Endophyte for rapid edgeworthia chrysantha seedling edgeworthia chrysantha and application of endophyte

Abstract

The invention provides an endophyte for rapid edgeworthia chrysantha seedling edgeworthia chrysantha. The preservation number of the endophyte is CGMCC (China General Microbiological Culture Collection Center) No.32263; the bacillus velezensis is classified and named as bacillus velezensis, and the bacillus velezensis is named as bacillus velezensis. The method specifically comprises the following steps: smearing or injecting a bacillus velezensis bacterial liquid into the wound surface of an annual or more-year-old machilus fraseri seedling, wherein the whole machilus fraseri seedling can produce agilawood after two years. The produced Huangchi Aquilaria agallocha is golden yellow or brown in appearance and soft in texture, the Huangchi Aquilaria agallocha has a feeling similar to that of cut meat skin when cut by a knife, grease at the broken part is fine and bright, and filaments can be drawn out. The aroma of the phoebe davidii is fresh and elegant, has obvious sweet and frankincense tastes, and is lasting, elegant and pleasant. The use of the endophyte for rapid agilawood formation of the phoebe fraseri seedlings enables the agilawood formation rate to be far higher than that of traditional wound induction.

Description

Technical Field

[0001] The present invention relates to a fungus and its application, and more specifically to an endophyte that induces agarwood formation on wounds of Aquilaria sinensis 'Huang Qinan', its preparation method and application. Technical Background

[0002] Aquilaria sinensis 'Huang Qinan' belongs to the high-grade variety of agarwood and is famous for its unique aroma and rarity. Its production is less, so it is precious in the market. The appearance of Aquilaria sinensis 'Huang Qinan' presents golden yellow or brown, with a soft texture. When cutting it with a knife, there is a feeling similar to cutting meat skin. The grease at the cut is delicate and shiny, and can pull out filaments. The aroma of Aquilaria sinensis 'Huang Qinan' is fresh and elegant, with obvious sweet and frankincense flavors. The aroma is long-lasting and elegant. Its fragrance gradually changes from strong to sweet, and there is a hint of milk fragrance in the aftertaste. The overall fragrance is gentle and soothing, giving a sense of tranquility like moonlight shining on the lake. Comparison with other Qinan agarwoods: Compared with other colored Qinan agarwoods, the aroma of Aquilaria sinensis 'Huang Qinan' is more restrained, delicate, with a sweet and mild feeling. Compared with Bai Qinan and Green Qinan, the aroma of Aquilaria sinensis 'Huang Qinan' is more restrained, with a milk fragrance in the aftertaste, and the overall fragrance is gentle and soothing.

[0003] Existing research reports show that the endophytes of Qinan agarwood mainly include those distributed in Penicillium ( Penicillium sp. ), Fusarium (Fusarium sp.) ), Diplodia seriata ( Botryodiplodia sp. ), Auricularia mesenterica ( Melanotus flavolives ), and Trichoderma ( Trichoderma sp. ). The representative bacteria mainly include Lasiodiplodia theobromae ( Lasiodiplodia pseudotheobrome ), Lasiodiplodia theobromae ( Botryosphaeria rhododendron ), Penicillium digitatum ( Penicillium italicum ), Fusarium solani ( Fusarium solani ), Penicillium italicum ( Penkillium spp. ), Trichoderma pseudokoningii ( Trichoderna koningiopsis ), Trichoderma atroviride ( Trichoderma atroviride ).

[0004] Among them, the dominant bacteria in the heartwood at the initial stage of agarwood formation are Brevibacterium laterosporum Brachybacterium paraconglomeratum and Mycoplasma pneumoniae Mycoplasma pneumoniae [[ID=For the heartwood at the middle stage of agarwood formation, the dominant bacteria are: Rhizobium tropici Rhizobiumtropici , Mycoplasma pneumoniae <For the heartwood at the later stage of agarwood formation, the dominant bacteria are: Rhizobium tropici <000Atroviride ( Trichoderma atroviride ).

