D30D and application thereof

Abstract

This invention discloses a strain of *Dendrobium nobile* D30D that promotes seed germination and seedling formation, as well as plant growth, and its applications. The strain D30D has the accession number CCTCC NO: M 20252856. Strain D30D can rapidly promote the germination of *Dendrobium nobile* seeds into protocorms and seedlings, promote the growth of *Dendrobium nobile*, and / or increase the medicinal components of *Dendrobium nobile*, demonstrating its significant comprehensive application potential.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a strain of *Dendrobium nobile* D30D that promotes seed germination and seedling growth and promotes plant growth, and its application. Background Technology

[0002] Dendrobium officinale (with curved stems) Dendrobium flexicaule ZHTsi, SCSum & L.G.Xu) is a perennial herb belonging to the genus Dendrobium in the family Orchidaceae. This plant is one of the sources of Dendrobium medicinal materials and has the effects of benefiting the stomach and promoting the production of body fluids, nourishing yin and clearing heat.

[0003] Currently, the Dendrobium nobile industry mainly relies on greenhouse cultivation, rock wall cultivation, and tree-attached cultivation, with most seedlings originating from tissue culture. The current industrialized seedling production of Dendrobium nobile primarily involves a three-step (seed germination → protocorm proliferation and differentiation → robust seedling and rooting) or even a four-step (seed germination → protocorm proliferation and differentiation → robust seedling → rooting) process. This process is lengthy, requires multiple manual transfers, and incurs high labor costs.

[0004] As an orchid, *Dendrobium nobile* has extremely small seeds without endosperm. In its natural environment, it requires a symbiotic fungus to germinate. In the early stages when nutrient resources are scarce, its dependence on this symbiotic fungus is very strong. Therefore, the symbiotic relationship is crucial in the seed germination and seedling formation stages of *Dendrobium nobile*.

[0005] In the conservation and artificial propagation of Dendrobium nobile, the choice of seed germination method has a crucial impact on its subsequent growth and adaptability. Currently, the commonly used method is non-symbiotic germination under sterile conditions using artificial culture media. Although the germination rate is high, in field reintroduction practices, these sterile-germinated seedlings generally suffer from low survival rates after transplanting to the natural environment. Because they fail to establish a symbiotic relationship with natural fungi, their subsequent growth is severely hindered. Therefore, screening for symbiotic fungi that can effectively promote seed germination has become the primary step in the conservation of rare and endangered orchid species and a key link in achieving their ecological cultivation. Summary of the Invention

[0006] This invention overcomes the shortcomings of the prior art and provides a strain of Tulasnella calospora D30D that promotes seed germination and seedling growth of Dendrobium nobile and promotes plant growth, as well as its applications.

[0007] The *Tulasnella calospora* D30D strain described in this invention, classified as *Tulasnella calospora* D30D, was deposited on December 11, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 20252856.

[0008] The present invention also provides a bacterial suspension comprising Tulasnella calospora D30D as described herein. The bacterial suspension can be prepared according to conventional methods in the art.

[0009] This application also provides the use of Tulasnella calospora D30D or a bacterial solution containing it as described in this invention in promoting seed germination, protocorm formation, seedling development, promoting plant growth, and / or enhancing the medicinal components of Dendrobium calospora.

[0010] In a specific example, the above-described application of this invention involves applying the *Tulasnella calospora* D30D or a bacterial solution containing this fungus to the seeds or roots of *Dendrobium nobile*. The *Tulasnella calospora* D30D described in this invention can form a symbiotic relationship with *Dendrobium nobile* seeds, thereby achieving the aforementioned function.

[0011] The present invention also provides a method for promoting the germination of Dendrobium nobile seeds by applying Tulasnella calospora D30D or a bacterial solution containing the fungus described in the present invention to Dendrobium nobile seeds.

[0012] The present invention also provides a method for promoting the formation of protocorms of Dendrobium calospora, by applying Tulasnella calospora D30D or a bacterial solution containing the fungus described in the present invention to Dendrobium calospora seeds.

[0013] The present invention also provides a method for promoting the development of Dendrobium officinale seedlings by applying Tulasnella calospora D30D or a bacterial solution containing the fungus to Dendrobium officinale seeds.

[0014] The present invention also provides a method for promoting the growth of Dendrobium calospora by applying the D30D bacterial solution of Tulasnella calospora or a bacterial solution containing the present invention to Dendrobium calospora seeds or roots.

