Artificial mycorrhizal seedling of boletus luteus and synthetic method thereof

Abstract

The invention discloses a yellow indigo bolete artificial mycorrhizal seedling and a synthesis method thereof, and relates to the field of fungus artificial cultivation. The synthetic method of the artificial mycorrhizal seedling of the boletus luteus comprises the following steps: S1, culturing a mother strain of the boletus luteus in a solid culture medium, and identifying through a molecular biological method after hyphae grow out; s2, after the hyphae obtained in the step S1 are determined to be boletus luteus, the hyphae are transferred into a liquid culture medium to be cultured, and boletus luteus liquid is obtained and identified through a molecular biological method; and S3, after hyphae in the boletus ocellatus liquid obtained in the step S2 are determined to be boletus ocellatus, the boletus ocellatus liquid is inoculated to the roots of the masson pine aseptic seedlings, and artificial mycorrhizal seedlings of the boletus ocellatus are cultured for 3-12 months. According to the invention, rapid proliferation of hyphae and formation of artificial mycorrhiza are realized, the purity of the strain is ensured through a molecular biological method, and a technical basis is provided for artificial cultivation of the yellow indigo boletus.

Description

Technical Field

[0001] The invention relates to the field of artificial cultivation of fungi, in particular to artificial mycorrhizal seedlings of Boletus edulis and a synthesis method thereof. Background Art

[0002] Butyriboletus roseoflavus belongs to the order Boletales, Boletaceae, class Agaricomycetes. It is a special mycorrhizal fungus in Qingyuan County, Zhejiang Province, and is also a delicious edible fungus loved by local people. Under natural conditions, Butyriboletus roseoflavus mainly forms ectomycorrhiza with Pinus taiwanensis. Because it is an ectomycorrhizal fungus, it must establish a symbiotic relationship with trees to complete its life cycle and form fruiting bodies in the aboveground part. Therefore, indoor cultivation has certain difficulties. In addition, the current serious resource destruction, the reduction of wild resources and the increasing demand of consumers have led to a shortage of supply of Butyriboletus roseoflavus. In order to solve this problem, on the one hand, it is necessary to protect wild resources and rationally develop and utilize wild resources; on the other hand, the study of new artificial cultivation technologies is the most fundamental way to solve the shortage of supply, but the study of artificial cultivation technology must first solve the problem of artificial synthesis of mycorrhiza.

[0003] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0004] Based on the above-mentioned deficiencies of the prior art, the object of the present invention is to provide artificial mycorrhizal seedlings of yellow indigo boletus and a synthesis method thereof, aiming to provide artificial mycorrhizal seedlings of yellow indigo boletus, thereby realizing the artificial cultivation of yellow indigo boletus.

[0005] The technical solution of the present invention is as follows:

[0006] A first aspect of the present invention provides a method for synthesizing artificial mycorrhizal seedlings of Boletus edulis, the method comprising the following steps:

[0007] S1. Cultivating the mother strain of Boletus edulis in a solid culture medium, growing hyphae, and identifying the hyphae by molecular biology methods;

[0008] S2. After determining that the hyphae obtained in step S1 are Boletus edulis, transferring the hyphae to a liquid culture medium for culturing to obtain a Boletus edulis bacterial solution, and identifying the hyphae in the Boletus edulis bacterial solution by a molecular biology method;

[0009] S3. After determining that the mycelium in the yellow indigo bolete liquid obtained in step S2 is yellow indigo bolete, inoculate the yellow indigo bolete liquid into the roots of sterile Masson pine seedlings, and cultivate artificial mycorrhizal seedlings of yellow indigo bolete after 3 to 12 months of cultivation.

