Mycorrhizal fungi and application thereof in promoting paphiopedilum seed germination and rapid seedling formation

Abstract

The invention relates to mycorrhizal fungi and application thereof in promoting paphiopedilum seed germination and rapid seedling, and belongs to the technical field of microorganisms. The mycorrhizal fungus is MLXY010JD, the taxonomic name of the mycorrhizal fungus is Tulasnella sp., the strain is preserved in Wuhan University, Wuhan, China on September 15, 2025, and the preservation number of the strain is CCTCC NO: M 20252049. The invention also provides a method for promoting seed germination and rapid seedling formation of paphiopedilum by using the strain. The method comprises the following steps: performing symbiotic culture on seeds and mycorrhizal fungi to induce germination to form seedlings; carrying out symbiotic culture on the MLXY010JD and the seedlings to promote the growth of the seedlings; transferring to a specific culture medium for continuous culture; finally, the whole seedling raising process is completed through seedling hardening and transplanting management. The method is suitable for various paphiopedilum species and provides support for artificial breeding and field regression of paphiopedilum.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a mycorrhizal fungus isolated from the roots of Paphiopedilum orchid, and its application and method in promoting the germination and rapid seedling growth of Paphiopedilum orchid seeds. Background Technology

[0002] Paphiopedilum ( Paphiopedilum *Pfitz.* is the flagship group of the Orchidaceae family and one of the most primitive groups within it, occupying a unique and important position in plant phylogenetic evolution. However, due to multiple factors such as over-harvesting, habitat destruction, and climate change, coupled with its highly dependent reproductive process on specific pollinators and its life history on the symbiotic fungi required for seed germination and growth, almost all wild *Pfitz.* species are endangered to varying degrees. On September 7, 2021, the *National Key Protected Wild Plants List* was published, listing *Pfitz.* as an endangered species, except for *Pfitz.* var. *hardleaf*. P. micranthum ) and Paphiopedilum ( P. hirsutissimum Except for one species listed as a Class II protected plant in China, all other species in the genus *Paphiopedilum* are listed as Class I protected species. Therefore, the effective protection of *Paphiopedilum* species has become one of the core tasks of biodiversity conservation, and scientific and efficient conservation measures are urgently needed. This is of great significance for maintaining ecological balance and promoting sustainable development.

[0003] Advances in tissue culture technology have facilitated the large-scale propagation of some orchid species. However, *Paphiopedilum* remains one of the most challenging groups in the orchid family for aseptic sowing and tissue culture, generally suffering from low seed germination rates, long growth cycles, and slow recovery of wild populations. Orchid seeds are tiny, lacking endosperm, and under natural conditions require specific fungal symbiotic relationships to provide nutrients for germination. Research shows that mycorrhizal fungi play an irreplaceable role in the entire life cycle of orchids, from seed germination and seedling development to mature plant propagation. Therefore, developing efficient mycorrhizal fungal inoculation techniques and screening superior strains with growth-promoting functions are of significant practical importance for overcoming the technical bottlenecks in the artificial propagation and reintroduction of *Paphiopedilum* into the wild.

[0004] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide a method for screening superior mycorrhizal fungi and to promote the germination of slipper orchid seeds and rapid seedling growth.

[0006] To achieve the above objectives, this invention provides a method for mycorrhizal propagation of Paphiopedilum orchids using the superior mycorrhizal fungus MLXY010JD screened by this invention, and verifies its application effect. The method sequentially includes screening of superior mycorrhizal fungi, activation of the mycorrhizal fungi, symbiotic germination of seeds, mycorrhizalization and seedling growth promotion, subculture of seedlings, induction of stress resistance, and hardening-off transplanting, as detailed below:

[0007] This invention provides a mycorrhizal fungus that promotes the growth of Paphiopedilum seedlings ( Tulasnella The strain MLXY010JD was deposited on September 15, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20252049.

[0008] The mycorrhizal fungus MLXY010JD provided by this invention can be used to promote seed germination and plant growth of Paphiopedilum plants.