[0004] Among them, the dominant bacteria in the heartwood at the initial stage of agarwood formation are Brevibacterium laterosporum Brachybacterium paraconglomeratum and Mycoplasma pneumoniae Mycoplasma pneumoniae . For the heartwood at the middle stage of agarwood formation, the dominant bacteria are: Rhizobium tropici Rhizobiumtropici , Mycoplasma pneumoniae lung cancer , Pseudomonas aeruginosa Pseudomonas aeruginosa , Methyloversatilis universalis Methylobacterium radiotolerans , Boletus sp. L Portuguese abrys and Sphingomonas sp. Hingomonas sp. CC-MHH0546 . For the heartwood at the later stage of agarwood formation, the dominant bacteria are: Rhizobium tropici Tropical Rhizobium , Mycoplasma pneumoniae Mycoplasma pneumoniae Polynucleobacter aromaticivorans <For the heartwood at the later stage of agarwood formation, the dominant bacteria are: Rhizobium tropici Tropical Rhizobium , Mycoplasma pneumoniae Mycoplasma pneumoniae Polynucleobacter aromaticivorans Sphingomonas polyaromaticivorans and Methyloversatilis universalis Methylobacterium radiotolerans.

[0005] The formation of agarwood substances is reportedly closely related to microorganisms such as fungi and bacteria. Open wounds in the tree's xylem are invaded by certain species of fungi and bacteria. These microorganisms promote metabolism in the xylem parenchyma cells and active cells, regulate enzyme activity, and stimulate signals, thereby inducing the tree's defense response and producing heartwood. Folk shandrapers also coat the trees with sugar or other sweeteners to attract ants, bees, and other insects that eat the trunks, making them porous and increasing their contact area with air, thereby promoting the production and accumulation of agarwood substances. However, this method has limited application and is difficult to promote on a large scale, so it has not yet been reported in the literature. Summary of the Invention

[0006] The present invention aims to provide a method for artificially introducing endophytes into wound surfaces of aurantia lutea plants, thereby inducing a defensive reaction on the wound surfaces of aurantia lutea seedlings through the elicitor of the endophytes, thereby promoting the formation of agarwood. The present invention also provides a method for achieving agarwood formation in aurantia lutea seedlings under wound conditions, and the endophytes used in the method.

[0007] The endophyte was submitted to the General Microbiology Center of the China Microorganism Culture Collection Administration on October 18, 2024, and was deposited. The deposit number of the endophyte is: CGMCC No.32263, the deposit time is: October 18, 2024, and it was detected as alive on October 18, 2024; the classification name is: Bacillus velezensis.

[0008] The preparation method of endophyte for rapid fragrance formation of Agarwood seedlings comprises the following steps: 1) Preparing agarwood powder; 2) Ultrasonic extraction of agarwood powder, filtering the extract to obtain an endophyte mixture; 3) Placing the endophytic bacterial liquid mixture on a solid culture medium for cultivation, and selecting bacteria to obtain an endophytic bacterial seed liquid; 4) The endophyte seed solution is placed in a liquid fermentation tank for fermentation. The liquid culture medium is added to the fermentation tank by batch feeding. The phosphorus concentration in the fermentation tank is controlled to be 0.03-0.06 g / L for the first 5 days. After 5 days, the phosphorus source is controlled to be less than 0.01 g / L and cultured for another 2 days to obtain the endophyte fermentation liquid; 5) diluting and centrifuging the endophytic bacteria fermentation liquid to obtain an endophytic bacteria liquid that can quickly produce fragrance in the yellow agarwood seedlings.

[0009] Among them, the preparation method of the agarwood powder described in step 1) is: the agarwood produced by agarwood is shaved into thin slices less than 1 mm by a flaker, sterilized in 60 mg / L ozone for 2 hours, crushed with a universal grinder in a sterile environment, passed through a 0.5 mm sieve, and then frozen in a liquid nitrogen freeze grinder for 10 minutes and crushed for 4 minutes to obtain pretreated agarwood powder.

[0010] The formula of the solid culture medium is: 40% bran, 30% bean cake powder, 15% yellow agarwood, and 15% corn kernels. The added components and inoculum amount ( / kg matrix) are 5g sucrose, 15g peptone, 3.00g NH4NO3, 3g KH2PO4, 3g CaSO4, 1g MgSO4, 10mg vitamin PP, and 500g distilled water.

[0011] The liquid is 8-10g beef peptone, 1.5-3g yeast powder, 4-6g glucose, 0.2-0.3g K2HPO4, 0.1-0.2g formic acid, 0.5g CaSO4, 0.05g FeSO4, 0.05g MgSO4, 0.05g NaCl, and 1L sterile water.

[0012] Bacillus Velez is inoculated on the wound surface of the agarwood seedlings to induce rapid fragrance formation; specifically, the Bacillus Velez bacterial liquid of the agarwood is smeared or injected into the wound surface of the agarwood seedlings that are one year old or older, and after two years, the entire plant can produce fragrance.

