Fungus for promoting growth and flavonoid content accumulation of dendrobium seedlings and application thereof

Abstract

The application discloses a fungus for promoting growth and flavonoid content accumulation of dendrobium officinale seedlings, wherein the strain is Serendipita sp. WX7, the preservation number is CGMCC NO. 40596, the preservation center is the General Microbiological Center of China, the preservation date is May 29, 2023, the preservation address is No. 3, Xibinxi Road, Chaoyang District, Beijing, and the classification name is Serendipita sp. The fungus has a significant promoting effect on the growth and development of dendrobium officinale seedlings and secondary metabolites, and provides an excellent strain resource for solving the bottleneck problem of low content of effective components of dendrobium officinale in artificial cultivation by using mycorrhizal symbiotic technology.

Description

Technical Field

[0001] This invention relates to the field of Dendrobium symbiotic fungi screening technology, and more specifically to a fungus that promotes the growth of Dendrobium seedlings and the accumulation of flavonoids, and its application. Background Technology

[0002] Dendrobium spp. is a large genus of epiphytic orchids, with over 1100 species discovered worldwide, widely distributed in tropical and subtropical Asia and Oceania. In my country, it is mainly distributed in provinces south of the Qinling Mountains, especially in southern Yunnan. Many Dendrobium species are used in traditional Chinese medicine, such as Dendrobium officinale, which is included in all editions of the Chinese Pharmacopoeia. Modern research shows that Dendrobium plants contain various types of chemical components, including polysaccharides and alkaloids, and possess anti-cancer, anti-aging, and anti-platelet aggregation effects. As an important medicinal plant resource in my country, Dendrobium plants have significant economic and scientific research value.

[0003] Dendrobium species grow slowly and have poor natural regeneration capabilities. Over-harvesting and habitat destruction by humans have led to the severe endangerment of wild resources for these species. Therefore, the regeneration and protection of Dendrobium species, especially medicinal Dendrobium resources, has become a focus of widespread attention.

[0004] Under natural conditions, the germination of orchid seeds, seedling growth, and reproduction of mature plants all depend on the symbiotic fungi they live with. Therefore, fungi, as a key limiting factor in the artificial cultivation, introduction, domestication, resource regeneration, and conservation of orchids, have attracted considerable attention from scholars both domestically and internationally. It has been reported that orchid mycorrhizal fungi are mostly classified into genera such as *Sebacina*, *Serendipita*, *Tulasnella*, and *Ceratobasidum*. Screening for culturable fungi capable of forming a symbiotic relationship with *Dendrobium* seedlings is a crucial prerequisite and guarantee for applying mycorrhizal symbiosis technology to the cultivation and resource regeneration of endangered medicinal orchids. Therefore, how to isolate and screen fungi that form a symbiotic relationship with *Dendrobium* seedlings is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a fungus that promotes the growth of Dendrobium seedlings and the accumulation of flavonoids, and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A fungus that promotes the growth of Dendrobium seedlings and the accumulation of flavonoids, the strain being *Serendipita sp.* WX7, with accession number CGMCC NO.40596, deposited at the China General Microbiological Culture Collection Center on May 29, 2023, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and classified as *Serendipita sp.*.

[0008] As an inventive concept with the same technical solution described above, this invention also seeks protection for the use of the aforementioned non-spore-forming cereus WX7 in the preparation of microbial preparations that promote the growth of Dendrobium seedlings and the accumulation of flavonoids.

[0009] Preferably, the non-spore-forming ...

[0010] Preferably, the non-spore-forming ...

[0011] As an inventive concept with the same technical solution described above, this invention also claims protection for a microbial preparation that promotes the growth of Dendrobium seedlings and the accumulation of flavonoids, including *Tetranychus cereus* WX7.

[0012] As an inventive concept with the same technical solution as above, this invention also claims protection for a method for preparing a microbial preparation that promotes the growth of Dendrobium seedlings and the accumulation of flavonoids. The process is as follows: fermenting strain WX7 for 2 weeks, filtering to obtain mycelium, freeze-drying the mycelium, pulverizing it, and obtaining the microbial preparation.

[0013] The mechanism of action of this fungus is currently unclear. However, based on our previous extensive research, inoculation with mycorrhizal fungi can effectively promote the high expression of genes related to carbohydrate and lipid metabolism, signal transduction, and defense responses in the host plant *Dendrobium*. In other words, the fungus can enhance the plant's absorption and utilization of nutrients, increase the content of plant hormones, and thus promote plant growth, development, and metabolite accumulation. The exact mechanism requires further experimental research.