[0009] Preferably, the above-mentioned Paphiopedilum genus includes Paphiopedilum 'white-flowered', Paphiopedilum 'small-leaved', and / or Paphiopedilum 'leaved'.

[0010] This invention also provides a method for promoting the germination and rapid seedling growth of Paphiopedilum seeds, comprising the following steps: Step 1: Paphiopedilum seeds and mycorrhizal fungus FQXY019 were co-cultured on PDA medium to germinate and form seedlings. The preservation number of FQXY019 is CCTCC NO: M 20211513. Step 2: The seedlings obtained in Step 1 are co-cultured with the above-mentioned mycorrhizal fungus MLXY010JD on PDA medium to obtain symbiotic seedlings; Step 3: Transfer the symbiotic seedlings obtained in Step 2 to a growth-promoting culture medium for subculture.

[0011] Preferably, the subculture in the above method includes two stages: the first stage uses culture medium ① and the second stage uses culture medium ②. Among them, culture medium ① contains: ¼ MS medium, Flower Treasure No. 1, Flower Treasure No. 2, glucose, activated carbon, potato and agar; Culture medium ② contains: ¼ MS medium, Flower Treasure No. 1, Flower Treasure No. 5, glucose, activated charcoal, peptone and agar.

[0012] Preferably, the above method further includes, after step 3, inoculating the symbiotic seedlings with mycorrhizal fungus FQXY019 to enhance stress resistance.

[0013] Preferably, the above method further includes seedling hardening and transplanting steps, and the cultivation substrate used is a mixture of dried pine bark, tree fern, volcanic rock, pumice and peat moss in a mass ratio of 35:10:30:10:15.

[0014] Preferably, the Paphiopedilum genus in the above method includes Paphiopedilum 'White Flower', Paphiopedilum 'Small Leaf', and / or Paphiopedilum 'Leafy Leaf'.

[0015] The combination of mycorrhizal fungi MLXY010JD and FQXY019 provided by this invention can be used to promote seed germination and seedling growth of Paphiopedilum plants. Among them, the preservation number of FQXY019 is CCTCC NO: M 20211513. It can also be used in the mycorrhizal seedling cultivation of Paphiopedilum, with high seed germination rate, fast seedling growth and high transplant survival rate, providing technical support for the artificial propagation of Paphiopedilum.

[0016] The present invention has the following advantages: This invention screened and obtained an excellent mycorrhizal fungus, MLXY010JD, which can effectively promote the growth of Paphiopedilum 'White Flower' (…). P. emersonii ), Paphiopedilum simonii ( P. barbigerum The germination of seeds and plant growth of Paphiopedilum species such as Paphiopedilum var. ...

[0017] This invention also provides a method for effectively promoting the germination and rapid seedling growth of various Paphiopedilum seeds using this strain. It has the advantages of high seed germination rate, rapid seedling growth, and high transplant survival rate. This method can not only be applied to the artificial propagation of Paphiopedilum, but also provides a new technical solution for the in-situ symbiotic germination and wild reintroduction of Paphiopedilum plants. Attached Figure Description

[0018] Figure 1 The colony morphology and hyphal microstructure of the screened strain MLXY010JD are shown in the image.

[0019] Figure 2 The growth of Paphiopedilum seeds inoculated on culture medium containing strain FQXY019-PDA.

[0020] Figure 3 Seedlings formed 60 days after inoculating Paphiopedilum seeds with strain FQXY019-PDA.

[0021] Figure 4 The effects of mycorrhizal fungi MLXY010JD and FQXY019 on the growth of Paphiopedilum symbiotic seedlings were investigated 90 days after inoculation.

[0022] Figure 5 The growth of seedlings formed by symbiotic germination of strain FQXY019 after inoculation with growth-promoting strain MLXY010JD for 90 days.

[0023] Figure 6 The growth of Paphiopedilum mycorrhizal seedlings after 90 days of culture on medium ①.

[0024] Figure 7 The growth of Paphiopedilum mycorrhizal seedlings after 90 days of culture on medium ②.

