Probiotic liquid for promoting edgeworthia chrysantha and application

Abstract

According to the probiotic liquid capable of promoting agilawood formation, the lasiodiplodia theobroma, the laetiporus sulphureus and the trichoderma viride are adopted to form the probiotic liquid, the biotransformation process in a tree body is promoted, the probiotic liquid can shorten the agilawood formation time, and the content of extracts in agilawood is high.

Description

Technical Field

[0001] The invention relates to the technical field of agarwood planting, and in particular to a probiotic liquid for promoting agarwood formation and its application. Background Art

[0002] Endophytic fungi infection is a key biological factor that induces a defensive response in damaged or stimulated agarwood. It can induce the xylem cells of agarwood plants to secrete a fragrant defensive black resin substance - agarwood. In recent years, through the separation and screening of agarwood fungi, the formation of medicinal agarwood has been accelerated, the production cycle of agarwood has been shortened, and good practical results have been achieved.

[0003] At present, the endophytic fungi that induce the formation of fragrance in soil agarwood come from the white agarwood plant. In recent years, researchers have isolated more than 20 active strains that can promote the formation of fragrance in the host from soil agarwood plants through tissue block separation. These fungi belong to 6 classes, 9 orders and 13 families of Ascomycota and Basidiomycota in taxonomy, with Ascomycota as the main species. For example, Huang Qiuwei et al. isolated 11 endophytic fungi from the fragrance-forming part of white agarwood, among which Fusarium was the dominant species, followed by Trichoderma; on this basis, the researchers used benzyl acetone, the main component of agarwood, as the resistance indicator of fragrance-forming fungi, and screened out a Fusarium fungus G7# strain that can induce the formation of agarwood.

[0004] Prior art CN106069147B, a method for producing artificial agarwood, discloses that Aspergillus niger, Schizophyllum or Scleropore can be beneficial to the production of agarwood. Chen Xuyu et al. found that the B1 and B2 strains of Lasiodiplodia theobromae can also induce the production of agarwood. Pestalo-tiopsis virgatula can also promote the production of white wood agarwood. Wang Dongguang found that the formation of sesquiterpenes, aromatics and 2-(2-phenylethyl) chromones was induced by the bacterial liquid of seven fungi, including Lasiodiplodia theobromae, Penicillium meleagrinum, Trichoderma atroviride, Trichoderma koningiiopsis, Penicillium italicum, Fusarium solani and Botryosphaeria rhodina. Artificial induction experiments have shown that Fusarium solani A2 strain can induce healthy white wood to produce agarwood, and Fimetariella rabenhorstii A20 strain can produce substances with a similar structure to agarwood. Endophytic fungi (Cytosphaera mangiferae), Bot-ryosphaeria rhodina, Hypocrea jecorina and Trichoderma have also been shown to promote the formation of agarwood. The shortest agarwood formation time in the above-mentioned prior art is 2 months, so a probiotic liquid is proposed to shorten the agarwood formation time. Summary of the invention

[0005] In view of this, the present invention proposes a probiotic liquid for promoting fragrance formation and its application to solve the above problems.

[0006] The technical solution of the present invention is achieved in this way:

[0007] The invention discloses a probiotic liquid for promoting aroma formation, wherein the probiotic strains include Lasiodiplodia theobromae, Laetiporus sulphureus and Trichoderma viride.

[0008] Furthermore, the probiotic strains include Diplodia theobromin, Sulphur Fungus and Trichoderma viride in a mass ratio of 0.5-0.9:1-1.8:0.8-1.3.

[0009] The invention discloses an application of a probiotic liquid for promoting agarwood formation. The probiotic strains are mixed with a culture medium to prepare the probiotic liquid, and the probiotic liquid is injected into an agarwood tree.

[0010] Furthermore, the mass ratio of the probiotics to the culture medium is 0.02-0.04:10.

[0011] Furthermore, the culture medium includes 7-9 g / L glucose, 15-20 g / L yeast extract, 0.7-0.8 g / L ammonium nitrate, 1-2 g / L potassium nitrate, 5-7 g / L copper sulfate, 11-13 mg / L streptomycin sulfate and 15-20 mg / L vitamin PP.

[0012] Furthermore, an agarwood tree with a tree diameter of more than 6 cm is selected, and a hole is drilled in the trunk 55-60 cm above the ground with a drilling depth of 2.5-3.2 cm, and the probiotic solution is injected into the agarwood tree.

[0013] Further, the aperture is 0.5-0.8 cm.

