Lucilaginoidea fungus strain SH1 and application thereof in promoting germination of dendrobium chrysanthum seeds

Abstract

The invention belongs to the technical field of plant protection, and particularly relates to a Tulasnella calospora fungus strain SH1 and application of the Tulasnella calospora fungus strain SH1 in promoting germination of dendrobium chrysanthum seeds. The preservation number of the fungus strain SH1 is GDMCC No: 66867. The fungus strain SH1 and mature dendrobium chrysanthum seeds are subjected to symbiotic germination, and the germination rate of the seeds can be remarkably increased. Results of the embodiment show that when the fungus strain SH1 is used for symbiotic germination of dendrobium chrysanthum seeds, the germination rate can reach 73.20% at most.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant protection, and particularly relates to a fungus strain SH1 of the genus Cortinarius and application of the fungus strain SH1 in promoting seed germination of Dendrobium chryseum. BACKGROUND

[0002] Dendrobium chryseum Dendrobium chrysanthum is mainly distributed in China (Yunnan, Guangxi, Guizhou, Tibet, etc.), northeast India, Nepal, Bhutan, Myanmar, Thailand, Laos and Vietnam. It usually grows on tree trunks or rocks in mountain open woods at an altitude of 500-1800 meters, and prefers warm, humid and semi-shady environment. Dendrobium chryseum has a cluster of golden yellow inflorescences, elegant plant type and autumn flowering period, and is an orchid with high ornamental value, which is loved by orchid lovers and horticultural cultivators. Due to its ornamental and medicinal value, Dendrobium chryseum has been over-harvested, and its natural habitat has been destroyed, so the number of wild populations has decreased sharply. At present, Dendrobium chryseum has been listed in the National Key Protected Wild Plant List (2021), and is a national second-class protected wild plant. International trade is also regulated by Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES).

[0003] Increasing the number of individuals by improving seed germination rate is an important measure to carry out in-situ conservation. Mature seed of orchid is extremely small, and has no endosperm tissue for storing nutrients. At present, researches on seed germination of orchid mainly focus on two ways, i.e. non-symbiotic germination and symbiotic germination. When artificial cultivation is carried out by using non-symbiotic germination, there are problems such as slow growth, low survival rate, genetic stability decline caused by continuous tissue culture, reduction of medicinal ingredient synthesis capacity such as dendrobium alkaloid, and increase of colletotrichum disease incidence, which cannot meet the market demand. Therefore, Dendrobium chryseum relies on symbiotic germination in the wild environment for cultivation, but no endophytic fungus capable of promoting seed germination of Dendrobium chryseum has been found. SUMMARY

[0004] The purpose of the present application is to provide a fungus strain SH1 of the genus Cortinarius and application of the fungus strain SH1 in promoting seed germination of Dendrobium chryseum, which can significantly improve the seed germination rate of Dendrobium chryseum.

[0005] The present application provides a fungus strain SH1 of the genus Cortinarius Tulasnella calospora The preservation number of the fungus strain SH1 is GDMCC No: 66867.

[0006] The present application provides a microbial inoculant comprising the fungus strain SH1.

[0007] The present invention provides a method for preparing the above-mentioned microbial agent, comprising: culturing fungal strain SH1 in a culture medium to obtain the microbial agent; wherein the culture medium includes: antibiotic PDA culture medium.

[0008] As a preferred embodiment, the culture temperature is 25~27℃.

[0009] This invention provides the application of the above-mentioned fungal strain SH1 or the above-mentioned microbial agent in promoting the germination of Dendrobium nobile seeds.

[0010] The present invention also provides a method for promoting the germination of Dendrobium nobile seeds, the method comprising the following steps: symbiotic germination culture of Dendrobium nobile seeds with the above-mentioned fungal strain SH1 or the above-mentioned microbial agent.

[0011] As a preferred embodiment, the culture medium for the symbiotic germination culture includes oat agar medium.

