Boletus brownii G8 as well as culture method and application thereof

Abstract

The invention provides boletus nigrum G8 as well as a culture method and application thereof, and belongs to the technical field of edible mushrooms. The preservation number of the boletus nigrum G8 is CGMCC (China General Microbiological Culture Collection Center) No.20233. The invention also provides a corresponding culture method of the boletus nigrum G8 and application of the boletus nigrum G8 in culture of boletus nigrum mycorrhizal seedlings and potting. Compared with other strains, the boletus brownii G8 has the advantages that the growth speed is higher, and the obvious competitive advantage and application value are achieved; sporocarp can be formed in a short time through potting, and continuous mushroom production is achieved.

Description

Technical Field

[0001] This invention belongs to the field of edible fungi technology, and in particular relates to a brown ring bolete G8, its culture method and application. Background Technology

[0002] Brown ring lactobacillus ( Yellow pig (L.:Fr.) Gray) belongs to the kingdom Fungi, phylum Basidiomycota, class Agaricomycetes, family Boletaceae, and genus Boletus. It is a nutritious and delicious edible mushroom with thick, fleshy flesh, a tender and delicious taste, excellent flavor, and an enticing aroma. Boletus brownii is rich in crude protein, crude fat, crude fiber, various amino acids, B vitamins, various minerals, active enzymes, polysaccharides, matsutake alcohol, isomatsutake alcohol, methyl cinnamate, polyphenols, alkaloids, flavonoids, and saponins, among other nutrients and bioactive substances. It has medicinal effects such as immunomodulation, antiviral, and antioxidant properties. It is one of the mushroom types used in prescriptions for treating Kashin-Beck disease and is an ideal multifunctional forest food. It can be eaten fresh or dried, has a large market capacity, and great development potential.

[0003] *Boletus circinus* is a symbiotic fungus that grows in association with pine trees, improving their growth indicators and stress resistance. It has been widely used in the production of mycorrhizal bio-fertilizers. As a mycorrhizal edible fungus, the cultivation of *Boletus circinus* has gradually gained attention, with studies focusing on its habitat, symbiotic characteristics and mechanisms, cultivation, fermentation, nutritional value, strain production, and cultivation techniques. For example, Chai Junhong et al. studied the polysaccharide extraction method and antioxidant activity of *Boletus circinus*, finding that its polysaccharides exhibited a scavenging rate of 48.30% for superoxide ion free radicals, 62.19% for hydroxyl free radicals, and 28.32% for DPPH free radicals, demonstrating strong antioxidant capacity and promising development potential. Yin Dachuan et al. explored the… Yellow pig The biological characteristics of *Boletus circinus* were studied. Tan Lingwen et al. screened the optimal culture medium for *Boletus circinus*. The products in applications 202210788690.2 ("A Special Bio-organic Fertilizer for Chestnut Mycorrhizae and Its Preparation Method") and 20241018492.0 ("A Strand of *Boletus circinus*, Growth Promoter and / or Biocontrol Agent and Application") both contain *Boletus circinus*. Application 200710164731.X ("Artificial Cultivation Method of *Boletus circinus*") describes the use of tissue isolation to obtain the mother culture of *Boletus circinus*, the preparation of liquid propagation culture on a liquid culture medium, the breaking up of the propagation culture into a mycelial slurry, and then dipping the resulting sterile seedlings in the mycelial slurry and densely planting them in a sterile nursery for fruiting management, focusing on strain isolation, mycelial agent preparation, and mycelial agent culture medium formulation. To date, there are no reports on superior strains and potted cultivation of *Boletus circinus*. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a *Boletus chalcogenide* G8 strain, its culture method, and its application, which has a faster growth rate than other strains. yellow pork G8 has a strong mycorrhizal formation ability and can form fruiting bodies through pot cultivation.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A type of brown ring lactobacillus ( Yellow pig (The accession number is CGMCC No.20233).

[0006] The present invention also provides a method for culturing the above-mentioned *Boletus globosum*, comprising the following steps: Brown ring bolete was cultured in PDA agar medium on a solid medium. The resulting mycelium was inoculated into a liquid medium and cultured in a shaker to obtain the brown ring bolete inoculum.