[0025] Figure 8 The stress resistance of Paphiopedilum seedlings after inoculation with fungus FQXY019.

[0026] Figure 9 The growth of mycorrhizal seedlings after transplanting to a greenhouse for 360 days.

[0027] Figure 10 This study compares the growth vigor of Paphiopedilum seedlings in a greenhouse with those inoculated with mycorrhizal fungus FQXY019 and those without inoculation. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0030] The present invention relates to some of the culture media: (1) Fungal culture medium PDA: 200 g / L potatoes, 20 g / L glucose, 10 g / L agar.

[0031] (2) Seed and fungal symbiotic culture medium: OMA medium: 20 g / L oats, 20 g / L glucose, 10 g / L agar; B5 medium: B5 medium 2 g·L, glucose 20 g·L, agar 10 g·L; PDA medium: potato 200 g·L, sucrose 20 g·L, agar 10 g·L; MS medium: ¼ MS medium, glucose 20 g·L, agar 10 g·L.

[0032] (3) Seedling symbiotic culture medium: Culture medium ①: ¼ MS + Flower Treasure No. 1 2 g·L + Flower Treasure No. 2 1 g·L + Glucose 20 g·L + Activated charcoal 1 g·L + Potato 50 g·L + Agar 6 g·L; Culture medium ②: ¼ MS + Flower Treasure No. 1 2 g·L + Flower Treasure No. 5 2 g·L + Glucose 20 g·L + Activated charcoal 1 g·L + Peptone 1 g·L + Agar 6 g·L.

[0033] Example 1: Screening of mycorrhizal fungi S1. Surface disinfection and mycelial release of root samples Fresh root segments from healthy Paphiopedilum orchids were cut and rinsed under running water to remove surface attachments. The root segments were then aliquoted into sterile culture dishes containing sterile water and transferred to a laminar flow hood for further processing. Root hairs, root coat, and other external appendages were removed using a sterile scalpel. Microscopic examination was then performed to select root segments containing endophytic hyphae within the cortical cells. After rinsing with sterile water, the roots underwent sequential surface disinfection: first, immersion in 75% ethanol solution for 2 minutes, followed by immersion in 0.1% mercuric chloride solution for 4-6 minutes. After disinfection, the root segments were rinsed at least 5 times with sterile water to thoroughly remove residual disinfectant. Root segments containing hyphae were cut into 3 cm long pieces, and their surfaces were scraped using a sterile scalpel and forceps to release the single hyphae encased within the cortical cells and diffuse them into 60 mm diameter sterile culture dishes containing 10 mL of sterile water. The culture dishes were then pre-cultured in a constant temperature incubator at 24±1℃ for 24 hours for later use.

[0034] S2. Primary isolation and purification of mycelial clusters Under a stereomicroscope, the bacterial suspension after pre-culturing in step S1 was observed in dark field to identify active hyphal clusters or hyphal knots that had sprouted new hyphae. The light source was then adjusted to bright field to locate the target hyphal cluster within the visible range. 45 μL of the bacterial suspension containing the target hyphal cluster was aspirated using a micropipette and transferred to a 1 cm² PDA agar plate. The inoculated agar plate was placed in a constant temperature incubator at 24±1℃ and cultured until hyphal growth was observed. The preliminarily purified strain was then transferred to PDA slant culture medium and cultured at 25±1℃. After microscopic examination confirmed the absence of microbial contamination, the pure culture was placed in a refrigerator at 4℃ for short-term preservation once the colonies had covered the entire slant.

[0035] S3. Primary isolation and purification of mycelial clusters Under a stereomicroscope, observe the small culture medium piece cultured in step S2 and locate the well-grown hyphal clusters within it. Use a sterile scalpel to cut the hyphal clusters from the original culture medium piece and transfer them to a new PDA culture medium piece, incubating at a constant temperature of 24±1℃. When the hyphae grow to a length of approximately 0.5 cm, use a sterile blade to cut off the tips of the most vigorous hyphae on the outermost edge of the hyphal community and transfer them to the center of a 90 mm diameter PDA plate for further purification. Once the colonies in the plate are uniformly grown and confirmed to be free of contamination, the purified target strain can be obtained. Transfer the purified strain to test tube slant culture medium and preserve the strain at 4℃ after incubation.

