Method for promoting germination of orchid seeds by using pteridium fungus

Abstract

The invention relates to the technical field of orchid seed germination, and discloses a method for promoting orchid seed germination by utilizing crispus fungi, which comprises the following steps of: selecting the root of a wild adult orchid seedling growing in Changbai Mountain, searching a root section with hypha mass by utilizing a bare-handed slicing technology and a CH3COOH black ink dyeing technology, and inoculating the hypha mass into the root section; the method comprises the following steps: washing a root segment containing a fungal filament cluster for 48 hours under a running water condition, clamping the root segment into a 2% sodium hypochlorite solution by tweezers after high-temperature sterilization, soaking for 30 seconds, then putting the root segment into a sterile water flask, cleaning, and absorbing excessive moisture. The method comprises the following steps: searching a root section with a hypha group from the root of an orchid seedling, culturing to grow hyphae under an illumination condition, transferring single-colony hyphae into an FIM culture medium, continuously culturing and continuously purifying to obtain a fragile mushroom fungus strain, inoculating the fragile mushroom fungus strain into an oat culture medium of orchid seeds, and setting a control group by using a sterile agar block. The germination rate and the protocorm formation rate of the orchid seeds are calculated, and the method can be applied to orchid seed breeding.

Description

Technical Field

[0001] The invention relates to the technical field of germination of mountain cymbidium seeds, and in particular to a method for promoting germination of mountain cymbidium seeds by utilizing Fragilis genus fungi. Background Art

[0002] Mountain orchid is a perennial herbaceous plant of the Orchidaceae family, with high ornamental and medicinal value. Orchidaceae plants and fungi form a typical orchidaceous mycorrhiza, and the mycorrhizal symbiotic relationship accompanies the entire life cycle of orchidaceae plants from seed germination to flowering and fruiting.

[0003] Orchid mycorrhiza is a symbiotic relationship between the rhizomes (protocorms) or roots of adult plants of the orchid family and specific fungi, which are defined as orchid mycorrhizal fungi. The symbiotic fungi species discovered so far are basically basidiomycetes and ascomycetes, and the common genera include: Gelatocystis, Rhizoctonia, Fusarium, Angiospermum, Fragilis, Ceroplastes, Aspergillus, etc.

[0004] Given that the germination of orchid seeds in the natural environment is highly dependent on the symbiotic relationship with specific fungi, it is particularly urgent and important to explore and cultivate excellent fungal strains, especially the genus Agaricus, that can significantly promote the germination of orchid seeds, a precious orchidaceae medicinal plant. This move is not only aimed at improving the germination rate of orchid seeds, but also hopes to optimize the efficiency of its protocorm formation, thereby building an efficient path to orchid seed conservation and sustainable resource utilization, and laying a solid foundation for protecting biodiversity and promoting the rational use of medicinal plant resources. Summary of the invention

