A fungus to promote seed germination and uses thereof

Abstract

This invention belongs to the field of microbial technology, specifically relating to a fungus that promotes seed germination and its uses. The fungus is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC NO.42374, and is classified as follows: Coprinellus pseudodisseminatus The present invention describes a symbiotic culture of fungi and Rhododendron seeds, which can colonize within the protocorm cells of Rhododendron in the form of mycelial clusters. After 30 days of culture, the germination rate of Rhododendron seeds can reach 41.40%, significantly improving the germination efficiency of Rhododendron seeds and possessing practical value for widespread application.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a fungus that promotes seed germination and its uses. Background Technology

[0002] Seed germination is a complex and ordered biological process involving multiple stages from dormancy to active growth, and it is crucial for plant reproduction and growth. Orchids (Orchidaceae) are among the more evolved groups of angiosperms. Their seeds are tiny, lack endosperm, and consist only of an immature embryo encased in a simple seed coat, lacking the nutrients required for germination. Under natural conditions, orchid seeds need to establish a symbiotic relationship with fungi, relying on the fungi to provide nutrients for germination and seedling formation. Selecting suitable fungi for symbiotic seed germination can effectively improve the adaptability of orchids, thereby ensuring the ecological restoration of artificially cultivated orchids and the wild populations of endangered species.

[0003] Currently, research on symbiotic fungi of orchids mainly focuses on three major groups: Tulasnellaceae, Ceratobasidiaceae, and Sebacinales. Developing new groups of fungi that promote seed germination in orchids is of great significance for broadening our understanding of orchid mycorrhizal fungi and for the conservation of orchid species. Zhangneng Pan et al. in... <Enhancing Seed Germination of Cremastra appendiculata:Screening and Identification of Four New Symbiotic Fungi in thePsathyrellaceae Family> A fungus was screened out. Coprinellus pseudodisseminatus However, its efficiency in promoting seed germination is low. Therefore, it is necessary to further explore and screen other potential high-efficiency germination-promoting fungal groups to improve the germination rate of orchid seeds and the survival rate of seedlings, thereby providing stronger scientific support for the protection and sustainable utilization of orchids. Summary of the Invention

[0004] To achieve the above objectives, this invention provides a fungus that promotes seed germination, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.42374. Coprinellus pseudodisseminatus .

[0005] The Coprinellus pseudodisseminatusIt was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 19, 2025, and classified and named as follows: Coprinellus pseudodisseminatus The address for storage is: Building 1, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing.

[0006] The present invention also provides the use of the aforementioned fungi in the preparation of seed germination agents that promote the germination of plant seeds.

[0007] Furthermore, the plant seeds include rhododendron seeds.

[0008] The present invention also provides a plant seed germination agent, which is a formulation made of the aforementioned fungus as the active ingredient and with the addition of acceptable excipients.

[0009] Furthermore, the formulation includes solutions, powders, granules, sprays, and blocks.

[0010] Furthermore, the plant seeds include rhododendron seeds.

[0011] Finally, this invention provides a method for promoting plant seed germination, which includes the following steps: Take the aforementioned fungi, inoculate them into PDA medium, cut off small pieces of fungal agar, and then inoculate them into germination medium. Finally, sow the sterilized plant seeds on the surface of the germination medium and culture them.

[0012] Furthermore, the fungus was cultured in PDA medium at a temperature of 28 ℃ and a humidity of 60%–70% for 7 days; the fungus and plant seeds were cultured in germination medium at a temperature of 23±2 ℃, a light intensity of 1600–2000 lx, and a light duration of 14 h / d.

[0013] Furthermore, the PDA culture medium is formulated as follows: each L of culture medium contains 200g potato juice, 20g glucose and 8g agar; The germination medium is formulated as follows: each L of medium contains 2.0g oat flour, 5g sucrose, 30 ml tomato juice and 8.0g agar.

[0014] Furthermore, the plant seeds include rhododendron seeds.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The fungus TC-6 that promotes seed germination in this invention has been identified as... Coprinellus pseudodisseminatusIt has a good promoting effect on the germination of Rhododendron seeds. Experiments have verified that when the fungus TC-6 and Rhododendron seeds are co-cultured in a germination medium supplemented with specific organic matter, the fungus TC-6 can colonize in the Rhododendron protocorm cells in the form of mycelial clusters. After 30 days of culture, the Rhododendron seed protocorm formation rate is high, and the germination rate can reach 41.40%, which significantly improves the germination efficiency and quality of Rhododendron seeds and has practical application value.

