A strain of Phialophora richardsiae CNOCC PP35 and its application in enhancing the photosynthesis of Paphiopedilum purpuratum

Abstract

The present invention relates to the field of microbial technology, and particularly to a Cladophialophora sp. CNOCC PP35 and its application in enhancing the photosynthesis of Paphiopedilum purpuratum. The Cladophialophora sp. CNOCC PP35 provided by the present invention is a strain of dark septate endophytic fungus isolated from the mycorrhizae of adult plants of Paphiopedilum purpuratum, with the preservation number of GDMCC NO: 65862. When co-cultured with the axenic seedlings of Paphiopedilum purpuratum, this strain can effectively promote the formation of chlorophyll a and total chlorophyll in the axenic seedlings of Paphiopedilum purpuratum, increasing by 122.35% and 98.42% respectively compared with the axenic control group, and can enhance the photosynthesis of the axenic seedlings of Paphiopedilum purpuratum; this strain can effectively promote the growth of the seedlings of Paphiopedilum purpuratum, with the fresh weight increasing by 127.78% compared with the control group, can realize the mycorrhization of the axenic seedlings of Paphiopedilum purpuratum, and shorten the artificial seedling raising time.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a Cladosporium CNOCC PP35 and application thereof in enhancing the photosynthesis of Paphiopedilum purpurogenum. Background Art

[0002] The purple-veined Paphiopedilum, a member of the genus Paphiopedilum in the Orchidaceae family, boasts exceptional ornamental value. Under natural conditions, orchids maintain a close relationship with fungi, with survival, from seed germination to adulthood, dependent to varying degrees on different types of symbiotic fungi. Under artificial conditions, sterile germination techniques are currently the most common method for growing orchid seedlings. However, due to the lack of symbiotic fungi involved, sterile cultivation of Paphiopedilum requires a lengthy process and the addition of plant growth regulators in varying proportions.

[0003] Dark-septate endophytes are a group of small soil fungi that extensively colonize plant roots and form dark-septate hyphae or microsclerotia. They have a wide ecological distribution and are known to promote plant growth and development and enhance plant resistance. However, there is currently a lack of dark-septate endophytes that promote growth and enhance photosynthesis in Paphiopedilum purpurogenum seedlings. Summary of the Invention

[0004] To address the above issues, the present invention provides a strain of Cladosporium (CNOCC PP35) and its use in enhancing photosynthesis in Paphiopedilum purpurogenum. The Cladosporium (CNOCC PP35) described in the present invention is a dark-septate endophyte fungus that promotes growth and enhances photosynthesis in Paphiopedilum purpurogenum seedlings. It can mycorrhize sterile Paphiopedilum purpurogenum seedlings, shortening artificial seedling cultivation time.

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

[0006] The present invention provides a strain of Cladosporium ( Cladophialophora sp.) CNOCC PP35, the deposit number is GDMCC NO: 65862.

[0007] The present invention provides a microbial preparation, comprising the Cladosporium CNOCC PP35 described in the above technical solution.

[0008] The present invention provides a method for preparing the microbial preparation described in the above technical solution, comprising:

[0009] The Cladosporium CNOCC PP35 described in the above technical solution is inoculated onto a culture medium and cultured in the dark to obtain the microbial preparation.

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

[0011] The present invention provides the use of Cladosporium CNOCC PP35 described in the above technical solution, or the microbial preparation described in the above technical solution, or the microbial preparation prepared by the preparation method described in the above technical solution in enhancing plant photosynthesis and / or promoting plant nutritional growth.

[0012] Preferably, the enhancing plant photosynthesis comprises: increasing the chlorophyll a content in plant leaves and / or increasing the total chlorophyll content in plant leaves.

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

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

[0015] The present invention provides a method for enhancing the photosynthesis of Paphiopedilum purpurogenum and / or promoting the nutritional growth of Paphiopedilum purpurogenum, comprising: co-culturing Paphiopedilum purpurogenum seedlings and a microbial preparation; the microbial preparation is the microbial preparation described in the above technical solution or the microbial preparation prepared by the preparation method described in the above technical solution.

