CNOCC V.coerule-41 strain and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a CNOCC V.coerule-41 strain and application thereof. According to the invention, a CNOCC V.coerulea-41 strain is obtained through separation and screening, and the preservation number of the CNOCC V.coerulea-41 strain is GDMCC No: 67766. The strain provided by the invention has a stable growth promoting effect on various orchid plants, the strain is inoculated into pre-cultured seedlings or explants of vanda macrantha, Chinese renanthera, anoectochilus formosanus or paphiopedilum bulbifer for symbiotic culture, the survival rate of tissue culture seedlings can be remarkably increased under the condition of completely no hormone, and the survival rate of the tissue culture seedlings can be increased. The problem that tissue culture seedlings are prone to death in hormone-free culture is effectively solved, the tissue culture seedlings inoculated with the strain can maintain normal growth, biomass accumulation is stable, and the method has the advantages of being easy and convenient to operate, low in cost and high in seedling survival rate.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a CNOCC V. coerulea-41 strain and its applications. Background Technology

[0002] Orchidaceae is a family within the order Asparagales, native to tropical and subtropical regions worldwide, and possesses extremely high medicinal and ornamental value. The mutualistic symbiotic relationship between orchid plants and mycorrhizal fungi is an indispensable biological basis for their survival and reproduction under natural conditions.

[0003] Currently, existing technologies typically involve screening for specific symbiotic fungi for particular orchid species and establishing symbiotic culture systems. For example, existing technology CN116925930A discloses a strain of *Farropis foliata* isolated from a living perennial herb of the genus *Dendrobium nobile* (Orchidaceae). Phyllosticta fallopiae Through co-cultivation with *Dendrobium nobile*, the fresh weight and alkaloid content of *Dendrobium nobile* were significantly increased. However, existing technologies suffer from bottlenecks such as time-consuming and costly screening processes, a narrow host range of obtained fungi hindering large-scale application, and the need to establish different symbiotic cultivation procedures for different orchid species, making it difficult to meet the needs of multi-species, high-efficiency conservation and production. Therefore, there is an urgent need in this field for a broad-spectrum symbiotic fungus that can overcome species limitations and provide stable growth-promoting effects on a variety of orchid species. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of CNOCC V. coerulea-41 and its application, which enables efficient symbiotic growth promotion of multi-species orchid tissue culture seedlings, significantly improves the survival rate of orchid tissue culture under hormone-free conditions, and effectively maintains the normal accumulation of biomass. It is suitable for large-scale propagation and germplasm resource protection of orchids.

[0005] This invention provides a strain of CNOCC V. coerulea-41, wherein the CNOCC V. coerulea-41 strain is... Nemania sp., accession number GDMCC No: 67766.

[0006] This invention provides a microbial agent comprising the CNOCC V. coerulea-41 strain described in the above technical solution.

[0007] Preferably, the inoculant is a solid inoculant, including a block of CNOCC V. coerulea-41 strain.

[0008] Preferably, the size of any one of the bacterial blocks is 0.3~0.7cm × 0.3~0.7cm.

[0009] This invention provides a method for preparing the microbial agent described in the above technical solution, comprising the following steps: The activated CNOCCV.coerulea-41 strain was inoculated into a solid culture medium and cultured. The mycelial blocks with newly formed hyphae on the periphery of the CNOCCV.coerulea-41 strain colony were selected to obtain the inoculum.

[0010] Preferably, the solid culture medium is PDA solid culture medium; the culture temperature is 26~30℃ and the time is 5~10 days.

[0011] This invention provides the application of the CNOCC V. coerulea-41 strain described in the above technical solution, or the fungal agent described in the above technical solution, or the fungal agent obtained by the preparation method described in the above technical solution, in the cultivation of orchid plants.

[0012] Preferably, the CNOCC V. coerulea-41 strain or inoculant is used to improve the survival rate of orchid culture and / or maintain normal biomass accumulation; The orchid species include one or more of the following: Vanda grandiflora, Flame Orchid of China, Anoectochilus roxburghii, and Paphiopedilum breviscapus. The culture includes hormone-free tissue culture.

