Plant endophytic fungal species, strains, and their isolation and application methods

Abstract

This invention belongs to the field of microbial preparation technology, specifically relating to a plant endophytic microbial community and its isolation and purification method. The plant endophytic microbial community consists of at least two types of plant endophytic microbes: (Parengyodontium SP.) MD313901 and (Purpureocillium SP.) MD313902. This plant endophytic microbial community is derived from natural medicinal plants; the two strains may originate from the same medicinal plant or different medicinal plants. The preparation method of this plant endophytic microbial community includes plant tissue pretreatment, endophytic microbial extraction from plant tissue, isolation of plant tissue endophytic microbes, and purification.

Description

Technical Field

[0001] This invention relates to the field of microbial preparation technology, and more specifically to a method for preparing a microbial community. Background Technology

[0002] Plant endophytes are a large class of microorganisms that live within the tissues and organs of healthy plants at certain or all stages of their life cycle, forming parasitic, symbiotic, and saprophytic relationships with the plant. Studies have found that endophytes can be isolated not only from various plants but also from the roots, stems, leaves, flowers, fruits, and seeds of the same plant species. Endophytes are numerous and diverse, including endophytic fungi, endophytic bacteria, and endophytic actinomycetes. Plant endophytes exhibit rich biodiversity; for a single plant species, several to dozens, and sometimes even hundreds, of endophytic fungi or bacteria can typically be isolated. Plant endophytes also represent a novel microbial resource with potential applications, and the search for new bioactive substances from them has become a research hotspot. Summary of the Invention

[0003] The primary objective of this invention is to provide a plant endophytic fungus strain (Parengyodontium SP.) MD313901.

[0004] The second objective of this invention is to provide a plant endophytic fungus strain (Purpureocillium SP.) MD313902.

[0005] The third objective of this invention is to provide a plant endophytic flora comprising strains (Parengyodontium SP.)MD313901 and (Purpureocillium SP.)MD313902.

[0006] The fourth objective of this invention is to provide a microbial agent comprising (Parengyodontium SP.)MD313901 strain and / or (Purpureocillium SP.)MD313902 strain.

[0007] The fifth objective of this invention is to provide a method for isolating plant endophytic fungi and plant endophytic flora as described in this invention.

[0008] The sixth objective of this invention is to provide a method for applying the plant endophytic fungi and plant endophytic flora described in this invention.

[0009] A plant endophytic fungus (Parengyodontium SP.) strain MD313901 (also referred to as MD313901 in this invention) has the accession number CCTCC M 2018256.

[0010] This invention provides a novel strain belonging to the endophytic fungi, named (Parengyodontium SP.)MD313901, which is deposited at the China Center for Type Culture Collection (CCTCC) in Wuhan on May 8, 2018; the accession number is CCTCC M 2018256.

[0011] In addition, the present invention provides another novel strain, the plant endophytic fungus (Purpureocillium SP.) strain MD313902 (also referred to as MD313902 in this invention), with accession number CCTCC M2018257.

[0012] The novel endophytic strain MD313902 provided by this invention belongs to endophytic fungi and is named (Purpureocillium SP.)MD313902. It is deposited at the China Center for Type Culture Collection in Wuhan on May 8, 2018, with accession number CCTCC M 2018257.

[0013] The MD313901 and MD313902 strains described in this invention have similar functions. Both strains can significantly promote the extraction of polysaccharide or glycoprotein components from natural medicinal materials through fermentation, thereby increasing the polysaccharide extraction rate.

[0014] The present invention also provides a plant endophytic flora comprising strains (Parengyodontium SP.)MD313901 and (Purpureocillium SP.)MD313902.

[0015] This invention innovatively discovers that the *Parengyodontium* SP. MD313901 and *Purpureocillium* SP. MD313902 strains described in this invention can unexpectedly improve the extraction rate of polysaccharides or glycoproteins from fermented natural medicinal materials. Furthermore, the inventors have also found that strains MD313901 and MD313902 exhibit good synergistic effects, which can further enhance the extraction rate of polysaccharide or glycoprotein components from natural medicinal materials.

[0016] The present invention also provides a plant endophytic fungal agent, comprising (Parengyodontium SP.) MD313901 strain and / or (Purpureocillium SP.) MD313902 strain, and further comprising excipients for which the (strains) are resistant to survival.

