WO2023109839A1 - Fungus for continuously inducing accumulation of agarwood and use thereof - Google Patents

Abstract

Provided are a fungus for continuously inducing accumulation of agarwood and the use thereof. The fungus Phaeoacremonium rubrigenum has a strain number of LX2018, and has a registration number of CGMCC No. 23266 in the China General Microbiological Culture Collection Center. In the implementations, functional verification is carried out on the Phaeoacremonium rubrigenum in three systems of Aquilaria sinensis seedlings, Aquilaria trees and Aquilaria sinensis callus tissues; and it is determined that the Phaeoacremonium rubrigenum has the effect of rapidly promoting the formation of agarwood in the Aquilaria plant Aquilaria sinensis and also has the function of sustainably promoting the formation of agarwood.

Description

A fungus that continuously induces the accumulation of agarwood and its application technical field

The invention relates to the field of biotechnology, in particular to a fungus that continuously induces the accumulation of agarwood and its application.

Background technique

Aquilaria is the resinous wood of Aquilaria and Gyrinops plants in the family Thymelaeaceae. Among them, domestically produced Aquilaria is derived from Aquilaria sinensis, which is a typical inductive medicinal plant. A healthy tree does not produce incense, and only after years or even decades of damage can it produce aloes. Since the formation of natural agarwood is extremely slow, and the artificial agarwood induced by the existing agarwood technology is extremely difficult to achieve the quality of natural agarwood, optimizing the agarwood technology and finding ways to promote the accumulation of agarwood are the core issues of research and application in this field. The agarwood produced by the existing artificial technology is thin in thickness and low in balsam content, which limits the trade and promotion of high-quality agarwood. Therefore, finding strains that can promote the continuous accumulation of agarwood has a vital role in promoting the agarwood industry and has extremely high commercial value. .

invention disclosure

The technical problem to be solved by the present invention is how to promote the continuous aroma formation of the saplings of the agarwood plant and/or how to promote the thickening of the agarwood layer of the agarwood plant and/or how to promote the aroma formation of the callus of the agarwood plant.

In order to solve the problems of the technologies described above, the present invention at first provides the fungus Phaeoacremonium rubrigenum. The strain number of the Phaeoacremonium rubrigenum is LX2018, and its registration number in the General Microbiology Center of China Microbiological Culture Collection Management Committee is CGMCC No.23266. Hereinafter referred to as LX2018 or Pmr.LX2018.

The colony growth rate of the Pmr.LX2018 on the MEA solid medium can be slower growth. It can have radial grooves and dark brown pigment rings on the surface of the colony, the texture can be fluffy, the height of the colony can be flat, and the colony can emit an aromatic smell. Its branched hyphae may have septa, may have abundant spores, and its spores may be oval.

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

The culture of Phaeoacremonium rubrigenum described above also belongs to the protection scope of the present invention. The culture of Phaeoacremonium rubrigenum can be the material obtained by culturing the above-mentioned Phaeoacremonium rubrigenum in a microbial culture medium.

In order to solve the above technical problems, the present invention also provides bacterial agents. The bacterial agent contains the above-mentioned Phaeoacremonium rubrigenum or/and the metabolites or/and cultures of the above-mentioned Phaeoacremonium rubrigenum.

The bacterial agent described above may have at least one of the following characteristics:

A1) promote the agarwood plant saplings to produce fragrance.

A2) Promote the thickening of the agarwood layer of the agarwood plant.

A3) Promote the agarwood plant callus to produce fragrance.

The aroma setting time of the agarwood plant saplings treated with the bacterial agent can be longer than that of the agarwood plant saplings not treated with the bacterial agent.

The active ingredient of the above-mentioned bacterial agent can be the metabolite of the Phaeoacremonium rubrigenum or/and the Phaeoacremonium rubrigenum, the active ingredient of the above-mentioned bacterial agent can also contain other biological components or non-biological components, other active ingredients of the above-mentioned bacterial agent are skilled in the art Personnel can be determined according to the effect of the bacterial agent.