[0013] The beneficial technical effects of the present invention are as follows: It provides a method and endophyte for inducing rapid agarwood formation under non-traumatic culture conditions. The yellow agarwood produced by this method has a golden or brown appearance and a soft texture. When cut with a knife, it feels like cutting meat skin. The oil at the cut is fine and shiny, and fine threads can be pulled out. The aroma of yellow agarwood is fresh and elegant, with a distinct sweet and frankincense note. The fragrance is long-lasting, elegant, and pleasant. The agarwood formation rate achieved by this method is far higher than that achieved by traditional trauma-induced agarwood production. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The fragrance effect of the yellow agarwood plant with the addition of Bacillus Velez of yellow agarwood; Figure 2 The incense is ready after three years. DETAILED DESCRIPTION

[0015] The agarwood produced by the agarwood was shaved into thin slices less than 1 mm by a flaker, sterilized in 60 mg / L ozone for 2 hours, crushed with a universal grinder in a sterile environment, passed through a 0.5 mm sieve, and then frozen in a liquid nitrogen freeze grinder for 10 minutes and crushed for 4 minutes to obtain pretreated agarwood powder; Pour the agarwood powder into sterile water at a weight ratio of 1:10, use a 40KHZ ultrasonic cleaner to perform ultrasonic extraction at room temperature for 1 hour, centrifuge at 3000 rpm for 5 minutes, and take the supernatant to filter through a 0.22 microporous filter membrane.

[0016] Place the microporous filter membrane in 50 ml of sterile water and ultrasonicate it at room temperature for 10 minutes using a 40KHZ ultrasonic cleaner to obtain a clarified endophyte mixture. The endosymbiotic mixture was inoculated onto a solid microbial culture medium for activation and culture; the solid microbial culture medium contained 40% bran, 30% soybean meal, 15% yellow agarwood, and 15% corn groats. The additional components and inoculum size (per kg of substrate) included 5 g of sucrose, 15 g of peptone, 3.00 g of NH4NO3, 3 g of KH2PO4, 3 g of CaSO4, 1 g of MgSO4, 10 mg of vitamin PP, 500 g of distilled water, and an inoculum size of 18 ml to obtain an endophyte seed solution. The endophyte seed solution was identified as Bacillus velezensis (see the "Acceptance Notice (Receipt) and Viability Report" of the Microbial Culture Collection of the General Microbiology Center of the China Culture Collection Administration Committee for Microorganisms for details). DNA sequencing by the Institute of Microbiology, Chinese Academy of Sciences, confirmed the bacterium to be Bacillus velezensis and named "Yellow agarwood Bacillus velezensis."

[0017] 1. Preparation of Bacillus cyperus 1 g of the endophyte seed solution of "Bacillus velezii" in Example 1 was added to a fermenter and cultured with a liquid culture medium to obtain a high-density endophyte. The liquid culture medium was composed of: 8-10 g beef peptone, 1.5-3 g yeast powder, 4-6 g glucose, 0.2-0.3 g K2HPO4, 0.1-0.2 g formic acid, 0.5 g CaSO4, 0.05 g FeSO4, 0.05 g MgSO4, 0.05 g NaCl, and 1 L sterile water. The liquid culture medium was added to the fermenter in a batch-fed manner. The phosphorus concentration in the fermenter was controlled at 0.03-0.06 g / L for the first 5 days. After 5 days, the phosphorus source was controlled to be less than 0.01 g / L and cultured for another 2 days to obtain an endophyte fermentation broth. The cultured endophyte fermentation broth was diluted 100-fold and centrifuged at 3000 rpm for 5 minutes to obtain a dilution of Bacillus velezii.

[0018] Study on the Activity of Bacillus Velez on Aurora chinensis Select 60 one-year-old or older agarwood seedlings that are not infected by Penicillium, Fusarium, Diplodia, Myrrha lutea, or Trichoderma. Use an iron drill bit set at 600-800°C to drill holes in the plants (along the trunk, with holes spaced 10-15 cm apart). Randomly select 20 of these agarwood seedlings and apply Bacillus Velez-like bacteria from agarwood to the holes drilled in the agarwood seedlings. Also, select another 20 agarwood seedlings and apply Bacillus Velez-like bacteria extracted from black agarwood using the same extraction method used for Bacillus Velez-like bacteria from agarwood. The process of agarwood formation was continuously observed. As time went by, the secondary xylem cells of the Agarwood seedlings thickened and lignified, and the content of chromone substances in the xylem increased significantly. As the new cells were infected by the mycelium, the hyphae in the old cells were decomposed. In addition, because the nutrient transport channels of the vessels and ray cells in the xylem of the Agarwood seedlings were blocked due to trauma, the hyphae received less nutrition and their activity became lower, which slowed down the speed of infecting new cells. From the second month, the agarwood formation phenomenon could be observed. Figure 1 shown.

