Fungus for continuously inducing accumulation of agarwood and application thereof

Abstract

The application discloses a fungus capable of continuously inducing accumulation of agarwood and application thereof. The strain number of the fungus Phaeoacremonium rubrigenum is LX2018, and the registration number thereof in the China General Microbiological Culture Collection Center is CGMCC No.23266. In the implementation of the application, the Phaeoacremonium rubrigenum is functionally verified in three systems of a Aquilaria sinensis seedling, an agarwood tree and Aquilaria sinensis callus, and it is determined that the Phaeoacremonium rubrigenum has the functions of rapidly promoting agarwood formation of the agarwood plant Aquilaria sinensis and continuously promoting agarwood formation. The application has good commercial value and development potential, and has important significance for improving artificial agarwood, optimizing agarwood formation technology and promoting industrial development in actual production.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a fungus that continuously induces the accumulation of agarwood and its applications. Background Technology

[0002] Agarwood is the resinous wood of plants in the genera *Aquilaria* and *Gyrinops* of the family Thymelaeaceae. Domestically produced agarwood originates from *Aquilaria sinensis*, a typical example of an induced agarwood plant. Healthy trees do not produce resin; it only develops after years or even decades of biological or abiotic damage. Because the formation of natural agarwood is extremely slow, and artificially induced agarwood produced using current techniques rarely reaches the quality of natural agarwood, optimizing resin-forming techniques and finding methods to promote agarwood accumulation are core issues in this field of research and application. Current artificially produced agarwood is thin and has low resin content, limiting the trade and promotion of high-quality agarwood. Therefore, finding strains that can promote the continuous accumulation of agarwood is crucial for the agarwood industry and has extremely high commercial value. Summary of the Invention

[0003] The technical problem to be solved by the present invention is how to promote the continuous resin formation of agarwood seedlings and / or how to promote the thickening of the agarwood layer in agarwood plants and / or how to promote the resin formation of callus tissue in agarwood plants.

[0004] To address the aforementioned technical problems, this invention first provides the fungus *Phaeoacremonium rubrigenum*. The strain number of *Phaeoacremonium rubrigenum* is LX2018, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 23266. Hereinafter referred to as LX2018 or Pmr.LX2018.

[0005] The colony growth rate of Pmr.LX2018 on MEA solid medium can be relatively slow. The colony surface may have radial grooves and dark brown pigment rings; the texture may be fluffy; the colony height may be flat; and the colonies may emit an aromatic odor. Its branched hyphae may be septate and may contain abundant spores, which may be elliptical.

[0006] The ITS of Phaeoacremonium rubrigenum mentioned above may contain the DNA molecule shown in SEQ ID NO.1 of the sequence listing.

[0007] The culture of Phaeoacremonium rubrigenum described above also falls within the scope of protection of this invention. The culture of Phaeoacremonium rubrigenum can be a substance obtained by culturing the Phaeoacremonium rubrigenum described above in a microbial culture medium.

[0008] To address the aforementioned technical problems, the present invention also provides a microbial agent. The microbial agent contains the metabolites and / or cultures of *Phaeoacremonium rubrigenum* as described above.

[0009] The above-mentioned microbial agent may have at least one of the following characteristics:

[0010] A1) Promotes resin formation in agarwood seedlings.

[0011] A2) Promotes the thickening of the agarwood layer in agarwood plants.

[0012] A3) Promotes the formation of resin in the callus tissue of agarwood plants.

[0013] The resin formation time of the agarwood seedlings treated with the microbial agent can be longer than that of the agarwood seedlings not treated with the microbial agent.

[0014] The active ingredient of the above-mentioned microbial agent may be the metabolites of Phaeoacremonium rubrigenum or / and Phaeoacremonium rubrigenum. The active ingredient of the above-mentioned microbial agent may also contain other biological or non-biological components. Other active ingredients of the above-mentioned microbial agent can be determined by those skilled in the art based on the effect of the microbial agent.