The bacterial agent may also include a carrier. The carrier can be a solid carrier or a liquid carrier. The solid carrier is a mineral material, a biological material; the mineral material can be at least one of peat, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomite; the biological material is At least one of stalks, pine shells, straw, peanut shells, corn flour, soybean flour, starch, peat and animal manure of various crops; the liquid carrier can be water; in the bacterial agent, the Phaeoacremonium rubrigenum or/and metabolites of said Phaeoacremonium rubrigenum may exist in the form of cultured living cells, fermentation broth of living cells, filtrate of cell culture or a mixture of cells and filtrate. The dosage form of the bacterial agent can be various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water-dispersible granule.

In the above, the metabolite of the Phaeoacremonium rubrigenum may be the fermentation liquid of the Phaeoacremonium rubrigenum. The fermented liquid of described Phaeoacremonium rubrigenum can be prepared according to the following method: cultivate described Phaeoacremonium rubrigenum in liquid fermentation medium, collect fermented liquid (containing described Phaeoacremonium rubrigenum and the material secreted in liquid medium), this fermented liquid is Metabolites of the Phaeoacremonium rubrigenum.

The following at least one application of the above-mentioned Phaeoacremonium rubrigenum also belongs to the protection scope of the present invention.

B1) the application of Phaeoacremonium rubrigenum described above in the preparation of a product that promotes the aroma formation of agarwood plant saplings.

B2) the application of Phaeoacremonium rubrigenum described above in the preparation of a product that promotes the thickening of the agarwood layer of the agarwood plant.

B3) application of Phaeoacremonium rubrigenum described above in the preparation of a product that promotes agarwood plant callus to produce fragrance.

At least one of the following applications of the bacterial agent described above also belongs to the protection scope of the present invention.

C1) Application of the bacteria agent described above in the preparation of a product that promotes the continuous aroma formation of agarwood plant saplings.

C2) Application of the bacteria agent described above in the preparation of a product that promotes the thickening of the agarwood layer of the agarwood plant.

C3) Application of the bacteria agent described above in the preparation of a product that promotes the continuous aroma formation of agarwood plant saplings.

In the bacterial agent mentioned above and/or the application mentioned above, the agarwood plant can be Aquilaria sinensis.

In the bacterial agent mentioned above and/or the application mentioned above, the aroma component of the incense can be sesquiterpene and/or chromone.

The sesquiterpene may be a sesquiterpene component similar to a core such as δ-guaiaene, α-clubene, α-guaiaene and/or nocaene.

In order to solve the above technical problems, the present invention also provides a method for preparing the bacterial agent described above. The method comprises the step of culturing the Phaeoacremonium rubrigenum described above in a microbial culture medium.

Preservation instructions

Latin name: Phaeoacremonium rubrigenum

Strain number: LX2018

Preservation institution: General Microbiology Center of China Committee for the Collection of Microorganisms

Depository institution abbreviation: CGMCC

Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing

Preservation date: September 27, 2021

Registration number of the collection center: CGMCC No.23266

Description of drawings

Figure 1 shows the microhistochemical detection of different layers of Agarwood. (a) Microhistochemistry experiment diagram of different layers of Agarwood. A represents the junction position of the agarwood layer and the transition layer. B stands for transition layer. C stands for white wood layer. A-1, B-1, C-1 represent I ₂ -KI staining to analyze the distribution of starch granules; A-2, B-2, C-2 represent PAS staining to analyze the distribution of polysaccharides; A-3, B- 3. C-3 represents DAPI staining to analyze cell viability.

Figure 2 is the classification tree of fungal species in different layers of Agarwood. The sectors of different colors in the circle represent different groups, corresponding to the legend on the left; the size of the sector represents the proportion of the relative abundance of the group in that category. In the figure, it can be seen that Phaeoacremonium rubrigenum has a high proportion in the transition layer (TL) and the agarwood layer (AL).

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

Figure 4 is a clustering tree of isolated and purified Pmr.LX2018 and all species of Phaeoacremonium.

Figure 5 shows Pmr.LX2018 treatment of Akiras saplings. The figure includes control and treatment groups.

Figure 6 is a line chart of the percentage content of total sesquiterpenes and total chromones in different days of Pmr. Percent content (%) = peak area of the target component╳100%/total peak area

Figure 7 shows Pmr.LX2018 treatment of Aquilaria sinensis for 30 years. The five-pointed star is the sample of the treatment group, and the triangle mark is the sample of the control group.