[0012] As a preferred embodiment, the light conditions for the symbiotic germination culture are alternating light and dark, with 10-14 hours of light and 14-10 hours of darkness.

[0013] As a preferred embodiment, the intensity of the illumination is 2000~2500 lux.

[0014] As a preferred embodiment, the temperature for the symbiotic germination culture is 25~28℃.

[0015] Beneficial Effects: This invention provides a fungal strain SH1 of the genus *Dendrobium*, with the preservation number GDMCC No. 66867. The fungal strain SH1 described in this invention can be co-sown with *Dendrobium* seeds for seedling raising in trays, pots, or seedbeds, or for field cultivation. It exhibits high germination promotion efficiency for *Dendrobium* seeds and can germinate symbiotically with *Dendrobium* seeds under natural environmental conditions, providing the necessary conditions for the efficient production of mycorrhizal seedlings. It has application value as an alternative to tissue culture for obtaining seedlings. The results of the examples show that when using fungal strain SH1 for symbiotic germination of *Dendrobium* seeds, the germination rate can reach up to 73.20%.

[0016] Biological Preservation Information Species of the genus Collagenae ( Tulasnella calospora The fungal strain SH1 was deposited on August 21, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 66867. Attached Figure Description

[0017] Figure 1This is a colony morphology diagram of fungal strain SH1 after 7 days of growth on PDA medium containing antibiotics. Figure 2 A phylogenetic tree diagram of fungal strain SH1 constructed based on its ITS sequence; Figure 3 The images show the germination of Dendrobium nobile; A represents Dendrobium nobile germinating after 25 days of ex-situ symbiotic growth; B represents Dendrobium nobile germinating after 25 days of symbiotic growth. Detailed Implementation

[0018] This invention provides a type of film-forming fungus ( Tulasnella calospora The fungal strain SH1, whose preservation number is GDMCC No: 66867.

[0019] Unless otherwise specified, the present invention does not have special requirements for the raw materials used, and commercially available products known to those skilled in the art can be used.

[0020] This invention isolates and purifies the genus *Dendrobium* from the protocorms of *Dendrobium nobile*. Tulasnella calospora The fungal strain SH1 (hereinafter referred to as fungal strain SH1) is described in this invention. The colonies of fungal strain SH1 are regularly round, milky white and opaque, with no pigment secretion or diffusion observed; the edges are smooth and neat, without wavy, serrated, or fibrous extensions; the surface is uniform and delicate, exhibiting a smooth, moist, membranous structure without wrinkles, cracks, or granular protrusions. No aerial hyphae, radial stripes, concentric rings, or other special morphological structures were observed.

[0021] In a preferred embodiment, the nrDNA ITS sequence of the fungal strain SH1 of the present invention is shown in SEQ ID NO.1: 5'--3'. The amplification primers for the nrDNA ITS sequence of the fungal strain SH1 of the present invention can be ITS4 and ITS5; the sequence of ITS4 is shown in SEQ ID NO.2: 5'-TCCTCCGCTTATTGATATGC-3'; the sequence of ITS5 is shown in SEQ ID NO.3: 5'-GGAAGTAAAAGTCGTAACAAGG-3'.In a specific embodiment of the present invention, the PCR reaction system (in 50 μL) for the nrDNA ITS sequence of the fungal strain SH1 includes: 2.0 μL DNA template, 25.0 μL 2×Rapid Taq Master Mix, 2 μL each primer (primer concentration of 10.0 μM), and 19.0 μL ddH2O; the PCR reaction conditions for the nrDNA ITS sequence of the fungal strain SH1 are: 95℃ pre-denaturation for 5 min; followed by 35 cycles, including 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s; and finally 72℃ final extension for 7 min.

[0022] The present invention provides a microbial inoculant, comprising the aforementioned fungal strain SH1.

