A composite substrate, device, and method for enhancing ectomycorrhizal symbiosis

Abstract

This invention discloses a combined substrate, device, and method for enhancing ectomycorrhizal symbiosis. The combined substrate comprises a fertilizer-attracting substrate, a fertilizer-isolating substrate, and a fungal-attracting substrate. The fertilizer-attracting substrate layer is composed of river sand and vermiculite; the fertilizer-isolating substrate layer is quartz sand; and the fungal-attracting substrate layer is vermiculite. The particle size of both the fertilizer-attracting and fungal-attracting substrate layers is smaller than that of the fertilizer-isolating substrate layer. The volume ratio of the fertilizer-attracting, fertilizer-isolating, and fungal-attracting substrate layers is (5-7):(1-2):(2-3). This invention addresses the lack of strictness in the symbiosis between ectomycorrhizal fungi and host trees. Based on the symbiotic characteristic that ectomycorrhizal fungi primarily assist host trees in absorbing mineral nutrients, it provides a simple, efficient, and highly feasible method that allows fungi to quickly adapt to the actual forest environment in a short period and maintain a long-term, non-degrading symbiotic relationship with the host trees. Later, only routine forest management of the ectomycorrhizae is required.

Description

Technical Field

[0001] This invention relates to the field of microbial cultivation, and more specifically, to a combined substrate, apparatus, and method for enhancing ectomycorrhizal symbiosis. Background Technology

[0002] Ectomycorrhizal fungi are a class of fungi that can establish ectomycorrhizal relationships with forest trees (such as pine and Fagaceae trees). Ectomycorrhizal fungi not only assist host trees in absorbing mineral nutrients but also increase their resistance to adverse conditions. However, most ectomycorrhizal fungi do not form obligate symbiotic relationships with forest trees, and establishing a stable symbiotic relationship between ectomycorrhizal fungi and forest seedlings requires a considerable amount of time.

[0003] Currently, traditional methods for establishing symbiotic relationships between ectomycorrhizal fungi and forest trees mostly involve first propagating artificially culturable ectomycorrhizal fungal hyphae into ectomycorrhizal fungal inoculants, then inoculating the propagated inoculants onto the root system of host forest trees, and finally obtaining a symbiotic relationship between the ectomycorrhizal fungi and the host forest trees through long-term symbiotic cultivation. For example, existing technologies such as a method for establishing symbiotic relationships between regenerated chestnut plants and ectomycorrhizal fungi (CN110367102A), a method for establishing symbiotic relationships between ectomycorrhizal fungi and pine plants (CN106171515B), a method for synthesizing ectomycorrhizae (CN105917972A), and a method for optimizing seedling cultivation using ectomycorrhizal fungi (CN105861315B) are mostly supplements to traditional methods for establishing symbiotic relationships between ectomycorrhizal fungi and forest trees, without addressing the enhanced management after the establishment of the ectomycorrhizal symbiosis.

[0004] Therefore, current technologies related to the co-cultivation of ectomycorrhizal fungi and their hosts mostly involve extensive seedling nutrient management, without considering the symbiotic characteristics of ectomycorrhizal fungi in seedling management. Furthermore, once a symbiotic relationship is established between the ectomycorrhizal fungi and the host seedling, there are no corresponding seedling management measures to specifically enhance the symbiotic relationship, taking into account the characteristics of ectomycorrhizal symbiosis. Summary of the Invention

[0005] In order to overcome the shortcomings of existing methods for cultivating ectomycorrhizal fungi in symbiosis with host trees, and to address the lack of forest land management methods that combine ectomycorrhizal symbiotic characteristics to enhance their symbiotic relationship with host trees, this invention provides a combined substrate, device, and method for strengthening ectomycorrhizal symbiosis.

[0006] The first objective of this invention is to provide a composite substrate that enhances ectomycorrhizal symbiosis.

[0007] A second objective of this invention is to provide the application of the above-described composite substrate in the cultivation of ectomycorrhizae.

[0008] A third objective of this invention is to provide a device for enhancing ectomycorrhizal symbiosis.

[0009] A fourth objective of this invention is to provide the application of the above-described apparatus in the cultivation of ectomycorrhizae.

[0010] The fifth objective of this invention is to provide a method for enhancing ectomycorrhizal symbiosis.

[0011] To achieve the above objectives, the present invention is implemented through the following solution:

[0012] This invention uses a small-pore fertilizer-attracting substrate to distribute plant nutrient solution evenly through capillary action, a large-pore fertilizer-isolating substrate to block capillary action and isolate the plant nutrient solution, and a large-pore fungal-attracting substrate to provide sufficient oxygen to attract fungal hyphae to grow and colonize.