Figure 8 is the cross-section of Aquilaria sinensis treated with Pmr.LX2018 for 30 years. The position of the arrow is the agarwood layer.

Figure 9 is a picture of callus of Aquilaria sinensis treated with Pmr.LX2018 for different days.

Figure 10 is a graph showing the relative contents of four sesquiterpenes in Aquilaria sinensis calli treated with Pmr.LX2018 for different days. Relative content = peak area of the target compound╳average internal reference peak area of all samples/internal reference peak area of this sample. "**" represents P<0.01.

The best way to practice the invention

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guideline for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Quantitative experiments in the following examples were all set up to repeat the experiments three times or more, and the results were averaged.

Embodiment 1, the screening and analysis of the fungus that promotes the continuous aroma formation of agarwood

1. Microhistochemical detection of different layers of Agarwood

1. Acquisition and stratification of agarwood materials

The agarwood material is collected from Dongguan, Guangdong Province. A 50-year-old white wood tree with a diameter at breast height of 15-25cm is taken. It is traditionally formed after 5 years of natural agarwood. The agarwood layer is 0.5-1.0cm thick. From outside to inside, the agarwood material is rotten layer (DL), transition (DA) between rotten layer and agarwood layer, agarwood layer (AL), transition (TL) between agarwood layer and white wood layer, and white wood layer (NL). Agarwood samples from different parts were taken, cut into 0.5-0.1cm thick, 0.5cm×0.5cm small pieces, and fixed in 10% formaldehyde solution.

2. Observation on the distribution of starch granules in different layers of Agarwood

The material samples in the fixative were taken and sliced at -20°C using a cryostat with a thickness of 10 μm. The slices were stained in Lugol's iodine solution for 3 min, washed and mounted for observation. The results showed that a large number of starch granules existed in the NL layer, a small amount existed in the TL layer, and the rest of the layers were not observed (Figure 1). At the same time, it can also be seen that agarwood brown oil exists in DA and AL in large quantities, and in TL in a small amount.

3. Observation on the distribution of polysaccharides in different layers of Agarwood

Take the material samples in the fixative solution and slice them with a cryostat at -20°C. The thickness of the slices is 10 μm. Using the PAS staining method, the slices are treated in 0.5% periodic acid solution for 10 minutes, rinsed with water, and then stained in Schiff reagent for 4 minutes. , After washing the color, mount the film for observation. The results showed that polysaccharides mainly existed in TL layer and NL layer, and more polysaccharides existed in TL layer (Fig. 1).

4. Comparison of cell viability in different layers of Agarwood

The material samples in the fixative solution were taken and sliced with a cryostat at -20°C with a thickness of 10 μm, stained with DAPI solution for 10 min, washed after staining, and mounted quickly for observation to reduce fluorescence quenching during observation. The results showed that there were a large number of viable cells in the NL layer, some viable cells in the TL layer, and a small number of nuclei were deformed, and there were no viable cells in AL, DA, and DL (Fig. 1).

Since the oils of Agarwood are mainly produced by the living cells of Agarwood, and the TL layer is the key position connecting the Agarwood layer and the Agarwood layer, it contains some living cells and the chemical substances (starch grains, polysaccharides, Agarwood brown oil) change significantly, which is The key part of agarwood to continue to develop fragrance.

2. Detection and analysis of fungal distribution in different layers of Agarwood

1. DNA extraction and detection

The DNA of different layered samples of Agarwood was extracted by CTAB method, and the concentration and purity of DNA were detected by 1% agarose gel electrophoresis, and an appropriate amount of sample was taken in a 1.5mL centrifuge tube, and the sample was diluted with sterile water to 1ng. μL ⁻¹ .

