Method for culturing ectomycorrhiza of brilliant lactarius deliciosus

Abstract

The invention discloses a method for culturing ectomycorrhiza of brilliant lactarius deliciosus, which comprises the following steps: S1, culturing brilliant lactarius deliciosus on a culture medium A to obtain a brilliant lactarius deliciosus colony; s2, disinfecting the seeds of the pinus massoniana; s3, the disinfected seeds are inoculated to a culture medium B to be cultured until the seeds germinate to form masson pine seedlings, after the masson pine seedlings grow lateral roots, the masson pine seedlings are moved to a culture medium C, the brilliant lactarius deliciosus colonies are inoculated to the edge of the roots, and ectomycorrhiza of the brilliant lactarius deliciosus on the masson pine root system is obtained after culture. Through the construction of a sterile in-vitro culture system, four defects of a traditional soil culture method are systematically solved, the research on a brilliant lactarius deliciosus-masson pine symbiosis mechanism can be accelerated, and efficient technical support is provided for mycorrhizal seedling production, forest ecological restoration and artificial domestication of edible mushrooms.

Description

Technical Field

[0001] The invention relates to the technical field of edible fungi cultivation, in particular to an in vitro cultivation method of the ectomycorrhiza of Pleurotus eryngii. Background Art

[0002] Bright milk mushroom (lactariusvividus ) is a famous wild edible mushroom belonging to the phylum Basidiomycota ( Basidiomycota ), Agaricus ( Agaricomycetes ), Russula family ( Russulaceae ), Lactarius ( Lactarius) , which is widely distributed in the central and southern regions of my country. Its fruiting bodies are tender and delicious, rich in nutrients, and have a large market demand. However, as a mycorrhizal fungus, the bright milk mushroom must form ectomycorrhizas with the roots of symbiotic tree species (such as Masson pine) to complete its life history. Ectomycorrhiza is a symbiotic body with specific structure and function formed by mycorrhizal fungi infecting the roots of trees. It plays an important role in improving the mineral nutrition and water absorption of host plants, enhancing plant resistance to stress and maintaining the stability of forest ecosystems. A large number of studies have shown that the formation of ectomycorrhizas is beneficial to the growth of Masson pine. Therefore, studying the principles, mechanisms and phenomena of the interaction between bright milk mushrooms and Masson pine roots has considerable research value and expected economic benefits. However, the ectomycorrhizas of bright milk mushrooms currently used for research are generally achieved by traditional soil cultivation methods, which have the following defects: (1) Difficulty in sterile pure culture: On the one hand, Pinus massoniana seeds often contain bacteria and fungi, which are difficult to sterilize and difficult to sterilize on plates. On the other hand, the traditional method of soil culture through substrate cannot eliminate the interference of other microorganisms, and the external environment has a greater impact on the root system and mycorrhizal fungi, causing interference and difficulties in the study of the interaction mechanism.

[0003] (2) Long cycle: The traditional soil culture method not only requires the sterilization of the culture soil matrix, but also takes up a lot of space. In addition, the experimental cycle is long, and it takes about 40 days to establish a mycorrhizal symbiotic relationship.

[0004] (3) It is not conducive to the observation of the establishment process of the mycorrhizal system: the traditional soil matrix culture of Pinus massoniana root system cannot intuitively observe the root distribution and the establishment process of mycorrhizal interaction.

[0005] (4) It is not conducive to the separation of mycorrhizal system: the process of separating the root system from the soil and other matrices is likely to cause damage to the root system, and the soil matrix adhering to the mycorrhizal structure is difficult to remove, which will affect the accuracy of subsequent experimental analysis. Summary of the Invention

[0006] The present invention provides an in vitro culture method of the ectomycorrhiza of Pleurotus eryngii that can realize soilless in vitro culture, so as to solve the technical problems existing in the existing soil culture method mentioned in the background technology.