[0023] This invention provides a method for preparing the aforementioned microbial inoculant, comprising: culturing fungal strain SH1 in a culture medium to obtain the microbial inoculant. As a preferred embodiment, the culture medium of this invention comprises: PDA medium containing antibiotics. The antibiotics of this invention include: ampicillin and streptomycin. In a specific embodiment of this invention, the composition of the PDA medium, per 1L, is 200 g / L potato, 20 g / L glucose, and 15 g / L agar. The culture temperature of this invention can be any value within the range of 25~27℃, for example, 25, 26, or 27℃.

[0024] This invention provides the application of the aforementioned fungal strain SH1 or the aforementioned microbial inoculant in promoting the germination of Dendrobium nobile seeds. The symbiotic germination of Dendrobium nobile seeds with the fungal strain SH1 or the microbial inoculant of this invention can significantly improve the germination rate of the seeds, thereby providing the necessary conditions for the efficient production of mycorrhizal seedlings.

[0025] This invention also provides a method for promoting the germination of Dendrobium nobile seeds, the method comprising the following steps: symbiotic germination culture of Dendrobium nobile seeds with the aforementioned fungal strain SH1 or the aforementioned microbial inoculant. The germination-promoting fungal strain SH1 isolated from Dendrobium nobile has a good germination-promoting effect only on itself.

[0026] In a preferred embodiment, the culture medium for symbiotic germination culture of the present invention comprises: oat agar medium (denoted as OMA medium). In a specific embodiment of the present invention, the OMA medium, per 1 L, consists of 4 g / L oats and 10 g / L agar, with a pH of 5.8. The light conditions for symbiotic germination culture of the present invention can be any value within the range of light-dark alternation, with light intensity of 10-14 h / darkness of 14-10 h, such as 10 h / 14 h, 11 h / 13 h, 12 h / 12 ​​h, 13 h / 11 h, or 14 h / 10 h. The light intensity of the present invention can be any value within the range of 2000-2500 lux, such as 2000, 2300, or 2500 lux. The temperature for symbiotic germination culture of the present invention can be any value within the range of 25-28°C, such as 25, 26, 27, or 28°C. Symbiotic cultivation conditions are more conducive to improving the germination rate of Dendrobium nobile seeds.

[0027] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a fungal strain SH1 of the genus Dendrobium and its application in promoting the germination of Dendrobium nobile seeds, should not be construed as limiting the scope of protection of the present invention.

[0028] Antibiotic-containing PDA medium (potato glucose agar medium) formula: 200 g / L potato, 20 g / L glucose and 15 g / L agar.

[0029] Ampicillin stock solution: 100 mg ampicillin dissolved in 1 mL distilled water; Streptomycin stock solution: 100 mg streptomycin dissolved in 1 mL distilled water The specific preparation method of antibiotic-containing PDA culture medium is as follows: Weigh 200g of fresh potatoes and add 1.2L of water. Boil and stir for about 15 minutes, then filter through two layers of gauze. Retain the filtrate and add water to 1L. Add 20.0g of glucose and 15.0g of agar to each liter of potato filtrate. Sterilize in an Erlenmeyer flask (121℃, 25min). After sterilization in a laminar flow hood and cooling to 60℃, add 500μm of ampicillin stock solution and streptomycin stock solution to each liter of culture medium. Mix well and dispense into sterile culture dishes with a diameter of 9cm and cool.

[0030] OMA (Oat Agar) medium formula: 4 g / L oats and 10 g / L agar, pH=5.8.

[0031] OMA culture medium preparation method: Weigh 4g of ultrafine oat flour (Beijing Jinclone Biotechnology Co., Ltd.) and 10g of agar, boil and stir, mix well, transfer to a beaker, add water to 1L, adjust pH to 5.8, pour into an Erlenmeyer flask for sterilization (121℃, 25min), after sterilization, dispense into 9cm diameter sterile petri dishes in a clean bench and cool.

[0032] Example 1: Induced isolation and purification of fungi 1. Seed collection and storage Mature, unopened fruits of *Dendrobium nobile* were collected from the wild. All fruits were obtained through natural pollination. After collection, the fruits were sterilized with 75% ethanol, washed three times with deionized distilled water, and dried on sterile filter paper. Finally, the seeds were dried in anhydrous calcium chloride in a sealed glass container for four days and then frozen at -20°C.