[0015] The microbial agent may further include a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier may be a mineral material or a biological material; the mineral material may be at least one of peat moss, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the biological material may be at least one of various crop straws, pine shells, rice straw, peanut shells, corn flour, soybean flour, starch, peat moss, and animal manure; the liquid carrier may be water; in the microbial agent, the metabolites of *Phaeoacremonium rubrigenum* and / or *Phaeoacremonium rubrigenum* may exist in the form of cultured live cells, fermentation broth of live cells, filtrate of cell culture, or a mixture of cells and filtrate. The microbial agent may be in various formulations, such as liquid, emulsion, suspension, powder, granules, wettable powder, or water-dispersible granules.

[0016] In the above text, the metabolites of *Phaeoacremonium rubrigenum* can be the fermentation broth of *Phaeoacremonium rubrigenum*. The fermentation broth of *Phaeoacremonium rubrigenum* can be prepared as follows: *Phaeoacremonium rubrigenum* is cultured in a liquid fermentation medium, and the fermentation broth (containing *Phaeoacremonium rubrigenum* and substances secreted into the liquid medium) is collected; this fermentation broth is the metabolite of *Phaeoacremonium rubrigenum*.

[0017] At least one of the following applications of Phaeoacremonium rubrigenum described above also falls within the scope of protection of this invention.

[0018] B1) The application of Phaeoacremonium rubrigenum as described above in the preparation of products that promote resin formation in agarwood seedlings.

[0019] B2) The application of Phaeoacremonium rubrigenum as described above in the preparation of products that promote the thickening of the agarwood layer in agarwood plants.

[0020] B3) The application of Phaeoacremonium rubrigenum as described above in the preparation of products that promote the formation of resin in the callus tissue of agarwood plants.

[0021] At least one of the following applications of the above-described microbial agent also falls within the scope of protection of this invention.

[0022] C1) The application of the above-mentioned microbial agent in the preparation of products that promote the continuous resin formation of agarwood seedlings.

[0023] C2) The application of the above-mentioned microbial agent in the preparation of products that promote the thickening of the agarwood layer of agarwood plants.

[0024] C3) The application of the microbial agent described above in the preparation of products that promote the continuous resin formation of agarwood seedlings.

[0025] In the above-described microbial agents and / or applications, the agarwood plant may be Aquilaria sinensis.

[0026] In the above-described fungal agents and / or applications, the aroma components of the resinous compound may be sesquiterpenes and / or chromones.

[0027] The sesquiterpenes may be sesquiterpenoid components with similar parent nuclei, such as δ-guaiacene, α-cubene, α-guaiacene, and / or nocarne.

[0028] To address the aforementioned technical problems, the present invention also provides a method for preparing the aforementioned microbial agent. The method includes the step of culturing *Phaeoacremonium rubrigenum* as described above in a microbial culture medium.

[0029] This invention isolated the fungal strain *Phaeoacremonium rubrigenum* from the transition layer between agarwood and osmanthus. Amplicon sequencing of agarwood revealed that *Phaeoacremonium rubrigenum* plays a crucial role in agarwood accumulation. The strain was successfully isolated and named *Phaeoacremonium rubrigenum* LX2018 (abbreviated as Pmr.LX2018). *Pmr.LX2018* belongs to the kingdom Fungi, class Sordariomycetes, order Diaporthales, and is a filamentous fungus. Experiments showed that *Pmr.LX2018* inoculated into *Aquilaria* plants produced agarwood sesquiterpenes within 5 days, with the total sesquiterpenes content gradually increasing. By day 15, the treated *Aquilaria* seedlings began producing chromones, and both the total chromone and total sesquiterpenes contents continued to increase. Furthermore, Pmr.LX2018 can promote the thickening of the agarwood layer and the formation of resin in the callus tissue of agarwood plants. In actual production, it is of great significance for improving artificial agarwood, optimizing agarwood formation technology, and promoting industrial development.

[0030] Preservation Instructions

[0031] Latin name: Phaeoacremonium rubrigenum

[0032] Strain number: LX2018

[0033] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0034] Collection institution abbreviation: CGMCC

[0035] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0036] Date of preservation: September 27, 2021

[0037] CGMCC Registration Number: CGMCC No. 23266 Attached Figure Description

[0038] Figure 1Microscopic histochemical analysis of different layers of agarwood. (a) Experimental diagram of microscopic histochemical analysis of different layers of agarwood. A represents the junction between the agarwood layer and the transition layer. B represents the transition layer. C represents the scleroderma layer. A-1, B-1, and C-1 represent I2-KI staining to analyze starch grain distribution; A-2, B-2, and C-2 represent PAS staining to analyze polysaccharide distribution; A-3, B-3, and C-3 represent DAPI staining to analyze cell viability.