[0016] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0017] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0018] Figure 1 Morphological feature diagram; Figure 2 Phylogenetic tree. Detailed Implementation

[0019] The raw materials, reagents, and equipment used in the specific embodiments of this invention were all obtained through commercial purchase.

[0020] Example 1: Method for germinating azalea seeds according to the present invention (1) Preparation of culture medium PDA medium: Take 200g potato juice, 20g glucose and 8g agar, add 1L water to dissolve, sterilize, dispense into petri dishes before solidification, and solidify to obtain the medium.

[0021] Germination medium: Take 2g oat flour, 5g sucrose, 30ml tomato juice and 8g agar, add 1L water to dissolve, sterilize, dispense into petri dishes before solidification, and solidify to obtain the germination medium.

[0022] (2) Plant seed germination Take fungus TC-6, inoculate it into PDA medium, and culture it for 7 days at 28 ℃ and 60%~70% humidity. Cut off small pieces of fungal agar and inoculate them into germination medium. Finally, sow sterilized rhododendron seeds on the surface of the germination medium and co-culture them at 23±2℃, light intensity of 1600-2000lx, and light duration of 14h / d.

[0023] The following experimental examples further illustrate the beneficial effects of the present invention. Experiment 1: Screening and efficacy experiment of fungi promoting the germination of Rhododendron seeds 1. Strain screening and bacterial culture preparation An endophytic fungus, TC-6, was isolated from the protocorm of *Rhododendron simsii* and identified as *Rhododendron simsii* based on morphological and phylogenetic analysis. Coprinellus pseudodisseminatus .

[0024] Its specific identification characteristics are as follows: (1) Morphological characteristics The colonies are milky white to light cream in color. Initially, they are round dots, which expand radially and creep as the culture time progresses. After 7 days of culture, the colony diameter can reach 52.50±2.12 mm, and after 10 days, they fill a 90 mm petri dish. The colony edges are neat, slightly wavy and curled, and they are tightly bound to the culture medium without obvious bulges. The texture is loose and cottony, with no secretions on the surface, no pigment production, and no color change on the back of the culture medium. Aerial hyphae are underdeveloped, and they mainly grow adherently to the culture medium.

[0025] Optical microscope: The hyphae are colorless, transparent, septate hyphae with few branches, and a diameter of 2.5–3.5 μm. They colonize the cortical cells at the base of the Rhododendron protocorm, forming dense mycelial knots (pelotons). These mycelial knots are nearly spherical, with a diameter of 15–25 μm, and are typical mycorrhizal structures of orchid symbiotic fungi. Figure 1 ).

[0026] (2) Physiological and biochemical characteristics Growth rate: PDA medium, natural pH, dark culture at 28℃, growth rate was 7.50 mm / d, and colonies filled a 90 mm petri dish on the 10th day of culture.

[0027] Optimal pH: When cultured in symbiosis with Rhododendron seeds, the optimal pH is 5.5, under which the seed germination rate reaches 41.40%. When pH < 5.0 or pH > 6.0, the germination-promoting effect of the strain decreases significantly, and the seed germination rate is only 18.50% at pH 4.5. Culture medium adaptability: The growth status in OMA medium (4 g / L oat flour + 8 g / L agar) is better than that in PDA and MS medium. When combined with tomato juice (30 mL / L), the growth-promoting effect is the best, which is the optimal culture medium combination for the symbiotic culture of Rhododendron.

[0028] (3) ITS sequence analysis ① Fungal DNA Extraction Fungi were inoculated onto PDA solid medium and cultured for 7 days. A small amount of hyphae was scraped off with a sterile toothpick, and fungal genomic DNA was extracted using a fungal genome extraction kit for PCR amplification.

[0029] ②PCR amplification and sequencing Primer design: Primers ITS1 (SEQ ID NO.1: 5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (SEQ ID NO.2: 5'-TCCTCCGCTTATTGATATGC-3') were used, with a target fragment of approximately 600 bp.

[0030] Reaction system: Total volume 50 μL, containing 2 μL DNA template (final concentration 0.8 ng / μL), 2.5 μL forward primer (final concentration 500 nM), 2.5 μL reverse primer (final concentration 500 nM), 25 μL 2X PCR Bestaq MasterMix (final concentration 1X) and 18 μL ultrapure water.