[0015] The present invention also provides a method for improving the medicinal components of Dendrobium nobile by applying Tulasnella calospora D30D or a bacterial solution containing the fungus to Dendrobium nobile seeds or roots.

[0016] In some embodiments, the medicinal components of Dendrobium nobile mentioned in this invention refer to the content of total flavonoids, total alkaloids and / or polysaccharides in Dendrobium nobile.

[0017] Advantages of this invention over existing technologies:

[0018] The fungus D30D described in this invention can solve the problems of difficult seed germination and low seedling rate of Dendrobium nobile, especially under poor soil conditions. It can significantly promote root development and plant growth, and greatly increase the content of medicinal active ingredients, especially polysaccharides, which is of great significance.

[0019] Using mycorrhizal fungus D30D to co-germinate Dendrobium officinale seeds not only shortens the seedling cycle and significantly reduces costs compared to traditional plant tissue culture methods, but also, by maintaining this symbiotic relationship during cultivation, can promote plant growth and increase the content of some metabolites, demonstrating its significant comprehensive application potential. Attached Figure Description

[0020] Figure 1 A frontal view of the colony morphology of D30D fungus of the genus *Collagenia*.

[0021] Figure 2 Reverse view of the colony morphology of D30D fungus of the genus *Collagenia*;

[0022] Figure 3 Micrograph of hyphae of D30D fungus of the genus *Morphozoa*;

[0023] Figure 4 The image shows the results of the promotion of seed germination of Dendrobium nobile by Tulasnella calospora D30D at 30 days.

[0024] Figure 5 The image shows the results of the promotion of seed germination of Dendrobium nobile by Tulasnella calospora D30D at 60 days.

[0025] Figure 6 The image shows the results of the promotion of seed germination of Dendrobium nobile by Tulasnella calospora D30D at 90 days.

[0026] Figure 7The figure shows the results of different fungal treatments promoting the germination of Dendrobium officinale seeds at 90 days. Among them, a is OMA (poor culture medium) treatment; b is MS (enriched culture medium) treatment; c is D12B fungal treatment; d is D62A fungal treatment; e is D27A fungal treatment; f is D53C fungal treatment; g is Si fungal treatment; and h is D30D fungal treatment. Detailed Implementation

[0027] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0028] Example 1: Obtaining and identifying Tulasnella calospora strain D30D

[0029] 1. Plant tissue isolation method

[0030] In May 2025, at the Dendrobium nobile semi-wild cultivation base in Xixia County, Nanyang City, Henan Province, mature roots of healthy, semi-wild Dendrobium nobile plants aged 3 to 5 years were collected, wrapped in moist moss, and brought back.

[0031] Freshly collected mature roots were rinsed thoroughly with running water. In a clean bench, root segments were selected for surface sterilization: first, treated with 75% ethanol for 30 seconds, rinsed three times with sterile water, and then disinfected with 2% sodium hypochlorite (NaClO) solution for 3 minutes. After rinsing five times with sterile distilled water, the surface moisture was blotted dry with sterile filter paper, and the roots were cut into 1–2 mm thick pieces. These pieces were then placed on PDA medium and incubated at 25±2℃ for 3–10 days. Once fungal hyphae had grown from the root pieces, the tips were cut with a sterile scalpel and transferred to fresh PDA medium for purification. After 3–5 purification cycles, pure colonies were obtained.

[0032] 2. Fungal Preservation: The purified fungi were preserved using the conventional test tube slant method. An appropriate amount of prepared PDA medium was poured into 18×20 mm glass test tubes, filling approximately 1 / 3 of the tube volume. The tubes were then sealed tightly with silicone stoppers and sterilized in an autoclave (121℃, 20 min). After sterilization, the test tubes were placed in a laminar flow hood and arranged as slant plates. On the laminar flow hood, the purified strain was cut into appropriately sized pieces using a sterile scalpel. These pieces were then inoculated onto the PDA slant using an inoculation loop, and labeled with the strain, number, and date. The inoculated test tubes were then placed in an artificial climate chamber at 25±2℃ for incubation. When the mycelium had almost completely covered the PDA slant, the test tubes were removed and stored in a laboratory medical refrigerator at 4℃.

[0033] The isolated strain was classified and named *Tulasnella calospora* D30D, and was deposited on December 11, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC NO: M 20252856.

