in the aqueous solution.

[0025] The soaking of seeds in an aqueous solution containing hydrogen peroxide may be carried out by immersing the seeds in a container such as a beaker containing an aqueous solution containing hydrogen peroxide, as shown in the soaking and sterilization step section of Figure 1. At this time, the seeds may float in the aqueous solution and only part of them may be immersed in the aqueous solution.

[0026] The aqueous solution containing hydrogen peroxide may be a hydrogen peroxide solution containing hydrogen peroxide and water, or may be an aqueous solution containing other components. The content of hydrogen peroxide in the aqueous solution containing hydrogen peroxide is not particularly limited.

[0027] The content of hydrogen peroxide in the aqueous solution should be 0.01 w / v (weight / volume)% or more from the viewpoint of sterilization ability, and 10.00 w / v% or less from the viewpoint of reducing damage to seeds.

[0028] The soaking time for soaking the seeds in the aqueous solution containing hydrogen peroxide is not particularly limited, but may be 10 minutes or more or 15 minutes or more from the viewpoint of sterilization ability, and may be 20 minutes or less or 15 minutes or less from the viewpoint of reducing damage to the seeds.

[0029] The seeds may be immersed in an aqueous solution containing hydrogen peroxide at room temperature or at outdoor ambient temperature. In this specification, room temperature refers to a temperature between 10°C and 25°C. The outdoor ambient temperature is not particularly limited, but may be, for example, between 5°C and 30°C.

[0030] In the seed soaking and sterilization process, it is preferable to soak the seeds in an aqueous solution containing hydrogen peroxide and then wash the seeds to remove the aqueous solution. The washing water used to wash the seeds is preferably water such as pure water or tap water, but may also be an aqueous solution containing a washing component such as a surfactant. Furthermore, the seeds may be washed by soaking the seeds in washing water or by pouring the washing water over the seeds.

[0031] The surfactant is not particularly limited, but examples thereof include nonionic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants, and combinations thereof are also acceptable. From the viewpoint of reducing damage to seeds, the surfactant is preferably a nonionic surfactant. Examples of nonionic surfactants include Tween 20, Tween 40, Tween 60, and Tween 80.

[0032] When such washing is performed, the set of soaking the seeds in an aqueous solution containing hydrogen peroxide and washing may be performed once or twice or more times in the seed soaking and sterilization process. By performing this set twice or more times, the sterilization efficiency of the seeds can be improved without increasing the hydrogen peroxide content in the aqueous solution containing hydrogen peroxide. This makes it easier to reduce damage to the seeds during the seed soaking and sterilization process.

[0033] In conventional methods for sterilizing seeds, for example, aqueous sodium hypochlorite solutions have been commonly used. While aqueous sodium hypochlorite solutions have very strong sterilizing properties, they also cause significant damage to seeds, reducing their germination rate. Therefore, when using aqueous sodium hypochlorite solutions, it has been difficult to sterilize seeds while maintaining a germination rate above an acceptable level.

[0034] This production method uses an aqueous solution containing hydrogen peroxide to sterilize seeds. While hydrogen peroxide has the ability to sterilize bacteria and other microorganisms, it also causes less damage to seeds than sodium hypochlorite. As a result, the seed germination rate can be improved compared to when sodium hypochlorite is used.

[0035] (Seeding process) The sowing step is a step of sowing Japanese black pine seeds in inorganic soil having a soil pH of 4 or more and 7 or less, which is held in a water-holding seedling container so that at least a portion of the soil is in contact with the water.

[0036] The seedling container is not particularly limited as long as it has a portion for holding water and a portion for holding inorganic soil, and is a container with a shape that allows at least a portion of the inorganic soil to come into contact with water. By using such a seedling container that allows contact between water and inorganic soil, a sufficient amount of water can be continuously supplied to seeds and seedlings compared to watering or sprinkling.