[0013] A composite substrate for enhancing ectomycorrhizal symbiosis, characterized in that it comprises a fertilizer-introducing substrate layer, a fertilizer-isolating substrate layer, and an introducing substrate layer; the fertilizer-introducing substrate layer is composed of river sand and vermiculite; the fertilizer-isolating substrate layer is quartz sand; and the introducing substrate layer is vermiculite.

[0014] The particle size of both the fertilizer-introducing matrix layer and the bacteria-introducing matrix layer is smaller than that of the fertilizer-isolating matrix layer.

[0015] The volume ratio of the fertilizer-introducing substrate layer, the fertilizer-isolating substrate layer, and the bacteria-introducing substrate layer is (5-7):(1-2):(2-3).

[0016] Preferably, the volume ratio of the fertilizer-introducing substrate layer, the fertilizer-isolating substrate layer, and the bacteria-introducing substrate layer is 7:1:2.

[0017] Preferably, the particle size of the fertilizer-introducing substrate layer is 1.8 mm to 2.2 mm; the particle size of the fertilizer-isolating substrate layer is 4.8 mm to 5.2 mm; and the particle size of the bacteria-introducing substrate layer is 2.8 mm to 3.2 mm.

[0018] More preferably, the fertilizer substrate layer is composed of river sand of 1.8 mm to 2.2 mm and vermiculite of 1.8 mm to 2.2 mm.

[0019] More preferably, the fertilizer substrate layer is composed of river sand of 1.8mm to 2.2mm and vermiculite of 1.8mm to 2.2mm in a volume ratio of 1:1.

[0020] More preferably, the fertilizer substrate layer is composed of river sand with an average particle size of 2 mm and vermiculite with an average particle size of 2 mm in a volume ratio of 1:1.

[0021] More preferably, the fertilizer-separating substrate layer is quartz sand with an average particle size of 4 mm.

[0022] More preferably, the initiation substrate layer is vermiculite with an average particle size of 3 mm.

[0023] Preferably, the fertilizer-isolating substrate layer is disposed between the fertilizer-introducing substrate layer and the bacteria-introducing substrate layer.

[0024] More preferably, the fertilizer-introducing substrate layer, the fertilizer-isolating substrate layer, and the bacteria-introducing substrate layer are stacked sequentially.

[0025] Most preferably, the composite matrix comprises a fertilizer-introducing matrix layer, a fertilizer-isolating matrix layer, and a microbial-introducing matrix layer. The fertilizer-introducing matrix is ​​composed of river sand with an average particle size of 2 mm and vermiculite with an average particle size of 2 mm in a volume ratio of 1:1. The fertilizer-isolating matrix is ​​quartz sand with an average particle size of 4 mm. The microbial-introducing matrix is ​​vermiculite with an average particle size of 3 mm. The volume ratio of the fertilizer-introducing matrix, the fertilizer-isolating matrix, and the microbial-introducing matrix is ​​7:1:2. The fertilizer-introducing matrix layer, the fertilizer-isolating matrix layer, and the microbial-introducing matrix layer are stacked sequentially.

[0026] The application of any of the above-mentioned combined substrates in the cultivation of ectomycorrhizae should also be within the scope of protection of this invention.

[0027] This invention provides a nutrient tube that intercepts the root system of host trees that coexist with ectomycorrhizal fungi, allowing only the mycelia of the ectomycorrhizal fungi to enter the tube and absorb nutrients. Furthermore, the nutrient tube, located away from the root end, guides the plant nutrient solution evenly into the nutrient-introducing substrate from bottom to top through capillary action. The middle section has an isolation design to block capillary action, while the root-contacting end is filled with a large-pore introducing substrate. This nutrient tube design introduces aerobic ectomycorrhizal fungal mycelia without causing nutrients to overflow from the nutrient tube to the root end.

[0028] A device for enhancing ectomycorrhizal symbiosis includes a nutrient tube, one end of which is provided with a first valve, and the opening at the other end is provided with a first root isolation net.

[0029] A second valve is provided on the side wall of the nutrient tube;

[0030] The nutrient tube is loaded with any of the above-mentioned combined substrates, wherein the end of the fertilizer-introducing substrate layer adjacent to the first valve does not extend beyond the second valve, the end of the bacteria-introducing substrate layer adjacent to the first root isolation net, and the fertilizer-isolating substrate layer is located between the fertilizer-introducing substrate layer and the bacteria-introducing substrate layer.

[0031] Preferably, the ratio of the distance from the second valve to the first valve to the distance from the second valve to the first root system isolation net is 1:(0.5~2).

