Angelica sinensis endophytic branchium and its isolation method and application

Abstract

This invention discloses an endophytic Cladosporium fungus of Angelica sinensis and its fermentation products. This endophytic fungus was isolated, purified, and cultured from the seeds of Angelica sinensis (Oliv.) Diels, a plant belonging to the Apiaceae family. Microbial ITS sequencing identified it as belonging to the genus Cladosporium sp. The strain is deposited at the China Center for Type Culture Collection (CCTCC), located at the School of Life Sciences, Wuhan University, Wuhan, Hubei, China, 430072, China, on June 9, 2022, with accession number CCTCCNO:M2022842. The liquid fermentation products of Cladosporium Z1 can produce flavonoids, phenols, and other active ingredients, exhibiting good DPPH free radical scavenging ability. It can also promote the germination of Angelica sinensis seeds and increase the accumulation of ligustrazine A in Angelica sinensis plants, making it suitable for the preparation of microbial fertilizers specifically for Angelica sinensis.

Description

Technical Field

[0001] This invention relates to an endophytic mycorrhizal fungus strain of Angelica sinensis, which has good antioxidant activity and promotes seed germination, plant growth and accumulation of active ingredients, belonging to the field of microbial technology. Background Technology

[0002] Endophytic fungi are a class of fungi that complete part or all of their life cycle within a plant, growing between plant tissue cells and distributed in roots, stems, leaves, flowers, and seeds, without causing any disease. Currently, over one million species of endophytic fungi have been discovered. Through long-term co-evolution, host plants and endophytic fungi have gradually formed a mutually beneficial survival relationship and a mutually dependent symbiotic structure. Endophytic fungi can exhibit multidimensional interactions within host plants. They can promote plant growth not only by producing essential nutrients (nitrogen, phosphorus, iron, etc.) or regulating plant hormone levels (auxins, cytokinins, and gibberellins, etc.), but also by stimulating the plant to produce bioactive metabolites, activating the host's defense system, and thus enhancing the host plant's resistance to biotic and abiotic stresses. Furthermore, endophytic fungi can directly or indirectly affect the content and distribution of active substances in medicinal plants by influencing the synthesis, content, and accumulation of secondary metabolites.

[0003] Endophytic fungi, widely found in the tissues and organs of medicinal plants, exhibit rich diversity. The bioactive substances produced by some endophytic fungi during their metabolism have shown great application potential and economic value in drug development and biocontrol of plant diseases, thus attracting widespread attention from scientists worldwide. The metabolites of endophytic fungi in medicinal plants are diverse, with varying biological activities, particularly antitumor, antibacterial, and antioxidant activities, which have garnered significant attention.

[0004] Angelica sinensis (Oliv.) Diels, a plant in the Apiaceae family, is a dried root that mainly contains volatile oils, ferulic acid, and water-soluble components. It possesses a wide range of pharmacological activities, including promoting blood circulation, regulating menstruation, relieving pain, and moistening the intestines to relieve constipation. In clinical treatment, Angelica sinensis plays an important role in regulating bodily functions, enhancing immunity, resisting hypoxia, inhibiting bacteria, fighting cancer, and combating arteriosclerosis. However, with the increasing market demand for Angelica sinensis, excessive use of chemical fertilizers and indiscriminate fertilization during cultivation have led to a series of problems such as declining soil quality, soil compaction, nutrient imbalance, and poor fertilizer storage capacity. These issues severely affect the yield and quality of Angelica sinensis, seriously threatening the sustainable development of the Angelica sinensis cultivation industry. Endophytic fungi not only promote the accumulation of secondary metabolites in the host but also produce various types of chemical components themselves, which is of great significance for resource protection, the development of new bio-fertilizers, and the implementation of ecological cultivation. Currently, researchers have isolated various endophytic fungi from Angelica sinensis, but research on endophytic fungi in Angelica sinensis and their applications is still in its early stages. Screening strains that can promote high-quality growth and increase the accumulation of active ingredients in Angelica sinensis is of great significance for the in-depth development of bio-fertilizers and the promotion of green and healthy planting. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to conduct in-depth screening of endophytic fungi in Angelica sinensis and cultivate a new strain of Angelica sinensis endophytic mycorrhizal fungi. This strain has good antioxidant activity and promotes seed germination, plant growth and accumulation of active ingredients.

