Menghai dendrobium nobile symbiotic germination bacterium and Menghai dendrobium nobile seedling culture method

Abstract

The invention relates to the technical field of microorganisms, in particular to a Menghai dendrobium nobile symbiotic germination bacterium and a Menghai dendrobium nobile culture method. The fungus is captured from the primary environment of Menghai dendrobium nobile, is MHSH, and is preserved in the China General Microbiological Culture Collection Center (CGMCC) on December 23, 2024, the preservation place is No. 3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 41766. The invention also provides a method for realizing symbiotic germination of Dendrobium buchneroides Menghai seeds by using the fungus, and the method has the advantages of high seed germination rate and high growth speed of germinated seedlings.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a symbiotic germination fungus of dendrobium officinale and a method for raising seedlings of dendrobium officinale. Background Art

[0002] Plant symbiotic fungi are microorganisms that parasitize in plant tissues during specific or all stages of the plant life cycle without causing harmful symptoms to the plant, including mycorrhizal fungi, endophytic fungi living on the surface of plants, and latent pathogens. In the long-term co-evolution process, symbiotic fungi and host plants have formed special physiological and metabolic pathways, producing active compounds of various structures, which can promote plant growth and help the host enhance its resistance to insects, diseases, drought, plant pathogens, etc., and improve plant stress resistance.

[0003] Menghai Dendrobium (Dendrobium sinominutiflorum SCChen, JJ Wood & H.P. Wood) is a plant of the genus Dendrobium in the Orchidaceae family. Dendrobium is an orchid plant with tiny seeds, only a spherical embryo, and no endosperm or other tissues. Orchid seeds cannot germinate normally to form seedlings without external assistance. At present, the seed germination technology of orchid plants is divided into non-symbiotic germination, that is, sterile sowing technology and symbiotic germination technology. Non-symbiotic germination is to achieve the germination of orchid seed embryos under the condition of artificial nutrition, so as to obtain a large number of seedlings; symbiotic germination technology is to form a symbiotic relationship between orchid seeds and suitable fungi, and obtain the substances and nutrients required for germination through the interaction between fungi and seeds, so as to achieve the process of seed germination and formation of seedlings.

[0004] Fungi of the genus Glechoma are common symbiotic fungi of orchids, and often play a positive role in promoting seed germination, plant growth, etc. However, the effectiveness of symbiotic fungi of different orchids on different species varies. Therefore, obtaining efficient symbiotic fungi of each species is of great significance to promoting the growth and development of Dendrobium. Summary of the invention

[0005] In view of the shortcomings of the existing Menghai Dendrobium seedling cultivation technology, the object of the present invention is to provide a symbiotic fungus of Menghai Dendrobium.

[0006] Furthermore, the Menghai Dendrobium Symbiotic Fungus (MHSH) was deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration on December 23, 2024, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 41766 and a classification name of Tulasnella sp.

[0007] Furthermore, the morphological characteristics of the symbiotic fungi are: the colonies are white, surrounded by a large number of aerial hyphae, flocculent, the front and back of the colonies are white, the edges of the colonies are relatively neat, the thickness is uneven, and the middle is relatively smooth.

[0008] Furthermore, the symbiotic fungi of the Menghai Dendrobium were identified by molecular analysis as belonging to the genus Tulasnellasp.

[0009] Furthermore, the ITS gene sequence of the symbiotic fungus of Dendrobium Menghaiensis is shown in SEQ ID No.1.

[0010] On the other hand, the present invention also provides a method for raising seedlings of Dendrobium officinale from Menghai, comprising:

[0011] S1: Cultivate MHSH strain to form hyphae;

[0012] S2: Evenly sow the mature seeds of Dendrobium Menghaiensis on the culture dish covered with mycelium;

[0013] S3: culture the culture dish obtained in S2 until the seeds germinate;

[0014] S4: culture the culture dish obtained in S3 to obtain seedlings;

[0015] S5: Transplant the seedlings obtained in S4 until they grow up.

[0016] Furthermore, the step S1 includes:

[0017] S11: The preserved MHSH strains were inoculated on PDA medium and cultured until a colony with a diameter of about 5 cm and a large number of aerial hyphae were formed on the surface of the medium;

[0018] S12: Cut the bacterial block from the S11 culture medium, inoculate it on the sterile oatmeal culture medium, and culture it until the surface of the culture medium is covered with hyphae.

[0019] Furthermore, the culture conditions of step S11 are: culture at 25-30° C. in the dark for 5-10 days.

[0020] Furthermore, in step S12, the size of the cut bacterial block is (0.3-0.8)*(0.3-0.8) cm, and the sterile oat culture medium is placed in a culture dish with a diameter of 5-10 cm.

