Endophytic fungus Pseudosperma yunnanense JMG086 from the roots of Cibotium barometz, its isolation method and application

Abstract

The application provides a Cibotium barometz root endophytic fungus Pseudosperma yunnanense JMG086 and a separation method and application thereof belong to the technical field of bacterial disease control. The Cibotium barometz root endophytic fungus Pseudosperma yunnanense JMG086 is named as Pseudosperma yunnanense , and the preservation number is CCTCC NO: M 20251771. It is found through experiments that the Cibotium barometz root endophytic fungus Pseudosperma yunnanense JMG086 liquid fermentation broth extract has a strong inhibitory effect on the growth of Agrobacterium tumefaciens, Pectobacterium carotovorum and Ralstonia solanacearum, and can effectively prevent and control plant diseases.

Description

Technical Field

[0001] This invention relates to the field of fungal pest control technology, specifically to the endophytic fungus Pseudosperma yunnanense JMG086 found in the roots of Cibotium barometz, its isolation method, and its applications. Background Technology

[0002] Cibotium barometz, belonging to the family Cibotaceae in the phylum Ceratophyllum, is known as "Gouji" in Traditional Chinese Medicine. It is widely distributed and extensively harvested in Southeast Asia, including China, Vietnam, Japan, Thailand, Indonesia, and Malaysia. Cibotium barometz is renowned for its medicinal benefits in ancient medicine. Even today, its hairs are a key ingredient in natural hemostatic ointments. Furthermore, the rhizome of Cibotium barometz possesses anti-inflammatory, analgesic, and kidney-tonifying properties, used to treat rheumatism, lower back pain, sciatica, dysuria, polyuria, and abnormal vaginal discharge in the elderly. According to Traditional Chinese Medicine theory, it helps strengthen bones and tendons. In this respect, Cibotium barometz is one of the most commonly used herbs in Chinese prescriptions for treating osteoporosis.

[0003] Plant endophytic fungi are a class of fungi that live within healthy plant tissues without causing obvious disease symptoms. Through long-term co-evolution, they have formed a mutually beneficial symbiotic relationship with their hosts. In biocontrol, endophytic fungi play a crucial role: they can produce various secondary metabolites with antibacterial and insecticidal activities, thereby enhancing plant resistance to pathogens and pests; simultaneously, they can induce systemic resistance in plants, improving their overall defense level. Furthermore, some endophytic fungi can help plants resist abiotic stresses (such as drought and heavy metal pollution), indirectly improving plant health and survival ability. Particularly noteworthy is the novel structure and diverse functions of terpenoids and other active substances synthesized by endophytic fungi, which not only have significant effects in antibacterial and insecticidal activity but also provide important resources for the development of biopesticides. Therefore, endophytic fungi have broad application prospects in green agriculture and eco-friendly disease management.

[0004] Pantoea agglomerans is a Gram-negative bacterium widely distributed in the natural environment, belonging to the family Enterobacteriaceae. This bacterium can act as a plant pathogen, an opportunistic pathogen in humans, and some strains are even beneficial to plant growth. As a plant pathogen, Pantoea agglomerans poses a serious threat to many crops, primarily affecting crop growth and development, reducing yield and quality. Pantoea agglomerans can infect various crops, including rice, eucalyptus, and pine. In rice, this bacterium mainly affects grain filling and development; rice is susceptible to this bacterium from the booting stage to the early heading stage.

[0005] *Pectobacterium carotovorum* (originally *Erwinia carotovora*) is an important plant pathogenic bacterium belonging to the genus *Pectobacterium*. This bacterium is known for its powerful tissue maceration ability, causing soft rot in various plants and resulting in devastating losses. The main symptoms of diseases caused by *Pectobacterium carotovorum* are soft rot and gumming. In 2014-2015, a devastating bacterial soft rot outbreak occurred on cucumber stems and leaves in Shandong, Shanxi, Hebei, Henan, and Liaoning provinces of China, causing severe economic losses to the cucumber industry.