[0010] The method for synthesizing artificial mycorrhizal seedlings of yellow indigo boletus provided by the present invention comprises the following steps: firstly culturing in a solid culture medium to reduce pollution, then transferring the obtained mycelium into a liquid culture medium for continuous culturing and proliferation, and finally inoculating the root of a sterile seedling of a Masson pine, thereby realizing rapid proliferation of mycelium and formation of artificial mycorrhiza, and ensuring the purity of the strain by a molecular biological method, thereby solving the contradiction between the shortage of wild resources of yellow indigo boletus and the growth of market demand. The present invention establishes a stable model for the synthesis of artificial mycorrhizal seedlings, which can be used as a basis for establishing an artificial mycorrhizal nursery and supporting a key technology for the simulated wild cultivation of yellow indigo boletus with reasonable transplantation, providing a technical basis for on-site conservation and artificial cultivation of yellow indigo boletus, and also providing a reference for the artificial cultivation of other fungi.

[0011] Optionally, the solid culture medium is an SMN solid culture medium, and the SMN solid culture medium comprises the following components in parts by weight:

[0012] 100-200 parts of pine needles, 10-20 parts of glucose, 1 / 50-4 / 50 parts of ferric chloride hexahydrate, 1 / 50-4 / 50 parts of sodium chloride, 1 / 20-4 / 20 parts of calcium chloride, 1 / 2-1 parts of potassium dihydrogen phosphate, 1 / 4-3 / 4 parts of ammonium dihydrogen phosphate, 3 / 20-7 / 20 parts of magnesium sulfate heptahydrate, 1 / 1000000-5 / 1000000 parts of vitamin B complex, 10-20 parts of agar powder, 3-6 parts of malt extract and 1000-2000 parts of water.

[0013] In the present invention, the SMN solid culture medium is more suitable for the cultivation and growth of yellow indigo boletus than other solid culture media (such as MMN solid culture medium, which differs from SMN solid culture medium only in that it does not contain 100-200 parts of pine needles; such as PMN solid culture medium, which differs from SMN solid culture medium only in that 100-200 parts of pine needles in SMN solid culture medium are replaced by 200-400 parts of potatoes; such as PDA solid culture medium, which includes the following components in parts by mass: 200-400 parts of potatoes, 20-40 parts of glucose, 18-40 parts of agar powder and 1000-2000 parts of water), is not easy to cause pollution, and can obtain mycelium with good growth, which provides a technical basis for the synthesis of artificial mycorrhizal seedlings of yellow indigo boletus.

[0014] Optionally, the liquid culture medium is an SMN liquid culture medium, and the SMN liquid culture medium comprises the following components in parts by weight:

[0015] 100-200 parts of pine needles, 10-20 parts of glucose, 1 / 50-4 / 50 parts of ferric chloride hexahydrate, 1 / 50-4 / 50 parts of sodium chloride, 1 / 20-4 / 20 parts of calcium chloride, 1 / 2-1 parts of potassium dihydrogen phosphate, 1 / 4-3 / 4 parts of ammonium dihydrogen phosphate, 3 / 20-7 / 20 parts of magnesium sulfate heptahydrate, 1 / 1000000-5 / 1000000 parts of vitamin B complex, 3-6 parts of malt extract and 1000-2000 parts of water.

[0016] In the present invention, the SMN liquid culture medium is more suitable for the cultivation and growth of yellow indigo bolete than other liquid culture media, and more hyphae with good growth can be obtained in a short time, the growth cycle is short, and the growth environment (such as temperature, pH, etc.) is easy to control. At the same time, the obtained bacterial liquid is convenient for inoculating yellow indigo bolete on the roots of plants to obtain artificial mycorrhizal seedlings of yellow indigo bolete. The SMN liquid culture medium provided by the present invention provides a technical basis for the synthesis of artificial mycorrhizal seedlings of yellow indigo bolete, which is conducive to the artificial cultivation of yellow indigo bolete.

