A CNOCC V. coerulea-13 strain and its application

Abstract

This invention belongs to the field of microbial technology, specifically relating to a strain CNOCC V. coerulea-13 and its applications. The present invention isolates and screens CNOCC V. coerulea-13, with the accession number GDMCC No:67765. When this strain is inoculated into pre-cultured orchid tissue culture seedlings or explants for symbiotic culture, under completely hormone-free conditions, the tissue culture seedlings maintain normal growth and stable biomass accumulation, significantly improving the survival rate of tissue culture seedlings of various orchid species. This effectively solves the problem of easy death of tissue culture seedlings in hormone-free culture and effectively avoids the side effects such as deformity and excessive growth that may be caused by the use of traditional hormones. This invention is simple to operate and low in cost. The resulting seedlings have a physiological balance and root development closer to the natural state, laying a good foundation for their subsequent transplanting and adaptation to the natural environment.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a CNOCC V. coerulea-13 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. Orchids are among the most difficult monocotyledonous plants to propagate. Traditional propagation of orchids is usually achieved through division; however, traditional asexual propagation methods such as division and cuttings have low survival rates, slow growth rates, and low reproduction rates, resulting in low levels of industrialization.

[0003] Tissue culture technology, with its high propagation coefficient and strong genetic stability, is a commonly used and effective propagation method for orchids in conservation and production. Tissue culture typically requires the addition of plant growth regulators under artificially controlled conditions to regulate plant growth and development, achieving high-efficiency production. However, the use of hormones also brings a series of problems: precise concentration is required, and improper preparation can easily lead to seedling deformities, vitrification, or abnormal development; hormone residues may affect the physiological state of seedlings, resulting in decreased adaptability after transplanting. Orchid tissue culture in hormone-free media often faces seedling growth stagnation and a significantly reduced survival rate. Therefore, how to improve the survival rate of orchid tissue culture and promote the healthy growth of orchid tissue-cultured seedlings without relying on exogenous hormones is particularly important in this field. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of CNOCC V. coerulea-13 and its application, which significantly improves the survival rate of orchid tissue culture under hormone-free conditions and effectively maintains the normal accumulation of biomass, making it suitable for large-scale propagation and germplasm resource protection of orchids.

[0005] This invention provides a strain of CNOCC V. coerulea-13, which is a hardened carbonaceous bacterium (CNOCC V. coerulea-13). Hypoxylon investiens The accession number is GDMCC No:67765, the accession date is January 29, 2026, and the depository institution is Guangdong Provincial Center for Microbial Culture Collection.

[0006] The present invention provides a microbial agent comprising the CNOCC V. coerulea-13 strain described in the above technical solution.

[0007] Preferably, the inoculant is a solid inoculant, including a block of CNOCC V. coerulea-13 strain.

[0008] Preferably, the size of the fungal block 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-13 strain was inoculated into a solid culture medium and cultured. The mycelial blocks with newly formed hyphae on the periphery of the CNOCCV.coerulea-13 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-13 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-13 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, Crane Crown Orchid, Dragon Boat Orchid, and Paphiopedilum. 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-13 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-13 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-13 strain, which was sequenced by ITS as follows: Hypoxylon investiens The strain CNOCC V. coerulea-13 was used to inoculate pre-cultured orchid tissue culture seedlings or explants for symbiotic culture. Under completely hormone-free conditions, this significantly improved the survival rate of various orchid tissue culture seedlings, effectively solving the problem of easy death of tissue culture seedlings in hormone-free culture. Furthermore, the seedlings inoculated with this strain maintained normal growth and stable biomass accumulation. This invention achieves robust growth of orchid tissue culture seedlings under "hormone-free" conditions, resulting in healthier plant morphology and effectively avoiding the side effects such as deformities and excessive growth that may be caused by traditional hormone use. Through the physiological regulation of fungal symbiosis replacing hormones, the resulting seedlings exhibit a physiological balance and root development closer to the natural state, laying a good foundation for subsequent transplanting and adaptation to the natural environment. It features simple operation, low cost, and high seedling survival rate, making it suitable for large-scale propagation and germplasm resource protection of orchids, and providing a new approach for orchid symbiotic research and reintroduction into the wild.

[0016] Biological Preservation Information CNOCC V. coerulea-13 strain, classified as *Cyclopyrum styracifolium*. Hypoxylon investiens It 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:67765. 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-13 strain after purification; Figure 2The growth status of different orchid species symbiotically cultured with CNOCC V. coerulea-13 strain for 6 months was observed; among them, A is Vanda grandiflora; B is Orchidia sinensis; C is Anoectochilus roxburghii; D is Orchidia cerana; E is Orchidia cerana; and F is Paphiopedilum breviscapus. Detailed Implementation

[0019] This invention provides a strain of CNOCC V. coerulea-13, which is a hardened carbonaceous bacterium (CNOCC V. coerulea-13). Hypoxylon investiens (The accession number is GDMCC No:67765).