[0032] More preferably, the ratio of the distance from the second valve to the first valve to the distance from the second valve to the first root system isolation net is 1:1.

[0033] Preferably, the first root isolation net is at an angle of 40° to 50° to the axial direction of the nutrient tube.

[0034] More preferably, the first root isolation net is at a 45° angle to the axial direction of the nutrient tube.

[0035] Preferably, the first root system isolation mesh is a filter mesh with a pore size of 20μm to 45μm.

[0036] More preferably, the first root system isolation mesh is a filter mesh with a pore size of 45 μm.

[0037] The present invention does not have any special limitation on the material of the first root system isolation net. Conventional filter nets, including but not limited to nylon net, plastic net, nylon net, stainless steel net and iron wire net, can achieve the purpose of the present invention.

[0038] Preferably, the first root system isolation mesh is a nylon mesh with a pore size of 20μm to 45μm.

[0039] More preferably, the first root system isolation mesh is a nylon mesh with a pore size of 45μm.

[0040] Preferably, a fertilizer-separating filter screen is provided between the fertilizer-introducing substrate and the fertilizer-separating substrate to separate the fertilizer-introducing substrate and the fertilizer-separating substrate.

[0041] More preferably, the pore size of the fertilizer-isolating filter is smaller than the particle size of the fertilizer-isolating substrate, so that the fertilizer-isolating substrate cannot fall into the fertilizer-introducing substrate through the fertilizer-isolating filter.

[0042] More preferably, the fertilizer-separating filter screen is a filter screen with a pore size of 0.3 mm to 1 mm.

[0043] More preferably, the fertilizer-separating filter screen is a filter screen with a pore size of 1 mm.

[0044] This invention does not have any special limitations on the material of the fertilizer filter screen. Conventional filter screens such as nylon mesh and stainless steel mesh can achieve the purpose of this invention.

[0045] Preferably, the fertilizer filter screen is a nylon mesh with a pore size of 0.3mm to 1mm.

[0046] More preferably, the fertilizer filter screen is a nylon mesh with a pore size of 1 mm.

[0047] Preferably, a second root isolation net is provided between the fertilizer-isolating substrate and the inoculum-introducing substrate to prevent the plant roots of ectomycorrhizae from coming into contact with the fertilizer-isolating substrate through the inoculum-introducing substrate.

[0048] More preferably, the second root system isolation mesh is a filter with a pore size of 20μm to 45μm.

[0049] More preferably, the second root system isolation mesh is a filter with a pore size of 45 μm.

[0050] The present invention does not have any special limitation on the material of the second root system isolation net. Conventional filter nets, including but not limited to nylon net, plastic net, nylon net, stainless steel net and iron wire net, can achieve the purpose of the present invention.

[0051] Preferably, the second root system isolation mesh is a nylon mesh with a pore size of 20μm to 45μm.

[0052] More preferably, the second root system isolation net is a nylon net with a pore size of 45μm.

[0053] Preferably, the plant nutrient solution is a modified Hoagland nutrient solution.

[0054] More preferably, the nitrogen concentration in the modified Hogrange nutrient solution is 10 mM to 20 mM.

[0055] More preferably, the nitrogen concentration in the modified Hogrange nutrient solution is 15 mM.

[0056] More preferably, the phosphorus concentration in the modified Hogrange nutrient solution is 1 mM to 4 mM.

[0057] More preferably, the phosphorus concentration in the modified Hogrange nutrient solution is 2 mM.

[0058] Most preferably, the nitrogen concentration in the modified Hogland nutrient solution is 15 mM and the phosphorus concentration is 2 mM.

[0059] After the topsoil is removed from bare forest grounds, soil nutrients become scarce. Furthermore, the sowing of non-mycorrhizal Chenopodiaceae herbaceous plants can further deplete the rhizosphere mineral nutrients of the host trees. To obtain these mineral nutrients, the host trees must strengthen their symbiosis with ectomycorrhizal fungi, thereby increasing their dependence on these fungi. This increases the colonization rate of ectomycorrhizal fungi on the host tree roots and expands their ecological niche under actual forest conditions, thus enhancing the symbiosis between ectomycorrhizal fungi and the host trees, and the corresponding mycorrhizal symbiotic effects.

[0060] The application of any of the above-mentioned devices in the cultivation of ectomycorrhizae should also be within the scope of protection of this invention.

[0061] A method for enhancing ectomycorrhizal symbiosis, using any of the above-described combined substrates or any of the above-described devices to enhance the cultivation of ectomycorrhizal symbiosis.

[0062] Preferably, any of the above-described devices are used to cultivate ectomycorrhizae.