2. PCR amplification and high-throughput sequencing

Use universal primers ITS5-1737F (5'-GGAAGTAAAGTCGTAACAAGG-3' (SEQ ID NO.2 in the sequence listing)) and ITS2-2043R (5'-GCTGCGTTTCTTCATCGATGC-3' (SEQ ID NO.3 in the sequence listing)) for PCR Amplify and purify, and use the gel extraction kit provided by QIAGEN to recover the product for the PCR product band. use

The DNA PCR-Free Sample Preparation Kit was used for library construction. The constructed library was quantified by Qubit and Q-PCR. After the library was qualified, the IonS5 ^TM XL platform was used for high-throughput sequencing.

3. Data processing analysis and species classification tree

According to the Barcode sequence and PCR amplification primer sequence, the data of each agarwood layered sample was obtained. After the Barcode and primer sequence were cut off, the reads of each sample were spliced using FLASH, and the spliced sequence obtained was the original tags data. Uparse software was used to cluster all the effective tags of all samples (operational taxonomic units) according to the similarity of 97%. The OTUs were annotated by comparing SILVA, Greengene, and RPD databases with QIIME software.

For the species classification results of each layered sample of Agarwood or each group, the top 20 species with the largest relative abundance were screened for species classification tree statistics. It was found that the fungus with the highest proportion of TL layer was Phaeoacremonium rubrigenum, and it was speculated that it could promote the continuous fragrance formation of agarwood (Figure 2).

Example 2, Acquisition of Agarwood Fungus Pmr.LX2018

1. Sample pretreatment

Rinse the agarwood sample with ultra-clean water for 5 minutes, divide according to different layers to obtain samples of different layers of agarwood (hereinafter referred to as samples), and carry out disinfection treatment in an ultra-clean workbench according to aseptic operation: soak in 75% ethanol for 1-2 minutes, Wash with sterile water twice, soak with 2% sodium hypochlorite for 10 minutes, wash with sterile water for 5 to 6 times, put the sample on sterile absorbent paper to filter the water after disinfection, and then carefully scrape off the surface of the resin wood with a sterile scalpel tissue, and finally cut the transition layer into wooden pieces with a size of 5mm×5mm×5mm and place them in the MEA medium, and place one sample in each plate; in order to test whether the surface of the sample is thoroughly disinfected, take the sterile water used for the last rinse Inoculated in MEA medium as blank control.

2. Isolation and purification of Agarwood fungus

Place the sample in a constant temperature incubator at 25°C for dark cultivation. When the agarwood transition layer sample is 3 to 4 days old, there will be hyphae visible to the naked eye at the edge. Use a sterile toothpick to inoculate the mycelium of a single colony into a new medium, and continue to inoculate After culturing under the same conditions, after more than 4 times of isolation and purification, a single colony can be obtained, and the strain is named LX2018.

3. Identification and naming of Agarwood fungus Pmr.LX2018

The LX2018 fungal colony obtained after purification 4 times in step 2 was first observed with the naked eye, and it was found that the colony grew slowly, and it took about 25 days to cover the plate. The surface of the colony had radial grooves, dark brown pigment rings, and a fluffy texture. shape, the height of the colony is flat (the left picture in Figure 3), and the colony emits an aromatic smell. The branched hyphae are septated and have abundant spores, which are oval in shape and do not produce fruiting body structures (right panel in Figure 3). According to the analysis of fungal colony morphological characteristics against the "Handbook of Fungal Identification", it was determined that the fungus belonged to the genus Phaeoacremonium.