[0031] Amplification program: 95℃ pre-denaturation for 5 min; 30 cycles (94℃ denaturation for 45 sec, 50℃ annealing for 30 sec, 72℃ extension for 1 min); final extension at 72℃ for 12 min. Electrophoresis detection: 1% agarose gel (LE agarose), 150 V for 20 min. Qualified purified products were sent to Chengdu Luoning Biotechnology Co., Ltd. for sequencing.

[0032] The sequence is as follows (SEQ ID NO. 3): TAAAGAATCCGGGGAGTCCTACCTGATTTGAGGTCAAATTGTCAAAAGTTGTCTTTGCAGACGGTTAGAAGCAAGTCTAAACCCCTTCCACGGCGTAGATAATTATCACACCAATAGACGGAGCTTAGTGAACTCGCTAATGCATTTCAGGGGAGCAGACCGCACAG AGGCAGCCTGCAAAAACCCCCACATCCAAGCCTTACACAGATTCATTACAAAACTGGTGAGGTTGAGAATTTAATGACACTCAAACAGGCATGCTCCTCGGAATACCAAGGAGCGCAAGGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTAC TTATCGCATTTCGCTGCGTTCTTCATCGATGCGAGAGCCAAGAGATCCGTTGCTGAAAGTTGTATTGTGTTTTATAGGCTTAGAGCCCATTGACTTCATTCTACATCATTCAATTGGAGTTTGTAATGACATAGACCTGGAAATTCAAGGAGAGCTGGTCTTGCGAC ACAGCAATCCCTGTTTCCGTCGTGAGAGCGAGAGTTATCCAGATCTACATTATGTGCACAGGTGGAAAGATAAGATGACGGGCGTGCACATGTCCCTAAGAGCCAGCTACAACCACATCAAGTTATTCATTAATGATCCTTCCGCAGGTTCCCCTAACGGGAAGGT The sequence was aligned in GenBank to determine the genus of the strain; ITS sequences of major species within the same genus were downloaded from the NCBI nucleic acid database, and a phylogenetic tree was constructed using mega7 software via neighbor-joining. The results are as follows: Figure 2 As shown. Phylogenetic analysis indicates that this fungus is a novel strain. Coprinellus pseudodisseminatus .

[0033] 2. Identification and Preservation After identification, the TC-6 strain was found to be... Coprinellus pseudodisseminatus It was deposited on November 19, 2025, at the China General Microbiological Culture Collection Center (CGMCC); the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing; the accession number is: CGMCC No. 42374.

[0034] 3. Study on the symbiotic germination of Rhododendron seeds with strain TC-6 ① Activation of TC-6 strain The TC-6 strain was inoculated onto PDA medium (200.0 g / L potato juice + 20.0 g / L glucose + 8.0 g / L agar) and cultured for 7 days at 28 ℃ and 60%–70% humidity. Small pieces of fungal agar were then cut off for later use.

[0035] ② Pretreatment of Rhododendron seeds Harvest mature, unopened capsules of azaleas. Brush off surface dust under running tap water with a soft brush, rinse three times with sterile water, blot dry with filter paper, and then place in a sterile laminar flow hood for surface disinfection. Disinfection steps: Immerse in 75% ethanol for 30 seconds, rinse twice with sterile water, shake with 0.1% mercuric chloride solution for 8 minutes, and finally rinse repeatedly with sterile water four times. Afterward, wipe dry with sterile filter paper and cut lengthwise with a scalpel.

[0036] ③ Symbiotic germination of TC-6 strain with Rhododendron seeds Germination medium (2.0 g / L oat flour + 5 g / L sucrose + 30 ml / L organic additive + 8.0 g / L agar, pH 5.5; organic additive was banana juice, tomato juice, or coconut juice) was used. The fungal agar block obtained in step ① was inoculated in the center of the medium, and then the Rhododendron seeds treated in step ② were evenly sown on the surface of the germination medium. Three replicates were set up and cultured at 23±2℃, light intensity of 1600-2000 lx, and light duration of 14 h / d. Meanwhile, the germination medium (2.0 g / L oat flour + 5 g / L sucrose + 8.0 g / L agar, pH 5.5) inoculated with the fungal agar block obtained in step ① and the Rhododendron seeds treated in step ② were sown as a control. Germination rate and protocorm formation rate were statistically analyzed 30 days after co-cultivation.