[0063] More preferably, it includes the following steps:

[0064] Trees with ectomycorrhizal symbiosis are planted in a plot of land where no mycorrhizal plants are grown; the first root isolation net of any of the above devices is placed near the ectomycorrhizae, plant nutrient solution is added through the second valve, and waste liquid is discharged through the first valve.

[0065] More preferably, the plot is further enclosed with a polyvinyl chloride sheet, leaving only one drainage outlet.

[0066] More preferably, the method also includes land preparation and clearing of the plot; the clearing includes removing dead branches and leaves and the humus layer from the surface.

[0067] More preferably, the method further includes dividing the plot into rows and digging ditches, the ditches being located between the rows and around the plot and connected to drainage outlets for drainage.

[0068] In a further preferred embodiment, the height difference between the top of the ridge and the bottom of the furrow is 40 cm.

[0069] More preferably, the cross-section of the ridge is an isosceles trapezoid with an upper base of 0.8m, a lower base of 1.6m, and a height of 0.4m.

[0070] More preferably, a burrow is constructed from bottom to top at an angle of 45 degrees to the horizontal at a distance of 15 cm from the bottom of the furrow, and the nutrient tube is placed in the burrow.

[0071] More preferably, the planting interval of the trees is 1m to 3m.

[0072] More preferably, the plot is also sown with non-mycorrhizal plants.

[0073] More preferably, the non-mycorrhizal plant is a plant of the Chenopodiaceae family. The Chenopodiaceae plants include, but are not limited to, root beets and spinach.

[0074] Most preferably, the non-mycorrhizal plant is spinach.

[0075] More preferably, the sowing density of the non-mycorrhizal plants is 30 plants / m². 2 .

[0076] After a period of cultivation, soil seepage and other factors cause some waste liquid to remain in the nutrient tubes, hindering root and fungal aeration. Therefore, it needs to be discharged through the first valve. At the same time, the nutrient solution in the nutrient tubes gradually depletes, requiring timely replenishment of fresh plant nutrient solution through the second valve.

[0077] More preferably, once the plant nutrient solution is depleted, the waste liquid is first discharged through the first valve, and then new plant nutrient solution is added through the second valve.

[0078] More preferably, waste liquid is discharged periodically through the first valve, and then new plant nutrient solution is added through the second valve.

[0079] More preferably, the periodicity is every 20 to 30 days.

[0080] Most preferably, the period is every 30 days.

[0081] More preferably, the plant nutrient solution is a modified Hoagland nutrient solution.

[0082] More preferably, the nitrogen concentration in the modified Hogrange nutrient solution is 10 mM to 20 mM.

[0083] More preferably, the nitrogen concentration in the modified Hogland nutrient solution is 15 mM.

[0084] More preferably, the phosphorus concentration in the modified Hogrange nutrient solution is 1 mM to 4 mM.

[0085] More preferably, the phosphorus concentration in the modified Hogrange nutrient solution is 2 mM.

[0086] Most preferably, the nitrogen concentration in the modified Hogland nutrient solution is 15 mM and the phosphorus concentration is 2 mM.

[0087] Specifically, the method for adding plant nutrient solution is as follows: add plant nutrient solution through the second valve until the liquid overflows from the second valve.

[0088] Preferably, the ectomycorrhizae are a symbiotic relationship between the roots of *Pyracantha fortuneana* and the ectomycorrhizal fungi of *Lycoperdon perlatum*.

[0089] More preferably, the method for establishing ectomycorrhizae includes the following steps: preparing one-year-old red cones, removing the original ectomycorrhizae from their roots, then inoculating the roots with a mycelium cake cultured on Modified Melin-Norkrans solid medium with colored puffball mycelium, using sterilized vermiculite and sterilized quartz sand in a 1:1 volume ratio as the seedling substrate, and cultivating them in seedling pots for a total of 6 months.

[0090] Compared with the prior art, the present invention has the following beneficial effects:

[0091] This invention addresses the lack of strictness in the symbiotic relationship between ectomycorrhizal fungi and host trees. Based on the symbiotic characteristic that ectomycorrhizal fungi mainly assist host trees in absorbing mineral nutrients, this invention provides a simple, efficient, and feasible method that allows fungi to quickly adapt to the actual forest growth environment in a short period of time and maintain a long-term, non-degenerative symbiotic relationship with host trees. In the later stages, only routine forest management is required for the trees with ectomycorrhizal fungi symbiosis. Attached Figure Description

[0092] Figure 1 This is a schematic diagram of the nutrient tube of the present invention; 1-nutrient tube; 101-first valve; 102-first root isolation net; 103-second valve.

