Coprinus nigrum strain for promoting germination of cremastra appendiculata seeds and application thereof

Abstract

The invention discloses a coprinus comatus strain for promoting seed germination of cremastra appendiculata and application of the coprinus comatus strain, the strain is separated and screened from root tissues of cremastra appendiculata, the strain is identified as coprinus comatus through ITS (internal transcribed spacer) sequencing, the Latin name is Coprinella micaceus, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) NO: 42051. The strain is inoculated into a liquid culture medium for shake culture, and mycelia are collected and mixed with sterile vermiculite and turfy soil to prepare a fungicide; the method comprises the following steps: laying a sterilized culture medium formed by mixing leaf mold, perlite and bark in a sterile culture container, spreading a fungicide, spreading the treated azalea seeds, covering the culture medium, and culturing by controlling the temperature, the illumination condition and the relative humidity. According to the method, the germination rate of the rhododendron simsii seeds can reach 60%-70%, the germination time is shortened to 15-20 days, the survival rate of seedlings is high, operation is easy and convenient, cost is low, the method is suitable for large-scale breeding and resource protection of the rhododendron simsii, and a new thought is provided for research on symbiotic germination of orchid plants.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicinal plant breeding, and particularly relates to a strain capable of promoting seed germination of Cremastra appendiculata and a method for symbiotic culture of seeds of Cremastra appendiculata by using the strain. The present application establishes a symbiotic relationship with the seeds of Cremastra appendiculata by screening, isolating and purifying the species of Craterellus cristatus, so as to improve the germination rate of the seeds of Cremastra appendiculata, and provides effective technical support for the large-scale artificial breeding and wild resource protection of Cremastra appendiculata. BACKGROUND

[0002] As a perennial rare medicinal herbaceous plant of Orchidaceae and Cremastra, Cremastra appendiculata (D. Don) Makino has dry pseudobulbs with the effects of clearing heat and resolving toxins, promoting blood circulation and removing blood stasis, and resisting tumors, and is widely used in the field of medicine and has a strong market demand. However, due to long-term predatory excavation and deterioration of the living environment, the wild resources of Cremastra appendiculata are close to exhaustion, and have been listed as a national key protected wild plant, so resource protection and sustainable utilization are imminent.

[0003] From the perspective of reproductive characteristics, the seeds of Cremastra appendiculata have significant defects: the seeds are extremely small (about 0.3-0.5 mm in length and about 0.05-0.1 mm in width), have no endosperm, contain only undifferentiated embryos, are difficult to germinate spontaneously under natural conditions, and the germination rate is usually less than 5%, and the germination period is as long as 6-12 months. The current mainstream breeding methods have obvious limitations: the efficiency of clonal propagation is extremely low, and each adult plant can only produce 1-2 new plants per year, which cannot meet the demand of large-scale production; the seed aseptic germination technology can improve the germination rate to a certain extent, but the seedlings have poor resistance to pathogenic microorganisms after being transplanted to soil, have a low survival rate, and are difficult to establish a symbiotic relationship with fungi in the natural environment, resulting in growth inhibition.

[0004] The seed germination of orchid plants depends on specific symbiotic fungi to provide carbon source, nitrogen source and other nutrients, so screening of high-efficiency seed germination promoting fungal strains of Cremastra appendiculata becomes the key. In existing research, the symbiotic fungi found have many problems: first, the germination rate is low and unstable, usually between 10% and 30%, and there are significant differences between different batches, which is difficult to stabilize production; second, the germination period is long, even under the best conditions, the seed germination to form protocorms takes 30-60 days, which seriously affects the breeding efficiency; third, the fungal culture is difficult, and some strains grow slowly under artificial culture conditions, which is difficult to reproduce in large quantities; fourth, the symbiotic conditions are complex, and the requirements for temperature, humidity, light and other environments are harsh, which is difficult to control in large-scale production.

[0005] In summary, the existing technology cannot effectively solve the problems of low seed germination rate, long period and limited application of symbiotic fungi of Cremastra appendiculata, and it is urgent to explore an efficient, stable and easy-to-promote technical solution to realize the effective protection and large-scale breeding of Cremastra appendiculata resources. SUMMARY

[0006] The present application aims to overcome the problems of low seed germination rate, long germination period and difficult cultivation of symbiotic fungi in the prior art, and provides a strain for symbiotic germination of Cymbidium seed with high germination rate, short germination period, simple cultivation, high efficiency and stability, and a specific method for symbiotic germination of Cymbidium seed using the strain.