[0037] An example of a seedling container is the seedling container 10 shown in Figure 2. The seedling container 10 has a first container 11 and a second container 13. The first container 11 is the portion of the seedling container 10 that holds the inorganic culture soil S, and has a water passage hole 12 formed on its bottom surface. It is preferable that the water passage hole 12 is formed on a portion other than the bottom surface of the first container 11. Furthermore, the first container 11 may have two or more water passage holes 12 formed therein.

[0038] The second container 13 is a portion of the seedling container 10 that holds water W. The seedling container 10 may be used by inserting the first container 11 into the internal space that holds the water W in the second container 13. The water W held in the second container 13 is supplied, for example, by permeating through the water passage holes 12 into the inorganic culture soil S held in the first container 11.

[0039] Another preferred example of a seedling container is the seedling container 20 shown in Figure 3. The seedling container 20 has a first container 21, a second container 23, and a lid 24. The first container 21 has a shape in which a plurality of first containers 11 of the seedling container 10 are connected side by side. With a first container 21 of this shape, a plurality of Japanese black pine seedlings can be individually raised in one seedling container 20.

[0040] The second container 23 differs from the second container 13 in that the second container 23 has an internal space large enough to allow the first container 21 to be inserted therein. The second container 23 holds water W, and the water W can pass through the water passage hole 22 formed in the first container 21.

[0041] The lid 24 is a cover member that covers the first container 21 from above. By covering the first container 21 with the lid 24, evaporation of water W from the first container 21 can be reduced. The lid 24 is also formed with an air vent 25. The air vent 25 allows fresh air to be supplied from outside the seedling raising container 20 to the Japanese black pine seedlings raised in the first container 21. The air vent 25 may be openable and closable.

[0042] The water held in these seedling containers is preferably tap water or well water. When tap water is used, it is preferable to leave it to stand for, for example, 12 hours or more after drawing it out to remove chlorine before use.

[0043] The amount of water held in the seedling container is not particularly limited, as long as the water held in the first container is in contact with at least a portion of the inorganic culture medium held in the second container. The amount of water held in the seedling container until germination occurs may be, for example, an amount that keeps the water level above the bottom of the second container and below the surface of the inorganic culture medium. More preferably, the water level is below the depth at which the seeds are sown in the inorganic culture medium.

[0044] The amount of water held in the seedling container may be changed depending on the growth stage of the seeds or seedlings. For example, if the amount of water is too high when the seedlings start to lignify, the progress of lignification tends to slow down. Therefore, for example, the amount of water held in the seedling container may be changed so that the water level is at the following position: Until seed germination: slightly below the surface of the mineral soil, After germination and until lignification begins: halfway between the surface and bottom of the inorganic soil After lignification begins: 1 / 3 or less between the surface and the bottom of the inorganic soil, After lignification: There should be almost no water in the seedling container and water once every three days.

[0045] The soil pH of inorganic culture soil is 4 or higher and 7 or lower, preferably 4 or higher and 6 or lower. The soil pH of inorganic culture soil can be measured in accordance with JIS Z 8802:2011. Specifically, for example, the soil pH of inorganic culture soil may be measured using a soil pH tester such as the HALO2 soil pH tester / HI9810302 (manufactured by Hana Instruments). For soil pH measurement, it is preferable to drop ion-exchanged water into the inorganic culture soil, insert the soil pH tester, wait until the fluctuations in the measured value stabilize, and then determine the soil pH. Inorganic culture soil with such a soil pH is suitable for germinating and raising Japanese black pine seedlings.

[0046] Inorganic soil is intended to be soil that is poor in organic fertilizer components such as nitrogen, phosphorus, and potassium, which are abundant in organic soil such as Akadama soil, compost, or plant nutrients, and is mainly composed of other inorganic components. Inorganic soil does not need to contain zero organic fertilizer components, but can be inorganic soil to which no organic fertilizer components have been intentionally added.

