Mycorrhizal fungus CNOCC PP26 from Paphiopedilum purpurogenum and its application in promoting plant root growth

Abstract

The present invention relates to the field of microbial technology, and in particular to a mycorrhizal fungus CNOCC PP26 from Paphiopedilum purpurogenum and its use in promoting plant root growth. The present invention provides a mycorrhizal fungus CNOCC PP26 from Paphiopedilum purpurogenum, with a deposit number of GDMCC NO: 65861. The mycorrhizal fungus CNOCC PP26 from the present invention is a strain of the genus Paphiopedilum genus isolated from the mycorrhizae of adult Paphiopedilum purpurogenum plants. When co-cultured with sterile Paphiopedilum purpurogenum seedlings, the strain can effectively promote the formation and growth of new roots in the seedlings, effectively resolving the problem of slow seedling growth during the seedling cultivation process, shortening the seedling cultivation time of Paphiopedilum purpurogenum, and providing technical support for the artificial propagation and restoration of Paphiopedilum purpurogenum.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to a mycorrhizal fungus CNOCC PP26 from Paphiopedilum purpurogenum and an application thereof in promoting the growth of plant roots. Background Art

[0002] The growth and development of orchids is closely linked to the interaction between mycorrhizal fungi. Research has focused on the role of mycorrhizal fungi in overcoming seed germination difficulties caused by endosperm deficiency, promoting mineral absorption, and enhancing plant growth and stress tolerance through the secretion of growth regulators. The genus Mycorrhizalus is a key member of the orchid mycorrhizal fungi family, playing a crucial role in physiological processes such as seed germination in many orchid species.

[0003] The purple-veined Paphiopedilum is the lowest-altitude species in the Chinese genus. Currently, the number of wild Paphiopedilum is small, making its efficient cultivation and reintroduction more important. However, there is currently a lack of nodular mycorrhizal fungi that can promote root formation in artificial Paphiopedilum seedlings. Summary of the Invention

[0004] To address the above problems, the present invention provides a mycorrhizal fungus CNOCC PP26 from Paphiopedilum purpurogenum and its use in promoting plant root growth. The mycorrhizal fungus CNOCC PP26 provided by the present invention can promote the formation of the root system of artificial Paphiopedilum purpurogenum seedlings and shorten the seedling raising time of Paphiopedilum purpurogenum.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a mycorrhizal fungus of Paphiopedilum purpurogenum ( Epulorhiza sp.) CNOCC PP26, the deposit number is GDMCC NO: 65861.

[0007] The present invention provides a growth-promoting bacterial agent, comprising the mycorrhizal fungus CNOCCPP26 of Paphiopedilum purpurogenum described in the above technical solution.

[0008] The present invention provides a method for preparing the growth-promoting bacterium agent described in the above technical solution, comprising:

[0009] The mycorrhizal fungus CNOCC PP26 described in the above technical solution is inoculated onto a culture medium and cultured in the dark to obtain the growth-promoting bacterial agent.

[0010] Preferably, the culture medium includes PDA culture medium; and the temperature of the light-proof culture is 25-28°C.

[0011] The present invention provides the use of the purple-veined Paphiopedilum mycorrhizal fungus CNOCC PP26 described in the above technical solution, or the growth-promoting agent described in the above technical solution, or the growth-promoting agent prepared by the preparation method described in the above technical solution in promoting the growth of plant roots.

[0012] Preferably, the promoting the growth of plant roots includes: promoting the formation and / or growth of new roots of the plant.

[0013] Preferably, the plant is a Paphiopedilum plant.

[0014] Preferably, the Paphiopedilum plant is Paphiopedilum purpurogenum.

[0015] The present invention provides a method for promoting plant root growth, comprising: co-culturing plant seedlings and a growth-promoting bacterium; the growth-promoting bacterium is the growth-promoting bacterium described in the above technical solution or the growth-promoting bacterium prepared using the preparation method described in the above technical solution.

[0016] Preferably, the plant seedlings include Paphiopedilum seedlings; the culture medium used for co-cultivation includes 1 / 2MS solid culture medium containing 4 g / L oatmeal; the co-cultivation conditions include: temperature of 23~27°C, light intensity of 1200 Lux, and light-dark ratio of 16h:8h.