[0007] To achieve the above purpose, the strain provided by the present application is named Coprinellus micaceus N6, and was preserved in the China General Microbiological Culture Collection Center on July 9, 2025, with the preservation number of CGMCC NO: 42051 and the preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing.

[0008] Further, the ITS sequence of the strain is shown in SEQ ID NO. 1, specifically as follows:

[0009] ggctttttga tatgcttaag ttcagcgggt agtcctacct gatttgaggt caaattgtca 60

[0010] aaggtattgt ccttgcggac ggttagaagc gagtctaaac cctatccacg gcgtagataa 120

[0011] ttatcacacc aatagacgga gctcagtacg aactcgctaa tgcatttcag gggagcagac 180

[0012] cgcgctgagg cagcctgcac aaacccccac atccaagcct cggagaaccg ttcagaaaac 240

[0013] gggtgaggtt gagaatttaa tgacactcaa acaggcatgc tcctcggaat accaaggagc 300

[0014] gcaaggtgcg ttcaaagatt cgatgattca ctgaattctg caattcacat tacttatcgc 360

[0015] atttcgctgc gttcttcatc gatgcgagag ccaagagatc cgttgctgaa agttgtatag 420

[0016] tgttttatag gcgatcaagc ccattgacta cattctatat catgcttttg gggtgtgtaa 480

[0017] aaagacgtag agcctggaaa ttcgaggaga gacacctccg agttgaaggg caatcctcgc 540

[0018] atccgcactc agagagcacg agagtcatcc agacctacag tcggtgcaca ggtggataga 600

[0019] taaaaatggc gggcgtgcac aatgctccga ggagccagct acaaccaaga caccatagtt 660

[0020] attcgttaat gatccttccg cggtcac 687

[0021] The method for obtaining the strain of this invention is as follows: Collect the root systems of healthy, disease-free Rhododendron plants, remove the soil attached to the roots, and then cut the root tissue into 1-2 cm segments. Disinfect the root segments with 75% alcohol for 30 seconds, then with 0.1% mercuric chloride solution for 5 minutes, rinse 5 times with sterile water, and then cut them into 0.5 cm long segments in a clean bench. Inoculate 3-5 segments onto PDA medium plates. Place the inoculated plates in a constant temperature incubator at 25-28℃ and incubate in the dark. Observe the colony growth daily. Once mycelia appear, promptly pick the pure mycelia from the edge and inoculate them onto a new PDA medium plate. Incubate at a constant temperature of 25-28℃ for 5-7 days. Repeat the purification culture 3-5 times until a single colony is obtained, thus obtaining the target strain.

[0022] The method for identifying the target strain is as follows:

[0023] (1) Morphological identification: the purified strain was cultured on PDA medium, and the colony morphology was observed, including the color, texture, edge characteristics, growth rate, etc. Meanwhile, the mycelium morphology and spore characteristics were observed under a microscope. The main morphological characteristics of the strain are as follows: the mycelium is white at the initial stage, and is gradually changed to gray or grayish yellow at the later stage; the mycelium is thin and has septa, with a diameter of 2-5 μm; the fruiting body is small, with a cap diameter of 0.5-3 cm, an initial ovate shape, and a later bell-shaped to flat shape, and the surface has a luster and fine scales; the spores are oval, black, smooth, and have a size of (8-12) μm × (5-7) μm.

[0024] (2) Molecular biological identification: the CTAB method was used to extract the genomic DNA of the strain, and PCR amplification was carried out by using ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') as primers. The PCR reaction system is 25 μL: 10 × PCR Buffer 2.5 μL, dNTPs (2.5 mmol / L) 2 μL, primer ITS1 (10 μmol / L) 1 μL, primer ITS4 (10 μmol / L) 1 μL, Taq DNA polymerase (5 U / μL) 0.2 μL, genomic DNA (50 ng / μL) 1 μL, and ddH2O 17.3 μL. The PCR reaction conditions are as follows: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 1 minute, a total of 35 cycles; and 72℃ final extension for 10 minutes. After the PCR product was detected by 1% agarose gel electrophoresis, it was sent to a sequencing company for sequencing. The ITS sequence obtained by sequencing was subjected to BLAST comparison with the known sequences in the GenBank database, and the molecular identification of the strain was carried out by taking similarity of 99% and above as the standard. The results show that the ITS sequence similarity of the strain with Coprinellus micaceus is 99.5%. Therefore, the strain is named as Coprinellus micaceus N6.

[0025] The application also provides an application of the above-mentioned Coprinellus micaceus N6 in promoting the seed germination of the Cymbidium goeringii, and the specific method comprises the following steps.

