An efficient method for artificial synthesis of Lactarius deliciosus mycorrhiza

Abstract

The present invention relates to an efficient method for artificially synthesizing Lactarius deliciosus mycorrhiza, which comprises the following steps: (1) Take the mycelium with a size of 1-3 mm at the connection of the stipe and cap of fresh Lactarius deliciosus fruit bodies, place it in a PDA medium, and conduct tissue culture at 20-25 °C for 20-30 days. Then, take the mycelium block and inoculate it onto the PDA medium, and continue to culture at 20-25 °C for 25-40 days. Subsequently, take the mycelium block again and inoculate it into a PDB medium for culturing for 30-50 days to obtain Lactarius deliciosus liquid spawn; (2) Transplant pine seedlings at a density of 6-15 seedlings per basket into a culture basket filled with mycorrhizal substrate, add the Lactarius deliciosus liquid spawn prepared in step (1) to the substrate 3-5 cm deep near the root of each seedling, and mycorrhiza can be formed after culturing for 10-14 days. The present invention scientifically screens the substrate and method for Lactarius deliciosus. By using the optimized medium formula and culture method, the mycelium of Lactarius deliciosus grows faster and the biomass of the obtained Lactarius deliciosus mycelium is larger; inoculating the plant roots with liquid spawn greatly improves the inoculation efficiency; multiple inoculated host plants are cultured in the same culture device, which greatly shortens the mycorrhiza formation time after inoculation and effectively improves the mycorrhiza synthesis rate; reducing the amount of peat soil in the mycorrhizal substrate reduces the proportion of mycorrhiza formed by miscellaneous bacteria and pine roots.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an efficient method for artificially synthesizing Lactarius deliciosus mycorrhiza. Background Art

[0002] Lactarius deliciosus, also known as delicious milk mushroom and pine mushroom, belongs to the Russulaceae family and the Lactarius genus. This mushroom is widely distributed around the world and is found in regions such as Northeast China, Yunnan, Guizhou, and Sichuan in China. It has important economic and ecological values. Lactarius deliciosus is one of the common ectomycorrhizal edible mushrooms. It has tender flesh, a unique and delicious taste, is rich in nutrients, and contains highly nutritious mineral elements and amino acids (such as glutamic acid, aspartic acid, etc.). It is regarded as a precious product in the mountains and is widely consumed and sold at home and abroad. It is an important source of income for many local people. At the same time, Lactarius deliciosus also has medicinal values such as anti-tumor, antibacterial, and immune-enhancing effects.

[0003] Lactarius deliciosus is an ectomycorrhizal edible mushroom, and its cultivation technology is more complex than that of non-ectomycorrhizal basidiomycetes (such as Ganoderma lucidum, Lentinula edodes, Pleurotus eryngii, etc.). The cultivation of Lactarius deliciosus can only rely on artificially synthesizing mycorrhiza and then transplanting it outdoors for artificial management to produce mushrooms. Existing methods for artificially synthesizing Lactarius deliciosus mycorrhiza have defects such as low inoculation efficiency, low mycorrhiza synthesis rate, long time required for mycorrhiza formation, and high contamination rate of miscellaneous bacteria, which limit the research on artificial cultivation of Lactarius deliciosus and the application of Lactarius deliciosus mycorrhiza in afforestation. Therefore, there is an urgent need to study an efficient method for artificially synthesizing Lactarius deliciosus mycorrhiza. Summary of the Invention

[0004] The purpose of the present invention is to provide an efficient method for artificially synthesizing Lactarius deliciosus mycorrhiza, which includes the following steps:

[0005] (1) Take the mycelium of 1 - 3 mm in size at the connection of the stipe and cap of a fresh Lactarius deliciosus fruiting body, place it in a PDA medium, and conduct tissue culture at 20 - 25 °C for 20 - 30 days. Then, take the mycelium block and inoculate it onto the PDA medium, and continue to culture at 20 - 25 °C for 25 - 40 days. Then, take the mycelium block again and inoculate it into a PDB medium, and culture it at 20 - 25 °C and 120 - 180 rpm for 30 - 50 days to obtain Lactarius deliciosus liquid spawn;

[0006] (2) Transplant pine seedlings at a density of 6 - 15 seedlings per basket into a culture basket filled with mycorrhizal substrate. Then, add the Lactarius deliciosus liquid spawn prepared in step (1) to the substrate at a depth of 3 - 5 cm near the root of each seedling at a concentration of 5 - 10 ml. Place the culture basket in an environment of 18 - 20 °C, 10000 - 20000 lx, and a relative humidity (RH) of 90 - 95% for cultivation, and mycorrhiza can be formed after 10 - 14 days.