[0006] Technical solution: To achieve the above objectives, the technical solution adopted by this invention is as follows:

[0007] A strain of endophytic cladosporium from Angelica sinensis has been deposited at the China Center for Type Culture Collection (CCTCC), School of Life Sciences, Wuhan University, Wuhan, Hubei, China, 430072, China. The deposit date was June 9, 2022, and the accession number is CCTCCNO:M 2022842. The classification name is Cladosporium sp.Z1.

[0008] A method for isolating endophytic mycorrhizal fungi of Angelica sinensis, comprising washing, inoculation, isolation, culture, and preservation, with the specific steps as follows:

[0009] a) Cleaning: Remove mud and impurities from the surface of the angelica root, then rinse the surface three times with sterile water in a laminar flow hood. Cut it into 3cm pieces with a sterilized blade, place them in sterile petri dishes, and immerse them sequentially in 75% ethanol for 1 minute, then in 5% sodium hypochlorite for 5 minutes. Rinse 4-6 times with sterile water, and blot off excess water with sterile filter paper. Spread the sterile water from the final rinse evenly onto PDA culture medium as a control for surface disinfection.

[0010] b) Isolation: Endophytic bacteria were isolated using two methods: direct cutting and grinding. Direct cutting: The contact surface of a 3cm sterilized Angelica sinensis segment was removed, and then the segment was placed into PDA medium using sterile forceps. Three samples were incubated per dish at 28°C. After hyphae grew from the edge of the tissue block, a small amount of hyphae was picked up with an inoculation loop and inoculated into fresh PDA medium. Purification was performed using the streak plating method, repeated multiple times until a single colony was formed. Grinding: The contact surface of a 3cm sterilized Angelica sinensis segment was removed, and then the sterilized segment was ground evenly using a sterile mortar and pestle. 1mL of sterile water was used to dilute the sample, and then 100μL of the diluted solution was evenly spread onto PDA medium. This treatment was repeated three times, and the samples were incubated at 28°C. Hyphae with different colors and morphologies were picked up and placed into fresh PDA medium. Purification was performed using the streak plating method, repeated multiple times until a single colony was formed.

[0011] c) Cultivation and Preservation: Pour PDA medium into test tubes, filling them approximately 1 / 5 full. Tilt the tubes and allow the medium to solidify. In a clean bench, use an inoculation loop to transfer the purified mycelia into the test tubes and inoculate using the streak method. Incubate at 28°C. Once the mycelia have grown, store in a 4°C freezer. Prepare a spore suspension from the purified fungi on the plates using sterile water. Mix the spore suspension with 50% glycerol at a 1:1 ratio in sterilized cryovials, mix thoroughly, seal with sealing film, and store in a -80°C cryovial.

[0012] A method for preparing a spore suspension of Cladosporium sp. Z1 strain from Angelica sinensis is characterized by inoculating the strain into PDA medium, activating it in a 28°C constant temperature biological incubator, and culturing for 5 days. After the culture is completed, 3 mL of sterile water is added to rinse the bacterial cells on the surface of the medium in a clean bench, and the liquid is collected to obtain the spore suspension. The concentration of the spore suspension is determined using a hemocytometer.

[0013] The fermentation culture method of Cladosporium sp. Z1 provided by the present invention includes the following steps: inoculating Cladosporium sp. Z1 into 50 mL of PDB liquid medium, activating it in a constant temperature shaker at 28°C and 160 r / min for 3 days, then taking 1 mL of the activated strain into an Erlenmeyer flask containing 500 mL of PDB liquid medium for fermentation, and culturing it in a shaker at 28°C and 160 r / min for 5 days; after the above fermentation culture, collecting the fermentation broth.

[0014] The fermentation products of Angelica sinensis endophytic spores were analyzed using fermentation broth as raw material. The broth was extracted three times with ethyl acetate, the ethyl acetate was evaporated, and the broth was redissolved with methanol. The solution was used to detect the fermentation products. The total flavonoid content was 37.35 μg / mL and the total phenol content was 393.84 μg / mL.

