Application of a fungus of the genus gloeophyllum in promoting synthesis of budlejastrin in budleja davidii

Abstract

The application discloses application of Tulasnella sp. in promoting synthesis of bletilline in bletilla striata, and relates to the technical field of microorganisms. The Tulasnella sp. BJ1 can significantly promote synthesis and accumulation of militarine in bletilla striata, is a special bacterial fertilizer for bletilla striata cultivation, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to the application of a type of *Bacillus* fungus in promoting the synthesis of leucoside in *Bletilla striata*. Background Technology

[0002] Bletilla striata (Thunb.) Reiehb.f., also known as Baiji or Baijizi, is a perennial terrestrial herb belonging to the genus Bletilla in the Orchidaceae family. It is mainly distributed in Hubei, Hunan, Guizhou, Yunnan, and coastal areas of my country. As a traditional Chinese medicine, Bletilla striata is used medicinally as its dried tuber, possessing astringent, hemostatic, anti-inflammatory, and tissue-regenerating properties. It is often used to treat external bleeding, hematemesis, chapped skin, swelling, and ulcers. The tuber of Bletilla striata contains over 150 compounds, among which polysaccharides are the most abundant, reaching up to 60%. These polysaccharides are also recognized as indicative components. However, the 2020 edition of the Chinese Pharmacopoeia has changed the indicative component of Bletilla striata from polysaccharides to 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutyl malate (C 34 H 46 O 17 Mitarine, also known as Bletilla striata glycoside (hereinafter referred to as militarine), has gradually become a subject of significant research interest. Modern studies have shown that militarine in Bletilla striata possesses functions such as improving cognitive function, delaying aging, and preventing Alzheimer's disease, and its content is relatively high among the main components of Bletilla striata. Therefore, how to increase the content of militarine, a medicinal component in Bletilla striata tubers, has attracted considerable attention and interest from researchers and the industry.

[0003] Mycorrhizal fungi and host plants have a mutually beneficial symbiotic relationship. Mycorrhizal fungi not only maintain the healthy growth of plants, but also promote the accumulation of secondary metabolites of host plants through symbiosis. Studies have found that mycorrhizal fungi can regulate the sugar accumulation of Polygonum cuspidatum after inoculation, causing changes in its secondary metabolites. There is also a report (Li Q, Ding G, Li B, et al. Transcriptome Analysis of Genes Involved in Dendrobine Biosynthesis in Dendrobium nobile Lindl. Infected with Mycorrhizal Fungus MF23 (Mycena sp.). Scientific Reports, 2017, 7(1): 316.) that a mycorrhizal fungus MF23 (Mycenasp.) isolated from Dendrobium roots can regulate the biosynthesis of sesquiterpenes and argentine in the medicinal plant Dendrobium. Chinese patent document CN110438011B discloses an endophytic fungus, Cladosporium tenuissimum DF11, belonging to the endophytic fungus *Cladosporium tenuissimum*, with accession number CGMCC No. 16883, which can promote the synthesis of tanshinone compounds. Therefore, exploring the effects of mycorrhizal fungi on plant growth, development, and the accumulation of active ingredients is of great significance. Summary of the Invention

[0004] The applicant isolated a mycorrhizal fungus from the roots of *Bletilla striata*, a plant in the Orchidaceae family, using fungal isolation and purification techniques. The fungus was identified as a strain of *Tulasnella* sp., named BJ1, and was found to promote seed germination and growth. However, no research reports have been found on its ability to promote the accumulation of secondary metabolites in *Bletilla striata*. Militarine is a quality control indicator and also an active ingredient in the traditional Chinese medicine *Bletilla striata*. Most studies have focused on its pharmacological effects and the determination and evaluation of its content in *Bletilla striata* samples from different origins, with no literature on the regulation of militarine accumulation by *Tulasnella* sp. fungi. Therefore, this patent discloses the use of strain BJ1 in promoting the synthesis and accumulation of militarine in *Bletilla striata* using uptake-cell pyrethroid (UPLC-MS) methods, laying the foundation for the cultivation of high-quality traditional Chinese medicine *Bletilla striata*.

[0005] On one hand, the present invention provides the application of Tulasnella sp. fungus in promoting the synthesis of leucoside in Bletilla striata, wherein the Tulasnella sp. fungus is BJ1, with accession number CGMCC No.40773.

[0006] This invention utilizes field co-culture of *Tulasnella* sp. BJ1 with *Bletilla striata* seedlings, revealing that this strain significantly promotes the synthesis of militarine in *Bletilla striata*. UPLC-MS analysis showed that BJ1 significantly increased the response value of militarine metabolites in *Bletilla striata*, thereby increasing the relative content of militarine.

