Umbelliferella type-2 fungus and application thereof in promoting germination of arundina graminifolia seeds

Abstract

The invention relates to the technical field of microorganisms, and discloses a type-2 umbelliflora fungus and application thereof in promoting germination of arundina graminifolia seeds, the type-2 umbelliflora fungus comprises arundina graminifolia root symbiotic bacteria and required seeds, the arundina graminifolia root symbiotic bacteria are type-2 umbelliflora, the seeds are arundina graminifolia seeds, and the specific culture process is as follows. According to the type-II umbelliferella sp. Fungus and the application thereof in promoting germination of arundina graminifolia seeds, ZhuHN-04 has a remarkable promoting effect on germination of the arundina graminifolia seeds and is the type-II umbelliferella sp. Fungus with arundina graminifolia growth promoting potential, and research on the growth promoting potential and the application method of the strain can effectively solve the problem that in artificial cultivation, the growth of the arundina graminifolia seeds cannot be promoted. According to the method, the problem that orchid seeds are difficult to germinate is solved, conservation and development of orchid resources are further promoted, industrial development of orchid is promoted, the orchid is better applied to arundina graminifolia cultivation and production practice, and theoretical and technical support is provided for promotion of arundina graminifolia industry development.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a type II umbrella fungus and its application in promoting the germination of *Cymbidium goeringii* seeds. Background Technology

[0002] Orchids, a large family widely distributed globally, are unusually endangered, primarily due to their tiny seeds, consisting only of undifferentiated proembryos, and their large roots with underdeveloped root hairs. Under natural conditions, their seed germination rate is extremely low, requiring symbiotic relationships with specific fungi to form endomycorrhizae and mycelial masses. These fungi provide nutrients to the embryoless seeds, promoting normal germination. Subsequent growth also necessitates interaction with symbiotic 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 from the seed stage. *Dendrobium nobile*, a terrestrial medicinal orchid belonging to the genus *Dendrobium*, is monotypic in China. Utilizing the diverse and specific symbiotic relationships between orchids and fungi, symbiotic germination techniques are of great significance for the conservation and industrialization of *Dendrobium nobile*.

[0003] Type II Apiaceae belongs to the class Apiaceae, order Apiaceales, family Apiaceae. 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, it is necessary to propose a type II umbrella fungus 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 type II umbrella fungus and its application in promoting the germination of *Cymbidium goeringii* seeds, which has the advantages of [the invention] and solves the problems mentioned in the background art.

[0006] This invention provides the following technical solution: a type II *Umbelliferae* fungus and its application in promoting the germination of *Cymbidium goeringii* seeds, comprising a root symbiotic fungus of *Cymbidium goeringii* and the desired seeds, wherein the root symbiotic fungus of *Cymbidium goeringii* is a type II *Umbelliferae* fungus, and the seeds are *Cymbidium goeringii* seeds. The specific cultivation process is as follows:

[0007] (1) Take out the seeds of Bamboo Leaf Orchid on the ultra-clean table and use a sterile spatula to make a seed suspension;

[0008] (2) Use a pipette to draw 5 mL of seed suspension and calculate the seed density on a hemocytometer (repeat 3 times);

[0009] (3) 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.

[0010] (4) Place them in an artificial climate chamber with conditions of 25±1℃, 75% humidity, 6000Lx light intensity, and 12h light / 12h dark for cultivation.

[0011] Preferably, the seed suspension is composed of *Cymbidium goeringii* seeds with 1 g / L of sterile agar solution.

[0012] Preferably, the OMA culture medium is an oat culture medium.

[0013] Preferably, the oat culture medium is as follows: 5g of pure oats are added to 900mL of deionized water and boiled for 30min. After filtration through 4 layers of gauze, 5g of agar powder is added, and deionized water is added to bring the volume to 1000mL. The pH value is 5.6-5.8. The mixture is then sterilized at 121℃ for 30min.

[0014] Preferably, the fungal cake is a 5mm diameter fungal cake of Umbelopsis dimorpha ZhuHN-04PDA culture.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This study investigated the application of a type II *Apis cerana* fungus in promoting seed germination of *Cymbidium goeringii*. ZhuHN-04 significantly promoted seed germination in *Cymbidium goeringii*, demonstrating its potential to enhance *Cymbidium goeringii* growth. Research on the growth-promoting potential and application methods 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, facilitating the industrialization of orchids, and enabling their better application in *Cymbidium goeringii* cultivation. This research provides theoretical and technical support for the development of the *Cymbidium goeringii* industry. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram illustrating the morphological identification of the ZhuHN-04 strain of the present invention.

[0019] Figure 2This is a schematic diagram showing the results of 90 days of symbiotic culture between strain ZhuHN-04 of this invention and seeds of *Cymbidium goeringii*. Detailed Implementation

[0020] 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.

[0021] Please see Figure 1 and Figure 2 A type II *Apigeninus* fungus and its application in promoting the germination of *Cymbidium goeringii* seeds, including the root symbiotic fungus of *Cymbidium goeringii* and the desired seeds, wherein the root symbiotic fungus of *Cymbidium goeringii* is a type II *Apigeninus* fungus, and the seeds are *Cymbidium goeringii* seeds. The specific culture process is as follows:

[0022] (1) Take out the seeds of Bamboo Leaf Orchid on the ultra-clean table and use a sterile spatula to make a seed suspension;

[0023] (2) Use a pipette to draw 5 mL of seed suspension and calculate the seed density on a hemocytometer (repeat 3 times);

[0024] (3) 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.