[0017] This invention also provides a method for isolating strains (Parengyodontium SP.) MD313901 and / or (Purpureocillium SP.) MD313902, including plant tissue pretreatment, leaching, isolation, and purification: the operation process is as follows:

[0018] (1) Plant tissue pretreatment

[0019] Plant tissue was cut into segments, soaked in ethanol solution, soaked in sodium hypochlorite solution, and then washed with sterile water to obtain pretreated plant tissue.

[0020] (2) Extraction of endophytic bacteria from plant tissues

[0021] The pretreated plant tissue and biological enzymes were ground together, and the supernatant in the grinding solution was separated.

[0022] (3) Isolation and purification of plant endophytic strains

[0023] The supernatant was spread onto a culture medium, and the strains or groups were selected after culturing.

[0024] The present invention has found that this method can effectively isolate the target strain or bacterial group.

[0025] Preferably, the plant tissue described in this invention is a natural medicinal plant.

[0026] The plant tissue contains at least one species of the microbial community.

[0027] Preferably, the plant tissue is at least one of the following: Polygonatum odoratum, Astragalus membranaceus, Hippophae rhamnoides, Portulaca oleracea, Polygonatum sibiricum, Platycodon grandiflorus, Sophora tonkinensis, Isatis indigotica, Phytolacca acinosa, Houttuynia cordata, Corydalis yanhusuo, Pinellia ternata, Arctium lappa root, Platycodon grandiflorus, Taraxacum mongolicum, Glycyrrhiza uralensis, Paeonia lactiflora, Dendrobium nobile, Angelica sinensis, and Acorus tatarinowii.

[0028] In step (1), the plant tissue is rinsed with water and then washed with sterile water. After drying the surface moisture, it is cut into sections with a length of 0.5-2.0 cm. The cut material is then disinfected with 70-80% ethanol for 1-2 minutes, rinsed with sterile water, and then soaked in 3-8% sodium hypochlorite for 5-10 minutes. After rinsing with sterile water and drying the surface moisture, it is the pretreated plant tissue.

[0029] In step (2), the pretreated plant tissue is placed in a sterilized mortar or grinder, and biological enzymes are added for grinding. The grinding liquid is transferred into an Erlenmeyer flask and shaken at 100-200 rpm for 1-2 hours at 25-40℃ to separate the supernatant and plant residue.

[0030] Preferably, the bioenzyme is one or more of pectinase, cellulase, and neutral protease.

[0031] Preferably, the amount of bio-enzyme used is 0.01%-5% of the weight of the medicinal material (plant tissue).

[0032] In step (3), the supernatant obtained from the leaching process is diluted 10-20 times with sterile water under aseptic conditions. 100-200 μL of each supernatant is spread on MS medium for culture. After a period of culture, colonies of different morphologies are selected. Fungi are cultured on PDA (potato dextrose agar) medium, and bacteria are cultured on beef extract peptone medium.

[0033] In step (3), the isolated fungi are purified by streak plating.

[0034] The separation method described in this invention first effectively avoids the introduction of contaminating bacteria through disinfection, thus preventing the separation effect of endophytic bacteria. The entire separation process is strictly controlled to maintain a sterile environment and avoid the introduction of foreign bacteria during operation.

[0035] The plant endophytic microbiota described in this invention are distributed in the same medicinal material from different origins and varieties, extracted using the same preparation method.

[0036] The present invention also provides an application of (Parengyodontium SP.)MD313901 strain and / or (Purpureocillium SP.)MD313902 strain for the fermentation of plant tissues to improve the extraction rate of polysaccharides from plant tissues.

[0037] The present invention has found that using the MD313901 strain and / or MD313902 strain of the present invention to ferment natural plants can significantly improve the extraction rate of total polysaccharides and glycoproteins from natural plants.

[0038] Preferably, in the aforementioned application, plant tissue is enzymatically hydrolyzed and then fermented by the (Parengyodontium SP.) MD313901 strain and / or (Purpureocillium SP.) MD313902 strain to extract plant tissue polysaccharides.

[0039] The method of this invention can further convert certain components other than polysaccharides in plant tissues into polysaccharides, thereby further increasing the polysaccharide content in the fermentation broth and improving the polysaccharide extraction rate.