[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. Hypoxylon investiens The strain, named CNOCC V. coerulea-13, with accession number GDMCCNo:67765, was used for symbiotic culture of pre-cultured orchid tissue culture seedlings or explants. This significantly improved the survival rate of tissue culture seedlings under hormone-free conditions and effectively maintained normal biomass accumulation. It is characterized by its simple operation, low cost, and high seedling survival rate, making it suitable for large-scale propagation and germplasm resource conservation of orchids. It also provides a new approach for orchid symbiotic research and reintroduction into the wild.

[0021] The present invention provides a microbial agent comprising the CNOCC V. coerulea-13 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-13 strain. In one embodiment, the size of the bacterial block is 0.3~0.7cm × 0.3~0.7cm; in another embodiment, the size of the bacterial block is 0.5cm × 0.5cm. The present invention limits the size of the bacterial block, ensuring that the block contains sufficient active mycelium, maintains 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-13 strain into a solid culture medium for culture, selecting the mycelial block with newly formed hyphae on the periphery of the CNOCC V. coerulea-13 strain colony, and obtaining the bacterial agent.

[0024] In one embodiment, the present invention activates the CNOCC V. coerulea-13 strain to obtain an activated CNOCC V. coerulea-13 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-13 strain, restoring its growth viability.

[0025] After obtaining the activated CNOCCV. coerulea-13 strain, the present invention inoculates the activated CNOCCV. coerulea-13 strain into a solid culture medium for cultivation. As one embodiment, the solid culture medium is PDA solid culture medium. As one embodiment, the cultivation temperature is 26-30℃; as another embodiment, the cultivation temperature is 28℃. As one embodiment, the cultivation time is 5-10 days; as another embodiment, the cultivation time is 7 days.

[0026] This invention provides the application of the CNOCC V. coerulea-13 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-13 strain or inoculant described in this invention is used to improve the survival rate of orchid culture 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, Orchidia cerana, Cymbidium goeringii, and Paphiopedilum breviscapus. In one embodiment, the culture described in this invention includes hormone-free tissue culture. Using the CNOCC V. coerulea-13 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-13 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. 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.

[0030] 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-13 strain or inoculum agent for continued culture. In one embodiment, the pre-culture time is 25-35 days, and the continued culture time is 160-200 days; in another embodiment, the pre-culture time is 30 days, and the continued 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.

[0031] In one embodiment, the inoculation amount of the CNOCC V. coerulea-13 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-13 strain or the inoculum agent described in the above-mentioned technical solution is inoculated into the center of the orchid tissue culture material.

[0032] 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.

[0033] This invention involves inoculating orchid tissue cultures with the CNOCC V. coerulea-13 strain, which effectively maintains and promotes the growth of tissue-cultured seedlings in a completely hormone-free medium, thus avoiding the risks and side effects associated with hormone use. Through a fungal-plant symbiotic mechanism, the CNOCC V. coerulea-13 strain can mimic the microbial regulatory role in natural growth, helping tissue-cultured seedlings establish a physiological balance and root development closer to that of wild plants, laying a good foundation for their transplantation into the natural environment. This method reduces dependence on chemical hormones and helps cultivate high-quality seedlings with strong resistance and adaptability, showing significant application prospects in the fields of rare orchid species conservation, ecological restoration, and sustainable horticultural production.

[0034] To further illustrate the present invention, the following detailed description of a CNOCCV.coerulea-13 strain and its applications, in conjunction with the accompanying drawings and embodiments, is provided but should not be construed as limiting the scope of protection of the present invention.

[0035] 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.

[0036] (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-13. The CNOCC V. coerulea-13 strain initially appears as a white, fluffy or cottony colony on PDA medium, with dense and vigorous hyphae that spread towards the edge of the plate. Later, it gradually changes color from the center, eventually turning olive-brown to dark brown. The colonies are firm, dense, and adhere tightly to the medium, making them difficult to pick up. Figure 1 ).

[0037] (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 for later 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.

[0038] 2. Molecular identification of strains The CNOCC V. coerulea-13 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).

[0039] The ITS sequences obtained from sequencing were 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 the fungus *Acanthophytum comosum* (…). Hypoxylon investiens The sequence similarity was 99.36%. CNOCC V. coerulea-13 strain is a hard carbonaceous bacterium, and it was biopreserved.

[0040] 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.

[0041] 2. Pre-culture of tissue culture seedlings: Aseptic seedlings of Vanda grandiflora propagated by sowing 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 pre-culture for 1 month for later use.

[0042] 3. Fungal activation culture: The slant test tubes of CNOCC V. coerulea-13 strain stored at -4℃ were taken out and transferred to a constant temperature incubator at 28℃ for 2-3 days to allow the mycelium to regain its growth vitality.

