Pleurotus ostreatus strain and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses a fragile-stem mushroom strain C0774 for promoting germination of Dangxia plant seeds, and the preservation number of the fragile-stem mushroom strain C0774 is CGMCC No. 41692. The invention also provides a biological preparation containing the strain C0774 and a method for promoting the germination of Dangxia plant seeds. The strain C0774 provided by the invention can promote germination of Young Dangxia cymbidium seeds, and has important application value for sexual propagation and resource protection of Dangxia cymbidium plants.

Description

Technical Field

[0001] The invention belongs to the field of applied microorganisms, and in particular relates to a Pleurotus eryngii strain capable of promoting the germination of seeds of Danxia orchid plants. Background Art

[0002] Orchidaceae is one of the major families of angiosperms, with about 700 genera and nearly 20,000 species worldwide, many of which are rare and endangered. All wild orchids are included in the scope of protection of the Convention on International Trade in Endangered Species of Wild Fauna and Flora, accounting for more than 90% of the plants to be protected by the Convention, and are the "flagship" group in plant protection. Danxiaorchis ) is a new genus of completely heterotrophic saprophytic orchidaceae discovered in 2013. Currently, there are only three species, namely Danxia orchid ( Danxiaorchis singchiana )、Yang's Danxia orchid( Danxiaorchis yangii ) and Mangdangshan Danxialan ( Danxiaorchis mangdangshanensis Among them, Yang's Danxia Orchid is a typical species with extremely small populations, with only about 200 individuals existing. It is only found in Jinggang Mountain, Jiangxi Province in southeastern China and is in a critically endangered state. It is of great significance to carry out population reproduction and resource protection of Yang's Danxia Orchid.

[0003] There are two main methods for orchid reproduction: sexual reproduction and asexual reproduction. In terms of asexual reproduction, tissue culture can be used to carry out large-scale cloning and reproduction of orchids, but the tissue culture method and conditions of Yang's Danxia orchid have not been studied, and the experience of tissue culture of other plants has found that tissue culture seedlings will degenerate after multiple subcultures, and are prone to diseases, reducing yield and quality. In terms of sexual reproduction, researchers have achieved relatively ideal results by controlling nutrition, ecology and other conditions to promote the germination and growth of orchid seeds. At present, some Dendrobium plants can obtain seedlings in large quantities through aseptic germination of seeds. However, this method of reproduction has the problem that the seedlings formed after seed germination are relatively fragile, and there are many difficulties that are difficult to overcome when transplanting from tissue culture conditions to field environments for continued growth. The seeds of orchids are tiny and cannot store nutrients by themselves. Their germination stage completely relies on mycorrhizal fungi to provide them with nutrition. Based on the characteristics that the germination stage of orchid seeds depends on fungi, it is of great significance to study the excellent fungal strains (commonly known as germination fungi) that promote the germination of orchid seeds. Currently, there have been studies on the germination fungi of some orchid plants such as Gastrodia elata and Dendrobium, and good results have been achieved. However, there has been no research on the symbiotic germination of Danxia orchid plants, especially Yang's Danxia orchid, a critically endangered species. In order to better protect this critically endangered orchid plant, it is urgent to study its germination fungi. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a fungal strain capable of promoting the germination of seeds of Danxia orchid plants, so as to promote the protection and artificial propagation of orchidaceae, especially Danxia orchid plant resources.

[0005] The purpose of the present invention and the solution to its technical problem can be achieved by adopting the following technical solutions.

[0006] In a first aspect, the present invention provides a small brittle mushroom ( Psathyrella sp. ) strain C0774. The strain was isolated from the tuber of Yang's Danxia orchid collected from the native habitat of Yang's Danxia orchid in Jinggangshan, Jiangxi Province in May 2024, and was deposited in the China General Microbiological Culture Collection Center on December 11, 2024, with the address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101; the deposit number is: CGMCC No. 41692, and the taxonomic name of the strain C0774 is: Psathyrella sp . .

[0007] In an embodiment of the first aspect of the present invention, the ITS sequence of strain C0774 is shown as SEQ ID NO: 1.

[0008] In an embodiment of the first aspect of the present invention, strain C0774 has the following morphological characteristics: the colonies on PDA medium are light yellow and round, with well-developed aerial hyphae, the colony diameter after growing at 25° C. for 3 days is 5.00 cm, and no spores are produced on conventional culture medium.

[0009] The present invention studies and analyzes the culture characteristics and ITS sequence of strain C0774 and determines that it is a Pleurotus eryngii ( Psathyrella sp. ).

