Method for highly growing agilawood based on agilawood trees

Abstract

A method for high fruiting of agilawood based on an agilawood tree belongs to the field of Daphneaceae, and comprises the following steps: 1) grafting local aquilaria sinensis as a stock with a scion of a mature agilawood seed tree by adopting a cleft grafting method; 2) after the grafted seedlings are planted for more than 3 years and the diameter at breast height is more than 8cm, chiseling fungus planting holes and infusion holes in the tree; (3) implanting the specific penicillium fungus filler into the tree body through the fungus planting holes, so that the specific penicillium fungus filler parasitizes and diffuses in the tree body, and the tree is stimulated to secrete more resin; and 4) inputting an agilawood tree inducer into the tree body through an infusion hole through an infusion method, inducing formation of sesquiterpenoids and chromone compounds, accompanying generation of other aromatic compounds, and producing agilawood with medicinal value.

Description

Technical Field

[0001] The present invention relates to the field of Thymelaeaceae, and particularly to a method for high-grade agarwood formation based on Aquilaria malaccensis trees. Background Art

[0002] Agarwood is the abbreviation of "submerged agarwood", which is a traditional precious medicinal material and spice with a strong and charming aroma. Since the resin formed is heavier than water, it is named "agarwood". Natural wild agarwood requires a harsh agarwood formation environment and a time span of decades to even over a hundred years. Natural wild native agarwood and Aquilaria sinensis in Yunnan have been included in the list of protected plants. With the development of technology and the continuous expansion of the market, the technology of artificially cultivating agarwood has been continuously innovated. However, due to the problem of adaptability to the planting environment, even if agarwood can be formed through grafted seedlings, the quality of the agarwood will decline, and its active ingredients are difficult to meet the medicinal standards. Summary of the Invention

[0003] The purpose of the present invention is to solve the above problems in the prior art and provide a method for high-grade agarwood formation based on Aquilaria malaccensis trees.

[0004] To achieve the above purpose, the present invention adopts the following technical solutions:

[0005] A method for high-grade agarwood formation based on Aquilaria malaccensis trees, comprising the following steps:

[0006] 1) Adopt the cleft grafting method to graft the local Aquilaria sinensis as the rootstock with the scions of mature Aquilaria malaccensis mother trees;

[0007] 2) After the grafted seedlings are planted for more than 3 years and the diameter at breast height exceeds 8 cm, drill inoculation holes and infusion holes on the tree;

[0008] 3) Insert specific Penicillium fungi fillers into the tree through the inoculation holes, enabling them to parasitize and spread in the tree, and stimulating the tree to secrete more resin;

[0009] 4) Input Aquilaria malaccensis tree inducer into the tree through the infusion method at the infusion holes, inducing the formation of sesquiterpenoids and chromones, accompanied by other aromatic compounds, to produce agarwood with medicinal value.

[0010] The specific steps of the grafting are as follows: Use a grafting knife to cut a bevel with an inclination of 43° - 46° and a length of 0.5 - 0.6 cm at 1 - 2 cm below the bud eye of the scion; Cut off the rootstock and retain a height of 15 - 20 cm, and split it straight along the axial direction of the reverse bevel for 1 - 1.2 cm; Insert the double wedge surface of the scion into the rootstock, and wrap and tie the joint tightly in circles from bottom to top with grafting agricultural film, and cover the grafted scion with a self-sealing bag.

[0011] The requirements for setting the inoculation holes are as follows: Drilling starts from 20 - 30 cm above the grafting joint, with a vertical spacing of 50 - 60 cm, a drilling inclination angle of 45° - 50°, and a depth of 1 / 3 of the tree trunk diameter; Subsequently, every 3 - 4 months, holes are drilled at 70 - 80 cm, 110 - 120 cm, and 160 - 167 cm respectively, in a spiral ascending arrangement.