[0040] Beneficial effects

[0041] This invention provides novel strains (Parengyodontium SP.) MD313901 and (Purpureocillium SP.) MD313902; and innovatively discovers that these novel strains help improve the extraction rate of total polysaccharides and glycoproteins from natural medicinal materials. Furthermore, this invention also found that the combined use of (Parengyodontium SP.) MD313901 and (Purpureocillium SP.) MD313902 strains has a synergistic effect, which helps to further improve the extraction rate of polysaccharides from natural medicinal materials.

[0042] The separation method of this invention has good screening temperature, reproducibility, and technical stability. Detailed Implementation

[0043] The following embodiments are specific descriptions of the present invention. These embodiments are intended to further illustrate the present invention and should not be construed as limiting the scope of protection of the invention. Improvements and modifications made by those skilled in the art based on this application are also within the scope of protection.

[0044] Example 1

[0045] The method for preparing plant endophytic microbiota involves the following steps:

[0046] (1) Plant tissue pretreatment

[0047] The plant tissues (using different origins and varieties of Polygonatum odoratum medicinal materials as shown in Table 1) were rinsed with tap water and then washed with sterile water. After drying the surface moisture, they were cut into sections of 0.5-2.0 cm in length. The cut materials were then disinfected with 70% ethanol for 2 minutes, washed with sterile water, soaked in 3% sodium hypochlorite for 10 minutes, rinsed with sterile water, and dried for later use.

[0048] (2) Extraction of endophytic bacteria from plant tissues

[0049] The sterilized Solomon's Seal tissue was placed in a sterilized mortar and ground with a biological enzyme (cellulase, 5%). The grinding liquid was transferred into an Erlenmeyer flask and shaken at 200 rpm for 2 hours at 25°C to obtain the supernatant and plant residue.

[0050] (3) Isolation and purification of plant endophytic strains

[0051] Under aseptic conditions, the supernatant obtained from the leaching process was diluted 10-fold with sterile water, and 200 μL of each supernatant was spread onto MS medium for incubation. After a period of incubation and colony formation, colonies of different morphologies were selected. Fungi were cultured on PDA (potato dextrose agar) medium, and bacteria were cultured on beef extract peptone medium. Purification was performed using the streak plating method.

[0052] The endophytic fungi of Polygonatum odoratum plants from different origins and varieties were isolated and are shown in Table 1.

[0053] Table 1

[0054]

[0055] The results in Table 1 show that two strains can be obtained from different varieties of Polygonatum odoratum from different origins using the isolation method of this patent.

[0056] This invention analyzes the 16SRNA fragment of strain MD313901, which contains a fungus-specific ITS2 fragment and has 99% homology with Parengyodontium SP., and is named MD313901. Strain (Parengyodontium SP.)MD313901 was deposited at the China Center for Type Culture Collection on May 8, 2018, with accession number CCTCCM 2018256.

[0057] This invention analyzes the 16SRNA fragment of strain MD313902, which contains a fungus-specific ITS2 fragment and has 99% homology with Purpureocillium SP., and is named MD313902. Strain (Purpureocillium SP.)MD313902 was deposited at the China Center for Type Culture Collection on May 8, 2018, with accession number CCTCCM 2018257.

[0058] Example 2

[0059] The method for preparing plant endophytic microbiota involves the following steps:

[0060] (1) Plant tissue pretreatment

[0061] The plant tissues (take different origins and varieties of Platycodon grandiflorus plant materials as shown in Table 2) were rinsed with tap water and then washed with sterile water. After drying the surface moisture, they were cut into sections with a length of 0.5-2.0 cm. The cut materials were then disinfected with 80% ethanol for 1 minute, washed with sterile water, soaked in 8% sodium hypochlorite for 5 minutes, rinsed with sterile water, and dried for later use.

[0062] (2) Extraction of endophytic bacteria from plant tissues

[0063] The sterilized plant tissue was placed in a sterilized mortar and ground with a biological enzyme (a 1:1 mixture of pectinase and cellulase, at a dosage of 2%). The grinding liquid was then transferred to an Erlenmeyer flask and shaken at 100 rpm for 1 hour at 40°C to obtain the supernatant and plant residue.

[0064] (3) Isolation and purification of plant endophytic strains

[0065] Under aseptic conditions, the supernatant obtained from the leaching process was diluted 20-fold with sterile water, and 100 μL of each supernatant was spread onto MS medium for incubation. After a period of incubation and colony formation, colonies of different morphologies were selected. Fungi were cultured on PDA (potato dextrose agar) medium, and bacteria were cultured on beef extract peptone medium. Purification was performed using the streak plating method.