[0039] Figure 2 This is a taxonomic tree of fungal species in different layers of agarwood. Different colored sectors within the circles represent different groups, corresponding to the legend on the left; the size of the sector indicates the proportion of relative abundance of that group within that taxonomy. The figure shows that *Phaeoacremonium rubrigenum* has a high proportion in the transition layer (TL) and agarwood layer (AL).

[0040] Figure 3 The image shows the isolated and purified Pmr. LX2018. The left image shows Pmr. strain LX2018 grown on MEA medium. The right image is a micrograph of the hyphae and spores of Pmr. LX2018.

[0041] Figure 4 This is a cluster tree for the isolated and purified Pmr.LX2018 and all species of the genus Phaeoacremonium.

[0042] Figure 5 Pmr.LX2018 was used to treat Aquilaria sinensis seedlings. The figure includes the control group and the treatment group.

[0043] Figure 6 Line graphs showing the percentage content of total sesquiterpenes and total chromones in *Aquilaria sinensis* seedlings treated with Pmr.LX2018 at different days. Percentage content (%) = Peak area of ​​target component × 100% / Total peak area

[0044] Figure 7 30-year-old Aquilaria sinensis were treated with Pmr.LX2018. The pentagrams represent the treated samples, and the triangles represent the control samples.

[0045] Figure 8 A cross-section of agarwood from 30-year-old Aquilaria sinensis treated with Pmr.LX2018. The arrow indicates the agarwood layer.

[0046] Figure 9 Images of callus tissue from Aquilaria sinensis processed with Pmr.LX2018 for different number of days.

[0047] Figure 10The relative contents of four sesquiterpenes in Aquilaria sinensis callus treated with Pmr.LX2018 for different days are shown in the figure. Relative content = target compound peak area × average internal reference peak area of ​​all samples / internal reference peak area of ​​this sample. "**" represents P < 0.01. Detailed Implementation

[0048] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0050] In the quantitative experiments in the following examples, three or more repeated experiments were set up, and the average value of the results was taken.

[0051] Example 1: Screening and Analysis of Fungi that Promote Continuous Agarwood Formation

[0052] I. Microscopic Histochemical Detection of Different Layers of Agarwood

[0053] 1. Acquisition and Layering of Agarwood Materials

[0054] The agarwood material was sourced from Dongguan, Guangdong Province, from 50-year-old Aquilaria sinensis trees with a diameter at breast height (DBH) of 15–25 cm. It was traditionally formed naturally over a period of 5 years, with the agarwood layer being 0.5–1.0 cm thick. From the outside in, the agarwood material consists of the decay layer (DL), the transition layer between the decay layer and the agarwood layer (DA), the agarwood layer (AL), the transition layer between the agarwood layer and the sapwood layer (TL), and the sapwood layer (NL). Samples from different parts of the agarwood were cut into small pieces, 0.5–0.1 cm thick and 0.5 cm × 0.5 cm in size, and fixed in a 10% formaldehyde solution.

[0055] 2. Observation of starch grain distribution in different layers of agarwood

[0056] Material samples in fixative were sectioned using a cryostat at -20°C to a thickness of 10 μm. Sections were stained in Lugol's iodine solution for 3 min, washed, and then mounted for observation. Results showed that starch granules were abundant in the NL layer, present in a small amount in the TL layer, and not observed in the other layers. Figure 1 At the same time, it can be seen that a large amount of brown agarwood oil exists in DA and AL, and a small amount exists in TL.

[0057] 3. Observation of polysaccharide distribution in different layers of agarwood

[0058] Material samples in fixative were sectioned using a cryostat at -20℃ to a thickness of 10 μm. PAS staining was performed; sections were treated in 0.5% periodic acid solution for 10 min, rinsed with water, and then stained in Schiff's reagent for 4 min. After washing, the sections were mounted for observation. Results showed that polysaccharides were mainly found in the TL and NL layers, with a higher concentration in the TL layer. Figure 1 ).