[0033] 2. Ex-situ symbiotic germination experiment Bark and moss were collected from within 25cm of the roots of mature *Dendrobium nobile* plants and crushed into fine granules. Half of the granular substrate was placed in a 12.5cm × 8.8cm × 5.7cm transparent plastic box, and sterile water was added for 10 minutes. Excess water was then drained, leaving the substrate moist. A layer of gauze was placed on top of the substrate, followed by a layer of nylon cloth with a pore size of 300 mesh. *Dendrobium nobile* seeds were thoroughly mixed with sterile water to prepare a seed suspension. 1mL of the seed suspension was evenly spread onto the nylon cloth to obtain the symbiotic germination substrate. The symbiotic germination substrate was then placed in an artificial climate incubator under the following conditions: temperature 25±2℃, light intensity 2000 lux, and a 12-hour light-12-hour dark cycle. Observations were made daily. After 25 days of cultivation, the seeds germinated and grew into seedlings. Figure 3 Type A can be used for the isolation and purification of fungi.

[0034] 3. Isolation and purification of fungi Fungi were isolated from seedlings using a tissue isolation method, as follows: Germinating seedlings were surface-sterilized on a clean bench. First, they were rinsed with sterile distilled water, then surface-sterilized with 75% ethanol for 30 seconds, rinsed three times with sterile distilled water, then surface-sterilized with 1% NaClO solution for 4 minutes, and finally washed three times with sterile distilled water. After sterilization, the seedlings were fixed with sterile forceps and transversely cut in half with a sterile scalpel. The cut surfaces were placed on the surface of antibiotic-containing PDA medium, covered, and quickly heated with an alcohol lamp. After cooling, the plates were sealed with sealing film and transferred to a fungal incubator at 28°C for several days in the dark. Once hyphae grew from the edges of the tissue blocks in the medium, these edge hyphae were picked and transferred to fresh antibiotic-containing PDA medium for fungal strain purification. The samples were then transferred to a fungal incubator and cultured until hyphae extending towards the edges were observed on the medium. When the fungal edges occupied 2 / 3 of the medium, the fungi were purified again. Edge hyphae were repeatedly picked and transferred to fresh PDA medium containing antibiotics for purification three times until pure colonies without contamination by other microorganisms were obtained. The purified fungi were then observed for colony morphology, hyphal structure, and other characteristics, and preliminary identification was performed according to the fungal classification system.

[0035] Fungal Preservation: Purified fungi were preserved using the conventional test tube slant method. The specific method is as follows: An appropriate amount of prepared antibiotic-containing PDA medium was poured into 18×20mm glass test tubes, filling approximately one-third of the tube volume. After sealing with silicone stoppers, the tubes were placed in an autoclave for sterilization (121℃, 25min). After sterilization, the test tubes were arranged in a laminar flow hood to form slant plates. On the laminar flow hood, the purified strain was inoculated onto the antibiotic-containing PDA medium slant plate using a sterilized bamboo stick, and labeled with the strain name, number, and date, designated as SH1. The inoculated test tubes were then incubated at 28℃ for 3 days. Once the mycelium had fully colonized the antibiotic-containing PDA medium slant plate, the test tubes were removed and stored at 4℃.