[0066] Different varieties and origins of Platycodon grandiflorus plants were isolated using the method described in this case study. The plants were purified by streak plating and the endophytic bacteria were isolated. The results are shown in Table 2.

[0067] Table 2

[0068] strains Platycodon grandiflorus from Anhui Platycodon grandiflorus from Yunnan Sichuan-grown Platycodon grandiflorus Platycodon grandiflorus from Heilongjiang MD313901 √ √ √ √

[0069] The results in Table 2 show that different varieties of Platycodon grandiflorus from different origins can also obtain a strain of bacteria using the isolation method of this patent.

[0070] Example 3

[0071] The method for preparing plant endophytic microbiota involves the following steps:

[0072] (1) Plant tissue pretreatment

[0073] The plant tissues (different varieties of Phytolacca acinosa shown in Table 2) were rinsed with tap water and then washed with sterile water. After drying the surface moisture, they were cut into sections of 0.5-2.0 cm in length. The cut materials were then disinfected with 75% ethanol for 1.5 min, washed with sterile water, soaked in 5% sodium hypochlorite for 8 min, rinsed with sterile water, and dried for later use.

[0074] (2) Extraction of endophytic bacteria from plant tissues

[0075] The sterilized plant tissue was placed in a sterilized mortar and ground with a biological enzyme (a mixture of pectinase, cellulase and neutral protease in a 1:1:1 ratio, at a dosage of 0.1%). The grinding liquid was transferred into an Erlenmeyer flask and shaken at 200 rpm for 1.5 hours at 35°C to obtain the supernatant and plant residue.

[0076] (3) Isolation and purification of plant endophytic strains

[0077] Under aseptic conditions, the supernatant obtained from the leaching process was diluted 20-fold with sterile water, and 200 μL of each supernatant was spread onto MS medium for incubation. After a period of incubation and the formation of colonies, colonies of different morphologies were selected. Fungi were cultured on PDA (potato dextrose agar) medium, and bacteria were cultured on beef extract peptone medium. Purification was performed using the streak plating method.

[0078] Different varieties of Phytolacca acinosa were purified by streak plasmid culture and their endophytic bacteria were isolated. The results are shown in Table 2.

[0079] Table 3

[0080]

[0081] The results in Table 3 show that two strains can be obtained from different varieties of pokeweed using the isolation method of this patent.

[0082] Application Example 1:

[0083] 1. Enzymatic hydrolysis and cell wall disruption of dandelion medicinal materials:

[0084] Take 10 kg of dandelion medicinal material, remove impurities and rinse it with tap water, pour it into a fermentation tank, add purified water to cover the medicinal material, add 0.1% pectinase (total raw material mass fraction) and 0.1% cellulase (total raw material mass fraction), stir and mix thoroughly, adjust the pH value to 6.8, control the temperature at 48℃, enzymatically hydrolyze for 2 hours, and then sterilize by steaming for 40 minutes.

[0085] 2. Fermentation

[0086] After cooling to room temperature, add 0.3% (total raw material mass fraction) of Parengyodontium SP.MD313901 strain, and let it ferment statically at 30℃ for 6 days. Filter and collect the liquid for further processing.

[0087] 3. Extraction and content determination of dandelion polysaccharides

[0088] After fermentation, the collected liquid was mixed with 8 times the amount of purified water, heated to 100°C and refluxed twice for 2 hours each time. The mixture was then filtered to obtain an extract, which was concentrated to an appropriate concentration. Free proteins were removed using the Sevag method. Anhydrous ethanol was then added to bring the solution concentration to 80%. The mixture was centrifuged twice to precipitate the precipitate, which was then dried to obtain total dandelion polysaccharides.

[0089] The polysaccharide content of dandelion was determined using the phenol-sulfuric acid method. The results showed that the polysaccharide content of dandelion after fermentation reached 60.4% of the dry weight of dandelion. Compared with the case that did not carry out the fermentation step 2 in this case (directly carrying out the extraction step 3 after the cell wall breaking treatment in step 1), the polysaccharide content was 2.7 times higher.