[0059] 4. Comparison of cell vitality in different layers of agarwood

[0060] Material samples in fixative were sectioned using a cryostat at -20℃ to a thickness of 10 μm. Sections were stained with DAPI solution for 10 min, washed, and quickly mounted for observation to minimize fluorescence quenching. Results showed that the NL layer contained a large number of viable cells, the TL layer contained some viable cells with some nuclei showing deformation, and no viable cells were observed in the AL, DA, and DL layers. Figure 1 ).

[0061] Since the oily substances in agarwood are mainly produced by the living cells of scleroderma, and the TL layer, as the key position connecting the agarwood layer and the scleroderma layer, contains some living cells and its chemical substances (starch grains, polysaccharides, and brown agarwood oil) change significantly, it is the key part for the continuous formation of agarwood.

[0062] II. Detection and Analysis of Fungal Distribution in Different Layers of Agarwood

[0063] 1. DNA extraction and detection

[0064] DNA was extracted from different layers of agarwood samples using the CTAB method. The concentration and purity of the DNA were determined by 1% agarose gel electrophoresis. An appropriate amount of sample was placed in a 1.5 mL centrifuge tube and diluted with sterile water to a final concentration of 1 ng / μL. -1 .

[0065] 2. PCR amplification and high-throughput sequencing

[0066] PCR amplification and purification were performed using universal primers ITS5-1737F (5′-GGAAGTAAAGTCGTAACAAGG-3′) and ITS2-2043R (5′-GCTGCGTTCTTCATCGATGC-3′). The PCR product bands were recovered using a gel extraction kit provided by QIAGEN. The DNA PCR-Free Sample Preparation Kit was used for library construction. The constructed libraries were then quantified using Qubit and Q-PCR. Once the libraries were deemed acceptable, IonS5 was used for their analysis. TM High-throughput sequencing was performed on the XL platform.

[0067] 3. Data processing and analysis and species taxonomy tree

[0068] Data for each agarwood stratified sample was obtained by splitting the barcode and PCR amplification primer sequences. After removing the barcode and primer sequences, the reads of each sample were assembled using FLASH, and the resulting assembled sequences were the original tag data. Uparse software was used to cluster all effective tags of all samples into OTUs (operational taxonomic units) based on a 97% similarity. OTUs were then annotated using QIIME software against the SILVA, Greengene, and RPD databases.

[0069] For each agarwood stratified sample or each group, the top 20 species with the highest relative abundance were selected for species taxonomic analysis. The fungus with the highest proportion in the TL layer was found to be *Phaeoacremonium rubrigenum*, suggesting its role in promoting sustained resin formation in agarwood. Figure 2 ).

[0070] Example 2: Obtaining Agarwood Fungus Pmr.LX2018

[0071] 1. Sample pretreatment

[0072] The agarwood samples were rinsed with ultrapure water for 5 minutes and then divided into different layers to obtain agarwood samples of different layers (hereinafter referred to as samples). The samples were sterilized in an ultrapure workbench under aseptic conditions: soaked in 75% ethanol for 1-2 minutes, rinsed twice with sterile water, soaked in 2% sodium hypochlorite for 10 minutes, and rinsed 5-6 times with sterile water. After sterilization, the samples were placed on sterile absorbent paper to drain the water. Then, the resin wood surface tissue was carefully scraped off with a sterile scalpel. Finally, the transition layer was cut into wood blocks of 5mm×5mm×5mm and placed in MEA medium, with one sample in each petri dish. To check whether the sample surface sterilization was thorough, the sterile water from the last rinse was inoculated into MEA medium as a blank control.

[0073] 2. Isolation and purification of agarwood fungi

[0074] The samples were placed in a 25°C constant temperature incubator for dark incubation. When the agarwood transition layer samples were incubated for 3-4 days, the hyphae on the edges were visible to the naked eye. The hyphae of a single colony were picked with a sterile toothpick and inoculated into a new culture medium. The samples were then cultured under the same conditions. After more than 4 separations and purifications, a single colony was obtained and the strain was named LX2018.