[0013] This invention provides a method for hormone-free tissue culture of orchid plants, comprising the following steps: Under aseptic conditions, orchid tissue culture materials are co-cultured with the CNOCC V. coerulea-41 strain described in the above technical solution, or the inoculum agent described in the above technical solution, or the inoculum agent obtained by the preparation method described in the above technical solution; After the co-cultivation is completed, the mycelium remaining on the roots of the orchid seedlings is removed, and the seedlings are transplanted and planted for growth management. The orchid tissue culture materials include orchid seedlings or orchid explants. The co-culture temperature was 23~27℃, the light intensity was 1800~2200 Lux, and the photoperiod was 12~16h / d.

[0014] Preferably, the co-culture method includes: After pre-culturing the orchid tissue culture material in a symbiotic medium for a period of time, it was inoculated with the CNOCC V. coerulea-41 strain or inoculum and cultured for a further period of time. The symbiotic culture medium comprises 3-5 g / L oat flour, 2-3 g / L 1 / 2 MS medium, 6-7 g / L agar powder, 4-6 g / L sucrose, and the remainder water; The pre-culture time is 25-35 days, and the continued culture time is 160-200 days.

[0015] Beneficial effects: This invention isolated and screened CNOCC V. coerulea-41 strain, which was sequenced by ITS as follows: Nemania sp., with accession number GDMCC No: 67766. The CNOCC V. coerulea-41 strain provided by this invention has a stable growth-promoting effect on various orchid species, featuring simple operation, low cost, and high seedling survival rate. It is suitable for large-scale propagation and germplasm resource protection of various orchid species, and provides a new approach for orchid symbiosis research and field reintroduction. The results of the examples show that inoculating CNOCC V. coerulea-41 strain into pre-cultured seedlings or explants of *Vanda grandiflora*, *Cymbidium sinense*, *Anoectochilus roxburghii*, or *Paphiopedilum buergerianum* for symbiotic culture significantly improves the survival rate of tissue culture seedlings under completely hormone-free conditions, effectively solving the problem of easy death of tissue culture seedlings in hormone-free culture. Furthermore, tissue culture seedlings inoculated with this strain can maintain normal growth and stable biomass accumulation.

[0016] Biological Preservation Information CNOCC V. coerulea-41 strain, classified and named Nemania sp. was deposited on January 29, 2026 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No:67766. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 The colony morphology of CNOCC V. coerulea-41 strain after purification; Figure 2 The growth status of different orchid species after 6 months of symbiotic culture with CNOCC V. coerulea-41 strain; among them, A is Vanda grandiflora; B is Orchidia sinensis; C is Anoectochilus roxburghii; and D is Paphiopedilum breviscapus. Detailed Implementation

[0019] This invention provides a strain of CNOCC V. coerulea-41, wherein the CNOCC V. coerulea-41 strain is... Nemania sp., accession number GDMCC No: 67766.

[0020] This invention isolated a fungus from the root tissue of a healthy, disease-free Vanda grandiflora orchid under ex-situ conservation, and sequenced it using ITS sequencing. Nemania The strain, named CNOCC V. coerulea-41, with accession number GDMCC No: 67766, was used to inoculate pre-cultured orchid seedlings or explants for symbiotic culture. This significantly improved the survival rate of tissue-cultured seedlings of various orchid species, including Vanda grandiflora, Flame Orchid of China, Anoectochilus roxburghii, and Paphiopedilum, under hormone-free conditions. It also effectively maintained normal biomass accumulation. The method is characterized by its simplicity, low cost, and high seedling survival rate, making it suitable for large-scale propagation and germplasm resource conservation of orchids. Furthermore, it provides a new approach for orchid symbiotic research and reintroduction into the wild.

[0021] This invention provides a microbial agent comprising the CNOCC V. coerulea-41 strain described in the above technical solution.

[0022] In one embodiment, the bacterial agent of the present invention is a solid bacterial agent, comprising a block of CNOCC V. coerulea-41 strain. In one embodiment, the size of any one of the bacterial blocks is 0.3~0.7cm × 0.3~0.7cm; in another embodiment, the size of any one of the bacterial blocks is 0.5cm × 0.5cm. The present invention limits the size of the bacterial blocks to ensure that the blocks contain sufficient active mycelium, maintain high initial viability and infection potential, while avoiding premature depletion of culture medium nutrients or excessive accumulation of local metabolic waste due to excessively large blocks, thus achieving a balance between viability and infection efficiency.

