CNOCC PP35 and application thereof in enhancing photosynthesis of paphiopedilum purpuratum

Abstract

The invention relates to the technical field of microorganisms, in particular to a mycospora sp. CNOCC PP35 and application thereof in enhancing photosynthesis of paphiopedilum purpuratum. The bottle mycospora CNOCC PP35 provided by the invention is a dark partitioned endophytic fungus separated from mycorrhiza of a paphiopedilum purpuratum adult plant, the preservation number is GDMCC NO: 65862, when the strain and a sterile seedling of paphiopedilum purpuratum are co-cultured, the formation of chlorophyll a and total chlorophyll of the sterile seedling of paphiopedilum purpuratum can be effectively promoted, and compared with a sterile control group, the chlorophyll a and total chlorophyll are respectively improved by 122.35% and 98.42%, and the chlorophyll a and total chlorophyll of the sterile seedling of paphiopedilum purpuratum can be effectively promoted. Photosynthesis of sterile seedlings of paphiopedilum purpuratum can be enhanced; the strain can effectively promote the growth of paphiopedilum purpuratum seedlings, the fresh weight is improved by 127.78% compared with that of a control group, mycorrhization of sterile seedlings of paphiopedilum purpuratum can be achieved, and the artificial seedling raising time is shortened.

Description

Technical Field

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

[0002] Purple-veined Paphiopedilum belongs to the genus Paphiopedilum of the Orchidaceae family and is extremely ornamental. Under natural conditions, there is a close connection between orchids and fungi, and the survival of orchids from seed germination to adult plants depends to varying degrees on different types of symbiotic fungi. Under artificial conditions, aseptic germination technology is one of the most common methods for raising orchid seedlings. Due to the lack of symbiotic fungi, the aseptic culture time of Paphiopedilum is long, and different proportions of plant growth regulators need to be added to complete the entire process.

[0003] Dark septate endophytes are a group of small soil fungi that widely colonize plant roots and form dark septate hyphae or microsclerotia. They have a wide ecological distribution and play a good role in promoting plant growth and development and improving resistance. However, there is currently a lack of dark septate endophytes that can promote the growth of Paphiopedilum purpurogenum and enhance the photosynthesis of seedlings. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a strain of Phiala cladosporium CNOCC PP35 and its application in enhancing the photosynthesis of Paphiopedilum purpurogenum. The Phiala cladosporium CNOCC PP35 of the present invention is a dark septate endophytic fungus that promotes the growth of Paphiopedilum purpurogenum and enhances the photosynthesis of seedlings, can realize the mycorrhization of sterile seedlings of Paphiopedilum purpurogenum, and shorten the artificial seedling raising time.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: The present invention provides a strain of Cladosporium Cladophialophora sp.) CNOCC PP35, the deposit number is GDMCC NO: 65862.

[0006] The present invention provides a microbial preparation, comprising the Cladosporium CNOCC PP35 described in the above technical solution.

[0007] The present invention provides a method for preparing the microbial preparation described in the above technical solution, comprising: 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.

[0008] Preferably, the culture medium is PDA culture medium; and the temperature of the light-proof culture is 25-28°C.

[0009] The present invention provides the use of the 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.

[0010] Preferably, the enhancing plant photosynthesis comprises: increasing the chlorophyll a content in plant leaves and / or increasing the total chlorophyll content in plant leaves.

[0011] Preferably, the plant is a Paphiopedilum plant.

[0012] Preferably, the Paphiopedilum plant is Paphiopedilum purpurogenum.

[0013] The present invention provides a method for enhancing the photosynthesis of Paphiopedilum purpurogenum and / or promoting the vegetative 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 scheme or the microbial preparation prepared by the preparation method described in the above technical scheme.

[0014] 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.

[0015] Beneficial effects: The present invention provides a Cladosporium phialariae CNOCC PP35, with a deposit number of GDMCC NO: 65862. The Cladosporium phialariae CNOCC PP35 of the present invention is a dark septate endophytic fungus isolated from the mycorrhiza of an adult plant of Paphiopedilum purpurogenum, and when the strain is co-cultured with sterile seedlings of Paphiopedilum purpurogenum, the formation of chlorophyll a and total chlorophyll of the sterile seedlings of Paphiopedilum purpurogenum can be effectively promoted, which are increased by 122.35% and 98.42% respectively compared with a sterile control group, and the photosynthesis of the sterile seedlings of Paphiopedilum purpurogenum can be enhanced; the strain can effectively promote the growth of the seedlings of Paphiopedilum purpurogenum, and the fresh weight is increased by 127.78% compared with the control group, and the mycorrhization of the sterile seedlings of Paphiopedilum purpurogenum can be realized, and the artificial seedling raising time can be shortened. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0017] Figure 1 This is a colony diagram of the fungus of the genus Cladosporium CNOCC PP35 of the present invention; Figure 2 It is an enlarged view of the colony of the fungus of the genus Cladosporium CNOCC PP35 of the present invention; Figure 3This is a physical picture of the Paphiopedilum purpurogenum seedlings cultivated for 5 months in Example 2; Figure 4 This is a real picture of the Paphiopedilum purpurogenum seedlings cultivated for 5 months in Comparative Example 1.

