Psathyrella gracilis Strains and Their Applications

Abstract

The present invention belongs to the field of microbial technology, and discloses a Psathyrella strain C0774 for promoting the seed germination of Cremastra plants, with the preservation number of CGMCC No. 41692. The present invention also provides a biological agent containing the strain C0774 and a method for promoting the seed germination of Cremastra plants. The strain C0774 of the present invention can promote the germination of Cremastra yangii seeds, and has great application value for the sexual reproduction and resource protection of Cremastra plants.

Description

Technical Field

[0001] The present invention belongs to the field of applied microbiology, and more specifically, relates to a Psathyrella strain that can promote the seed germination of Cremastra plants. Background Art

[0002] Orchidaceae is one of the large families of angiosperms, with approximately 700 genera and nearly 20,000 species worldwide, many of which are rare and endangered plants. All wild Orchidaceae plants 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 protected by this convention, and are the "flagship" group in plant protection. Cremastra Danxiaorchis is a newly discovered fully heterotrophic saprophytic orchid genus in 2013, and currently there are only 3 species, namely Cremastra appendiculata Danxiaorchis singchiana , Cremastra yangii Danxiaorchis yangii , and Cremastra mendongensis Danxiaorchis mangdangshanensis . Among them, Cremastra yangii is a typical species with an extremely small population. The existing individual number is only about 200, and it is only found in Jinggangshan, Jiangxi Province in the southeast of China, and is in an extremely endangered state. It is of great significance to carry out population reproduction and resource protection for Cremastra yangii.

[0003] The propagation methods of Orchidaceae plants mainly include sexual propagation and asexual propagation. In terms of asexual propagation, the method of tissue culture can be used to carry out large-scale clonal propagation of Orchidaceae plants, but the tissue culture method and conditions of Cremastra yangii have not been studied. And according to the tissue culture experience of other plants, the tissue culture seedlings will show the degradation phenomenon of reduced yield and quality after multiple subcultures, and are prone to diseases, reducing the yield and quality. In terms of sexual propagation, researchers have achieved relatively ideal results by controlling conditions such as nutrition and ecology to promote the seed germination and growth of Orchidaceae plants. At present, some Dendrobium plants can obtain a large number of seedlings through the axenic germination of seeds. However, this propagation method has problems such as the seedlings formed after seed germination being relatively fragile and having many insurmountable difficulties when transplanted from the tissue culture conditions to the field environment for continued growth. The seeds of Orchidaceae plants are tiny and cannot store nutrients by themselves, and their germination stage completely depends on mycorrhizal fungi to provide nutrients for them. Based on the characteristics that the seed germination stage of Orchidaceae plants depends on fungi, it is of great significance to study excellent fungal strains (commonly known as germination fungi) that can promote the seed germination of Orchidaceae plants. At present, there have been studies on germination fungi for some Orchidaceae plants such as Gastrodia elata and Dendrobium, and good results have been achieved, but there is no research on the symbiotic germination of Cremastra plants, especially Cremastra yangii, an extremely endangered species. To better protect this extremely endangered Orchidaceae plant, it is urgent to study its germination fungi. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a fungal strain that can promote the seed germination of the genus Danxiaorchis, so as to promote the protection and artificial propagation of the resources of the Orchidaceae family, especially the genus Danxiaorchis.

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

[0006] In the first aspect, the present invention provides a Psathyrella ( Psathyrella sp. ) strain C0774 that can promote the seed germination of the genus Danxiaorchis. This strain was isolated from the tubers of Danxiaorchis yangii collected from the original habitat of Danxiaorchis yangii in Jinggangshan, Jiangxi in May 2024, and was deposited in the China General Microbiological Culture Collection Center on December 11, 2024. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode 100101; the deposit number is: CGMCC No. 41692. The taxonomic naming of this strain C0774 is: Psathyrella sp . .

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

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

[0009] The present invention determines it as Psathyrella ( Psathyrella sp. ) by studying and analyzing the culture characteristics and ITS sequence of strain C0774.