[0007] In order to solve the above technical problems, the technical solution proposed by the present invention is: A method for cultivating the ectomycorrhiza of bright-colored milk mushroom comprises the following steps: S1. Cultivating the bright-colored milk mushroom on culture medium A to obtain a bright-colored milk mushroom colony; S2. Disinfecting the seeds of Pinus massoniana; S3, the sterilized seeds are inoculated onto culture medium B and cultured until germination forms Pinus massoniana seedlings, after the Pinus massoniana seedlings grow lateral roots, the Pinus massoniana seedlings are moved onto culture medium C, and the bright-colored Lactarius colony is inoculated at the root edge to obtain the ectomycorrhiza of bright-colored Lactarius on the root system of Pinus massoniana after cultivation.

[0008] The present invention eliminates interference from other microorganisms through sterile culture medium and seed disinfection steps, thus achieving a pure environment for studying mycorrhizal interaction mechanisms. The in vitro culture system does not require soil sterilization and complex space, which can shorten the mycorrhizal symbiotic relationship establishment cycle and research time. In addition, in vitro culture allows direct observation of root distribution and the dynamic process of mycorrhizal formation. During subsequent separation, there is no soil matrix adhesion, which avoids root damage and facilitates subsequent mycorrhizal structure separation and analysis.

[0009] As a further preferred embodiment of the above technical solution, in S2, when the seeds of the masson pine are disinfected, a disinfectant, a fungicide and sterile water are used to treat the seeds in sequence, wherein the disinfectant includes Tween 20 and potassium permanganate, and the fungicide includes streptomycin. In soilless in vitro culture, how to effectively disinfect the seeds of the masson pine is one of the key factors to ensure that the research can be successfully implemented. The present invention uses a disinfectant and a fungicide to treat the seeds in sequence, and uses sterile water for cleaning, uses Tween 20 to enhance the surface wettability of the seeds, uses potassium permanganate to efficiently kill surface fungi / bacteria, and uses streptomycin to specifically inhibit residual bacteria, breaking through the problem of sterilization of masson pine seeds, improving the aseptic germination rate, and reducing experimental interference.

[0010] As a further preferred embodiment of the above technical solution, the volume fraction of Tween 20 in the disinfectant is 0.01%~0.02%, the addition amount of potassium permanganate in the disinfectant is 0.005g / mL~0.01g / mL; and the addition amount of streptomycin in the bactericide is 50μg / mL~200μg / mL.

[0011] As a further preferred embodiment of the above technical solution, in S2, after the Masson pine seeds germinate to form Masson pine seedlings, the Masson pine seedlings are re-inoculated onto culture medium B with a sterile filter paper on the surface, and culture is continued until the Masson pine seedlings grow lateral roots.

[0012] As a further preferred embodiment of the above technical solution, in S3, the surface of the culture medium C is covered with cellophane. The sterile filter paper and cellophane can prevent the Masson pine from growing into the culture medium.

[0013] As a further preferred embodiment of the above technical solution, the culture media A, B, and C are sterilized before use; the sterilization process includes the following steps: treating the culture media A, B, and C with high-temperature steam at 121°C for 20 minutes, followed by drying, and pouring the culture media into a mold for molding when the temperature reaches 50°C. This sterilization process ensures thorough sterilization of the culture media, maintains the stability of the sterile culture environment, and prevents high temperatures from damaging heat-sensitive components.

[0014] As a further preference of the above technical solution, the culture medium A and / or culture medium C is an MMN optimized culture medium, and the MMN optimized culture medium comprises the following components in parts by weight: 30 parts of malt extract powder, 100 parts of glucose, 5 parts of potassium dihydrogen phosphate, 2.5 parts of diammonium hydrogen phosphate, 1.5 parts of magnesium sulfate heptahydrate, 0.5 parts of calcium chloride, 0.25 parts of sodium chloride, 0.12 parts of ferric chloride, 0.101 parts of vitamin B, 1 part of myristic acid, 10 parts of mycopeptone, 90 parts of agar powder and distilled water.