[0047] Examples of inorganic soil include Kanuma soil, diatomite, vermiculite, perlite, zeolite, imogolite, rock wool, Kiryu sand, mountain sand, river sand, sea sand, pumice, and sintered earthen materials. Among these, the inorganic soil is preferably a microporous inorganic soil having micropores such as Kanuma soil, diatomite, vermiculite, perlite, zeolite, imogolite, and rock wool, and more preferably Kanuma soil.

[0048] The inorganic soil may contain only one of these, or two or more. When the inorganic soil contains two or more types of inorganic soil, these inorganic soils may be mixed, or may have a multi-layer structure in which multiple types of inorganic soil are layered.

[0049] It is preferable to sterilize the inorganic culture medium before use. There are no particular limitations on the method for sterilizing the inorganic culture medium, but for example, high-pressure heating using an autoclave (high-pressure steam sterilizer) is used.

[0050] In this production method, inorganic culture soil is held in the first container of the seedling container, as shown in Figure 1. Here, before placing the inorganic culture soil in the first container, bottom stones may be placed in the bottom of the first container. By placing bottom stones in the first container, clogging of the water passage holes formed in the first container with inorganic culture soil can be prevented, making it easier to maintain contact between the water and the inorganic culture soil. It also prevents the first container from becoming unstable due to the buoyancy of the water held in the second container. It is preferable that the bottom stones are sterilized in the same way as the inorganic culture soil. However, it is not necessary to place bottom stones in the first container.

[0051] The step of putting the inorganic culture medium into the first container may be carried out before the step of soaking and sterilizing the seeds, after the step of soaking and sterilizing the seeds, or may be carried out in parallel with the step of soaking and sterilizing the seeds.

[0052] In the sowing process, seeds soaked in an aqueous solution containing hydrogen peroxide in the soaking and sterilization process are sown in inorganic culture medium. Seeding into inorganic culture medium can be accomplished by burying the seeds in the inorganic culture medium, or by forming a depression in the surface of the inorganic culture medium and placing the seeds in the depression. In this case, it is preferable to sow Japanese black pine seeds to a depth of 1.5 cm or less from the surface of the inorganic culture medium. Sowing seeds at such a depth tends to germinate toward the surface of the inorganic culture medium, improving the normal seed germination rate and seedling growth rate.

[0053] The seeds sown in the soaking and sowing process germinate and become seedlings. Seedlings refer to seedlings obtained by germinating seeds and are also called own-root seedlings. In this specification, seedlings refer to the state after the seeds have germinated and at least the roots have become visible. Furthermore, hereafter in this specification, when simply referring to "seedlings," it refers to Japanese black pine seedlings. This production method, which involves soaking and sterilizing the seeds for a specified period of time, and sterilizing them with an aqueous solution containing hydrogen peroxide, has a better seed germination rate than conventional methods.

[0054] The average ambient temperature during the period from sowing of black pine seeds in inorganic soil to germination should be 35°C or less, and preferably 30°C or less. It has been known that, in order to germinate seeds, the average ambient temperature during the period from sowing to the first brewing of the seeds should be between 5°C and 35°C. Furthermore, the daily average ambient temperature during this period, rather than the entire period from sowing to germination, should preferably be 35°C or less on every day, more preferably 30°C or less, and even more preferably within the room temperature range. By maintaining such an ambient temperature, the seed germination rate can be improved.

[0055] (contact process) The contacting step is a step of bringing the roots of seedlings germinated from black pine seeds into contact with at least one of the spores and mycelium of Camphor Tree. By bringing the roots of the seedlings into contact with at least one of the spores and mycelium of Camphor Tree in this way, mycorrhizae of Camphor Tree can be formed on the roots of the seedlings.

[0056] At least one of the spores and mycelia of Camphorata is at least one of Camphorata spores, a suspension of Camphorata spores, Camphorata mycelia, or a suspension of Camphorata mycelia. The suspension of Camphorata spores or mycelia is preferably prepared, for example, as a water suspension, based on a known method.