[0005] The object of the present invention is to provide a method for promoting the germination of Cymbidium seeds by utilizing the genus Fragilis fungus, so as to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for promoting the germination of mountain orchid seeds by using the genus Fragilis fungus, comprising the following steps: S1: Isolation and characterization of Fragilis fungal strains suitable for promoting germination of Cyperus rotundus seeds The roots of wild adult mountain orchid seedlings growing in the Changbai Mountain area were selected, and the root segments with mycelium were found under a high-resolution optical microscope using the freehand sectioning technique and CH3COOH black ink staining technique. The root segments containing mycelium were rinsed under running water for 48 hours, and the excess water of the root segments was absorbed with sterile absorbent paper and placed on a clean bench. The root segments were clamped with high-temperature sterilized tweezers and soaked in 2% sodium hypochlorite solution for 30 seconds, and then transferred to a beaker containing 75% alcohol, gently shaken for 1 minute, and then washed in a sterile water flask, and excess water was absorbed and set aside. In a clean bench, use a high-temperature sterilized blade and tweezers to cut the dried root segments into slices of about 1 mm, spread the root slices flat on FIM medium, and culture them at 28°C and 12 h of light. When hyphae grow around the root slices, transfer the single colony hyphae to FIM medium for continued culture and purification until a single colony strain is obtained; Add 300 μL of 80 mmol / L Tris lysis buffer and 2 ml of CTAB to the 2 ml EP tube containing the fungus, shake at high frequency for 3 min, and heat in a 80°C water bath for 10 min to melt. The purpose is to release the DNA in the fungal cell wall into the solution as much as possible. Add 500 μL of equal volumes of chloroform and isoamyl alcohol to the EP tube in turn, shake manually for 3 min, centrifuge at 8000g for 8 min, aspirate 500 μL of the supernatant, place in a refrigerator at 4°C for 12 h, select the precipitated DN, wash the precipitate twice with 75% alcohol, centrifuge at 12000g for 3 min, let the precipitate stand, wait for the alcohol in it to completely evaporate, take 50 μL of TE buffer and add it to the precipitate to completely dissolve the DNA for later use; The PCR amplification reaction system (50 μL) is shown in Table 1; PCR amplification reaction procedure is as follows Figure 1 ; After rDNA-ITS molecular identification, the results were compared and analyzed using NBCI software. The isolated endophytic fungi were identified as Psathyrella fungi based on the fungal category with the highest sequence similarity in the reference comparison. The ITS comparison structure of mycorrhizal fungi is shown in Table 2.

[0007] S2: Morphological observation and biological characteristics determination of selected Fragilis fungi strains Observation of morphological characteristics of mycorrhizal fungi: The selected fungi were inoculated into FIM medium. When the hyphae grew to 2 / 3 of the surface of the medium, the morphological characteristics of the hyphae were observed using an optical microscope. From the time the hyphae were inoculated into the medium, the growth rate and changes of the hyphae were statistically analyzed every 8 hours. At the same time, the colony morphology was observed and statistically analyzed. The selected strain began to grow rapidly on the third day after being inoculated into the culture medium, and covered the entire culture medium dish after 10 days. The fastest growth rate was on the 7th day, with an average growth rate of 0.56 cm / d. The front of the fungal colony was covered with regular aerial hairy hyphae and was white and opaque, and the back was yellow-white concentric knots, and no spores were produced. The average diameter of the hyphae was 9.32 μm, with septa and no right-angle branches. Determination of biological characteristics of mycorrhizal fungi: After activating the isolated and identified Pseudomonas Psa-2 strain, take a 0.3*0.3 cm fungal agar block with the fungus side facing up and place it on FIM medium containing carbon (sucrose, glucose, lactose, fructose, maltose, CK without carbon source), nitrogen (yeast, beef extract, peptone, ammonium sulfate, potassium nitrate, CK without nitrogen source), pH (4-8), temperature (10-35°C, 6 groups of treatments) to statistically analyze the growth and development data of the fungi (6 groups of treatments, 10 replicates), draw lines on the back of the culture dish, and statistically analyze the mycelial growth rate; The biological characteristics of the Psaeocantha strain Psa-2 are shown in Table 3.

[0008] S3: Morphological observation of Cymbidium seeds and delineation of different germination stages Morphological observation of mountain orchid seeds. The overall morphology and microstructure of mountain orchid seeds were observed using a stereo microscope and an electron microscope. The mountain orchid seeds with water were brown in color. The seeds in a dry state were dark brown, spindle-shaped, with one end slender and the other slightly round. The endosperm was at the thicker end, about 1000μm long and 250μm wide. The surface of the seed coat was grid-like, and each grid had a flocculent structure inside. Observation of different germination stages of mountain orchid seeds: After washing, select unopened and full capsules, rinse with running water for 24 hours, soak in 2% sodium hypochlorite solution for 3 minutes, and then rinse with sterile water for 3 to 5 times in sequence. In the clean bench, use tweezers and scalpels to cut the peel longitudinally along the diaphragm line, shake the fruit gently, and spread the seeds evenly on the surface of 4.0g / L, 8.0g / L, and 12.0g / L oat culture medium. Cut the fragilis strain into 0.3*0.3cm fungal agar blocks and inoculate them into oat culture medium covered with mountain orchid seeds. Set up a control group with sterile agar blocks to eliminate contamination interference. Set up 15 replicates. After inoculation, observe and count the germination of seeds every day, calculate the germination rate and protocorm formation rate of mountain orchid seeds, and observe and statistically analyze the germination process of mountain orchid seeds. The dynamic process of mountain orchid seed germination is divided into 5 stages. The dynamic germination process of Cymbidium orchid seeds is shown in Table 4.