[0034] 3. Identification of Tulasnella calospora strain D30D

[0035] The PDA culture of strain D30D was sent to General Biotechnology (Anhui) Co., Ltd. for sequencing. The characteristic nrDNA ITS sequence of strain D30D is shown in SEQ ID NO.1. The nrDNA ITS sequence of strain D30D was then submitted to the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov / ) database, with Genbank number PX625526. BLAST comparison showed that this strain was most similar to the fungus PV581120.1, Tulasnella calospora, with a maximum similarity of 99.31%.

[0036] Morphological characteristics: After 8 days of culture on PDA plates, strain D30D produced white, concentric colonies without rings, relatively thick colonies, dense, fluffy hyphae, and radially spreading white color. The colony morphology was as follows. Figure 1 and Figure 2 As shown.

[0037] Physiological and biochemical characteristics: The microscopic morphological characteristics of the strain were observed using the coverslip culture method. The strain was cultured in an artificial climate chamber at 25±2℃ for 4–6 days, and slides were prepared using standard methods. Under an optical microscope, the hyphae were septate, with a relatively uniform diameter of approximately 2.13–5.68 μm. The cell length was 20.0–58.0 μm, with branching at approximately 45°. Older hyphae were clustered at the base with more developed branching, while new hyphae were sparsely distributed. The cell walls were thin, and there were numerous septa. Figure 3 As shown.

[0038] Based on morphological, physiological, biochemical, nucleotide sequence, and phylogenetic analysis, it was identified as *Tulasnella calospora*, a fungus belonging to the genus *Tulasnella*.

[0039] Example 2: Experiment on the promotion of seed symbiotic germination and seedling formation of Dendrobium officinale by strain D30D

[0040] This study investigated the effects of different fungi on the germination of *Dendrobium nobile* seeds within the same timeframe using a symbiotic culture of seeds and fungi. The germination-promoting effect of strain D30D isolated in this invention on *Dendrobium nobile* seeds was compared with that of other fungi. The different fungi were derived from other fungi isolated from the fibrous roots of *Dendrobium nobile* during the same period, as well as strains purchased from the China Agricultural Microbial Culture Collection Center.

[0041] Isolated from Dendrobium nobile ( Dendrobium flexicaule The fungus Tulasnella sp. D12B (ZHTsi, SCSum & L.G.Xu), Genbank accession number PX625523, NCBI accession number OM728188.1.

[0042] Isolated from Dendrobium nobile ( Dendrobium flexicaule The fungus Tulasnella sp. D62A (ZHTsi, SCSum & L.G.Xu) is listed under Genbank accession number PX625528 and NCBI accession number FJ613252.1.

[0043] Isolated from Dendrobium nobile ( Dendrobium flexicaule The fungus *Sebacinales* sp. D27A (ZHTsi, SCSum & L.G.Xu) is listed under GenBank accession number PX625531 and NCBI accession number MN918490.1.

[0044] Isolated from Dendrobium nobile ( Dendrobium flexicaule The fungus Tulasnella sp. D53C (ZHTsi, SCSum & L.G.Xu), Genbank accession number PX625529, NCBI accession number LC175326.1.

[0045] Purchased strain: *Piriformospora indica* strain *Serendipita indica* (Si), NCBI: KF061284.1, belonging to the phylum Basidiomycota, class Hymenomycetes, order Sebacinales, family Sebacinaceae, genus *Piriformospora*. Isolated in 1998 from the rhizosphere of shrubs in the Thar Desert, it can colonize the surface of plant roots, epidermal cells, and intercellular spaces, forming typical pear-shaped chlamydospores that survive on plant roots. It has a wide host range, capable of symbiotic relationships with over 200 monocotyledonous and dicotyledonous plant species. By enhancing the absorption of nutrients such as N and P, it promotes plant growth and strengthens systemic resistance, making it a multifunctional plant endophytic fungus with broad application potential.

[0046] The specific experimental steps for the symbiotic germination and seedling formation experiment with Dendrobium nobile seeds are as follows:

[0047] (1) Preparation of culture medium

[0048] Prepare a symbiotic germination medium, which is oat agar (OMA) containing 4 g / L of oat agar. -1 Oatmeal and 15g·L -1 Agar, culture medium pH 5.8–6.0; 120 plates of oat medium were prepared and sterilized for later use, for a total of 6 treatment groups; each treatment group had 20 replicates.