[0057] The spores or mycelia of Camphorata can be obtained by artificially cultivating Camphorata or by harvesting it from natural Camphorata. The mycelia of Camphorata can be obtained from any stage other than the spore stage in the life cycle of Camphorata, but from the viewpoint of the efficiency of mycorrhizal formation, it is preferable to use mycelia from a stage other than the spore stage or the fruiting body stage.

[0058] The contact between the roots of the seedlings and at least one of the spores and mycelia of Camphora japonica is preferably direct, for example, by applying at least one of the spores and mycelia to the roots of the seedlings. Alternatively, a suspension of at least one of the spores and mycelia of Camphora japonica may be infiltrated into the inorganic culture medium around the roots of the seedlings so that the suspension comes into contact with the roots of the seedlings. From the viewpoint of the efficiency of mycorrhizal formation, the above-mentioned direct contact method is preferred.

[0059] The timing and frequency of contact between the roots of the seedlings and at least one of the spores and mycelium of Camphor are not particularly limited, but it is preferable to carry out the contact at least once immediately after the seeds germinate. Regarding "immediately after germination" of the seeds, for example, if the seeds are observed daily after sowing and the contact is carried out within the day when seed germination is first observed, this may be included in the range of "immediately after germination."

[0060] In addition, it is preferable to contact the roots of the seedlings with at least one of the spores and mycelia of Camphor tree multiple times at predetermined intervals. By contacting the roots of the seedlings with at least one of the mycelia and spores of Camphor tree multiple times, it is possible to promote mycorrhizal formation of Camphor tree on the roots of the seedlings.

[0061] When the above-mentioned contact is performed multiple times, the predetermined interval is not particularly limited, and may be, for example, one day or more, one week or more, two weeks or more, four weeks or more, or eight weeks or more. Furthermore, when the contact is performed three or more times, the predetermined intervals for each contact may be the same or different. For example, the contact may be performed at a predetermined interval of one week for the first month after germination, and then at a predetermined interval of four weeks thereafter. From the viewpoint of promoting mycorrhizal formation in Camphor, it is preferable to perform the above-mentioned contact about once a month. To form Camphor mycorrhizal formation, the above-mentioned contact may be performed between March and September, and preferably between June and August.

[0062] (Cultivation process) The cultivation process involves cultivating seedlings in inorganic soil for at least three months after germination. In the case of Japanese black pine, lignification is observed in the stems of seedlings approximately three months after germination. Lignification, also known as lignification, refers to the deposition of lignin in the cell walls of plants. As lignification progresses, the stems of seedlings are observed to change in appearance, for example, from green to brown.

[0063] As seedlings become lignified, their resistance to pests, mold, etc. increases. Conversely, seedlings before lignification do not have sufficient resistance, so reducing the occurrence of pests, mold, etc. as much as possible is important for improving seedling growth rates.

[0064] In the cultivation step, seedlings are cultivated in the inorganic culture medium described above for at least three months after germination. As described above, inorganic culture medium is substantially free of organic fertilizer components, so seedlings can be cultivated while reducing the occurrence of pests, mold, and the like during the period when inorganic culture medium is used. Therefore, the cultivation step of this production method can improve the seedling growth rate during the period when seedlings are less tolerant, at least up to three months after germination, when lignification begins to be observed.

[0065] In the cultivation step, the method for replenishing the water held in the seedling container is not particularly limited, but it is preferable to maintain the water held in the seedling container at a predetermined level or more by adding water to the seedling container. After the initial contact between the roots of the seedling and at least one of the spores or mycelia of Camphorata in the contact step, at least one of the spores or mycelia of Camphorata may remain in the water held in the seedling container. This water is continuously absorbed by the roots of the seedling, and can promote mycorrhizal formation of Camphorata in the seedlings.