[0009] S4: Screening of conditions for promoting germination of Cymbidium seeds by Agaricus spp. During the germination of mountain orchid seeds, the state between the appearance of the apical meristem and the formation of the first true leaf is called the protocorm period. After this, the seeds will become seedlings and gradually develop into plants. In the early stage of mountain orchid seed germination, this experiment used a stereo microscope to observe and statistically analyze the seed growth and development on the surface of 4.0g / L, 8.0g / L, and 12.0g / L oatmeal culture media. When the symbiotic culture lasted for more than 2 weeks, the mycelium would cover the surface of the culture medium, and the excess mycelium needed to be regularly removed in the clean bench; The data related to the promotion of germination of Cymbidium seeds by the strains of the genus Fragilis are as follows: the germination began after 100 days of co-culture with the Cymbidium seeds (a large number of seeds obviously absorbed water and became larger), and the maximum germination rate was reached at 165 days. The statistical calculation results showed that the Cymbidium seeds in the treatment group inoculated with the fungus showed signs of germination, while the seeds in the control group inoculated with sterile agar blocks showed no signs of germination. The germination rates of the treatment groups were significantly higher than those of the control group, and there was a significant difference. In the experiment of promoting the germination of Cymbidium orchid seeds, the germination rates of Cymbidium orchid seeds under the culture conditions of 4.0g / L and 12.0g / L oats were 20.11% and 31.07% respectively. The seeds all absorbed water and swelled, the seed coats broke and swelled to a spherical shape, and the final stage of development of seeds symbiotically germinated with the genus Fragilis was swelling to a transparent white spherical shape, i.e. germination stage 1; the seed germination rate under the culture conditions of 8.0g / L oats was as high as nearly 55.26%, of which most seeds were in germination stage 2, the seed coats were completely broken, the embryos absorbed water and swelled into transparent white spheres, a small number of seeds developed to stages 3~4, grew the first true leaf, and the protocorm formation rate was about 30%.

[0010] Preferably, in step S1, the FIM medium includes: 0.35 g sodium nitrate, 0.30 g potassium dihydrogen phosphate, 0.10 g magnesium sulfate, 0.15 g potassium chloride, 5.00 g yeast, 2.50 g sucrose, and 8.00 g agar.

[0011] Preferably, in step S1, the internal transcribed spacer (ITS) region ITS1 and ITS2 of ribosomal rDNA are amplified by PCR using universal primers ITS-1 (5'-TCCTAGGTGAACCTGCGG-3') and ITS-4 (5'TCCTCCGTTATTGATATGC 3'). PCR reaction conditions and PCR amplification procedures for fungal DNA, and the PCR products are sequenced using Sanger sequencing technology.

[0012] Preferably, in step S1, after rDNA-ITS molecular identification, the results are compared and analyzed using NBCI software, and the isolated endophytic fungus is identified as a fungus of the genus Fragilis by referring to the fungal category with the highest sequence similarity in the comparison.

[0013] Preferably, in step S3, the germination rate and protocorm formation rate of Cymbidium orchid seeds are calculated according to the following formula: Seed germination rate: (total number of germinated seeds / total number of sowings) × 100% Protocorm formation rate: (total number of protocorms formed / total number of germinated seeds) × 100.

[0014] Preferably, in step S4, there are three stages of germination process of mountain orchid seeds: The first stage was co-cultivation for 100 days, which was at germination stage 0-1; The second stage is symbiosis for 100-130 days, which is in the germination stage 1-2; The third stage is 165 days of symbiosis, which is completely in the germination stage 3-4.

