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.32264. The bacillus velezensis is classified and named as bacillus velezensis, and the bacillus velezensis is named as bacillus velezensis. The invention relates to a method for inducing rapid agilawood formation of the strain under a wound culture condition and endophyte. The black aquilaria agallocha wood produced through the method is hard in texture, deep in color and luster, attractive in texture, soft and gentle in fragrance, moist in first taste, light in sweet taste, clear and soft in middle taste, light in medicine fragrance revealed in tail fragrance, not thick but solid and not strong and mellow. And the added endophyte is far higher than the traditional wound induction in agilawood formation rate.

Description

Technical Field

[0001] The invention relates to a fungus and application thereof, and more particularly to an endophytic fungus for inducing fragrance formation on a wound surface of black agarwood, a preparation method and application thereof. Technical Background

[0002] Black agarwood is a precious incense material. It mainly grows in the primeval forests in the east and southwest of Hainan Island in my country. It is one of the local specialties. Its wood is hard, deep in color, beautiful in texture, and has a certain decorative beauty. The aroma of black agarwood is unique and rich, with a light sweetness and vanilla flavor. It contains rich oil components, mainly volatile oil components such as α-agarone. After long-term fumigation and preservation, its fragrance is deeper and longer, and it is known as "the fragrance that never dissipates." In addition, black agarwood has certain medicinal value and is widely used in traditional Chinese medicine formulas. It can cool blood and disperse blood stasis, relieve pain and itching, remove dampness and detoxify, and harmonize qi and blood. It also has a certain therapeutic effect on some chronic diseases such as arthritis and skin diseases. Therefore, it has a wide range of applications and promotion in traditional Chinese medicine and folk prescriptions.

[0003] Existing research reports show that the endophytes of agarwood mainly include Penicillium sp., Fusarium sp., Botryodiplodia sp., Melanotus flavolives and Trichoderma sp., among which the representative bacteria are Lasiodiplodia pseudotheobrome, Botryosphaeria rhodina, Penicillium italicum, Fusarium solani, Penkillium naeleagrinum, Trichodernaa koningiopsis and Trichoderma atroviride.

[0004] Among them, the dominant bacteria in the heartwood at the early stage of incense formation are Brachybacterium paraconglomeratum and Mycoplasma pulmonis. The dominant bacteria in the heartwood at the middle stage of incense formation are: Rhizobium tropici, Mycoplasma pulmonis, Pseudomonas aeruginosa, Methylobacterium radiotolerans, Labrysportucalensis and Sphingomonas sp.CC-MHH0546. The dominant bacteria in the heartwood at the late stage of incense formation are: Rhizobium tropici, Mycoplasma pulmonis, Sphingomonas polyaromaticivorans and Methylobacterium radiotolerans.

[0005] It is reported that the formation of agarwood substances is closely related to microorganisms such as fungi and bacteria. The open wounds in the xylem of the tree are invaded by certain types of fungi and bacteria. These microorganisms promote the metabolism of xylem parenchyma cells and active cells, regulate enzyme activity and stimulate signals, thereby inducing tree defense responses and producing heartwood. Folk shannons also promote the production and accumulation of agarwood substances by applying sugar or other sweeteners on the tree to attract ants, bees and other insects to eat the agarwood trunks, making the trunks porous and increasing the contact area with the air. However, this method has a limited scope of 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 purpose of the present invention is to provide a method for artificially introducing endophytes on the wound surface of black agarwood plants, and causing a defense reaction on the wound surface of black agarwood seedlings through the elicitor of the endophytes, so as to produce fragrance. A method for achieving fragrance production of black agarwood seedlings under wound conditions and the endophytes thereof.

[0007] The deposit number of the endophyte is: CGMCC No.32264; the classification name is: Bacillus velezensis, the depository is the General Microbiology Center of China Microbiological Culture Collection Administration, located in Beijing, China, and the deposit date is October 18, 2024.