[0007] In the preferred technical solution of the present invention, the PDA medium contains 20 - 25% potatoes, 0.5 - 1% glucose, 1 - 5% agar, and the rest is distilled water.

[0008] In the preferred technical solution of the present invention, the PDB medium contains 20 - 25% potatoes, 2 - 5% soluble starch, 0.1 - 1% potassium nitrate, 0.002 - 0.01% vitamin B1, and the rest is distilled water.

[0009] In the preferred technical solution of the present invention, the mycelial blocks inoculated into the PDA medium are mycelial blocks without contamination by miscellaneous bacteria, with strong growth, and not aged.

[0010] In the preferred technical solution of the present invention, the mycelial blocks inoculated into the PDB medium are mycelial blocks with a length and width of 1 mm, and the number is 8 - 10 pieces.

[0011] In the preferred technical solution of the present invention, the cultivation method of the pine genus plant seedlings is as follows: after soaking the pine genus plant seeds in clean water for 36 - 48 h, then soaking the seeds in 30% hydrogen peroxide for 30 - 60 min, filtering, rinsing the seeds with sterile water, and then evenly placing them in a cultivation basket. A 1 - 3 cm thick seed germination substrate is covered on the surface of the seeds, watering regularly, and culturing for 50 - 60 days under the conditions of a day length of 14 - 16 h, a night length of 8 - 10 h, and a temperature of 16 - 20 °C, thus obtaining the pine genus plant seedlings. The seed culture substrate is uniformly mixed by perlite and vermiculite according to a volume ratio of 1 - 3:1.

[0012] In the preferred technical solution of the present invention, the mycorrhizal culture substrate in step (2) is uniformly mixed by peat soil and vermiculite according to a volume ratio of 0.5 - 2:1.

[0013] In the preferred technical solution of the present invention, the planting spacing between the pine genus plant seedlings ≥ 8 cm × 8 cm.

[0014] In the preferred technical solution of the present invention, the transplanting density of the pine genus plant seedlings is 8 - 12 plants / basket.

[0015] In the preferred technical solution of the present invention, the upper mouth of the cultivation basket is 25 cm * 35 cm; the lower mouth is 20 cm * 30 cm.

[0016] In the preferred technical solution of the present invention, 20 - 30 holes are punched in the bottom surface of the cultivation basket, preferably 25 - 28 holes are punched.

[0017] In the preferred technical solution of the present invention, the mycorrhizal synthesis rate of Lactarius deliciosus is greater than 95%, preferably greater than 99%, and more preferably 100%.

[0018] In the preferred technical solution of the present invention, the proportion of miscellaneous bacteria forming mycorrhizae with Pinus plants is less than 5%, preferably less than 1%, and more preferably 0.

[0019] Another object of the present invention is to provide the application of the method for efficiently artificially synthesizing Lactarius deliciosus mycorrhizae of the present invention in the afforestation of Pinus plant seedlings.

[0020] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between a liquid and a solid, the percentage is volume / weight percentage; when the present invention relates to the percentage between a solid and a liquid, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0021] Unless otherwise specified, the following detection methods are adopted in the present invention:

[0022] 1. Mycorrhiza identification: Extract mycorrhiza DNA with a fungal genomic DNA extraction kit, perform PCR amplification using ITS4 and ITS5 as primers, and perform Blast alignment after sequencing the PCR products to confirm that the synthesized mycorrhiza is Lactarius deliciosus mycorrhiza.