[0015] The present invention relates to the application of Angelica sinensis endophytic spore fungus in the preparation of antioxidant agents and in promoting the germination of Angelica sinensis seeds and plant growth. The spore suspension is obtained by the preparation method described above and co-cultured with Angelica sinensis seeds and sterile seedlings of Angelica sinensis. The germination of Angelica sinensis seeds and the growth of sterile seedlings are recorded.

[0016] Beneficial effects: Compared with existing research, the present invention has the following advantages:

[0017] (1) The Z1 strain of Cladosporium provided by this invention can directly metabolize and produce flavonoids, phenols and other components, which have good antioxidant activity and are of great significance for further research on novel antioxidants in microbial fermentation.

[0018] (2) The Z1 strain of Cladosporium provided by the present invention can promote the germination of Angelica sinensis seeds and increase the content of Angelica sinensis lactone A. It can be used to develop low-cost, pollution-free, stable-yield bio-fertilizer that can effectively improve the soil, and then applied to the green and healthy planting industry of Angelica sinensis. Attached Figure Description

[0019] Figure 1 Front view of a colony of Cladosporium Z1 strain;

[0020] Figure 2 Back view of a colony of Cladosporium Z1 strain;

[0021] Figure 3 Phylogenetic tree of Cladosporium Z1 ITS sequence;

[0022] Figure 4 Analysis of antioxidant activity of fermentation products from Cladosporium Z1;

[0023] Figure 5 Effects of Cladosporium Z1 strain on the growth of Angelica sinensis (A: plant height; B: fresh weight);

[0024] Figure 6 Effect of Cladosporium Z1 on the content of secondary metabolites of Angelica sinensis (A: Ligusticum lactone A; B: Ferulic acid). Detailed Implementation Plan

[0025] The endophytic fungus of the present invention is a strain isolated from Angelica sinensis from the Angelica sinensis Research Institute of Minxian County, Dingxi City, Gansu Province. The present invention can be better understood according to the following examples.

[0026] Example 1

[0027] A method for isolating endophytic mycorrhizal fungi of Angelica sinensis, comprising the following steps:

[0028] (1) Cleaning: Remove mud and impurities from the surface of Angelica sinensis, then rinse the surface three times with sterile water in a laminar flow hood. Cut it into 3cm pieces with a sterilized blade, place them in sterile petri dishes, and soak them in 75% ethanol for 1 minute, then in 5% sodium hypochlorite for 5 minutes. Rinse with sterile water 4-6 times, and blot off excess water with sterile filter paper. Spread the sterile water from the last rinse evenly on PDA culture medium as a control for surface disinfection.

[0029] (2) Separation: Endophytic bacteria were separated by two methods: direct cutting and grinding.

[0030] Direct segmentation: The contact surface of a 3cm sterilized segment of Angelica sinensis was removed, and then the segment was placed into PDA medium with sterile forceps, 3 samples per plate, and incubated at 28℃. After hyphae grew from the edge of the tissue block, a small amount of hyphae was picked up with an inoculation loop and inoculated into a new PDA medium. The mixture was purified by streak plating, and repeated several times until a single colony was obtained to obtain the Angelica sinensis endophytic spore strain Cladosporium sp. Z1.

[0031] Grinding treatment: The contact surface of the 3cm sterilized Angelica sinensis segment was removed, and then the sterilized Angelica sinensis segment was ground evenly with a sterile mortar and pestle. 1mL of sterile water was taken to dilute the sample, and then 100μL of the diluted solution was evenly spread on PDA medium. The treatment was repeated three times and incubated at 28℃. Hyphae with different colors and morphologies were picked and purified in new PDA medium by streak plating. The process was repeated multiple times until a single colony was obtained to obtain the Angelica sinensis endophytic spore strain Cladosporium sp.Z1.

[0032] (3) Preparation of spore suspension of Cladosporium sp. Z1 strain: Cladosporium sp. Z1 strain was inoculated into PDA medium and activated in a 28℃ constant temperature biological incubator. After 5 days of culture, the bacterial cells on the surface of the medium were rinsed with 3 mL of sterile water in a clean bench and the liquid was collected to obtain the spore suspension.