[0017] Optionally, in step S1, the step of culturing the mother strain of Boletus edulis in a solid culture medium to grow mycelium specifically includes:

[0018] S11, culturing the mother strain of yellow indigo boletus in a PDA test tube slant medium, and after growing mycelium, transferring it to a culture dish containing cellophane and SMN solid medium, and culturing it in the dark in an incubator at 20-30° C. (for example, 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C. or 30° C., etc., preferably 25° C.);

[0019] S12, selecting an area where mycelium grows well, and under sterile conditions, transferring the mycelium to a culture dish containing cellophane and SMN solid culture medium at a temperature of 20 to 30° C. (for example, 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C. or 30° C., etc., preferably 25° C.) to obtain pure mycelium;

[0020] Wherein, the PDA test tube slant culture medium includes the following components in parts by weight:

[0021] 200-400 parts of potatoes, 20-40 parts of glucose, 18-40 parts of agar powder and 1000-2000 parts of water.

[0022] Optionally, in step S2, the step of transferring the mycelium to a liquid culture medium for culturing to obtain a culture solution of Boletus edulis specifically comprises:

[0023] When the size of the yellow indigo boletus colony grown on the SMN solid culture medium in step S12 grows to 1 / 3 to 2 / 3 of the size of the culture dish, select the yellow indigo boletus colonies with good growth condition and no pollution as inoculation materials, cut and transfer them to SMN liquid culture medium, place them in a shaking constant temperature incubator with a temperature of 20 to 30°C (for example, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C, etc., preferably 25°C) and a rotation speed of 150 to 180rpm (for example, 150rpm, 160rpm, 170rpm or 180rpm, etc.) for dark culture, and further proliferate the mycelium to obtain the yellow indigo boletus bacterial liquid.

[0024] Optionally, in step S3, the step of inoculating the yellow indigo boletus liquid into the roots of the sterile Masson pine seedlings, and cultivating the yellow indigo boletus artificial mycorrhizal seedlings after 3 to 12 months of cultivation specifically includes:

[0025] Provide sterile Masson pine seedlings grown in nutrient soil;

[0026] Under sterile conditions, the yellow indigo boletus bacterial liquid is injected into the nutrient soil around the roots of the sterile Masson pine seedlings. After culturing for 5 to 10 days under the conditions of sterility, sufficient water, a temperature of 20 to 25° C. (for example, it can be 20° C., 21° C., 22° C., 23° C., 24° C. or 25° C., etc., preferably 25° C.), a humidity of 60% to 70% and light, the yellow indigo boletus bacterial liquid is injected into the nutrient soil around the roots of the sterile Masson pine seedlings again. After continuing to culture for 3 to 12 months (for example, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, etc.) under the conditions of sterility, sufficient water, a temperature of 20 to 25° C. (for example, it can be 20° C., 21° C., 22° C., 23° C., 24° C. or 25° C., etc., preferably 25° C.), a humidity of 60% to 70% and light, artificial mycorrhizal seedlings of yellow indigo boletus are cultivated.

[0027] Optionally, the step of providing sterile Masson pine seedlings grown in nutrient soil specifically comprises:

[0028] After soaking the Masson pine seeds in sterile water at 25 to 30° C. (for example, 25° C., 26° C., 27° C., 28° C., 29° C. or 30° C., etc.) for 1 to 3 days (for example, 1 day, 2 days or 3 days, etc.), the seeds are sterilized with a potassium permanganate solution for 30 to 60 minutes (for example, 30 minutes, 40 minutes, 50 minutes or 60 minutes, etc.), and the sterilized Masson pine seeds are repeatedly rinsed with sterile water for 2 to 3 times under sterile conditions, and the Masson pine seeds repeatedly rinsed with sterile water are sown on sterilized (for example, autoclaved) sand, and then Covering with a layer of sand 1 to 2 cm thick (for example, 1 cm, 1.2 cm, 1.5 cm, 1.8 cm or 2 cm, etc.), and then placing in a sterile environment at a temperature of 20 to 30° C. (for example, 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C. or 30° C., etc., preferably 25° C.) and a humidity of 60% to 80%, and culturing for 15 to 20 days (for example, 15 days, 16 days, 17 days, 18 days, 19 days or 20 days, etc.), the seeds can germinate to obtain Masson pine seedlings;

[0029] Under sterile conditions, the Masson pine seedlings are transplanted into a sterile culture container containing nutrient soil and cultured for three months to obtain sterile Masson pine seedlings grown in the nutrient soil.