[0007] Preferably, the liquid culture medium comprises component A and component B; Component A is: 200-300g of potatoes, boiled in 1500-3000mL of water for 1 hour, filtered, and the filtrate is diluted to 1000mL. Component B consists of: calcium chloride dihydrate 0.05-0.1 g / L, potassium dihydrogen phosphate 0.2-0.5 g / L, magnesium sulfate heptahydrate 0.3-0.5 g / L, inositol 0.05-1 mg / L, folic acid 0.05-0.1 mg / L, vitamin B1 0.05-0.1 mg / L, manganese sulfate 0.005-0.01 g / L, zinc sulfate heptahydrate 0.005-0.01 g / L, ferric chloride hexahydrate 0.005-0.01 g / L, glucose 20-30 g / L, peptone 2-3 g / L, niacin 0.6-1.2 mg / L, and jasmonic acid 0.08-0.12 mg / L; The volume ratio of component A to component B is 20%-40%:60%-80%.

[0008] Preferably, the temperature of the shaker liquid culture is 18-28℃, the rotation speed is 140-220 r / min, and the time is 14-24 days; the initial pH value of the liquid culture medium is 5.5-6.5, and the liquid volume is 800-1400mL / 2000mL.

[0009] The present invention also provides the application of the above-mentioned *Boletus circinus* in the cultivation of *Boletus circinus* mycorrhizal seedlings and potted plants.

[0010] Preferably, the cultivation of *Boletus globosum* mycorrhizal seedlings includes the following steps: Prepare seedling substrate and inoculum of *Boletus circinus*, and cultivate pine seedlings. When the pine seedlings have been cultivated for 30-45 days, inject the diluted inoculum into the roots of the seedlings. After inoculation, cultivate for 180-270 days to obtain *Boletus circinus* mycorrhizal seedlings.

[0011] More preferably, the preparation method of the *Boletus circinus* inoculum includes: breaking up the *Boletus circinus* culture broth that has been cultured in liquid for 14-24 days to obtain a mycelial suspension with a mycelial ball diameter of less than 1 mm, and adjusting the concentration to 1-2 mg dry mycelium / mL to obtain the inoculum.

[0012] More preferably, the pine family plants include Masson pine, Chinese pine, Scots pine, foreign pine, Chinese pine, Yunnan pine, Korean pine and alpine pine.

[0013] More preferably, the inoculum is diluted 8-10 times; and the amount of diluted inoculum is 2-5 mL / strain.

[0014] Preferably, the cultivation of *Boletus edulis* in pots includes the following steps: Transplant the cultivated *Boletus glomeratus* mycorrhizal seedlings into flowerpots and cultivate them in a greenhouse; or cultivate pine seedlings directly in flowerpots, inoculate them with *Boletus glomeratus* inoculant, and then place them in a greenhouse for cultivation.

[0015] More preferably, the seedling substrate formula and preparation for *Boletus glomeratus* mycorrhizal seedlings is as follows: the volume ratio of peat moss, vermiculite, perlite, and yellow clay is 4:3:2:1, 3:3:3:1, 4:2:3:1, or 4:3:1:2; each component is crushed separately and then mixed in the proportion, water is added to maintain a humidity of 60%-70%, and sterilized under high pressure or normal pressure for later use.

[0016] More preferably, the substrate formula and preparation for the potted Boletus chamaejasminoides are as follows: the volume ratio of yellow soil, peat moss, vermiculite and perlite is 5:3:1:1 or 4:3:2:1; each component is crushed separately and then mixed in proportion, sterilized under high pressure or normal pressure, and set aside for later use.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The *Boletus globosum* strain provided by this invention (preservation number: CGMCC No. 20233, strain number G8) is an excellent and specific strain. Its growth rate and biomass are much higher than those of other strains in the same genus. It can rapidly accumulate mycelium, laying a material basis for subsequent inoculum preparation and mycorrhizal synthesis. The strain has neat colony edges, dense and white mycelium on PDA medium, and emits a natural mushroom fragrance. It has no risk of contamination by other microorganisms, strong stability, and is suitable for large-scale propagation.

[0018] The present invention demonstrates a high symbiotic efficiency between *Boletus circinus* and pine plants. After inoculation, the mycorrhizalization rate of pine seedlings reaches 94.77%, and the mycorrhizal infection rate reaches 89.34%. Furthermore, the mycorrhizal synthesis and host growth promotion effects are significant, and the seedling height, taproot length, ground diameter, and total biomass of pine seedlings are significantly increased, making it suitable for field transplanting.