[0018] Biological Deposit Description Cladosporium CNOCC PP35, classified as Cladophialophora sp., was deposited in the Guangdong 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

[0019] The invention provides a Cladosporium phage CNOCC PP35, whose deposit number is GDMCC NO:65862.

[0020] The Cladosporium phialariae CNOCC PP35 of the present invention is a dark septate endophytic fungus isolated from the mycorrhiza of an adult plant of Paphiopedilum purpurogenum, with a black colony and short grayish-white aerial hyphae densely formed into felt-like hairs; the colony is uniform, the edges are neat, and the culture medium is invaded for growth, and the average growth rate of hyphae at 25°C is 0.44 mm / d. When the strain is co-cultured with sterile seedlings of Paphiopedilum purpurogenum, the formation of chlorophyll a and total chlorophyll of the sterile seedlings of Paphiopedilum purpurogenum can be effectively promoted, which is increased by 122.35% and 98.42% respectively compared with the sterile control group, and the photosynthesis of the sterile seedlings of Paphiopedilum purpurogenum can be enhanced; the strain can effectively promote the growth of the seedlings of Paphiopedilum purpurogenum, and the fresh weight is increased by 127.78% compared with the control group, and the mycorrhization of the sterile seedlings of Paphiopedilum purpurogenum can be realized, and the artificial seedling raising time can be shortened.

[0021] Based on the above advantages, the present invention provides a microbial preparation, including the Cladosporium CNOCC PP35 described in the above technical solution.

[0022] The present invention provides a method for preparing the microbial preparation described in the above technical solution, comprising: 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.

[0023] 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.

[0024] Based on the above advantages, the present invention provides the use of the 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. As an embodiment, the enhancing plant photosynthesis includes: increasing the chlorophyll a content of plant leaves and / or increasing the total chlorophyll content of plant leaves. As an embodiment, the plant is a Paphiopedilum plant. As another embodiment, the Orchidaceae plant is Paphiopedilum purpurogenum. The Cladosporium CNOCC PP35 provided by the present invention can enhance plant photosynthesis and promote plant nutritional growth. As an embodiment, the promoting plant nutritional growth is to increase the fresh weight of the plant.

[0025] Based on the above advantages, the present invention provides a method for enhancing the photosynthesis of Paphiopedilum purpurogenum and / or promoting the vegetative 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. As an embodiment, the enhancing plant photosynthesis comprises: increasing the chlorophyll a content of plant leaves and / or increasing the total chlorophyll content of plant leaves. As an embodiment, the promoting plant vegetative growth is to increase the fresh weight of the plant.

[0026] As an embodiment, the purple-veined Paphiopedilum seedlings are sterile purple-veined Paphiopedilum seedlings; the culture medium used for the co-cultivation is a 1 / 2MS solid culture medium containing 4g / L oatmeal powder. As an embodiment, the co-cultivation temperature is 25±2°C, the light intensity is 1200Lux, and the light-dark ratio time is 16h:8h. The method provided by the present invention can achieve mycorrhization of sterile seedlings of purple-veined Paphiopedilum and shorten the time of artificial seedling cultivation.

[0027] In order 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 in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1 The isolation and identification process of strain CNOCC PP35 is as follows: 1) Collect the roots of adult Paphiopedilum purpurogenum plants, which should be intact and free of rot; remove the root cover with an iron spoon and tweezers, and rinse them under clean water; cut the processed root segments into small segments of 2-3 cm, and use the manual slicing method to cut thin slices from one end of the root segment, and then examine them under an optical microscope to select the root segments containing mycelium.

[0029] 2) In a clean bench, soak the selected root segments with mycelium in 75% alcohol for 30 seconds, sterilize the root surface with 1% NaClO solution (immerse and shake) for 4 minutes, wash with sterile water 3 times, and then scrape the cortical cells layer by layer with a scalpel to release the mycelium; use a pipette to draw 10 µL of mycelium suspension and inoculate it on a PDA medium plate supplemented with penicillin and streptomycin (final concentration of 50 µg / mL).

[0030] 3) Place the plate at 25°C in the dark and observe every 2 days; pick the germinated mycelium and transfer it to a new PDA medium for purification until a pure strain is obtained, which is recorded as CNOCC PP35.