[0075] 3. Identification and nomenclature of the agarwood fungus Pmr.LX2018

[0076] After four purification cycles in step 2, the LX2018 fungal colonies were first observed visually. It was found that the colonies grew slowly, taking approximately 25 days to fully colonize an agar plate. The colony surface exhibited radial grooves, dark brown pigment rings, a velvety texture, and a flattened colony height. Figure 3 (Left image) The colony emits a fragrant aroma. The branching hyphae are septate and contain abundant spores. The spores are oval-shaped and do not produce fruiting bodies. Figure 3 (Right image in the middle). By analyzing the colony morphology of the fungus in accordance with the "Handbook of Fungal Identification", the fungus was identified as a fungus of the genus *Phaeoacremonium*.

[0077] Furthermore, the DNA of this fungus was extracted using the CTAB method. PCR amplification of the fungal DNA was performed using primers ITS1F (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4R (5′-TCCGTAGGTGAACCTGCGG-3′). The reaction mixture consisted of 50 μL, 10 μL of 5× buffer, 4 μL of dNTPs (2.5 mM), 1 μL each of primers ITS1F and ITS4R, 1 μL of DNA template, 1 μL of DNA polymerase, and sterile distilled water to a final volume of 50 μL. The PCR amplification parameters were: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; 72℃ for 10 min. After the reaction, 5 μL of product was added to 1 μL of 6× Loading Buffer, vortexed, and observed using a 1.5% agarose gel electrophoresis and gel imaging system. A clear and bright band was found at approximately 600 bp, which was the ITS sequence of Pmr. LX2018. Sanger sequencing (Beijing Ruiboxingke Biotechnology Co., Ltd.) was used to perform bidirectional sequencing and assembly of this band sequence. BLAST analysis was used to compare sequence similarity. The ITS (internal transcribed spacer region) of the ribosomal rRNA gene of this strain (SEQ ID NO. 1 in the sequence listing) showed 100% similarity to the ITS of Phaeoacremonium rubrigenum. Furthermore, phylogenetic analysis of this sequence with sequences from other species in the same genus revealed that this strain clustered with Phaeoacremonium rubrigenum in the same clade, with a support rate of 98%. Therefore, combining morphological and molecular identification, it can be concluded that the strain LX2018 isolated in this invention is *Phaeoacremonium rubrigenum*, and is named *Phaeoacremonium rubrigenum stain LX2018*. *Phaeoacremonium rubrigenum stain LX2018* was deposited on September 27, 2021, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 23266 and strain number LX2018, hereinafter abbreviated as Pmr.LX2018. Figure 4 ).

[0078] Example 3: Pmr.LX2018 promotes continuous resin formation in agarwood seedlings (agarwood plant seedlings).

[0079] 1. Pmr.LX2018 treatment for Aquilaria sinensis seedlings

[0080] Under aseptic conditions, Pmr.LX2018 was inoculated and activated using the corresponding MEA solid medium (prepared from 2% malt extract and 1.5% agar), and cultured in the dark at 25°C. When the fungus produced spores, a 0.5 cm diameter mycelial cake was taken as the strain for treating the seedlings. One-year-old healthy seedlings of Aquilaria sinensis (laboratory-preserved, related literature: Liu et al. Volatile organic compound and endogenous phytohormone characteristics during callus browning in Aquilaria sinensis. Industrial Crops & Products, 2021:168. Available to the public from the applicant, for the purpose of replicating this invention only, and not for other uses) were randomly selected. Pmr.LX2018 was inoculated into the seedlings using a 45° oblique cut method. Specifically, the cutting tool and stem were disinfected with 75% ethanol. A 45° oblique cut was made into the stem at approximately 5cm, 7cm, and 9cm above the ground, to a depth of 1 / 3 of the tree's diameter. The mycelium was inoculated into the wound and fixed with sealing film. Figure 5 (Middle treatment group). Additionally, *Aquilaria sinensis* seedlings that were not inoculated with strain Pmr.LX2018 after oblique cutting served as a negative control. Figure 5 The interaction time was (0 days, 5 days, 10 days, 15 days, 20 days, 25 days, and 30 days), with 6 biological replicates for each experimental group. Samples were collected promptly according to the specified interaction time, freeze-dried, and then powdered for later use.