[0066] By adding water to the seedling container, the loss of at least one of the spores and mycelium of Camphorata present in the water held in the seedling container can be reduced compared to when the water is replaced. Therefore, replenishing water by adding water promotes the formation of mycorrhizae in Camphorata in the seedlings.

[0067] In the cultivation step, the duration of light irradiation on the seedlings is not particularly limited as long as light irradiation is performed. The light irradiation duration may be continuous without interruption, or a dark period during which light irradiation is not performed may be provided on a daily basis. When a dark period is provided, the duration of the dark period may be, for example, 1 hour / day or less, 2 hours / day or less, 3 hours / day or less, 4 hours / day or less, 5 hours / day or less, or 6 hours / day or less.

[0068] The seedlings may be irradiated with artificial light such as LEDs (light-emitting diodes) or fluorescent lamps, sunlight, or a combination of these. From the viewpoint of ease of irradiation control, it is preferable to irradiate the seedlings with artificially generated light.

[0069] (Replanting process) The transplanting process is a process of transplanting the seedlings after the cultivation process into organic soil. From the perspective of growth efficiency, it is preferable to continue raising the seedlings in organic soil, which is richer in nutrients than inorganic soil, after lignification. In other words, the transplanting process makes it easier to obtain large, mature seedlings. However, the transplanting process is not essential for producing black pine seedlings with rhizosaccharomycorrhizae.

[0070] Organic soil is a soil rich in organic fertilizer components such as nitrogen, phosphorus, and potassium. Such organic soil is useful for further growing seedlings with rhizal mycorrhizae after they have been obtained. Organic soil is preferably sterilized before use. The sterilization method may be the same as that used for inorganic soil.

[0071] Examples of organic soil include Akadama soil, Arakida soil, Kuroboku soil, peat moss, and coco peat. The organic soil may contain one of these or two or more. Bark compost is a good organic soil. The organic soil may also contain inorganic soil such as Kanuma soil or vermiculite.

[0072] In particular, the organic soil preferably has a layer containing Akadama soil, a layer containing Kiryu sand, and a layer containing bark compost and vermiculite. After extensive research, the present inventors have found that such a three-layer organic soil is particularly suitable for raising Japanese black pine seedlings.

[0073] In this way, it is preferable that the organic soil has a multi-layer structure in which multiple types of soil are layered. In this case, the soil to be replanted in the repotting process only needs to have at least one layer containing organic soil, or in other words, it may also contain a layer of inorganic soil.

[0074] 〔summary〕 A method for producing black pine seedlings having Peronospora mycorrhizae according to aspect 1 of the present invention comprises the following steps: a soaking and sterilizing step of soaking black pine seeds in running water for a predetermined period of time and then immersing them in an aqueous solution containing hydrogen peroxide; a sowing step of sowing the seeds in mineral culture soil, the soil pH of which, measured in accordance with JIS Z8802:2011, is between 4 and 7 and is held in a seedling container that holds water so that at least a portion of the seedlings is in contact with the water; a contacting step of contacting the roots of seedlings germinated from the seeds with at least one of Peronospora mycorrhizae; and a cultivation step of cultivating the seedlings in the mineral culture soil for at least three months after germination.

[0075] The production method according to aspect 2 of the present invention may further include a transplanting step in which the seedlings after the cultivation step are transplanted into organic soil having a layer containing Akadama soil, a layer containing Kiryu sand, and a layer containing bark compost and vermiculite, in accordance with aspect 1.

[0076] In the production method of aspect 3 of the present invention, in aspect 1 or 2, it is preferable that in the cultivation step, the amount of water held in the seedling container is maintained at a predetermined amount or more by adding water to the seedling container.

[0077] In the production method according to aspect 4 of the present invention, in any one of aspects 1 to 3, the seeds are preferably sown to a depth of 1.5 cm or less from the surface of the inorganic culture soil in the sowing step.