[0036] S4. Preparation of mycorrhizal fungi-PDA medium The pure culture of mycorrhizal fungi obtained by isolation and purification as in step S3 was inoculated into the center of a freshly prepared PDA agar plate with a diameter of 90 mm. At least three replicates were set up for each strain. The inoculated petri dishes were placed in a constant temperature incubator at 25±1℃ and incubated in the dark for 2 weeks, until the mycelium had basically covered the entire surface of the agar plate. This yielded the mycorrhizal fungi-PDA medium for subsequent symbiotic germination experiments.

[0037] S5. Artificial pollination and pod harvesting Select robust, disease-free Paphiopedilum plants as hybrid parents. During the flowering period, follow standard artificial pollination procedures, transferring pollen masses from the earlier-flowering plants to the stigmas of the later-flowering plants. For 180 to 360 days after pollination, provide continuous water and fertilizer management, closely observe pod development, and harvest when the pods turn slightly greenish-yellow but have not yet split open.

[0038] S6. Disinfection treatment of explants in fruit pods The collected pods were immersed in a 1% (w / w) neutral soap solution for 15 minutes, with gentle agitation. They were then rinsed under running water for 30 minutes to thoroughly remove surface residue. The pretreated pods were then transferred to a laminar flow hood for aseptic processing: first, they were immersed in a 75% ethanol solution for 5 minutes; after discarding the ethanol, they were then immersed in a 0.1% mercuric chloride solution for 20 minutes. After sterilization, the pods were removed using sterile dissecting instruments and rinsed at least three times with sterile distilled water to obtain sterile pods for later use.

[0039] S7. Symbiotic germination culture Inside a clean bench, using a sterile scalpel and forceps, the pods, sterilized in step S6, were carefully dissected along the longitudinal suture. The pods were gently shaken to evenly distribute the extremely fine seeds inside onto the surface of a pre-prepared mycorrhizal fungus-PDA medium. The sowing density was controlled, with approximately 100 seeds inoculated per petri dish. The inoculated petri dishes were placed in a light incubator with the following conditions: temperature 24±2℃, light intensity 1200 lux, and a photoperiod of 18 hours light / 6 hours dark. Symbiotic germination culture of the Paphiopedilum seeds was then performed under these conditions.

[0040] S8. Screening and Identification of Superior Mycorrhizal Fungi Through the above symbiotic germination comparison experiment, the germination status of Paphiopedilum seeds and the seedling development process in each treatment group were observed and statistically analyzed regularly. The seed germination rate and protocorm formation and conversion rate were used as the core evaluation indicators: when the seed germination rate of a certain mycorrhizal fungus treatment group was significantly higher than that of the control group, reaching 30% to 50% or higher, and it could successfully induce seeds to form protocorms, and further promote the development of protocorms into healthy seedlings with roots and leaves, then the strain was identified as an excellent mycorrhizal fungus for promoting Paphiopedilum seed germination and seedling growth.

[0041] Using this screening system, this invention successfully screened a specific and highly efficient bacterial strain, named MLXY010JD. This strain exhibits remarkable effects in promoting the symbiotic germination of Paphiopedilum seeds and the establishment of seedlings.

[0042] The selected strains were sent to a biotechnology company for ITS sequence testing, as detailed below.

[0043] ITS sequence (SEQ ID NO.1) .