[0026] Step 1: Preparation of the Coprinellus micaceus agent

[0027] (1) Liquid strain culture

[0028] The crystal grain small ghost umbrella (Coprinellus micaceus) N6 with the preservation number of CGMCC NO: 42051 in claim 1 is inoculated into a liquid PDB culture medium at an inoculation amount of 3% to 5%, and is placed on a shaking bed for shaking culture, with a culture temperature of 25 to 28 DEG C and a culture time of 5 to 7 days.

[0029] (2) Mycelium collection and treatment

[0030] The liquid culture is filtered with sterile gauze, the mycelium is washed with sterile water to remove residual culture medium, and the washed mycelium is placed in a sterile culture dish and dried in a super-clean bench until the surface is free of water.

[0031] (3) Preparation of the microbial agent

[0032] The dried mycelium is mixed with sterile vermiculite and grass carbon under sterile conditions at a volume ratio of 1:1 to 2:2 to 3:2 to 3, to obtain the crystal grain small ghost umbrella microbial agent.

[0033] Step 2: Treatment of Cymbidium seed

[0034] (1) Capsule collection and surface disinfection

[0035] Healthy Cymbidium plants free of pests and diseases are selected, and the capsules are collected when they are mature but have not yet cracked. The collected capsules are surface disinfected in a super-clean bench.

[0036] (2) Seed extraction and treatment

[0037] The disinfected capsules are vertically cut under sterile conditions using a sterilized scalpel, and the seeds are poured into a sterile culture dish and gently stirred with a sterile brush to disperse the seeds evenly. The seeds are then evenly spread on sterile filter paper and dried in a super-clean bench for 10 to 15 minutes until the surface is free of water.

[0038] Step 3: Symbiotic germination culture

[0039] (1) Substrate laying

[0040] A layer of sterilized culture medium with a thickness of 2 to 3 cm is laid at the bottom of a sterilized culture container, and a sterile glass rod is used to compact the culture medium to make the surface flat. The culture medium is composed of rotten leaf soil, perlite and tree bark at a volume ratio of 3:1 to 1.5:1 to 1.5:1. The culture medium is sterilized by spraying with 1% potassium permanganate solution and left to stand for 24 to 36 hours, and then washed with sterile water until neutral to obtain the sterilized culture medium.

[0041] (2) Inoculation of the microbial agent

[0042] The crystal grain small ghost fungus agent in step 1 is evenly scattered on the surface of the culture medium, and the dosage of the crystal grain small ghost fungus agent is 5-10 g per 100 cm 2 The culture medium is scattered on the surface of the culture medium, and the culture medium is scattered on the surface of the culture medium.

[0043] (3) Seed sowing

[0044] The treated dujuanlan seeds in step 2 are evenly scattered on the surface of the mixed layer of the crystal grain small ghost fungus agent and the culture medium with a sterile brush, and the sowing density is 50-100 seeds per 100 cm 2 Scattered 50-100 particles.

[0045] (4) Covering and culture

[0046] After sowing, a layer of sterile culture medium with a thickness of 0.5-1 cm is covered on the surface of the dujuanlan seeds, and a sterile glass rod is used to gently compact the culture medium and the dujuanlan seeds to make them closely contact; then, 3-5 small holes with a diameter of 0.5-1.0 cm are drilled on the cover of the culture container for ventilation; the culture container is placed in an artificial climate chamber for culture, and the culture conditions are as follows: temperature 20-25℃, light intensity 1000-1500lx, light time 12-14 hours / day, and relative humidity 70%-80%.

[0047] Further, in the above step 1, the dried mycelium is mixed with sterile vermiculite and grass charcoal in a volume ratio of 1:2:3 under sterile conditions to obtain the crystal grain small ghost fungus agent.

[0048] Further, in the above step 2, the surface disinfection method is as follows: first, wipe the surface with 75% alcohol cotton ball 2-3 times, then put it in a sterile beaker, add 75% alcohol and soak for 30-60 seconds, pour out the alcohol, add 0.1% mercury solution and soak for 5-10 minutes; pour out the mercury solution, rinse the capsule 3-5 times with sterile water to completely remove the residual mercury.

[0049] Further, in the above step 3, the culture medium is mixed by 3:1:1 in volume ratio of rotten leaf soil, perlite and bark.

[0050] Further, in the above step 3, the rotten leaf soil is selected from the soil of broad-leaved tree fallen leaves after sufficient composting, the perlite is sterilized by high-pressure steam at 121-125℃ for 2-2.5 hours, and the bark is selected from pine bark, which is crushed into small pieces of 0.5-1 cm and dried at 155-165℃ for 4-4.5 hours.