[0017] Beneficial effects:

[0018] The present invention provides a mycorrhizal fungus strain of Paphiopedilum purpurogenum, CNOCC PP26, with a deposit number of GDMCC NO: 65861. The mycorrhizal fungus strain of Paphiopedilum purpurogenum, CNOCC PP26, is isolated from the mycorrhizae of adult Paphiopedilum purpurogenum plants. When co-cultured with sterile Paphiopedilum purpurogenum seedlings, the strain can effectively promote the formation and growth of new roots in the seedlings, effectively resolving the problem of slow seedling growth during the cultivation process, shortening the seedling cultivation time, and providing technical support for the artificial propagation and restoration of Paphiopedilum purpurogenum. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0020] Figure 1 This is a comparative photo of the root system of Paphiopedilum purpurogenum seedlings;

[0021] Figure 2 This is a colony image of the mycorrhizal fungus CNOCC PP26 of Paphiopedilum purpurogenum on PDA culture medium.

[0022] Biological Deposit Description

[0023] The mycorrhizal fungus CNOCC PP26 of Paphiopedilum purpurogenum is classified as Epulorhiza sp., was deposited in the Guangdong Provincial Microbial Strain Collection Center (GDMCC) on February 7, 2025. The deposit address is 5th Floor, Laboratory Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, and the deposit number is GDMCC NO: 65861. DETAILED DESCRIPTION

[0024] The invention provides a mycorrhizal fungus of Paphiopedilum purpurogenum CNOCC PP26, with a preservation number of GDMCC NO: 65861.

[0025] The mycorrhizal fungus CNOCC PP26, described in this invention, is a strain of the genus Paphiopedilum purpurogenum, isolated from the mycorrhizae of adult Paphiopedilum purpurogenum plants. Its colonies are creamy yellow, with slightly wrinkled central cakes and irregularly serrated edges. During the growth phase, its hyphae are fine and weak, appearing cloud-like; during the mature phase, their hyphae are twisted and form cords or granular projections. When co-cultured with sterile Paphiopedilum purpurogenum seedlings, this strain can effectively promote the formation and growth of new roots in these seedlings, effectively resolving the issue of slow seedling growth and shortening the seedling cultivation process.

[0026] Based on the above advantages, the present invention provides a growth-promoting bacterial agent, including the purple-veined Paphiopedilum mycorrhizal fungus CNOCC PP26 described in the above technical solution.

[0027] The present invention provides a method for preparing the growth-promoting bacterium agent described in the above technical solution, comprising:

[0028] The mycorrhizal fungus CNOCC PP26 described in the above technical solution is inoculated onto a culture medium and cultured in the dark to obtain the growth-promoting bacterial agent.

[0029] In one embodiment, the culture medium is PDA culture medium. In one embodiment, the temperature of the dark culture is 25-28°C; in another embodiment, the temperature of the dark culture is 25-27°C; in another embodiment, the temperature of the dark culture is 25-26°C. In one embodiment, the dark culture period is 3 weeks. In one embodiment, the growth-promoting agent can be a fungal mass containing mycelium obtained by culture.

[0030] Based on the above advantages, the present invention provides the use of the purple-veined Paphiopedilum mycorrhizal fungus CNOCC PP26 described in the above technical solution, or the growth-promoting agent described in the above technical solution, or the growth-promoting agent prepared by the preparation method described in the above technical solution in promoting plant root growth.

[0031] In one embodiment, promoting plant root growth includes promoting the formation and / or growth of new plant roots. In one embodiment, the plant is a Paphiopedilum plant; in another embodiment, the Orchidaceae plant is Paphiopedilum purpurogenum. The Paphiopedilum purpurogenum mycorrhizal fungus CNOCC PP26 provided by the present invention can promote the formation and growth of new roots in Paphiopedilum purpurogenum seedlings.

[0032] Based on the above advantages, the present invention provides a method for promoting plant root growth, comprising: co-cultivating purple-veined Paphiopedilum seedlings and a growth-promoting bacterium; the growth-promoting bacterium is the growth-promoting bacterium described in the above technical scheme or the growth-promoting bacterium prepared using the preparation method described in the above technical scheme.