[0036] Example 2: Symbiotic Culture 1. Activation and culture of bacterial strains Fungal strain SH1 was selected from the fungi isolated in Example 1 for a screening test to promote germination. The fungal strain was placed in a clean bench, and the culture medium containing mycelia of the test strain was picked up with a sterilized disposable bamboo stick and placed in the center of fresh antibiotic-containing PDA medium for strain activation. The culture dish was sealed with sealing film and placed in an artificial climate chamber at 28±2℃ for incubation. After the fungal mycelia had fully grown in the culture dish, it was transferred back to the clean bench, and holes were punched in the culture medium in the above-mentioned culture dish with a sterilized 5mm diameter pipette tip to create mycelial cakes. The mycelial cakes were transferred to the center of prepared OMA medium using sterile forceps to obtain the culture dish inoculated with the strain. The above steps were repeated for each strain to prepare 4 culture dishes. The blank control was inoculated with the same size PDA medium without fungi. The culture dishes were sealed with sealing film and transferred to a constant temperature 28℃ biochemical incubator for incubation in the dark for several days. After the fungal mycelia of the strain grew in the culture dish containing OMA medium, seeds could be sown.

[0037] 2. The promoting effect of fungal strain SH1 on the germination of mature Dendrobium nobile seeds. 1) OMA medium was selected. Fungi can grow on OMA medium, but orchid seeds cannot germinate on it due to the lack of carbon source. OMA medium was prepared as a symbiotic germination medium for fungi and seeds. Using a sterile bamboo stick, mycelia from the edge of colonies on antibiotic-containing PDA medium were picked and placed in the center of the OMA medium surface. The medium was sealed, labeled, and incubated in a 28°C incubator for 7 days for subsequent experiments.

[0038] 2) Seed sowing In a clean bench, remove the syringe plunger, add an appropriate amount of seeds, seal the plunger tightly, disinfect with 1% NaClO solution and shake well for 5 minutes, drain the disinfectant, transfer the seeds and liquid to a funnel lined with nylon cloth to drain the solution, rinse three times with distilled water, then transfer the nylon cloth to an Erlenmeyer flask, and suspend the seeds in sterile water. Then, pipette 1 mL of the suspension (containing approximately 500 seeds) and evenly sow it onto the surface of OMA medium inoculated with fungal strain SH1. Cover the petri dish, seal with sealing film, and label. In the artificial incubator, maintain alternating light and dark conditions (12 hours light / 12 hours dark), a light intensity of 2000 lux, and a constant temperature of 25±2℃.

[0039] 3) Observation and statistical analysis of data In the blank control, seeds not cultured with the isolated fungus failed to germinate; however, after co-culturing with fungal strain SH1 for 25 days, the seeds began to germinate. Figure 3 In section B, the petri dish was opened, and the germination status of *Dendrobium nobile* seeds was recorded according to the description in Table 1. The number of germinating seeds and the total number of seeds were counted. In this scheme, germination stage 3 and above were considered germination. Four fields of view were randomly selected on the 9cm petri dish, and the total number of seeds and the number of germinating seeds were recorded under a 1x magnified dissecting environment. The germination rate was calculated as (number of germinating seeds ÷ total number of seeds) × 100%, and the results are shown in Table 2.

[0040] Table 1. Description of the germination stages of Dendrobium nobile seeds.

[0041] Table 2. Germination rate of Dendrobium nobile seeds and fungal strain SH1

[0042] As shown in Table 2, the average germination rate of fungal strain SH1 reached 69.70%, with the highest germination rate reaching 73.20%.

[0043] Example 3: Identification of fungal strain SH1 1) Inoculate fungal strain SH1 onto PDA medium containing antibiotics and grow for 7 days. Observe the colony morphology characteristics, such as... Figure 1 As shown.

[0044] from Figure 1 As can be seen, the colonies of fungal strain SH1 are regularly round, and the entire colony is milky white and opaque, with no pigment secretion or diffusion observed. The edges are smooth and neat, without wavy, serrated, or fibrous extensions. The surface is uniform and delicate, with a smooth, moist, membranous structure, without wrinkles, cracks, or granular protrusions. No aerial hyphae, radial stripes, concentric rings, or other special morphological structures were observed.