[0051] Further, in the above-mentioned step 3, preferably, the culture container is placed in an artificial climate box for culture, and the culture conditions are as follows: temperature 23℃ in the daytime and 20℃ at night, light intensity 1200lx, light time 13 hours per day, and relative humidity 75%.

[0052] Further, in the above-mentioned step 3, during the culture process, the moisture condition of the culture medium is observed every day, and when dry marks appear on the surface of the culture medium, sterile water is sprayed by using a sterile sprayer to keep the culture medium moist, but water accumulation is avoided.

[0053] The present application realizes rapid and efficient germination of the Cymbidium kanran seeds by screening high-efficiency Coprinus cinereus strains, optimizing the preparation process of the microbial agent and the symbiotic culture conditions, and provides technical support for the large-scale breeding and wild resource protection of the Cymbidium kanran. Compared with the prior art, the present application has the following beneficial effects:

[0054] 1. Improved germination rate and stability: The present application ensures the purity and activity of the Coprinus cinereus strain through a strict strain purification process, so that the symbiotic action of the strain and the Cymbidium kanran seeds is more stable and efficient. By using the strain and the method, the germination rate of the Cymbidium kanran seeds can reach 60% to 70%, and the germination rate difference of different batches is small, and the stability is significantly better than that of the prior art.

[0055] 2. Shortened germination period: The purified Coprinus cinereus strain can more quickly establish a symbiotic relationship with the Cymbidium kanran seeds to provide timely nutritional support for seed germination, so that the seeds can start to germinate 15 to 20 days after sowing, which shortens the germination time and improves the breeding efficiency compared with the prior art.

[0056] 3. Simple and easy operation: The strain purification process adopts a continuous transfer purification method, which is simple and easy to master; and the symbiotic germination culture conditions are mild and have low requirements for equipment, so it is convenient to popularize and apply in actual production.

[0057] 4. Improved seedling quality: The Cymbidium kanran seedlings germinated by the method have developed root systems and grow healthily, and the transplanting survival rate can be more than 80%, which lays a good foundation for the subsequent growth of the Cymbidium kanran and is beneficial to improve the success rate of artificial breeding. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 is a colony morphology diagram of the purified Coprinus cinereus N6 strain (cultured for 7 days).

[0059] Figure 2 is a schematic diagram of different stages of symbiotic germination of the Cymbidium kanran seeds and the Coprinus cinereus N6 strain in Example 2.

[0060] Figure 3 is the seed germination condition of the Cymbidium kanran seeds after 30 days of symbiotic germination culture in Example 2.

[0061] Figure 4 This is the seedling growth after 150 days of symbiotic germination culture of Rhododendron seeds in Example 2. Detailed Implementation

[0062] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0063] Example 1

[0064] Isolation, purification, and identification of bacterial strains

[0065] Sample Collection: Root systems from healthy, disease-free Rhododendron plants were collected in mountainous areas where Rhododendrons thrive. Soil adhering to the roots was removed, and the root tissue was cut into 1-2 cm segments and placed in sterile sampling bags. Spring and autumn are the best times for collection, as soil moisture is suitable and fungal activity is high. Collected samples must be stored at 4℃ and brought back to the laboratory for processing within 24 hours.

[0066] Isolation and purification of the strain: Root segments were disinfected with 75% alcohol for 30 seconds, then with 0.1% mercuric chloride solution for 5 minutes, rinsed 5 times with sterile water, and then cut into 0.5 cm long segments. These segments were inoculated onto PDA medium plates, with 3-5 segments inoculated per plate. The inoculated plates were placed in a 25°C incubator in the dark for daily observation of colony growth. After 5 days of incubation, mycelia appeared. Pure mycelia from the edges were picked and inoculated onto new PDA medium plates. Purification was carried out at 26°C for 6 days, and the purification process was repeated 4 times to obtain single colonies, thus obtaining the target strain.

[0067] The purified target strain was identified using the following methods:

[0068] 1. Morphological identification of the strain

[0069] The purified strain was inoculated onto solid PDA medium and cultured at 28°C for 7 days. Colony morphology was then observed, including colony color, texture, edge characteristics, and growth rate. Simultaneously, hyphae morphology and spore characteristics were observed under a microscope. Figure 1 As shown, the initial mycelium is white, fluffy, with neat edges and good growth; in the later stage of growth, a large number of gray or grayish-yellow mycelial bundles are produced. The mycelium is slender, septate, and has a diameter of 2-5 μm; the fruiting body is small, with a cap diameter of 0.5-3 cm. It is initially oval, and later becomes bell-shaped to flat. The surface is glossy and has small scales; the spores are elliptical, black, smooth, and have a size of (8-12) μm × (5-7) μm.