[0008] The preparation method of endophytic bacteria for rapid fragrance formation of black agarwood seedlings comprises the following steps:

[0009] 1) preparing agarwood powder;

[0010] 2) Ultrasonic extraction of agarwood powder, filtering the extract to obtain an endophytic bacteria mixture;

[0011] 3) placing the endophytic bacteria liquid mixture on a solid culture medium for cultivation, and selecting bacteria to obtain an endophytic bacteria seed liquid;

[0012] 4) placing the endophyte seed liquid in a liquid fermentation tank for fermentation, and adding the liquid culture medium into the fermentation tank in a batch feeding manner, controlling the phosphorus concentration in the fermentation tank to be 0.03-0.06 g / L in the first 5 days, and controlling the phosphorus source to be less than 0.01 g / L after 5 days, and then culturing for another 2 days to obtain the endophyte fermentation liquid;

[0013] 5) diluting and centrifuging the endophytic bacteria fermentation liquid to obtain the endophytic bacteria liquid that can quickly produce fragrance in black agarwood seedlings.

[0014] Among them, the preparation method of the agarwood powder described in step 1) is: 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 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.

[0015] The formula of the solid culture medium is: 40% bran, 30% bean cake powder, 15% black agarwood, 15% corn crumbs, and the added components and inoculation amount ( / kg matrix) are 5g sucrose, 15g peptone, 3.00g NH4NO3, 3g KH2PO4, 3g CaSO4, 1g MgSO4, 10mg vitamin PP, and 500g distilled water.

[0016] 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.

[0017] The black agarwood seedlings are inoculated with Bacillus Velez subtilis to induce rapid fragrance formation; specifically, the black agarwood seedlings are smeared or injected with the bacterial liquid of Bacillus Velez subtilis to the wound surface of the black agarwood seedlings over one year old, and the whole plant can produce fragrance after two years.

[0018] The beneficial technical effect of the present invention is: the present invention provides a method and endophyte for inducing rapid incense formation under wound culture conditions. The black agarwood produced by the method has a hard texture, a deep color, a beautiful texture, a soft and gentle aroma, a moist top taste with a light sweetness, a clear and soft middle taste, and a light medicinal fragrance in the tail fragrance, which is not strong but thick, not strong but mellow. The incense formation rate of the method is much higher than that of traditional wound induction. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The aroma formation of plants with Bacillus Velez-Black Qinan added;

[0020] Figure 2 The finished product is 2 years after the incense is formed. DETAILED DESCRIPTION

[0021] Example 1 Isolation of Endophytes

[0022] The agarwood produced by black agarwood is planed into thin slices less than 1 mm by a flaker, sterilized in 60 mg / L ozone for 2 hours, crushed by 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;

[0023] 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 and filter it through a 0.22 microporous filter membrane.

[0024] The microporous filter membrane was placed in 50 ml of sterile water and ultrasonicated at room temperature for 10 minutes using a 40KHZ ultrasonic cleaner to obtain a clarified endophyte mixture.

[0025] The endosymbiotic bacteria mixture is inoculated on a solid microbial culture medium for culture activation; the solid microbial culture medium is formulated as 40% bran, 30% bean cake powder, 15% black agarwood, and 15% corn crumbs; the added components and inoculation amount ( / kg matrix) are 5g sucrose, 15g peptone, 3.00g NH4NO3, 3g KH2PO4, 3g CaSO4, 1g MgSO4, 10mg vitamin PP, 500g distilled water, and 18m inoculation amount, to obtain an endophytic bacteria seed solution; the endophytic bacteria seed solution is identified as Bacillus velezensi; see the microbial strain preservation report of the General Microbiological Center of the China Microbiological Culture Collection Administration for details; at the same time, the bacteria is identified as Bacillus velezensi through DNA sequencing by the Institute of Microbiology, Chinese Academy of Sciences, and is named "Black agarwood Bacillus velezensi".