[0023] 2. Lactarius deliciosus mycorrhiza synthesis rate = the number of Pinus plant seedlings with synthesized Lactarius deliciosus mycorrhizae in the culture basket / the total number of all Pinus plant seedlings in the culture basket × 100%.

[0024] 3. Contamination rate of miscellaneous bacteria mycorrhiza = the number of Pinus plant seedlings with synthesized miscellaneous bacteria mycorrhizae in the culture basket / the total number of all Pinus plant seedlings in the culture basket × 100%.

[0025] Compared with the prior art, the beneficial technical effects of the present invention include:

[0026] 1. The present invention scientifically screens the culture medium and method for Lactarius deliciosus strains. The Lactarius deliciosus hyphae grow faster, and the biomass of the obtained Lactarius deliciosus hyphae is larger. Multiple inoculated host plants are cultured in the same culture device. After the Lactarius deliciosus hyphae germinate near the roots of different plants, they can infect each other, improving the inoculation efficiency, greatly shortening the time for the formation of Lactarius deliciosus mycorrhizae after inoculation, effectively increasing the mycorrhiza synthesis rate, and reducing the proportion of miscellaneous bacteria forming mycorrhizae with the roots of Pinus plants.

[0027] 2. The operation of the present invention is simple, the proportion of Pinus plants forming mycorrhizae with miscellaneous bacteria is low, the cost is low, and it is suitable for large-scale production. Moreover, the method for mycorrhizalization of pine trees in the present invention can also improve the survival rate of Pinus plant seedlings after afforestation, which is beneficial to green ecological protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Growth conditions of Lactarius deliciosus mycorrhizae in Examples 1 - 3

[0029] Figure 2Cultivation conditions of Pinus seedlings after artificial synthesis of mycorrhizae in Examples 1-3 Detailed implementation mode

[0030] The present invention will be described below with reference to the examples. However, the present invention is not limited to the examples.

[0031] Tested strains: Lactarius deliciosus, from Huidong County, Liangshan Prefecture, Sichuan Province.

[0032] Pinus seeds: Pinus armandii or Pinus massoniana, purchased from Huidong County, Liangshan Prefecture, Sichuan Province and Dazhou City, Sichuan Province respectively.

[0033] Preparation of Pinus seedlings: Soak Pinus seeds in clear water for 48 h, then soak the seeds in 30% hydrogen peroxide for 30 min, filter, and rinse the seeds with sterile water 3 times. Bury the treated seeds in the seed germination medium, 1 cm deep from the surface; set the light source and temperature according to the plant photoperiod of 16 h during the day at 18 °C and 8 h at night at 16 °C, water every day, and cultivate the seedlings for 2 months to obtain Pinus seedlings.

[0034] Peat soil, perlite and vermiculite: Commercially available.

[0035] Polypropylene culture basket: The upper opening is 25 cm wide and 35 cm long; the lower opening is 20 cm wide and 30 cm long, with a height of 19 cm, and 25 holes are drilled at the bottom, with a hole diameter of 4 mm.

[0036] PDA medium: 200 g potatoes, 20 g glucose, 15 g agar, 1 L distilled water.

[0037] PDB medium: 200 g potatoes, 20 g soluble starch, 1 g potassium nitrate, 20 mg vitamin B1, add distilled water to 1 L.

[0038] Seed germination medium: After mixing perlite and vermiculite evenly according to the volume ratio of 1:1, place it in an autoclave at 121 °C for 2 h, cool, and reserve.

[0039] Mycorrhizal medium: Mix peat soil and vermiculite evenly according to the volume ratio of 2:1, place it in an autoclave at 121 °C for 2 h, cool, and reserve.

