CN113207552A - Seedling mycorrhization method taking mycorrhizal fungi hypha culture medium as seedling culture medium and application of seedling mycorrhization method - Google Patents
Seedling mycorrhization method taking mycorrhizal fungi hypha culture medium as seedling culture medium and application of seedling mycorrhization method Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/20—Culture media, e.g. compost
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/50—Inoculation of spawn
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Abstract
The invention provides a seedling mycorrhization method taking a mycorrhizal fungi hypha culture medium as a seedling culture medium and application thereof, belonging to the technical field of seedling mycorrhization. The specific method comprises the following steps: inoculating the ectomycorrhizal fungi liquid into the pretreated hypha culture medium for culture, crushing, uniformly mixing, filling into a seedling culture plug tray for continuous culture, and obtaining a seedling culture medium containing active mycelia; and (3) placing the pretreated seeds in the seedling culture substrate containing the active mycelium for culture. The invention provides a simple, rapid and efficient mycorrhization method, which is of great significance for popularizing the application of mycorrhizal fungi and realizing industrial production of fine variety mycorrhization of forest flowers.
Description
Technical Field
The invention belongs to the technical field of seedling mycorrhization, and particularly relates to a seedling mycorrhization method taking a mycorrhizal fungi hypha culture medium as a seedling culture medium and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Mycorrhiza is a plant symbiosis phenomenon commonly existing in nature, is a composite absorption organ formed by soil beneficial fungi (mycorrhizal fungi) and higher plant root systems, and is a reciprocal symbiont. More than 85% of terrestrial plant species can establish a mutual-benefit symbiotic relationship with various types of mycorrhizal fungi to form mycorrhizal symbionts, wherein the most common ones are ectomycorrhiza, endophytic mycorrhiza and ectomycorrhiza. In recent years, with the gradual improvement of forest mycorrhizal biological technology, many countries in the world are dedicated to the research and application of efficient mycorrhizal fungi preparation and the research and application of mycorrhizal technology on the basis of effective mycorrhizal fungi screening. The seedling mycorrhization refers to a process of artificially providing a certain amount of effective propagules of effective mycorrhizal fungi with strong competitiveness for the root of a seedling, and controlling a suitable growing environment to enable the root system of the seedling to form mycorrhiza. The mycorrhization mainly has the effects of enlarging the absorption area of a seedling root system, promoting the seedling to absorb soil water and fertilizer, improving the rhizosphere environment of the seedling, secreting growth regulators and enhancing the resistance of the seedling to plant diseases and insect pests and adversity, so that the growth of the seedling is promoted, the quality of the seedling is improved, and the productivity level of a nursery land and the survival rate of forestation are improved. The application of the mycorrhizal nursery stocks can reduce the use of chemical fertilizers and pesticides, avoid environmental pollution and promote ecological balance; the soil activity is enhanced, the physical and chemical properties of the soil are improved, the soil fertility decline and the water and soil loss are prevented and controlled, and the soil resources are protected; the technological content of forestry production is increased, and the forestry production value is improved; provides a feasible biological measure for preventing wind and fixing sand, preventing and controlling desertification, transforming waste mining areas and industrial polluted areas, accelerating afforestation of barren mountains and improving and beautifying ecological environment. In addition, mycorrhizal fungi fruiting bodies produced on nursery lands and forestation lands have edible, medicinal and industrial values, and potential economic benefits are remarkable. Therefore, the mycorrhization of the nursery stock is a new ecological technology which accords with the natural law, and has remarkable ecological benefit, environmental benefit, social benefit and economic benefit.