[0016] Preferably, the Paphiopedilum purpurogenum seedlings are sterile Paphiopedilum purpurogenum seedlings; and the culture medium used for co-cultivation is a 1 / 2MS solid culture medium containing 4 g / L oatmeal.

[0017] Beneficial effects:

[0018] The present invention provides a Cladosporium phialariae CNOCC PP35, with a deposit number of GDMCC NO: 65862. The Cladosporium phialariae CNOCC PP35 described in the present invention is a dark-septate endophytic fungus isolated from the mycorrhizae of adult Paphiopedilum purpurogenum plants. When co-cultured with sterile Paphiopedilum purpurogenum seedlings, the strain can effectively promote the formation of chlorophyll a and total chlorophyll in the sterile Paphiopedilum purpurogenum seedlings, increasing them by 122.35% and 98.42%, respectively, compared to the sterile control group, thereby enhancing photosynthesis in the sterile Paphiopedilum purpurogenum seedlings. The strain can also effectively promote the growth of the Paphiopedilum purpurogenum seedlings, increasing their fresh weight by 127.78% compared to the control group, thereby achieving mycorrhization of the sterile Paphiopedilum purpurogenum seedlings and shortening the artificial seedling cultivation time. 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 colony diagram of the fungus Cladosporium CNOCC PP35 of the present invention;

[0021] Figure 2 This is an enlarged view of the colony of the fungus Cladosporium CNOCC PP35 of the present invention;

[0022] Figure 3 This is a photo of the Paphiopedilum purpurogenum seedlings cultured for 5 months in Example 2;

[0023] Figure 4 This is a photo of the Paphiopedilum purpurogenum seedlings cultured for 5 months in Comparative Example 1.

[0024] Biological Deposit Description

[0025] Cladosporium CNOCC PP35, classified as Cladophialophora 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: 65862. DETAILED DESCRIPTION

[0026] The invention provides a Cladosporium CNOCC PP35, with a preservation number of GDMCC NO: 65862.

[0027] The Cladosporium phialariae strain CNOCC PP35 described in the present invention is a dark, septate endophytic fungus isolated from the mycorrhizae of adult Paphiopedilum purpurogenum plants. Its colonies are black and produce short, dense, off-white aerial hyphae that form dense, felt-like hairs. The colonies are uniform, with neat edges, and they invade the culture medium for growth. The average hyphae growth rate at 25°C is 0.44 mm / day. When co-cultured with sterile Paphiopedilum purpurogenum seedlings, this strain effectively promotes the formation of chlorophyll a and total chlorophyll in these seedlings, increasing them by 122.35% and 98.42%, respectively, compared to the sterile control group. This enhances photosynthesis in these seedlings. This strain also effectively promotes the growth of these seedlings, increasing their fresh weight by 127.78% compared to the control group. This strain can achieve mycorrhization in these sterile seedlings and shorten artificial seedling cultivation time.

[0028] Based on the above advantages, the present invention provides a microbial preparation, comprising the Cladosporium CNOCC PP35 described in the above technical solution.

[0029] The present invention provides a method for preparing the microbial preparation described in the above technical solution, comprising:

[0030] The Cladosporium CNOCC PP35 described in the above technical solution is inoculated onto a culture medium and cultured in the dark to obtain the microbial preparation.

[0031] In one embodiment, the culture medium is PDA culture medium; 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.

[0032] Based on the above advantages, the present invention provides the use of Cladosporium CNOCC PP35 described in the above technical solution, or the microbial preparation described in the above technical solution, or the microbial preparation prepared using the preparation method described in the above technical solution, for enhancing plant photosynthesis and / or promoting plant vegetative growth. In one embodiment, enhancing plant photosynthesis includes increasing the chlorophyll a content and / or total chlorophyll content of plant leaves. In one embodiment, the plant is a Paphiopedilum plant. In another embodiment, the Orchidaceae plant is Paphiopedilum purpurogenum. The Cladosporium CNOCC PP35 provided by the present invention can enhance plant photosynthesis and promote plant vegetative growth. In one embodiment, promoting plant vegetative growth includes increasing plant fresh weight.

