Schizophyllum strain and use thereof

Abstract

This invention belongs to the field of microbial technology and discloses a strain of *Schizophyllum commune* that promotes seed germination in plants of the *Schizophyllum* genus. Schizophyllum commune The present invention provides strain pb5, with accession number CGMCC No. 42595. It also provides a microbial preparation containing strain pb5 and a method for promoting seed germination of *Gynostemma* species. The strain pb5 of this invention can promote the germination of *Gynostemma* species, especially *Gynostemma* seeds, and has significant application value for the sexual reproduction and resource conservation of *Gynostemma* species.

Description

Technical Field

[0001] This invention belongs to the field of applied microbiology, and specifically relates to a strain of Schizophyllum commune that can promote the germination of seeds of plants in the genus *Schizophyllum*. Background Technology

[0002] Orchidaceae is one of the largest families of angiosperms, with approximately 700 genera and nearly 20,000 species worldwide, a significant number of which are rare and endangered. All wild orchid species are listed under the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), making them an important group for biodiversity conservation. *Orchidaceae 'Single Garlic Orchid' (*Orchidaceae 'Single Garlic Orchid') Pleione bulbocodioides (Franch.) Rolfe is a plant belonging to the genus *Franch.* of the Orchidaceae family. It is one of the important original plants of the traditional Chinese medicine *Cremastra appendiculata*, possessing both medicinal and ornamental value. In recent years, due to over-harvesting of wild resources and habitat destruction, the wild population of *Franch.* has declined sharply, and its endangered status has continued to worsen. Therefore, it is of great significance to carry out artificial breeding and germplasm resource protection.

[0003] Orchids primarily reproduce asexually and sexually. Asexual propagation can be achieved through division or tissue culture, but these methods generally suffer from low propagation coefficients, long cycles, and the risk of germplasm degradation and disease accumulation after multiple generations, making them unsuitable for large-scale, stable production of *Orchidonia spp.* Sexual propagation, through controlled nutrition and culture conditions to achieve aseptic germination of orchid seeds, has found some application in some orchid species. However, seedlings obtained this way often exhibit weak growth, poor environmental adaptability, and low survival rates when transplanted from aseptic culture conditions to natural or production environments, limiting its practical application value.

[0004] Orchid seeds are tiny, lack endosperm, and have almost no nutrient reserves of their own. Their germination and early development under natural conditions depend entirely on a symbiotic relationship with specific mycorrhizal fungi, which provide them with carbon sources and inorganic nutrients. Therefore, screening and utilizing fungal strains (commonly known as germination fungi) that can promote orchid seed germination and seedling growth is an important technical approach to achieving efficient sexual reproduction of orchids.

[0005] Currently, research on germination-promoting fungi for some orchid species, such as Gastrodia elata and Dendrobium, has made some progress and shown good results in production practice. However, research on seed germination-promoting fungi resources for Cymbidium species, especially Cymbidium goeringii, is still relatively limited, which seriously restricts the development of sexual reproduction technology for Cymbidium goeringii and its application in resource conservation and artificial breeding. Summary of the Invention

[0006] In view of this, the present invention provides a fungus that can promote the germination of seeds of plants in the genus *Schizophyllum* (or *Schizophyllum*). Schizophyllum commune ( ) strains to promote the conservation and artificial propagation of orchid resources, especially the genus *Gnaphalium*.

[0007] The objectives of this invention and the solutions to its technical problems can be achieved by the following technical solutions.

[0008] On one hand, this invention provides a *Schizophyllum commune* strain pb5 capable of promoting seed germination in plants of the *Pleurotus* genus. This strain was isolated from *Pleurotus* plants collected in May 2025 from the summit of Longtoushan Mountain in Ludian County, Zhaotong City, Yunnan Province, and was deposited on March 16, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China; accession number: CGMCC No. 42595. The suggested classification name for strain pb5 is... Schizophyllum commune .