[0093] Figure 2 This is a schematic diagram of the nutrient pipes arranged on the side of the ridge according to the present invention; 1-nutrient pipe; 101-first valve; 102-first root isolation net; 103-second valve; 2-combined substrate; 201-fertilizer-introducing substrate; 202-fertilizer-isolating substrate; 203-bacterial-introducing substrate; 204-fertilizer-isolating filter; 205-second root isolation net; 5-ridge.

[0094] Figure 3 A schematic diagram of the plot setup for this invention; 1-nutrient pipe; 3-polyvinyl chloride board; 4-drainage outlet; 5-ridge; 6-ditch; 7-spinach.

[0095] Figure 4 The images show the growth of the ectomycorrhizal fungus *Calvatia variegata* in a nutrient tube; A shows the nutrient substrate and mycelium intertwined and clustered together in the nutrient tube; B shows the nutrient substrate remaining attached to the mycelium even after being removed from the nutrient tube; C shows the nutrient substrate connected to the mycelium.

[0096] Figure 5 The growth of the ectomycorrhizal fungus *Callotus variegata* hyphae in the soil surrounding the roots of *Pyracantha fortuneana* in contact with the nutrient tube. Detailed Implementation

[0097] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0098] Example 1: Combined substrate for enhanced ectomycorrhizal symbiosis

[0099] The combined matrix provided by this invention comprises three components: fertilizer-introducing matrix, fertilizer-isolating matrix, and bacteria-introducing matrix.

[0100] 1. Determination of raw materials

[0101] (1) Candidate raw materials

[0102] Candidate raw materials for fertilizer substrate include: river sand with an average particle size of 2 mm (particle size 1.8 mm to 2.2 mm), vermiculite with an average particle size of 2 mm (particle size 1.8 mm to 2.2 mm), and ceramsite with an average particle size of 2 mm (particle size 1.8 mm to 2.2 mm).

[0103] Candidate raw materials for fertilizer-separating substrates include: quartz sand with an average particle size of 4 mm (particle size 3.8 mm to 4.2 mm) and glass beads with an average particle size of 4 mm (particle size 3.8 mm to 4.2 mm).

[0104] Candidate raw materials for the initiation substrate include: vermiculite with an average particle size of 3 mm (particle size 2.8 mm to 3.2 mm) and perlite with an average particle size of 3 mm (particle size 2.8 mm to 3.2 mm).

[0105] (2) Screening criteria

[0106] The particle size of the fertilizer-introducing substrate, fertilizer-isolating substrate, and bacteria-introducing substrate must be stable.

[0107] The fertilizer-attracting substrate needs to have a capillary effect to attract the culture medium and disperse it evenly; the fertilizer-blocking substrate can block the capillary effect of the fertilizer-attracting substrate and retain the culture medium in the fertilizer-attracting substrate; the fungal-attracting substrate needs to provide sufficient oxygen to guide the growth of ectomycorrhizal fungi.

[0108] (3) Results

[0109] Perlite, due to its brittle nature, cannot be used as a target matrix. Considering both the wide availability and price of raw materials, glass beads and ceramsite of specified sizes are relatively expensive and are no longer considered as a target matrix.

[0110] In summary, the raw materials for the fertilizer-introducing substrate are determined to be river sand with an average particle size of 2 mm and vermiculite with an average particle size of 2 mm (volume ratio 1:1), the raw material for the fertilizer-separating substrate is quartz sand with an average particle size of 4 mm, and the raw material for the bacteria-introducing substrate is vermiculite with an average particle size of 3 mm.

[0111] All fertilizer-introducing substrates, fertilizer-isolating substrates, and bacteria-introducing substrates are sterilized before use.

[0112] 2. Determination of the proportions and combinations

[0113] (1) Proportion

[0114] After sterilizing all the fertilizer-introducing substrate, fertilizer-isolating substrate, and bacteria-introducing substrate selected in the previous step, combine them according to different volume ratios, as follows:

[0115] Fertilizer substrate: fertilizer barrier substrate: bacteria introduction substrate = 5:2:3;

[0116] Fertilizer substrate: fertilizer barrier substrate: bacteria introduction substrate = 6:1:3;

[0117] Fertilizer substrate: fertilizer barrier substrate: bacteria substrate = 7:1:2.

[0118] (2) Combination method

[0119] The combination methods of each matrix are as follows:

[0120] From bottom to top, the substrate consists of a fertilizer-introducing substrate, a fertilizer-isolating substrate, and a bacteria-introducing substrate. The fertilizer-introducing substrate and the fertilizer-isolating substrate are separated by a filter screen with a pore size of 0.5 mm. The fertilizer-isolating substrate and the bacteria-introducing substrate are separated by a filter screen with a pore size of 45 μm.