[0006] Ralstonia solanacearum (originally Pseudomonas solanacearum) is one of the most destructive plant pathogens in the world, belonging to the class Betaproteobacteria. It causes bacterial wilt, resulting in enormous economic losses globally, particularly affecting solanaceous crops. This bacterium severely damages solanaceous crops worldwide (tomatoes, potatoes, peppers, etc.), with losses reaching up to 90% in tropical / subtropical regions.6 Typical symptoms of bacterial wilt have been observed in eggplants and tomatoes in four villages in eastern Sikkim, India (Assam Rinzai, Padmchen, Sazong, and Sintang, the state's main vegetable-growing areas), leading to significant yield reductions of 10-90%.

[0007] In crop cultivation, long-term reliance on chemical pesticides has led to increasing resistance in pathogens, significantly reducing the effectiveness of chemical control. Against this backdrop, biological control has demonstrated significant application value and development potential. In particular, the development of biopesticides using endophytic fungi has become a highly promising control strategy in green agriculture. Many endophytic fungi can live in symbiosis with host plants and produce antibiotic-like active substances, significantly enhancing the plant's resistance to pathogens. These natural products not only effectively inhibit plant pathogenic fungi and bacteria but also have inhibitory effects on some protozoa, exhibiting diverse mechanisms of action and good environmental compatibility. Therefore, screening and developing novel antibiotic-like substances from endophytic fungi has become an important direction in the creation of biopesticides, with profound significance for promoting sustainable agricultural development, ensuring food safety, and reducing environmental pollution. It also shows broad application prospects in the medical and food industries. Summary of the Invention

[0008] The purpose of this invention is to propose an endophytic fungus *Pseudosperma yunnanense* JMG086 from the roots of *Cibotium barometz*, its isolation method, and its application. *Pseudosperma yunnanense* JMG086 can effectively inhibit the growth of *Pantoea agglomerans*, *Pectobacterium carotovorum*, and *Ralstonia solanacearum*, thus achieving a certain degree of biological control.

[0009] The technical solution of this invention is implemented as follows:

[0010] This invention provides an endophytic fungus from the roots of Cibotium barometz, Pseudosperma yunnanense JMG086. The fungus is classified as Pseudosperma yunnanense, deposited at the China Center for Type Culture Collection (CCTCC) on August 4, 2025, with accession number CCTCC NO: M 20251771.

[0011] This invention further protects a method for isolating the endophytic fungus Pseudosperma yunnanense JMG086 from the roots of Cibotium barometz, comprising the following steps:

[0012] S1. After disinfecting and drying the root hairy part of the perennial root of Cibotium barometz, cut it into tissue blocks. Place the tissue blocks into centrifuge tubes, add small steel balls and sterile water, and crush them. Then dilute the crushed solution, spread it on CMNMK solid medium, and culture it. After the colonies grow, select single colonies.

[0013] S2. Single colonies were transferred to PDA solid medium and cultured. After incubation, the strain was molecularly identified, and the endophytic fungus Pseudosperma yunnanense JMG086 from the roots of Cibotium barometz was obtained.

[0014] As a further improvement of the present invention, the formula of the CMNMK culture medium in step S1 is as follows: 1-3 g / L of Cibotium barometz root powder, 15-25 g / L of maltose, 0.2-1 g / L of ammonium sulfate, 0.5-1.5 g / L of magnesium sulfate, 0.5-1.5 g / L of potassium dihydrogen phosphate, 80-120 μg / mL of kanamycin, 80-120 μg / mL of tetracycline, 80-120 μg / mL of gentamicin, 10-20 g / L of agar, and the balance being water.

[0015] As a further improvement of the present invention, the formula of the PDA solid culture medium in step S2 is: 3-8 g / L potato extract, 15-25 g / L glucose, 10-20 g / L agar, and the balance is water.

[0016] This invention further protects the application of the above-mentioned endophytic fungus Pseudosperma yunnanense JMG086 from the roots of Cibotium barometz in the prevention and control of crop diseases.

[0017] As a further improvement of the present invention, the crop diseases are caused by Pantoea agglomerans, Pectobacterium carotovorum, and Ralstonia solanacearum.

[0018] As a further improvement of the present invention, the application method is to use the crude extract of fermentation broth of Pseudosperma yunnanense JMG086, an endophytic fungus in the roots of Cibotium barometz, to prepare a bacterial agent or drug composition for the prevention and control of crop diseases.