[0021] Furthermore, the culture conditions of step S12 are: culture at 25-30° C. in the dark for 8-13 days.

[0022] Furthermore, the mature seeds of Dendrobium Menghaiense in step S2 need to be disinfected.

[0023] Furthermore, the disinfection treatment includes chemical treatment, physical treatment and biological treatment.

[0024] Furthermore, the chemical treatment method is to use fungicides, bactericides, inorganic chemical reagents, plant growth regulators, etc. to treat seeds; the physical treatment method is to use heat, freezing, drying, electromagnetic waves, ultrasound, microwaves and other means to inhibit, passivate or kill pathogens; the biological treatment method includes the use of beneficial microorganisms, rhizosphere bacteria that promote plant growth, and biological pesticides that have a preventive and control effect on plant diseases to be used as seed soaking, seed dressing or microbial coating agents.

[0025] Furthermore, the culture conditions of step S3 are: placing the culture dish in S2 in a light-proof culture room at a temperature of 20-28° C. for 12-18 days.

[0026] Furthermore, the culture conditions of step S4 are: placing the culture dish in S3 in a culture room at a temperature of 20-28° C. and a light intensity of 1500-2000 lx for 50-70 days; the seedlings are young plants that have grown a second leaf.

[0027] Furthermore, the transplanting in step S5 is to transplant the seedlings in step S4 to the surface of broad-leaved wood chips and / or coconut husks with a particle size of not less than 10 meshes and culture them until they grow up.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) A new strain is provided. The strain disclosed in the present invention belongs to the genus Glechoma, is isolated from the native environment of Dendrobium Menghaiensis, has the advantages of higher compatibility with Dendrobium Menghaiensis and higher efficiency, can promote the germination of Dendrobium Menghaiensis seeds, and shorten the cultivation time of Dendrobium Menghaiensis seedlings.

[0030] (2) An efficient method for using the strain is provided. The strain provided by the present invention can interact efficiently with the Menghai Dendrobium seeds. Compared with the conventional aseptic sowing and seedling raising technology used for orchid plants, the symbiotic germination and seedling raising process is significantly faster than aseptic sowing at each germination stage, and the operational difficulty of symbiotic germination and seedling raising is much lower than aseptic sowing; secondly, using the method of the present invention, after the symbiotic seedlings are formed, they can be directly transplanted to open seedling culture conditions, which greatly simplifies the aseptic seedling hardening and transplanting process compared to aseptic sowing, and the germination rate and transplanting survival rate of Dendrobium seeds are much higher than aseptic sowing technology. DETAILED DESCRIPTION

[0031] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments. 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. The present invention is specifically introduced below in conjunction with the specific embodiments.

[0032] The embodiment of the present invention provides a symbiotic fungus of Dendrobium Menghaiensis.

[0033] Specifically, the Menghai Dendrobium symbiotic fungus (MHSH) was deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration on December 23, 2024. The deposit location is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC No. 41766.

[0034] Specifically, the morphological characteristics of the symbiotic fungi are: the colony is white, surrounded by a large number of aerial hyphae, flocculent, the front and back of the colony are white, the edges of the colony are relatively neat, the thickness is uneven, and the middle is relatively smooth.

[0035] Specifically, the symbiotic fungi of Menghai Dendrobium are identified as belonging to the genus Tulasnellasp. by molecular identification.

[0036] Specifically, the ITS gene sequence of the symbiotic fungus of Dendrobium Menghaiensis is shown in SEQ ID No.1.

[0037] (SEQ ID No.1).

[0038] The second aspect of the embodiment of the present invention further provides a method for growing seedlings of Dendrobium officinale in Menghai, comprising:

[0039] S1: Cultivate MHSH strain to form hyphae;

[0040] S2: Evenly sow the mature seeds of Dendrobium Menghaiensis on the culture dish covered with mycelium;

[0041] S3: culture the culture dish obtained in S2 until the seeds germinate;

[0042] S4: culture the culture dish obtained in S3 to obtain seedlings;

[0043] S5: Transplant the seedlings obtained in S4 until they grow up.

[0044] Specifically, the step S1 includes:

[0045] S11: The preserved MHSH strains were inoculated on PDA medium and cultured until a colony with a diameter of about 5 cm and a large number of aerial hyphae were formed on the surface of the medium;

[0046] S12: Cut the bacterial block from S1 culture medium, inoculate it on sterile oatmeal culture medium, and culture it until the surface of the culture medium is covered with hyphae.

[0047] Specifically, the PDA medium described in S11 is a potato, glucose, and agar medium. Potato extract powder helps the growth of various molds and yeasts, glucose provides energy, and agar is a coagulant for the medium. The PDA medium used in this embodiment was purchased from Beijing Solebow Technology Co., Ltd. The preparation method is to weigh 46 grams of the reagent, add it to 1000 milliliters of distilled water, heat it until it is completely dissolved, and sterilize it at 121°C for 15 minutes for standby use.