[0037] The effects of different organic additions on symbiotic germination are shown in Table 1. Stage 0+1 represents the non-germinating stage with intact seed coat and swollen embryo after water absorption; Stage 2 represents the germination stage when the embryo breaks through the seed coat; and Stage 3+4 represents the stage of protocorm emergence and leaf primordia differentiation. Germination rate (%) = (Stage 2 + Stage 3 + Stage 4) / total number of seeds × 100%; Protocorm formation rate (%) = (Stage 3 + Stage 4) / total number of seeds × 100.

[0038] Table 1. Germination statistics of Rhododendron simsii in germination media with different organic additives Note: Different lowercase letters in the same column indicate the significance of the difference at the 0.05 level.

[0039] As shown in Table 1, in the symbiotic germination system of TC-6 strain and Rhododendron seeds, the seed germination rate was represented by the proportion of (Stage 2 + Stage 3 + Stage 4). The effects of different organic additives varied significantly: Tomato juice significantly reduced the proportion of non-germinating seeds, specifically from 62.1% to 58.6%; it increased the protocorm formation rate (19.00%) by 79.25% compared to the control group (10.60%), and the seed germination rate (41.40%) was the highest, making it the optimal additive; Banana juice also reduced the proportion of non-germinating seeds, specifically from 62.1% to 58.8%, but had no significant promoting effect on protocorm formation; Coconut juice significantly inhibited the germination initiation stage (Stage 2 proportion 19.20%), and although it increased the protocorm formation rate, the overall germination efficiency was lower than that of the control group; The control group had the highest proportion of non-germinating seeds (62.10%), while the germination rate and protocorm formation rate were the lowest among all groups.

[0040] These results confirm that exogenous organic matter plays a crucial role in supplementing the symbiotic germination system of TC-6 strain and Rhododendron seeds, with tomato juice showing the best promoting effect, significantly improving the germination efficiency and quality of TC-6 strain. Based on this, the optimal culture medium formula was determined to be: 2.0 g oat flour, 5 g sucrose, 30 ml tomato juice, and 8.0 g agar per L.

[0041] In summary, the fungus of the present invention Coprinellus pseudodisseminatus TC-6 exhibits excellent promoting effects on the germination of Rhododendron seeds. Co-culturing Rhododendron seeds with the fungus TC-6 in a germination medium supplemented with specific organic matter resulted in a high protocorm formation rate and a germination rate reaching 41.40% after 30 days of cultivation. This significantly improved the germination efficiency and quality of Rhododendron seeds, demonstrating its practical value for widespread application.

Claims

1. A fungus that promotes seed germination, characterized in that: It is deposited with China General Microbiological Culture Collection Center (CGMCC) under the accession number CGMCC NO. 42374 Coprinellus pseudodisseminatus .

2. Use of the fungus of claim 1 in the preparation of a seed germination agent that promotes the germination of plant seeds.

3. The use as described in claim 2, characterized in that: The plant seeds include rhododendron seeds.

4. A plant seed germination agent, characterized in that: It is a formulation made with the fungus described in claim 1 as the active ingredient, plus acceptable excipients.

5. The plant seed germination agent according to claim 4, characterized in that: The formulations include solutions, powders, granules, sprays, and blocks.

6. The plant seed germination agent as described in claim 4, characterized in that: The plant seeds include rhododendron seeds.

7. A method for promoting plant seed germination, characterized in that: It includes the following steps: Take the fungus described in claim 1, inoculate it into PDA medium, cut off small pieces of fungal agar, inoculate them into germination medium, and finally sow the sterilized plant seeds on the surface of the germination medium for culture.

8. The method according to claim 7, characterized in that: The fungus was cultured in PDA medium at a temperature of 28 ℃ and a humidity of 60%–70% for 7 days; the fungus and plant seeds were cultured in germination medium at a temperature of 23±2 ℃, a light intensity of 1600–2000 lx, and a light duration of 14 h / d.

9. The method according to claim 7 or 8, characterized in that: The formula for the PDA medium is: each L of medium contains 200g potato juice, 20g glucose and 8g agar; The germination medium is formulated as follows: each L of medium contains 2.0g oat flour, 5g sucrose, 30 ml tomato juice and 8.0g agar.

10. The method according to claim 7, characterized in that: The plant seeds include rhododendron seeds.

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