[0019] As a further improvement of the present invention, the method for preparing the crude extract of the fermentation broth includes the following steps:

[0020] (1) The endophytic fungus Pseudosperma yunnanense JMG086 from the roots of Cibotium barometz was activated;

[0021] (2) The activated endophytic fungus Pseudosperma yunnanense JMG086 from the roots of Cibotium barometz was inoculated into CMMD liquid medium and then cultured to obtain fermentation broth;

[0022] (3) Centrifuge the fermentation broth in a centrifuge tube to separate the cells and the fermentation broth. Add ethyl acetate to the fermentation broth and sonicate. Then pour it into a separatory funnel and let it stand to separate the layers. Take the supernatant of ethyl acetate and dry it by reflux using a rotary evaporator to obtain the crude extract of the fermentation broth.

[0023] As a further improvement of the present invention, the cultivation conditions in step (2) are as follows: 20-30 days in a constant temperature shaker at 20-30℃ and 100-200 rpm.

[0024] As a further improvement of the present invention, the formula of the CMMD liquid culture medium in step (2) is as follows: 1-3 g / L of Cibotium barometz root powder, 5-15 g / L of maltose, 0.2-1 g / L of ammonium sulfate, 0.5-1.5 g / L of magnesium sulfate, 0.5-1.5 g / L of potassium dihydrogen phosphate, 30-70 μg / L of 5-azacytidine, and the remainder is water.

[0025] The present invention has the following beneficial effects:

[0026] This invention isolated and screened endophytic fungi from the roots of Cibotium barometz to obtain a fungus with antibacterial activity, named *Pseudosperma yunnanense* JMG086, with accession number CCTCC NO: M 20251771. Experiments showed that the liquid fermentation extract of *Pseudosperma yunnanense* JMG086 exhibited strong inhibitory effects on the growth of *Pantoea agglomerans*, *Pectobacterium carotovorum*, and *Ralstonia solanacearum*, effectively controlling plant diseases. Attached Figure Description

[0027] 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 some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a diagram showing the mycelial growth of the endophytic fungus *Pseudosperma yunnanense* JMG086 in the root of *Cibotium barometz*, as described in Example 1 of this invention.

[0029] Figure 2 Micrograph of the hyphae of Pseudosperma yunnanense JMG086, an endophytic fungus in the root of Cibotium barometz, as described in Example 1 of this invention.

[0030] Figure 3 This is a diagram illustrating the phylogenetic tree of endophytic fungi constructed based on ITS in Embodiment 1 of the present invention.

[0031] Figure 4This invention presents a diagram illustrating the inhibitory effect of the endophytic fungus *Pseudosperma yunnanense* JMG086 from the roots of *Cibotium barometz* on *Plasmodium clumps*. 1: Mycelial extract of the endophytic fungus *Pseudosperma yunnanense* JMG086 from the roots of *Cibotium barometz*; 2: Crude extract of the endophytic fungus *Pseudosperma yunnanense* JMG086 from liquid fermentation; 3: Crude extract from CMMD liquid culture medium; 4: DSMO.

[0032] Figure 5 This invention presents a diagram illustrating the inhibitory effect of the endophytic fungus *Pseudosperma yunnanense* JMG086 from the roots of *Cibotium barometz* on *Bacillus sclerotiorum*, a type of *Carrot Soft Rot*. The diagram shows: 1: Mycelial extract of the endophytic fungus *Pseudosperma yunnanense* JMG086 from the roots of *Cibotium barometz*; 2: Crude extract of the endophytic fungus *Pseudosperma yunnanense* JMG086 from liquid fermentation; 3: Crude extract from CMMD liquid culture medium; 4: DSMO.

[0033] Figure 6 The diagram illustrates the inhibitory effect of the endophytic fungus *Pseudosperma yunnanense* JMG086 from the roots of *Cibotium barometz* on *Ralstonia solanacearum*. 1: Mycelial extract of the endophytic fungus *Pseudosperma yunnanense* JMG086 from the roots of *Cibotium barometz*; 2: Crude extract of the endophytic fungus *Pseudosperma yunnanense* JMG086 from liquid fermentation; 3: Crude extract from CMMD liquid culture medium; 4: DSMO. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0035] The endophytic fungus *Pseudosperma yunnanense* JMG086 from the roots of *Cibotium barometz* provided by this invention is deposited under the name *Pseudosperma yunnanense* JMG086, classified as *Pseudosperma yunnanense*, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on August 4, 2025, with accession number CCTCC NO: M 20251771.