[0023] The present invention provides a method for preparing the bacterial agent described in the above technical solution, comprising the following steps: inoculating the activated CNOCC V. coerulea-41 strain into a solid culture medium for culture, selecting the mycelial block with newly formed hyphae on the periphery of the CNOCC V. coerulea-41 strain colony, and obtaining the bacterial agent.

[0024] In one embodiment, the present invention activates the CNOCC V. coerulea-41 strain to obtain an activated CNOCC V. coerulea-41 strain. In one embodiment, the activation culture temperature is 26-30°C; in another embodiment, the activation culture temperature is 28°C. In one embodiment, the activation culture time is 2-3 days. In one embodiment, PDA medium is used for the activation culture. The present invention activates the CNOCC V. coerulea-41 strain, restoring its growth viability.

[0025] After obtaining the activated CNOCCV. coerulea-41 strain, the present invention inoculates the activated CNOCCV. coerulea-41 strain into a solid culture medium for cultivation. In one embodiment, the solid culture medium is PDA solid culture medium. In one embodiment, the cultivation temperature is 26-30℃; in another embodiment, the cultivation temperature is 28℃. In one embodiment, the cultivation time is 5-10 days; in another embodiment, the cultivation time is 7 days.

[0026] This invention provides the application of the CNOCC V. coerulea-41 strain described in the above technical solution, or the fungal agent described in the above technical solution, or the fungal agent obtained by the preparation method described in the above technical solution, in the cultivation of orchid plants.

[0027] In one embodiment, the CNOCC V. coerulea-41 strain or inoculant described in this invention is used to improve the survival rate of orchid cultures and / or maintain normal biomass accumulation. In one embodiment, the orchids described in this invention include one or more of Vanda grandiflora, Orchidia sinensis, Anoectochilus roxburghii, and Paphiopedilum breviscapus. In one embodiment, the culture described in this invention includes hormone-free tissue culture. Using the CNOCC V. coerulea-41 strain or inoculant described in this invention for hormone-free tissue culture of orchids can significantly improve the survival rate of tissue-cultured seedlings under hormone-free conditions and effectively maintain normal biomass accumulation.

[0028] This invention provides a method for hormone-free tissue culture of orchid plants, comprising the following steps: Under aseptic conditions, orchid tissue culture materials are co-cultured with the CNOCC V. coerulea-41 strain described in the above technical solution, or the inoculum agent described in the above technical solution, or the inoculum agent prepared by the above technical solution. After the co-culture is completed, the mycelium remaining on the roots of the orchid seedlings is removed, and the seedlings are transplanted and managed for growth. The orchid tissue culture materials include orchid seedlings or orchid explants. The co-culture temperature is 23~27℃, the light intensity is 1800~2200 Lux, and the photoperiod is 12~16h / d.

[0029] In one embodiment, the orchid explant of the present invention includes an orchid stem segment.

[0030] In one embodiment, the co-culturing temperature of the present invention is 25°C. In one embodiment, the light intensity of the co-culturing is 2000 Lux. In one embodiment, the photoperiod of the co-culturing is 14 h / d. In one embodiment, the co-culturing time is 210 days.

[0031] In one embodiment, the co-culture method of the present invention includes: inoculating the orchid tissue culture material into a symbiotic culture medium for a pre-culture period of time, and then inoculating it with the CNOCC V. coerulea-41 strain or inoculum agent for further culture. In one embodiment, the pre-culture time is 25-35 days, and the further culture time is 160-200 days; in another embodiment, the pre-culture time is 30 days, and the further culture time is 180 days. In one embodiment, the symbiotic culture medium of the present invention comprises 3-5 g / L oat flour, 2-3 g / L 1 / 2 MS medium, 6-7 g / L agar powder, 4-6 g / L sucrose, and the remainder water; in another embodiment, the symbiotic culture medium comprises 4 g / L oat flour, 2.47 g / L 1 / 2 MS medium, 6.5 g / L agar powder, 5 g / L sucrose, and the remainder water. This invention utilizes a symbiotic culture medium to pre-culture orchid tissue culture materials for a period of time before inoculation. This has the advantage of eliminating exogenous pollution or stress reactions introduced by the change of culture medium, and helps tissue culture seedlings gradually adapt to the symbiotic culture environment from the conventional culture medium.