[0014] As can be seen from the above technical solution, compared with the prior art, this invention isolated multiple strains of mycorrhizal fungi and dermal fungi from *Dendrobium officinale*, among which two fungi were identified as M4 (*Tulasnella sp.*) and WX7 (*Serendipita sp.*). Through co-culture experiments with *Dendrobium officinale* seedlings, the promoting effect of mycorrhizal fungi on the growth and development of *Dendrobium officinale* was verified. In addition to morphological changes, this experiment verified the accumulation effect of orchid fungi on the active compounds of *Dendrobium officinale* by measuring the content of flavonoids, the main active chemical substance in *Dendrobium officinale* symbiotic with orchid fungi. By analyzing the intrinsic relationship between fungi and the growth and development of Dendrobium officinale and related metabolic compounds, we discovered that a fungus, WX7 (Serendipita sp.), has a significant promoting effect on the growth and development of Dendrobium officinale seedlings and the production of secondary metabolites. This provides an excellent fungal resource for solving the bottleneck problem of low content of effective components in the artificial cultivation of Dendrobium officinale using mycorrhizal symbiosis technology. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0016] Figure 1 The attached image shows the morphological colony diagram of *Aspora cereus* WX7.

[0017] Figure 2 The attached figure is a phylogenetic tree of the non-sporangiogenic cereus WX7.

[0018] Figure 3 The attached image shows a fungal infection.

[0019] Figure 4 The attached diagram shows the symbiotic culture of *Dendrobium officinale* seedlings with *Aspora cereus* WX7; a. Symbiotic culture for 0 days; b. Symbiotic culture for 2 months; c. Symbiotic culture for 5 months;

[0020] Figure 5 The attached figure shows the effects of different fungal strains on the content of flavonoids chaftaside and rutin, the main active components of Dendrobium officinale; * indicates significant differences at the 0.05 level; ** indicates significant differences at the 0.01 level. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] culture medium

[0023] PDA medium: Peel 200g of potatoes, cut them into appropriately sized small pieces, put them in a pot, add an appropriate amount of distilled water, boil for 20 minutes, and filter. Add 20g of glucose and 12g of agar to the filtrate, and after it is fully dissolved, bring the volume to 1L. Autoclave at 121℃ for 22 minutes.

[0024] 1 / 2MS medium: To prepare 1L of medium, you will need 50ml of stock solution I, 10ml of stock solution II, 10ml of stock solution III, 0.2ml of stock solution IV, 100mg of inositol, 30g of sucrose, 7g of agar, and 200g of potato extract. After preparation, autoclave at 121℃ for 22 minutes. The preparation method for the stock solutions is as follows:

[0025] Fungal materials

[0026] Mycorrhizal fungi isolated from the roots of Dendrobium using the tissue block isolation method. The fungi, designated M4 and WX7, were identified as the mucilaginous fungus *Tulasnella* sp. and the waxy fungus *Serendipita* sp., respectively.

[0027] Example 1: Screening and identification of symbiotic fungi in Dendrobium seedlings

[0028] 1. Symbiotic culture of Dendrobium officinale seedlings and fungi

[0029] Under aseptic conditions, about 4-5 Dendrobium officinale seedlings were inserted into the culture medium, and 4-5 1.5×1.5cm mycelial blocks were added to the culture medium as the inoculation experimental group (test strains WX-7, M4). The control group was not added with mycelial blocks.

[0030] Fifteen bottles were placed in each of the control and experimental groups and cultured in a light culture chamber (12h light / 12h darkness, 24℃±1℃, light intensity 1500Lx).

[0031] Record the height of fresh seedlings, stem internode length, and stem diameter at the beginning of cultivation and 5 months after co-cultivation, and calculate the growth.