The conventional seedling mycorrhization method comprises the steps of sowing mycorrhization seedling culture (namely, mycorrhizal fungi preparation is applied while sowing, seed dressing, furrow application, hole application or seed coating), vegetative propagation mycorrhization seedling culture (mycorrhizal fungi preparation is applied while cuttage is performed, and a microbial inoculum can be applied in a hole or mixed into a cuttage matrix according to a certain proportion), mycorrhization seedling culture of transplanting mycorrhization seedling culture (namely, mycorrhizal fungi preparation is applied while transplanting seedlings or seedling changing beds, and can be dipped in slurry and applied), mycorrhization seedling cutting root and seedling culture (firstly, seeds are treated and then sowed on a sand bed, when the seeds germinate and grow out of seedlings and seed cases are about to fall off, the tips of the radicles are cut off from 1/4-1/2 of the length of the radicles, then a small amount of mycorrhizal fungi preparation is placed at the bottom of transplanting holes of the seed beds, and then the sprouts are planted in the holes, so that the cuts of the tips of the roots directly contact with the mycorrhizal fungi preparation), And (3) container bag mycorrhizal seedling raising (container seedling raising substrate hole application, or mycorrhizal fungi preparation is mixed into the substrate according to a certain proportion and then bagged for direct sowing), and the like. The method is characterized in that a proper amount of mycorrhizal fungi preparation is put into a seedling growth substrate during sowing, cuttage, transplanting or bed changing so as to realize the mycorrhization of the seedlings. However, the mycorrhizal fungi grow slowly, are difficult to realize pure culture, have the characteristics of high symbiosis with plants and obligate nutrition, and the mycorrhizal formation of the seedlings is influenced by various factors (fungal characteristics, host characteristics, environmental factors and the like), so that the growth and the propagation of high-efficiency mycorrhizal fungi propagules (mycelia) are greatly restricted by a new inoculation carrier (seedling culture substrate) and a seedling culture environment, the growth speed of the mycelia is not matched with the growth speed of roots of seeds, cuttage branches, transplanting seedlings or root breaking seedlings, the contact area capable of infecting is reduced, the mycorrhizal formation time is prolonged, the mycorrhizal effect is influenced, and even the mycorrhizal formation failure is caused. The method for cultivating the seedlings by the mycorrhizal interception slows down the growth speed of primary roots by root cutting, ensures that a fungus invasion mode is mainly cross-section wounds, enlarges the colonization area, ensures that colonization and infection simultaneously occur, improves the colonization strength and obtains a more ideal mycorrhizal effect; however, the method needs seedling pulling, root breaking, mycorrhizal transformation and transplantation, is complex in process, time-consuming and labor-consuming, easily causes mechanical damage to the seedlings, and is high in cost.
Disclosure of Invention
Aiming at the defects of the conventional seedling mycorrhization method, the invention provides a seedling mycorrhization method taking a mycorrhizal fungi hypha culture medium as a seedling culture medium and application thereof.
Specifically, the invention relates to the following technical scheme:
in a first aspect of the present invention, there is provided a seedling mycorrhization method using a mycorrhizal fungi hypha culture substrate as a seedling raising substrate, the method comprising:
inoculating the ectomycorrhizal fungi liquid into the pretreated hypha culture medium for culture, crushing, uniformly mixing, filling into a seedling culture plug tray for continuous culture, and obtaining a seedling culture medium containing active mycelia; and (3) placing the pretreated seeds in the seedling culture substrate containing the active mycelium for culture.
Wherein the ectomycorrhizal fungus may be suillus luteus (Xerocomus basilus) which is capable of rapid growth in a culture medium.
The hypha culture medium is prepared from wood chips, turf, cow dung, calcium carbonate and calcium oxide.
In a second aspect of the invention, the application of the seedling mycorrhizalization method in seedling cultivation is provided.
The beneficial technical effects of one or more technical schemes are as follows:
1. the seedling culture substrate formula in the technical scheme can simultaneously meet the growth conditions of the ectomycorrhizal fungi X.badius, the germination of seedling seeds and the growth of seedlings, and hypha metabolites can promote the seed germination, lateral root generation and seedling growth.
2. According to the technical scheme, when the hyphae grow for 5-7 days, the hyphae are directly sown in the seedling culture substrate containing the active hyphae, the defect of slow growth of mycorrhizal fungi is overcome, the growth speeds of the hyphae and radicles (radicles) are matched, the colonization contact surface of the fungi on the root systems of the radicles is enlarged, the mycorrhization strength is improved, and the mycorrhization time is shortened.
3. The germinated mycelia are mutually wound in the matrix, and matrix particles are wrapped and bonded to form a whole, so that the matrix transplanting of seedlings is facilitated, the seedling revival time is shortened, and the transplanting survival rate is improved.
In conclusion, the technical scheme is simpler, faster and more efficient, and has important significance for popularizing the application of the mycorrhizal fungi and realizing the industrial production of the fine variety mycorrhizal of the forest flowers.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions, or according to the conditions recommended by the sales companies; materials, reagents and the like used in examples were commercially available unless otherwise specified.
In a typical embodiment of the present invention, there is provided a seedling mycorrhization method using a mycorrhizal fungi hypha culture substrate as a seedling substrate, the method comprising:
inoculating the ectomycorrhizal fungi liquid into the pretreated hypha culture medium for culture, crushing, uniformly mixing, filling into a seedling culture plug tray for continuous culture, and obtaining a seedling culture medium containing active mycelia; and (3) placing the pretreated seeds in the seedling culture substrate containing the active mycelium for culture.
Wherein the ectomycorrhizal fungus may be suillus luteus (Xerocomus basilus) which is capable of rapid growth in a culture medium.