[0044] After comparison, the ITS sequence of this strain was obtained ( Tulasnella The similarity is highest for sp.) The strain was inoculated and cultured on PDA medium. The colony morphology and hyphal microstructure of the strain are shown in the figures below. Figure 1 The results are shown in sections a and b. The colonies are milky white, waxy, and slightly thick, growing adherently to the surface of the culture medium. The surface is smooth with no aerial hyphae; the marginal hyphae are filamentous and thinner. The hyphal growth rate is 0.167-0.270 mm / h, and the hyphae are branched, septate, and 1.46-4.63 μm in diameter. The bead-like cells are elliptical, measuring 11.7-19.52 × 27.39-39.15 μm, and consist of 6-10 or more cells, clustering into dense chains to form a clustered structure. This strain was deposited on September 15, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20252049.

[0045] Example 2: Verification of the application effect of mycorrhizal fungus MLXY010JD in the mycorrhizal propagation of Paphiopedilum. Mycorrhizal culture, also known as mycorrhizal seedling cultivation, hinges on the inoculation and culture conditions of the mycorrhizal seedlings. 1. Activation and symbiotic germination culture medium for mycorrhizal fungi The superior mycorrhizal fungus MLXY010JD screened in this invention, and existing strains used as comparative examples, were compared. Epulorhiza sp. FQXY019 (deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M20211513) was inoculated into tissue culture flasks (e.g., 250 mL Erlenmeyer flasks) containing 30 mL of PDA medium, with inoculation blocks measuring 0.5 cm × 1.0 cm. The flasks were then incubated in the dark at 28 ± 1℃ for 2 weeks until the mycelium completely covered the surface of the medium. This yielded the MLXY010JD-PDA medium and FQXY019-PDA medium for subsequent symbiotic germination experiments.

[0046] 2. Aseptic symbiotic germination of Paphiopedilum seeds Mature pods of *Paphiopedilum blazei*, *Paphiopedilum microphyllum*, or *Paphiopedilum florida* were selected and surface-sterilized (specifically: 75% ethanol solution for 5 min, 0.1% mercuric chloride solution for 20 min, and rinsing with sterile water at least 3 times). The pods were then opened with a sterile scalpel in a laminar flow hood, and the seeds were evenly sown onto the surface of MLXY010JD-PDA and FQXY019-PDA media. Uninoculated PDA media served as a blank control (CK). Approximately 100 seeds were sown in each treatment group, with 10 replicates. After inoculation, the culture bottles were sealed with a special sealing film and placed in a tissue culture room under the following conditions: temperature 23 ± 2℃, relative humidity 75%-80%, light intensity 1000-1200 lux, and a photoperiod of 8 h light / 16 dark.

[0047] 3. Growth-promoting effects of mycorrhizal fungi on seedlings Paphiopedilum seedlings with 2-3 true leaves, formed through symbiotic germination with strain FQXY019 in step 2 above, were transferred to MLXY010JD-PDA and FQXY019-PDA media for symbiotic culture, with uninoculated PDA medium serving as a control (CK). Ten bottles were inoculated for each treatment. The culture conditions were: temperature 25 ± 2℃, relative humidity 75%-80%, light intensity 1200-1500 lux, photoperiod 12 h light / 12 h dark, and cultured for 90 days.

[0048] 4. Subculture of symbiotic seedlings to promote growth 4.1 First Stage of Subgeneration Healthy seedlings that had undergone 90 days of symbiotic culture with strain MLXY010JD in step 3 above were transferred to subculture medium ①. This medium consisted of: ¼ MS basal medium 2 g / L + Flower Power No. 1 2 g / L + Flower Power No. 2 1 g / L + glucose 20 g / L + activated charcoal 1 g / L + mashed potato 50 g / L + agar 6 g / L, with the addition of plant growth regulators KT 0.5 mg / L and NAA 0.2 mg / L. Each bottle contained 5-6 seedlings, and the culture conditions were the same as in step 3, for 90 days.

[0049] 4.2 Second Stage Subgeneration After 90 days of culture on medium ①, the seedlings were transferred to subculture medium ②. This medium consisted of: 2 g / L ¼ MS basal medium + 2 g / L Flower Power No. 1 + 2 g / L Flower Power No. 5 + 20 g / L glucose + 1 g / L activated charcoal + 1 g / L peptone + 6 g / L agar, with the addition of plant growth regulators KT 0.5 mg / L and NAA 0.5 mg / L. Each bottle contained 3-4 seedlings, and the culture conditions were the same as in step 3, for 90 days.