[0015] Currently, domestic and international research has shown that the mycelium of Lactarius variegatus can grow on symbiotic culture media such as MMN (Modified Melin-Norkrans Medium). Therefore, existing techniques often use ordinary MMN medium to culture Lactarius variegatus. However, the mycelium grows slowly on this medium, requiring approximately 40 days to form large colonies, significantly increasing the research cycle. This invention, for the first time, adds the fatty acid myristic acid as a carbon source for the growth of Lactarius variegatus, and adds peptone to optimize the nitrogen source. By leveraging the advantages and synergistic effects of these two, the culture cycle of Lactarius variegatus mycelium is shortened, as is the time required for the mycelium to infect the root system, thereby reducing the time cost of research experiments.

[0016] As a further preferred embodiment of the above technical solution, in S1, before culturing the bright milk mushroom, taking the bright milk mushroom strain and inoculating it on a common MMN culture medium, and culturing it in a dark environment at 25° C. for 30 days to form a large colony as a raw material for culturing the bright milk mushroom.

[0017] As a further preferred embodiment of the above technical solution, the colonies formed by activation culture are evenly inoculated on culture medium A. After inoculation, the lid is covered and sealed with a sealing film. The culture is carried out in a dark environment at 25° C. for 3 weeks to obtain bright-colored milk mushroom colonies.

[0018] As a further preferred embodiment of the above technical solution, in S3, after inoculating the Lactarius variegata colony at the root margin, the root portion of the Masson pine is shaded with aluminum foil. The culture is cycled at 25°C for four weeks, with a cycle of 16 hours in the light and 8 hours in the dark, to obtain ectomycorrhizae of Lactarius variegata on the Masson pine root system. This culture method simulates the natural photoperiod, promoting seedling photosynthesis and coordinated hyphal growth. Shading the root system can reduce root light stress and optimize the mycorrhizal symbiotic environment.

[0019] The present invention has the following beneficial effects: The present invention systematically solves the four major defects of traditional soil culture methods by constructing a sterile in vitro culture system: Sterility assurance: Multi-stage disinfection strategy (seed surface treatment + culture medium sterilization) and closed culture environment eliminate microbial interference and achieve precise control of mycorrhizal interaction mechanism research.

[0020] The cycle is greatly shortened: the culture medium formula and in vitro culture conditions are optimized to significantly improve the mycorrhizal symbiosis efficiency and shorten the establishment cycle of the symbiotic system.

[0021] Visualization and non-destructive operation: Cellophane media supports dynamic observation of the root system without the need for soil separation steps, avoiding root damage and ensuring the reliability of experimental data.

[0022] Standardization and scalability: The full-process modular design (with clear division of labor between culture medium A / B / C) is suitable for high-throughput experimental scenarios, providing a technical foundation for the study of mycorrhizal interaction mechanisms and large-scale applications.

[0023] This method can accelerate the research on the symbiotic mechanism of Lactarius variegata and Pinus massoniana, and provide efficient technical support for mycorrhizal seedling production, forest ecological restoration and artificial domestication of edible fungi. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The following are photographs comparing the colorful milk mushrooms of Example 1 grown on MMN medium and MMN optimized medium as a control medium for 21 days.

[0025] Figure 2 This is a photo of the Masson pine seedlings inoculated onto WPM medium in Example 1.

[0026] Figure 3 This is a photo of the bright milk mushroom inoculated on the root edge of Masson pine in Example 1.

[0027] Figure 4 This is a photo of the co-cultivation operation of Pinus massoniana and Lactarius glabra in Example 1.

[0028] Figure 5 This is a photo of the ectomycorrhiza of the brightly colored milk mushroom in Example 1. DETAILED DESCRIPTION

[0029] The present invention will be described in detail below with reference to the embodiments thereof, but the present invention can be implemented in many different ways as defined and covered by the claims.