[0015] Compared with the prior art, the present invention provides a method for promoting the germination of mountain orchid seeds by utilizing the genus Fragilis fungi, which has the following beneficial effects: the present invention uses the freehand slicing technique and the CH3COOH black ink staining technique from the roots of wild adult mountain orchid seedlings growing in the Changbai Mountain area to find root segments with mycelium masses under a high-resolution optical microscope, cultivates mycelium around the root segment slices under light conditions, transfers the single colony mycelium to a FIM culture medium for continued cultivation and continuous purification until a strain of the genus Fragilis fungi is obtained, the cut Fragilis fungus strain is a 0.3*0.3 cm fungal agar block, which is inoculated into oat culture media of different concentrations covered with mountain orchid seeds, and a control group is set up with sterile agar blocks, and the germination rate and protocorm formation rate of the mountain orchid seeds are calculated, which can be applied in mountain orchid seed breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The present invention provides a schematic diagram of the PCR amplification reaction procedure. DETAILED DESCRIPTION

[0017] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] In the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0019] The present invention provides the following technical solutions: A method for promoting the germination of mountain orchid seeds by using the genus Fragilis fungus comprises the following steps: S1: Isolation and characterization of Fragilis fungal strains suitable for promoting germination of Cyperus rotundus seeds The roots of wild adult mountain orchid seedlings growing in the Changbai Mountain area were selected, and the root segments with mycelium were found under a high-resolution optical microscope using the freehand sectioning technique and CH3COOH black ink staining technique. The root segments containing mycelium were rinsed under running water for 48 hours, and the excess water of the root segments was absorbed with sterile absorbent paper and placed on a clean bench. The root segments were clamped with high-temperature sterilized tweezers and soaked in 2% sodium hypochlorite solution for 30 seconds, and then transferred to a beaker containing 75% alcohol, gently shaken for 1 minute, and then placed in a sterile water flask for washing and absorbing excess water for standby use; In a clean bench, use a high-temperature sterilized blade and tweezers to cut the dried root segments into slices of about 1 mm, spread the root slices flat on FIM medium, and culture them at 28°C and 12 h of light. When hyphae grow around the root slices, transfer the single colony hyphae to FIM medium for continued culture and purification until a single colony strain is obtained; Add 300 μL of 80 mmol / L Tris lysis buffer and 2 ml of CTAB to the 2 ml EP tube containing the fungus, shake at high frequency for 3 min, and heat in a 80°C water bath for 10 min to melt. The purpose is to release the DNA in the fungal cell wall into the solution as much as possible. Add 500 μL of equal volumes of chloroform and isoamyl alcohol to the EP tube in turn, shake manually for 3 min, centrifuge at 8000g for 8 min, aspirate 500 μL of the supernatant, place in a refrigerator at 4°C for 12 h, select the precipitated DNA, wash the precipitate twice with 75% alcohol, centrifuge at 12000g for 3 min, let the precipitate stand, wait for the alcohol in it to completely evaporate, take 50 μL of TE buffer and add it to the precipitate to completely dissolve the DNA, and set aside; FIM medium: 0.35g sodium nitrate, 0.30g potassium dihydrogen phosphate, 0.10g magnesium sulfate, 0.15g potassium chloride, 5.00g yeast, 2.50g sucrose, 8.00g agar. In step S1, PCR amplified the internal transcribed spacer (ITS) region ITS1 and ITS2 of ribosomal rDNA, and amplified with universal primers ITS-1 (5'-TCCTAGGTGAACCTGCGG-3') and ITS-4 (5'TCCTCCGTTATTGATATGC 3'). PCR reaction conditions and PCR amplification procedures for fungal DNA, and sequencing of PCR products using Sanger sequencing technology. In step S1, after rDNA-ITS molecular identification, the results were compared and analyzed using NBCI software, and the fungal category with the highest similarity sequence in the comparison was referred to, and the isolated endophytic fungus was identified as a fungus of the genus Fragilis. The PCR amplification reaction system (50μL) is shown in Table 1;