[0050] 5. Induction of stress resistance in seedlings by mycorrhizal fungi The superior mycorrhizal fungus FQXY019 was prepared into FQXY019-PDA medium according to the method in step 1. The seedlings obtained in step 4.2 were inoculated onto the surface of this medium, with uninoculated PDA medium as the control (CK). Seven seedlings were inoculated per bottle, with 10 replicates. The culture conditions were the same as in step 3, and the culture was carried out for 90 days to evaluate the effect of mycorrhizalization on enhancing the stress resistance of the seedlings.

[0051] 6. Hardening off seedlings and transplanting 6.1 Seedling hardening treatment After completing the above culture, the seedlings were transferred from the tissue culture room to a transition room with ventilation to the outside environment. They were left unopened for one day to acclimatize to the temperature change. Then, the caps were removed, and the seedlings were hardened off indoors for three days. The seedlings were then removed, and the culture medium adhering to the roots was gently washed off with running water. The roots were then disinfected by soaking them in a 1000-fold diluted solution of methyl thiophanate or carbendazim. After disinfection, the seedlings were placed in a well-ventilated, shady place to air dry until the surface moisture of the roots evaporated, the color turned white, and the texture softened slightly.

[0052] 6.2 Preparation and disinfection of cultivation substrate The cultivation substrate is composed of dried pine bark (0.5 cm × 0.5 cm), tree fern fiber (2-3 cm in length), volcanic rock (3-6 mm in diameter), pumice (3-6 mm in diameter), and peat moss in a mass ratio of 35:10:30:10:15. The substrate is first sterilized by autoclaving at 121℃ for 2-3 hours, then thoroughly moistened by spraying with a 0.3% potassium permanganate solution. The sterilized substrate is then dispensed into seedling trays (54 cm × 28 cm × 5 cm) that have been pre-sterilized by soaking in a 0.3% potassium permanganate solution for 60 minutes.

[0053] 6.3 Transplanting and Management Select robust mycorrhizal seedlings with at least 3 taproots, 3-5 leaves, and leaf length greater than 3 cm for transplanting. Use tweezers to transplant the dried seedlings into seedling trays, ensuring that the leaves above the rootstock junction are exposed above the substrate surface. Plant 50 seedlings per tray. Water thoroughly immediately after transplanting.

[0054] The following are the post-transplantation greenhouse cultivation management procedures: Cultivation environment: Greenhouse with good air circulation, with shading controlled at 60%-70%.

[0055] Temperature: Maintain an optimal growth temperature of 15-28℃. In summer, use shading and misting to cool down the temperature, and the maximum temperature should not exceed 32℃. In winter, pay attention to keeping warm and preventing frost.

[0056] Humidity: Maintain a relative humidity of 75%-85% by using a spray system to keep the substrate moist but avoid spraying directly onto the leaf axils.

[0057] Watering: Water thoroughly every 3-4 days in the early morning during summer; every 7 days in spring and autumn; and every 15 days in winter.

[0058] Fertilization: Apply slow-release orchid fertilizer (NPK 14-13-13) to the substrate surface every 4-5 months, avoiding direct contact between the fertilizer and the roots; spray the leaves with a 1000-fold diluted general-purpose foliar fertilizer every 2 weeks.

[0059] Pest and disease control: Follow the local standard DB52 / T 1569; pesticide use shall comply with the provisions of the agricultural industry standard NY / T1276.

[0060] 7. Application Effect Verification Results The application effect of strain MLXY010JD was verified using the above system: Figure 2 This paper presents the statistical results of germination rates of Paphiopedilum seeds inoculated with MLXY010JD, FQXY019, and the control (CK). Analysis of variance shows that different letters indicate the mean values ​​among the treatments. pSignificant differences were observed at the <0.05 level. The results indicate that MLXY010JD significantly improved the seed germination rate of Paphiopedilum.