[0030] Example 1: The method for cultivating the ectomycorrhiza of the bright-colored milk mushroom of the present embodiment comprises the following steps: S1. Prepare MMN medium, MMN optimized medium, and WPM (Woody Plant medium); MMN optimized medium contains the following components: 3000 mg malt extract, 10,000 mg glucose, 500 mg potassium dihydrogen phosphate, 250 mg diammonium hydrogen phosphate, 150 mg magnesium sulfate heptahydrate, 50 mg calcium chloride, 25 mg sodium chloride, 12 mg ferric chloride, 0.1 mg vitamin B1, 0.1 g myristic acid, 1 g mycopeptone, 9 g agar powder, pH 5.7; add distilled water to 1 L. Compared to MMN optimized medium, MMN medium lacks myristic acid and mycopeptone.

[0031] S2. Culture medium sterilization: The above-mentioned MMN optimized culture medium and WPM culture medium were sterilized with high-pressure steam at 121°C for 20 min, placed in an electric blast drying oven for drying, and when the temperature dropped to about 50°C, poured into a 13 cm*13 cm square plate in a clean bench.

[0032] S3. Activation culture of fungi: The bright-colored Lactarius fungi stored in the Fungal Herbarium of Hunan Normal University (MHHNU) were inoculated on MMN medium and cultured in the dark at 25°C for about 30 days until they grew into large colonies for subsequent experiments.

[0033] S4. Culture of Mycelium of Lactarius variegatus: In a cleanroom, use an agar borer (0.5 cm diameter) to obtain colonies of Lactarius variegatus grown in S3. Use tweezers to remove the spores from the borer and inoculate them evenly onto MMN optimized medium. After inoculation, cover the plate and seal with parafilm. Incubate the inoculated medium in the dark at 25°C for 3 weeks.

[0034] In order to explore the effect of the MMN optimized culture medium used in this example, the above-mentioned bacteria were inoculated in the same way on MMN culture medium and control culture medium. The control culture medium included MMN culture medium containing 0.1g myristic acid (named control 1) and MMN culture medium containing 1g mycopeptone (named control 2). The actual photos after culture are as follows: Figure 1(The scale bar is 1 cm. A, B, C, and D in the figure are MMN medium, control 1, control 2, and MMN optimized medium, respectively). The colony diameters were measured using the cross-hatch method. The colony diameters on MMN medium were 9.25±1.77 mm, the colony diameters on control 1 were 16.38±1.77 mm, the colony diameters on control 2 were 23.50±2.12 mm, and the colony diameters on MMN optimized medium were 8.13±9.72 mm. Statistics showed that the colony diameter on MMN optimized medium was 3.1 times that on MMN medium, and the mycelium density was the highest.

[0035] S5. Sterilization of Pinus massoniana seeds: Add an appropriate amount of Pinus massoniana seeds to a 50ml centrifuge tube, add 3 μL Tween20, 0.15g KMnO4, add water to 30mL, mix well and let it stand for 2 hours; rinse the seeds with sterile water for 5 minutes each time until the washing water is clear and colorless, then add streptomycin (final concentration 100 μg / mL), mix well, and let it stand for 15 minutes; rinse the seeds with sterile water 3 times for 5 minutes each time.

[0036] S6. Seed germination: Use sterile tweezers to select fuller seeds and evenly inoculate them on WPM medium. Incubate them in the dark at 25℃ for about 5 days. Inoculate the germinated Masson pine seedlings on a square plate of WPM medium covered with sterile filter paper. Figure 2 shown.

[0037] S7. Establishment of ectomycorrhiza: Masson pine seedlings were cultured in a 25°C incubator with 16 hours of light and 8 hours of darkness for about two weeks. After the lateral roots grew, the Masson pine seedlings were moved to MMN optimized square culture medium with cellophane and inoculated with bright-colored milk mushroom blocks (such as Figure 3 As shown), cover the lower half of the culture dish containing the Masson pine roots with aluminum foil (as shown Figure 4 As shown), 25℃ light for 16 hours and dark for 8 hours, cultured for about 4 weeks, observed the formation of mycorrhiza, and obtained bright-colored mushroom ectomycorrhizae such as Figure 5 As shown (scale bar is 3 mm).