[0033] In one embodiment, the plant seedling is a Paphiopedilum seedling. In one embodiment, the Paphiopedilum seedling is a Paphiopedilum purpurogenum seedling. In one embodiment, the Paphiopedilum purpurogenum seedling is a sterile Paphiopedilum purpurogenum seedling. In one embodiment, the sterile Paphiopedilum purpurogenum seedling is obtained by artificial sterile sowing and germination culture of mature Paphiopedilum purpurogenum seeds. In one embodiment, the sterile Paphiopedilum purpurogenum seedling is a sterile Paphiopedilum purpurogenum seedling with 3 to 4 leaves. In one embodiment, the culture medium used for the co-cultivation is a 1 / 2MS solid culture medium containing 4 g / L oatmeal. In one embodiment, the co-cultivation temperature is 25±2°C, the light intensity is 1200 Lux, and the light-dark ratio is 16h:8h. In one embodiment, the co-cultivation period is 2 to 3 months. The method provided by the present invention can promote the formation and growth of new roots in Paphiopedilum purpurogenum seedlings and shorten the artificial seedling cultivation time.

[0034] To further illustrate the present invention, the purple-veined Paphiopedilum mycorrhizal fungus CNOCC PP26 provided by the present invention and its application in promoting plant root growth are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] The isolation and identification process of strain CNOCC PP26 is as follows:

[0037] The roots of Paphiopedilum purpurogenum with intact appearance, no rot and fullness were picked, the surface of the roots was cleaned with water, disinfected with 1% sodium hypochlorite aqueous solution for 15 minutes, and the root epidermis was gently scraped off with a scalpel. The part of the root segment that was different in color from the majority of the area was observed and cut, and sliced ​​into 1 mm thick slices and inoculated on PDA medium supplemented with streptomycin (the medium was purchased from Guangdong Huankai Microbial Technology Co., Ltd.). 1 to 3 slices were inoculated per culture dish and cultured in a constant temperature box at 28°C for 3 days. The grown mycelium was picked and transferred to a new culture medium for purification until a pure strain was obtained.

[0038] A pure bacterial strain was cut and inoculated in the center of a PDA culture dish, then placed in a 28°C incubator and observed for the initial morphology of the colony. Preliminary morphological identification of this mycorrhizal fungus revealed a milky yellow colony with slightly wrinkled central cakes and irregularly serrated edges. During the growth phase, the hyphae were thin and weak, appearing cloud-like. During the mature phase, the hyphae were twisted and cord-like or granular. Figure 2 ).

[0039] Molecular identification of fungi: ITS ( Internal Transcribed Space ) genes, including ITS1 and ITS2 The two parts evolve rapidly and are polymorphic, making them suitable for distinguishing and identifying closely related strains. Primers were designed using conserved rDNA sequences to PCR amplify the ITS region of the unknown fungus. After sequencing, homology was compared with known sequences in GenBank to obtain species information for identification. The nucleotide sequence of the ITS region of the unknown fungus is shown in SEQ ID NO: 1, which is as follows:

[0040] 5'--3'.

[0041] In summary, strain CNOCC PP26 was identified as a fungus of the genus Paphiopedilum mycorrhizum by morphology and ITS sequencing and named Paphiopedilum purpurogenum mycorrhizum ( Epulorhiza sp.) CNOCC PP26, deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC), with the deposit number GDMCC NO: 65861.

[0042] Example 2

[0043] 1) The mycorrhizal fungus CNOCC PP26 of Paphiopedilum purpurogenum with the deposit number GDMCC NO: 65861 in Example 1 was inoculated on PDA medium. 25 mL of the medium was placed in a 9 mm culture dish and cultured in a fungus incubator at 28° C. in the dark for 3 weeks until the mycelium covered the culture dish and set aside.

[0044] 2) Prepare 1 / 2MS solid medium supplemented with 4g / L oatmeal in tissue culture flasks to meet the growth requirements of fungi and seedlings.

[0045] 3) Select healthy, sterile Paphiopedilum purpurogenum seedlings with 3-4 leaves and transplant them into tissue culture bottles in a laminar flow hood, with one plant per bottle. Then, culture them in a plant culture room at 25±2°C, 1200 Lux, and a light-dark ratio of 16h:8h for 3 weeks until the Paphiopedilum purpurogenum seedlings have fully recovered.