[0033] Based on the above advantages, the present invention provides a method for enhancing photosynthesis and / or promoting vegetative growth in Paphiopedilum purpurogenum, comprising: co-culturing Paphiopedilum purpurogenum seedlings with a microbial preparation; the microbial preparation is the microbial preparation described in the above technical solution or a microbial preparation prepared using the preparation method described in the above technical solution. In one embodiment, the enhancing plant photosynthesis includes increasing the chlorophyll a content and / or the total chlorophyll content in the plant leaves. In one embodiment, the promoting plant vegetative growth includes increasing the plant fresh weight.

[0034] In one embodiment, the Paphiopedilum purpurogenum seedlings are sterile Paphiopedilum purpurogenum seedlings; the co-cultivation medium is a 1 / 2MS solid medium containing 4g / 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. The method provided by the present invention can achieve mycorrhization of sterile Paphiopedilum purpurogenum seedlings, shortening the artificial seedling cultivation time.

[0035] To further illustrate the present invention, a strain of Cladosporium CNOCC PP35 provided by the present invention and its application in enhancing the photosynthesis of Paphiopedilum purpurogenum 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.

[0036] Example 1

[0037] The isolation and identification process of strain CNOCC PP35 is as follows:

[0038] 1) Collect roots from adult Paphiopedilum purpurogenum plants, ensuring they are intact and free of rot. Remove the root integument with an iron spatula and tweezers, then rinse thoroughly under clean water. Cut the treated root segments into 2-3 cm segments. Manually slice thin slices from one end of the root segment, then examine under an optical microscope to identify those containing mycelial masses.

[0039] 2) In a laminar flow hood, soak the selected root segments with mycelial masses in 75% alcohol for 30 seconds. Sterilize the root surface with 1% NaClO solution (immerse and shake) for 4 minutes, rinse three times with sterile water, and then scrape the cortical cells layer by layer with a scalpel to release the mycelial masses. Use a pipette to draw up 10 µL of the mycelial suspension and inoculate it onto a plate containing PDA medium supplemented with penicillin and streptomycin (final concentration 50 µg / mL).

[0040] 3) Incubate the plates at 25°C in the dark and observe every two days. Pick the germinated mycelium and transfer it to new PDA medium for purification until a pure strain is obtained, which is designated as CNOCC PP35.

[0041] 4) Cut a piece of the strain CNOCC PP35 into the center of the PDA medium plate, place it in a 25℃ incubator and observe the morphology of the colony. The results are shown in Figure 1 and Figure 2 The results showed that the colony characteristics of strain CNOCC PP35 on PDA medium were: black colonies with short, off-white aerial hyphae densely packed into a felt-like pattern; uniform colonies with neat edges and invasion of the culture medium. During cultivation, the hyphae of strain CNOCC PP35 grew slowly, with an average growth rate of 0.44 mm / day at 25°C.

[0042] 5) Molecular identification of fungi: Primers were designed using the conserved sequences of rDNA to PCR amplify the ITS (Internal Transcribed Space) fragment of the fungus. After sequencing, the homology was compared with the known sequences in GenBank to obtain the species information of the fungus. CladophialophoraThe similarity is above 95%. The ITS sequence is shown in SEQ ID NO. 1, specifically as follows: 5'--3'.

[0043] In summary, strain CNOCC PP35 was identified as Cladosporium sp. ( Cladophialophora sp.), deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC) with the deposit number GDMCC NO: 65862.

[0044] Example 2

[0045] 1) Cladosporium CNOCC PP35 with the deposit number GDMCC NO: 65862 in Example 1 was inoculated onto PDA culture medium and cultured in a fungus incubator at 25° C. in the dark until mycelium filled the culture dish and set aside.

[0046] 2) Prepare 1 / 2MS solid medium supplemented with 4g / L oatmeal in tissue culture flasks to meet the growth requirements of fungi and seedlings respectively.

[0047] 3) Select healthy, uniformly sized sterile Paphiopedilum purpurogenum seedlings (with 2-3 leaves) and transplant them into tissue culture bottles in a laminar flow hood, with one plant per bottle. The bottles are then cultured in a plant culture room at 25 ± 2°C, 1200 Lux, and a light-dark ratio of 16 h:8 h for two months, until the Paphiopedilum purpurogenum seedlings have fully recovered.