[0010] In the second aspect, the present invention provides a biological agent for promoting the seed germination of the genus Danxiaorchis, and this biological agent contains the culture of the Psathyrella strain.

[0011] In the embodiment of the second aspect, the Psathyrella strain is strain C0774.

[0012] In the third aspect, the present invention provides a method for promoting the seed germination of the genus Danxiaorchis, including treating the seeds of the genus Danxiaorchis with the culture of the Psathyrella strain.

[0013] In the embodiment of the third aspect, the Psathyrella strain is strain C0774.

[0014] In the embodiment of the third aspect of the present invention, the dosage of the Psathyrella strain C0774 is 5×5 mm mycelial blocks grown on 4 PDA media for 10 days.

[0015] In a fourth aspect, the present invention provides the use of the Psathyrella candolleana strain in the preparation of a biological agent for promoting the seed germination of plants of the genus Danxiaorchis.

[0016] In an embodiment of the fourth aspect, the Psathyrella candolleana strain is strain C0774.

[0017] In an embodiment of the present invention, the plants of the genus Danxiaorchis can be Danxiaorchis sinica D. singchiana , Danxiaorchis yangii D. yangii or Danxiaorchis mangdangshanensis D. mangdangshanensis . In a preferred embodiment of the present invention, the plants of the genus Danxiaorchis are Danxiaorchis yangii.

[0018] In a fifth aspect, the present invention provides the use of the Psathyrella candolleana strain in promoting the seed germination of plants of the genus Danxiaorchis.

[0019] In an embodiment of the fifth aspect, the Psathyrella candolleana strain is strain C0774.

[0020] The present invention proves that the Psathyrella candolleana strain, especially strain C0774, can promote the seed germination of plants of the genus Danxiaorchis, and has great application value for the sexual reproduction and resource protection of Orchidaceae plants, especially plants of the genus Danxiaorchis.

[0021] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly, it can be implemented according to the content of the description. And in order to make the above and other purposes, features and advantages of the present invention more obvious and understandable, the following specific preferred embodiments are given, and in conjunction with the accompanying drawings, the details are described as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 shows the morphological characteristics of strain C0774 on PDA medium. Figure 1 The left figure in shows the front characteristics of the colony, with well-developed white floccose mycelium. Figure 1 The right figure in shows the back characteristics of the colony, with light yellowish-brown pigment produced.

[0023] Figure 2 shows the seed morphological characteristics of symbiotic germination (left) and non-symbiotic germination (right) of Danxiaorchis yangii seeds. DETAILED DESCRIPTION OF THE EMBODIMENTS

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

[0025] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained through commercial channels.

[0026] Example 1. Isolation, purification, culture and identification of strain C0774

[0027] 1.1 Obtaining tubers of Yang's Danxiaorchis

[0028] In May 2024, tubers of Yang's Danxiaorchis were collected from the original habitat in Jinggangshan, Jiangxi, packed in 50 mL centrifuge tubes and brought back to the laboratory.

[0029] 1.2 Isolation, purification and culture of endophytic fungi from tubers

[0030] The collected tubers were washed with running water to remove the surface soil, blotted dry with filter paper, then the blotted dry tubers were disinfected in 75% ethanol for 30 s, rinsed once with sterile water, disinfected with 5% NaClO for 3 min, rinsed once with sterile water, and then the tubers were soaked in sterile water containing 150 μg / mL streptomycin sulfate and 150 μg / mL potassium penicillin at 10 mL per 10 mL for 10 min, then rinsed 3 times with sterile water, and finally blotted dry with sterile filter paper. 1 mL of the last rinse solution was taken and placed in a PDA medium for incubation in a dark incubator at 18 - 20 °C. Five replicates were set up and observed after 3 days to check whether colonies appeared to test whether the disinfection was thorough. In the laminar flow hood, the disinfected tubers were placed in a sterile glass dish and cut into 2 mm small pieces with a sterile scalpel, and placed on 1 cm³ PDA medium squares containing double antibiotics (containing 100 μg / mL streptomycin sulfate and 100 μg / mL potassium penicillin). Nine PDA medium squares were placed in each 9 cm petri dish, and each square was inoculated with one 2 mm small piece, and incubated in a dark incubator at 18 - 20 °C. After fungal hyphae grew on the medium, the hyphae were continuously picked and inoculated onto a new PDA medium for serial transfer. After 3 - 5 transfers, pure colonies could be obtained.