[0032] After 5 months of cultivation, the Dendrobium officinale seedlings showed a clear growth trend, which was manifested in the plants growing taller, the stems elongating, and the leaves and roots developing. The average height of plants inoculated with M4 (Tulasnella sp.) was 10.65 cm, the average stem segment length was 11.48 mm, and the average diameter was 3.60 mm, with growth rates of 44.62%, 53.25%, and 9.43%, respectively. The average height of the uninoculated control group was 9.70 cm, the average stem segment length was 11.35 mm, and the average diameter was 3.57 mm, with growth rates of 33.58%, 53.02%, and 9.06%, respectively. The average height of plants inoculated with WX7 (Serendipita sp.) was 12.83 cm, the average stem segment length was 12.58 mm, and the average diameter was 4.28 mm, with growth rates of 57.41%, 57.98%, and 41.98%, respectively. The average height of the uninoculated control group was 8.80 cm, the average stem segment length was 7.05 mm, and the average diameter was 3.38 mm, with growth rates of 46.59%, 29.07%, and 29.36%, respectively. The stem segment length of Dendrobium officinale seedlings symbiotic with WX7 (Serendipita sp.) showed the greatest change in growth rate relative to the control group.

[0033] Strain identification:

[0034] 1. Morphological characteristics of mycorrhizal fungi

[0035] Basic characteristics of strain WX7: Slow colony growth on PDA medium; milky white flocculent growth on the front of the colony; irregular colony edges and uneven thickness. Figure 1 BLAST alignment of the ITS sequence (MW432200) of this strain showed 97.66% sequence similarity to *Uncultured Serendi pitaceae* (MG707407.1). Based on morphological observation and molecular identification, it was preliminarily identified as a species of *Uncultured Serendi*. The intraribosome transcribed spacer (ITS) sequence of this strain has been deposited in the NCBI International Nucleic Acid Database, sequence number: MW432200.

[0036] Based on the BLAST alignment results, we downloaded orchid mycorrhizal fungi sequences with high affinity from GenBank and constructed a phylogenetic tree using megalinking. Figure 2 )

[0037] PCR primers for amplifying the rDNA-ITS region:

[0038] The forward primer ITS1OF-C:5'-AACTCGGCCATTTAGAGGAAG-3' is shown in SEQ ID NO.1;

[0039] The reverse primer ITS4-OF:5'-GTTACTAGGGGAATCCTTGTT-3' is shown in SEQ ID NO.2.

[0040] Example 2: Symbiotic culture of Dendrobium officinale seedlings and fungi

[0041] Under aseptic conditions, about 4-5 Dendrobium officinale seedlings were inserted into the culture medium. 4-5 mycelial blocks of about 1.5×1.5cm were placed into the culture medium as the inoculation experimental group, and the control group was not added.

[0042] Fifteen bottles were placed in each of the control and experimental groups and cultured in a light culture chamber (12h light / 12h darkness, 24℃±1℃, light intensity 1500Lx).

[0043] Record the height of fresh seedlings, stem internode length, and stem diameter at months 0 and 5 of cultivation, and calculate the growth.

[0044] After about 3 weeks of cultivation, the fungus covered the surface of the culture medium, and the infection status of the root segments was as follows. Figure 3 As shown. After 5 months of cultivation, the Dendrobium officinale seedlings showed a clear growth trend, manifested in increased plant height, elongated stem nodes, and improved leaf and root development, etc. Figure 4 As shown, the morphology of *Dendrobium officinale* seedlings in the inoculated group and the uninoculated control group differed significantly after 5 months of symbiotic culture. Specifically, the inoculated seedlings had thicker stems and darker leaves; the roots of the inoculated seedlings changed from green or light green to yellowish-brown, while the roots of the uninoculated control group seedlings remained green. Based on the morphological records of the *Dendrobium officinale* seedlings (Table 1), the average growth rate of plant height, diameter, and stem segment length of the inoculated seedlings was higher than that of the uninoculated seedlings. The average height of plants inoculated with M4 (Tulasnella sp.) was 10.65 cm, the average stem segment length was 11.48 mm, and the average diameter was 3.60 mm, with growth rates of 44.62%, 53.25%, and 9.43%, respectively. The average height of plants inoculated with WX7 (Serendipita sp.) was 12.83 cm, the average stem segment length was 12.58 mm, and the average diameter was 4.28 mm, with growth rates of 57.41%, 57.98%, and 41.98%, respectively. The average height of the uninoculated control group was 8.80 cm, the average stem segment length was 7.05 mm, and the average diameter was 3.38 mm, with growth rates of 46.59%, 29.07%, and 29.36%, respectively. Among these, the growth rate of stem segment length in Dendrobium officinale seedlings symbiotic with WX7 (Serendipita sp.) showed the greatest variation compared to the control group. Analysis of the morphological changes revealed that the plant height, stem internode length, and seedling diameter of the Dendrobium officinale seedlings in the experimental group inoculated with WX7 (Serendipitasp.) were significantly higher than those in the uninoculated control group.