[0038] Example 2: The method for cultivating the ectomycorrhiza of the bright-colored milk mushroom of the present embodiment comprises the following steps: S1. Prepare MMN medium, MMN optimized medium and WPM medium; MMN optimized medium contains the following components: 3000 mg malt extract, 10,000 mg glucose, 500 mg potassium dihydrogen phosphate, 250 mg diammonium hydrogen phosphate, 150 mg magnesium sulfate heptahydrate, 50 mg calcium chloride, 25 mg sodium chloride, 12 mg ferric chloride, 0.1 mg vitamin B1, 0.1 g myristic acid, 1 g mycopeptone, 9 g agar powder, pH 5.7; add distilled water to 1 L. Compared to MMN optimized medium, MMN medium lacks myristic acid and mycopeptone.

[0039] S2. Culture medium sterilization: The above-mentioned MMN optimized culture medium and WPM culture medium were sterilized with high-pressure steam at 121°C for 20 min, placed in an electric blast drying oven for drying, and when the temperature dropped to about 50°C, poured into a 13 cm*13 cm square plate in a clean bench.

[0040] S3. Activation culture of fungi: The bright-colored Lactarius fungi stored in the Fungal Herbarium of Hunan Normal University (MHHNU) were inoculated on MMN medium and cultured in the dark at 25°C for about 30 days until they grew into large colonies for subsequent experiments.

[0041] S4. Culture of Mycelium of Lactarius variegatus: In a cleanroom, use an agar borer (0.5 cm diameter) to obtain colonies of Lactarius variegatus grown in S3. Use tweezers to remove the spores from the borer and inoculate them evenly onto MMN optimized medium. After inoculation, cover the plate and seal with parafilm. Incubate the inoculated medium in the dark at 25°C for 3 weeks.

[0042] S5. Sterilization of Pinus massoniana seeds: Add an appropriate amount of Pinus massoniana seeds to a 50ml centrifuge tube, add 5 μL Tween20, 0.2 g KMnO4, add water to 30mL, mix well and let it stand for 2 hours; rinse the seeds with sterile water for 5 minutes each time until the washing water is clear and colorless, then add streptomycin (final concentration 100 μg / mL) and mix well, and let it stand for 15 minutes; rinse the seeds with sterile water 3 times for 5 minutes each time.

[0043] S6. Seed germination: Use sterile tweezers to select fuller seeds and evenly inoculate them onto WPM medium. Incubate at 25°C in the dark for approximately 5 days. Germinated Masson pine seedlings are inoculated onto square plates of WPM medium covered with sterile filter paper.

[0044] S7. Establishment of ectomycorrhiza: Masson pine seedlings were cultured in a 25°C incubator with 16 h of light and 8 h of darkness for about two weeks. After lateral roots grew, the seedlings were moved to an MMN optimized square culture medium containing cellophane. Bright-colored milk mushroom clumps were inoculated at the root edge. The lower half of the culture dish containing the Masson pine roots was covered with aluminum foil. The culture was carried out at 25°C with 16 h of light and 8 h of darkness for a total of about 4 weeks, and the mycorrhizal formation was observed.

[0045] The above are only preferred embodiments of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. For those skilled in the art, improvements and modifications obtained without departing from the technical concept of the present invention should also be considered as the scope of protection of the present invention.

[0046] Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for cultivating the ectomycorrhiza of Lactarius variegata, characterized in that: The following steps are involved: S1. Cultivating the bright-colored milk mushroom on culture medium A to obtain a bright-colored milk mushroom colony; S2. Disinfecting the seeds of Pinus massoniana; S3, the sterilized seeds are inoculated onto culture medium B and cultured until germination forms Pinus massoniana seedlings, after the Pinus massoniana seedlings grow lateral roots, the Pinus massoniana seedlings are moved onto culture medium C, and the bright-colored Lactarius colony is inoculated at the root edge to obtain the ectomycorrhiza of bright-colored Lactarius on the root system of Pinus massoniana after cultivation.