[0009] In an embodiment of the first aspect of the present invention, the ITS sequence of strain pb5 is shown in SEQ ID NO: 1.

[0010] In an embodiment of the first aspect of the present invention, strain pb5 has the following morphological characteristics: When cultured on PDA medium, the colony surface is covered with dense white fluffy hyphae, which are thick and have well-developed aerial hyphae. The overall appearance is cottony or felt-like, and the back of the colony is yellowish-white. The growth rate is relatively fast.

[0011] In a second aspect, the present invention provides a microbial preparation for promoting the germination of seeds of plants in the genus *Gnaphalium*, the microbial preparation comprising a strain of *Schizophyllum commune*.

[0012] In an embodiment of the present invention, the Schizophyllum commune strain is strain pb5.

[0013] In a third aspect, the present invention provides a method for promoting the germination of seeds of the genus *Gnaphalium*, comprising applying a strain of *Schizophyllum commune* or the microbial preparation described in the second aspect to the seeds of *Gnaphalium*.

[0014] In an embodiment of the third aspect of the present invention, the Schizophyllum commune strain is strain pb5.

[0015] In an embodiment of the third aspect of the present invention, the amount of *Schizophyllum commune* strain pb5 is at least one 5 mm × 5 mm PDA substrate. In a specific embodiment of the present invention, the amount of *Schizophyllum commune* strain pb5 can be 1, 2, 3, 4, 5, 6, 7, 8, or 9 5 mm × 5 mm PDA substrates, preferably 4 5 mm × 5 mm PDA substrates.

[0016] In a fourth aspect, the present invention provides the use of Schizophyllum commune strains in the preparation of biological agents for promoting the germination of seeds of the genus *Hylocereus*.

[0017] In an embodiment of the fourth aspect of the present invention, the Schizophyllum commune strain is strain pb5.

[0018] In a fifth aspect, the present invention provides the use of *Schizophyllum commune* strains or microbial preparations implemented in the second aspect in promoting the germination of seeds of *Hylocereus* species.

[0019] In an embodiment of the fifth aspect of the present invention, the strain of Schizophyllum commune is strain pb5.

[0020] In an embodiment of the present invention, *Gnaphalium* species can be *Gnaphalium dwarfum* (…). P. humilis ), White-flowered Garlic Orchid ( P. albiflora ), Baoshan single garlic orchid ( P. baoshanensis ), Southern Tibet's single garlic orchid ( P. arunachalensis ), Long-necked Garlic Orchid ( P. autumnalis ), Chen's Single Garlic Orchid ( P. chunii Spring-flowering single garlic orchid ( P. kohlsii ), Large-flowered Garlic Orchid ( P. grandiflora ), Dali Single Garlic Orchid ( P. taliensis ), Yunnan's unique garlic orchid ( P. christianii ), single garlic orchid ( P. bulbocodioides Two-leaved single-clove orchid ( P. scopulorum ), fragrant garlic orchid ( P. confusa ), Yellow-flowered single-garlic orchid ( P. forrestii ), Jinhua single garlic orchid ( P. jinhuana ), Garlic of Caryophyllum ( P. kaatiae ), Cat Mountain Garlic Orchid ( P. maoershanensis ), Hairy-lipped Garlic Orchid ( P. hookeriana ), Beautiful Garlic Orchid ( P. pleionoides ), Autumn-flowered single-garlic orchid ( P. maculata ), Sichuan single garlic orchid ( P. limprichtii ), Taiwan Garlic Orchid ( P. formosana ), Vesuvius garlic ( P. 'Vesuvius' ), Small-leaved single-clove orchid ( P. microphylla ), Xiushan single garlic orchid ( P. xiushanensis ), Rock-grown Garlic Orchid ( P. saxicola ), and the beautiful single-flowered garlic orchid ( P. aurita ), Garlic Orchid with Warts ( P. praecox ), Yunnan single-garlic orchid ( P. yunnanensis In a preferred embodiment, the plant in the genus *Hylocereus* is *Hylocereus*.