[0045] Table 1 Growth rate of Dendrobium officinale seedlings after inoculation culture

[0046]

[0047] Different letters 'a' and 'b' indicate significant differences in diversity indices between regions.

[0048] Example 3: HPLC determination of flavonoids

[0049] Processing of Dendrobium officinale material and preparation of test samples: Take dried Dendrobium officinale stem samples, grind them into powder, and pass them through a No. 3 sieve. Accurately weigh 1 g of the Dendrobium officinale sample powder dried to constant weight, place it in a 500 mL round-bottom flask, accurately add 100 mL of methanol, reflux extract in an 85℃ water bath for 1 h, filter, place the filtrate in an evaporating dish, collect the residue, add another 100 mL of methanol, reflux extract in an 85℃ water bath for 1 h, filter, combine the filtrates, and evaporate to dryness in an 85℃ water bath. Dissolve the residue in methanol, make up to 5 mL, filter through a 0.22 μm microporous membrane, and use the filtrate as the test sample solution.

[0050] Preparation of mixed standards: Dissolve appropriate amounts of the standards separately in methanol to prepare concentrations of 0.537 mg / mL for shampooside and 0.506 mg / mL for rutin. Take different volumes of the single standard solutions and dilute them together to prepare a series of mixed standard solutions with varying concentrations: shampooside: 5.37–85.92 μg / mL, rutin: 5.06–253.00 μg / mL. The peak positions in the mixed standard solutions were kept relatively stable compared to the original single standard peaks.

[0051] Chromatographic conditions: Agelaitechnologies Venusil ASBC18 column (4.6 mm × 250 mm, 5 μm); mobile phase A (acetonitrile): mobile phase B (0.2% formic acid solution) gradient elution: 0-45 min: 13-16% A, 45-55 min: 16-38% A, 55-65 min: 38-45% A; detection wavelength: 340 nm, flow rate: 1 ml / min, injection volume: 5.0 μL; column temperature: 30 °C.

[0052] After 5 months of symbiotic culture between fungi and *Dendrobium officinale* seedlings, the flavonoid content was determined by HPLC. The results are shown in Table 2. The flavonoid and rutin contents of *Dendrobium officinale* seedlings symbiotic with WX7 (*Serendipitasp.*) were 115.44 ug / g and 54.99 ug / g, respectively, compared to 41.47 ug / g and 32.05 ug / g, respectively, in the uninoculated control group. The flavonoid and rutin contents of *Dendrobium officinale* seedlings symbiotic with M4 (*Tulasnella sp.*) were 26.56 ug / g and 17.04 ug / g, respectively. The significant differences in flavonoid content between the inoculated and uninoculated control groups were compared using SPSS 26.0. Figure 5 The contents of flavonoid compounds shamtoside and rutin in Dendrobium officinale seedlings symbiotic with WX7 (Serendipitasp.) were significantly higher than those in Dendrobium officinale seedlings not inoculated with the fungus. Furthermore, the contents of shamtoside and rutin in Dendrobium officinale seedlings symbiotic with WX7 (Serendipitasp.) were significantly higher than those in Dendrobium officinale seedlings symbiotic with M4 (Tulasnellasp.).

[0053] Table 2. Determination of Flavonoid Content in Inoculated Vaccines

[0054]

[0055] * indicates a significant difference at the 0.05 level; ** indicates a significant difference at the 0.01 level.

[0056] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0057] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A fungus for promoting the growth of Dendrobium seedlings and the accumulation of Shuanghu and rutin content, characterized in that, The fungus is a sporocystidia. (Serendipita sp .) WX7, with accession number CGMCC NO.40596, is deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 29, 2023, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named as follows: Serendipita sp . .

2. The use of the non-spore-forming cereus WX7 as described in claim 1 in the preparation of a microbial preparation that promotes the growth of Dendrobium seedlings and the accumulation of shampooside and rutin.

3. Use according to claim 2, characterized in that, The non-spore-forming spore-forming spore-forming spore-forming fungus WX7 increases the stem internode length and seedling diameter growth of Dendrobium seedlings.

4. A microbial preparation for promoting the growth of Dendrobium seedlings and the accumulation of Shuanghu and rutin content, characterized in that, Includes the non-spore-forming cereus WX7 as described in claim 1.

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