[0070] 2. Molecular biological identification

[0071] Genomic DNA of the strain was extracted using a fungal DNA extraction kit by CTAB method, and PCR amplification was performed using fungal universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), and the PCR reaction system was 25 μL: 10 x PCR Buffer 2.5 μL, dNTPs (2.5 mmol / L) 2 μL, primer ITS1 (10 μmol / L) 1 μL, primer ITS4 (10 μmol / L) 1 μL, Taq DNA polymerase (5 U / μL) 0.2 μL, genomic DNA (50 ng / μL) 1 μL, ddH2O 17.3 μL. The PCR reaction conditions were as follows: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 1 min, a total of 35 cycles; 72 ℃ final extension for 10 min. After the PCR product was detected by 1% agarose gel electrophoresis, it was sent to a sequencing company for sequencing. The ITS sequence obtained by sequencing was subjected to BLAST comparison with known sequences in the GenBank database, and the similarity was 99% or above, and the molecular identification of the strain was performed. The results showed that the ITS sequence similarity of the strain with Coprinellus micaceus was 99.5%. Therefore, the strain was named Coprinellus micaceus N6. The ITS sequence of the strain is shown as SEQ ID NO. 1, and specifically:

[0072] ggctttttga tatgcttaag ttcagcgggt agtcctacct gatttgaggt caaattgtca 60

[0073] aaggtattgt ccttgcggac ggttagaagc gagtctaaac cctatccacg gcgtagataa 120

[0074] ttatcacacc aatagacgga gctcagtacg aactcgctaa tgcatttcag gggagcagac 180

[0075] cgcgctgagg cagcctgcac aaacccccac atccaagcct cggagaaccg ttcagaaaac 240

[0076] gggtgaggtt gagaatttaa tgacactcaa acaggcatgc tcctcggaat accaaggagc 300

[0077] gcaaggtgcg ttcaaagatt cgatgattca ctgaattctg caattcacat tacttatcgc 360

[0078] atttcgctgc gttcttcatc gatgcgagag ccaagagatc cgttgctgaa agttgtatag 420

[0079] tgttttatag gcgatcaagc ccattgacta cattctatat catgcttttg gggtgtgtaa 480

[0080] aaagacgtag agcctggaaa ttcgaggaga gacacctccg agttgaaggg caatcctcgc 540

[0081] atccgcactc agagagcacg agagtcatcc agacctacag tcggtgcaca ggtggataga 600

[0082] taaaaatggc gggcgtgcac aatgctccga ggagccagct acaaccaaga caccatagtt 660

[0083] attcgttaat gatccttccg cggtcac 687

[0084] 3. Preservation of bacterial strains

[0085] The Coprinellus micaceus N6 strain was inoculated onto PDA slant medium and grown at 28°C for 7 days. It was deposited on July 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 42051. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0086] Example 2

[0087] Application of Coprinellus micaceus N6 in promoting the germination of Rhododendron seeds

[0088] Step 1: Preparation of crystalline *Coprinus comatus* inoculant

[0089] (1) Liquid culture

[0090] The Coprinellus micaceus N6 strain is inoculated into a liquid PDB medium with a formula of 200 g of potato, 20 g of glucose, 5 g of yeast extract, 3 g of KH2PO4, 1.5 g of MgSO4·7H2O, and 1000 mL of water, and the pH is adjusted to 6.0-6.5. The culture conditions are as follows: a 250 mL triangular flask is used, the liquid volume is 100 mL, the inoculation volume is 5% (v / v, i.e., 5 mL of liquid strain is inoculated into 100 mL of culture medium), the shaking culture is performed on a shaking table, the rotation speed is 180 r / min, the culture temperature is 26°C, and the culture time is 6 days. During the culture process, the mycelium growth is observed every day, and when the mycelium forms uniform spherical bodies and the culture solution is turbid, the culture is stopped.

[0091] (2) Mycelium collection and treatment

[0092] The liquid culture is filtered with sterile gauze, the mycelium is washed with sterile water for 3 times to remove the residual culture medium. The washed mycelium is placed in a sterile culture dish and dried on an ultra-clean bench until the surface is free of water.