[0032] In one embodiment, the inoculation amount of the CNOCC V. coerulea-41 strain or the inoculum agent described in the above-mentioned technical solution is based on the number of mycelial blocks, with one mycelial block inoculated for each orchid tissue culture material. In another embodiment, the CNOCC V. coerulea-41 strain or the inoculum agent described in the above-mentioned technical solution is inoculated into the center of the orchid tissue culture material.

[0033] In one embodiment, after removing residual mycelium from the roots of orchid seedlings, the present invention transplants them into a cultivation substrate for establishment. In another embodiment, the cultivation substrate comprises bark and humus. In one embodiment, the volume ratio of bark to humus is 1:(0.5~1.5); in yet another embodiment, the volume ratio is 1:1. The present invention does not impose strict requirements on the growth management methods; conventional methods in the art can be used.

[0034] This invention involves inoculating orchid tissue cultures with the CNOCC V. coerulea-41 strain for symbiotic culture. This effectively promotes the growth of tissue-cultured seedlings in a completely hormone-free medium, enhancing their environmental adaptability and physiological tolerance. By mimicking the symbiotic mechanism between orchids and microorganisms in their natural habitat, the CNOCC V. coerulea-41 strain assists tissue-cultured seedlings in establishing a physiological homeostasis and healthy root structure closer to that of wild growth, thus laying a solid foundation for successful transplantation and adaptation to the natural environment. The CNOCC V. coerulea-41 strain described in this invention breaks through the high species specificity of traditional orchid-fungus symbiotic relationships. It shows significant growth-promoting effects on tissue-cultured seedlings of various important ornamental and medicinal orchids, including Paphiopedilum, Vanda, Anoectochilus, and Flame Orchid, achieving "one strain for multiple uses." This solves the technical problem of individually screening and preserving strains for different orchid species. It not only helps to cultivate high-quality orchid seedlings with strong resistance and adaptability, but also provides a new and effective approach for the protection and rejuvenation of rare orchid species, the restoration of degraded ecosystems, and the sustainable development of the horticulture industry. It has significant scientific research value and application prospects.

[0035] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a CNOCCV.coerulea-41 strain and its applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1 1. Isolation, purification, identification and preservation of bacterial strains (1) Root sample collection: Select healthy, disease-free Vanda orchids that have been ex-situ protected, cut healthy root segments and place them in sterile sampling bags. After bringing them back to the laboratory, remove the bark attached to the root surface and cut the root segments into small sections of 5-8 cm for later use.

[0037] (2) Isolation and purification of the strain: After cleaning the surface of the root segments with detergent, rinse them under running water for 1-2 hours. Then transfer them to a clean bench and disinfect them sequentially with 75% alcohol for 1 minute, 20% sodium hypochlorite solution for 20 minutes, and then rinse them with sterile water 5-6 times. After that, use a sterile blade to cut the root segments into small segments of about 0.5 cm and inoculate them onto PDA medium plates, with 1-3 segments per plate. Place the inoculated PDA plates in a 28℃ constant temperature incubator for dark incubation and observe the colony growth. After about 2 days of incubation, mycelia begin to appear. Cut the pure mycelial blocks from the edge and inoculate them onto fresh PDA medium and continue to incubate at 28℃. Repeat the above operation more than 3 times until a single strain is obtained and named CNOCC V. coerulea-41. CNOCC V. coerulea-41 strain exhibits concentric diffusion growth on PDA medium, forming a distinct concentric ring structure. The color ranges from milky white to pale yellow, with the central area being slightly darker and denser, gradually becoming thinner and more velvety towards the outside. Figure 1 ).

[0038] (3) Preservation of bacterial strain: Dispense the prepared PDA medium into 18×180mm glass test tubes, 6ml per tube. After high-temperature moist heat sterilization, arrange them on the lab bench as slant and let them solidify before use. In a clean bench, inoculate the purified strain onto the PDA slant and incubate at 28℃ until the mycelium covers the slant. Then transfer it to a 4℃ refrigerator for low-temperature storage.

[0039] 2. Molecular identification of strains The CNOCC V. coerulea-41 strain sample obtained in step 1 was sent to Guangzhou Aiji Biotechnology Co., Ltd. for genomic DNA extraction, PCR amplification of the ITS sequence, and sequencing. The sequence is as follows: (SEQ ID NO:1).

[0040] The ITS sequence obtained from sequencing was compared with known sequences in the GenBank database using BLAST. Molecular identification of the bacterial species was performed based on a similarity of 99% or higher. The identification results showed that this strain is similar to fungi. Nemania The sequence similarity of sp. (MN844430.1) is 100%. CNOCC V. coerulea-41 strain is... Nemania sp., for biological preservation.