[0026] Example 2 Investigation of Endophytic Bacterial Activity

[0027] 1. Preparation of Bacillus Velezii

[0028] 1 g of the endophyte seed solution of "Black China Bacillus Velez" in Example 1 and a liquid culture medium are added to a fermenter for cultivation to obtain a high-density endophyte; the components of the liquid culture medium include: 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, 1 L sterile water, the liquid culture medium is added to the fermenter in a batch feeding manner, the phosphorus concentration in the fermenter is controlled to be 0.03-0.06 g / L in the first 5 days, and the phosphorus source is controlled to be less than 0.01 g / L after 5 days, and then cultured for 2 days to obtain an endophyte fermentation liquid; the cultured endophyte fermentation liquid is diluted 100 times, and centrifuged at 3000 rpm for 5 minutes to obtain a dilution of Black China Bacillus Velez.

[0029] 2. Activity investigation of Bacillus velezinensis

[0030] Select 40 black agarwood seedlings that are over one year old and not infected by Penicillium, Fusarium, Coccidioides, Mycelium luteum and Trichoderma, and use an iron drill at a temperature of 600-800℃ to drill holes in the plants (along the trunk of the plant, with a distance of 10-15cm between each hole). Randomly select 20 black agarwood seedlings and apply Bacillus Velez of black agarwood to the holes of the black agarwood seedlings. And keep observing the fragrance process. As time goes by, it can be observed that the secondary xylem cells of the black agarwood seedlings are thickened and lignified, and the content of chromone substances in the xylem increases significantly. As the new cells are infected by the mycelium, the mycelium in the old cells will be completely decomposed. Because the nutrient transport channels of the vessels and ray cells in the xylem of the black agarwood seedlings are blocked due to trauma, the mycelium gets less nutrition and the activity becomes low, which slows down the infection of new cells. From the second month, the fragrance phenomenon can be observed. Figure 1 shown.

[0031] Two years later, all 20 strains of the above-mentioned Bacillus Velez added to the black agarwood produced fragrance, of which 18 produced fragrance as a whole plant; the wood has a hard texture, deep color, beautiful grain, soft and gentle aroma, the top taste is moist with a hint of sweetness, the middle taste is clear and soft, and the aftertaste reveals a faint medicinal aroma, which is not strong but thick, not strong but mellow.

[0032] It should also be noted that in a comparative experiment conducted by the applicant, it was found that adding the Bacillus Velez extracted from black agarwood to yellow agarwood plants could not effectively improve the yield and effect of agarwood.

Claims

1. An endophyte for rapidly producing fragrance in black agarwood seedlings, characterized in that: The preservation number of the endophyte is: CGMCC No.32264; the classification name is: Bacillus velezensis.

2. A method for preparing an endophyte for rapidly producing fragrance in black agarwood seedlings, characterized in that The following steps are involved: 1) preparing black agarwood agarwood powder from black agarwood agarwood; 2) Ultrasonic extraction of black agarwood powder, filtering the extract to obtain an endophytic bacteria mixture; 3) placing the endophytic bacteria liquid mixture on a solid culture medium for cultivation, and selecting bacteria to obtain an endophytic bacteria seed liquid; 4) placing the endophyte seed liquid in a liquid fermentation tank for fermentation, and adding the liquid culture medium into the fermentation tank in a batch feeding manner, controlling the phosphorus concentration in the fermentation tank to be 0.03-0.06 g / L in the first 5 days, and controlling the phosphorus source to be less than 0.01 g / L after 5 days, and then culturing for another 2 days to obtain the endophyte fermentation liquid; 5) diluting and centrifuging the endophytic bacteria fermentation liquid to obtain the Bacillus Velez black aurantium bacterial liquid.

3. The method for preparing the endophytic bacteria for rapid incense formation of black agarwood seedlings as described in claim 2 is characterized in that: the method for preparing the black agarwood agarwood powder described in step 1) is: the agarwood produced by black agarwood is planed 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 the pretreated agarwood agarwood powder.

4. The method for preparing the endophytic bacteria for rapid fragrance formation of black agarwood seedlings as described in claim 2 is characterized in that: the formula of the solid culture medium is: 40% bran, 30% bean cake powder, 15% black agarwood, 15% corn crumbs, and 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.

5. The method for preparing the endophytic bacteria for rapid aromatic formation of black agarwood seedlings as described in claim 2 is 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 endophytic bacteria that cause rapid fragrance formation in black agarwood seedlings induce rapid fragrance formation under wound conditions.

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

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