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

Abstract

The invention relates to the technical field of microorganisms, and discloses a mortierella fungus and application thereof in promoting germination of arundina graminifolia seeds, the mortierella fungus comprises arundina graminifolia root symbiotic bacteria and seeds, the arundina graminifolia root symbiotic bacteria are the mortierella fungus ZhuHN-09, 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; the method comprises the following steps: preparing seeds into a seed suspension by using a sterile medicine spoon, sucking 5mL of the seed suspension by using a pipette, and calculating the seed density on a blood cell counting plate (repeating for three times); the mortierella fungus and the application thereof in promoting germination of arundina graminifolia seeds can promote growth of roots after seed germination and is a mortierella fungus strain with orchid growth promoting potential, the ability of the strain in promoting germination of arundina graminifolia seeds is explored, the problem that orchid seeds are difficult to germinate in artificial cultivation can be effectively solved, and the yield of orchids is increased. The conservation and development of orchid plant resources are further promoted, and the industrial development of orchid plants is promoted.

Description

Technical Field

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

[0002] Orchids, a large family widely distributed globally, are unusually endangered. The main reason is that orchid seeds are extremely small, consisting only of an undifferentiated proembryo. While their roots are large, root hairs are underdeveloped, resulting in a very low germination rate in the wild. They require symbiotic fungi to form endomycorrhizae and mycelial masses, providing nutrients to the embryoless seeds and promoting normal germination. During subsequent growth, they also need to interact with symbiotic fungi to meet the plant's needs for water and nutrients. Symbiotic fungi play a crucial role in the life cycle of orchids, providing the necessary C and N for seed germination from the seed stage onwards. Compounds containing elements such as phosphorus and phosphorus (P) are present. *Arundina basilica*, a terrestrial medicinal orchid belonging to the genus *Arundina* of the family Orchidaceae, is monotypic in China. Utilizing the diverse and specific symbiotic relationships between orchids and fungi, symbiotic germination technology is of great significance for the conservation and industrialization of *Arundina basilica*. *Mortierella* fungi belong to the phylum Mucoromycota, class Zygomycetes, order Mortierellales, and family Mortierellaceae. These fungi are widely distributed, and numerous studies have focused on the diverse chemical substances they produce to obtain substances with multiple biological activities. However, research on the use of this fungus to promote orchid seed germination and plant growth is relatively rare.

[0003] Based on this, the present invention provides a strain of the genus *Morchella* and its application in promoting the germination of *Cymbidium goeringii* seeds. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a strain of the genus *Morchella* and its application in promoting the germination of *Cymbidium goeringii* seeds. It possesses the advantages of using *Morchella* fungi to promote the germination of *Cymbidium goeringii* seeds and solves the problems mentioned in the background art.

[0005] This invention provides the following technical solution: a strain of *Morchella* fungus 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 *Morchella* fungus ZhuHN-09, 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.

[0006] Preferably, the seed is a bamboo orchid seed.

[0007] Preferably, the seed suspension is composed of *Cymbidium goeringii* seeds with 1 g / L of sterile agar solution.

[0008] Preferably, the OMA culture medium is an oat culture medium, which is made by boiling 5 g of pure oats in 900 mL of deionized water for 30 min, filtering with 4 layers of gauze, adding 5 g of agar powder, adding deionized water to make up to 1000 mL, setting the pH value to 5.6-5.8, and then sterilizing at 121°C for 30 min.

[0009] Preferably, the fungal cake is a 5mm diameter fungal cake of the genus *Morchella* ZhuHN-09PDA.

[0010] Compared with the prior art, the present invention has the following beneficial effects: This study investigated the application of a *Morchella* strain, ZhuHN-09, in promoting the germination of *Orchidonia spp.* seeds. ZhuHN-09 significantly promoted seed germination and root growth after germination, making it a *Morchella* strain with potential for promoting orchid growth. Exploring the ability of this strain to promote *Orchidonia spp.* seed germination can effectively address the difficulty in seed germination in artificial cultivation of orchids, further promoting the conservation and development of orchid resources and facilitating the industrialization of orchid cultivation. Attached Figure Description

[0011] Figure 1 This is a morphological identification diagram of the ZhuHN-09 strain of the present invention; Figure 2 This image shows the results of 90 days of symbiotic culture between strain ZhuHN-09 of this invention and seeds of *Cymbidium goeringii*. Detailed Implementation

[0012] 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.