[0090] Application Example 2:

[0091] 1. Enzymatic hydrolysis and cell wall disruption of burdock root:

[0092] Take 10 kg of burdock root, remove impurities, rinse with tap water, pour into a fermentation tank, add purified water to cover the root, add 1% pectinase (total raw material mass fraction) and 2% cellulase (total raw material mass fraction), stir and mix thoroughly, adjust the pH to 6.8, control the temperature at 50℃, enzymatically hydrolyze for 1.5 hours, and then sterilize with steam for 30 minutes.

[0093] 2. Fermentation:

[0094] After cooling to room temperature, add 0.3% (total raw material mass fraction) of Purpureocillium SP.MD002 strain, and let it ferment statically at 30℃ for 5 days. Filter and collect the liquid for further processing.

[0095] 3. Extraction and content determination of burdock root polysaccharides

[0096] After fermentation, the collected liquid was mixed with 10 times the amount of purified water, heated to 90°C and refluxed twice for 2 hours each time. The mixture was then filtered to obtain an extract, which was concentrated to an appropriate concentration. Free proteins were removed using the Sevag method. Anhydrous ethanol was then added to bring the solution concentration to 80%. The mixture was centrifuged twice to precipitate the precipitate, which was then dried to obtain burdock root polysaccharide.

[0097] The polysaccharide content of burdock root was determined using the phenol-sulfuric acid method. The results showed that the polysaccharide content of burdock root after fermentation reached 28.3% of the dry weight of burdock root. Compared with the case that did not perform step 2 of the fermentation step (which directly proceeded to step 3 extraction after the cell wall breaking treatment in step 1), the polysaccharide content was 2.1 times higher.

[0098] Application Example 3:

[0099] 1. Enzymatic hydrolysis and cell wall disruption of the composition

[0100] Weigh out 5 kg of Dendrobium and 5 kg of Portulaca oleracea as a combination, remove impurities and rinse with tap water, pour into a fermentation tank, add purified water to cover the herbs, add 3% pectinase (total raw material mass fraction) and 2% cellulase (total raw material mass fraction), stir and mix thoroughly, adjust the pH to 7.0, control the temperature at 45℃, enzymatically hydrolyze for 3 hours, and then sterilize by steaming for 30 minutes.

[0101] 2. Fermentation:

[0102] After cooling to room temperature, add 1% (total raw material mass fraction) of Parengyodontium SP.MD313901 strain and 1% (total raw material mass fraction) of Purpureocillium SP.MD313902 strain, and let it ferment statically at 30℃ for 10 days. Filter and collect the liquid for further processing.

[0103] 3. Extraction and content determination of polysaccharides in the composition

[0104] After fermentation, the collected liquid was mixed with 10 times the amount of purified water, heated under vacuum at 65°C twice for 2 hours each time, filtered to obtain the extract, concentrated to an appropriate concentration, and the free protein was removed by the Sevag method. Then anhydrous ethanol was added to make the solution concentration reach 80%, centrifuged twice to precipitate, and the precipitate was dried to obtain the polysaccharide composition.

[0105] The polysaccharide content of the composition was determined using the phenol-sulfuric acid method. The results showed that the polysaccharide content of the composition after fermentation reached 30.2% of the dry weight of the composition. Compared with the case that did not perform step 2 of the fermentation step (which directly proceeded to step 3 extraction after the cell wall breaking treatment in step 1), the polysaccharide content was twice as high.

[0106] Application Example 4:

[0107] 1. Enzymatic hydrolysis and cell wall disruption of the composition:

[0108] Take 5 kg of Astragalus membranaceus and 5 kg of Codonopsis pilosula, remove impurities and rinse them with tap water, pour them into a fermentation tank, add purified water to cover the herbs, add 4% pectinase (total raw material mass fraction) and 1% cellulase (total raw material mass fraction), stir and mix thoroughly, adjust the pH value to 7.0, control the temperature at 45℃, enzymatically hydrolyze for 3 hours, and then sterilize by steaming for 30 minutes.

[0109] 2. Fermentation:

[0110] After cooling to room temperature, add 2% (total raw material mass fraction) of Parengyodontium SP.MD313901 strain and 2% (total raw material mass fraction) of Purpureocillium.SP.MD313902 strain, and let it ferment statically at 30℃ for 10 days. Filter and collect the liquid for further processing.