[0078] In the production method according to aspect 5 of the present invention, in any one of aspects 1 to 4, in the contact step, it is preferable to contact the roots with at least one of the spores and mycelia of the camphor tree multiple times, with each contact being at a predetermined interval.

[0079] A sixth aspect of the present invention relates to the production method of any one of the first to fifth aspects, and it is preferable that in the sowing step, the average atmospheric temperature during the period from the sowing to the germination is set to 30° C. or less.

[0080] [Additional Notes] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Example]

[0081] An embodiment of the present invention will now be described.

[0082] [1. Preliminary study of seed sterilization conditions] First, a preliminary study was conducted on the relationship between the sterilization conditions for Japanese black pine seeds and the germination rate. Two sterilization solutions were tested: a sodium hypochlorite solution with 10% available chlorine (Reference Example 1) and an aqueous solution containing 0.9 w / v% hydrogen peroxide (Reference Example 2). The aqueous solution containing 0.9 w / v% hydrogen peroxide was prepared by diluting hydrogen peroxide (3.00 w / v% hydrogen peroxide solution) to 30% by volume.

[0083] In Reference Example 1, seeds were soaked in running tap water for three days. Then, the seeds were sterilized by immersing them in a sterilizing solution for 10 minutes twice, and then washed by immersing them in a 0.1% by mass Tween 80 aqueous solution for 10 minutes. This set was carried out twice. In Reference Example 2, two conditions were tested: one in which the sterilization and washing set was carried out once (Reference Example 2-1) and one in which it was carried out twice (Reference Example 2-2). A condition in which the sterilization of seeds with a sterilizing solution was omitted (Reference Example 3) was also tested.

[0084] The seeds after these sterilization treatments were sown in a culture medium with a vermiculite:bark compost ratio of 1:1 and watered until the culture medium was completely submerged in water. The culture medium was sterilized by heating before use. 50 seeds were sown in each of Reference Examples 1, 2-1, and 2-2.

[0085] In Reference Example 1, no germination was observed even two months after sowing. On the other hand, in Reference Example 2-2, germination was observed in almost all (80 to 100%) of the seeds one month after sowing. Also, in Reference Example 2-1, germination of 70 to 80% of the seeds was observed one month after sowing. Note that in Reference Example 3, germination of 30 to 60% of the seeds was observed one month after sowing.

[0086] The above results show that when sterilization was performed using a solution containing hydrogen peroxide, as in the soaking and sterilization process of this production method, the germination rate was higher than when sterilization was omitted.It was also shown that the germination rate was significantly higher than when sterilization was performed using a sodium hypochlorite solution.

[0087] In conventional methods, sodium hypochlorite solution is sometimes used, but in order to sterilize without reducing the germination rate, it is thought that very strict adjustment of conditions such as sodium hypochlorite concentration or soaking time is required. On the other hand, if an aqueous solution containing hydrogen peroxide is used as in the present production method, it is thought that such condition settings can be set relatively widely and easily.

[0088] [2. Preliminary study of germination conditions] (2-1. Consideration of ambient temperature) The effect of ambient temperature on germination rates during the period leading up to seed germination was examined. In the summer (end of July), organic soil A or organic soil B, each with the following composition, was placed in a planter, and 50 seeds were sown in each. The values ​​in the composition of each organic soil indicate mass ratios.

[0089] Organic soil A: a 1:1 mixture of bark compost and vermiculite Organic soil B: The first layer on the surface is a mixture of Kiryu sand and Akadama soil (1.3:3.8), the second layer below that is Kanuma soil, the third layer below that is a mixture of bark compost and vermiculite (1.5:1.5), and the fourth layer at the bottom is gravel. The ratio of the first layer: the second layer: the third layer: the fourth layer = 5.1:1.3:3:0.6. The planter containing organic soil A was placed indoors, near a window where it was exposed to direct sunlight and had a high ambient temperature (Reference Example 4). The planter containing organic soil B was placed away from a window where it was less exposed to direct sunlight and had a relatively low ambient temperature (Reference Example 5). Figure 4 shows the ambient temperature (°C) and humidity (%) for each day from the day the seeds were sown in Reference Example 4. Figure 5 shows the ambient temperature (°C) and humidity (%) for each day from the day the seeds were sown in Reference Example 5. In Figures 4 and 5, the dashed line labeled "germination limit temperature" indicates the temperature of 35°C, at which germination of black pine seeds is generally known to be inhibited.