[0049] To prepare a nutrient-rich culture medium, MS medium, also known as nutrient-rich medium (MS), contains 4.74 g·L⁻¹. -1 MS, 20 g·L -1 Sucrose and 15g·L -1 Agar. Prepare 20 nutrient-rich culture media, sterilize, and use for one treatment group with 20 replicates.

[0050] Prepare a medium for the isolation and purification of fungi in the laboratory. PDA medium, also known as potato dextrose solid medium (PDA), 200 g / L. -1 Potatoes, 20g·L -1 glucose and 15 g·L -1 Agar, culture medium pH 5.8–6.0.

[0051] (2) Activation of microbial strains

[0052] The six strains of fungi stored in test tube slants at 4°C were taken out and inoculated onto different PDA media. They were then placed in an artificial climate chamber and cultured at 25±2°C. The desired symbiotic germination strains were obtained when the fungal hyphae covered the culture dish.

[0053] (3) Seed disinfection and preparation of seed suspension

[0054] Prepare a 0.1% agar suspension, cut 400-mesh nonwoven fabric into 1.5 cm × 1.5 cm squares, and sterile water. After autoclaving and cooling, place the mixture in a clean bench. Inside the clean bench, open a brand new disposable syringe, remove the needle, and insert nonwoven fabric at the junction of the needle and syringe. Pour the pre-weighed seeds into the syringe, draw up a 1% NaClO solution, and continuously shake the syringe for 5 minutes to ensure thorough mixing and sterilization. Discard the NaClO solution, and rinse with sterile water, shaking 3–5 times. Drain the liquid to obtain sterile seeds.

[0055] Remove the needle and non-woven fabric, aspirate the agar slurry, and inject all the seeds from the syringe into a 0.1% agar suspension. After mixing, pipette 200 μl onto a glass slide and count the seeds to determine the number of seeds per unit volume of Dendrobium officinale seed suspension. Control the seed concentration to approximately 80 Dendrobium officinale seeds per 200 μl unit volume.

[0056] (4) Inoculation and sowing

[0057] Pure cultures of single fungi (D30D, D12B, D62A, D27A, D53C, and Si) with a diameter of 1 cm were inoculated into the center of OMA medium. 200 μl of seed suspension was pipetted and evenly distributed around the fungal blocks. Each treatment was replicated 20 times. For the blank control, PDA agar blocks were inoculated onto nutrient-rich MS medium, and PDA agar blocks were inoculated onto nutrient-poor OMA medium. After inoculation, the plates were sealed with paraffin wax and then wrapped with plastic wrap 2-3 times for labeling.

[0058] (5) Symbiotic germination culture

[0059] Place all culture dishes in a light incubator or tissue culture rack, and incubate at a constant temperature of 25±2℃ for 12 h of light / 12 h of darkness. The light intensity is 2500–3000 Lx.

[0060] (6) Seed germination status was statistically analyzed after 30, 60 and 90 days of symbiotic culture.

[0061] Germination rate was observed and statistically analyzed every 30 days. Seed germination and seedling growth and development were described in four stages: non-germination, germination (embryo swells and turns green after absorbing water, but the seed coat remains), protocorm formation (embryo continues to swell and breaks through the seed coat), development (color continues to turn green, protometrial tissue appears), seedling differentiation (first leaf appears), and developmental stage (two leaves appear and rooting occurs). Based on these definitions, the seed germination rate (G), protocorm formation rate (C), and seedling rate (K) were calculated.

[0062] Seed germination, protocorm formation, and seedling development: The total number of seeds (z), the number of germinated seeds (m), the number of protocorms (q), and the number of seedlings (y) were recorded at 30, 60, and 90 days after germination. The seed germination rate (G), protocorm formation rate (C), and seedling formation rate (K) were calculated as follows:

[0063] G = (m + q + y) / z × 100%;

[0064] C = (q + y) / z × 100%;

[0065] K = y / z × 100%.

[0066] At the end of the 90-day co-culture experiment, the results of promoting the germination of Dendrobium nobile seeds by the 30-day, 60-day, and 90-day *Mammillaria rubra* D30D strains described in this invention were obtained (see the image below). Figure 4 , Figure 5 , Figure 6 (As shown). And the results of different fungal inoculation treatments and a blank control promoting the germination of Dendrobium officinale seeds (Figure 1). Figure 7 Data were subjected to one-way ANOVA statistical tests, using parametric tests and one-way ANOVA post-hoc multiple comparisons to compare the effects of different fungal inoculation treatments on seed germination, protocorm formation, and seedling development of Dendrobium nobile, and to compare whether there were significant differences in the mean values ​​among the treatment groups.