Further, the DNA of the fungus was extracted by CTAB method. Use ITS1F (5'-TCCGTAGGTGAACCTGCGG-3' (SEQ ID NO.4 in the sequence listing)) and ITS4R (5'-TCCGTAGGTGAACCTGCGG-3' (SEQ ID NO.5 in the sequence listing)) primers to carry out PCR amplification to the fungal DNA The reaction system is 50 μL, 5× buffer 10 μL, dNTP (2.5 mM) 4 μL, primers ITS1F and ITS4R 1 μL, DNA template 1 μL, DNA polymerase 1 μL, add sterile distilled water to make up to 50 μL. The parameters of the PCR amplification reaction system were: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 1 min, 35 cycles; 10 min at 72°C. After the reaction, take 5 μL of the product and add 1 μL 6×Loading Buffer, vortex and mix well, use 1.5% agarose gel electrophoresis detection and gel imaging system to observe, it is found that there is a clear and bright band at about 600bp, which is Pmr .ITS sequence of LX2018 bacteria. Sanger sequencing method (Beijing Ruibo Xingke Biotechnology Co., Ltd.) was used for bidirectional sequencing and splicing of the band sequence, and the sequence similarity was compared and analyzed by BLAST method. The ribosomal rRNA gene ITS (internal Transcribed spacer) (SEQ ID NO.1 in the sequence listing) has 100% similarity with the ITS of Phaeoacremonium rubrigenum bacteria. At the same time, the phylogenetic tree analysis of this sequence and the sequences of other species of this genus found that this strain and Phaeoacremonium rubrigenum were clustered into one branch, with a support rate of 98. Therefore, in combination with morphological identification and molecular identification, it can be drawn that the bacterial strain LX2018 isolated by the present invention is Phaeoacremonium rubrigenum, named Phaeoacremonium rubrigenum stain LX2018. Phaeoacremonium rubrigenum stain LX2018 was deposited in the General Microbiology Center of China Committee for Microbial Culture Collection on September 27, 2021, with the preservation number CGMCC No. 23266, the strain number LX2018, and the abbreviation below is Pmr.LX2018 (Figure 4).

Embodiment 3, Pmr.LX2018 promotes Agarwood seedlings (Agarwood plant saplings) to continue to produce fragrance

1. Pmr.LX2018 treatment of Acacia saplings

Under sterile conditions, Pmr.LX2018 was inoculated and activated with the corresponding MEA solid medium (prepared from 2% malt extract and 1.5% agar), cultured in the dark at 25°C, and the diameter was taken when the fungus produced spores. The bacterium cake that is 0.5cm is used as processing sapling bacterial strain. Aquilaria sinensis (Aquilaria sinensis) was randomly selected (preserved in the laboratory, related literature: Liu et al. Volatile organic compound and endogenous phytohormone characteristics during callus browning in Aquilaria sinensis. Industrial Crops & Products, 2021: 168. The public can obtain from the applicant , only for repeating the present invention, can not be used for other purposes) 1-year-old healthy saplings, Pmr.LX2018 is inoculated in the saplings by oblique cutting 45 ° method, the specific method is: use 75% ethanol to surface the cutters and stems used Sterilize, use a scalpel to cut obliquely at 45° from the tree stems of about 5cm, 7cm and 9cm from the ground, deep to 1/3 of the tree body diameter, inoculate the bacterium cake in the wound, and fix it with parafilm (treatment group in Fig. 5 ). In addition, the Akiras saplings that were not inoculated with the strain Pmr.LX2018 after oblique cutting were used as a negative control (control group in Figure 5); the interaction time was respectively (0 day, 5 days, 10 days, 15 days, 20 days, 25 days , 30 days), all experimental groups were 6 biological repetitions. Samples were collected in time according to a certain interaction time, freeze-dried and powdered for later use.

2. Determination of aroma components of A. sinensis saplings treated with Pmr.LX2018

The two types of aroma components of Agarwood are sesquiterpenes and chromones, respectively. Therefore, the present invention uses GC-MS to detect the volatile components of the stems of Agarwood saplings treated with Pmr.LX2018.

Preparation of the test solution: accurately weigh 0.1g of the powder of the stem of Akiras sinensis, place it in an imported 2ml centrifuge tube, add 1.5ml ethyl acetate (analytical pure) precisely with a micropipette, seal the tube mouth, and place it at room temperature Soak overnight. Ultrasonic (40kHz) at low temperature (4°C) for 45min, centrifuge at 12000rpm for 10min, weigh, add ethyl acetate to make up for weight loss, transfer the supernatant to a new 2ml centrifuge tube, use nitrogen blowing to concentrate until the solvent is completely evaporated, then add precisely 200 μL of ethyl acetate, vortex to mix well, use 0.22 μm polytetrafluoroethylene (PTFE) filter membrane to filter, put the subsequent filtrate in a gas phase vial with an inner insert, seal it with an unopened cap, and use it as the test solution , stored at low temperature (4°C) for later use.