The hypha culture medium is prepared from wood chips, turf, cow dung, calcium carbonate and calcium oxide.
Specifically, the preparation method of the hypha culture substrate comprises the following steps: mixing 40-70 parts of sawdust, 20-40 parts of grass carbon, 0-20 parts of cow dung, 1-2 parts of calcium carbonate and 0-2 parts of calcium oxide in parts by mass, adding water to adjust the water content to 60-65%, and uniformly mixing to obtain the hypha culture medium. By optimizing the hypha culture medium, the invention is beneficial to the growth of the ectomycorrhizal fungi and can effectively promote the growth of the seedling seeds for subsequent cultivation.
The pretreatment method comprises the following steps: bagging the prepared mycelium culture medium, sterilizing (such as wet heat sterilization at 121 deg.C for 30min), and cooling to room temperature.
The ectomycorrhizal fungi liquid is obtained by activating and proliferating ectomycorrhizal fungi. In one embodiment of the present invention, the activation and proliferation method is specifically: inoculating the ectomycorrhizal fungi mother strain to a fungi solid culture medium (such as a PDA solid culture medium) for plate activation and proliferation, and then placing the hypha block in a liquid culture medium (such as a PDB liquid culture medium) for light-proof culture (25 +/-1 ℃, 55-65.00% RH, 150-200 r/min).
Inoculating the ectomycorrhizal fungi liquid into the pretreated hypha culture substrate for culture under the conditions that the culture temperature is 25-30 ℃ under the aseptic condition, the culture environment humidity is 60-65%, and the culture time is 2-3 days.
The conditions for continuous culture were: and (3) culturing under an aseptic condition at the temperature of 25-30 ℃ and the environment humidity of 60-65% for 5-7 days, and controlling and optimizing the culture condition to obtain the seedling culture substrate containing the active mycelia.
The seed pretreatment method comprises the steps of disinfecting the seedling seeds, and then washing the seedling seeds with sterile water for later use. The disinfection may be by conventional disinfectant treatment (e.g. 10% sodium hypochlorite).
The specific method for culturing the pretreated seeds in the seedling culture substrate containing the active mycelia comprises the following steps: punching a seedling substrate, filling 0.5-2 cm of filler into the dibbling holes, placing the pretreated seeds into the dibbling holes of the seedling substrate containing the active mycelia, and then covering the surface with the filler with the thickness of 0.5-1 cm. And performing conventional seedling management. In one embodiment of the invention, the temperature is kept at 25-30 ℃ and the humidity is kept at 60-80%.
Wherein the filler comprises a mixture of turf, perlite and vermiculite. In one embodiment of the present invention, the filler preparation method comprises: mixing 45-55 parts of turf, 20-30 parts of perlite and 20-30 parts of vermiculite in parts by mass, adding water to adjust the water content to 55-65%, and uniformly mixing to obtain the filler.
In a second aspect of the invention, the application of the seedling mycorrhizalization method in seedling cultivation is provided. Through the mycorrhization treatment, the survival rate of seeds is effectively improved, and the growth of the seeds and seedlings is promoted.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1: application of mycorrhizal seedling raising method with hypha growth matrix as seedling raising matrix
In the embodiment, the ectomycorrhizal fungi Xerocomus badius is inoculated with different formula matrixes, the growth condition of the fungi is observed, ryegrass is sown by taking the fungi as a seedling culture matrix, and the seed germination and seedling growth conditions are observed. Badius is purchased from China forestry microbiological collection center with the preservation number cfcc 5946. The specific experimental steps are as follows:
1. the x.badius stock was inoculated on PDA solid medium (pH 6.5), plate-activated and proliferation-cultured, and incubated under constant temperature and humidity (25 ± 1 ℃, 60.00%) in the dark for 1 week. And (3) punching holes on the edges of the activated colonies by using a sterile puncher with the diameter of 5mm, selecting a mycelium block, placing the mycelium block in a PDB liquid culture medium, and placing the PDB liquid culture medium in a constant-temperature constant-humidity rotary incubator (25 +/-1 ℃, 60.00% RH, 150-200 rpm) to culture in the dark for 7-10 days to obtain a liquid strain.
2. Adding water into the matrixes with different formulas in the table 1, adjusting the water content to 60-65%, uniformly mixing, respectively filling into sterilization bags, placing into an autoclave for moist heat sterilization at 121 ℃ for 30min, and then cooling to room temperature for later use.
Table 1 different formulations of seedling substrate (mass parts,%)
3. Under the aseptic condition, liquid strains are inoculated into sterilized substrate bags with different formulas, the substrate bags are moved to an aseptic culture room for culture (25-30 ℃ and 60-65%), and the growth conditions of hyphae of substrates with different formulas are continuously observed in the process.