[0020] After rDNA-ITS molecular identification, the results were compared and analyzed using NBCI software. The isolated endophytic fungi were identified as Psathyrella fungi based on the fungal category with the highest similarity in the comparison. The ITS comparison structure of mycorrhizal fungi is shown in Table 2:

[0021] S2: Morphological observation and biological characteristics determination of selected Fragilis fungi strains Observation of morphological characteristics of mycorrhizal fungi: The selected fungi were inoculated into FIM medium. When the hyphae grew to 2 / 3 of the surface of the medium, the morphological characteristics of the hyphae were observed using an optical microscope. From the time the hyphae were inoculated into the medium, the growth rate and changes of the hyphae were statistically analyzed every 8 hours. At the same time, the colony morphology was observed and statistically analyzed. The selected strain began to grow rapidly on the third day after being inoculated into the culture medium, and covered the entire culture medium dish after 10 days. The fastest growth rate was on the 7th day, with an average growth rate of 0.56 cm / d. The front of the fungal colony was covered with regular aerial hairy hyphae and was white and opaque, and the back was yellow-white concentric knots, and no spores were produced. The average diameter of the hyphae was 9.32 μm, with septa and no right-angle branches. Determination of biological characteristics of mycorrhizal fungi: After activating the isolated and identified Pseudomonas Psa-2 strain, take a 0.3*0.3 cm fungal agar block with the fungus side facing up and place it on FIM medium containing carbon (sucrose, glucose, lactose, fructose, maltose, CK without carbon source), nitrogen (yeast, beef extract, peptone, ammonium sulfate, potassium nitrate, CK without nitrogen source), pH (4-8), temperature (10-35°C, 6 groups of treatments) to culture and statistically analyze the growth and development data of fungi (6 groups of treatments, 10 replicates), draw lines on the back of the culture dish, and statistically analyze the mycelial growth rate; Determination of biological characteristics of Pseudomonas Psa-2 strain is shown in Table 3:

[0022] S3: Morphological observation of Cymbidium seeds and delineation of different germination stages Morphological observation of mountain orchid seeds. The overall morphology and microstructure of mountain orchid seeds were observed using a stereo microscope and an electron microscope. The mountain orchid seeds with water were brown in color. The seeds in a dry state were dark brown, spindle-shaped, with one end slender and the other slightly round. The endosperm was at the thicker end, about 1000μm long and 250μm wide. The surface of the seed coat was grid-like, and each grid had a flocculent structure inside. Observation of different germination stages of mountain orchid seeds: After washing, select unopened and full capsules, rinse with running water for 24 hours, soak in 2% sodium hypochlorite solution for 3 minutes, and then rinse with sterile water for 3 to 5 times in sequence. In the clean bench, use tweezers and scalpels to cut the peel longitudinally along the diaphragm line, shake the fruit gently, and spread the seeds evenly on the surface of 4.0g / L, 8.0g / L, and 12.0g / L oat culture medium. Cut the fragilis strain into 0.3*0.3cm fungal agar blocks and inoculate them into oat culture medium covered with mountain orchid seeds. Set up a control group with sterile agar blocks to eliminate contamination interference. Set up 15 replicates. After inoculation, observe and count the germination of seeds every day, calculate the germination rate and protocorm formation rate of mountain orchid seeds, and observe and statistically analyze the germination process of mountain orchid seeds. The dynamic process of mountain orchid seed germination is divided into 5 stages. The germination rate and protocorm formation rate of mountain orchid seeds were calculated using the following formula: Seed germination rate: (total number of germinated seeds / total number of sowings) × 100% Protocorm formation rate: (total number of protocorms formed / total number of germinated seeds) × 100; The dynamic germination process of mountain orchid seeds is shown in Table 4:

[0023] S4: Screening of conditions for promoting germination of Cymbidium seeds by Agaricus spp. During the germination of mountain orchid seeds, the state between the appearance of the apical meristem of the seeds and the formation of the first true leaf is called the protocorm period. After this, the seeds will become seedlings and gradually develop into plants. In the early stage of mountain orchid seed germination, this experiment used a stereo microscope to observe and statistically analyze the growth and development of seeds on the surface of 4.0g / L, 8.0g / L, and 12.0g / L oatmeal culture media (when the symbiotic culture exceeds 2 weeks, the mycelium will cover the surface of the culture medium, and the excess mycelium needs to be regularly removed in the clean bench); The data related to the promotion of germination of Cymbidium seeds by the strains of the genus Fragilis are as follows: the germination began after 100 days of co-culture with the Cymbidium seeds (a large number of seeds obviously absorbed water and became larger), and the maximum germination rate was reached at 165 days. The statistical calculation results showed that the Cymbidium seeds in the treatment group inoculated with the fungus showed signs of germination, while the seeds in the control group inoculated with sterile agar blocks showed no signs of germination. The germination rates of the treatment groups were significantly higher than those of the control group, and there was a significant difference. In the test of promoting the germination of Cymbidium orchid seeds, the germination rates of Cymbidium orchid seeds under the conditions of 4.0 g / L and 12.0 g / L oats were 20.11% and 31.07% respectively. The seeds all absorbed water and swelled, and the seed coats broke and expanded to a spherical shape. The final stage of the development of the seeds symbiotically germinated with the genus Fragilis was swelling to a transparent white spherical shape, i.e., germination stage 1. The germination rate of seeds under the conditions of 8.0 g / L oats was the highest, close to 55.26%, of which most of the seeds were in germination stage 2, with the seed coats completely broken, the embryos absorbed water and expanded into transparent white spheres, and a small number of seeds developed to stages 3-4, grew the first true leaf, and the rate of protocorm formation was about 30%. The germination process of mountain orchid seeds has three stages: The first stage was co-cultivation for 100 days, which was at germination stage 0-1; The second stage is symbiosis for 100-130 days, which is in the germination stage 1-2; The third stage is symbiosis for 165 days, which is completely in the germination stage 3-4.

[0024] (1) Isolate and identify Fragilis fungi strains suitable for promoting the germination of Cymbidium seeds; (2) Conduct morphological observations and determine biological characteristics of the selected Fragilis fungi strains; (3) Conduct morphological observations on Cymbidium seeds and define different germination stages; (4) Screen conditions for Fragilis fungi to promote the germination of Cymbidium seeds; A strain of the genus Fragilis was isolated from the roots of wild adult Cymbidium seedlings in the Changbai Mountain area. It can effectively promote the germination of Cymbidium seeds when co-cultivated with Cymbidium seeds under different concentrations of oat culture conditions.

[0025] By using the freehand slicing technique and CH3COOH black ink staining technique from the roots of wild adult Cymbidium seedlings growing in the Changbai Mountain area, root segments with mycelium masses were found under a high-resolution optical microscope, and mycelium was grown around the root segment slices under light conditions. The single colony mycelium was transferred to FIM culture medium for continued culture and purification until a strain of the genus Fragilis was obtained. The cut Fragilis strain was a 0.3*0.3cm fungal agar block, which was inoculated into oatmeal culture media of different concentrations covered with Cymbidium seeds, and a control group was set up with sterile agar blocks. The germination rate of Cymbidium seeds and the protocorm formation rate were calculated, which can be applied in Cymbidium seed breeding.

[0026] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the statement "comprise a ..." do not exclude the presence of other identical elements in the process, method, article or device including the elements.