2. The method for cultivating the ectomycorrhiza of the bright-colored milk mushroom according to claim 1, characterized in that: In S2, when the seeds of the masson pine are disinfected, a disinfectant, a fungicide and sterile water are used to treat the seeds in sequence, wherein the disinfectant includes Tween 20 and potassium permanganate, and the fungicide includes streptomycin.

3. The method for cultivating the ectomycorrhiza of the bright-colored milk mushroom according to claim 2, characterized in that: The volume fraction of Tween 20 in the disinfectant is 0.01% to 0.02%, the amount of potassium permanganate added to the disinfectant is 0.005 g / mL to 0.01 g / mL; and the amount of streptomycin added to the bactericide is 50 μg / mL to 200 μg / mL.

4. The method for cultivating the ectomycorrhiza of the bright-colored milk mushroom according to claim 1, characterized in that: In S2, after the Masson pine seeds germinate to form Masson pine seedlings, the Masson pine seedlings are re-inoculated onto culture medium B with a sterile filter paper on the surface, and culture is continued until the Masson pine seedlings grow lateral roots.

5. The method for cultivating the ectomycorrhiza of the bright-colored milk mushroom according to claim 1, characterized in that: In S3, the surface of the culture medium C is covered with cellophane.

6. The method for cultivating the ectomycorrhiza of the bright-colored milk mushroom according to claim 1, characterized in that: The culture medium A, culture medium B and culture medium C are all sterilized before use; the sterilization process includes the following operations: treating the culture medium A, culture medium B and culture medium C with high-temperature steam at 121°C for 20 minutes and then drying them. When the temperature of the culture medium reaches 50°C, pouring the culture medium into a template for molding.

7. The method for cultivating the ectomycorrhiza of the bright-colored milk mushroom according to any one of claims 1 to 5, characterized in that: The medium A and / or medium C is an MMN optimized medium, which comprises the following components in parts by weight: 30 parts of malt extract powder, 100 parts of glucose, 5 parts of potassium dihydrogen phosphate, 2.5 parts of diammonium hydrogen phosphate, 1.5 parts of magnesium sulfate heptahydrate, 0.5 parts of calcium chloride, 0.25 parts of sodium chloride, 0.12 parts of ferric chloride, 0.101 parts of vitamin B, 1 part of myristic acid, 10 parts of mycopeptone, 90 parts of agar powder and distilled water.

8. The method for cultivating the ectomycorrhiza of Lactarius variegata according to any one of claims 1 to 5, characterized in that: In S1, before culturing the bright milk mushroom, taking the bright milk mushroom strain and inoculating it on a common MMN culture medium, activating and culturing it in a dark environment at 25° C. for 30 days, and using the formed larger colonies as raw materials for culturing the bright milk mushroom.

9. The method for cultivating the ectomycorrhiza of the bright-colored milk mushroom according to claim 8, characterized in that: In S1, the colonies formed by the activation culture were taken and evenly inoculated on the culture medium A. After the inoculation was completed, the lid was covered and sealed with a sealing film. The culture was carried out in a dark environment at 25° C. for 3 weeks to obtain bright-colored milk mushroom colonies.

10. The method for cultivating the ectomycorrhiza of Lactarius variegata according to any one of claims 1 to 5, characterized in that: In S3, after the bright milk mushroom colony is inoculated on the root edge, the root part of the masson pine is covered with aluminum foil, and the culture is carried out at 25° C. for four weeks with a cycle of culturing for 16 hours in a light environment and 8 hours in a dark environment to obtain the ectomycorrhiza of the bright milk mushroom on the root system of the masson pine.

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