[0033] (4) Cultivation and Preservation: Pour PDA medium into test tubes, filling about 1 / 5 of the tubes. Place the tubes at an angle and allow the medium to solidify. In a clean bench, use an inoculation loop to pick up the purified mycelia into the test tubes and inoculate them using the streak method. Cultivate in a 28°C incubator. After the mycelia have grown, store them in a 4°C freezer for short-term preservation. Prepare a spore suspension from the purified fungi on the plates using sterile water. Mix the spore suspension with 50% glycerol at a 1:1 ratio in sterilized cryovials, mix thoroughly, seal with sealing film, and store long-term in an ultra-low temperature freezer at -80°C.

[0034] The PDA medium used in the isolation and culture of the strains described above consists of 200 g / L potato, 20 g / L glucose and 15 g / L agar.

[0035] Example 2

[0036] The molecular identification of Angelica sinensis endophytic fungi was performed using the following methods:

[0037] The Cladosporium sp. Z1 strain cultured in Example 1 was sent to Sangon Biotech (Shanghai) Co., Ltd. for ITS sequencing. The upstream primer ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and the downstream primer ITS4 (5'-TCCTCCGCTTATTGATATGC-3') were used. The ITS sequences were compared with those in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). Blast sequence alignment analysis showed that the Cladosporium sp. Z1 strain shared 88% homology with Cladosporium, and the strain was identified as Cladosporium.

[0038] Sequence listing

[0039]

[0040] Table 1. Sequence alignment results of strain Z1

[0041]

[0042] Example 3

[0043] Fermentation culture method of Cladosporium sp. Z1 strain of Angelica sinensis endophytic mycorrhizal fungus

[0044] (1) Take the single colony obtained by repeated purification in step (2) of Example 1 and inoculate it into 50 mL of PDB liquid medium (200 g / L potato, 20 g / L glucose). Activate and culture it in a constant temperature shaker at 28℃ and 160 r / min for 3 days. After 3 days, take 1 mL of the activated strain into an Erlenmeyer flask containing 500 mL of PDB liquid medium for fermentation. Culture in a shaker at 28℃ and 160 r / min for 5 days. After the above fermentation culture, collect the fermentation broth. Extract the fermentation broth sample three times with an equal volume of ethyl acetate, evaporate the ethyl acetate, and redissolve it in methanol.

[0045] (2) The total flavonoid content in the fermentation broth was determined to be 37.35 μg / mL using the aluminum nitrate colorimetric method with rutin standard as the reference solution.

[0046] (3) The total phenol content in the fermentation broth was determined to be 393.84 μg / mL using the Folin-Ciocalteu colorimetric method with gallic acid standard as reference solution.

[0047] Example 4 Antioxidant Activity Experiment

[0048] (1) The antioxidant activity of the fermentation products was evaluated using the DPPH free radical scavenging rate experiment. The fermentation broth of Cladosporium sp. Z1, an endophytic spore of Angelica sinensis, was serially diluted with methanol solution to prepare sample solutions with volume fractions of 100%, 50%, 25%, 12.5%, 6.25%, and 3.125%, respectively. An appropriate amount of ascorbic acid powder was accurately weighed and dissolved in ultrapure water to prepare a 1 mg / mL ascorbic acid solution as a positive control. A 0.25 mmol / L DPPH solution was prepared with anhydrous ethanol (protected from light). 50 μL of sample solutions of different concentrations and 150 μL of DPPH solution were placed in 96-well plates (50 μL methanol + 150 μL DPPH solution as blank, 50 μL sample solution + 150 μL anhydrous ethanol as control), with 3 parallel wells for each well. The plates were incubated in the dark at 37℃ for 30 min, and the absorbance was measured at 517 nm.

[0049] DPPH free radical scavenging rate (%) = [1 - (Sample A - Control A) / Blank A] × 100%

[0050] (2) Results of antioxidant activity experiments, such as Figure 4 As shown, the fermentation broth of Cladosporium sp. Z1 can achieve a 100% scavenging rate of DPPH free radicals, which is comparable to that of a 62.5 μg / mL VC solution.

[0051] Example 5: Experiment on the promotion of Angelica sinensis seed germination by Cladosporium sp. Z1, an endophytic fungus of Angelica sinensis.