[0019] Two years later, all 20 strains of the above-mentioned Bacillus Velez-Bélez yellow agarwood produced fragrance, of which 16 strains produced fragrance as a whole; the fragrance was more restrained, with a milky aftertaste, and the overall fragrance was gentle and soothing. The fragrance of yellow agarwood was more restrained, delicate, and had a sweet and gentle feeling. Figure 2 As shown in the figure, only 11 of the seedlings without the addition of Bacillus Velez-like bacteria produced fragrance, and 2 of them all produced fragrance, but the quality of the fragrance was far lower than that of the plants with the addition of Bacillus Velez-like bacteria.

[0020] Furthermore, all 20 yellow agarwood plants inoculated with Bacillus Velez strains isolated from black agarwood produced aroma, but not all of them produced aroma at all. This set of experiments suggests that aroma induction by Bacillus Velez strains is somewhat specific; the specific reasons for this need further study.

Claims

1. An endophyte for rapidly producing fragrance in Aurora chinensis seedlings, characterized by: The preservation number of the endophyte is CGMCC No. 32263; the classification name is Bacillus velezensis.

2. A method for preparing an endophyte for rapidly producing fragrance in yellow agarwood seedlings, characterized in that The following steps are involved: 1) preparing agarwood powder from yellow agarwood; 2) Ultrasonic extraction of Aquilaria sinensis powder, filtering the extract to obtain an endophyte mixture; 3) culturing the endophyte mixture on a solid culture medium and selecting the bacteria to obtain an endophyte seed solution; 4) placing the endophyte seed solution in a liquid fermentation tank for fermentation, and adding the liquid culture medium to the fermentation tank in a batch-fed manner. During the first five days, the phosphorus concentration in the fermentation tank was controlled to be 0.03-0.06 g / L. After five days, the phosphorus source was controlled to be less than 0.01 g / L, and the culture was continued for another two days to obtain the endophyte fermentation liquid. 5) diluting and centrifuging the endophyte fermentation broth to obtain a Bacillus velezii culture broth.

3. The method for preparing the endophyte that causes rapid incense formation in yellow agarwood seedlings according to claim 2 is characterized in that: the method for preparing the yellow agarwood agarwood powder described in step 1) is as follows: the agarwood produced by agarwood is planed into thin slices less than 1 mm by a flaker, sterilized in 60 mg / L ozone for 2 hours, crushed in a sterile environment with a universal grinder to pass through a 0.5 mm sieve, and then frozen in a liquid nitrogen freeze grinder for 10 minutes and crushed for 4 minutes to obtain the pretreated agarwood agarwood powder.

4. The method for preparing the endophyte for rapid fragrance formation of yellow agarwood seedlings according to claim 2 is characterized in that: the formula of the solid culture medium is: 40% bran, 30% bean cake powder, 15% yellow agarwood, 15% corn kernels, and the added components and inoculum amount ( / kg substrate) are 5g sucrose, 15g peptone, 3.00g NH4NO3, 3g KH2PO4, 3g CaSO4, 1g MgSO4, 10mg vitamin PP, and 500g distilled water.

5. The method for preparing the endophyte for rapid fragrance formation of yellow agarwood seedlings according to claim 2, characterized in that: the liquid is 8-10g beef peptone, 1.5-3g yeast powder, 4-6g glucose, 0.2-0.3g K2HPO4, 0.1-0.2g formic acid, 0.5g CaSO4, 0.05g FeSO4, 0.05g MgSO4, 0.05g NaCl, and 1L sterile water.

6. The endophyte that causes rapid fragrance formation in yellow agarwood seedlings induces rapid fragrance formation under wound conditions.

7. The endophyte for rapidly producing fragrance in agarwood seedlings according to claim 1, wherein the endophyte induces rapid fragrance production in agarwood seedlings under non-wounding conditions, characterized in that: Apply or inject the Bacillus Velez bacteria solution of Agarwood to the wound surface of Agarwood seedlings that are one year old or older. After two years, the whole plant will produce fragrance.

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