[0030] Optionally, the mycelium is identified by a PCR method, wherein the primer pair used includes primer ITS1-F and primer ITS4b, the nucleotide sequence of primer ITS1-F is shown in SEQ ID NO: 1 (specific sequence: 5′-CTTGGTCATTTAGACGAAGTAA-3′), and the nucleotide sequence of primer ITS4b is shown in SEQ ID NO: 2 (specific sequence: 5′-CAGGAGACT TGTACACGGTCCAG-3′).

[0031] In steps S1 and S2, the PCR method can be used to identify the hyphae, the amplification of the hyphae DNA is achieved by the above-mentioned primer pair, and then the PCR product is sequenced, and the obtained result is compared with the gene sequence on the NCBI website to determine that the strain is yellow indigo bolete, and then the subsequent steps are performed. In addition, when it is measured that the hyphae in step S1 is not yellow indigo bolete, the following steps will not be continued, and when it is measured that the liquid strain (i.e., the bacterial liquid) in step S2 is not yellow indigo bolete, first check whether the liquid strain is contaminated, and then identify the hyphae that has been determined to be yellow indigo bolete in step S1 again to see if it is yellow indigo bolete. If not, stop the subsequent steps, if so, continue to culture the liquid strain, and identify the subsequent cultured liquid strain again, until it is determined to be yellow indigo bolete, and then continue the subsequent steps.

[0032] Optionally, the nutrient soil comprises the following components in parts by weight:

[0033] 50-60 parts of peat soil, 15-20 parts of perlite and 15-20 parts of rice husk.

[0034] The nutrient soil provided by the invention is suitable for the growth of Masson pine seedlings.

[0035] The second aspect of the present invention provides an artificial mycorrhizal seedling of Boletus edulis, which is obtained by the synthesis method of the present invention as described above.

[0036] Beneficial effects: The method for synthesizing artificial mycorrhizal seedlings of yellow indigo boletus provided by the present invention first cultivates in a solid culture medium to reduce pollution, and then transfers the obtained mycelium to a liquid culture medium for continued cultivation and proliferation, which can achieve rapid proliferation of mycelium and formation of artificial mycorrhizae, and ensure the purity of the strains through molecular biology technology, thereby solving the contradiction between the shortage of wild resources of yellow indigo boletus and the growth of market demand. The present invention establishes a stable model for the synthesis of artificial mycorrhizal seedlings, which can be used as a basis to establish an artificial mycorrhizal nursery and a key technology for the simulated wild cultivation of yellow indigo boletus with reasonable transplantation, providing a technical basis for the on-site conservation and artificial cultivation of yellow indigo boletus, and can also provide a reference for the artificial cultivation of other fungi. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The figure is a schematic diagram of the preparation process of artificial mycorrhizal seedlings of Boletus edulis, wherein (a) is a physical picture of Boletus edulis grown in SMN solid culture medium, (b) is a physical picture of Boletus edulis culture liquid, and (c) is a physical picture of Masson pine sterile seedlings.

[0038] Figure 2 This is a comparison chart between the DNA identification results of Boletus edulis in Example 1 and NCBI.

[0039] Figure 3 The present invention is a schematic diagram and result diagram of the identification of the artificial mycorrhizal seedlings of yellow indigo bolete in Example 1, wherein (a) is a real picture of the artificial mycorrhizal seedlings of yellow indigo bolete growing in nutrient soil, (b) is a real picture of the artificial mycorrhizal seedlings of yellow indigo bolete taken out of the nutrient soil, (c) is an enlarged picture of the roots of the artificial mycorrhizal seedlings of yellow indigo bolete, and (d) is a comparison picture of the DNA identification results of the roots of the artificial mycorrhizal seedlings of yellow indigo bolete with NCBI. DETAILED DESCRIPTION

[0040] The present invention provides artificial mycorrhizal seedlings of Boletus edulis and a synthesis method thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0041] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0042] The present invention will be further described below by means of specific examples.