[0007] On the other hand, the present invention also provides the application of Tulasnella sp. fungus in the preparation of a formulation that promotes the synthesis of leucoside in Bletilla striata, wherein the Tulasnella sp. fungus is BJ1, with accession number CGMCC No. 40773.

[0008] On the other hand, the present invention also provides a formulation for promoting the synthesis of leucoside in Bletilla striata, the active ingredient of which includes Tulasnella sp. fungus BJ1, accession number CGMCC No. 40773.

[0009] Specifically, after inoculating the Tulasnella sp. fungus BJ1 into the fermentation medium and culturing it, the resulting fermentation broth is the preparation used to promote the synthesis of leucoside in Bletilla striata.

[0010] The incubation temperature is 25℃, and the incubation time is 5-7 days.

[0011] On the other hand, the present invention also provides a method for promoting the synthesis of baicalin in Bletilla striata, by co-culturing Tulasnella sp. fungus BJ1 with Bletilla striata seedlings, or by applying the preparation to the roots of Bletilla striata seedlings.

[0012] The fungus BJ1, belonging to the genus Tulasnella sp., has the accession number CGMCC No. 40773.

[0013] The application method is spraying. The amount sprayed is 165 mL / bag.

[0014] The experimental subjects were Bletilla striata seedlings (selected from those with similar growth (20-22cm height), uniform tuber size (mature tuber length 2.1-2.8cm, stem diameter 0.5-0.6cm, stem length 5-6cm, tuber diameter 0.7-0.9cm, fresh weight 8-10g, mature tuber weight 1-2g), and vigorous plants) were co-cultured with a strain of *Bletilla striata* in field nursery soil (substrate ratio: coconut coir: pearl powder: peat = 1:1:1). Natural light was used in the greenhouse; when the light was too strong, 1-2 layers of 70% shade netting were added to control the light intensity below 15000lx. Watering was done 2-3 times per week. Two groups were designed: a control group and group BJ1. Samples were taken approximately once a month, for a total of 4 samplings. Assuming 30 days per month, the processed Bletilla striata tubers were stored at -80℃ for later use.

[0015] This invention uses liquid chromatography-mass spectrometry (UPLC-MS) to compare the metabolites of different months to determine the changes in the content of militarine, and performs qualitative and quantitative analysis to reveal the relationship between Tulasnellasp. BJ1 and the synthesis of 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutyl malate.

[0016] 1,4-Bis[4-(glucoseoxy)benzyl]-2-isobutylmalate, also known as: militarine, English name: militarine, English alternative name: militarin, molecular formula: C 34 H 46 O 17 CAS: 58139-23-4, chemical structure as follows:

[0017]

[0018] The Tulasnella sp. BJ1 strain described in this invention can significantly promote the synthesis and accumulation of militarine in Bletilla striata, making it a candidate strain for Bletilla striata cultivation-specific microbial fertilizer with good application prospects. Attached Figure Description

[0019] Figure 1 The images show the colony and fibrous structure characteristics of the fungus BJ1 (Tulasnella sp.); where A shows the colony morphology of BJ1 after 8 days of culture (scale bar = 1 cm); B shows the morphology of mycelium and basidiospores under a microscope (scale bar = 100 μm (10 × 40)); C shows the morphology of mycelium and spores under a scanning electron microscope (scale bar = 50 μm); and D shows the interweaving and right-angled branching of mycelium under a scanning electron microscope (scale bar = 50 μm).

[0020] Figure 2A phylogenetic tree for the genus *Tulasnella* was constructed based on ITS sequence analysis results.

[0021] Figure 3 The total ion chromatogram for Bletilla striata in QC anion mode.

[0022] Figure 4 The QC of Bletilla striata is the total ion chromatogram in cation mode.

[0023] Figure 5 The change in relative militarine content is represented by *, where * indicates p < 0.05. Detailed Implementation

[0024] Example 1: Isolation, culture and identification of Bletilla striata mycorrhizal fungus BJ1

[0025] The Bletilla striata used in this invention was collected in Qingyuan County, Lishui City, Zhejiang Province, China in June 2019.

[0026] (1) After washing the roots and tubers of Bletilla striata without any wounds or diseases, cut them into three sections, examine them under a microscope, and select samples with mycelial colonies for preservation for subsequent mycorrhizal fungal isolation.