Gas chromatography-mass spectrometry (GC-MS) was used to detect the components of the test sample. The GC part came from Thermo Company model Trace 1310, and the MS part came from Thermo Company model TSQ8000. GC-MS method: automatic sample injection (1 μL, splitless), programmed temperature rise, initially at 50°C, rising to 15°C at 10°C·min ^-1 , maintaining for 15 min, rising to 280°C at 8°C·min ^-1 , maintaining 10 minutes; the temperature of the injection port is 250°C, the electron energy is 70eV, the temperature of the ion source is 250°C, and the mass scanning range of the sample is 50-600m/z.

Trance Finder 3.3 workstation (Thermo Company) was used to process the total ion chromatogram of material samples, background interference was deducted, information comparison was performed using NIST 2.2 standard mass spectrum library, chromatographic peaks were analyzed and each compound was identified, peak area normalization method and statistical The relative mass fraction of each compound is calculated by the method, and finally the sum of all sesquiterpene components is the total sesquiterpene content, and the sum of all chromone components is the total chromone content. As can be seen from Figure 6, after 5 days of treatment, the saplings of the treatment group have begun to produce sesquiterpene components, and as the treatment time increases, the total sesquiterpene content gradually increases; to the 15th day, the saplings of the treatment group Chromone components began to be produced, and the total chromone content and total sesquiterpene content were all increasing. The results indicated that Pmr.LX2018 could continuously stimulate the production of sesquiterpenes and chromones in A. argentina saplings to promote continuous aroma formation, while no sesquiterpenes and chromones were detected in the untreated control group.

Example 4, Pmr.LX2018 promotes the thickening of the Agarwood plant Agarwood layer

1.Pmr.LX2018 processing 30-year-old Arboria argentina

Under sterile conditions, Pmr.LX2018 was inoculated and activated with the corresponding MEA solid medium (prepared from 2% malt extract and 1.5% agar), cultured in the dark at 25°C, and the diameter was taken when the fungus produced spores. The bacterium cake that is 0.5cm is used as the bacterial strain of processing white woody tree. Six 30-year-old healthy Aquilaria sinensis trees with the same DBH in the base were randomly selected (preserved in the laboratory, related literature: Liu et al. Agarwood wound locations provide insight into the association between fungal diversity and volatile compounds in Aquilaria sinensis. R.Soc.Open Sci, 6:190211. The public can obtain from the applicant, only for repeating the invention, not for other purposes), use 75% ethanol to carry out surface disinfection of the saw and stem used, and use the saw in the Saw on the main stem about 1.5m from the ground, as deep as 1/2 of the diameter of the tree body, inoculate the fungus cake into the wound, and fix it with parafilm (treatment group in Figure 7); Pmr.LX2018 was not inoculated after sawing As a negative control (control group in Figure 7). Both the control group and the experimental group had 3 biological repetitions. Sampling after 1 year of interaction.

2. Detect the thickness of the agarwood layer

The agarwood layer was detected on the Pmr.LX2018-treated stems of Akiras sinensis (shown in the Pmr.LX2018 group in Figure 8), and the thickness of the agarwood layer was found to be 0.4±0.07cm; while the control did not treat the Akiras sinensis stems with Pmr.LX2018 (Figure 8 Shown in the control group) the thickness of the agarwood layer is 0.2 ± 0.08cm. The results showed that the strain Pmr.LX2018 had a significant thickening effect on the agarwood layer of the A. argentina compared with the control group.

Example 5, Pmr.LX2018 promotes Agarwood plant callus to produce fragrance

1. Induction and culture of A. chinensis callus

Take healthy and young leaves of Aquilaria sinensis in a tissue culture bottle, rinse them with pure water for 2-3 times, then wipe the water on the surface of the leaves with absorbent paper and set aside. Disinfection work is carried out in an ultra-clean workbench: soak the freshly taken leaves in 75% ethanol for 30 seconds, take them out immediately, wash them with sterile water for 2 to 3 times, and transfer them to 2% sodium hypochlorite solution for 10 minutes to make the leaves fully Contact solution for disinfection. Immediately after sterilization, rinse with sterile water for 4 to 5 times, each time for 2 to 3 minutes. After cleaning, filter the surface water with sterile absorbent paper, cut off the edge of the leaf, and inoculate the leaf with a size of 0.5cm×0.5cm. In MS+1.0mg·L ^-1 6-BA+0.5mg·L ^-1 NAA medium (sucrose 30g·L ^-1 , agar 7.0g·L ^-1 , pH=5.7～5.8), 25°C, dark nourish. The induced callus was subcultured every 20-30 days to obtain the callus of A. sinensis.