[0043] The information of the culture medium used in the following examples is as follows:

[0044] The PDA test tube slant medium includes the following components in parts by weight:

[0045] 200 parts of potatoes, 20 parts of glucose, 20 parts of agar powder and 1000 parts of water.

[0046] SMN solid medium includes the following components in parts by weight:

[0047] 100 parts of pine needles, 10 parts of glucose, 1 / 50 part of ferric chloride hexahydrate, 1 / 50 part of sodium chloride, 1 / 20 part of calcium chloride, 1 / 2 part of potassium dihydrogen phosphate, 1 / 4 part of ammonium dihydrogen phosphate, 3 / 20 part of magnesium sulfate heptahydrate, 1 / 1000000 part of vitamin B complex, 20 parts of agar powder, 3 parts of malt extract and 1000 parts of water.

[0048] SMN liquid medium includes the following components by weight:

[0049] 100 parts of pine needles, 10 parts of glucose, 1 / 50 part of ferric chloride hexahydrate, 1 / 50 part of sodium chloride, 1 / 20 part of calcium chloride, 1 / 2 part of potassium dihydrogen phosphate, 1 / 4 part of ammonium dihydrogen phosphate, 3 / 20 part of magnesium sulfate heptahydrate, 1 / 1000000 part of vitamin B complex, 3 parts of malt extract and 1000 parts of water.

[0050] The nutrient soil includes the following components by weight:

[0051] 60 parts peat soil, 20 parts perlite and 20 parts rice husk.

[0052] Example 1

[0053] A method for synthesizing artificial mycorrhizal seedlings of Boletus edulis, such as Figure 1 As shown, the following steps are included:

[0054] S1. Cultivate the mother strain of Boletus edulis in SMN solid culture medium, and after growing mycelium, identify the mycelium by molecular biology method to ensure that it is Boletus edulis. Specifically, the steps include:

[0055] (1) Cultivation of mother strain and mycelium transfer

[0056] In a strict aseptic environment, the mother strain of Boletus edulis (obtained from the Edible Fungi Industry Center of Qingyuan County, Zhejiang Province) was placed in a PDA test tube slant medium, and after the mycelium grew, it was transferred to a culture dish containing cellophane and SMN solid medium, and then placed in a 25°C incubator for dark culture until abundant mycelium grew. The area with good mycelium growth was selected, and the mycelium was transferred to a culture dish containing cellophane and SMN solid medium under aseptic conditions to obtain pure mycelium, such as Figure 1 As shown in (a) in .

[0057] (2) Identification of mycelium

[0058] (a) Mycelium extraction and preservation: A small amount of mycelium was extracted and placed in a 0.2 mL centrifuge tube, and 20 μL of ES solution (i.e., ethyl acetate solution) was added.

[0059] (b) Pretreatment before DNA extraction: Heat-treat the mycelium at 95°C for 10 min using a PCR instrument.

[0060] (c) DNA stabilization and extraction: 3 volumes of BSA solution (i.e., bovine serum albumin solution) were added to the heat-treated mycelium and allowed to stand at 4° C. for one day to obtain a DNA extract of Boletus edulis.

[0061] (d) Preparation of PCR reaction system: Take a 0.2 mL PCR reaction tube, then add 1 μL of Boletus edulis DNA extract, 0.375 μL of primer ITS4b (its sequence is: 5′-CAGGAGACT TGTACACGGTCCAG-3′), 0.375 μL of primer ITS1-F (its sequence is: 5′-CTTGGTCATTTAGACGAAGTAA-3′), 6.25 μL of gold medal mix and 4 μL of sterile deionized water (ddH2O) to prepare a PCR reaction system with a total volume of 12 μL.