[0121] (3) Results

[0122] The ratio of the three substrates was determined by observing and measuring the mycelial density of ectomycorrhizal fungi that entered the fertilizer substrate.

[0123] Since the hyphae of ectomycorrhizal fungi enter the inoculum substrate in a disordered manner, by comparing substrate combinations with different volume ratios, it was found that when the volume ratio of the inoculum substrate and the fertilizer barrier substrate was increased, the hyphae could not come into contact with the fertilizer inoculum substrate as soon as possible, resulting in a low hyphae density in the fertilizer inoculum substrate.

[0124] The optimal volume ratio of the combined substrate was finally determined to be 7:1:2, where fertilizer-introducing substrate : fertilizer-isolating substrate : bacteria-introducing substrate.

[0125] Example 2: Nutrient tubes for enhanced ectomycorrhizal symbiosis

[0126] This invention provides a nutrient tube 1 for enhancing ectomycorrhizal symbiosis, the structure of which is as follows: Figure 1 As shown.

[0127] One end of the nutrient tube 1 is closed and equipped with a first valve 101 for discharging waste liquid from the tube; the other end is a 45° beveled opening and equipped with a filter screen with a pore size of 45μm as a first root isolation net 102 to prevent the host roots from extending into the tube. The first root isolation net 102 used here is a nylon mesh, but it can also be replaced with a plastic mesh, nylon mesh, stainless steel mesh or wire mesh with the same pore size; a second valve 103 is provided on the side wall of the nutrient tube 1 for adding plant nutrient solution to the tube. The ratio of the distance from the second valve 103 to the first valve 101 to the distance from the second valve 103 to the first root isolation net 102 is 1:1.

[0128] like Figure 2 As shown, the nutrient tube 1 is loaded with the combined substrate 2 of Example 1. The fertilizer-introducing substrate layer 201 is adjacent to the first valve 101 and does not extend beyond the second valve 103. The bacteria-introducing substrate layer 203 is adjacent to the first root isolation net 102. The fertilizer-isolating substrate layer 202 is located between the fertilizer-introducing substrate layer 201 and the bacteria-introducing substrate layer 203. The fertilizer-introducing substrate layer 201 and the fertilizer-isolating substrate layer 202 are separated by a fertilizer-isolating filter 204 with a pore size of 1 mm. The fertilizer-isolating filter 204 used here is a nylon mesh, which can be replaced with a stainless steel mesh with the same pore size. The fertilizer-isolating substrate layer 202 and the bacteria-introducing substrate layer 203 are separated by a second root isolation net 205 with a nylon mesh with a pore size of 45 μm. The second root isolation net 205 used here is a nylon mesh, which can also be replaced with a plastic mesh, nylon mesh, stainless steel mesh or wire mesh with the same pore size.

[0129] Example 3: Method for Enhancing Ectomycorrhizal Symbiosis

[0130] I. Operating Procedures

[0131] 1. Establishment of a symbiotic system

[0132] Prepare one-year-old Castanopsis hystrix seedlings. Use conventional methods to wash the roots and remove fine roots to maximize the removal of residual ectomycorrhizae in the seedlings. Then, inoculate the roots of the seedlings with a pure culture of the ectomycorrhizal fungus Pisolithus tinctorius on MMN (Modified Melin-Norkrans) solid medium. Use sterilized vermiculite and sterilized quartz sand in a 1:1 volume ratio as the seedling substrate. Cultivate the seedlings in seedling pots for 6 months to establish an ectomycorrhizal symbiotic system between the ectomycorrhizal fungus Pisolithus tinctorius and Castanopsis hystrix.

[0133] 2. Land preparation

[0134] Select a 10×10m bare area under the forest canopy suitable for the growth of *Pinus thunbergii*.

[0135] like Figure 3 As shown, in order to create a relatively independent growing environment for the seedlings of the ectomycorrhizal symbiotic system of colored puffball and red cone and to isolate them from the interference of external water and fertilizer conditions, a 50cm deep polyvinyl chloride board 3 was used to seal the exposed plot around the perimeter, and a 30cm wide drainage outlet 4 was left in one corner.

[0136] Conventional methods were used for land preparation, removing dead branches, fallen leaves, and the humus layer from the surface.

