Cladosporium fungus and application thereof in promoting germination of arundina graminifolia seeds

Abstract

The invention relates to the technical field of microorganisms, and discloses a cladosporium fungus and application thereof to arundina graminifolia seed germination promotion, the cladosporium fungus comprises arundina graminifolia root symbiotic bacteria and seeds, the arundina graminifolia root symbiotic bacteria are the cladosporium fungus ZhuHN-06, and the specific culture process is as follows: (1) taking out the arundina graminifolia seeds on a super clean bench, and culturing the arundina graminifolia seeds on the super clean bench; preparing the seeds into a seed suspension by using a sterile medicine spoon; according to the cladosporium fungus and application of the cladosporium fungus in promoting germination of arundina graminifolia seeds, ZhuHN-06 has a remarkable promoting effect on germination of the arundina graminifolia seeds, can promote growth of roots after seed germination, and is a cladosporium fungus strain with orchid growth promoting potential. The arundina graminifolia seed germination promoting capability of the strain is researched, the problem that orchid seeds are difficult to germinate in artificial cultivation can be effectively solved, conservation and development of orchid resources are further promoted, and industrial development of orchid is promoted.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a fungus of the genus Cladosporium and its application in promoting the germination of Oryza sativa seeds. Background Art

[0002] Orchids, a large family distributed globally, are unusually endangered. This is primarily due to the tiny size of their seeds, which consist only of undifferentiated protoembryos. While their roots are large, root hairs are underdeveloped, resulting in a very low germination rate in their natural state. They require symbiotic relationships with specific fungi to form endomycorrhizae and mycelial masses, providing nutrients to the embryoless seeds and promoting normal germination. Subsequent growth also necessitates interaction with these fungi to meet the plant's water and nutrient needs. Symbiotic fungi play a crucial role in the life cycle of orchids, providing essential elements like C, N, and P for germination from the seed stage onwards. *Arundina basilica*, a terrestrial medicinal orchid belonging to the genus *Arundina* of the Orchidaceae family, exemplifies this. Utilizing the diverse and specific symbiotic relationships between orchids and fungi, symbiotic germination techniques are of great significance for the conservation and industrialization of *Arundina basilica*.

[0003] Cladosporium sp. belongs to the phylum Ascomycota, class Dothideomycetes, order Capnodiales, and family Cladosporiaceae. This fungus is widely distributed, and many studies have focused on the diverse chemical substances it produces to obtain substances with various biological activities. However, research on its use in promoting orchid seed germination and plant growth is relatively rare.

[0004] Based on this, the present invention provides a fungus of the genus Cladosporium and its application in promoting the germination of Oryza sativa seeds. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a fungus of the genus Cladosporium and its application in promoting the germination of Oryza sativa seeds, which has the advantage of promoting Oryza sativa seed germination and solves the problems mentioned in the background art.

[0006] This invention provides the following technical solution: a fungus of the genus *Cladosporium* and its application in promoting the germination of *Cymbidium goeringii* seeds, comprising a root symbiotic fungus of *Cymbidium goeringii* and seeds, wherein the root symbiotic fungus of *Cymbidium goeringii* is *Cladosporium* fungus ZhuHN-06, and its specific culture process is as follows: (1) Take out the seeds of Oryza sativa on the clean bench, use a sterile spatula to make a seed suspension, use a pipette to draw 5 mL of the seed suspension, and calculate the seed density on a hemocytometer (repeat 3 times). (2) Pour the OMA medium into a 9cm plate, use a 0.5cm punch to punch the mycelium cake in the center of the OMA medium, and then use a pipette to draw 5mL of seed suspension and evenly inject it around the mycelium cake. (3) Place them in an artificial climate chamber with conditions of 25±1℃, humidity of 75%, light intensity of 6000Lx, and 12h light / 12h dark for cultivation.

[0007] Preferably, the seed is a bamboo orchid seed.

[0008] Preferably, the seed suspension is composed of *Cymbidium goeringii* seeds with 1 g / L of sterile agar solution.