[0045] 3) Molecular biological identification Genomic DNA was extracted from fresh fungal mycelia using the EZNASP Fungal DNA Kit (brand: Omega Bio-Tek) according to the manufacturer's instructions. The primers used for PCR amplification were ITS4 and ITS5. The sequence of ITS4 is shown in SEQ ID NO.2: 5'-TCCTCCGCTTATTGATATGC-3'; the sequence of ITS5 is shown in SEQ ID NO.3: 5'-GGAAGTAAAAGTCGTAACAAGG-3'. The PCR reaction system (50 μL) included: 2.0 μL DNA template, 25.0 μL 2×RapidTaq Master Mix, 2 μL of each primer (primer concentration of 10.0 μM), and 19.0 μL ddH2O. The amplification reaction was performed on a PCR instrument (brand: Perkin Elmer) using the following PCR cycles: 95℃ pre-denaturation for 5 min; followed by 35 cycles, including 95℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 30 s; and a final extension at 72℃ for 7 min. The PCR amplification products were sent to Kunming Qingke Biotechnology Co., Ltd. for sequencing. The sequenced ITS was submitted to the National Center for Biotechnology Information (NCBI) database for comparison. BLAST analysis showed that the fungal strain SH1 was most similar to *Tulasnella calospora*, with a maximum similarity of 99.84%, preliminarily confirming its taxonomic position. Tulasnella (Melaleuca spp.). Further, representative nrDNA-ITS sequences of orchid mycorrhizal fungi will be downloaded from GenBank and compared with the sequences obtained in this study for phylogenetic relationship analysis. The phylogenetic tree constructed based on the ITS sequences is shown below. Figure 2 As shown.

[0046] Comparative Example 1: Germination-promoting effect of fungal strain SH1 on other orchid seeds The co-culture was carried out in accordance with the method of Example 2, except that Dendrobium nobile was replaced with seeds of Dendrobium chrysanthum, Dendrobium nobile var. jinshajiangensis, Dendrobium rosenbergii, Dendrobium nobile var. chinense, and Dendrobium tibetica. After sterilization, the seeds were co-cultured with fungal strain SH1 on OMA medium for 25 days, and the germination of the seeds was observed.

[0047] The results showed that *Dendrobium nobile*, *Dendrobium jinshajiangense*, *Bulbus rubrotinctum*, *Cymbidium faberi*, and *Cymbidium tibetica* failed to germinate into seedlings, meaning they did not reach germination stages 3 and 4. This indicates that the fungal strain SH1 only had a good germination-promoting effect on *Dendrobium nobile*.

[0048] This demonstrates that symbiotic germination of Dendrobium nobile seeds with the fungal strain SH1 can significantly improve the germination rate of the seeds, thus providing the necessary conditions for the efficient production of mycorrhizal seedlings.

[0049] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A species of film-forming bacteria ( Tulasnella calospora Fungal strain SH1, characterized in that, The preservation number of the fungal strain SH1 is GDMCC No: 66867.

2. A microbial inoculant, characterized in that, Includes the fungal strain SH1 as described in claim 1.

3. The method for preparing the microbial inoculant according to claim 2, characterized in that, include: The fungal strain SH1 was cultured in a culture medium to obtain the microbial inoculum; The culture medium includes: antibiotic-containing PDA culture medium.

4. The preparation method according to claim 3, characterized in that, The culture temperature is 25~27℃.

5. The application of the fungal strain SH1 of claim 1 or the microbial agent of claim 2 in promoting the germination of Dendrobium nobile seeds.

6. A method for promoting the germination of Dendrobium nobile seeds, characterized in that, The process includes the following steps: symbiotic germination culture of Dendrobium nobile seeds with the fungal strain SH1 described in claim 1 or the microbial agent described in claim 2.

7. The method according to claim 6, characterized in that, The culture medium for the symbiotic germination culture includes oat agar medium.

8. The method according to claim 6, characterized in that, The light conditions for the symbiotic germination culture are alternating light and dark, with 10-14 hours of light and 14-10 hours of darkness.

9. The method according to claim 8, characterized in that, The intensity of the illumination is 2000~2500 lux.

10. The method according to any one of claims 6 to 9, characterized in that, The temperature for the symbiotic germination culture is 25~28℃.

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