[0013] Please see Figure 1 and Figure 2 A strain of *Morchella* fungus and its application in promoting the germination of *Dendrobium nobile* seeds, including the root symbiotic fungus and seeds of *Dendrobium nobile*. The root symbiotic fungus of *Dendrobium nobile* is *Morchella* fungus ZhuHN-09, 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.

[0014] The seeds are those of the Chinese bamboo orchid.

[0015] The seed suspension consisted of *Cymbidium goeringii* seeds with 1 g / L of sterile agar solution.

[0016] Among them, OMA medium is oat medium. The oat medium is made by boiling 5 g of pure oats in 900 mL of deionized water for 30 min, filtering it through 4 layers of gauze, adding 5 g of agar powder, adding deionized water to make up to 1000 mL, pH value 5.6-5.8, and then sterilizing at 121℃ for 30 min.

[0017] Among them, the fungal cake is a 5mm diameter fungal cake of the fungus ZhuHN-09PDA of the genus *Morchella*.

[0018] 1. Activation of strain ZhuHN-09: The mycelium of the genus *Morchella* ZhuHN-09, 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-09: Morphological observation was performed on the above strains after culturing them on culture plates for 5 days. The main body of the ZhuHN-09 mycelium was white, with vegetative hyphae growing into the substrate and aerial hyphae connecting to form a thin, net-like colony. Figure 1 A), conidiophores are spherical, both branched and unbranched, and no soluble pigments are observed on the reverse side of the colony ( Figure 1 B); 3. Molecular identification of strain ZhuHN-09: To perform molecular identification of the fungus ZhuHN-09, it is necessary to obtain its DNA. The total genomic DNA of the bacteria is extracted using the CTAB method, and the strain ITS sequence of ZhuHN-09 is amplified using the universal primers ITS1 and ITS4 for fungi. Primer sequences:

[0019] Sequencing of the PCR products showed that the ITS sequence of strain ZhuHN-09 consisted of 577 bases. BLAST (NCBI) alignment analysis showed that the ITS sequence of strain ZhuHN-09 (as shown in SEQ ID NO.1) had 99% homology with *Morchella* 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-09 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. Table 1. Evaluation Criteria for Seed Germination

[0020] In terms of germination rate statistics, the germination rate of the control group (CK) was 36%. Figure 2 A), strain ZhuHN-09 had a germination rate as high as 92%, significantly promoting seed germination. Figure 2 B). Regarding the Standardized Growth Index (GI) values, strain ZhuHN-09 had the highest GI value of 2.52±0.07, while the control group had only 1.37±0.05, showing a highly significant difference (P<0.01) (Table 2). Table 2 Results of 90-day co-culture of strain ZhuHN-09 with *Orchidonia javanica* seeds

[0021] 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.

[0022] 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 fungus of the genus *Morchella* and its application in promoting the germination of *Cymbidium goeringii* seeds, characterized in that... This includes the symbiotic fungus and seeds of *Cymbidium goeringii* roots. The symbiotic fungus is *Morchella* fungus ZhuHN-09, and its 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 *Morchella* fungus according to claim 1 and its application in promoting the germination of *Orchidonia spp.* seeds, characterized in that: The seeds are those of the Chinese bamboo orchid.

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

4. The *Morchella* fungus according to claim 1 and its application in promoting the germination of *Orchidonia spp.* seeds, characterized in that: The OMA medium is an oat medium, which is made by boiling 5 g of pure oats in 900 mL of deionized water for 30 min, filtering with 4 layers of gauze, adding 5 g of agar powder, adding deionized water to make up to 1000 mL, setting the pH to 5.6-5.8, and then sterilizing at 121℃ for 30 min.

5. The *Morchella* fungus according to claim 1 and its application in promoting the germination of *Orchidonia spp.* seeds, characterized in that: The fungal cake was a 5mm diameter fungal cake of the genus *Morchella* ZhuHN-09PDA.

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