[0036] The following examples use Pantoea agglomerans, strain number: CGMCC 1.2244, which can be purchased via the following link:

[0037] https: / / www.cgmcc.net / resources / details?uuid=78d6ba11-8ef2-11f0-94c6-78ac44479d74.

[0038] The strain used, *Pectobacterium carotovorum*, strain number CGMCC1.15539, can be purchased via the following link:

[0039] https: / / www.cgmcc.net / resources / details?uuid=78f1dc0d-8ef2-11f0-94c6-78ac44479d74.

[0040] The Ralstonia solanacearum strain used, strain number: CGMCC 1.12711, can be purchased via the following link:

[0041] https: / / www.cgmcc.net / resources / details?uuid=78ef8851-8ef2-11f0-94c6-78ac44479d74.

[0042] The link to purchase the Cibotium barometz root powder used is:

[0043] https: / / item.taobao.com / item.htm?ali_refid=a3_420434_1006%3A1689132817%3AH%3AGxXPLGhLZQQp9TY6KCgG0Q%3D%3D%3Af136692fabc201c07ea7e7699e085198&ali_trackid=282_f136692fabc201c07ea7e7699e085198&id=776850745795&mi_id=0000fPYrirIiCEbQU NcHQaaTOxZhTvzWudRCL5oXbnCTZBw&mm_sceneid=1_0_5466978989_0&priceTId=2147bfb817586175901505139e113a&skuId=548273169 5859&spm=a21n57.1.hoverItem.34&utparam=%7B%22aplus_abtest%22%3A%22fe20e41f9b7c7102e23c1249509a17f2%22%7D&xxc=ad_ztc Example 1:

[0044] 1. Isolation of the endophytic fungus *Pseudosperma yunnanense* JMG086 from the roots of *Cibotium barometz*

[0045] (1) After collecting the rootstock of Cibotium barometz, place it on ice and transport it to the laboratory at low temperature. Rinse it with tap water for 5 hours, then rinse it again with sterile water for 5 minutes on a clean bench. Then cut off the part of the rootstock containing root hairs, disinfect it with 10% hydrogen peroxide for 4 minutes, rinse it with sterile water for 2 minutes, and then dry the disinfected rootstock for later use.

[0046] (3) Cut the sterilized rootstock of Cibotium barometz with root hairs into tissue blocks with a length and width of 0.5 cm, place them in centrifuge tubes, add steel balls and 0.7 mL of sterile water, and crush for 2 minutes (180 rpm). Dilute 1000 times and spread on 10 CMNMK solid medium plates (CMNMK: Cibotium barometz root powder 1-3 g / L, maltose 15-25 g / L, ammonium sulfate 0.2-1 g / L, magnesium sulfate 0.5-1.5 g / L, potassium dihydrogen phosphate 0.5-1.5 g / L, kanamycin 80-120 μg / mL, tetracycline 80-120 μg / mL, gentamicin 80-120 μg / mL, agar 10-20 g / L, balance water). Incubate for 20 days. After the colonies grow, select single colonies and identify them.

[0047] After the mycelium was transferred to PDA solid medium and cultured for 15 days, the strain was molecularly identified, yielding the endophytic fungus *Pseudosperma yunnanense* JMG086 from the roots of *Cibotium barometz*. The PDA solid medium consisted of the following components: 5 g / L potato extract; 20 g / L glucose; 15 g / L agar (the remainder being water).

[0048] 2. Identification of endophytic fungi in the roots of Cibotium barometz

[0049] (1) Morphological identification: This strain grew well on PDA solid medium, and a large number of white aerial hyphae formed on the surface of the colonies. Morphological characteristics are shown in [reference needed]. Figure 1 .

[0050] (2) DNA extraction: Genomic DNA was extracted from the mycelium using the CTAB method. The simplified procedure included: grinding the mycelium with liquid nitrogen, treating with CTAB and PVP, cleaving with β-mercaptoethanol, and water bath at 65°C; removing proteins by phenol-chloroform extraction, purifying DNA by ethanol precipitation, and finally dissolving the DNA sample in EB buffer.