[0021] The *Schizophyllum commune* strain pb5 provided by this invention can promote seed germination in *Orchidaceae* species, especially *Orchidaceae*, and has significant application value for the sexual reproduction and resource conservation of orchids, especially *Orchidaceae* species.

[0022] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description

[0023] Figure 1 The frontal morphology of strain pb5 cultured on a PDA plate is shown.

[0024] Figure 2 The reverse morphology of strain pb5 cultured on a PDA plate is shown.

[0025] Figure 3 The growth status of *Gynostemma pentaphyllum* seeds co-cultured with strain pb5 and cultured with blank PDA slides for 2 to 7 weeks is shown.

[0026] Figure 4 The results showed that *Cymbidium goeringii* seeds reached grade 4 after 12 weeks of co-cultivation with strain pb5, and grade 5 after 17 weeks.

[0027] Schizophyllum commune strain pb5 was deposited on March 16, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China; accession number: CGMCC No. 42595. The recommended classification name for strain pb5 is... Schizophyllum commune . Detailed Implementation

[0028] The preferred embodiments of the present invention will now be described in detail with reference to examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and intent, and all such modifications and substitutions fall within the scope of protection claimed in the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] Unless otherwise specified, all examples were performed under standard experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2012), or as recommended by the manufacturer’s instructions.

[0031] Unless otherwise specified or obvious from the context, the term "about" as used herein shall be understood to mean within the normal tolerance range in the art, such as within two standard deviations of the average. "About" may be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the specified value. Unless the context clearly indicates otherwise, all numerical values ​​provided in this specification and claims may be modified by the term "about".

[0032] Example 1. Isolation, purification, culture and identification of strain pb5

[0033] 1.1 Obtaining Plant Materials In May 2025, *Cymbidium goeringii* plants were collected from the top of Longtoushan Mountain in Ludian County, Zhaotong City, Yunnan Province, along with a small amount of soil, and transported to the laboratory for mycotocin separation.

[0034] 1.2 Isolation, purification and culture of endophytic fungi Select healthy, fresh, robust, dark brown *Gnaphalium affine* roots, thoroughly rinse with running water to remove soil and debris, and air dry. Soak root segments in 75% ethanol for 30 seconds, then in 5% NaClO (Beilian, Tianjin, China) for 3-5 minutes, rinse with sterile water, and soak in sterile water containing 100 μg / mL streptomycin and 100 μg / mL potassium penicillin (Gibco, Grand Island, NY, USA) for 10 minutes. Wash three times with sterile water. Collect the solution after the last rinse, place 1 mL on a PDA agar plate, and incubate at 25°C in the dark for 3 days. Repeat five times. The absence of colony growth indicates effective sterilization of the root surface.

[0035] Place the surface-sterilized root segments in a sterile 60 mm petri dish, and add a small amount of sterile water until the water level just covers the root segments (approximately 35 mL). Gently scrape the darker-colored surface of the root segments with a dissecting needle until you reach the vascular bundles in the middle of the root, then turn the scraping direction and continue scraping. After all the root segments have been scraped, discard the remaining parts, and transfer the mixture of root tissue fragments and cells in the petri dish to an optical microscope to observe whether intracellular single hyphae have been successfully released.

[0036] Under a microscope, locate a single hyphal cluster in the liquid and aspirate it using a 2.5 μL pipette. Transfer the aspirate to a pre-prepared 60 mm PDA medium. After the single hyphal cluster is aspirated, seal the culture dish and transfer it to a 25°C dark incubator to observe for hyphal growth. If hyphal growth occurs, pick the newly formed hyphae and purify them in fresh PDA medium. After isolation and purification, pure culture strain pb5 is obtained on PDA plates.