Claims

1. A method for promoting the germination of mountain orchid seeds by using Agaricus fragilis fungi, It is characterized in that The following steps are involved: S1: Isolation and characterization of Fragilis fungal strains suitable for promoting germination of Cyperus rotundus seeds The roots of wild adult orchid seedlings growing in the Changbai Mountain area were selected and sliced ​​by hand using CH 3 COOH black ink staining technique was used to find the root segments with hyphae under a high-resolution optical microscope. The root segments with fungal hyphae were rinsed under running water for 48 hours. The excess water of the root segments was absorbed with sterile absorbent paper and then placed on a clean bench. The root segments were clamped with high-temperature sterilized tweezers and soaked in 2% sodium hypochlorite solution for 30 seconds. The root segments were then transferred to a beaker containing 75% alcohol, gently shaken for 1 minute, and then placed in a sterile water flask for washing and excess water was absorbed for later use. In a clean bench, use a high-temperature sterilized blade and tweezers to cut the dried root segments into slices of about 1 mm, spread the root slices flat on FIM medium, and culture them at 28°C and 12 h of light. When hyphae grow around the root slices, transfer the single colony hyphae to FIM medium for continued culture and purification until a single colony strain is obtained; Add 300 μL of 80 mmol / L Tris lysis buffer and 2 ml of CTAB to the 2 ml EP tube containing the fungus, shake at high frequency for 3 min, and heat in a 80°C water bath for 10 min to melt. The purpose is to release the DNA in the fungal cell wall into the solution as much as possible. Add 500 μL of equal volumes of chloroform and isoamyl alcohol to the EP tube in turn, shake manually for 3 min, centrifuge at 8000g for 8 min, aspirate 500 μL of the supernatant, place in a refrigerator at 4°C for 12 h, select the precipitated DN, wash the precipitate twice with 75% alcohol, centrifuge at 12000g for 3 min, let the precipitate stand, wait for the alcohol in it to completely evaporate, take 50 μL of TE buffer and add it to the precipitate to completely dissolve the DNA for later use; S2: Morphological observation and biological characteristics determination of selected Fragilis fungi strains Observation of morphological characteristics of mycorrhizal fungi: The selected fungi were inoculated into FIM medium. When the hyphae grew to 2 / 3 of the surface of the medium, the morphological characteristics of the hyphae were observed using an optical microscope. From the time the hyphae were inoculated into the medium, the growth rate and changes of the hyphae were statistically analyzed every 8 hours. At the same time, the colony morphology was observed and statistically analyzed. The selected strain began to grow rapidly on the third day after being inoculated into the culture medium, and covered the entire culture medium dish after 10 days. The fastest growth rate was on the 7th day, with an average growth rate of 0.56 cm / d. The front of the fungal colony was covered with regular aerial hairy hyphae and was white and opaque, and the back was yellow-white concentric knots, and no spores were produced. The average diameter of the hyphae was 9.32 μm, with septa and no right-angle branches. Determination of biological characteristics of mycorrhizal fungi: After activating the isolated and identified Pseudomonas Psa-2 strain, take a 0.3*0.3 cm fungal agar block, with the fungus side facing up, and place it in FIM medium containing carbon and nitrogen, pH 4-8, and temperature 10-35°C to statistically analyze the growth and development data of the fungus. Draw a line on the back of the culture dish to statistically analyze the mycelial growth rate. S3: Morphological observation of Cymbidium seeds and delineation of different germination stages Morphological observation of mountain orchid seeds. The overall morphology and microstructure of mountain orchid seeds were observed using a stereo microscope and an electron microscope. The mountain orchid seeds with water were brown in color. The seeds in a dry state were dark brown, spindle-shaped, with one end slender and the other slightly round. The endosperm was at the thicker end, about 1000μm long and 250μm wide. The surface of the seed coat was grid-like, and each grid had a flocculent structure inside. Observation of different germination stages of mountain orchid seeds: select unopened and full capsules, wash them, rinse them with running water for 24 hours, soak them in 2% sodium hypochlorite solution for 3 minutes, and then rinse them with sterile water for 3 to 5 times in sequence. In a clean bench, use tweezers and a scalpel to cut the peel longitudinally along the diaphragm line, shake the fruit gently and spread the seeds evenly on the surface of 4.0g / L, 8.0g / L, and 12.0g / L oat culture medium. Cut the fragilis strain into 0.3*0.3cm fungal agar blocks and inoculate them into oat culture medium covered with mountain orchid seeds. Set up a control group with sterile agar blocks to eliminate contamination interference. Set up 15 repetitions. Observe and count the germination of seeds every day after inoculation, calculate the germination rate and protocorm formation rate of mountain orchid seeds, observe and statistically analyze the germination process of mountain orchid seeds, and divide the dynamic process of mountain orchid seed germination into 5 stages. S4: Screening of conditions for promoting germination of Cymbidium seeds by Agaricus spp. During the germination of mountain orchid seeds, the state between the appearance of the apical meristem and the formation of the first true leaf is called the protocorm period. After this, the seeds will become seedlings and gradually develop into plants. In the early stage of mountain orchid seed germination, this experiment used a stereo microscope to observe and statistically analyze the seed growth and development on the surface of 4.0g / L, 8.0g / L, and 12.0g / L oatmeal culture media. When the symbiotic culture lasted for more than 2 weeks, the mycelium would cover the surface of the culture medium, and the excess mycelium needed to be regularly removed in the clean bench; The data related to the promotion of germination of Cymbidium seeds by the strains of the genus Fragilis are as follows: the germination began after the fungus and Cymbidium seeds were co-cultured for 100 days, and a large number of seeds absorbed water and became larger. The maximum germination rate was reached at 165 days. The statistical calculation results showed that the Cymbidium seeds in the treatment group inoculated with the fungus showed signs of germination, while the seeds in the control group inoculated with sterile agar blocks showed no signs of germination. The germination rates of the treatment groups were significantly higher than those of the control group, and there was a significant difference. In the experiment of promoting the germination of mountain orchid seeds, the germination rates of mountain orchid seeds under the conditions of 4.0g / L and 12.0g / L oats were 20.11% and 31.07% respectively. The seeds all absorbed water and swelled, the seed coats broke and swelled to a sphere, and the final stage of development of seeds symbiotically germinated with the genus Fragilis was swelling to a transparent white sphere, i.e. germination stage 1; the seed germination rate under the conditions of 8.0g / L oats was the highest, close to 55.26%, of which most seeds were in germination stage 2, the seed coats were completely broken, the embryos absorbed water and swelled into transparent white spheres, a small number of seeds developed to stages 3 to 4, grew the first true leaf, and the protocorm formation rate was about 30%.