[0093] (3) Preparation of the microbial agent

[0094] The dried mycelium is mixed with sterile vermiculite and grass carbon soil at a volume ratio of 1:2:3 to obtain the Coprinellus micaceus microbial agent. The mixing process is performed under sterile conditions, and a sterile stirrer or manual stirring (the tools need to be sterilized before operation) can be used. The Coprinellus micaceus microbial agent is packed into sterile plastic bags, sealed and stored in a 4°C refrigerator, and the storage period is not more than 1 month.

[0095] Step 2: Treatment of Cymbidium goeringii seeds

[0096] (1) Capsule collection and surface disinfection

[0097] Healthy and disease-free Cymbidium goeringii plants are selected, and the capsules are collected when they are mature but have not yet cracked. The mature capsules are yellow-green in color and have obvious ridges on the surface, and have slight elasticity when pinched by hand. When collecting, the capsules are cut from the fruit stalks with scissors and placed in sterile sampling bags to avoid squeezing and collision. The collected capsules are placed on an ultra-clean bench, first wiped with 75% alcohol cotton balls for 3 times, then placed in a sterile beaker, soaked with 75% alcohol for 30 seconds, poured out the alcohol, and then soaked with 0.1% mercury solution for 8 minutes, constantly shaking the beaker gently during the process to ensure sufficient disinfection. Pour out the mercury solution and rinse the capsules with sterile water for 5 times, each time for not less than 1 minute to completely remove the residual mercury.

[0098] (2) Seed extraction and treatment

[0099] Sterilized capsules were cut open with a sterilized scalpel under sterile conditions, and the seeds were poured into a sterile petri dish. The seeds were gently stirred with a sterile brush to ensure even distribution, and then evenly spread on sterile filter paper. The seeds were dried for 10-15 minutes in a clean bench until the surface was dry.

[0100] Step 3: Symbiotic germination culture

[0101] (1) Substrate preparation

[0102] Transparent plastic culture boxes (20 cm x 15 cm x 10 cm) were selected as the culture container and sterilized at 121°C for 2 hours before use. A layer of sterilized culture medium with a thickness of 2.5 cm was placed at the bottom of the sterilized culture box, and a sterile glass rod was used to compact the culture medium to make the surface flat. The culture medium was composed of leaf soil, perlite, and tree bark in a volume ratio of 3:1:1. The leaf soil was made from fully decomposed leaf litter of broad-leaved trees, the perlite was sterilized at 121°C for 2 hours, and the tree bark was pine bark crushed into 0.5-1 cm pieces and sterilized at 160°C for 4 hours. The culture medium was sterilized with 1% potassium permanganate solution and washed with sterile water to neutralize (pH 6.0-7.0) after 24 hours.

[0103] (2) Inoculation of microbial agents

[0104] The crystal grain small ghost mushroom agent in step 1 was evenly spread on the surface of the culture medium, and the amount of crystal grain small ghost mushroom agent was 8g±1g per 100cm 2 After spreading, the surface of the culture medium was gently stirred with a sterile glass rod to ensure that the crystal grain small ghost mushroom agent was fully contacted with the surface layer of the culture medium, with a contact depth of 0.4-0.6cm.

[0105] (3) Seed sowing

[0106] The treated Cymbidium seed in step 2 was evenly spread on the surface of the mixed layer of crystal grain small ghost mushroom agent and culture medium with a sterile brush, and the sowing density was 80 seeds±10 seeds per 100cm 2 The seeds should be spread evenly to ensure that each seed is in full contact with the crystal grain small ghost mushroom agent.

[0107] (4) Covering and culture

[0108] After sowing, a layer of sterile culture medium with a thickness of 0.8 cm was covered on the surface of the Cymbidium seed, and the culture medium was gently compacted with a sterile glass rod to make the culture medium in close contact with the Cymbidium seed. Then, 3-5 small holes with a diameter of 0.5 cm were drilled on the cover of the culture box for ventilation. The culture box was placed in an artificial climate box for culture, and the culture conditions were as follows: temperature 23°C in the daytime and 20°C at night, light intensity 1200 lx, light time 13 hours per day, and relative humidity 75%. During the culture process, the moisture content of the culture medium was observed every day, and sterile water was sprayed with a sterile sprayer when the surface of the culture medium showed signs of drying to keep the culture medium moist, but avoid water accumulation.