2. Strain Pmr.LX2018 to treat the callus of A. sinensis

Under sterile conditions, Pmr.LX2018 was inoculated and activated with the corresponding MEA solid medium (prepared from 2% malt extract and 1.5% agar), cultured in the dark at 25°C, and the diameter was taken when the fungus produced spores. The bacterium cake of 0.5cm is used as the treatment of the callus of A. sinensis. Put the bacteria cake with a diameter of 0.5cm into the medium (MS+1.0mg·L ^-1 6-BA+0.5mg·L ^-1 NAA medium (sucrose 30g·L ^-1 , agar 7.0g·L ^-1 , pH=5.7～5.8)) The center is 2.0 cm away from the 4 pieces of A. sinensis callus respectively ( FIG. 9 ). Then, callus samples treated with Pmr.LX2018 were collected on day 0, day 3 and day 18, respectively.

3. Detection of Aromatic components in Agarwood callus treated with Agarwood Pmr.LX2018 strain

The volatile components of the Pmr.LX2018-treated callus obtained in step 2 were detected by GC-MS (the GC part was derived from Thermo Company model Trace 1310, and the MS part was derived from Thermo Company model TSQ8000). Preparation of the test solution: accurately weigh 0.1 g of the powder of the callus of A. chinensis, place it in an imported 2ml centrifuge tube, use a micropipette to precisely add 1.5ml of ethyl acetate (analytical grade), seal the mouth of the tube, and place it in the Soak overnight at room temperature. Ultrasonic (40kHz) at low temperature (4°C) for 45min, centrifuge at 12000rpm for 10min, weigh, add ethyl acetate to make up for weight loss, transfer the supernatant to a new 2ml centrifuge tube, use nitrogen blowing to concentrate until the solvent is completely evaporated, and then add precisely 200 μL of ethyl acetate, vortex to mix well, use 0.22 μm polytetrafluoroethylene (PTFE) filter membrane to filter, put the subsequent filtrate in a gas phase vial with an inner insert, seal it with an unopened cap, and use it as the test solution , stored at low temperature (4°C) for later use. GC-MS method: automatic sample injection (1 μL, splitless), programmed temperature rise, initially at 50°C, rising to 15°C at 10°C·min ^-1 , maintaining for 15 min, rising to 280°C at 8°C·min ^-1 , maintaining 10 minutes; the temperature of the injection port is 250°C, the electron energy is 70eV, the temperature of the ion source is 250°C, and the mass scanning range of the sample is 50-600m/z.

Trance Finder 3.3 (Thermo) workstation was used to process the total ion chromatogram of material samples, background interference was deducted, information was compared using NIST 2.2 standard mass spectrum library, chromatographic peaks were analyzed and each compound was identified, and peak area normalization method and statistical method were used Calculate the relative mass fraction of each compound. It can be seen from Figure 10 that the callus of A. chinensis treated by the strain Pmr. alkene. After 3 days of strain Pmr.LX2018 treatment, these four components all had an increasing trend, and increased significantly by 18 days. The results showed that the strain Pmr.LX2018 had the effect of promoting the aroma formation of the callus of Agarwood plant.

To sum up, the fungus Pmr.LX2018 provided by the present invention can promote the accumulation and continuous formation of agarwood, which is of great significance for improving artificial agarwood, optimizing the technology of agarwood formation, and promoting industrial development.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application. Applications of some of the essential features are possible within the scope of the appended claims below.

sequence listing

SEQ ID NO.1 (ITS (internal transcribed spacer) of the ribosomal rRNA gene of bacterial strain):

SEQ ID NO.2(ITS5-1737F):

SEQ ID NO.3(ITS2-2043R):

SEQ ID NO.4(ITS1F):