[0046] 4) Cut the fungal colony cultured in step 1) into 5mm×5mm small pieces and inoculate them into the culture bottle cultured for 3 weeks in step 3) and co-cultivate them with the sterile seedlings of Paphiopedilum purpurogenum. After 2 months of co-cultivation, the occurrence and rapid growth of new roots of the seedlings of Paphiopedilum purpurogenum can be clearly seen ( Figure 1 The co-culture conditions were as follows: temperature 25±2°C, light intensity 1200 Lux, and light-dark ratio 16h:8h.

[0047] Comparative Example 1

[0048] 1) Prepare 1 / 2 MS solid medium supplemented with 4 g / L oatmeal in a tissue culture flask.

[0049] 2) Select healthy, sterile Paphiopedilum purpurogenum seedlings with 3-4 leaves and transplant them into tissue culture bottles in a laminar flow hood, with one plant per bottle. Then, culture them in a plant culture room at 25±2°C, 1200 Lux, and a light-dark ratio of 16h:8h for 3 weeks until the Paphiopedilum purpurogenum seedlings have fully recovered.

[0050] 3) Cut the sterile PDA solid culture medium into 5mm×5mm small pieces, inoculate them into culture bottles, and co-cultivate them with sterile Paphiopedilum purpurogenum seedlings. After 2 months of co-cultivation, the occurrence and growth of new roots of Paphiopedilum purpurogenum seedlings slowed down ( Figure 1 The co-culture conditions were as follows: temperature 25±2°C, light intensity 1200 Lux, and light-dark ratio 16h:8h.

[0051] Test Example 1

[0052] The number and length of newly grown roots of Paphiopedilum purpurogenum seedlings co-cultured for 3 months in Example 2 and Comparative Example 1 were measured, with three replicates per group, each with 7 seedlings. The results are shown in Table 1.

[0053] Table 1 Results of new root determination of Paphiopedilum purpurogenum seedlings

[0054]

[0055] The results show that the mycorrhizal fungus CNOCC PP26 provided by the present invention can effectively promote the formation and growth of new roots of Paphiopedilum purpurogenum seedlings, effectively solve the problem of slow growth of seedlings during the seedling cultivation process of Paphiopedilum purpurogenum, and shorten the seedling cultivation time of Paphiopedilum purpurogenum.

[0056] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A mycorrhizal fungus of Paphiopedilum purpurogenum ( Epulorhiza sp.) CNOCC PP26, characterized by The deposit number is GDMCC NO: 65861.

2. A growth-promoting agent, characterized in that It includes the purple-veined Paphiopedilum mycorrhizal fungus CNOCC PP26 described in claim 1.

3. The method for preparing the growth-promoting bacterium agent according to claim 2, characterized in that: include: The mycorrhizal fungus CNOCC PP26 of Paphiopedilum purpurogenum according to claim 1 is inoculated onto a culture medium and cultured in the dark to obtain the growth-promoting bacterial agent.

4. The preparation method according to claim 3, characterized in that The culture medium includes PDA culture medium; the temperature of the light-proof culture is 25-28°C.

5. Use of the mycorrhizal fungus CNOCC PP26 of Paphiopedilum purpurogenum according to claim 1, the growth-promoting agent according to claim 2, or the growth-promoting agent prepared by the preparation method according to claim 3 or 4 in promoting plant root growth; the plant is Paphiopedilum purpurogenum.

6. The use according to claim 5, characterized in that The promoting the growth of plant roots includes: promoting the formation and / or growth of new roots of plants.

7. A method for promoting plant root growth, characterized in that: include: The plant seedlings and the growth-promoting bacteria agent are co-cultivated; the growth-promoting bacteria agent is the growth-promoting bacteria agent according to claim 2 or the growth-promoting bacteria agent prepared by the preparation method according to claim 3 or 4; the plant seedlings are Paphiopedilum purpurogenum seedlings.

8. The method according to claim 7, characterized in that The culture medium used for the co-cultivation includes a 1 / 2MS solid culture medium containing 4g / L oatmeal. The co-cultivation conditions include: a temperature of 23-27°C, a light intensity of 1200 Lux, and a light-dark ratio of 16h:8h.

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