[0019] This invention is the first to realize the large-scale mushroom production of Boletus globosum in pots. It can achieve continuous mushroom production through two modes: "mycorrhizal seedling transplanting into pots" (mushroom production after 12 months) or "direct seedling inoculation in flower pots" (mushroom production after 18-36 months). No large-scale nursery facilities are required. Attached Figure Description

[0020] Figure 1 It is *Boletus globosum* ( yellow pork G8 growth on PDA solid medium; Figure 2 It is *Boletus globosum* ( yellow pork G8 forms mycorrhizae, mycorrhizal seedlings, and fruiting bodies with Masson pine; Figure 3 It is *Boletus globosum* ( yellow pork G8 potted fruiting bodies; Figure 4 It is *Boletus globosum* ( yellow pork The evolutionary tree of G8.

[0021] Biological Preservation Information The brown ring bolete of the present invention ( yellow pork G8 belongs to the genus *Boletus globosum*. pork yellow It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on July 31, 2020, with accession number CGMCC No. 20233. Detailed Implementation

[0022] This invention provides a brown ring lactobacillus ( Yellow pig (The accession number is CGMCCNo.20233).

[0023] The present invention also provides a method for culturing the above-mentioned *Boletus globosum*, comprising the following steps: Brown ring bolete was cultured in PDA agar medium on a solid medium. The resulting mycelium was inoculated into a liquid medium and cultured in a shaker to obtain the brown ring bolete inoculum.

[0024] In this invention, the liquid culture medium preferably comprises component A and component B; Component A is: 200-300g of potatoes, boiled in 1500-3000mL of water for 1 hour, filtered, and the filtrate is brought to a final volume of 1000mL; more preferably, 250g of potatoes, boiled in 2500mL of water for 1 hour, filtered, and the filtrate is brought to a final volume of 1000mL.

[0025] Component B consists of: calcium chloride dihydrate 0.05-0.1 g / L, potassium dihydrogen phosphate 0.2-0.5 g / L, magnesium sulfate heptahydrate 0.3-0.5 g / L, inositol 0.05-1 mg / L, folic acid 0.05-0.1 mg / L, and vitamin B1. 0.05-0.1 mg / L, manganese sulfate 0.005-0.01 g / L, zinc sulfate heptahydrate 0.005-0.01 g / L, ferric chloride hexahydrate 0.005-0.01 g / L, glucose 20-30 g / L, peptone 2-3 g / L, niacin 0.6-1.2 mg / L, and jasmonic acid 0.08-0.12 mg / L; further preferably, calcium chloride dihydrate 0.07 g / L, potassium dihydrogen phosphate 0.4 g / L, magnesium sulfate heptahydrate 0.4 g / L, inositol 1 mg / L, folic acid 0.08 mg / L, and vitamin B1 0.08 mg / L, manganese sulfate 0.006 g / L, zinc sulfate heptahydrate 0.08 g / L, ferric chloride hexahydrate 0.007 g / L, glucose 25 g / L, peptone 2.5 g / L, nicotinic acid 1 mg / L and jasmonic acid 0.1 mg / L.

[0026] The volume ratio of component A to component B is 20%-40%:60%-80%, and more preferably component A:B (v / v) = 3:7.

[0027] In this invention, the preferred temperature for liquid culture on a shaker is 18-28°C, more preferably 20-25°C; the preferred rotation speed is 140-220 r / min, more preferably 160-200 r / min; the preferred culture time is 14-24 days, more preferably 18-20 days; the preferred initial pH of the liquid culture medium is 5.5-6.5, more preferably 6.0; and the preferred liquid volume is 800-1400 mL / 2000 mL, more preferably 1000-1200 mL / 2000 mL.

[0028] The present invention also provides the application of the above-mentioned *Boletus circinus* in the cultivation of *Boletus circinus* mycorrhizal seedlings and potted plants.

[0029] In this invention, the preferred method for cultivating *Boletus glomeratus* mycorrhizal seedlings includes the following steps: (1) Preparation of seedling substrate: The optimal substrate formula and preparation for mycorrhizal seedlings of *Boletus glomeratus* is as follows: the volume ratio of peat moss, vermiculite, perlite, and yellow soil is 4:3:2:1, 3:3:3:1, 4:2:3:1, or 4:3:1:2. After crushing each component separately, mix them in the specified proportions, add water to maintain a humidity of 60%-70%, and sterilize under high pressure or normal pressure for later use.