[0041] Example 2 1. Preparation of symbiotic culture medium: Weigh 4g of oat flour, 2.47g of 1 / 2 MS medium, 6.5g of agar powder, and 5g of sucrose. Add an appropriate amount of deionized water, heat and stir continuously until completely dissolved. Then, bring the volume to 1L with deionized water and mix well. Dispense the prepared culture medium into tissue culture flasks, autoclave at 120℃ for 20 minutes, and allow to cool and solidify before use. This completes the preparation of the symbiotic culture medium.

[0042] 2. Pre-culture of tissue culture seedlings: Aseptic seed propagation of Vanda grandiflora seedlings were inoculated into the above-mentioned symbiotic culture medium and placed in a culture temperature of 25±2℃, light intensity of 2000 Lux, and photoperiod of 14h / d for one month for pre-culture.

[0043] 3. Fungal activation culture: Remove the slant test tubes of CNOCC V. coerulea-41 strain stored at -4℃ and transfer them to a 28℃ constant temperature incubator for 2-3 days to allow the mycelium to regain its growth vitality.

[0044] 4. Transplantation of strain: Select mycelia of strain CNOCC V. coerulea-41 after activation culture in slant tubes at -4℃, inoculate onto prepared PDA solid medium, and incubate in a constant temperature incubator at 28℃ for 7 days.

[0045] 5. Symbiotic culture: Take a 0.5×0.5cm PDA agar block with newly formed mycelium from the outermost part of the activated colony and transfer it to the middle of the pre-cultured Vanda grandiflora tissue culture seedling for symbiotic culture. The culture temperature is 25±2℃, the light intensity is 2000Lux, and the photoperiod is 14h / d.

[0046] 6. Transplanting and planting: After 180 days of symbiotic culture in step 5, the roots of the obtained Vanda grandiflora tissue culture seedlings were washed and transplanted into a cultivation substrate made of bark and humus in a 1:1 volume ratio for further cultivation and growth management in a greenhouse.

[0047] Comparative Example 1 The large-flowered Vanda orchids were cultured in accordance with the method of Example 2, the only difference being that 0.5×0.5cm PDA agar blocks were selected for transfer and co-culture.

[0048] Example 3 Following the method in Example 2, the aseptically propagated Vanda grandiflora seedlings were replaced with aseptically propagated Chinese Flame Orchid tissue culture seedlings to carry out symbiotic culture of Chinese Flame Orchid and CNOCC V. coerulea-41 strain.

[0049] Comparative Example 2 The *Cymbidium sinense* was cultured in accordance with the method described in Example 3, except that a 0.5 × 0.5 cm PDA agar block was selected for transfer and co-culture.

[0050] Example 4 Following the method in Example 2, aseptically propagated Vanda grandiflora seedlings were replaced with aseptically propagated Anoectochilus roxburghii stem segments for symbiotic culture of Anoectochilus roxburghii and CNOCC V. coerulea-41 strain.

[0051] Comparative Example 3 The *Anoectochilus roxburghii* was cultured in accordance with the method described in Example 4, except that a 0.5 × 0.5 cm PDA agar block was selected for transfer and co-culture.

[0052] Example 5 Following the method in Example 2, the tissue culture seedlings of Vanda grandiflora propagated by aseptic sowing were replaced with tissue culture seedlings of Paphiopedilum breviscapus propagated by aseptic sowing, and symbiotic culture of Paphiopedilum breviscapus and CNOCC V. coerulea-41 strain was carried out.

[0053] Comparative Example 4 The Paphiopedilum orchid was cultured in accordance with the method of Example 5, except that a 0.5×0.5cm PDA agar block was selected for transfer and co-culture.

[0054] Test Example 1 180 days after inoculation with CNOCC V. coerulea-41, the growth status of different orchid species in Examples 2-5 and Comparative Examples 1-4 was observed; survival rate and biomass were statistically analyzed, and the results are shown in Tables 1-2 and 2. Figure 2 As shown.

[0055] Table 1. Survival rates of different orchid species

[0056] Note: This represents a highly significant difference; This indicates a significant difference, and the same applies below.