[0037] Figure 1 and Figure 2 The front and back morphologies of strain pb5 grown on PDA medium are shown, respectively. From Figure 1 and Figure 2 It can be seen that the colonies exhibit white, fluffy mycelial growth, with a uniform and dense surface. The mycelia cover the entire surface of the culture medium, and the back of the colonies is pale yellow.

[0038] 1.3 Molecular biological identification of strain pb5 Total DNA was extracted from strain pb5 using the CTAB method. PCR amplification was performed on the extracted DNA using primers ITS1 and ITS4 (ITS1: 5′-TCCGTAGGTGAACCTGCGG-3′ / ITS4: 5′-TCCTCCGCTTATTGATATGC-3′). The PCR reaction mixture consisted of 8.5 μL ddH2O, 1 μL ITS1, 1 μL ITS4, 12.5 μL Taq polymerase, and 2 μL DNA template, for a total reaction volume of 25 μL. PCR cycling was as follows: 95℃ pre-denaturation for 5 min, 1 cycle; 94℃ denaturation for 1 min, 52℃ annealing for 50 s, 72℃ extension for 1 min, 35 cycles; and a final extension at 72℃ for 7 min. The amplified products were sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing. The ITS sequence of strain pb5 is shown in SEQ ID NO: 1.

[0039] The taxonomic position of strain pb5 was determined by BLAST comparison of the sequenced data in the National Center for Biotechnology Information (NCBI) database and combined with morphological characteristics.

[0040] Sequence alignment revealed that the ITS sequence of strain pb5 is identical to that of strain OR678730.1 in GenBank. Schizophyllum commune The sequence was most similar, with a similarity of 100.00%. Combined with morphological characteristics, strain pb5 was identified as *Schizophyllum commune*. S. commune ).

[0041] Example 2. Symbiotic germination of *Gynostemma pentaphyllum* seeds with PB5

[0042] 2.1 Single Garlic Orchid Seed Material The capsules of *Gnaphalium affine* were collected in Zhaotong, Yunnan Province in November 2024. After surface disinfection with cotton soaked in 75% ethanol, they were soaked in 1% NaClO for 10 minutes, washed three times with sterile water, and thoroughly dried. They were then placed on sterile A4 weighing paper, the capsules were cut open with a knife, the seeds inside were scraped out, wrapped in A4 weighing paper, and placed in a silicone resealable bag for later use.

[0043] 2.2 Preparation of culture medium Oatmeal Agar (OMA): Add 5 g of oat flakes to an appropriate amount of distilled water and boil for 15 min, then filter. Make up the volume of the filtrate to 1 L, boil the filtrate again, add 10 g of agar powder, and after it is fully dissolved, adjust the pH to 5.0-5.2. Dispense into Erlenmeyer flasks and autoclave at 121°C for 30 min. After sterilization, add 100 μg / mL streptomycin and 100 μg / mL potassium penicillin (Gibco, Grand Island, NY, USA), pour into plates, and use as a basal medium for seed symbiotic germination.

[0044] PDA (potato dextrose agar): Cut 200 g of peeled potatoes into appropriately sized pieces and place them in a pot. Add an appropriate amount of distilled water and boil for 20 minutes. Filter. Add 20 g of glucose and 12 g of agar to the filtrate. After fully dissolving, bring the volume to 1 L and dispense. Autoclave at 121°C for 30 minutes. After sterilization, add 100 μg / mL streptomycin and 100 μg / mL potassium penicillin (Gibco, Grand Island, NY, USA). Pour into plates. Use sterile PDA plates as blank controls.

[0045] 2.3 Seed sowing Soak the pre-separated seeds in an appropriate amount of sterile water for 12-24 hours, remove any floating seeds, sterilize with 1% NaClO for 5 minutes, aspirate the supernatant, and wash three times with sterile water to prepare a seed-water suspension. Use a disposable plastic pipette to transfer the seed-water suspension to OMA culture medium, being careful not to add too much water. Gently shake the petri dish to evenly disperse the seeds. Then, inoculate 5 × 5 mm mycelial blocks from four PDA media at the four corners of the plate for co-cultivation. The control group is not inoculated with mycelial blocks.