[0062] (e) DNA amplification: Place the prepared PCR reaction system into a PCR instrument and perform DNA amplification according to a certain procedure. The specific procedure is as follows:

[0063] Pre-denaturation at 95°C for 5 min;

[0064] Denaturation at 95°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 1 min were performed for a total of 35 cycles;

[0065] Finally, extension was performed at 72°C for 7 min;

[0066] Store at 4°C and terminate the reaction.

[0067] (f) Gene sequencing and identification: The PCR product was sequenced and the obtained detection result was compared with the gene sequence on the NCBI website to determine that the strain was Boletus edulis.

[0068] S2, transferring the hyphae obtained in step S1 to SMN liquid culture medium for culturing to obtain a yellow indigo bolete culture solution, and identifying the hyphae in the yellow indigo bolete culture solution by molecular biological methods to ensure that it is a yellow indigo bolete. Specifically comprising the following steps:

[0069] When the size of the yellow indigo boletus colony grown on the SMN solid culture medium in step S1 grows to 2 / 3 of the size of the culture dish, select the yellow indigo boletus colony with good growth condition and no pollution as the inoculation material, cut it with a scalpel and an inoculation spatula and transfer it to the SMN liquid culture medium, place it in a shaking constant temperature incubator with a temperature of 25°C and a rotation speed of 180rpm for dark culture, and further proliferate the mycelium until the mycelium is visible to the naked eye and relatively dense. Inoculation can be performed, and the yellow indigo boletus bacterial liquid (that is, the yellow indigo boletus liquid strain, such as Figure 1 , as shown in (b) in the figure).

[0070] Under sterile conditions, the mycelium of Boletus edulis cultured in SMN liquid medium was taken, rinsed with sterile water 4 times, and excess water on the mycelium of Boletus edulis was absorbed by absorbent paper. The mycelium DNA was extracted, amplified, and sequenced using the ES extraction method combined with PCR technology (for specific methods, see the mycelium identification part in step S1). The results are as follows Figure 2 As shown, it is determined to be yellow indigo boletus. That is, this step is verified again by molecular biological methods to ensure that it is yellow indigo boletus.

[0071] S3, inoculating the yellow indigo boletus bacterial liquid into the roots of the sterile Masson pine seedlings, and cultivating artificial mycorrhizal seedlings of the yellow indigo boletus after 8 months of cultivation. Specifically comprising the following steps:

[0072] Select mature and healthy Masson pine seeds, soak the Masson pine seeds in 25°C sterile water for 2 days, then use potassium permanganate solution (wherein the mass content of potassium permanganate is 0.5%) to disinfect for 30 minutes, and under sterile conditions, repeatedly rinse the sterilized Masson pine seeds with sterile water for 3 times, and evenly sow the Masson pine seeds repeatedly rinsed with sterile water on high-pressure sterilized sand, and then cover them with a layer of 1.5 cm thick sand, and then place them in a sterile environment with a temperature of 25°C and a humidity of 60% to 80%. After culturing for 20 days, the seeds can germinate and obtain Masson pine seedlings.

[0073] Under sterile conditions, transplant the Masson pine seedlings into new sterile seedling culture containers, ensure that the nutrient soil in the culture container has sufficient nutrients and appropriate moisture, and culture for three months to obtain sterile Masson pine seedlings grown in the nutrient soil (such as Figure 1 , as shown in (c) in the figure).

[0074] Under sterile conditions, use a syringe to draw 10 mL of yellow indigo boletus liquid (obtained from step S2), and accurately inject the yellow indigo boletus liquid into the nutrient soil around the roots of the above-mentioned Masson pine sterile seedlings to ensure that the yellow indigo boletus is in full contact with the roots. After the inoculated Masson pine sterile seedlings are cultured under sterile conditions, sufficient water, a temperature of 25°C, a humidity of 60% to 70%, and light (sufficient light) for 10 days, the Masson pine sterile seedlings inoculated with the yellow indigo boletus liquid are re-inoculated with the yellow indigo boletus liquid. The inoculation steps are the same as described above. The Masson pine seedlings that have been inoculated with the yellow indigo boletus liquid are cultured under sterile conditions, sufficient water, a temperature of 25°C, a humidity of 60% to 70%, and light (sufficient light) for 8 months to obtain artificial mycorrhizal seedlings of yellow indigo boletus. Figure 3 As shown, the mycorrhizal seedlings were sampled and identified by molecular biology method (for specific methods, see the part of identifying mycelium in step S1), and the result was yellow indigo boletus.