[0030] (2) Preparation of *Boletus globosum* inoculum: The preferred method for preparing the inoculum includes: breaking up the culture medium of *Boletus circinus* that has been cultured in liquid for 14-24 days to obtain a mycelial suspension with a mycelial ball diameter of less than 1 mm, further culturing for 20 days, and adjusting the concentration to 1-2 mg dry mycelium / mL to obtain the inoculum, with a further preferred concentration of 1.5 mg dry mycelium / mL.

[0031] (3) Cultivating seedlings of pine trees: Preferred pine species include Masson pine, Chinese pine, Scots pine, foreign pine, Chinese pine, Yunnan pine, Korean pine, and alpine pine.

[0032] The optimal cultivation process includes germination and sowing; Further optimization of germination methods includes: soaking host seeds in sterile water for 24-48 hours, removing the host seeds after soaking and placing them in 10%-15% H2O2 and shaking for 20-30 minutes, rinsing the host seeds 4-5 times with sterile water after shaking and soaking, placing the rinsed host seeds in a cotton bag and germinating them in an environment of 24-26℃, and spraying water daily during the germination period; Further optimization of sowing methods includes: when the germination rate of the host seeds is ≥80%-85%, the germinated host seeds are sown into the seedling substrate and placed in a greenhouse for seedling cultivation.

[0033] (4) When the seedlings of Pinaceae plants are cultured for 30-45 days, the diluted inoculum is injected into the roots of the seedlings. After inoculation, the seedlings are cultured for 180-270 days to obtain mycorrhizal seedlings of Boletus pulmonarius.

[0034] The preferred dilution concentration of the inoculum is 8-10 times, more preferably 9 times; the preferred inoculation amount of the diluted inoculum is 2-5 mL / strain, more preferably 3-4 mL / strain.

[0035] In this invention, the preferred method for cultivating *Boletus globosum* in pots includes the following steps: Method I: Transplant the cultivated *Boletus fulva* mycorrhizal seedlings into flowerpots and cultivate them in a greenhouse. Mushroom production begins after 12 months; or Method II: Cultivate pine seedlings directly in flowerpots, inoculate with Boletus circinus inoculum, and then place them in a greenhouse for cultivation. Mushroom production begins after 18-36 months.

[0036] Further optimization of the substrate formula and preparation for potted Boletus globosum: the volume ratio of yellow soil, peat moss, vermiculite and perlite is 5:3:1:1 or 4:3:2:1; each component is crushed separately and then mixed in proportion, sterilized under high pressure or normal pressure, and set aside for later use.

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1 Isolation of *Boletus globosum* strains During the peak mushroom production season from September to November 2017, at the Boletus lucida cultivation base in Daowushan Jili Street, Liuyang City, Hunan Province, healthy, disease-free, and unopened fresh Boletus lucida fruiting bodies with a cap diameter of 4-6 cm were selected from the canopy of host trees with a single production of more than 20 Boletus lucida fruiting bodies and dense mushroom production. Only 5-6 Boletus lucida fruiting bodies meeting the requirements were selected from each host tree and brought back to the laboratory at low temperature for tissue isolation to isolate the strains.

[0039] The specific steps are as follows: On a clean bench, gently wipe the surface of the fruiting body with a cotton swab or absorbent cotton soaked in 75% alcohol, then use sterile filter paper to absorb the moisture from the surface of the fruiting body. Cut off the cap, stipe, and inner tissue at the junction of the cap and stipe, place them on a PDA plate, and after the colonies have grown, remove the contaminated colonies. Select the hyphae from the edge of the uncontaminated colonies and culture them in a fresh PDA plate. The isolates obtained from each fruiting body of each tree are numbered with the same number, one fruiting body per number, such as G1-1, G1-2, G1-3, G1-4; G2-1, G2-2, G2-3, G2-4; G3-1, G3-2, G3-3, G3-4; G4-1... The successfully isolated and most vigorous strains from each tree are retained, and the retained trees are numbered G1, G2, G3, G4, G5, G6...

[0040] Example 2 Take the strain provided in Example 1.