SEQ ID NO.5(ITS4R):

industrial application

The present invention isolates and obtains the fungus Phaeoacremonium rubrigenum strain in the transition layer from agarwood to white wood, and through sequencing the amplicon of agarwood, it is found that the Phaeoacremonium rubrigenum plays a key role in the accumulation of agarwood, and the strain is successfully isolated and named Phaeoacremonium rubrigenum LX2018 strain (abbreviated as Pmr.LX2018). The Pmr.LX2018 fungus belongs to the kingdom Fungi (Fungi), class Ascomycetes (Sordariomycetes), and a filamentous fungus of the order Diaporthales. Experiments have shown that Pmr.LX2018 can produce agarwood sesquiterpenoids within 5 days after being inoculated with plants of the genus Agarwood, and the total sesquiterpene content gradually increases; on the 15th day, the Agarwood seedlings in the treatment group began to produce chromone components, and the total color Ketone content and total sesquiterpene content are increasing. And Pmr.LX2018 can promote the thickening of the agarwood layer of the agarwood plant and the aroma formation of the agarwood plant callus. In actual production, it is of great significance to improve the artificial agarwood, optimize the agarwood formation technology, and promote the development of the industry.

Claims (14)

1. Fungus Phaeoacremonium rubrigenum, the strain number of Phaeoacremonium rubrigenum is LX2018, and its registration number in the General Microbiology Center of China Microbiological Culture Collection Management Committee is CGMCC No.23266.
2. Phaeoacremonium rubrigenum according to claim 1, characterized in that: the ITS of said Phaeoacremonium rubrigenum contains the DNA molecule shown in SEQ ID NO.1 in the sequence listing.
3. The culture of Phaeoacremonium rubrigenum described in claim 1 or 2 is the material obtained by cultivating Phaeoacremonium rubrigenum described in claim 1 or 2 in a microbial culture medium.
4. A bacterial agent, characterized in that: the bacterial agent contains the Phaeoacremonium rubrigenum described in claim 1 or 2 or/and the metabolite of Phaeoacremonium rubrigenum described in claim 1 or 2 or/and the metabolite of claim 3 described cultures.
5. The bacterial agent according to claim 4, characterized in that: the bacterial agent has at least one of the following characteristics:

   A1) promote the fragrance formation of agarwood plant saplings;

   A2) promote the thickening of the agarwood layer of the agarwood plant;

   A3) Promote the agarwood plant callus to produce fragrance.
6. The microbial agent according to claim 5, characterized in that: the agarwood plant is agarwood.
7. The microbial agent according to claim 5 or 6, characterized in that: the aroma components of the incense are sesquiterpenes and/or chromones.
8. The following at least one application of Phaeoacremonium rubrigenum described in claim 1 or 2:

   B1) the application of Phaeoacremonium rubrigenum described in claim 1 or 2 in the product that promotes agarwood plant saplings to form fragrance;

   B2) application of Phaeoacremonium rubrigenum described in claim 1 or 2 in the product that promotes the thickening of the agarwood plant agarwood layer;

   B3) the application of Phaeoacremonium rubrigenum described in claim 1 or 2 in the product that promotes the agarwood plant callus knot in the preparation.
9. The application according to claim 8, characterized in that: the agarwood plant is agarwood.
10. The application according to claim 8 or 9, characterized in that: the aroma components of the incense are sesquiterpenes and/or chromones.
11. The following at least one application of the bacterial agent described in claim 4 or 5:

    C1) the application of the bacterial agent described in claim 4 or 5 in the product that promotes the continuous aroma formation of agarwood plant saplings;

    C2) The application of the bacteria agent described in claim 4 or 5 in the preparation of a product that promotes the thickening of the agarwood layer of the agarwood plant;

    C3) The application of the bacterial agent described in claim 4 or 5 in the preparation of a product that promotes the continuous aroma formation of agarwood plant saplings.
12. The application according to claim 11, characterized in that: the agarwood plant is agarwood.
13. The application according to claim 11 or 12, characterized in that: the aroma components of the incense are sesquiterpenes and/or chromones.
14. The method for preparing the bacterial agent described in claim 4 or 5, comprising the step of cultivating Phaeoacremonium rubrigenum described in claim 1 or 2 in a microbial culture medium.

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