[0051] (3) ITS sequence amplification and sequencing

[0052] The ITS region (including the ITS1, 5.8S rRNA, and ITS2 regions) in the rDNA was amplified using the fungal universal primers ITS1 and ITS4 to obtain the sequence shown in SEQ ID No. 1.

[0053] SEQ ID No. 1:

[0054] (SEQ ID NO.1).

[0055] (4) Phylogenetic analysis

[0056] Based on ITS sequences, a phylogenetic tree was constructed using MEGA software. Figure 2 Based on morphological characteristics and molecular systematics results, this strain was identified as *Pseudosperma yunnanense*. Example 2:

[0057] Preparation of crude extract from liquid fermentation of endophytic fungi in the roots of Cibotium barometz:

[0058] 1. Liquid culture

[0059] The mycelium of the endophytic fungus Pseudosperma yunnanense JMG086 from the roots of Cibotium barometz was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of CMMD liquid medium (2 g / L Cibotium barometz root powder, 10 g / L maltose, 0.5 g / L ammonium sulfate + 1 g / L magnesium sulfate + 1 g / L potassium dihydrogen phosphate; 50 μg / L 5-azacytidine, with the remainder being water). The flask was then placed in a constant temperature shaker at 25 °C and 150 rpm for 25 days.

[0060] 2. Preparation of crude extract from liquid culture

[0061] Place the fermentation broth into a 50 ml centrifuge tube, then centrifuge at 3000 g for 3 min to separate the cells and the fermentation broth.

[0062] Ethyl acetate was added to the fermentation broth at a ratio of 1:1.5, followed by sonication for 30 min. The mixture was then poured into a separatory funnel and allowed to stand for 12 h to allow for layering. The supernatant was then extracted. The extract was dried by reflux using a rotary evaporator to obtain the crude extract of the endophytic fungus *Pseudosperma yunnanense* JMG086 from the roots of *Cibotium barometz*.

[0063] 3. Preparation of crude bacterial extract

[0064] The fermentation broth was placed in a 50 ml centrifuge tube and centrifuged at 3000 g for 3 min to obtain the endophytic fungus *Pseudosperma yunnanense* JMG086 from the roots of *Cibotium barometz*. The *Pseudosperma yunnanense* JMG086 cells were then placed in an Erlenmeyer flask and submerged in 95% ethanol. After sonication for 30 min and standing for 12 h, the extract was filtered through filter paper. The extract was then dried by reflux using a rotary evaporator to obtain the crude extract of *Pseudosperma yunnanense* JMG086 from the roots of *Cibotium barometz*.

[0065] 4. Preparation of crude extract of CMMD liquid culture medium

[0066] A 250 mL Erlenmeyer flask containing 100 mL of CMMD liquid culture medium was placed in a constant temperature shaker at 25 °C and 150 rpm for 25 days. Ethyl acetate was added to the liquid culture medium at a volume ratio of 1:1.5, and the mixture was stirred thoroughly and sonicated for 45 min. The mixture was then poured into a separatory funnel and allowed to stand for 12 h to allow for layering. The supernatant was then extracted. The extract was dried under reflux using a rotary evaporator to obtain the crude extract of CMMD liquid culture medium. Example 3:

[0067] Antibacterial activity assay of crude extract from liquid fermentation of endophytic fungi in the roots of Cibotium barometz:

[0068] The crude extracts of liquid fermentation of Pseudosperma yunnanense JMG086, the crude extracts of Pseudosperma yunnanense JMG086, and the crude extracts of CMMD liquid culture medium prepared in Example 2 above were used as samples.

[0069] 1. Activation of pathogenic bacteria

[0070] Take 5 μL of each of the following bacteria: Pantoea agglomerans, Pectobacterium carotovorum, and Ralstonia solanacearum, and add them to 2 mL centrifuge tubes respectively. Then add 750 μL of LB liquid medium (10 g / L tryptone + 10 g / L sodium chloride + 5 g / L yeast extract) to each centrifuge tube. Incubate the centrifuge tubes in a constant temperature shaker at 37°C and 180 rpm for 12 h to obtain the pathogenic bacterial suspension. Store the suspension in a 4°C refrigerator for later use. Before use, remove the suspension and dilute the activated bacteria to 1 / 10.