[0041] 9cm glass petri dishes were used, with 10mL of sterilized PDA medium poured into each dish. After activating and culturing all strains in the same batch for the same time, tissue blocks of the same size were removed from the plates using a 0.5cm diameter punch and inoculated onto the prepared plates. Three plates were cultured for each strain, with three replicates. The plates were incubated at 23-27℃ after inoculation. Colony diameter was assessed every 3 days using the cross-crossing method to observe the growth of different strains. It was found that G8 had a higher growth rate and biomass than the other strains. Figure 1As shown, G8 colonies on universal fungal PDA medium have neat, round edges, abundant hyphae, and emit a mushroom aroma. They grow faster than other strains, exhibiting a higher competitive advantage and possessing significant application value. Its phylogenetic tree is shown below. Figure 4 As shown.

[0042] Table 1 Comparison of growth of G8 with other strains on PDA medium

[0043] Example 3 Using *Pinus massoniana* as the host, the mycorrhizal formation ability of *Boletus glomeratus* G8 was tested. Substrate formulation and preparation of sterilized substrate: peat moss:vermiculite:perlite:yellow core soil = 3:3:3:1 (volume ratio), moisture content 65%, mixed thoroughly and then subjected to high temperature and high pressure (121℃, 1.5 kg·cm³). -2 Sterilize for 2 hours, then cool before use.

[0044] Germination and sowing of Masson pine seeds: Select plump, pest-free Masson pine seeds. Indoors, first soak in sterile water for 16-24 hours, then soak in 10% hydrogen peroxide solution with shaking for 15 minutes. Rinse 4-5 times with sterile water, then place in a petri dish or transparent plastic box lined with sterile, damp gauze, and germinate in an incubator at 24-26℃. Spray with a suitable amount of sterile water daily to keep the gauze moist. When the seed germination rate reaches over 80%, sow the germinated seeds into 32-cell seedling trays containing sterile substrate and place them in a greenhouse for seedling management.

[0045] Preparation of inoculum: On a sterile operating table, the mycelia of various *Boletus globosum* strains cultured in liquid for 14-18 days were filtered through sterile gauze and rinsed with the filtrate. The mycelia were placed in a sterile beaker, and an appropriate amount of filtrate was added. The mycelia were broken up with a tissue mixer for about 1 minute. The mycelial suspension was mixed with the filtrate to obtain the inoculum, which was then stored in a refrigerator at 4°C.

[0046] Mycorrhizal synthesis of *Boletus edulis* and *Pinus massoniana*: After culturing *Pinus massoniana* seedlings in a sterile substrate for 25-40 days, a 10-fold diluted mycorrhizal agent was injected into the roots of the seedlings using a sterile medical syringe, with an inoculation volume of 5 mL per seedling. During the cultivation period, the substrate was kept moist with sterile water at field volume. 160 seedlings of each strain were inoculated, with three replicates. The control (CK) group maintained the same conditions except for no inoculation. Seven months after inoculation, 30 seedlings were randomly selected from each treatment, and seedling height, taproot length, ground diameter, total biomass, mycorrhizalization rate, and mycorrhizal infection rate were investigated.

[0047] Mycorrhizalization rate = (Number of mycorrhizal plants formed / Number of inoculated plants) × 100%; Mycorrhizal infection rate = number of root segments with mycorrhizae / total number of root segments × 100%.

[0048] Experimental results: As shown in Table 2, the mycorrhizal formation rate and mycorrhizal infection rate of G8 strain were significantly higher than those of other strains, reaching 94.77% and 89.34%, respectively; the CK group, which was not inoculated, showed no mycorrhizal formation. The seedling height, taproot length, ground diameter, and total biomass of *Boletus pulmonarius* G8-inoculated *Boletus pulmonarius* seedlings were all higher than those of the uninoculated control. *Boletus pulmonarius* G8 strain can be used to cultivate mycorrhizal seedlings suitable for field planting.

[0049] Ten months after the investigation, fruiting bodies were found to have grown in the seedling trays of strain G8. The growth of the mycorrhizae, mycorrhizal seedlings, and fruiting bodies was as follows. Figure 2 As shown.

[0050] Table 2 Comparison of mycorrhizal formation ability between G8 and other strains

[0051] Example 4 Mix yellow soil, peat moss, vermiculite, and perlite in a ratio of 4:3:2:1 with a moisture content of 70%. Sterilize the mixture, cool it, and then place it in a tray. Germinate and sow the seeds of the host plant, Masson pine, into the seedling tray.