[0010] In a second aspect, the present invention provides a biological preparation for promoting germination of seeds of a plant of the genus Danxialan, the biological preparation comprising a culture of a strain of Pleurotus eryngii.

[0011] In an embodiment of the second aspect, the Fragilis gracilis strain is strain C0774.

[0012] In a third aspect, the present invention provides a method for promoting germination of seeds of a Danxia orchid plant, comprising treating the seeds of the Danxia orchid plant with a culture of a Fragilis pygium strain.

[0013] In an embodiment of the third aspect, the Fragilis gracilis strain is strain C0774.

[0014] In an embodiment of the third aspect of the present invention, the dosage of Agaricus fragilis strain C0774 is 5×5 mm mushroom blocks grown on four PDA medium for 10 days.

[0015] In a fourth aspect, the present invention provides a use of a Pleurotus eryngii strain in preparing a biological agent for promoting seed germination of a plant of the genus Danxia orchid.

[0016] In an embodiment of the fourth aspect, the Fragilis gracilis strain is strain C0774.

[0017] In an embodiment of the present invention, the plant of the genus Danxia Orchid may be Danxia Orchid ( D. singchiana )、Yang's Danxia orchid( D. yangii ) or Mangdangshan Danxialan ( D. mangdangshanensis ). In a preferred embodiment of the present invention, the plant of the genus Danxia orchid is Yang's Danxia orchid.

[0018] In a fifth aspect, the present invention provides the use of a Pleurotus eryngii strain in promoting seed germination of Danxia orchid plants.

[0019] In an embodiment of the fifth aspect, the Fragilis gracilis strain is strain C0774.

[0020] The invention proves that the Pleurotus eryngii strain, especially the strain C0774, can promote the germination of seeds of Danxia orchid plants, and has great application value for the sexual reproduction and resource protection of orchid plants, especially Danxia orchid plants.

[0021] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the following specifically cites a preferred embodiment and describes it in detail with the accompanying drawings as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The morphological characteristics of strain C0774 on PDA medium are shown. Figure 1 The left picture in the figure shows the front features of the colony, with well-developed white woolly hyphae; Figure 1 The right picture shows the characteristics of the back of the colony, with light yellow-brown pigment produced.

[0023] Figure 2 The seed morphology of Danxia orchid seeds inoculated with fungi in symbiotic germination (left) and non-symbiotic germination (right) is shown. DETAILED DESCRIPTION

[0024] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of protection of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention, and all of these modifications and substitutions fall within the scope of the claims of the present invention.

[0025] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0026] Example 1. Isolation, purification, cultivation and identification of strain C0774 1.1 Obtaining tubers of Danxia orchid

[0027] In May 2024, tubers of Yang's Danxia Orchid were collected from the native habitat of Yang's Danxia Orchid in Jinggangshan, Jiangxi Province, packed in 50 mL centrifuge tubes, and brought back to the laboratory.

[0028] 1.2 Isolation, purification and cultivation of tuber endophytic fungi

[0029] Wash the collected tubers under running water to remove the surface soil, and use filter paper to absorb the water. Place the tubers that have absorbed the water in 75% ethanol for disinfection for 30 seconds, then rinse once with sterile water, disinfect with 5% NaClO for 3 minutes, rinse once with sterile water, and then soak the tubers in sterile water containing 150 μg / mL streptomycin sulfate and 150 μg / mL penicillin potassium for 10 minutes per 10 mL, then rinse three times with sterile water, and finally use sterile filter paper to absorb the water. Take 1 mL of the last rinse solution, place it in PDA medium and culture it in a dark culture room at 18-20°C, set up 5 replicates, and observe whether there are colonies after 3 days to check whether the disinfection is thorough. Place the sterilized tubers in a sterile glass dish in a clean bench, cut them into 2 mm pieces with a sterile surgical blade, and place them on a 1 cm³ PDA culture medium block containing double antibiotics (containing 100 μg / mL streptomycin sulfate and 100 μg / mL penicillin potassium). Place 9 PDA culture medium blocks per 9 cm plate, inoculate each block with a 2 mm piece, and culture in a dark culture room at 18-20°C. After fungal hyphae grow on the culture medium, continuously pick the hyphae and inoculate them on new PDA culture medium for serial transfer. After 3 to 5 transfers, pure colonies can be obtained.

[0030] Through macroscopic morphological observation, on PDA medium, the colonies were light yellow, round, with well-developed aerial hyphae, and the diameter of the colonies grown at 25°C for 3 days was 5.00 cm. On conventional medium, no spores were produced. Figure 1 shown.