[0031] 4) Cut a piece of the strain CNOCC PP35 and inoculate it in the center of the PDA medium plate, place it in a 25℃ incubator and observe the morphology of the colonies. 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: the colony was black, produced short gray-white aerial hyphae, densely packed into felt-like hairs; the colony was uniform, with neat edges, and invaded the culture medium for growth. During the culture process, it was found that the hyphae of strain CNOCC PP35 grew slowly, with an average growth rate of 0.44 mm / d at 25°C.

[0032] 5) Molecular identification of fungi: Primers were designed using the conservative sequence of rDNA, and PCR was used to 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. After sequencing, the ITS sequence of CNOCC PP35 was consistent with that of Phiala spp. CladophialophoraThe similarity is above 95%. The ITS sequence is shown in SEQ ID NO.1, and is specifically as follows: 5'--3'.

[0033] In summary, strain CNOCC PP35 was identified as Cladosporium sporophialum by morphology and ITS sequencing. Cladophialophora sp.), deposited in Guangdong Microbiological Collection Center (GDMCC) with the deposit number GDMCCNO: 65862.

[0034] Example 2 1) The Cladosporium spp. CNOCC PP35 with the deposit number of GDMCC NO: 65862 in Example 1 was inoculated on PDA medium, placed in a fungal incubator at 25° C. in the dark and cultured until the mycelium filled the culture dish, and then set aside.

[0035] 2) Prepare 1 / 2MS solid culture medium supplemented with 4g / L oatmeal powder in tissue culture bottles to meet the growth requirements of fungi and seedlings respectively.

[0036] 3) Select healthy and uniform-sized sterile Paphiopedilum purpurogenum seedlings (with 2-3 leaves), transplant them into tissue culture bottles in a clean bench, one plant per bottle, and then culture them in a plant culture room at 25±2℃, 1200Lux light intensity, and 16h:8h light-dark ratio for 2 months until the Paphiopedilum purpurogenum seedlings fully resume growth.

[0037] 4) The fungal colonies cultured in step 1) were cut into small pieces of 5 mm × 5 mm, inoculated into culture bottles, 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 ).

[0038] Comparative Example 1 1) Prepare 1 / 2MS solid culture medium supplemented with 4g / L oatmeal in a tissue culture bottle.

[0039] 2) Select healthy and uniform-sized sterile Paphiopedilum purpurogenum seedlings (with 2-3 leaves), transplant them into tissue culture bottles in a clean bench, one plant per bottle, and then culture them in a plant culture room at 25±2℃, 1200Lux, and a light-dark ratio of 16h:8h for 2 months until the Paphiopedilum purpurogenum seedlings fully resume growth.

[0040] 3) Cut the sterile PDA solid culture medium into small pieces of 5 mm × 5 mm, 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 ).

[0041] Test Example 1 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: The seedlings of Paphiopedilum purpurogenum were taken out, the culture medium was washed off, and then the fresh weight was measured; 95% ethanol and acetone were mixed in a volume ratio of 1:1, and the obtained mixed solution was used as an extractant to extract chlorophyll in the leaves; the absorbance value 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.

[0042] Table 1 Comparison of biomass (fresh weight) and chlorophyll content of Paphiopedilum purpurogenum seedlings

[0043] Among them, * is p <0.05, ** p <0.01.

[0044] The results show that the Cladosporium spp. CNOCC PP35 provided by the present invention can effectively promote the growth of Paphiopedilum purpurogenum seedlings, and the fresh weight is increased by 127.78% ( Figure 3 and Figure 4 ); When co-cultured, the contents of chlorophyll a and total chlorophyll in the seedlings increased significantly, increasing by 122.35% and 98.42% respectively compared with the control group, but the content of chlorophyll b did not change significantly. The above results show that CNOCC PP35 can enhance the photosynthesis of Paphiopedilum purpurogenum seedlings by promoting the formation of chlorophyll a, thereby accelerating the vegetative growth of the plants.

[0045] Although the above embodiment describes the present invention in detail, 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 protection scope 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 invention comprises the Cladosporium CNOCC PP35 described in claim 1.

3. The method for preparing the microbial preparation according to claim 2, characterized in that: include: The Cladosporium CNOCC PP35 described in claim 1 is inoculated onto a 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. Use of the Cladosporium CNOCC PP35 according to claim 1, 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.

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. The use according to claim 5 or 6, characterized in that: The plant is a Paphiopedilum plant.

8. The use according to claim 7, characterized in that: The Paphiopedilum plant is Paphiopedilum purpurogenum.

9. 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.

10. The method according to claim 9, 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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