[0027] (2) Roots found to contain mycelial clusters during microscopic examination were surface-sterilized in a laminar flow hood. The steps were as follows: immersion in 75% alcohol, followed by immersion in 1% NaClO (10% available chlorine) for 4 minutes, repeated rinsing with sterile water, and then transferring to a sterile petri dish. The epidermis was scraped off with a sterile scalpel, rinsed with sterile water, and the roots and tubers were cut into thin slices and inoculated onto PDA medium containing antibiotics (50 mg / L Ampicillin and Streptomycin sulfate). The slices were sealed, inverted, and incubated in a 25°C incubator, with observations every 2 days. Once mycelia emerged, they were isolated and purified using an inoculation needle.

[0028] (3) Some of the obtained mycorrhizal fungi were cultured in PDA liquid medium. After growing for 5-7 days, the mycorrhizal fermentation broth was obtained and sprayed onto the substrate soil containing substances that promote the germination and growth of Bletilla striata seeds. Finally, Bletilla striata seeds were sown on the substrate and allowed to germinate. The effect of promoting the germination of Bletilla striata seeds was observed. The effective Bletilla striata protocorms were isolated and purified using the endophytic fungus isolation method to obtain the mycorrhizal fungus BJ1, which has a high efficiency in promoting the growth and germination of Bletilla striata seeds.

[0029] The strains were observed for their microscopic morphology using the naked eye, a 400x microscope, and a scanning electron microscope. After incubation at 25±2℃ for 7–10 days, the colonies appeared white, loosely leathery or gelatinous, growing in a divergent, circumferential pattern, closely adhering to the culture medium. After 7 days of incubation, the average growth rate was 0.11–0.13 mm·h. -1 Using tweezers, hyphae were picked up to prepare temporary slides for observation and measurement under a microscope. Some hyphae produced spherical basidiospore clusters at their tips, and the initial hyphae grew in a beaded pattern. Scanning electron microscopy results showed that the strain contained elliptical beaded spores, with branching mostly at right or acute angles, and multiple intertwined hyphae were frequently observed (morphological images of mycorrhizal fungi on PDA plates are shown in [image missing]). Figure 1 A. Microscopic images of mycelium and basidiospore morphology are shown below. Figure 1 B, under scanning electron microscopy, the morphology of hyphae and spores, the interweaving of hyphae, and the right-angled branching are as follows: Figure 1 (C and D). Preliminary identification suggests this strain is a mycorrhizal fungus belonging to the genus *Tulasnella*.

[0030] The PDA culture medium formula of the present invention is: 200g potato, 20g glucose, and 1000mL deionized water.

[0031] (4) Molecular identification: DNA of the obtained strain BJ1 was extracted using the CTAB method and then sequenced by ITS.

[0032] 1) Extract total DNA:

[0033] 1. First, preheat the sample with 2% CTAB extraction buffer in a 65°C water bath. Use a knife to scrape the fungal hyphae from the culture medium, being careful not to scrape the culture medium. Place the sample in a sterile mortar and grind it thoroughly with liquid nitrogen in small amounts several times until it becomes powder. Then, put the sample into a 1.5ml sterile tube.

[0034] 2. Add 700 μL of 2% CTAB extraction buffer and gently stir to mix.

[0035] 3. Place in a 65℃ water bath, gently shake every 10 minutes, and remove after 40 minutes.

[0036] 4. After cooling for 2 minutes, add 700 μL of chloroform-isoamyl alcohol (24:1) and shake for 2-3 minutes.

[0037] 5. Centrifuge at 12000 rpm at 4℃ for 10 min (Steps 4 and 5 can be repeated twice).

[0038] 6. After centrifuging at 12000 rpm at 4℃ for 10 min, gently aspirate the supernatant with a pipette and transfer it into a centrifuge tube containing 600 μL of isopropanol (pre-cooled at -20℃). Gently shake the centrifuge tube up and down for 30 seconds to fully mix the isopropanol with the water layer until DNA flocculent matter is visible. Precipitate the DNA at -20℃ for 20 minutes.

[0039] 7. Centrifuge at 12000 rpm at 4℃ for 10 minutes, then immediately discard the liquid. Do not pour out the white DNA.

[0040] 8. Add 600 μL of 70% ethanol (pre-cooled at -20℃) and invert the container.

[0041] 9. Centrifuge at 12000 rpm at 4℃ for 30 seconds, then immediately discard the liquid. Allow the DNA to air dry naturally or dry it with a hairdryer. Add 30 μL of ddH2O to dissolve the DNA and store it at -20℃ for later use.