[0031] Through macroscopic morphological observation, on the PDA medium, the colony was light yellow, round, with well-developed aerial hyphae. The diameter of the colony after growing at 25 °C for 3 days was 5.00 cm, and no spores were produced on the conventional medium, as Figure 1 shown.

[0032] 1.4 Molecular biological identification of strain C0774

[0033] The total DNA of strain C0774 was extracted by the CTAB method [1], and the extracted DNA was subjected to PCR amplification using primers ITS1F and ITS4 (ITS1F: 5’-CTTGGTCATTTAGAGGAAGTAA-3’ / ITS4: 5’-TCCTCCGCTTATTGATATGC-3’). The PCR reaction system included: 8.5 μL ddH 2 O, 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: pre-denaturation at 95°C for 5 min, 1 cycle; denaturation at 94°C for 1 min, annealing at 52°C for 50 s, extension at 72°C for 1 min, 35 cycles; finally, extension at 72°C for 7 min. The amplified product was sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing; the sequenced sequence was BLAST compared in the National Center for Biotechnology Information (NCBI) database, and the taxonomic status of strain C0774 was determined by combining morphological characteristics.

[0034] Through sequence comparison and analysis, it was found that strain C0774 was most similar to the strain Candolleomyces candolleanus (formerly known as Psathyrella candolleana ) of Acession No. MW740407 in the GenBank database, with a similarity of 99.68%. Combining with the morphological characteristics of the colony, strain C0774 was tentatively identified as a species in the genus Psathyrella sp.).

[0035] Example 2. Germination effect of strain C0774 on the seeds of

[0036] 2.1 Preparation of symbiotic germination medium for seeds

[0037] The preparation method of the symbiotic germination medium for seeds is as follows: Add appropriate distilled water to 5 g of oatmeal, boil for 15 min, filter, make the filtrate up to 1 L, boil again, add 10 g of agar powder, adjust the pH to 5.0 - 5.2 after full melting, dispense, and sterilize at 121°C under high-pressure steam for 30 min to obtain the symbiotic germination medium for seeds.

[0038] 2.2 Sowing

[0039] The seeds of *Danxiaorchis yangii* were surface sterilized with 1% NaClO solution for 1 min, washed three times with sterile water, and diluted with sterile water to form a seed suspension for sowing. 200 μL of the seed suspension was pipetted onto the symbiotic germination medium, and then 5 × 5 mm mycelial blocks grown on four PDA media were inoculated at the four corners of the plate for co-culture. The control group was not inoculated with mycelial blocks. The seeded petri dishes were placed in a dark incubator at a temperature of 18 - 20 °C, and the seed germination was investigated after six months. According to the grading criteria for the developmental stages of Orchidaceae seeds shown in Table 1, the germination of *Danxiaorchis yangii* seeds was graded.

[0040] The results are as Figure 2 shown. The seeds of *Danxiaorchis yangii* inoculated with strain C0774 were able to germinate to stage 3, i.e., the apical meristem appeared ( Figure 2 the left image in Figure 2 ). In contrast, the seeds in the control group were still at stage 0, i.e., the seeds did not germinate and no further development was observed (

[0041] the right image in

[0042]

[0043] 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 foregoing embodiments, those of ordinary skill in the art should understand that: without departing from the concept, spirit, and scope of the present invention, the technical solutions described in the foregoing embodiments can be modified, or some or all of the technical features can be equivalently replaced; and these modifications or replacements all fall within the scope of 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 the Agaricus gracilis strain according to claim 1.

4. A method for promoting seed germination of Danxia orchid plants, characterized in that: The method comprises treating seeds of Danxia orchid plants with the Pleurotus eryngii strain C0774 described in claim 1.

5. The method according to claim 4, 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 ).

6. The method according to claim 4, 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.

7. 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.

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

Images