[0031] 1.4 Molecular biological identification of strain C0774 The total DNA of strain C0774 was extracted by the CTAB method [1], and the extracted DNA was amplified by PCR using primers ITS1F and ITS4 (ITS1F: 5'-CTTGGTCATTTAGAGGAAGTAA-3' / ITS4: 5'-TCCTCCGCTTATTGATATGC-3'). The PCR reaction system included: 8.5 μL ddH2O, 1 μL ITS1F, 1 μL ITS4, 12.5 μL Taq enzyme, 2 μL DNA template, and the total reaction volume was 25 μL. PCR cycle: 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 finally 72℃ extension for 7 min. The amplified products were sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing; the sequences were compared by BLAST in the National Center for Biotechnology Information (NCBI) database, and the taxonomic status of strain C0774 was determined in combination with morphological characteristics.

[0032] Sequence comparison analysis revealed that strain C0774 was similar to the strain Pleurotus eryngii with accession number MW740407 in the GenBank database ( Candolleomyces candolleanus (Original name Psathyrella candolleana )) with a similarity of 99.68%. Combined with the morphological characteristics of the colonies, strain C0774 was temporarily identified as a species of the genus Agaricus ( Psathyrella sp.).

[0033] Example 2. Effect of strain C0774 on germination of Danxia orchid seeds 2.1 Preparation of seed symbiotic germination medium

[0034] The preparation method of seed symbiotic germination medium is as follows: add appropriate amount of distilled water to 5 g of oatmeal, boil for 15 minutes, filter, dilute the filtrate to 1 L of filtrate, boil again, add 10 g of agar powder, adjust the pH to 5.0-5.2 after it is fully melted, divide into portions, and sterilize with high pressure steam at 121°C for 30 minutes to obtain seed symbiotic germination medium.

[0035] 2.2 Seeding

[0036] The seeds of Yang's Danxia orchid were surface disinfected with 1% NaClO solution for 1 min, washed with sterile water 3 times, and diluted with sterile water to form a seed suspension for sowing. 200 μL of seed suspension was sown on the symbiotic germination medium, and then 5 × 5 mm-sized bacterial blocks on 4 PDA medium were inoculated in the four corners of the plate for co-cultivation. The control group was not inoculated with bacterial blocks. The sown plates were placed in a dark culture room for culture at a temperature of 18-20°C, and the seed germination was investigated at six months. The germination of Yang's Danxia orchid seeds was graded according to the grading standards for orchid seed development levels shown in Table 1.

[0037] The results are as follows Figure 2 As shown, the seeds of D. yangyi inoculated with strain C0774 were able to germinate to the third stage, i.e., the emergence of apical meristem ( Figure 2 In contrast, the seeds in the control group were still at stage 0, meaning that the seeds had not germinated and showed no further development ( Figure 2 , right image in the figure).

[0038] Table 1. Orchidaceae seed development level classification standard

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents without departing from the concept, spirit and scope of the present invention; and these modifications or replacements fall within the scope of protection claimed in the claims of the present invention.

Claims

1. Small crispy mushroom ( Psathyrella sp. ) strain C0774, whose deposit number is CGMCC No. 41692.

2. The Agaricus fragilis strain C0774 according to claim 1, characterized in that: The ITS sequence of the Pleurotus eryngii strain C0774 is shown in SEQ ID NO:

1.

3. A biological agent for promoting seed germination of Danxia orchid plants, characterized in that: The biological preparation comprises a culture of Agaricus fragilis. The biological preparation according to claim 3 , wherein the Agaricus fragilis is strain C0774.

5. A method for promoting seed germination of Danxia orchid plants, characterized in that: The method comprises treating seeds of a plant of the genus Danxia with a culture of the Agaricus gracilis strain C0774.

6. The method according to claim 5, characterized in that The plant of the genus Danxia Orchid is Danxia Orchid ( D. singchiana )、Yang's Danxia orchid( D. yangii ) or Mangdangshan Danxialan ( D. mangdangshanensis ).

7. The method according to claim 5, characterized in that The dosage of the Agaricus fragilis strain C0774 is 4 5×5 mm mushroom blocks grown on PDA medium for 10 days.

8. Use of the Agaricus fragilis strain C0774 according to claim 1 in the preparation of a biological agent for promoting seed germination of Danxia orchid plants.

9. Use of the Agaricus fragilis strain C0774 according to claim 1 in promoting seed germination of plants of the genus Danxia orchid.

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