[0109] From the day of sowing, the seed germination was observed every day. The germination rate was counted at 10 days, 20 days and 30 days after sowing, and the germination rate = (the number of germinated seeds / the total number of sowed seeds) x 100%. According to the classification standard of seed germination stages of other orchid plants, the Cymbidium seed germination process was divided into 6 stages according to Table 1. The seed development to the first stage, i.e. the seed embryo swelling, was regarded as seed germination, and the seed in the 0th stage was considered as seed germination failure. The germination rate (%) = (N1+N2+N3+N4+N5) / (N0+N1+N2+N3+N4+N5) x 100%, wherein N0 is the number of seeds in the 0th stage, and N1, N2, N3, N4 and N5 are the number of seeds in the 1st, 2nd, 3rd, 4th and 5th stages, respectively.

[0110] Table 1 Characteristics of different development stages of orchid seeds

[0111] Stage Feature 0 Ungerminated seed, no change in embryo 1 Embryo absorbs water and swells (considered germination) 2 Embryo breaks through seed coat 3 Embryo develops into prothallus 4 Primordia appear 5 First true leaf appears or rhizoids appear

[0112] From Figure 2 It can be seen that there was no obvious change in the Cymbidium seed before it was co-cultured with the N6 strain of the crystal grain small ghost for 5 days, and the seed was still in the 0th stage without germination ( Figure 2 5 d), at this time, the Cymbidium seed was slender and yellow, and the seed embryo was small; when the seed was co-cultured for 10 days, the seed embryo began to swell, but had not yet broken through the seed coat, reaching the 1st stage of germination ( Figure 2 10 d); when the seed was co-cultured for 20 days, the volume of the Cymbidium embryo increased obviously, one side of the seed coat was opened, and the embryo was exposed, reaching the 2nd stage of germination ( Figure 2 20 d); when the seed was co-cultured for 30 days, the embryo developed into a visible protocorm, and the seed coat in the apical meristem region had fallen off and disappeared, reaching the 3rd stage of germination ( Figure 2 30 d); when the seed was co-cultured for 40 days, the seed coat completely fell off, the apical meristem formed a dorsal ridge, and the base turned green, reaching the 4th stage of germination ( Figure 2 40 d). During this period, the seeds in the control group without infection had no germination signs ( Figure 2The germination rate of seeds sowed for 10 days, 20 days and 30 days was calculated respectively. With the extension of culture time, the germination rate increased slightly. The germination rate of seeds sowed for 10 days was 42.17%±0.92%, the germination rate of seeds sowed for 20 days was 56.32%±1.64%, and the germination rate of seeds sowed for 30 days was 65.06%±1.32%.

[0113] As shown in FIG. 2, the total germination rate of seeds of Cymbidium goeringii Rchb.f. reached 65%±2% after 30 days of symbiotic germination of C. goeringii Rchb.f. seeds and Coprinellus micaceus N6, and most of the embryos continued to develop after breaking through the seed coat, and then developed into stems and leaves. Figure 3 As shown in FIG. 2, the total germination rate of seeds of Cymbidium goeringii Rchb.f. reached 65%±2% after 30 days of symbiotic germination of C. goeringii Rchb.f. seeds and Coprinellus micaceus N6, and most of the embryos continued to develop after breaking through the seed coat, and then developed into stems and leaves.

[0114] Figure 4 As shown in FIG. 2, the total germination rate of seeds of Cymbidium goeringii Rchb.f. reached 65%±2% after 30 days of symbiotic germination of C. goeringii Rchb.f. seeds and Coprinellus micaceus N6, and most of the embryos continued to develop after breaking through the seed coat, and then developed into stems and leaves.​

Claims

1. Coprinellus micaceus N6 was deposited at the China General Microbiological Culture Collection Center on July 9, 2025, with accession number CGMCC NO: 42051.

2. The Coprinellus micaceus N6 grain according to claim 1, characterized in that: The ITS sequence of the strain is shown in SEQ ID NO.

1.