[0057] Table 2 Biomass of different orchid species

[0058] according to Figure 2 It can be seen that after symbiotic culture with CNOCC V. coerulea-41, the leaves of *Vanda grandiflora*, *Cymbidium sinense*, and *Paphiopedilum* were healthy green, the plants grew vigorously, and the plants were upright. While the older leaves of *Anoectochilus roxburghii* turned yellow and withered, the new leaves were dark green, expansive, and had clear veins. These phenomena indicate that CNOCC V. coerulea-41 can establish a symbiotic relationship with various orchid species, providing nutritional support to the tissue-cultured seedlings without significantly inhibiting their healthy growth.

[0059] As shown in Table 1, the control group without CNOCC V. coerulea-41 strain had a low survival rate in hormone-free medium, while the control group inoculated with CNOCC V. coerulea-41 strain had a significantly higher survival rate, showing a highly significant difference from the control group. P <0.01). Further analysis in Table 2 shows that, compared to the control group, tissue-cultured seedlings inoculated with CNOCC V. coerulea-41 improved the normal biomass accumulation of several orchid species, including *Vanda grandiflora*, *Cymbidium sinense*, *Anoectochilus roxburghii*, and *Paphiopedilum buxiflorus*. Specifically, the biomass accumulation of *Cymbidium sinense* was significantly higher than that of the control group, while the biomass accumulation of *Anoectochilus roxburghii* was extremely significantly higher than that of the control group. In conclusion, these results indicate that CNOCC V. coerulea-41 can effectively improve the survival rate of various orchid species in hormone-free culture media and effectively maintain normal biomass accumulation.

[0060] 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 CNOCC V. coerulea-41 strain, characterized in that, The CNOCC V. coerulea-41 strain is Nemania sp., accession number GDMCC No: 67766.

2. A microbial agent, characterized in that, The microbial agent includes the CNOCC V. coerulea-41 strain as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent is a solid bacterial agent, including a block of CNOCCV.coerulea-41 strain.

4. The microbial agent according to claim 3, characterized in that, The size of any one of the said mycelial blocks is 0.3~0.7cm × 0.3~0.7cm.

5. The method for preparing the microbial agent according to any one of claims 2 to 4, characterized in that, Includes the following steps: The activated CNOCCV.coerulea-41 strain was inoculated into a solid culture medium and cultured. The mycelial blocks with newly formed hyphae on the periphery of the CNOCCV.coerulea-41 strain colony were selected to obtain the inoculum.

6. The preparation method according to claim 5, characterized in that, The solid culture medium is PDA solid culture medium; the culture temperature is 26~30℃ and the time is 5~10 days.

7. The application of the CNOCC V. coerulea-41 strain according to claim 1, or the fungal agent according to any one of claims 2 to 4, or the fungal agent obtained by the preparation method according to claim 5 or 6, in the cultivation of orchid plants.

8. The application according to claim 7, characterized in that, The CNOCC V. coerulea-41 strain or inoculant is used to improve the survival rate of orchid culture and / or maintain normal biomass accumulation. The orchid species include one or more of the following: Vanda grandiflora, Flame Orchid of China, Anoectochilus roxburghii, and Paphiopedilum breviscapus. The culture includes hormone-free tissue culture.

9. A method for hormone-free tissue culture of orchid plants, characterized in that, Includes the following steps: Under aseptic conditions, orchid tissue culture materials are co-cultured with the CNOCC V. coerulea-41 strain as described in claim 1, or the fungal agent as described in any one of claims 2 to 4, or the fungal agent obtained by the preparation method described in claim 5 or 6; After the co-cultivation is completed, the mycelium remaining on the roots of the orchid seedlings is removed, and the seedlings are transplanted and planted for growth management. The orchid tissue culture materials include orchid seedlings or orchid explants. The co-culture temperature was 23~27℃, the light intensity was 1800~2200 Lux, and the photoperiod was 12~16h / d.

10. The method according to claim 9, characterized in that, The co-culture method includes: After pre-culturing the orchid tissue culture material in a symbiotic culture medium for a period of time, it was inoculated with the CNOCCV.coerulea-41 strain or inoculum and cultured for a further period of time. The symbiotic culture medium comprises 3-5 g / L oat flour, 2-3 g / L 1 / 2 MS medium, 6-7 g / L agar powder, 4-6 g / L sucrose, and the remainder water; The pre-culture time is 25-35 days, and the continued culture time is 160-200 days.

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