[0048] Specifically, the culture conditions of step S11 are: culture at 25-30° C. in the dark for 5-10 days.

[0049] Specifically, the sterile oat culture medium described in S12 has oats as a main component, which is rich in carbon sources, nitrogen sources and other nutrients, and is conducive to the reproduction and expansion of MHSH strains.

[0050] Specifically, in step S12, the size of the cut bacterial block is (0.3-0.8)*(0.3-0.8) cm, and the sterile oat culture medium is placed in a culture dish with a diameter of 5-10 cm.

[0051] Specifically, the culture conditions of step S12 are: culture at 25-30° C. in the dark for 8-13 days.

[0052] Specifically, the mature seeds of Dendrobium Menghaiense in step S2 need to be disinfected.

[0053] Specifically, many diseases and insect pests in the process of plant growth are spread by bacteria in seeds. Some plant seeds have bacteria on the surface or even inside. These seeds will directly transmit the bacteria to seedlings and adult plants, causing diseases, especially the occurrence of seedling diseases. Therefore, in order to reduce the diseases and insect pests caused by bacteria in seeds, this embodiment disinfects the seeds before sowing.

[0054] Specifically, the disinfection treatment includes chemical treatment, physical treatment and biological treatment.

[0055] Specifically, the chemical treatment method is to use fungicides, bactericides, inorganic chemical reagents, plant growth regulators, etc. to treat seeds; the physical treatment method is to use heat, freezing, drying, electromagnetic waves, ultrasound, microwaves and other means to inhibit, passivate or kill pathogens; the biological treatment method includes the use of beneficial microorganisms, rhizosphere bacteria that promote plant growth, and biological pesticides that have a preventive and control effect on plant diseases to be used as seed soaking, seed dressing or microbial coating agents.

[0056] Specifically, the culture conditions of step S3 are: placing the culture dish in S2 in a light-proof culture room at a temperature of 20-28° C. for 12-18 days.

[0057] Specifically, the culture conditions of step S4 are: placing the culture dish in S3 in a culture room with a temperature of 20-28° C. and a light intensity of 1500-2000 lx for 50-70 days; the seedlings are young plants that have grown a second leaf.

[0058] Specifically, the transplanting in step S5 is to transplant the seedlings in step S4 to the surface of broad-leaved wood chips and / or coconut husks with a particle size of not less than 10 meshes and culture them until they grow up.

[0059] Specifically, the broad-leaved wood chips refer to the wood chips of poplar, oak, elm, etc.; the coconut bran is the coconut shell fiber powder. Both have good water retention and air permeability, are conducive to the growth of seedlings, and are good planting substrates.

[0060] The following is explained in conjunction with specific embodiments:

[0061] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified, and the techniques not described in detail are all performed according to standard methods well known to those skilled in the art.

[0062] Example 1

[0063] S11: inoculate the preserved MHSH strains on PDA medium, place it in a dark place at 28°C for 7 days, until a colony with a diameter of about 5 cm and a large number of aerial hyphae are formed on the surface of the medium, and then set aside;

[0064] S12: Cut a 0.5×0.5 cm bacterial block from the S11 medium, inoculate it on a 9 cm diameter petri dish filled with sterile oatmeal medium, and culture it at 28°C in the dark for 10 days until the surface of the medium is covered with hyphae;

[0065] S2: Take the sterilized mature seeds of Dendrobium officinale from Menghai and evenly spread them on the oatmeal medium plate covered with mycelium;

[0066] S3: Place the sown culture dish in a dark culture room at 25°C for 15 days until the seeds germinate;

[0067] S4: Place the culture dish in a culture room at 25°C and 1800 lx for 60 days until the second leaf grows out;

[0068] S5: The seedlings in the culture dish are transplanted to the surface of broad-leaved wood chips and / or coconut husks with a particle size of not less than 10 mesh and cultured until they grow up, with a survival rate of 100%.

[0069] Comparative Example 1

[0070] Prepare N6 medium, sterilize by high pressure to make sterile medium, pour into a 9 cm sterile culture dish, cool and solidify for later use;

[0071] Evenly sowing the sterilized mature Menghai Dendrobium seeds on the surface of the sterile culture medium;

[0072] The culture dish after sowing is placed in a clean culture room for cultivation, with a constant temperature of 25°C, a humidity of 50%, and a light intensity of 1800lx;

[0073] The sprouted protocorms of Dendrobium candidum in the culture dish were transferred to a culture bottle filled with N6 culture medium, and the culture bottle was placed in a constant temperature of 25°C, a humidity of 50%, and a light of 1800lx for cultivation until they grew into seedlings about 1 cm high; the seedlings were transferred and cultivated again, with 10 plants in each bottle, until single plant clustered seedlings were formed, about 5 cm high, and the seedlings were taken out of the bottle for hardening and transplanted into a closed greenhouse with a temperature of 20-35°C, a humidity of 70%, and natural light under 50% shading, and the transplant survival rate was 57%.