[0046] Using a 0.5 cm diameter punch that has been heated to red-hot and cooled, holes were punched in the PDA plates containing the pb5 strain. Four 5 × 5 mm pb5 bacterial discs were picked up with a heated and cooled inoculation needle and placed in the four corners of the culture dish containing the seeded bacteria for co-culture. For the control group, sterile PDA discs were picked up, and the date, strain name, and culture medium name were written on them. The discs were then sealed. Each group had 12 replicates, and an OMA blank control (CK) was set up.

[0047] 2.4 Seed germination rate statistics Orchid seed germination is divided into six stages. Stage 0: The embryo is transparent, the seed coat is intact, and the seed has not germinated. Stage 1: The seed absorbs water, swells, turns green, and develops epidermal hairs or rhizoids. Stage 2: The seed continues to absorb water and swells; the seed coat ruptures. Stage 3: Apical meristem appears. Stage 4: The apical meristem continues to grow, elongating and bending to form the first leaf. Stage 5: The first leaf elongates, grows curved, and the second leaf appears. Orchid seeds from the appearance of apical meristem to the formation of seedlings are usually called protocorms. Seeds germinating to stages 3-4 are called protocorms, while seeds germinating to stages 4-5 are called seedlings. Seed germination is observed every two weeks and graded according to the above criteria; seed germination data is collected promptly.

[0048] Seed germination rate = germinating seeds at each observed level / total number of observed seeds.

[0049] from Figure 3 It can be seen that the seeds of *Gynostemma pentaphyllum* inoculated with strain Pb5 can germinate to grade IV in large numbers after 9 weeks and can grow further, while the uninoculated CK group mostly remained at grade III from week 7.

[0050] from Figure 4 It can be seen that the seeds of *Cymbidium goeringii* inoculated with strain Pb5 can germinate in large numbers to level 5 or higher within 12 to 17 weeks.

[0051] Table 1 summarizes the germination and growth of *Orchidonia scabra* seeds inoculated with strain pb5 and uninoculated seeds after 9 weeks of culture on OMA petri dishes. pb5 significantly promoted seed germination to grade 4, while uninoculated *Orchidonia scabra* seeds mostly remained at grade 3.

[0052] Table 1. Comparison of the effects of strain pb5 and the control group on *Hylocereus undatus* seeds after 9 weeks of treatment.

[0053] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Schizophyllum commune ( Schizophyllum commune The strain pb5 has the accession number CGMCC No. 42595.

2. The *Schizophyllum commune* strain pb5 according to claim 1, characterized in that, The ITS sequence of the *Schizophyllum commune* strain pb5 is shown in SEQ ID NO:

1.

3. A microbial preparation for promoting seed germination of plants in the genus *Gynostemma*, characterized in that, The microbial preparation comprises the Schizophyllum commune strain as described in claim 1.

4. The microbial preparation according to claim 3, wherein the Schizophyllum commune strain is the strain pb5 according to claim 1.

5. A method for promoting seed germination of plants in the genus *Gnaphalium*, characterized in that, The method comprises applying the Schizophyllum commune strain pb5 of claim 1 or the microbial preparation of claim 3 to seeds of plants of the genus Garliclium.

6. The method according to claim 5, characterized in that, The species mentioned are *Symplocos moniliforme* (or *Symplocos moniliforme*). P. bulbocodioides ).

7. The method according to claim 5, characterized in that, The amount of the *Schizophyllum commune* strain pb5 used is at least one 5mm × 5mm PDA substrate.

8. The use of the *Schizophyllum commune* strain pb5 according to claim 1 in the preparation of a biological agent for promoting seed germination of plants in the genus *Hylocereus*.

9. The application of the *Schizophyllum commune* strain pb5 according to claim 1 or the microbial preparation according to claim 3 in promoting seed germination of *Hylocereus* species.

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