[0040] Example 1

[0041] The artificial synthesis method of the Lactarius deliciosus mycorrhizae of the present invention comprises the following steps:

[0042] (1) After disinfecting the collected Lactarius deliciosus fruit bodies, take a 4-mm piece of flesh from the junction of the stipe and the cap, transfer it to a PDA medium, and culture it at 25 °C for 20 days. Then, take a mycelium block without contamination by miscellaneous bacteria, growing vigorously, and not aging, inoculate it onto the PDA medium, and culture it for about 20 days until the colony diameter is greater than 2 cm. Use a surgical blade to cut it into inoculation blocks with a length and width of about 1 mm, and inoculate them again into a PDB medium. Culture them at 23 °C and 120 rpm for 40 days to obtain the Lactarius deliciosus liquid spawn;

[0043] (2) Transfer 9 pine seedlings into 1 culture basket filled with mycorrhizal substrate, and inject 5 ml of the Lactarius deliciosus liquid spawn obtained in step (1) into the substrate about 5 cm away from the surface near the root system of each seedling. Culture them at a temperature of 16 - 18 °C, with a light intensity of 10000 lx, and a humidity of 90 - 95% for 10 days, and obvious mycorrhizal formation can be seen.

[0044] Example 2

[0045] The method for artificially synthesizing the mycorrhiza of Lactarius deliciosus of the present invention includes the following steps:

[0046] (1) After disinfecting the collected Lactarius deliciosus fruit bodies, take a 4-mm piece of flesh from the junction of the stipe and the cap, transfer it to a PDA medium, and culture it at 25 °C for 20 days. Then, take a mycelium block without contamination by miscellaneous bacteria, growing vigorously, and not aging, inoculate it onto the PDA medium, and culture it for about half a month until the colony diameter is greater than 2 cm. Use a surgical blade to cut it into inoculation blocks with a length and width of about 1 mm, and inoculate them again into a PDB medium. Culture them at 23 °C and 120 rpm for 30 days to obtain the Lactarius deliciosus liquid spawn;

[0047] (2) Transfer 10 pine seedlings into 1 culture basket filled with mycorrhizal substrate, and inject 8 ml of the Lactarius deliciosus liquid spawn obtained in step (1) into the substrate about 5 cm away from the surface near the root system of each seedling. Culture them at a temperature of 16 - 18 °C, with a light intensity of 10000 lx, and a humidity of 90 - 95% for 12 days, and obvious mycorrhizal formation can be seen.

[0048] Example 3

[0049] The method for artificially synthesizing the mycorrhiza of Lactarius deliciosus of the present invention includes the following steps:

[0050] (1) After disinfecting the collected Lactarius deliciosus fruit bodies, take a 4-mm mycelium piece at the connection between the stipe and the cap, transfer it to a PDA medium, and culture it at 25 °C for 20 days. Then, take a mycelium block without contamination by miscellaneous bacteria, with strong growth and not aging, and inoculate it onto the PDA medium. After culturing for about half a month until the colony diameter is greater than 2 cm, use a surgical blade to cut it into inoculation blocks with a length and width of about 1 mm, and inoculate them again into a PDB medium. Culture them under the conditions of 23 °C and 120 rpm for 30 days to obtain the Lactarius deliciosus liquid spawn;

[0051] (2) Transfer 12 Pinus seedlings into a cultivation basket filled with mycorrhizal substrate, and inject 10 ml of the Lactarius deliciosus liquid spawn obtained in step (1) into the substrate about 5 cm from the surface near the root system of each seedling. After culturing at a temperature of 16 - 18 °C, a light intensity of 10000 lx, and a humidity of 90 - 95% for 10 days, obvious mycorrhizal formation can be seen.

[0052] Experimental Example 1

[0053] Take some of the mycorrhizae prepared in Examples 1 - 3, extract the mycorrhizal DNA using a fungal genomic DNA extraction kit, perform PCR amplification using ITS4 and ITS5 as primers, and conduct Blast alignment after sequencing the PCR products to confirm that the mycorrhizae are Lactarius deliciosus mycorrhizae. The mycorrhizal synthesis rate of the Lactarius deliciosus mycorrhizae prepared in Examples 1 - 3 is greater than 99%, and the proportion of miscellaneous bacteria forming mycorrhizae with Pinus plants is less than 1%.

[0054] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or deformations according to the present invention. As long as they do not depart from the spirit of the present invention, they shall fall within the scope of protection of the claims of the present invention.