[0052] (1) Select 200 plump and uniform Angelica sinensis seeds, place 100 seeds in each culture bottle, label them 1 and 2, and disinfect them sequentially with 75% ethanol for 1 min, mercuric chloride for 9 min, rinse 5 times with sterile water, and discard the sterile water. Obtain Cladosporium sp. Z1 spore suspension (4×10⁻⁶) according to step (3) in Example 1. 5 (cfu / mL) The spores were diluted and counted. The diluted Cladosporium sp. Z1 spore suspension was poured into culture flask #1, and sterile water was poured into culture flask #2. The spores were soaked for 24 hours. Afterward, the seeds were transferred to petri dishes lined with three layers of sterile filter paper, with 30-35 seeds neatly arranged in each dish. Each sample was treated three times. Approximately 5 mL of sterile water was added to fully saturate the seeds and filter paper. The petri dishes were sealed with sealing film to prevent contamination. The weight of the petri dishes and seeds was weighed and recorded.

[0053] (2) The above culture dishes were then placed in a light incubator for cultivation. The culture dishes were weighed every 24 hours to replenish the original weight, and the number of germinating seeds was recorded. The results are shown in Table 2. The spore suspension of Cladosporium sp. Z1 obtained by the present invention can promote the germination of Angelica sinensis seeds.

[0054] Table 2. Short-term germination potential and final germination rate of Angelica sinensis seeds.

[0055] 6d 8d 9d 10d 11d 13d 16d Final germination rate control group 0％ 13％ 39％ 59％ 62％ 65％ 65％ 65％ Cladosporium Z1 0％ 20％ 45％ 66％ 68％ 71％ 76％ 80％

[0056] Example 6: Effect of Cladosporium sp. Z1 strain on the growth promotion of Angelica sinensis.

[0057] Select plump and uniform-quality Angelica sinensis seeds, disinfect them sequentially with 75% ethanol for 1 min, mercuric chloride for 9 min, rinse 5 times with sterile water, discard the sterile water, and evenly place 5 seeds into MS medium per bottle. Inoculate the bottles and place them in an artificial climate chamber at 23℃, 70% humidity, 14 h / d light, and 2000 lx light intensity. After approximately 30 days of culture, Angelica sinensis seedlings with true leaves of relatively uniform size are randomly divided into two groups: a control group and a Z1 strain group, with five bottles per group and six seedlings per bottle. Add 20 μL of either blank PDB medium or Z1 strain spore suspension (4 × 10⁻⁶) to the roots of each seedling. 5 (cfu / mL). The bacterial culture was co-cultured with sterile seedlings for 7 days, and the fresh weight and plant height of the sterile Angelica sinensis seedlings were measured. Results are as follows: Figure 5 As shown, the spore suspension of Cladosporium sp. Z1 obtained by screening in this invention can promote the growth of Angelica sinensis and increase its fresh weight.

[0058] Example 7: Effect of Cladosporium sp. Z1 strain on secondary metabolites of Angelica sinensis

[0059] (1) The test solution was prepared as follows: Angelica seedlings cultured in blank PDB medium as described in Example 6 and Angelica seedlings cultured in Cladosporium sp. Z1 spore suspension were taken. The same weight of the two types of Angelica seedlings were weighed and homogenized separately. Then, 10 times the amount of 70% methanol was added to each, and the mixture was vortexed and ultrasonically extracted at 220W and 80Hz for 20 min. The mixture was then removed, centrifuged at 12000rpm for 10 min, and the supernatant was collected. The test solutions of Angelica seedlings cultured in blank PDB medium and Angelica seedlings cultured in Cladosporium sp. Z1 spore suspension were obtained.

[0060] (2) The preparation method of the reference solution is as follows: Take appropriate amounts of ferulic acid and ligustrazine A reference standards, accurately weigh them, and add 70% methanol to prepare a mixed reference solution containing 105.61 μg of ferulic acid and 154.74 μg of ligustrazine A per 1 mL.