[0137] After land preparation, the land is divided into ridges and trenches are dug. A total of 3 ridges 5, each 8m long, are set up. The cross-section of ridge 5 is an isosceles trapezoid with an upper base of 0.8m × a lower base of 1.6m × a height of 0.4m. A trench 6, 10m long, 1.3m wide, and 0.4m deep, is dug between each ridge 5. The height difference between the top of the ridge and the bottom of the trench is 40cm. The trenches are connected to the drainage outlet 4 to ensure that excess water after irrigation and rainfall flows through the trench 6 to the drainage outlet 4 and is discharged from the plot. No water is stored in the ridges and trenches.

[0138] In large-scale ectomycorrhizal symbiotic systems, to achieve the same isolation effect, a relatively clean, bare area that is not easily disturbed by surrounding water and fertilizer can be prepared and enclosed with PVC boards or other non-corrosive boards that can isolate water and fertilizer, leaving drainage outlets of appropriate size. Then, similar methods can be used to prepare the land, divide it into rows, and dig ditches.

[0139] 3. Transplanting

[0140] Plant the red pine seedlings that have already established a symbiotic relationship with the ectomycorrhizal fungus *Calvatia spp.* in the seedling pots in the middle of ridge 5, with 6 seedlings planted per ridge and the seedlings on the same ridge spaced 1.2m apart from each other.

[0141] 4. Installation of nutrient tubes

[0142] To prevent the nutrient tube 1 from contacting the bottom of the furrow, a cylindrical hole with a diameter of 4cm is dug from bottom to top at a 45-degree angle to the horizontal line on the side wall of the furrow, 15cm from the bottom of the furrow, reaching the middle of the furrow. The nutrient tube 1 of Example 2 is inserted into the hole with the end equipped with the first root isolation net 102 as the near root end, so that the sloping surface of the opening is perpendicular to the horizon and contacts the roots of the red cone seedling; the second valve 103 and the first valve 101 on the side wall are kept exposed outside the soil.

[0143] 5. Add fertilizer

[0144] A modified Hoagland's nutrient solution containing 15 mM nitrogen (N) and 2 mM phosphorus (P) was used as the plant nutrient solution. The plant nutrient solution was added through the second valve 103 until the liquid overflowed from the second valve 103, and then the valve was sealed.

[0145] Around the planted saplings, space 30 trees / m² 2 Sow the seeds of Spinacia oleracea, a non-mycorrhizal herbaceous plant belonging to the Chenopodiaceae family, at a density of [amount not specified]. After sowing, compact the topsoil and spray an appropriate amount of water to keep the soil moist.

[0146] 6. Subsequent Management

[0147] Every 30 days, once the plant nutrient solution in the tube is depleted, open the first valve 101 of the nutrient tube 1 to drain the waste liquid inside, and add plant nutrient solution through the second valve 103 until the liquid overflows from the second valve 103, then seal the tube.

[0148] During the cultivation period, simply keep the soil on the ridges moist by watering regularly, but avoid waterlogging.

[0149] II. Effects

[0150] 1. Infection rate

[0151] After 120 days of transplanting and cultivation, the infection rate of the ectomycorrhizal fungus *Lycorrhiza rubra* in the root system of *Pinus koraiensis* seedlings without nutrient tubes was only 23%–30%; while in the *Pinus koraiensis* seedlings with nutrient tubes, the infection rate of the ectomycorrhizal fungus *Lycorrhiza rubra* in the root system of *Pinus koraiensis* seedlings was significantly increased to 62%–70%, an increase of 2 to 3 times.

[0152] 2. Mycelial growth

[0153] After 120 days of transplanting and cultivation, the mycelium of *Puffballonia 'Colored Bean'* covered the entire nutrient tube in the *Pinus sylvestris* seedlings. For example... Figure 4 As shown in A, the mycelium is intertwined and aggregated with the fertilizer substrate; as... Figure 4 As shown in B and C, after the fertilizer substrate was removed from the nutrient tube, it can be seen that the fertilizer substrate remained compact and did not disperse under the fixation of the colored puffball mycelium, with the mycelium closely connected to the fertilizer substrate. Figure 5 As shown, in the soil around the pine seedlings with nutrient tubes, there are a large number of white mycelium on the roots of the pine trees near the nutrient tubes, and a large number of mycelia are also attached to the surrounding soil.

[0154] The above results indicate that the colored puffball can rapidly adapt to the actual forest growth environment in a short period of time and successfully establish an infectious symbiotic relationship with its host, the red pinnatifida.

[0155] 3. Seedling growth status

[0156] After 120 days of transplanting and cultivation, the height of the *Pinus koraiensis* seedlings without nutrient tubes ranged from 34cm to 63cm, with an average height of 48cm, and the ground diameter ranged from 3.2mm to 8.5mm, with an average ground diameter of 5.8mm. In contrast, the height of the *Pinus koraiensis* seedlings with nutrient tubes ranged from 42cm to 133cm, with an average height of 76cm, and the ground diameter ranged from 5.3mm to 13.4mm, with an average ground diameter of 9.2mm. Therefore, using the nutrient tubes of this invention increased seedling growth by 1.5 to 2 times.