Claims

1. An efficient method for artificially synthesizing Lactarius deliciosus mycorrhiza, characterized in that, It includes the following steps: (1) Take the mycelium at the connection of the stipe and cap of fresh Lactarius deliciosus fruit bodies with a size of 1 - 3 mm, place it in a PDA medium, and conduct tissue culture at 20 - 25 °C for 20 - 30 days. Then, take the mycelium block and inoculate it onto the PDA medium, and continue to culture at 20 - 25 °C for 25 - 40 days. Next, take the mycelium block and inoculate it into a PDB medium, and culture it at 20 - 25 °C and 120 - 180 rpm for 30 - 50 days to obtain the Lactarius deliciosus liquid spawn; (2) Transplant the seedlings of Pinus plants into culture baskets filled with mycorrhizal substrate at a density of 6 - 15 seedlings / basket. Then, add the Lactarius deliciosus liquid spawn prepared in step (1) to the substrate at a depth of 3 - 5 cm near the root of each seedling at a concentration of 5 - 10 ml. Place the culture baskets in an environment with a temperature of 16 - 22 °C, a light intensity of 10000 - 20000 lx, and a relative humidity (RH) of 90 - 95% for culture. Mycorrhiza can be formed after 10 - 14 days; The mycorrhizal substrate in step (2) is composed of peat soil: vermiculite in a volume ratio of 0.5 - 2: 1 and is evenly mixed; The PDA medium contains 20 - 25% potatoes, 0.2 - 1% glucose, 1.5 - 2.5% agar, and the rest is distilled water; The PDB medium contains 20 - 25% potatoes, 2 - 5% soluble starch, 0.1 - 1% potassium nitrate, 0.002 - 0.01% vitamin B1, and the rest is distilled water; The cultivation method of the Pinus plant seedlings is as follows: soak the seeds of Pinus armandii or Pinus massoniana in clean water for 36 - 48 h, then soak the seeds in 30% hydrogen peroxide for 30 - 60 min, filter, rinse the seeds with sterile water, and then evenly place them in the culture baskets. Cover the surface of the seeds with 1 - 3 cm thick seed germination substrate, water regularly, and set the day length to 14 - 16 h and the night length to 8 - 10 h, and raise seedlings at 18 - 20 °C for 2 - 3 months to obtain them. The seed substrate is composed of perlite: vermiculite in a volume ratio of 1 - 3:1 and is evenly mixed; The planting spacing between the Pinus plant seedlings is ≥8 cm × 8 cm.

2. The method according to claim 1, wherein The mycelium block inoculated onto the PDA medium is a mycelium block without contamination by miscellaneous bacteria, with strong growth and not aging.

3. The method according to claim 1, wherein The mycelium block inoculated onto the PDB medium is a mycelium block with a length and width of 1 mm, and the number is 8 - 10 pieces.

4. The method according to claim 1, wherein The transplanting density of the Pinus plant seedlings is 8 - 12 seedlings / basket.

5. The method according to claim 1, characterized in that, The upper mouth of the culture basket is 25 cm * 35 cm in width and length; the lower mouth is 20 cm * 30 cm in width and length.

6. The method according to claim 1, wherein Twenty to thirty holes are drilled on the bottom surface of the culture basket.

7. The method according to claim 6, wherein Twenty-five to twenty-eight holes are drilled on the bottom surface of the culture basket.

8. The method according to any one of claims 1 to 7, characterized in that The mycorrhizal synthesis rate of Lactarius deliciosus is greater than 95%.

9. The method according to claim 8, wherein The mycorrhizal synthesis rate of Lactarius deliciosus is greater than 99%.

10. The method according to claim 9, characterized in that, The mycorrhizal synthesis rate of Lactarius deliciosus is 100%.

11. The method according to any one of claims 1 to 7, characterized in that, The proportion of miscellaneous bacteria forming mycorrhiza with Pinus plants is less than 5%.

12. The method according to claim 11, wherein The proportion of miscellaneous bacteria forming mycorrhiza with Pinus plants is less than 1%.

13. The method according to claim 12, wherein The proportion of miscellaneous bacteria forming mycorrhiza with Pinus plants is 0.

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