[0081] 2. Detection of resin components in Aquilaria sinensis seedlings treated with Pmr.LX2018

[0082] The two main types of resin-forming components in agarwood are sesquiterpenes and chromones. Therefore, this invention uses GC-MS to detect the volatile components of stem segments of Aquilaria sinensis seedlings treated with Pmr.LX2018.

[0083] Preparation of the test solution: Accurately weigh 0.1 g of Aquilaria sinensis stem powder and place it in a 2 ml imported centrifuge tube. Accurately add 1.5 ml of ethyl acetate (analytical grade) using a micropipette, seal the tube, and soak overnight at room temperature. Sonicate at 4℃ (40 kHz) for 45 min, centrifuge at 12000 rpm for 10 min, weigh, and add ethyl acetate to make up for any weight loss. Transfer the supernatant to a new 2 ml centrifuge tube, concentrate with nitrogen blowing until the solvent is completely evaporated, then accurately add 200 μL of ethyl acetate, vortex thoroughly, and filter using a 0.22 μm polytetrafluoroethylene (PTFE) filter membrane. Place the filtrate in a gas chromatography vial containing an inner tube, seal with an unopened cap, and store as the test solution at 4℃ for later use.

[0084] The components of the test samples were determined using gas chromatography-mass spectrometry (GC-MS). GC fractions were obtained from a Thermo Fisher Scientific Trace 1310 spectrometer, and MS fractions were obtained from a Thermo Fisher Scientific TSQ8000 spectrometer. GC-MS method: autosampler (1 μL, splitless), programmed temperature ramp, initial at 50 °C, increments of 10 °C / min. -1 Rise to 15℃, hold for 15 minutes, 8℃·min -1 The temperature was raised to 280℃ and held for 10 minutes; the injection port temperature was 250℃, the electron energy was 70eV, the ion source temperature was 250℃, and the mass scan range of the sample was 50~600m / z.

[0085] The total ion chromatogram of the material samples was processed using a Trance Finder 3.3 workstation (Thermo Scientific). Background interference was removed, and information was compared using the NIST 2.2 standard mass spectrometry library. Chromatographic peaks were analyzed and each compound was identified. The relative mass fraction of each compound was calculated using peak area normalization and statistical methods. Finally, the total sesquiterpene content was obtained by summing all sesquiterpene components, and the total chromone content was obtained by summing all chromone components. Figure 6 It can be seen that after 5 days of treatment, the treated Aquilaria sinensis seedlings began to produce sesquiterpenes, and the total sesquiterpenes content gradually increased with the increase of treatment time; by day 15, the treated Aquilaria sinensis seedlings began to produce chromones, and the total chromone and total sesquiterpenes contents continued to increase. The results indicate that Pmr.LX2018 can continuously stimulate the production of sesquiterpenes and chromones in Aquilaria sinensis seedlings, thereby promoting continuous resin formation, while no sesquiterpenes or chromones were detected in the untreated control group.

[0086] Example 4: Pmr.LX2018 promotes the thickening of the agarwood layer in agarwood plants.

[0087] 1. Pmr.LX2018 treatment of a 30-year-old Aquilaria sinensis tree

[0088] Under aseptic conditions, Pmr.LX2018 was inoculated and activated using the corresponding MEA solid medium (prepared from 2% malt extract and 1.5% agar), and cultured in the dark at 25°C. When the fungus produced spores, a 0.5 cm diameter mycelial cake was collected as the strain for treating Aquilaria sinensis trees. Six healthy 30-year-old Aquilaria sinensis trees with uniform diameter at breast height (DBH) were randomly selected from the site (laboratory-preserved, related literature: Liu et al. Agarwood woundlocations provide insight into the association between fungal diversity and volatile compounds in Aquilaria sinensis. R.Soc. Open Sci, 6:190211. Available to the public from the applicant, for the purpose of repeating this invention only, and not for other uses). The saw and stem were surface-sterilized with 75% ethanol. The saw was used to cut into the main stem at a depth of about 1.5 m from the ground, to half the diameter of the tree. The mycelial cake was inoculated into the wound and fixed with sealing film. Figure 7 The treatment group was used as a negative control (Pmr.LX2018 was not inoculated after sawing). Figure 7 (Control group). Both the control and experimental groups consisted of three biological replicates. Sampling was performed one year after the interaction.