[0042] 2) PCR amplification

[0043] Amplification primers:

[0044] ITS1-F: 5'-TCCGTAGGTGAACCTGCGG-3';

[0045] ITS4-R: 5'-TCCTCCGCTTATTGATATGC-3';

[0046] PCR amplification of rDNA-ITS region reaction system (50.0 μL): total DNA of strain 2.0 μL, primer ITS1-F 2.0 μL, primer ITS4-R 2.0 μL, ddH2O 19 μL, Taq polymerase 25 μL.

[0047] PCR amplification of the rDNA-ITS region reaction program: pre-denaturation, 94℃ for 3 min; amplification cycles (34 cycles): 94℃ for 30 s, annealing, 50℃ for 30 s, extension, 72℃ for 90 s, extension; 72℃ for 8 min; storage, 4℃.

[0048] The qualified PCR products were sent to Shanghai Bioengineering Co., Ltd. for sequencing. The ITS gene sequence of the mycorrhizal fungi of the genus *Mycorrhiza* described in this invention was obtained as shown in SEQ ID No. 1. The sequence was then submitted to the NCBI database (blast.ncbi.nlm.nih.gov / Blast.cgi) for comparison. The phylogenetic tree of the mycorrhizal fungi described in this invention, constructed using the neighbor-joining method, is shown below. Figure 2This strain clusters in the same clade as *Epulorhiza* sp. (a genus of asexual *Epulorhiza*), with a coverage rate of 99% and a similarity of 99.84%. Based on morphological characteristics such as colonies, hyphae, and spores, strain BJ1 is classified as a fungus belonging to the genus *Tulasnella* sp.

[0049] The newly screened strain was named *Tulasnella* sp. fungus, strain number BJ1, and deposited on July 19, 2023, at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 40773, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. The nrDNA ITS sequence was submitted to the National Center for Biotechnology Information (NCBI) database (http: / / www.ncbi.nlm.nih.gov / ), sequence number OR245559.

[0050] Example 2: Cultivation of Bletilla striata seedlings in field experiments

[0051] The Bletilla striata seedlings used in the field experiment were obtained through the following steps:

[0052] Field experiments were conducted in a greenhouse. Transplanting containers were selected from greenhouse bags measuring 80cm long, 25cm wide, and 25cm high, ensuring adequate warmth and moisture retention. Natural light was used in the greenhouse; when sunlight was too intense, 1-2 layers of 70% shade netting were added to control the light intensity below 15000 lx. Watering was done 2-3 times per week. Temperature and relative humidity were automatically recorded using a temperature and humidity recorder, and daily averages were calculated. The substrate soil used was seedling soil (coconut coir: pearl powder: peat moss = 1:1:1), thoroughly mixed, and then transferred to the greenhouse bags. The experimental group's substrate soil was sprayed with 165mL / bag of BJ1 bacterial solution and thoroughly mixed. Each group had 12 seedling bags, with 12 Bletilla striata seedlings transplanted into each bag (selected seedlings with similar growth (20-22cm height), uniform tuber size (mature tuber length 2.1-2.8cm, stem diameter 0.5-0.6cm, stem length 5-6cm, tuber diameter 0.7-0.9cm, fresh weight 8-10g, mature tuber weight 1-2g), and vigorous plants as experimental subjects). The plants were spaced 10cm apart. Two groups were designed, including a control group and group BJ1. Samples were taken once a month for a total of 6 samplings, calculated based on 30 days per month, with 10 plants sampled each time. Finally, the Bletilla striata tuber samples were stored at -80℃ for later use.

[0053] Example 3: Analysis of changes in the content of militarine in Bletilla striata using UPLC-MS technology

[0054] The specific steps for the metabolomics assay are as follows:

[0055] 1) Metabolomics analysis: 100 mg of freeze-dried Bletilla striata samples from the control group and BJ1 group were placed in 15 mL centrifuge tubes, 4 mL of 50% ethanol was added, and the mixture was thoroughly mixed. After standing for 30 min, ultrasonic extraction was performed for 15 min, followed by centrifugation (4℃, 12000 rpm for 5 min). 2 mL of the clear supernatant was transferred to a liquid chromatography vial for UPLC-MS analysis. Equal volumes of samples were mixed as quality control (QC) samples, with 50% ethanol as the blank sample.