[0067] Table 1

[0068]

[0069] Note: All data are summarized as mean ± standard error (SE), n=20.

[0070] The results are shown in Table 1. After 90 days of symbiotic culture, it can be seen that seeds in the uninoculated group germinated and formed protocorms, but the seedling formation rate was generally low, with less than 50% of seedlings formed on the enrichment medium. D30D, isolated from the roots of mature *Dendrobium nobile* plants, significantly promoted seed germination (90.25% ± 0.55%) and protocorm formation (87.13% ± 0.49%) on day 90. Simultaneously, most of the protocorms continued to develop into seedlings (84.38% ± 0.65%). The seedling formation rate of other strains was not significantly better than that of seedlings formed on the enrichment medium. Based on the above data, it is clear that D30D can effectively promote the growth and development of *Dendrobium nobile* seeds to the seedling stage in a short period, and is an effective strain for promoting rapid seed germination and seedling formation.

[0071] Example 3: Symbiotic culture of *Dendrobium nobile* D30D with *Dendrobium officinale* seedling substrate

[0072] (1) Preparation of D30D suspension of *Bacillus spp.*

[0073] Materials: sterile water, PDA plates covered with *Morchella spp.* D30D, and PDB potato broth.

[0074] Methods: The fungal culture plates were cut into small pieces using a sterile scalpel and inoculated into PDB medium. 6-7 fungal agar blocks were inoculated per bottle of PDB medium, and 10 bottles were inoculated per fungal strain. The culture was placed in a constant-temperature shaker at 25℃ and 180 rpm for 10 days. After a large amount of mycelium had grown, the fermentation broth was filtered through non-woven fabric to obtain clumps of mycelium. The water in the mycelium was squeezed out, and the mycelium was rinsed with sterile water. Then, the mycelium was placed in a juicer, and an appropriate amount of sterile water was added. The mixture was then blended to prepare a bacterial solution (crystal volume to sterile water ratio 1:2).

[0075] (2) Preparation of Dendrobium officinale seedlings by one-step tissue culture:

[0076] Materials: Dendrobium officinale seeds, bananas, potatoes, 0.4 mg / L NAA, Flower Treasure No. 1, MS, N6, sucrose, agar.

[0077] The formula for the original bulb liquid culture medium is: N6 + 0.4 mg / L NAA + 30 g / L sucrose + 1 g / L agar.

[0078] One-step seedling solid culture medium formula: MS + 0.4 mg / L NAA + 1 g / L Flower Treasure No. 1 + 100 g / L potato + 100 g / L banana + 30 g / L sucrose + 6.5 g / L agar.

[0079] Method: Liquid culture of protocorms: Sterilized Dendrobium nobile seeds (sterilization method is the same as in Example 2) were squeezed into protocorm liquid culture medium, shaken evenly and placed in a culture room for culture. The culture room conditions were: temperature 24±1°C, humidity 80±5%, light intensity 2000~2500 Lx, light intensity 12h / d, and culture time 30d.

[0080] Inoculation by spot sowing: Dilute the protocorm suspension with sterile water. Use a pipette to take 250 μl of the protocorm suspension and place it on a glass slide to observe the number of protocorms. Observe 5 times and take the average value. Dilute to 25 protocorms per 250 μl. Then use a pipette to take 500 μl of the protocorm suspension and spot sow it evenly on the one-step seedling solid culture medium. Place it in the culture room for 270 days.

[0081] (3) Preparation of co-culture substrate

[0082] Materials: Fir wood chips (fine), pine bark (9-12mm), sphagnum moss

[0083] Method: Mix the above-mentioned fir sawdust, pine bark, and sphagnum moss in a volume ratio of 3:1:1 to obtain a substrate. Soak the mixed substrate in water for more than 24 hours. After draining the soaked substrate, put it into a high-temperature sterilization bag, filling the bag to no more than 80% capacity. Leave the sterilization bag open and place it in a high-temperature sterilizer for moist heat sterilization at a temperature of 121℃ for 20 minutes. Repeat the sterilization operation 2-3 times to fully eliminate fungi and bacteria in the substrate.