[0089] 2. Detect the thickness of the agarwood layer

[0090] Agarwood layer detection was performed on the stems of Aquilaria sinensis treated with Pmr.LX2018. Figure 8 As shown in the Pmr.LX2018 group, the agarwood layer thickness was found to be 0.4±0.07cm; while the control group, the untreated Aquilaria sinensis stem (…),… Figure 8 The agarwood layer thickness of the control group was 0.2 ± 0.08 cm. The results showed that strain Pmr.LX2018 significantly thickened the agarwood layer of Aquilaria sinensis compared with the control group.

[0091] Example 5: Pmr.LX2018 promotes resin formation in agarwood plant callus tissue.

[0092] 1. Induction and culture of Aquilaria sinensis callus

[0093] Take healthy, young leaves of *Aquilaria sinensis* and place them in tissue culture bottles. Rinse 2-3 times with purified water, then wipe the surface of the leaves dry with absorbent paper. Sterilization is performed in a laminar flow hood: immerse the freshly taken leaves in 75% ethanol for 30 seconds, then immediately remove and rinse 2-3 times with sterile water. Transfer to a 2% sodium hypochlorite solution and immerse for 10 minutes, ensuring full contact with the solution for sterilization. Immediately after sterilization, rinse 4-5 times with sterile water for 2-3 minutes each time. After cleaning, dry the surface with sterile absorbent paper, trim the leaf edges, and cut the leaves into 0.5cm × 0.5cm cubes. Inoculate onto MS medium containing 1.0 mg / L. -1 6-BA + 0.5 mg·L -1 NAA culture medium (sucrose 30 g·L⁻¹) -1 7.0 g / L agar -1 Cultured at 25°C in the dark (pH = 5.7–5.8). The induced callus was subcultured every 20–30 days to obtain Aristolochia debilis callus.

[0094] 2. Treatment of Aquilaria sinensis callus with strain Pmr.LX2018

[0095] Under aseptic conditions, Pmr.LX2018 was inoculated and activated using the appropriate MEA solid medium (prepared from 2% malt extract and 1.5% agar), and incubated in the dark at 25°C. When the fungus produced spores, 0.5 cm diameter mycelial discs were collected for use in treating Aristolochia debilis callus. The 0.5 cm diameter mycelial discs were placed in MS medium (MS + 1.0 mg·L⁻¹). -1 6-BA + 0.5 mg·L -1 NAA culture medium (sucrose 30 g·L⁻¹) -1 7.0 g / L agar -1 (pH = 5.7–5.8)) center, 2.0 cm away from the center of each of the four pieces of Aquilaria sinensis callus tissue. Figure 9 Then, Pmr.LX2018-treated callus samples were collected at 0, 3 and 18 days, respectively.

[0096] 3. Detection of resinous components in *Aquilaria sinensis* callus treated with *Pmr. LX2018* strain

[0097] The volatile components of the *Aquilaria sinensis* callus tissue treated with Pmr.LX2018 obtained in step 2 were detected by GC-MS (GC fraction from Thermo Trace 1310, MS fraction from Thermo TSQ8000). Preparation of the test solution: Accurately weigh 0.1 g of *Aquilaria sinensis* callus tissue powder and place it in a 2 ml imported centrifuge tube. Accurately add 1.5 ml of ethyl acetate (analytical grade) using a micropipette, seal the tube, and incubate overnight at room temperature. Sonicate at 40 kHz for 45 min at low temperature (4℃), centrifuge at 12000 rpm for 10 min, weigh, and add ethyl acetate to make up for weight loss. Transfer the supernatant to a new 2 ml centrifuge tube, concentrate with nitrogen blowing until the solvent is completely evaporated, then precisely add 200 μL of ethyl acetate, vortex thoroughly, filter using a 0.22 μm polytetrafluoroethylene (PTFE) filter membrane, and place the filtrate in a gas chromatography vial with an inner tube, seal with an unopened cap, and store as the test solution at low temperature (4℃) for later use. GC-MS method: Autosampler (1 μL, splitless), temperature programmed, initial at 50℃, increments at 10℃·min. -1 Rise to 15℃, hold for 15 minutes, 8℃·min -1 The temperature was raised to 280℃ and held for 10 minutes; the injection port temperature was 250℃, the electron energy was 70eV, the ion source temperature was 250℃, and the mass scan range of the sample was 50~600m / z.