[0012] The preparation method of the specific Penicillium fungus filler includes:

[0013] Mother liquid cultivation: 1 - 2 g of freeze - dried powder of the Penicillium fungus colony collected from the moldy corn cobs is used for artificial cultivation of the mother liquid. 150 - 200 g of potatoes are sliced, added to distilled water and boiled for 15 - 30 minutes, filtered to obtain the filtrate and made up to 100 ml, 15 - 20 g of glucose and 15 - 20 g of agar are added, stirred and melted, then filled into test tubes and sterilized; Inoculation is carried out under sterile conditions and placed in a constant temperature environment of 25 - 27 °C for 10 - 15 days until the hyphae cover the inclined plane;

[0014] Filler preparation: By weight, 80 - 85 parts of Aquilaria sinensis sawdust, 12 - 15 parts of wheat bran, 0.5 - 1 part of gypsum, and 1 - 2 parts of glucose are taken, stirred evenly and filled into shallow trays. The mass ratio of the filler to water is controlled at 1.0 - 1.2:1.3 - 1.5, and then autoclaved for 2 - 3 hours; The mother liquid is inoculated under sterile conditions and placed in a dark environment with a constant temperature of 25 - 27 °C and a relative humidity of 60% - 70% for 25 - 30 days until the hyphae grow full.

[0015] The preparation method of the Aquilaria malaccensis tree inducer is: Mix 980 - 985 ml of distilled water, 1.5 - 2.2 ml of methyl jasmonate, and 2 - 3 ml of 0.01% 24 - epi - brassinolide and stir until completely dissolved.

[0016] Compared with the prior art, the beneficial effects achieved by the technical solution of the present invention are:

[0017] The present invention implants the Aquilaria malaccensis tree into the fungal filler and inputs the inducer for specific binding to form agarwood; The present invention can significantly improve the agarwood production and quality of Aquilaria malaccensis in this region, effectively make up for the shortage of agarwood used in medicine in the market, and strongly promote the development of the agarwood industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the high - performance liquid chromatography control chart of the Chinese Pharmacopoeia 2020 edition;

[0019] Figure 2 It is the high - performance liquid chromatography chart of the sample of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0020] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0021] Example 1

[0022] The present embodiment provides a method for producing agarwood based on the Malayan Aquilaria tree, comprising the following steps:

[0023] 1) Using the cleft grafting method, the local white agarwood was used as the rootstock and grafted onto the cuttings of the mature Malay Aquilaria mother tree;

[0024] 2) After the grafted seedlings have been planted for more than 3 years and their DBH exceeds 8 cm, holes for planting and infusion should be drilled on the trees;

[0025] 3) Implanting a specific Penicillium fungus filler into the tree through the phytopores, allowing it to parasitize and spread inside the tree, stimulating the tree to secrete more resin;

[0026] 4) The Aquilaria sinensis inducer is injected into the tree through the infusion hole to induce the formation of sesquiterpenoid compounds and chromone compounds, accompanied by other aromatic compounds, to produce agarwood with medicinal value.

[0027] In step 1), the seedlings are grafted as soon as they are harvested. The grafting season is from late October to the end of April of the following year. After grafting, shade treatment is carried out in a 75% shade fence. Supplement fertilizer and water appropriately half a month after grafting. When the buds of the scion grow to 1-2 cm, remove the sealed bag. When the shoots grow to more than 15 cm, remove the shade fence. Move to the nursery for hardening for half a month before planting.

[0028] The grafting steps are specifically as follows: using a grafting knife to cut a 43° to 46° inclination and 0.5 to 0.6 cm long bevel at 1 to 2 cm below the bud of the scion; cutting the rootstock and retaining a height of 15 to 20 cm, and axially splitting 1 to 1.2 cm along the reverse bevel; aligning the double wedge surface of the scion with the rootstock and inserting it, wrapping the joint tightly with grafting agricultural film from bottom to top, and covering the grafted scion with a 10×15 ziplock bag.

[0029] In step 2), healthy plants are selected to drill inoculation holes and infusion holes. Two to three months before agarwood formation, for the plants to form agarwood, the side branches with a diameter less than 2 cm are all pruned by the method of dry pruning against the trunk; a hand drill equipped with twist drills with diameters of 5 cm, 9 cm, 13 cm, and 19 cm is used to drill inoculation holes starting from a position 20 - 30 cm above the interface, with an upper and lower spacing of 50 - 60 cm, a drilling depth of 1 / 3 of the trunk diameter, an inclination angle of 45º, and the agarwood formation time is selected in early spring or autumn and winter. Subsequently, every 3 - 4 months, holes are drilled at 70 - 80 cm, 110 - 120 cm, and 160 - 167 cm respectively, with a spiral ascending arrangement. In practice, the size and number of inoculation holes are determined according to the size of the inoculated tree. The position of the infusion hole is selected at a distance of 30 - 50 cm from the ground, a drill bit with a diameter of 4.5 - 5 mm is used for drilling, the drilling depth is about 5 cm, and the downward inclination angle is 45º.