[0084] Symbiotic culture method: After acclimatization and cleaning, select uniformly growing Dendrobium officinale seedlings (5-6 leaves, 2-3 tillers, 2-3 roots, root length 5-6 cm, plant height 3-3.5 cm, stem diameter 2.5-3 mm), and group 3-5 seedlings into a clump. Cover with sterile substrate and plant in seedling trays. For the inoculated group, evenly drip 5 ml of bacterial solution around the base of the seedlings, repeating every 10 days. For the uninoculated group, maintain routine management. Each treatment has 30 seedling trays. Place in an artificial climate chamber at a temperature of 24±1℃, humidity of 80±5%, light intensity of 2000-2500 Lx, and a light intensity of 12 h / d for 90 days of symbiotic culture.

[0085] Fungal colonization detection: After symbiotic culture, roots of *Dendrobium nobile* plants were taken every 30 days for freehand sectioning. The colonization of fungi in *Dendrobium nobile* plants was observed under a microscope to determine whether the colonization was successful. The criterion for successful colonization was the presence of mycelial clusters observed in the roots of *Dendrobium nobile*. No mycelial clusters were found in the uninoculated group on days 30, 60, and 90. In the inoculated group, successful fungal colonization was observed on days 30, 60, and 90, proving that D30D can successfully colonize the roots of *Dendrobium nobile* in the cultivation substrate (mycelial clusters were observed).

[0086] Example 4: Effect of inoculum on the growth of Dendrobium nobile.

[0087] To clarify the effect of strain D30D on the growth of *Dendrobium nobile*, in Example 3, after 90 days of cultivation, the fresh weight of stems, dry weight of stems, tillers, number of roots, root length, plant height, and stem diameter of *Dendrobium nobile* seedlings were measured, with 30 plants measured in each treatment group. For fresh stem weight: at 90 days, the seedlings were cleaned, leaves and roots removed, and allowed to air dry naturally until no moisture remained, then weighed. For dry weight: at 90 days, the fresh stems were placed in a 60℃ oven to dry, and weighed during this period to check for changes in dry weight until the dry weight of the stems no longer changed; all stem dry weights were then measured. The number of roots, root length, plant height, and stem diameter were measured using a ruler and vernier calipers.

[0088] Table 2. Effects of inoculation with fungal D30D culture on growth indicators of Dendrobium nobile.

[0089]

[0090] Note: All data are summarized as mean ± standard deviation (SD); * indicates significant difference (P < 0.05); ** indicates extremely significant difference (P < 0.01).

[0091] As shown in Table 2, the group inoculated with D30D fungal solution outperformed the uninoculated group in all growth-related indicators, especially in root development. The most significant effect was the promotion of root development, with the inoculated group showing a 58.3% increase in root number compared to the uninoculated group. This also enhanced the ability of *Dendrobium nobile* to absorb water and nutrients, leading to a greater allocation of photosynthetic products to the above-ground parts, resulting in taller, thicker plants with more tillers.

[0092] Example 5: Effect of inoculation with fungal D30D culture on the biomass of Dendrobium nobile.

[0093] In Example 3, after 90 days of co-cultivation, the fresh weight of the stem of each seedling was measured and the seedlings were placed in an oven at 105℃ for 3 minutes to kill the green, and then dried in an oven at 60℃ until constant weight. The dried sample was then pulverized and passed through a 60-mesh sieve to obtain the dried sample.

[0094] Polysaccharide content determination:

[0095] (1) Weigh 3 mg of the sieved fine powder into a 2 mL EP tube, add 2 mL of distilled water, heat in a boiling water bath at 95°C for 2 h, and gently shake to mix a few times with protective gloves every 20 min. After heating, remove and place at room temperature, and finally centrifuge at 8000 rpm at room temperature for 5 min. Set aside the supernatant.

[0096] (2) Take 0.2 mL of the supernatant after centrifugation in the previous step into an EP tube, add 1 mL of ethanol and mix well. Place at 4℃ for 1 h, take it out and centrifuge at 8000 rpm for 5 min, then discard the supernatant and keep the precipitate.

[0097] (3) Add 1 mL of 80% ethanol to the precipitate obtained in the second step, mix well, centrifuge at 8000 rpm for 5 min, discard the supernatant and keep the precipitate;

[0098] (4) Add 2 mL of distilled water to the precipitate obtained in step 3 and heat in a boiling water bath at 95°C until the precipitate is completely dissolved.

[0099] The absorbance was read at a wavelength of 488 nm using the phenol-sulfuric acid method on an ELISA reader.