[0074] Table 1 shows that the germination process of Dendrobium seeds is divided into 6 stages. The present invention evaluates the advantages and disadvantages of the method of the present invention and the traditional aseptic sowing technology by comparing the time experienced in each stage, seed germination rate and transplanting seedling rate of the embodiment and the comparative example. The seedling raising parameters are shown in Table 2.

[0075] Table 1 Developmental stages of Dendrobium officinale seed germination

[0076]

[0077] Table 2 Comparison of parameters between fungal symbiotic germination and aseptic seeding and seedling raising of Dendrobium officinale in Menghai

[0078]

[0079] As can be seen from the above table, using the fungal symbiotic germination method of the present invention, the seed is significantly shorter than aseptic sowing from the second stage of germination, and each stage of germination has a significant time acceleration until the seedling is formed. In addition, it can be seen from the specific implementation methods of the reference examples and comparative examples that the present invention can be directly removed from the culture dish after the seedling is formed to carry out relatively extensive management in the cultivation container, and the seedling strengthening and hardening processes of the aseptic sowing technology have greatly simplified operations and reduced costs. The use of symbiotic bacteria makes the Menghai Dendrobium seedlings have stronger adaptability and stress resistance to the external environment, and its seedling rate is much higher than the aseptic sowing technology.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A symbiotic fungus of Dendrobium Menghaiensis, named MHSH, has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, Beijing, with a deposit number of CGMCC No.41766.

2. The symbiotic fungus according to claim 1, characterized in that The symbiotic fungus colony is white, surrounded by a large number of aerial hyphae in a flocculent state, and both the front and back sides of the colony are white. The colony edge is relatively neat, the thickness is uneven, and the middle is relatively smooth.

3. The symbiotic fungus according to claim 1, characterized in that The symbiotic fungus belongs to the genus Tulasnella sp., and its ITS gene sequence is shown in SEQ ID No.

1.

4. A method for cultivating Dendrobium officinale from Menghai, comprising: S1: Cultivate MHSH strain to form hyphae; S2: Evenly sow the mature seeds of Dendrobium Menghaiensis on the culture dish covered with mycelium; S3: culture the culture dish obtained in S2 until the seeds germinate; S4: culture the culture dish obtained in S3 to obtain seedlings; S5: Transplant the seedlings obtained in S4 until they grow up.

5. The method according to claim 4, characterized in that The step S1 comprises: S11: inoculate the preserved MHSH strains on PDA medium, and culture at 25-30°C in the dark for 5-10 days until colonies with a diameter of about 5 cm and a large number of aerial hyphae are formed on the surface of the medium; S12: Cut a (0.3-0.8)*(0.3-0.8) cm bacterial block from the S1 culture medium, inoculate it on a sterile oatmeal culture medium, and culture it at 25-30°C in the dark for 8-13 days until the surface of the culture medium is covered with hyphae.

6. The method according to claim 4, characterized in that The mature seeds of Dendrobium officinale of step S2 need to be disinfected before sowing; the disinfection includes at least one of chemical treatment, physical treatment and biological treatment; the chemical treatment is to treat the seeds with at least one of a fungicide, a fungicide, an inorganic chemical reagent and a plant growth regulator; the physical treatment is to inhibit, passivate or kill pathogens by using at least one of heat, freezing, drying, electromagnetic waves, ultrasound and microwaves; the biological treatment includes using beneficial microorganisms, rhizosphere bacteria that promote plant growth and one of biological pesticides that have a preventive and control effect on plant diseases as a seed soaking, seed dressing or microbial coating agent.

7. The method according to claim 4, characterized in that The culture conditions of step S3 are as follows: placing the culture dish in S2 in a light-proof culture room at a temperature of 20-28° C. and culturing for 12-18 days.

8. The method according to claim 4, characterized in that The culture conditions of step S4 are: placing the culture dish in S3 in a culture room at a temperature of 20-28° C. and a light intensity of 1500-2000 lx for 50-70 days; the seedlings are young plants that have grown a second leaf.

9. The method according to claim 4, characterized in that The transplanting in step S5 is to transplant the seedlings in step S4 to the surface of broad-leaved wood chips and / or coconut husks with a particle size of not less than 10 meshes and culture them until they grow up.

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