3. A method for promoting the germination of Rhododendron seeds, characterized in that: The method includes the following steps: Step 1: Preparation of crystalline *Coprinus comatus* inoculant (1) Liquid culture The Coprinellus micaceus N6 specimen with accession number CGMCC NO: 42051 as described in claim 1 was inoculated into liquid PDB medium at an inoculation amount of 3% to 5%, and cultured on a shaker at a temperature of 25 to 28°C for 5 to 7 days. (2) Collection and treatment of mycelium Filter the liquid culture with sterile gauze, collect the mycelium, rinse with sterile water to remove residual culture medium; place the rinsed mycelium in a sterile petri dish and air dry in a laminar flow hood until there is no moisture on the surface. (3) Preparation of microbial agents Under aseptic conditions, the dried mycelium was mixed evenly with aseptic vermiculite and peat moss in a volume ratio of 1:1 to 2:2 to 3 to obtain crystalline small coccinea fungicide. Step 2: Treatment of Rhododendron Seeds (1) Capsule collection and surface disinfection Select healthy, disease-free Rhododendron plants and collect them when the capsules are mature but not yet split open. Place the collected capsules in a clean bench for surface disinfection. (2) Seed extraction and treatment Under aseptic conditions, use a sterile scalpel to cut the sterilized capsules longitudinally, pour the seeds into a sterile petri dish, gently stir the seeds with a sterile brush to disperse them evenly, then spread the seeds evenly on sterile filter paper and air dry them in a laminar flow hood for 10-15 minutes until there is no moisture on the seed surface. Step 3: Symbiotic Germination Culture (1) Substrate laying A layer of sterile culture medium with a thickness of 2-3 cm is laid at the bottom of a sterile culture container. The culture medium is compacted with a sterile glass rod to make the surface of the culture medium smooth. The culture medium is composed of leaf mold, perlite and bark mixed in a volume ratio of 3:1-1.5:1-1.

5. The culture medium is sprayed with 1% potassium permanganate solution for disinfection. After standing for 24-36 hours, it is rinsed with sterile water until neutral to obtain sterile culture medium. (2) Inoculation with microbial agents Evenly sprinkle the crystalline *Coprinus comatus* inoculant from step 1 onto the surface of the culture medium. The dosage of the crystalline *Coprinus comatus* inoculant is [per 100 cm³]. 2 Sprinkle 5-10g of the culture medium surface; after sprinkling, gently turn the surface of the culture medium with a sterile glass rod to ensure that the crystalline Coprinus comatus agent is in full contact with the surface of the culture medium, with a contact depth of 0.4-0.6cm. (3) Seed sowing Using a sterile brush, evenly sow the treated azalea seeds from step 2 onto the surface of the mixture of crystalline *Coprinus comatus* fungicide and culture medium, at a sowing density of 100 cm². 2 Sow 50-100 seeds; (4) Covering and cultivation After sowing, cover the azalea seeds with a 0.5–1 cm thick layer of sterilized culture medium, and gently press it down with a sterile glass rod to ensure close contact between the culture medium and the azalea seeds. Then, drill 3–5 small holes with a diameter of 0.5–1.0 cm in the lid of the culture container for ventilation. Place the culture container in an artificial climate chamber for cultivation under the following conditions: temperature 20–25℃, light intensity 1000–1500 lx, light duration 12–14 hours / day, and relative humidity 70%–80%.

4. The method for promoting the germination of Rhododendron seeds according to claim 3, characterized in that: In step 1, under aseptic conditions, the dried mycelium is mixed evenly with aseptic vermiculite and peat moss in a volume ratio of 1:2:3 to obtain crystalline small coprinus coccinea agent.

5. The method for promoting the germination of Rhododendron seeds according to claim 3, characterized in that: In step 2, the surface disinfection method is as follows: first, wipe the surface 2-3 times with a cotton ball soaked in 75% alcohol, then put it into a sterile beaker, add 75% alcohol and soak for 30-60 seconds, pour off the alcohol, then add 0.1% mercuric chloride solution and soak for 5-10 minutes; pour off the mercuric chloride solution, and rinse the capsules 3-5 times with sterile water to completely remove residual mercuric chloride.

6. The method for promoting the germination of Rhododendron seeds according to claim 3, characterized in that: In step 3, the culture medium is composed of leaf mold, perlite, and bark mixed in a volume ratio of 3:1:

1.

7. The method for promoting the germination of Rhododendron seeds according to claim 3 or 6, characterized in that: In step 3, the leaf mold is selected from the soil after the leaves of broad-leaved trees have been fully decomposed. The perlite is sterilized by high-pressure steam at 121-125℃ for 2-2.5 hours. The bark is selected from pine bark, which is crushed into small pieces of 0.5-1cm and sterilized by dry heat at 155-165℃ for 4-4.5 hours.

8. The method for promoting the germination of Rhododendron seeds according to claim 3, characterized in that: In step 3, the culture container is placed in an artificial climate chamber for cultivation. The cultivation conditions are: temperature 23℃ during the day and 20℃ at night, light intensity 1200lx, light duration 13 hours / day, and relative humidity 75%.

9. The method for promoting the germination of Rhododendron seeds according to claim 3, characterized in that: In step 3, during the cultivation process, observe the humidity of the culture medium daily. When signs of dryness appear on the surface of the culture medium, spray sterile water with a sterile sprayer to keep the culture medium moist, but avoid water accumulation.

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