[0111] 3. Extraction and content determination of polysaccharides in the composition

[0112] After fermentation, the combined medicinal liquid was added to 10 times the amount of purified water, heated under vacuum at 70°C twice for 2 hours each time, filtered to obtain the extract, concentrated to an appropriate concentration, and free proteins were removed by the Sevag method. Then anhydrous ethanol was added to make the solution concentration reach 80%, centrifuged twice to precipitate, and the precipitate was dried to obtain the combined polysaccharide.

[0113] The polysaccharide content of the composition was determined by the phenol-sulfuric acid method. The results showed that the polysaccharide content of the composition after fermentation reached 51.4% of the dry weight of the medicinal material. Compared with the case that did not carry out the fermentation step 2 in this case (directly carried out the extraction step 3 after the cell wall breaking treatment in step 1), the polysaccharide content was 1.4 times higher.

[0114] Application Example 5:

[0115] 1. Enzymatic hydrolysis and cell wall disruption of the composition:

[0116] Weigh out 5 kg of Polygonatum sibiricum, 5 kg of Dioscorea opposita, 5 kg of Phytolacca acinosa, and 5 kg of Pueraria lobata as a mixture. Remove impurities and rinse with tap water. Pour the mixture into a fermentation tank, add purified water to cover the herbs, add 1% pectinase (total raw material mass fraction) and 4% cellulase (total raw material mass fraction), stir and mix thoroughly, adjust the pH to 7.0, control the temperature at 50℃, enzymatically hydrolyze for 4 hours, and then sterilize with steam for 60 minutes.

[0117] 2. Fermentation:

[0118] After cooling to room temperature, add 3% (total raw material mass fraction) of Parengyodontium SP.MD313901 strain and 3% (total raw material mass fraction) of Purpureocillium SP.MD313902 strain, and let it ferment statically at 25℃ for 15 days. Filter and collect the liquid for further processing.

[0119] 3. Extraction and content determination of polysaccharides in the composition

[0120] After fermentation, the combined medicinal liquid was added to 10 times the amount of purified water, heated under vacuum at 70°C twice for 2 hours each time, filtered to obtain the extract, concentrated to an appropriate concentration, and free proteins were removed by the Sevag method. Then anhydrous ethanol was added to make the solution concentration reach 80%, centrifuged twice to precipitate, and the precipitate was dried to obtain the combined polysaccharide.

[0121] The polysaccharide content of the composition was determined using the phenol-sulfuric acid method. The results showed that the polysaccharide content of the composition after fermentation reached 42.0% of the dry weight of the composition. Compared with the case that did not perform step 2 of the fermentation step (which directly proceeded to step 3 extraction after the cell wall breaking treatment in step 1), the polysaccharide content was 2.1 times higher.

Claims

1. A plant endophytic fungus (MD313901 strain) characterized by, Parengyodontium SP . ) MD313901 strain, characterized by, The preservation number thereof is CCTCC M 2018256.

2. A plant endophytic fungus ( Purpureocillium SP. MD313902 strain, characterized in that, The preservation number thereof is CCTCC M 2018257.

3. A plant endophyte population, characterized in that comprising Parengyodontium SP the MD313901 strain and Purpureocillium SP. the MD313902 strain wherein, Parengyodontium SP The preservation number of the MD313901 strain is CCTCC M 2018256, Purpureocillium SP. The preservation number of the MD313902 strain is CCTCC M 2018257.

4. A plant endophyte inoculant characterized in that, include( Parengyodontium SP .) MD313901 strain and / or ( Purpureocillium SP. MD313902 strain, including its survival-resistant excipients; wherein, Parengyodontium SP The preservation number of the MD313901 strain is CCTCC M 2018256, Purpureocillium SP. The preservation number of the MD313902 strain is CCTCC M 2018257.

5. A kind of ( Parengyodontium SP .) MD313901 strain and / or ( Purpureocillium SP. The application of strain MD313902 is characterized by, The plant tissue is used for fermentation to improve the polysaccharide extraction rate of the plant tissue; The plant tissue is at least one of burdock root, dendrobium, spiny amaranth, milk vetch, codonopsis, turmeric, yam, pokeberry, and kudzu root; ( Parengyodontium SP The preservation number of strain MD313901 is CCTCC M 2018256. Purpureocillium SP. The preservation number of strain MD313902 is CCTCC M 2018257.

6. The use according to claim 5, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The plant tissue is subjected to enzymatic treatment and then fermentation.

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