[0056] 2) The sample was injected into an ACQUITY ultra-high performance liquid chromatograph (Waters Corporation, Milford, MA, USA). The sample was separated by passing it through a Waters ACQUITY UPLC Cortecs T3 column (2.1 mm × 100 mm, 1.7 μm, Waters, UK) at a column temperature of 30 °C and a flow rate of 0.35 mL / min, using acetonitrile (A) and 0.1% formic acid (B) as solvents. The gradient elution program was as follows: 0–4 min, 5%–20% A; 4–7 min, 20%–25% A; 7–8.5 min, 25% A; 8.5–17 min, 25%–42% A; 17–27 min, 42%–85% A; 27–28 min, 85%–100% A; 28–30 min, 100% A; 30–31 min, 100%–5% A; 31–33 min, 5% A. Data were collected using a SYNAPT G2 Si QTOF mass spectrometer (Waters Corporation, Manchester, UK).

[0057] 3) Raw data were processed using Progenesis QI v2.3 (Nonlinear, Dynamics, Newcastle, UK). The LC-MS data matrix was imported into SIMCA14.1 software for principal component analysis (PCA), followed by (orthogonal) partial least squares discriminant analysis (OPLS-DA). Based on the OPLS-DA model, the variable importance inprojection (VIP) values ​​were obtained, where metabolites with VIP>1 were considered potentially differentially expressed metabolites. Further validation using t-tests was conducted to determine whether the differences in metabolites between groups were significant (P<0.05). Finally, relative quantification of identified militarine was performed to detect and evaluate the effect of strain BJ1 on the accumulation of militarine.

[0058] This invention employs both positive ion mode (POS) and negative ion mode (NEG) for UPUPLC-MS / MS detection. The extraction and detection of metabolites in each sample show good repeatability under both modes, the instrument exhibits good stability, and the established chromatographic conditions demonstrate good overall separation between signal peaks. Therefore, it can be used for subsequent experiments. Figure 3 and Figure 4 Systematic analysis of the QC samples revealed 9802 valid signal values, with 7454 compounds detected in positive ion mode and 2348 in negative ion mode. The positive ion mode showed a higher number of peak signal values. Further normalization of the total ion current allowed for preliminary screening to exclude duplicates and low-scoring compounds, while retaining those with high fragment ion consistency. Ultimately, 537 compounds (including militarine) were identified, with 432 in positive ion mode and 105 in negative ion mode.

[0059] The mycorrhizal fungus BJ1 described in this invention can effectively promote the accumulation of militarine in Bletilla striata. From Figure 5 The data show that, compared with the control group, the response value of militarine in the BJ1 treatment group was significantly increased, and its relative content was also significantly higher than that in the control group, especially in June, July and September.

Claims

1. A species of film-forming bacteria ( Tulasnella Application of *Bletilla striata* fungi in promoting the synthesis of leucoside in *Bletilla striata*, wherein the *Bletilla striata* fungi are *Bletilla striata* (sp.) Tulasnella The fungus BJ1 (sp.) has the accession number CGMCC No. 40773.

2. The use of a fungus of the genus *Colletotrichum* in the preparation of a formulation that promotes the synthesis of leucoside in *Bletilla striata*, wherein the fungus is a species of *Colletotrichum*. Tulasnella The fungus BJ1 (sp.) has the accession number CGMCC No. 40773.

3. A preparation for promoting the synthesis of bletilins in Bletilla striata, characterized by, The active ingredients include the genus *Collagenia* (…). Tulasnella The fungus BJ1 (sp.) has the accession number CGMCC No. 40773.

4. The preparation method of the preparation for promoting the synthesis of gynoside in gynoside according to claim 3, wherein the preparation is prepared by mixing the gynoside and the excipient. The film-forming bacteria ( Tulasnella After the fungus BJ1 (sp.) is inoculated into the fermentation medium and cultured, the resulting fermentation broth is the preparation used to promote the synthesis of leucoside in Bletilla striata.

5. The production method according to claim 4, wherein The culture temperature is 25℃, and the culture time is 5-7 days.

6. A method for promoting the synthesis of budlejafranchetii glycosides in budlejafranchetii, characterized in that, Use of the genus *Collagenus* ( Tulasnella The fungus BJ1 (sp.) was co-cultured with Bletilla striata seedlings, or the preparation according to any one of claims 3 to 5 was applied to the roots of Bletilla striata seedlings. The genus *Colletobacter* ( Tulasnella The fungus BJ1 (sp.) has the accession number CGMCC No. 40773.

7. The method of claim 6, wherein the method is for promoting the synthesis of the gypsophila glycosides in the gypsophila. The application mode is spraying.

8. The method of claim 6, wherein the method is for promoting the synthesis of the gypsophila glycosides in the gypsophila. The illumination intensity of the co-culture is lower than 15000lx.

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