[0100] Determination of total flavonoid content:

[0101] Weigh 0.03 g of dried sample, add 1.5 mL of 60% ethanol, extract by shaking at 60℃ for 2 h, centrifuge at 25℃ and 12000 rpm for 10 min, and take the supernatant for testing.

[0102] The total flavonoid content was determined by the NaNO2-AlCl3-NaOH colorimetric method, and the absorbance was read at 510 nm using an ELISA reader.

[0103] Determination of total alkaloid content:

[0104] Weigh 0.05 g of dried sample into a 2 mL EP tube, add 1 mL of extraction buffer (Total Alkaloid Content Assay Kit G0150W), and sonicate for 30 min. After a 3-min interval, remove the tube, shake for 1 min, and then continue sonicating. Finally, add extraction buffer to bring the volume to 1 mL. Centrifuge at 4000 rpm for 10 min at room temperature, and take the supernatant for analysis. Develop the absorbance using bromocresol green and read the absorbance at 415 nm using a microplate reader.

[0105] Table 3 Measurement and analysis of biomass indicators

[0106]

[0107] Note: All data are summarized as mean ± standard deviation (SD), n=6; * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01).

[0108] The data in Table 3 show that the inoculated solution had higher contents of total flavonoids, total alkaloids, and polysaccharides compared to the uninoculated solution, with the polysaccharide content showing a significant increase.

[0109] The total flavonoid content increased by approximately 5.14%, indicating that this fungus can effectively activate the phenylpropanoid metabolic pathway and enhance the plant's antioxidant and stress resistance capabilities. The total alkaloid content increased by approximately 10.62%. As defensive secondary products of Dendrobium species, the increased content of alkaloids further confirms that fungal symbiosis induces a systemic defense response in plants. The polysaccharide content increased by as much as 45.2%, directly reflecting that the fungus promotes the synthesis and storage of carbohydrates in Dendrobium nobile. This also reflects the significant growth-promoting and quality-enhancing effects of this fungus.

[0110] The inoculated fungi can act as a highly efficient biological agent, establishing a favorable symbiotic relationship with Dendrobium nobile. Their effects extend beyond simply promoting biomass growth; their deeper value lies in significantly driving the synthesis and accumulation of medicinal active ingredients by regulating the plant's metabolic network.

Claims

1. A type of *Morphozoa* ( ) Tulasnella calospora D30D, characterized in that, The accession number is: CCTCCNO: M 20252856.

2. A bacterial suspension containing the *Morchella esculenta* D30D as described in claim 1.

3. The application of the *Dendrobium nobile* D30D as described in claim 1 or the bacterial solution as described in claim 2 in promoting seed germination, protocorm formation, seedling development, or improving the medicinal components of *Dendrobium nobile*, wherein the medicinal components refer to one or more of the total flavonoids, total alkaloids, or polysaccharides in *Dendrobium nobile*.

4. The application according to claim 3, characterized in that, Apply the *Dendrobium nobile* D30D as described in claim 1 or the bacterial solution as described in claim 2 to the seeds or roots of *Dendrobium nobile*.

5. A method for promoting the germination of Dendrobium officinale seeds, characterized in that, Apply the *Dendrobium nobile* seed solution of claim 1 (D30D) or claim 2 to *Dendrobium nobile* seeds.

6. A method for promoting the formation of protocorms in Dendrobium nobile, characterized in that, Apply the *Dendrobium nobile* seed solution of claim 1 (D30D) or claim 2 to *Dendrobium nobile* seeds.

7. A method for promoting the development of Dendrobium officinale seedlings, characterized in that, Apply the *Dendrobium nobile* D30D as described in claim 1 or the bacterial solution as described in claim 2 to the seeds or roots of *Dendrobium nobile*.

8. A method for promoting the growth of Dendrobium nobile with curved stems, characterized in that, Apply the *Dendrobium nobile* D30D as described in claim 1 or the bacterial solution as described in claim 2 to the seeds or roots of *Dendrobium nobile*.

9. A method for improving the medicinal components of Dendrobium officinale, characterized in that, The *Dendrobium nobile* D30D of claim 1 or the bacterial solution of claim 2 is applied to the seeds or roots of *Dendrobium nobile*, wherein the medicinal components of *Dendrobium nobile* refer to one or more of the total flavonoids, total alkaloids, or polysaccharides in *Dendrobium nobile*.

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