[0090] As shown in Figure 4, in Reference Example 4, although the germination limit temperature of 35°C was not reached, the temperature exceeded 30°C for a long period of time, and there were several days when the daily average atmospheric temperature exceeded 30°C. On the other hand, as shown in Figure 5, in Reference Example 5, the atmospheric temperature rarely exceeded 30°C. There were also no days when the average atmospheric temperature exceeded 30°C.

[0091] In Reference Example 4, no germination was observed, resulting in a germination rate of 0%. On the other hand, in Reference Example 5, germination was observed in 41 out of 50 seeds, resulting in a germination rate of 93%. These results demonstrate that an average atmospheric temperature of 30°C or less is important for improving the germination rate of seeds.

[0092] (2-2. Consideration of sowing depth) The effect of seed sowing depth on germination rate was examined. Kanuma soil, an inorganic soil, was placed in a water-holding seedling container. Seeds that had been soaked and sterilized were sown in the Kanuma soil. 100 seeds were sown at a depth of 1.5 cm or less from the surface of the Kanuma soil (Reference Example 6), and 100 seeds were sown at a depth of 2 cm to 3 cm from the surface of the Kanuma soil (Reference Example 7).

[0093] 110 days after sowing, 92 out of 100 seeds germinated in Reference Example 6, for a germination rate of 92%. In Reference Example 7, 56 out of 100 seeds germinated, for a germination rate of 56%. These results showed that sowing seeds at a depth of 1.5 cm or less improved the germination rate.

[0094] The seedling growth rate of the germinated seedlings in Reference Examples 6 and 7 was also examined. After germination, the number of seedlings that could be evaluated as having taken root in the inorganic soil without suffering from leaf wither or root rot was confirmed. This confirmation was carried out 130 days after germination in Reference Example 6 and 110 days after germination in Reference Example 7.

[0095] In Reference Example 6, 86 out of 92 germinated seedlings were evaluated as having taken root, resulting in a seedling growth rate of 94%. In Reference Example 7, 46 out of 56 germinated seedlings were evaluated as having taken root, resulting in a seedling growth rate of 82%. These results demonstrate that sowing seeds to a depth of 1.5 cm or less improves not only the germination rate but also the seedling growth rate.

[0096] [3. Trial of this production method] Seedling production was attempted using this production method (invention example) and a conventional method (comparison example). In the invention example, sterilized bottom gravel and Kanuma soil were first placed in the first container of the seedling raising container, as shown in Figure 1. Next, the seeds were sterilized by the same seed soaking and sterilization process as in Reference Example 2-2 above. The sterilized seeds were then sown in the Kanuma soil placed in the first container to a depth of 1.5 cm or less from the surface, and the sowing process was carried out. Of the 100 seeds sown, 92 germinated, resulting in a germination rate of 92%.

[0097] The sown seeds were cultivated in Kanuma soil until 161 days after germination. At 161 days after germination, 86 out of 92 germinated seedlings were evaluated as having taken root in Kanuma soil, giving a seedling growth rate of 93%.

[0098] On the other hand, in the comparative example, 50 seedlings germinated by the method shown in Reference Example 2-2 above were transplanted into a seedling raising box 30 days after germination. The seedling raising box has a water passage hole, but differs from the seedling raising container according to one embodiment of the present invention in that it does not have a second container for retaining water. The seedling raising box has a water passage hole to drain excess sprayed water.