[0009] Preferably, the OMA culture medium is oat culture medium (5 g of pure oats, added to 900 mL of deionized water and boiled for 30 min, then filtered through 4 layers of gauze, 5 g of agar powder added, and deionized water added to a final volume of 1000 mL, pH 5.6-5.8. Sterilized at 121℃ for 30 min).

[0010] Preferably, the fungal cake is a 5mm diameter fungal cake of the fungus ZhuHN-06 PDA of the genus Cladosporium.

[0011] Compared with the prior art, the present invention has the following beneficial effects: This study investigated the effects of a Cladosporium strain (ZhuHN-06) on the germination promotion of *Anoectochilus roxburghii* seeds. ZhuHN-06 significantly promoted seed germination and root growth after germination, making it a Cladosporium strain with potential for promoting orchid growth. Exploring the germination-promoting ability of this strain can effectively address the difficulty in seed germination of orchids in artificial cultivation, further promoting the conservation and development of orchid resources and facilitating the industrialization of orchid cultivation. Attached Figure Description

[0012] Figure 1 Morphological identification of the ZhuHN-06 strain of this invention; Figure 2 This image shows the results of 90 days of symbiotic culture between strain ZhuHN-06 of this invention and seeds of *Cymbidium goeringii*. Detailed Implementation

[0013] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0014] Please see Figure 1and Figure 2 This study investigated a *Cladosporium* fungus and its application in promoting seed germination in *Phyllostachys edulis*, including the symbiotic fungus on the roots of *Phyllostachys edulis* and the seeds. The symbiotic fungus on the roots of *Phyllostachys edulis* was *Cladosporium* fungus ZhuHN-06, and its specific culture process is as follows: (1) Take out the seeds of Oryza sativa on the clean bench, use a sterile spatula to make a seed suspension, use a pipette to draw 5 mL of the seed suspension, and calculate the seed density on a hemocytometer (repeat 3 times). (2) Pour the OMA medium into a 9cm plate, use a 0.5cm punch to punch the mycelium cake in the center of the OMA medium, and then use a pipette to draw 5mL of seed suspension and evenly inject it around the mycelium cake. (3) Place them in an artificial climate chamber with conditions of 25±1℃, humidity of 75%, light intensity of 6000Lx, and 12h light / 12h dark for cultivation.

[0015] The seeds are those of the Chinese bamboo orchid.

[0016] The seed suspension consisted of *Cymbidium goeringii* seeds with 1 g / L of sterile agar solution.

[0017] The OMA medium was oat medium (5 g of pure oats, added to 900 mL of deionized water and boiled for 30 min, then filtered through 4 layers of gauze, 5 g of agar powder was added, and deionized water was added to bring the volume to 1000 mL, pH 5.6-5.8. After sterilization at 121℃ for 30 min).

[0018] Among them, the fungal cake is a 5mm diameter fungal cake of the fungus ZhuHN-06 PDA of the genus Cladosporium.