[0025] (4) Place them in an artificial climate chamber with conditions of 25±1℃, 75% humidity, 6000Lx light intensity, and 12h light / 12h dark for cultivation.

[0026] As a preferred embodiment of the present invention, the seed suspension is composed of *Cymbidium goeringii* seeds with 1 g / L of sterile agar solution.

[0027] As a preferred embodiment of the present invention, the OMA culture medium is an oat culture medium, which is specifically prepared as follows: 5g of pure oats are added to 900mL of deionized water and boiled for 30min. After filtration through 4 layers of gauze, 5g of agar powder is added, and deionized water is added to bring the volume to 1000mL. The pH value is 5.6-5.8, and the mixture is sterilized at 121℃ for 30min.

[0028] As a preferred embodiment of the present invention, the fungal cake is a 5mm diameter fungal cake of Umbelopsis dimorpha ZhuHN-04PDA culture.

[0029] Among them; 1. Activation of strain ZhuHN-04

[0030] The mycelium of Umbelopsis dimorpha ZhuHN-04, which was stored in a -80℃ glycerol cryopreservation tube, was inoculated onto a PDA medium plate and placed in a 25℃ incubator for activation. After 3 days, a 5mm 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.

[0031] 2. Morphological identification of strain ZhuHN-04

[0032] Morphological observation was performed on the above strains after culturing them on PDA plates for 5 days. The hyphae of ZhuHN-04 were white, slightly raised in the center. Colonies were round, smooth, moist, and contained water droplets. A ring of sticky substance formed in the center of the colony, and the colonies were clear and transparent (see details). Figure 1 A) No soluble pigments are produced on the reverse side of the colony (see details). Figure 1 B);

[0033] 3. Molecular identification of ZhuHN-04

[0034] To perform molecular identification of the fungus ZhuHN-04, its DNA needs to be obtained. Total genomic DNA of the bacteria is extracted using the CTAB method, and the ITS sequence of the ZhuHN-04 strain is amplified using the universal fungal primers ITS1 and ITS4.

[0035] 4. Obtaining Bamboo Leaf Orchid Seeds and Seed Suspension

[0036] Select plump and mature capsules of *Cymbidium goeringii*, collect them in tea bags, and put them in a sealed bag 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 seeds / mL.

[0037] 5. Co-culture of seed suspension and fungi

[0038] Five days after the culture in step 1 above was taken, a 5 mm diameter mycelium was taken from the edge of the colony and inoculated into the center of the OMA medium. Then, 5 mL of seed suspension was taken with a pipette and evenly injected around the mycelium. The medium was then placed in an artificial climate chamber with conditions of 25±1℃, 75% humidity, 6000 Lx light intensity, and 12 h light / 12 h dark for cultivation.

[0039] 6. The promoting effect of ZhuHN-04 on the germination of Oryza sativa seeds

[0040] 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:

[0041] Table 1. Evaluation Criteria for Seed Germination

[0042]

[0043] In terms of germination rate statistics, the germination rate of the control group (CK) was 36% (see details). Figure 2 A) The germination rate of strain ZhuHN-04 is as high as 90%, significantly promoting seed germination (see details). Figure 2 B) Regarding the Standardized Growth Index (GI) value, strain ZhuHN-04 had the highest GI value of 2.57±0.12, while the control group had only 1.37±0.05, showing a significant difference (P<0.01).

[0044] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A type II umbrella-shaped fungus and its application in promoting the germination of *Cymbidium goeringii* seeds, comprising *Cymbidium goeringii* root symbiotic fungi and desired seeds, characterized in that: The symbiotic fungus on the roots of *Bamboo Leaf Orchid* is *Umbelliferae* type II, and the seeds are *Bamboo Leaf Orchid* seeds. The specific cultivation process is as follows: (1) Take out the seeds of Oryza sativa on the clean bench and make a seed suspension using a sterile spatula; (2) Use a pipette to draw 5 mL of seed suspension and calculate the seed density on a hemocytometer (repeat 3 times). (3) 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. (4) 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 type II umbrella-shaped 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 of sterile agar solution to the seeds of *Cymbidium goeringii*.

3. The type II umbrella-shaped fungus according to claim 1 and its application in promoting the germination of *Cymbidium goeringii* seeds, characterized in that: The OMA medium is oat medium.

4. The type II umbrella-shaped fungus according to claim 3 and its application in promoting the germination of *Cymbidium goeringii* seeds, characterized in that: The oat culture medium is specifically prepared as follows: 5g of pure oats are added to 900mL of deionized water and boiled for 30 minutes. After filtration through 4 layers of gauze, 5g of agar powder is added, and the volume is adjusted to 1000mL with deionized water. The pH value is 5.6-5.

8. The mixture is then sterilized at 121℃ for 30 minutes.

5. The type II umbrella-shaped fungus according to claim 1 and its application in promoting the germination of *Cymbidium goeringii* seeds, characterized in that: The mycelium was a 5mm diameter mycelium from a culture of Umbelopsis dimorpha ZhuHN-04PDA.

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