[0071] 2. Detection of antibacterial activity using the filter paper disc method

[0072] The filtered mycelium was extracted with ethanol, and dried by reflux using a rotary evaporator to obtain a crude extract of the mycelium of the endophytic fungus *Pseudosperma yunnanense* JMG086 from the roots of *Cibotium barometz*. 0.01 g of the liquid fermentation crude extract of *Pseudosperma yunnanense* JMG086, the crude mycelium extract, and the crude extract of *Pseudosperma yunnanense* JMG086 from pure fermentation broth were placed in a 2 mL centrifuge tube, and 500 μL of DMSO (dimethyl sulfoxide) solution was added to dilute to 50 mg / mL. The activated pathogenic bacterial solution was evenly spread onto LB solid medium (tryptone 10g / L + sodium chloride 10g / L + agar 15g / L + yeast extract 5g / L). After drying, 5mm filter paper discs containing the dissolved crude extract of the fermentation broth were placed at the corresponding marked positions on the medium. The medium was then incubated upright in a 37℃ constant temperature incubator for 12 hours. The presence of inhibition zones was observed, and their diameter was measured using the cross-sectional method. The antibacterial activity experiment results showed that the crude extract of the fermentation broth of the endophytic fungus *Pseudosperma yunnanense* JMG086 from the roots of *Cibotium barometz* exhibited an inhibition zone of 30.3±0.2 mm against *Pseudosperma yunnanense*. Figure 4The inhibition zone against *Streptomyces carotensis* was 28.5 ± 0.1 mm. Figure 5 The inhibition zone against Ralstonia solanacearum was 25.1 ± 0.2 mm. Figure 6 The results indicate that the crude extract of the fermentation broth of the endophytic fungus Pseudosperma yunnanense JMG086 from the roots of Cibotium barometz has significant antibacterial activity against Pantotheca acuminata, Bacillus carotenoides, and Ralstonia solanacearum. The crude extracts of the fungus cells and the crude extracts of CMMD liquid culture medium did not have obvious inhibition zones, and therefore had no antibacterial activity.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of Pseudosperma yunnanense JMG086 in the prevention and treatment of crop disease fungi, characterized in that, The taxonomic name of the Pseudosperma yunnanense JMG086 is Pseudosperma yunnanense, the preservation unit is China Center for Type Culture Collection, the preservation date is August 4, 2025, and the preservation number is CCTCC NO: M 20251771. The crop disease bacteria are Pantoea agglomerans, Pectobacterium carotovorum or Ralstonia solanacearum.

2. Use according to claim 1, characterized in that, The application mode is to apply the bacterial agent prepared from the fermentation broth extract of Pseudosperma yunnanense JMG086 to the crop disease bacteria.

3. Use according to claim 2, characterized in that, The preparation method of the fermentation broth extract comprises the following steps: (1) activating the Pseudosperma yunnanense JMG086; (2) inoculating the activated Pseudosperma yunnanense JMG086 into a CMMD liquid medium, and then culturing to obtain a fermentation broth; The formula of the CMMD liquid medium is: 1-3 g / L of Sanguinaria canadensis root powder, 5-15 g / L of maltose, 0.2-1 g / L of ammonium sulfate, 0.5-1.5 g / L of magnesium sulfate, 0.5-1.5 g / L of potassium dihydrogen phosphate, 30-70 μg / L of 5-azacytidine, and the rest is water; (3) centrifuging the fermentation broth in a centrifuge tube to separate the bacteria and the fermentation broth, adding ethyl acetate into the fermentation broth, and then performing ultrasonic treatment; immediately pouring into a separatory funnel, and then standing to separate the layers; taking the supernatant of the ethyl acetate, and then condensing and reflux drying by using a rotary evaporator to obtain the fermentation broth extract. The culture condition in step (2) is that the culture is carried out in a constant-temperature shaker at 20-30 °C and 100-200 rpm for 20-30 days. ​ 4. Use according to claim 3, characterized in that, ​

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