[0061] Figure 3 The phenotypic changes in seedlings formed by seed-strain symbiosis 60 days after inoculation with strain FQXY019 are shown.

[0062] Figure 4 The study showed the effects of mycorrhizal fungi MLXY010JD and FQXY019 on growth indicators (a: fresh weight; b: leaf length) of Paphiopedilum symbiotic seedlings 90 days after inoculation, indicating that MLXY010JD has a significant growth-promoting advantage.

[0063] Figure 5 The results show the vigorous growth of seedlings formed by symbiotic germination of strain FQXY019 and then cultured with growth-promoting strain MLXY010JD.

[0064] Figure 6 and Figure 7 The growth of mycorrhizal seedlings after 90 days of subculture in medium ① and medium ② are shown respectively.

[0065] Figure 8 This study demonstrates the enhanced stress resistance phenotype of seedlings after inoculation with fungus FQXY019 compared to the control group.

[0066] Figure 9 and Figure 10 The growth phenotypes of mycorrhizal seedlings after 360 days of transplanting to a greenhouse are shown. Figure 9 ) and statistics of key growth indicators ( Figure 10 (a. survival rate; b. maximum leaf width; c. longest leaf length) proves that this method can effectively cultivate robust Paphiopedilum seedlings with a high survival rate.

[0067] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A mycorrhizal fungus that promotes the growth of Paphiopedilum seedlings ( Tulasnella sp.)MLXY010JD, characterized in that: The strain has been deposited in China Center for Type Culture Collection on September 15, 2025, and the deposit address is Wuhan University in Wuhan, China, and the deposit number is CCTCC NO: M 20252049.

2. The use of the mycorrhizal fungus MLXY010JD of claim 1 in promoting seed germination and plant growth of Phalaenopsis.

3. Use according to claim 2, characterized in that, The Phalaenopsis comprises Phalaenopsis amabilis, Phalaenopsis equestris or / and Phalaenopsis pallens.

4. A method for promoting seed germination and rapid seedling development of Paphiopedilum, characterized in that, The method comprises the following steps: Step 1: symbiotic culture of Phalaenopsis seeds and mycorrhizal fungus FQXY019 on PDA medium to germinate and form seedlings, wherein the deposit number of the FQXY019 is CCTCC NO: M 20211513; Step 2: symbiotic culture of the seedlings obtained in step 1 and the mycorrhizal fungus MLXY010JD of claim 1 on PDA medium to obtain symbiotic seedlings; Step 3: subculture of the symbiotic seedlings obtained in step 2 in a growth-promoting medium.

5. The method of claim 4, wherein, The subculture comprises two stages, wherein the first stage adopts No. 1 medium, and the second stage adopts No. 2 medium; The No. 1 medium comprises ¼ MS medium, Hua Bao No. 1, Hua Bao No. 2, glucose, activated carbon, potato and agar; The No. 2 medium comprises ¼ MS medium, Hua Bao No. 1, Hua Bao No. 5, glucose, activated carbon, peptone and agar.

6. The method of claim 4, wherein: After step 3, the symbiotic seedlings are inoculated with the mycorrhizal fungus FQXY019 to enhance stress resistance.

7. The method of claim 4, wherein: The method further comprises a seedling hardening and transplanting step, wherein the cultivation substrate used is a mixture of dry pine bark, snake wood, volcanic rock, plant gold stone and peat soil in a mass ratio of 35:10:30:10:

15.

8. The method according to any one of claims 4-7, characterized in that, The Phalaenopsis comprises Phalaenopsis amabilis, Phalaenopsis equestris or / and Phalaenopsis pallens.

9. The use of the mycorrhizal fungus MLXY010JD in combination with the fungus FQXY019 of claim 1 in promoting seed germination and seedling growth of Phalaenopsis plants, wherein, The deposit number of the FQXY019 is CCTCC NO: M 20211513.

10. The use of the combination of the mycorrhizal fungus MLXY010JD of claim 1 and the fungus FQXY019 in Phalaenopsis mycorrhizal seedling raising.

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