[0075] In summary, the method for synthesizing artificial mycorrhizal seedlings of yellow indigo bolete provided by the present invention can achieve rapid proliferation of hyphae and formation of mycorrhizae through specific culture medium and culture conditions, and ensure the purity of strains through molecular biology technology, thereby solving the contradiction between the shortage of wild resources of yellow indigo bolete and the growth of market demand. The present invention establishes a stable model for the synthesis of artificial mycorrhizal seedlings, which can be used as a basis to establish an artificial mycorrhizal nursery and a key technology for the simulated wild cultivation of yellow indigo bolete with reasonable transplantation, providing a technical basis for the on-site conservation and artificial cultivation of yellow indigo bolete, and can also provide a reference for the artificial cultivation of other fungi.

[0076] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for synthesizing artificial mycorrhizal seedlings of Boletus edulis, characterized in that: The method for synthesizing the artificial mycorrhizal seedlings of Boletus edulis comprises the following steps: S1. Cultivating the mother strain of Boletus edulis in a solid culture medium, growing hyphae, and identifying the hyphae by molecular biology methods; S2. After determining that the hyphae obtained in step S1 are Boletus edulis, transferring the hyphae to a liquid culture medium for culturing to obtain a Boletus edulis bacterial solution, and identifying the hyphae in the Boletus edulis bacterial solution by a molecular biology method; S3. After determining that the mycelium in the yellow indigo bolete liquid obtained in step S2 is yellow indigo bolete, inoculate the yellow indigo bolete liquid into the roots of sterile Masson pine seedlings, and cultivate artificial mycorrhizal seedlings of yellow indigo bolete after 3 to 12 months of cultivation.

2. The synthesis method according to claim 1, characterized in that The solid culture medium is an SMN solid culture medium, and the SMN solid culture medium comprises the following components in parts by weight: 100-200 parts of pine needles, 10-20 parts of glucose, 1 / 50-4 / 50 parts of ferric chloride hexahydrate, 1 / 50-4 / 50 parts of sodium chloride, 1 / 20-4 / 20 parts of calcium chloride, 1 / 2-1 parts of potassium dihydrogen phosphate, 1 / 4-3 / 4 parts of ammonium dihydrogen phosphate, 3 / 20-7 / 20 parts of magnesium sulfate heptahydrate, 1 / 1000000-5 / 1000000 parts of vitamin B complex, 10-20 parts of agar powder, 3-6 parts of malt extract and 1000-2000 parts of water.

3. The synthesis method according to claim 2, characterized in that The liquid culture medium is an SMN liquid culture medium, and the SMN liquid culture medium comprises the following components in parts by weight: 100-200 parts of pine needles, 10-20 parts of glucose, 1 / 50-4 / 50 parts of ferric chloride hexahydrate, 1 / 50-4 / 50 parts of sodium chloride, 1 / 20-4 / 20 parts of calcium chloride, 1 / 2-1 parts of potassium dihydrogen phosphate, 1 / 4-3 / 4 parts of ammonium dihydrogen phosphate, 3 / 20-7 / 20 parts of magnesium sulfate heptahydrate, 1 / 1000000-5 / 1000000 parts of vitamin B complex, 3-6 parts of malt extract and 1000-2000 parts of water.