[0099] The condition of the seedlings was observed 10 days after transplanting into the seedling raising boxes. Approximately 60% of the seedlings had fallen over and begun to wither. The seedlings of these comparative examples had thinner roots than the seedlings obtained by the present invention, and are therefore more likely to fall over and wither.

[0100] Next, these seedlings that had fallen over and were beginning to wither were thinned out, and the location of the seedling boxes was changed from a location indoors that was not exposed to much sunlight to a window where sunlight was available. After thinning out, 30 seedlings remained. Seedlings were continued to be raised in this condition, and the condition of the seedlings was observed three months after thinning out. The number of seedlings remaining that had not suffered from leaf wither or root rot was 6. Of the 50 seedlings that germinated, only 6 remained, giving a seedling survival rate of 12%.

[0101] Thus, while the comparative example using the conventional method had a very low seedling growth rate of 12%, the example of the present invention using the present production method achieved an excellent result of 93%. In other words, compared with the conventional method, the present production method was shown to be more suitable for producing black pine seedlings with rhizal mycorrhizae.

[0102] [4. Consideration of organic soil used in the transplanting process] In the case of raising Japanese black pine seedlings, we investigated the difference in seedling growth rate depending on the type of organic fertilization medium into which the seedlings were transplanted during the transplanting process. Two-year-old Japanese black pine seedlings with rhizal roots (hereinafter referred to as "two-year-old seedlings") that had been germinating for two years were raised in planters. The above-mentioned organic fertilization medium A and organic fertilization medium B were used for the study.

[0103] Indoors, 400 two-year-old seedlings were grown in organic fertilization medium A for 60 days. Outdoors in July, 25 two-year-old seedlings were grown in organic fertilization medium B for 240 days. As a result, 336 of the 400 seedlings grown in organic fertilization medium A suffered leaf withering, with a survival rate of 16% after cultivation. On the other hand, when organic fertilization medium B was used, only 5 of the 25 seedlings suffered leaf withering, with a survival rate of 80%.

[0104] The results of this study showed that organic soil B is suitable as an organic soil for transplanting seedlings after the cultivation process. [Industrial Applicability]

[0105] The present invention can be used, for example, in the cultivation of Japanese black pine and the artificial cultivation of Chinese elm fruiting bodies. [Explanation of symbols]

[0106] 10, 20 Seedling container 11, 21 1st container 12, 22 Water passage holes 13, 23 Second container 24 Lid 25 Ventilation S Inorganic soil W water

Claims

1. A soaking and sterilizing process in which black pine seeds are soaked in running water for a predetermined period of time and then soaked in an aqueous solution containing hydrogen peroxide; A sowing step of sowing the seeds in inorganic soil having a soil pH of 4 or more and 7 or less as measured in accordance with JIS Z8802:2011, which is held in a seedling container holding water so that at least a portion of the soil is in contact with the water; A contacting step of contacting roots of seedlings germinated from the seeds with at least one of spores and mycelia of Camphor; and a cultivation step of cultivating the seedlings in the inorganic culture soil for at least three months after germination.

2. 2. The production method according to claim 1, further comprising a transplanting step of transplanting the seedlings after the cultivation step into organic soil having a layer containing Akadama soil, a layer containing Kiryu sand, and a layer containing bark compost and vermiculite.

3. 2. The production method according to claim 1, wherein in the cultivation step, the amount of water held in the seedling container is maintained at a predetermined amount or more by adding water to the seedling container.

4. The method according to claim 1 , wherein in the sowing step, the seeds are sown to a depth of 1.5 cm or less from the surface of the inorganic soil.

5. The production method according to claim 1, wherein in the contacting step, the roots are contacted with at least one of the spores and mycelia of the camphor tree multiple times at predetermined intervals.

6. The method according to claim 1 , wherein in the sowing step, an average atmospheric temperature during the period from the sowing to the germination is set to 30° C. or less.

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