2. A method for promoting the germination of mountain orchid seeds using the genus Fragilis fungus according to claim 1, Features: In step S1, the FIM medium includes: 0.35 g sodium nitrate, 0.30 g potassium dihydrogen phosphate, 0.10 g magnesium sulfate, 0.15 g potassium chloride, 5.00 g yeast, 2.50 g sucrose, and 8.00 g agar.

3. A method for promoting the germination of mountain orchid seeds by using the genus Fragilis fungus according to claim 1, Features: In the step S1, the internal transcribed spacer (ITS) region ITS1 and ITS2 of the ribosomal rDNA are amplified by PCR using universal primers ITS-1 and ITS-4, the PCR reaction conditions and PCR amplification procedure of fungal DNA are used, and the PCR products are sequenced using Sanger sequencing technology.

4. The method for promoting the germination of mountain orchid seeds by using the genus Fragilis fungus according to claim 1, Features: In the step S1, after rDNA-ITS molecular identification, the results are compared and analyzed using NBCI software, and the isolated endophytic fungus is identified as a fungus of the genus Fragilis by referring to the fungal category with the highest sequence similarity in the comparison.

5. The method for promoting the germination of mountain orchid seeds by using the genus Fragilis fungus according to claim 1, Features: In step S3, the germination rate and protocorm formation rate of Cymbidium orchid seeds are calculated according to the following formula: Seed germination rate: (total number of germinated seeds / total number of sowings) × 100% Protocorm formation rate: (total number of protocorms formed / total number of germinated seeds)×100.

6. The method for promoting the germination of mountain orchid seeds by using the genus Fragilis fungus according to claim 1, Features: In step S4, there are three stages of the germination process of mountain orchid seeds: The first stage was co-cultivation for 100 days, which was at germination stage 0-1; The second stage is symbiosis for 100-130 days, which is in the germination stage 1-2; The third stage is symbiosis for 165 days, which is completely in the germination stage 3-4.

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