[0048] 4) The fungal colony cultured in step 1) was cut into 5 mm × 5 mm pieces, inoculated into a culture bottle, and co-cultured with sterile Paphiopedilum purpurogenum seedlings; the co-culture conditions were the same as those in step 3), and the co-culture time was 5 months ( Figure 3 ).

[0049] Comparative Example 1

[0050] 1) Prepare 1 / 2 MS solid medium supplemented with 4 g / L oatmeal in a tissue culture flask.

[0051] 2) Select healthy, uniformly sized sterile Paphiopedilum purpurogenum seedlings (with 2-3 leaves) and transplant them into tissue culture bottles in a laminar flow hood, one plant per bottle. Culture the bottles in a plant culture room at 25 ± 2°C, 1200 Lux, and a light-dark ratio of 16 h:8 h for 2 months, until the Paphiopedilum purpurogenum seedlings have fully recovered.

[0052] 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; the co-cultivation conditions are the same as those in step 2), and the co-cultivation time is 5 months ( Figure 4 ).

[0053] Test Example 1

[0054] The fresh weight of Paphiopedilum purpurogenum co-cultivated for 5 months in Example 2 and Comparative Example 1 and the chlorophyll content in the leaves were measured, with 7 plants in each group being measured, and the method was as follows:

[0055] Paphiopedilum purpurogenum seedlings were removed, the culture medium was rinsed off, and their fresh weight was measured. Chlorophyll was extracted from the leaves using a mixture of 95% ethanol and acetone in a 1:1 volume ratio. The absorbance was measured using a spectrophotometer, and the contents of chlorophyll a, chlorophyll b, and total chlorophyll in the leaves were calculated. The results are shown in Table 1.

[0056] Table 1 Comparison of biomass (fresh weight) and chlorophyll content of Paphiopedilum purpurogenum seedlings

[0057]

[0058] Among them, * is p <0.05, ** p <0.01.

[0059] The results showed that the Cladosporium PP35 provided by the present invention can effectively promote the growth of Paphiopedilum purpurogenum seedlings, and the fresh weight was increased by 127.78% compared with the control group ( Figure 3 and Figure 4 When co-cultured with the fungus, the chlorophyll a and total chlorophyll contents of the seedlings increased significantly, by 122.35% and 98.42%, respectively, compared to the control group, while chlorophyll b content remained unchanged. These results suggest that CNOCC PP35 enhances photosynthesis by promoting chlorophyll a production in Paphiopedilum purpurogenum seedlings, thereby accelerating their vegetative growth.

[0060] 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 strain of Cladosporium Cladophialophora sp. CNOCC PP35, characterized in that The deposit number is GDMCCNO:65862.

2. A microbial preparation, characterized in that: The method comprises the Cladosporium described in claim 1 Cladophialophora sp. CNOCC PP35.

3. The method for preparing the microbial preparation according to claim 2, characterized in that: include: The Cladosporium described in claim 1 Cladophialophora sp. CNOCC PP35 is inoculated into the culture medium and cultured in the dark to obtain the microbial preparation.

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

5. The Cladosporium according to claim 1 Cladophialophora sp. CNOCC PP35 or the microbial preparation according to claim 2 or the microbial preparation prepared by the preparation method according to claim 3 or 4 in enhancing plant photosynthesis and / or promoting plant nutritional growth; the plant is Paphiopedilum purpurogenum.

6. The use according to claim 5, characterized in that: The enhancing of plant photosynthesis comprises: increasing the chlorophyll a content of plant leaves and / or increasing the total chlorophyll content of plant leaves.

7. A method for enhancing the photosynthesis of Paphiopedilum purpurogenum and / or promoting the vegetative growth of Paphiopedilum purpurogenum, characterized in that: include: The purple-veined Paphiopedilum seedlings and the microbial preparation are co-cultured; the microbial preparation is the microbial preparation according to claim 2 or the microbial preparation prepared by the preparation method according to claim 3 or 4.

8. The method according to claim 7, characterized in that The Paphiopedilum purpurogenum seedlings are sterile Paphiopedilum purpurogenum seedlings; the culture medium used for co-cultivation is a 1 / 2MS solid culture medium containing 4g / L oatmeal powder.

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