[0061] (3) The content determination was performed under the following liquid chromatography conditions: Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm), mobile phase 0.1% formic acid water (A) - acetonitrile (B), gradient elution (0–5 min, 5%–43% B; 5–9 min, 43%–43% B; 9–12 min, 43%–70% B; 12–14 min, 70%–95% B; 14–15 min, 95%–5% B; 15–16 min, 5%–5% B. Flow rate 0.4 mL / min; column temperature 30 °C; injection volume: 2 μL.

[0062] (4) Content determination

[0063] Take the two test solutions from step (1) and the mixed reference solution from step (2) respectively, inject them into the high performance liquid chromatograph, and inject them under the chromatographic conditions of step (3). Calculate the contents of ferulic acid and ligustrol A using the one-point external standard method.

[0064] Experimental results are as follows Figure 6 As shown: The Z1 spore suspension of Cladosporium obtained by screening in this invention can increase the content of ligustilide A and ferulic acid, indicating that it has a regulatory effect on the active components of Angelica sinensis, ligustilide A and ferulic acid.

[0065] The above results demonstrate that this invention has screened and obtained a novel endophytic fungus of Angelica sinensis—Cladosporium Z1. A method for its isolation, cultivation, preservation, metabolite analysis, and growth-promoting function has been established. Cladosporium Z1 can directly metabolize and produce abundant flavonoids and phenolic components, exhibiting good antioxidant activity, providing a new source of ingredients for the preparation of pharmaceutical raw materials and functional products. Simultaneously, Cladosporium Z1 has a good function in promoting Angelica sinensis seed germination, plant growth, and regulating the accumulation of secondary metabolites, which can be used to develop related products such as bio-fertilizers, possessing significant practical application value.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of Angelica sinensis endophytic mycorrhizal fungus ( Cladosporium sp. Z1 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2022842.

2. A method of fermenting Angelica archangelica endophytic branch fungus (Epiphyton angelicae) Z1, characterized by, Cladosporium sp. comprising the steps of: ​ The Angelica endophytic spores as described in claim 1 ( Cladosporium sp. Z1 strain was inoculated into PDB liquid medium and activated in a shaker at 28°C. After culturing, a small amount of activated bacterial solution was transferred to a bottle containing PDB liquid medium for fermentation and shaker culture to obtain fermentation broth.

3. The endophytic branch fungus of Angelica sinensis of claim 2, wherein the endophytic branch fungus of Angelica sinensis is a fungus of the genus Cladosporium. Cladosporium sp. ) The fermentation method of Z1, characterized in that, comprising the steps of: The endophytic branch mycoplasma (Endophyton angelicae) of claim 1 Cladosporium sp. ) Z1 strain was inoculated in 50 mL PDB liquid medium, and activated at 28°C constant temperature shaker at 160 r / min. After 3 days, 1 mL activated bacterial solution was taken to a triangular flask containing 500 mL PDB liquid medium for fermentation, and cultured at 28°C, 160 r / min shaker for 5 days. After the above fermentation culture, the fermentation broth was collected.

4. The endophytic branch mycoplasma of Angelica sinensis of claim 2 or 3, Cladosporium sp. ) The fermentation method of Z1, characterized in that, The fermentation liquor is extracted with ethyl acetate, the ethyl acetate is evaporated, and methanol is used for redissolution to obtain a fermentation product containing 37.35 μg / mL total flavonoids and 393.84 μg / mL total phenols.

5. Use of the fermentation broth obtainable by the fermentation according to claim 2 or 3 for the preparation of an antioxidative preparation, characterized in that, The fermentation liquor is used for preparing an oxidation preparation of anti-DPPH free radicals.

6. The endophytic branch fungus (Acaulospora moriformis) of Angelica sinensis of claim 1 for use in the preparation of microbial fertilizer for promoting the germination of Angelica sinensis seeds, increasing the fresh weight of Angelica sinensis plants, and increasing the content of the ligustilide A component of Angelica sinensis. Cladosporium sp. ) Z1 in the preparation of microbial fertilizer for promoting the germination of Angelica sinensis seeds, increasing the fresh weight of Angelica sinensis plants, and increasing the content of the ligustilide A component of Angelica sinensis.

7. Use according to claim 6, characterized in that, The endophytic branch mold of Angelica sinensis is co-cultured with seeds or aseptic seedlings of Angelica sinensis.

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