[0030] In step 3), filling the drilled holes of the aquilaria tree with fungal fillers is an important measure to increase the yield of agarwood. The fungi in the fillers can form a symbiotic relationship with the aquilaria tree. After filling the fillers, they are sealed with absorbent cotton to facilitate regular inspection and adding distilled water to improve the vitality of the fungi.

[0031] The preparation method of the specific penicillium genus fungal filler includes:

[0032] Mother liquid cultivation: 1 - 2 g of the freeze-dried powder of the penicillium group collected from the moldy corn cobs is used for artificial cultivation of the mother liquid. 150 - 200 g of potatoes are sliced, added to distilled water and boiled for 15 - 30 minutes, filtered to obtain the filtrate and made up to 100 ml, 15 - 20 g of glucose and 15 - 20 g of agar are added, stirred and melted, then filled into test tubes and sterilized; inoculated under sterile conditions and placed in a constant temperature environment of 25 - 27℃ for 10 - 15 days until the hyphae cover the inclined plane;

[0033] Filler preparation: By weight, take 80 - 85 parts of aquilaria sinensis sawdust, 12 - 15 parts of wheat bran, 0.5 - 1 part of gypsum, and 1 - 2 parts of glucose, stir evenly and put into a shallow tray, control the mass ratio of the filler to water to be 1.0 - 1.2:1.3 - 1.5, then autoclave for 2 - 3 hours; inoculate the mother liquid under sterile conditions and place it in a dark environment with a constant temperature of 25 - 27℃ and a relative humidity of 60% - 70% for 25 - 30 days until the hyphae grow full to obtain the required fungal filler for aquilaria malaccensis.

[0034] In step 4), the preparation method of the aquilaria malaccensis inducer is: mix 980 - 985 ml of distilled water, 1.5 - 2.2 ml of methyl jasmonate, and 2 - 3 ml of 24-epibrassinolide with a concentration of 0.01% and stir until completely dissolved.

[0035] The infusion step of the Malay Agarwood inducer is as follows: the prepared Malay Agarwood inducer is diluted with water 18 to 20 times and filled into an infusion bag, 480 to 500 ml per bag, and hung on the tree. The needle is inserted into the pre-punched infusion hole, and the flow rate control valve is used to control the slow flow into the infusion hole. The agarwood tree transports the liquid through the wood and conducts it to various parts of the tree body by transpiration; after a period of time, the filling and infusion are re-drilled upwards, and the old holes are sealed with yellow mud. The treatment lasts for 8 to 12 months, repeatedly stimulating the trunk defense system to produce antibodies and antigen-specific binding. After 1 to 2 years, it can form agarwood, and the agarwood part can occupy 1% to 2% of the wood, and then collect and obtain high-quality medicinal agarwood.

[0036] The agarwood trees filled with fungal fillers and induced according to the examples of the present invention were tested for 2 years.

[0037] 1. Detection of alcohol-soluble extract content: Collect samples, air-dry and grind into powder 100g, using ethanol as solvent. The alcohol-soluble extract (linalool) of the embodiment of the present invention is between 11% and 13%, while the agarwood tree without filler and induction needs hundreds of years and the tree is naturally damaged by lightning strikes, insect bites, etc. to trigger the formation of resin, and the resin reacts with fungi to form agarwood.

[0038] 2. Color reaction: After the alcohol-soluble extract (agarwood alcohol) is heated, the vanillin-concentrated hydrochloric acid color reaction turns cherry red. The highly oxidized 5,6,7,8-tetrahydroxy-2 (2-phenylethyl) chromone components are specific to agarwood.

[0039] 3. Characteristic spectrum of agarwood HPLC: The samples of the present invention are measured according to the general rule 0512 standard, see Figure 2 , 6 peaks appear in succession on the spectrum, Figure 1 Comparison Figure 1 It meets the industry standards for medicine.