[0157] The above results indicate that the colored puffball has a stable symbiotic relationship with the host plant, *Pyracantha fortuneana*, assisting *Pyracantha fortuneana* in absorbing mineral nutrients and promoting its growth.

[0158] 4. Subsequent growth

[0159] After 120 days of transplanting and cultivation, the nutrient tubes were removed, and the seedlings of the Red Cone continued to be managed in the field as usual. The colored puffballs could continue to be established on the roots of the Red Cone and maintain a symbiotic relationship with the host Red Cone.

[0160] In summary, this invention successfully established a stable obligate symbiotic relationship between the ectomycorrhizal fungus *Calvatia variegata* and its host, *Cephalotaxus fortunei*, and enhanced the symbiotic effect between them, which can be maintained for a long time without degradation.

[0161] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A device for enhancing ectomycorrhizal symbiosis, characterized in that, Includes a nutrient tube (1), one end of which is provided with a first valve (101), and the opening at the other end is provided with a first root isolation net (102). A second valve (103) is provided on the side wall of the nutrient tube (1). The nutrient tube (1) is loaded with a combined substrate (2), which includes a fertilizer-introducing substrate layer (201), a fertilizer-isolating substrate layer (202), and a microbial-introducing substrate layer (203). The fertilizer-introducing substrate layer (201) is composed of river sand and vermiculite. The fertilizer-isolating substrate layer (202) is composed of quartz sand. The microbial-introducing substrate layer (203) is composed of vermiculite. The particle size of the fertilizer-introducing substrate layer (201) and the microbial-introducing substrate layer (203) is smaller than that of the fertilizer-isolating substrate layer (202). The volume ratio of the fertilizer-introducing substrate layer (201), the fertilizer-isolating substrate layer (202), and the microbial-introducing substrate layer (203) is (5-7):(1-2):(2-3). The fertilizer-introducing substrate layer (201), the fertilizer-isolating substrate layer (202), and the microbial-introducing substrate layer (203) are stacked sequentially. The fertilizer-introducing substrate layer (201) is located at one end near the first valve (101) and does not extend beyond the second valve (103). The bacteria-introducing substrate layer (203) is located at one end near the first root isolation net (102). The fertilizer-isolating substrate layer (202) is located between the fertilizer-introducing substrate layer (201) and the bacteria-introducing substrate layer (203). A second root isolation net (205) is provided between the fertilizer-isolating substrate layer (202) and the bacteria-introducing substrate layer (203).

2. The apparatus according to claim 1, characterized in that, A fertilizer-separating filter (204) is provided between the fertilizer-introducing substrate layer (201) and the fertilizer-separating substrate layer (202).

3. The apparatus according to claim 1, characterized in that, The particle size of the fertilizer-introducing substrate layer (201) is 1.8 mm to 2.2 mm; the particle size of the fertilizer-isolating substrate layer (202) is 4.8 mm to 5.2 mm; and the particle size of the bacteria-introducing substrate layer (203) is 2.8 mm to 3.2 mm.

4. The apparatus according to claim 3, characterized in that, The fertilizer substrate layer (201) is composed of river sand of 1.8 mm to 2.2 mm and vermiculite of 1.8 mm to 2.2 mm.

5. The apparatus according to claim 1, characterized in that, The ratio of the distance from the second valve (103) to the first valve (101) to the distance from the second valve (103) to the first root system isolation net (102) is 1:(0.5~2).

6. The apparatus according to claim 1, characterized in that, The first root isolation net (102) is at an angle of 40° to 50° to the axial direction of the nutrient tube (1).

7. The use of the apparatus according to any one of claims 1 to 6 in the cultivation of ectomycorrhizae.

8. A method for enhancing ectomycorrhizal symbiosis, characterized in that, Ectomycorrhizae are cultivated using the apparatus described in any one of claims 1 to 6.

9. The method according to claim 8, characterized in that, Includes the following steps: Trees with ectomycorrhizal symbiosis are planted in a plot of land where no mycorrhizal plants are grown; the first root isolation net (102) of the device described in any one of claims 1 to 6 is placed near the ectomycorrhiza, waste liquid is discharged through the first valve (101), and plant nutrient solution is added through the second valve (103).

10. The method according to claim 9, characterized in that, The plot was also planted with non-mycorrhizal plants.

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