[0098] The total ion chromatograms of the material samples were processed using a Trance Finder 3.3 (Thermo) workstation. Background interference was removed, and information was compared using the NIST 2.2 standard mass spectrum library. Chromatographic peaks were analyzed and each compound was identified. The relative mass fraction of each compound was calculated using peak area normalization and statistical methods. Figure 10 It can be seen that the callus tissue of *Aquilaria sinensis* treated with strain Pmr.LX2018 contained four sesquiterpenoids: δ-guaiacol, α-cubene, α-guaiacol, and nocaene. After 3 days of treatment with strain Pmr.LX2018, all four components showed an increasing trend, and by 18 days, they had increased significantly. These results indicate that strain Pmr.LX2018 has the effect of promoting resin formation in the callus tissue of *Aquilaria sinensis*.

[0099] In summary, the fungus Pmr.LX2018 provided by this invention can promote the accumulation and continuous resin formation of agarwood, which is of great significance for improving artificial agarwood, optimizing agarwood resin formation technology, and promoting industrial development.

[0100] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. sequence list <110> Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences <120> A fungus that continuously induces agarwood accumulation and its applications <130> GNCSQ212415 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 595 <212> DNA <213> Phaeoacremonium rubrigenum <400> 1 aacctgcgga gggatcatta acgagtttcg tactccaaac cctttgtgaa catacctgtt 60 ttcgttgctt cggcaggtga aggcggacgg cctccgggcc tgaagccgcc gccgggcgga 120 cccctcgcgg ggcgctgccg ggtgggcctg ccggagggca cagactctgt attacaacgt 180 acctctctga gttatatttt acaaacaagt aaaaactttc aacaacggat ctcttggttc 240 tggcatcgat gaagaacgca gcgaaatgcg ataagtaatg tgaattgcag aattcagtga 300 atcatcgaat ctttgaacgc acattgcgcc cgccagtatt ctggcgggca tgcctgtccg 360 agcgtcattt caaccctcag gccctggttg cctggtgttg gggcgccgcg caccctcagc 420 gggcgcgggc cccgaaagtc agtggcgggc tcgccaggac tccgagcgca gtaattttct 480 ctcgctgtgg agcgcctggt gggttaccgg ccgtaaaaca ccccaaattc taaaggttga 540 cctcggatca ggtaggaata cccgctgaac ttaagcatat caataagcgg aggaa 595

Claims

1. The fungus Phaeoacremonium rubrigenum, strain number LX2018, which is registered at the China General Microbiological Culture Collection Center with the registration number CGMCCNo.23266.

2. The Phaeoacremonium rubrigenum according to claim 1, characterized in that: The ITS of Phaeoacremonium rubrigenum contains the DNA molecule shown in SEQ ID NO.1 of the sequence listing.

3. At least one of the following applications of Phaeoacremonium rubrigenum as described in claim 1 or 2: B1) The use of Phaeoacremonium rubrigenum as described in claim 1 or 2 in the preparation of products that promote resin formation in agarwood seedlings; B2) The use of Phaeoacremonium rubrigenum as described in claim 1 or 2 in the preparation of products that promote the thickening of the agarwood layer in agarwood plants; B3) The use of Phaeoacremonium rubrigenum as described in claim 1 or 2 in the preparation of products that promote the formation of resin in callus tissue of agarwood plants; The agarwood plant in question is Aquilaria sinensis.

4. The application according to claim 3, characterized in that: The aroma components of the resin are sesquiterpenes and / or chromones.

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