4. And when the hyphae grow to 7 days, transferring the fungus bags to a sterile room, taking out, crushing, and respectively filling into special sterilized seedling culture trays for later use. Selecting ryegrass seeds with uniform size and no plant diseases, insect pests and mechanical damage, carrying out surface disinfection by using 10% sodium hypochlorite, and washing the ryegrass seeds clean by using sterile water. And then respectively dibbling the sterilized seeds in different substrate seedling-raising trays. And (3) dibbling 10 seeds in each seedling raising tray hole, and setting 6 repeats for each treatment by taking 1 seedling raising tray hole as 1 repeat.
5. After sowing for 1 week, calculating the cumulative germination rate of the seeds; seedlings were removed 2 weeks after sowing and the plant height and biomass were measured.
As a result, as shown in table 2, x.badius could not grow on the commonly used seedling substrate; the germination rate, seedling height, diameter and biomass of ryegrass seeds on an X.badius common growth substrate are obviously lower than those of the common seedling substrate; badius can grow normally in the substrate of the embodiment, and the germination rate, seedling height, ground diameter and biomass of the ryegrass seeds in the substrate of the embodiment are obviously higher than those of other two substrates. Therefore, the substrate of the embodiment not only can meet the requirement of normal growth of X.badius, but also can obviously improve the germination rate of seeds and promote the growth of seedlings.
TABLE 2 influence of different formula substrates on the growth of Xerocomus badius hyphae and the germination rate of ryegrass seeds, the growth of seedlings and the infection rate of root systems
Example 2: application of mycorrhizal seedling raising method with hypha growth matrix as seedling raising matrix
In this example, the example of ryegrass is compared with the colonization of the root system of ryegrass by the ectomycorrhizal fungus Xerocomus badius under different inoculation methods. Badius is purchased from China forestry microbiological collection center with the preservation number cfcc 5946. The specific experimental steps are as follows:
1. the x.badius stock was inoculated on PDA solid medium (pH 6.5), plate-activated and proliferation-cultured, and incubated under constant temperature and humidity (25 ± 1 ℃, 60.00%) in the dark for 1 week. And (3) punching holes on the edges of the activated colonies by using a sterile puncher with the diameter of 5mm, selecting a mycelium block, placing the mycelium block in a PDB liquid culture medium, and placing the PDB liquid culture medium in a constant-temperature constant-humidity rotary incubator (25 +/-1 ℃, 60.00% RH, 150-200 rpm) to culture in the dark for 7-10 days to obtain a liquid strain.
2. By adopting the formula of the seedling substrate, 60 parts of wood chips, 20 parts of grass carbon, 16 parts of cow dung, 2 parts of calcium carbonate and 2 parts of calcium oxide are mixed by mass fraction, water is added to adjust the water content to 60-65%, and the mixture is uniformly mixed to obtain the seedling substrate. By adopting the formula of the covering material, 50 parts of turf, 25 parts of perlite and 25 parts of vermiculite are mixed in percentage by mass, water is added to adjust the water content to 55-65%, and the mixture is uniformly mixed to obtain the filler. Placing the seedling culture substrate into a sterilization bag, placing into an autoclave, performing damp-heat sterilization at 121 deg.C for 30min, and cooling to room temperature for use.
3. Selecting ryegrass seeds with uniform size and no plant diseases, insect pests and mechanical damage, carrying out surface disinfection by using 10% sodium hypochlorite, and washing the ryegrass seeds clean by using sterile water for later use.
4. Mycorrhization of fungi
(1) Comparison method: and (3) filling the sterilized seedling raising matrix in the step (2) into a sterilized special seedling raising tray under aseptic conditions for standby. And (3) placing the sterilized seeds in the step (3) into a triangular flask filled with the liquid strains in the step (1), co-culturing for 6 hours in a rotary incubator (26 +/-1 ℃, 60.00% RH and 150 rpm), inoculating, and then dibbling in a seedling culture plate.
(2) The method comprises the following steps: under the aseptic condition, inoculating the liquid strains in the step 1 to the sterilized seedling culture substrate in the step 2 by using an injector according to the volume ratio of 1:4, and placing the substrate in an aseptic culture room for culture; and after 3 days of culture, taking out, crushing and uniformly mixing the mixture under an aseptic condition, filling the mixture into a sterilized special seedling culture plug tray, compacting the mixture, covering a layer of plastic film on the surface of the mixture, and continuously culturing the mixture for 5 to 7 days (the temperature is 25 to 30 ℃, and the humidity is 75 to 80 percent) to obtain the seedling culture substrate containing the active mycelia. And (3) punching the seedling raising matrix by adopting a sterilized puncher under an aseptic condition, adding a filler with the thickness of 1cm into the dibbling hole, dibbling the seeds prepared in the step (3) into the dibbling hole, and covering the surface with the filler with the thickness of 0.5-1 cm.