[0040] 4. Under 365nm ultraviolet light, the agarwood cross section in this embodiment will show a unique biological metabolic trajectory. Fluorescent annual rings with blue-green halos can be seen on its surface, and the halo spacing is 0.2mm, corresponding to a 2-year bacterial community activity cycle. At the same time, radial golden thread patterns are also shown on the cross section, and at the end of each branch, there are fluorescent nodes formed by hyphae calcification. These optical characteristics record the rise and fall of microbial communities. Among them, the bright section represents the active period of the fungus, when the fungal metabolism is vigorous; while the dark band indicates that under environmental stress, the fungus has entered a metabolic dormant state. By observing the dendritic hyphae network formed by the Penicillium community on the agarwood section, it can be clearly found that this phenomenon is based on the natural law of microbial activity in the process of high-yield agarwood formation of Malay agarwood in a short period of time.

Claims

1. A method for producing agarwood based on Aquilaria sinensis, characterized in that: The following steps are involved: 1) Using the cleft grafting method, the local white agarwood was used as the rootstock and grafted onto the cuttings of the mature Malay Aquilaria mother tree; 2) After the grafted seedlings have been planted for more than 3 years and their DBH exceeds 8 cm, holes for planting and infusion should be drilled on the trees; 3) Inserting specific Penicillium fungus fillers into the tree through the phytopores, allowing them to parasitize and spread inside the tree, stimulating the tree to secrete more resin; 4) The Aquilaria sinensis inducer is injected into the tree through the infusion hole to induce the formation of sesquiterpenoid compounds and chromone compounds, accompanied by other aromatic compounds, to produce agarwood with medicinal value.

2. A method for producing agarwood based on Malayan agarwood trees as claimed in claim 1, characterized in that: The grafting steps are specifically as follows: using a grafting knife to cut a 43° to 46° inclination and 0.5 to 0.6 cm long bevel at 1 to 2 cm below the bud eye of the scion; cutting the rootstock and retaining a height of 15 to 20 cm, and axially splitting 1 to 1.2 cm along the reverse bevel; aligning the double wedge surface of the scion with the rootstock and inserting it, wrapping the joint tightly with grafting agricultural film from bottom to top, and covering the grafted scion with a ziplock bag.

3. A method for producing agarwood based on Malayan agarwood trees as claimed in claim 1, characterized in that: The requirements for setting the planting holes are as follows: start drilling holes 20~30cm above the grafting point, with an upper and lower spacing of 50~60cm, a drilling angle of 45º~50º, and a depth of 1 / 3 of the trunk diameter; every 3~4 months thereafter, drill holes at 70~80cm, 110~120cm, and 160~167cm, respectively, using a spiral ascending arrangement.

4. A method for producing agarwood based on Aquilaria sinensis as claimed in claim 1, characterized in that: The preparation method of the specific Penicillium fungus filler comprises: Mother liquid cultivation: 1-2g of freeze-dried powder of Penicillium collected from moldy corn cobs is used for artificial cultivation of mother liquid. 150-200g of potato is cut into thin slices, added with distilled water and boiled for 15-30 minutes, filtered to obtain the filtrate and made up to 100ml, 15-20g of glucose and 15-20g of agar are added, stirred and dissolved, and then put into a test tube and sterilized; inoculated under sterile conditions, placed in a constant temperature environment of 25-27℃ and cultured for 10-15 days until the mycelium covers the slope; Preparation of filler: Take 80-85 parts of white agarwood sawdust, 12-15 parts of wheat bran, 0.5-1 part of gypsum, and 1-2 parts of glucose by weight, stir evenly and put them on a plate, control the mass ratio of filler to water to be 1.0-1.2:1.3-1.5, and then sterilize by high pressure for 2-3 hours; add the mother liquor under sterile conditions, place it in a dark environment with a constant temperature of 25-27°C and a relative humidity of 60%-70% for culturing for 25-30 days until the mycelium is fully grown.

5. The method for producing agarwood based on Aquilaria sinensis as claimed in claim 1, characterized in that: The preparation method of the Aquilaria sinensis inducer is as follows: 980-985 ml of distilled water, 1.5-2.2 ml of methyl jasmonate, and 2-3 ml of 0.01% 24-epicassinolide are mixed and stirred until they are completely dissolved.

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