[0043] 4. Transplantation of strains: Select mycelia of CNOCC V. coerulea-13 strain after activation culture in slant tubes at -4℃, inoculate them onto prepared PDA solid medium, and incubate in a constant temperature incubator at 28℃ for 7 days.

[0044] 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.

[0045] 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 mixed in a 1:1 volume ratio for further cultivation and growth management in a greenhouse.

[0046] 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.

[0047] Example 3 Following the method in Example 2, the tissue culture seedlings of Vanda grandiflora propagated by aseptic sowing were replaced with tissue culture seedlings of Oryza sativa propagated by aseptic sowing, and symbiotic culture of Oryza sativa and CNOCC V. coerulea-13 strain was carried out.

[0048] 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.

[0049] Example 4 Following the method in Example 2, the tissue culture seedlings of Vanda grandiflora propagated by aseptic sowing were replaced with stem segments of Anoectochilus roxburghii propagated by aseptic sowing, and a symbiotic culture of Anoectochilus roxburghii and CNOCC V. coerulea-13 strain was carried out.

[0050] 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.

[0051] 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 Cercidiphyllum 'Crane' propagated by aseptic sowing, and symbiotic culture of Cercidiphyllum 'Crane' and CNOCC V. coerulea-13 strain was carried out.

[0052] Comparative Example 4 The *Cymbidium goeringii* was cultured in accordance with the method described in Example 5, except that a 0.5 × 0.5 cm PDA agar block was selected for transfer and co-culture.

[0053] Example 6 Following the method in Example 2, the tissue culture seedlings of Vanda grandiflora propagated by aseptic sowing were replaced with tissue culture seedlings of Cymbidium sinense propagated by aseptic sowing, and symbiotic culture of Cymbidium sinense and CNOCC V. coerulea-13 strain was carried out.

[0054] Comparative Example 5 The *Cymbidium sinense* was cultured in accordance with the method described in Example 6, except that a 0.5×0.5cm PDA agar block was selected for co-culture.

[0055] Example 7 Following the method of 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-13 strain was carried out.

[0056] Comparative Example 6 The Paphiopedilum orchid was cultured in accordance with the method of Example 7, except that a 0.5×0.5cm PDA agar block was selected for transfer and co-culture.

[0057] Test Example 1 180 days after inoculation with CNOCC V. coerulea-13, the growth status of different orchid species in Examples 2-7 and Comparative Examples 1-6 was observed; survival rate and biomass were statistically analyzed, and the results are shown in Tables 1-2 and 2. Figure 2 As shown.

[0058] Table 1. Survival rates of different orchid species

[0059] Note: This represents a highly significant difference.

[0060] Table 2 Biomass of different orchid species

[0061] Note: This represents a significant difference.

[0062] according to Figure 2 It can be seen that after symbiotic culture with CNOCCV.coerulea-13, the leaves of *Vanda grandiflora*, *Cymbidium sinense*, *Cymbidium ensifolium*, and *Paphiopedilum buergerianum* remained green, and the plants exhibited vigorous growth and upright posture. While the older leaves of *Cymbidium goeringii* and *Anoectochilus roxburghii* turned yellow and withered, the new leaves were a deep green and had a graceful shape. These phenomena indicate that CNOCCV.coerulea-13 can establish a symbiotic relationship with various orchid species, providing nutritional support to orchid tissue culture seedlings without significantly inhibiting their healthy growth.

[0063] As shown in Table 1, the control group without CNOCC V. coerulea-13 strain had a low survival rate in hormone-free medium, while the control group inoculated with CNOCC V. coerulea-13 strain had a significantly higher survival rate, showing a highly significant difference from the control group. P <0.01). Table 2 further shows that, under completely hormone-free conditions, tissue culture seedlings inoculated with CNOCC V. coerulea-13 strain maintained normal biomass accumulation in various orchid species, including Vanda grandiflora, Anoectochilus roxburghii, Cynoglossum 'Crown of the Crane', and Cymbidium goeringii. Among these, the biomass accumulation of Cynoglossum 'Chinese Flame Orchid' was significantly higher than that of the control group. In summary, these results indicate that CNOCC V. coerulea-13 strain can effectively improve the survival rate of various orchid species in hormone-free culture media and effectively maintain normal biomass accumulation.

[0064] 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-13 strain, characterized in that, The CNOCC V. coerulea-13 strain is a hardened carbonaceous bacterium ( Hypoxylon investiens (The accession number is GDMCC No:67765).

2. A microbial agent, characterized in that, The microbial agent includes the CNOCC V. coerulea-13 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-13 strain.

4. The microbial agent according to claim 3, characterized in that, The size of the fungal block 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-13 strain was inoculated into a solid culture medium and cultured. The mycelial blocks with newly formed hyphae on the periphery of the CNOCCV.coerulea-13 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-13 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-13 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, Crane Crown Orchid, Dragon Boat Orchid, and Paphiopedilum. 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-13 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-13 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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