And (3) dibbling 10 seeds in each hole of the seedling raising hole tray, wherein 1 hole is used as 1 repeat, and each treatment is provided with 6 repeats. And (3) placing the seedling culture plug tray inoculated by the two methods in a greenhouse for conventional seedling culture management, and keeping the temperature of the greenhouse at 25-30 ℃ and the humidity at 60-80%.
5. After sowing for 1 week, calculating the cumulative germination rate of the seeds; and taking out the seedlings after sowing for 2 weeks, measuring the plant height and biomass, simultaneously adopting a root system, manufacturing a paraffin section by adopting a safranin fast green staining method, observing a mycorrhizal sheath structure under a scanning electron microscope, and calculating the infection rate.
The results are shown in Table 3: compared with a comparison method, the method obviously improves the infection rate and the seed germination rate of X.badius in the root system of the ryegrass, and increases the height, the ground diameter and the biomass of seedlings. Therefore, the method is beneficial to mycorrhization of the seedlings and promotes seed germination and seedling growth. TABLE 3 influence of Xerocomus badius on the seed germination rate, seedling growth and root infection rate of ryegrass under different inoculation methods
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A seedling mycorrhization method taking a mycorrhizal fungi hypha culture medium as a seedling culture medium is characterized by comprising the following steps:
inoculating the ectomycorrhizal fungi liquid into the pretreated hypha culture medium for culture, crushing, uniformly mixing, filling into a seedling culture plug tray for continuous culture, and obtaining a seedling culture medium containing active mycelia; and (3) placing the pretreated seeds in the seedling culture substrate containing the active mycelium for culture.
2. The nursery stock mycorrhizal method of claim 1, wherein the ectomycorrhizal fungus is suillus luteus.
3. The nursery stock mycorrhization method according to claim 1, wherein the hypha culture medium is prepared from wood chips, turf, cow dung, calcium carbonate and calcium oxide.
4. The nursery stock mycorrhizal method according to claim 3, wherein the preparation method of the hypha culture substrate comprises the following steps: mixing 40-70 parts of sawdust, 20-40 parts of grass carbon, 0-20 parts of cow dung, 1-2 parts of calcium carbonate and 0-2 parts of calcium oxide in parts by mass, adding water to adjust the water content to 60-65%, and uniformly mixing to obtain the hypha culture medium.
5. The nursery stock mycorrhizal method according to claim 1, wherein the ectomycorrhizal fungi liquid is obtained by activating and proliferating ectomycorrhizal fungi;
preferably, the activation and proliferation method is specifically: inoculating the mother strain of ectomycorrhizal fungi to a solid culture medium of fungi, performing plate activation and proliferation, and placing the hypha block in a liquid culture medium for light-tight culture.
6. The seedling mycorrhization method according to claim 1, wherein the culture conditions for inoculating the ectomycorrhizal fungi liquid into the pretreated hypha culture substrate for culture are controlled to be 25-30 ℃ under aseptic conditions, 60-65% of culture environment humidity and 2-3 days.
7. The seedling mycorrhization method according to claim 1, wherein the continuous culture conditions are as follows: the culture temperature is 25-30 ℃ under the aseptic condition, the culture environment humidity is 60-65%, and the culture time is 5-7 days.
8. The nursery stock mycorrhization method according to claim 1, wherein the pretreatment method of the seeds comprises the steps of sterilizing the nursery stock seeds, and then washing the nursery stock seeds clean with sterile water for later use.
9. The nursery stock mycorrhization method according to claim 1, wherein the specific method for culturing the pretreated seeds in the seedling culture substrate containing the active mycelium comprises the following steps: punching a seedling substrate, filling 0.5-2 cm of filler into the dibbling holes, placing the pretreated seeds into the dibbling holes of the seedling substrate containing active mycelia, and then covering the surface with the filler with the thickness of 0.5-1 cm;
preferably, the filler comprises a mixture of turf, perlite and vermiculite;
further preferably, the filler preparation method comprises the following steps: mixing 45-55 parts of turf, 20-30 parts of perlite and 20-30 parts of vermiculite in parts by mass, adding water to adjust the water content to 55-65%, and uniformly mixing to obtain the filler.
10. Use of the seedling mycorrhization method of any one of claims 1-9 in seedling cultivation.
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