4. The synthesis method according to claim 3, characterized in that In step S1, the mother strain of Boletus edulis is cultured in a solid culture medium, and the step of growing mycelium specifically includes: S11, culturing the mother strain of Boletus edulis in a PDA test tube slant medium, and after growing hyphae, transferring the mother strain to a culture dish containing cellophane and SMN solid medium, and culturing in a dark incubator at 20-30°C; S12, selecting an area where mycelium grows well, and under sterile conditions, transferring the mycelium to a culture dish containing cellophane and SMN solid culture medium and culturing in the dark at a temperature of 20 to 30° C. to obtain pure mycelium; Wherein, the PDA test tube slant culture medium includes the following components in parts by weight: 200-400 parts of potatoes, 20-40 parts of glucose, 18-40 parts of agar powder and 1000-2000 parts of water.

5. The synthesis method according to claim 4, characterized in that In step S2, the mycelium is transferred to a liquid culture medium for culturing to obtain a yellow indigo boletus bacterial liquid, which specifically includes: When the size of the yellow indigo boletus colony grown on the SMN solid culture medium in step S12 grows to 1 / 3 to 2 / 3 of the size of the culture dish, the yellow indigo boletus colonies with good growth conditions and no pollution are selected as inoculation materials, cut and transferred to the SMN liquid culture medium, and placed in a shaking constant temperature incubator with a temperature of 20 to 30°C and a rotation speed of 150 to 180 rpm for dark culture to further proliferate the mycelium and obtain the yellow indigo boletus culture liquid.

6. The synthesis method according to claim 1, characterized in that In step S3, the steps of inoculating the yellow indigo boletus liquid into the roots of the sterile Masson pine seedlings and cultivating the yellow indigo boletus artificial mycorrhizal seedlings after 3 to 12 months of cultivation specifically include: Provide sterile Masson pine seedlings grown in nutrient soil; Under aseptic conditions, a fungus solution of yellow indigo boletus is injected into the nutrient soil around the roots of the sterile Masson pine seedlings, and the soil is cultured for 5 to 10 days under the conditions of aseptic conditions, sufficient water, a temperature of 20 to 25° C., a humidity of 60% to 70% and light. Then, the fungus solution of yellow indigo boletus is injected into the nutrient soil around the roots of the sterile Masson pine seedlings again, and the soil is cultured for 3 to 12 months under the conditions of aseptic conditions, sufficient water, a temperature of 20 to 25° C., a humidity of 60% to 70% and light, thereby cultivating artificial mycorrhizal seedlings of yellow indigo boletus.

7. The synthesis method according to claim 6, characterized in that The step of providing the aseptic seedlings of Masson pine growing in the nutrient soil specifically comprises: After soaking Masson pine seeds in sterile water at 25-30° C. for 1-3 days, the seeds are sterilized with potassium permanganate solution for 30-60 minutes, and the sterilized Masson pine seeds are repeatedly rinsed with sterile water for 2-3 times under sterile conditions. The Masson pine seeds repeatedly rinsed with sterile water are sown on sterilized sand, and then covered with a layer of sand 1-2 cm thick, and then placed in a sterile environment with a temperature of 20-30° C. and a humidity of 60%-80% and cultured for 15-20 days, the seeds can germinate, and Masson pine seedlings are obtained; Under sterile conditions, the Masson pine seedlings are transplanted into a sterile culture container containing nutrient soil and cultured for three months to obtain sterile Masson pine seedlings grown in the nutrient soil.

8. The synthesis method according to claim 1, characterized in that The hyphae were identified by PCR method, wherein the primer pair used included primer ITS1-F and primer ITS4b, the nucleotide sequence of primer ITS1-F was shown in SEQ ID NO: 1, and the nucleotide sequence of primer ITS4b was shown in SEQ ID NO:

2.

9. The synthesis method according to claim 6, characterized in that: The nutrient soil comprises the following components in parts by weight: 50-60 parts of peat soil, 15-20 parts of perlite and 15-20 parts of rice husk.

10. An artificial mycorrhizal seedling of Boletus edulis, characterized in that: Obtained by the synthesis method according to any one of claims 1 to 9.

Images