[0019] 1. Activation of strain ZhuHN-06: The mycelium of the genus *Cladosporium* ZhuHN-06, preserved in a -80℃ glycerol cryopreservation tube, was inoculated onto a PDA medium plate using the mycelium picking method and placed in a 25℃ incubator for activation. After 3 days, a 5 mm diameter mycelial cake was taken from the edge of the colony using a punch and inoculated into a new PDA medium, which was then placed in a 25℃ incubator for cultivation. 2. Morphological identification of strain ZhuHN-06: Morphological observation was performed on the above strains after culturing them on PDA plates for 5 days. The hyphae of ZhuHN-06 were white, smooth, dense, and opaque. Figure 1 A). The colonies are large and thick, with no obvious aerial hyphae. A bright red soluble pigment is produced on the reverse side of the colonies (…). Figure 1 B); 3. Molecular identification of ZhuHN-06: Molecular identification of the fungus ZhuHN-06 requires obtaining its DNA. Total bacterial genomic DNA was extracted using the CTAB method. The ITS sequence of strain ZhuHN-06 was amplified using universal primers ITS1 and ITS4 for fungi. Primer sequences: Sequencing of the PCR products showed that the ITS sequence of strain ZhuHN-06 consisted of 520 bases. BLAST (NCBI) alignment analysis showed that the ITS sequence of strain ZhuHN-06 (as shown in SEQ ID NO.1) had 99% homology with Cladosporium fungi. 4. Obtaining Bamboo Leaf Orchid Seeds and Seed Suspension: Select plump and mature capsules of *Cymbidium goeringii*, collect them in tea bags, and put them in a sealed bag together with silica gel desiccant. Take out the *Cymbidium goeringii* seeds on a clean bench, add the seeds to a 1 g / L sterile agar solution with a sterile spatula and shake well to make a seed suspension. Use a pipette to take 5 mL of the seed suspension and calculate the seed density on a hemocytometer. Adjust the concentration to 1×104 cells / mL. 5. Co-culture of seed suspension and fungi: Five days after taking culture 1 above, take a 5 mm diameter mycelial disc from the edge of the colony and inoculate it in the center of OMA medium. Then, use a pipette to draw 5 mL of seed suspension and evenly pour it around the mycelial disc. Incubate in an artificial climate chamber with conditions of 25±1℃, 75% humidity, 6000 Lx light intensity, and 12 h light / 12 h dark. 6. The promoting effect of ZhuHN-06 on the germination of *Cymbidium goeringii* seeds: The observation of *Cymbidium goeringii* seeds and endophytic fungi after 90 days of co-culture on OMA medium. Seed germination criteria for *Cymbidium goeringii* are shown in Table 1 below. Stage Description Stage 0 No embryo growth Stage 1 The embryo grows larger, and the seed coat ruptures. Stage 2 Appearance of promeristem Stage 3 Forming a protocorm Stage 4 Rhizoids begin to form on the surface of the protocorm. Stage 5 The protocorm differentiates into a seedling. In terms of germination rate statistics, the germination rate of the control group (CK) was 36%. Figure 2 A), strain ZhuHN-06 had a germination rate as high as 93%, significantly promoting seed germination. Figure 2 B). Regarding the Standardized Growth Index (GI) values, strain ZhuHN-06 had the highest GI value of 2.62±0.02, while the control group had only 1.37±0.05, showing a highly significant difference (P<0.01).

[0020] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0021] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A fungus of the genus Cladosporium and its application in promoting the germination of *Cymbidium goeringii* seeds, characterized in that... This includes the symbiotic fungi and seeds of *Cymbidium goeringii* roots. The symbiotic fungi are *Cladosporium* fungi ZhuHN-06, and the specific cultivation process is as follows: (1) Take out the seeds of Oryza sativa on the clean bench, use a sterile spatula to make a seed suspension, use a pipette to draw 5 mL of the seed suspension, and calculate the seed density on a hemocytometer (repeat 3 times). (2) Pour the OMA medium into a 9cm plate, use a 0.5cm punch to punch the mycelium cake in the center of the OMA medium, and then use a pipette to draw 5mL of seed suspension and evenly inject it around the mycelium cake. (3) Place them in an artificial climate chamber with conditions of 25±1℃, humidity of 75%, light intensity of 6000Lx, and 12h light / 12h dark for cultivation.

2. The *Cladosporium* fungus according to claim 1 and its application in promoting the germination of *Cymbidium goeringii* seeds, characterized in that: The seeds are those of the Chinese bamboo orchid.

3. The *Cladosporium* fungus according to claim 1 and its application in promoting the germination of *Cymbidium goeringii* seeds, characterized in that: The seed suspension is made by adding 1 g / L sterile agar solution to the seeds of *Cymbidium goeringii*.

4. The *Cladosporium* fungus according to claim 1 and its application in promoting the germination of *Cymbidium goeringii* seeds, characterized in that: The OMA medium was oat medium (5 g of pure oats, added to 900 mL of deionized water and boiled for 30 min, then filtered through 4 layers of gauze, 5 g of agar powder was added, and deionized water was added to bring the volume to 1000 mL, pH 5.6-5.

8. After sterilization at 121℃ for 30 min).

5. The *Cladosporium* fungus according to claim 1 and its application in promoting the germination of *Cymbidium goeringii* seeds, characterized in that: The fungal cake was a 5mm diameter fungal cake of the fungus ZhuHN-06 PDA of the genus Cladosporium.

Images