[0052] The brown ring lactic acid bacteria that can produce fruiting bodies in seedling trays ( yellow pork G8 was cultured on PDA plates in a solid state, and the resulting mycelium was inoculated into a liquid culture medium. Liquid culture with shaking in a flask yielded *Boletus purpureus* (*Lactobacillus brownii*). yellow pork G8 inoculum. When the pine seedlings have 4-10 needles, inject the inoculum diluted 10 times into the roots of the seedlings and cultivate them in a greenhouse. After 180 days of cultivation, a mycorrhizal seedling is randomly transplanted into a foam box. One year later, the foam box produces 4 fruiting bodies, and mushrooms are produced continuously for the next 3 years. Figure 3 As shown.

[0053] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of *Boletus lucida* (Brown ring lactis) Suillus luteus ), characterized in that, The preservation number of the Suillus luteus is CGMCC No.20233.

2. The cultivation method of the Boletus rufofuscus according to claim 1, characterized by, The method comprises the following steps: The Suillus luteus of claim 1 is solidly cultured in PDA flat plate medium, and the obtained mycelium is inoculated in a liquid culture medium to perform liquid culture by shaking flask oscillation to obtain the Suillus luteus inoculation agent.

3. The culturing method according to claim 2, wherein, The liquid culture medium comprises component A and component B. The component A is: 200-300g of potatoes are boiled with 1500-3000mL of water for 1h, and after filtration, the filtrate is diluted to 1000mL. The component B is: 0.05-0.1g / L of calcium chloride dihydrate, 0.2-0.5g / L of potassium dihydrogen phosphate, 0.3-0.5g / L of magnesium sulfate heptahydrate, 0.05-1mg / L of myo-inositol, 0.05-0.1mg / L of folic acid, 0.05-0.1mg / L of vitamin B1, 0.005-0.01g / L of manganese sulfate, 0.005-0.01g / L of zinc sulfate heptahydrate, 0.005-0.01g / L of ferric chloride hexahydrate, 20-30g / L of glucose, 2-3g / L of proteose peptone, 0.6-1.2mg / L of nicotinic acid and 0.08-0.12mg / L of jasmonic acid. The volume ratio of the component A to the component B is 20%-40%:60%-80%.

4. The culturing method according to claim 2, wherein The temperature of the shaking bed oscillation liquid culture is 18-28℃, the rotating speed is 140-220r / min, and the time is 14-24 days; the initial pH value of the liquid culture medium is 5.5-6.5, and the liquid loading amount is 800-1400mL / 2000mL.

5. The application of the Suillus luteus of claim 1 in cultivating Suillus luteus mycorrhizal seedlings and pot culture.

6. Use according to claim 5, characterized in that, The method for cultivating the Suillus luteus mycorrhizal seedlings comprises the following steps: The seedling substrate, the Suillus luteus inoculation agent and the conifer seedlings are prepared, when the conifer seedlings are cultured for 30-45 days, the diluted inoculation agent is injected into the root of the seedlings, the seedlings are cultured for 180-270 days after inoculation, and the Suillus luteus mycorrhizal seedlings are obtained.

7. Use according to claim 6, characterized in that, The preparation method of the Suillus luteus inoculation agent comprises: the Suillus luteus culture solution after liquid culture for 14-24 days is broken to obtain a mycelium suspension with a mycelium ball diameter less than 1mm, and the concentration is adjusted to 1-2mg of dry mycelium / mL, thereby obtaining the inoculation agent.

8. Use according to claim 6, characterized in that, The conifers comprise Pinus massoniana, Pinus tabulaeformis, Pinus sylvestris var. mongolica, Pinus taiwanensis, Pinus yunnanensis, Pinus armandii and Pinus densata.

9. Use according to claim 6, characterized in that, The dilution concentration of the inoculation agent is 8-10 times, and the inoculation amount of the diluted inoculation agent is 2-5mL / plant.

10. Use according to claim 5, characterized in that, The method for cultivating the Suillus luteus pot culture comprises the following steps: The Suillus luteus mycorrhizal seedlings cultivated by the application of any one of claims 6-9 are transplanted into a flowerpot and cultured in a greenhouse; or the conifer seedlings are directly cultivated in a flowerpot, inoculated with the Suillus luteus inoculation agent and then cultured in a greenhouse.

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