[0014] Furthermore, Diplodia theobromin was purchased from the Plant Protection Center of the Coconut Research Institute, Chinese Academy of Tropical Agricultural Sciences; Bacillus thuringiensis was purchased from Beijing Biobowei Biotechnology Co., Ltd.; and Trichoderma viride was purchased from Shanghai Huzheng Biotechnology Co., Ltd.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The invention adopts Lasiodiplodia theobromae, Laetiporus sulphureus and Trichoderma viride to form a probiotic liquid, and the probiotic liquid forms a dominant bacterial species in the tree, changes the fungal community composition in the tree, further regulates the secondary metabolic pathway of the agarwood tree, and promotes the biological transformation process in the tree. The probiotic liquid can shorten the agarwood formation time, and the agarwood has a high extract content. DETAILED DESCRIPTION

[0017] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0018] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0019] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0020] Example 1

[0021] The culture medium consists of 8 g / L glucose, 18 g / L yeast extract, 0.9 g / L ammonium nitrate, 1.5 g / L potassium nitrate, 6 g / L copper sulfate, 12 mg / L streptomycin sulfate, 18 mg / L vitamin PP and the remainder water.

[0022] The probiotic strains are composed of Lactodiplodia theobromin, Sulphur Fungus and Trichoderma viride in a mass ratio of 0.7:1.4:1.

[0023] The probiotic solution was prepared by mixing the probiotics and the culture medium in a mass ratio of 0.03:10.

[0024] The probiotic strain is mixed with culture medium to prepare a probiotic solution, which is then injected into the agarwood tree.

[0025] Select agarwood trees with a diameter of more than 6 cm, drill holes in the trunk more than 60 cm above the ground, with a hole diameter of 0.7 and a drilling depth of 3 cm, and inject probiotic liquid into the agarwood tree.

[0026] Example 2

[0027] The culture medium consists of 7 g / L glucose, 15 g / L yeast extract, 0.7 g / L ammonium nitrate, 1 g / L potassium nitrate, 5 g / L copper sulfate, 11 mg / L streptomycin sulfate, 15 mg / L vitamin PP and the balance water.

[0028] The probiotic strains are composed of Diplodia theobromin, Sulphur Fungus and Trichoderma viride in a mass ratio of 0.5:1:0.8.

[0029] The probiotic solution was prepared by mixing the probiotics and the culture medium in a mass ratio of 0.02:10.

[0030] The probiotic strain is mixed with culture medium to prepare a probiotic solution, which is then injected into the agarwood tree.

[0031] Select agarwood trees with a diameter of more than 6 cm, drill holes in the trunk more than 60 cm above the ground, with a hole diameter of 0.7 and a drilling depth of 3 cm, and inject probiotic liquid into the agarwood tree.

[0032] Example 3

[0033] The culture medium consists of 9 g / L glucose, 20 g / L yeast extract, 0.8 g / L ammonium nitrate, 2 g / L potassium nitrate, 7 g / L copper sulfate, 13 mg / L streptomycin sulfate, 20 mg / L vitamin PP and the balance water.

[0034] The probiotic strains are composed of Diplodia theobromin, Sulphur Fungus and Trichoderma viride in a mass ratio of 0.9:1.8:1.3.

[0035] The probiotic solution was prepared by mixing the probiotics and the culture medium in a mass ratio of 0.04:10.

[0036] The probiotic strain is mixed with culture medium to prepare a probiotic solution, which is then injected into the agarwood tree.

[0037] Select agarwood trees with a diameter of more than 6 cm, drill holes in the trunk more than 60 cm above the ground, with a hole diameter of 0.7 and a drilling depth of 3 cm, and inject probiotic liquid into the agarwood tree.

[0038] Comparative Example 1

[0039] The culture medium consists of 8 g / L glucose, 18 g / L yeast extract, 0.9 g / L ammonium nitrate, 1.5 g / L potassium nitrate, 6 g / L copper sulfate, 12 mg / L streptomycin sulfate, 18 mg / L vitamin PP and the remainder water.

[0040] The probiotic strain is Lactodiplodia theobromin.

[0041] The probiotic solution is prepared by mixing the probiotics and the culture medium in a mass ratio of 0.02-0.04:10.

[0042] The probiotic strain is mixed with culture medium to prepare a probiotic solution, which is then injected into the agarwood tree.

[0043] Select agarwood trees with a diameter of more than 6 cm, drill holes in the trunk more than 60 cm above the ground, with a hole diameter of 0.7 and a drilling depth of 3 cm, and inject probiotic liquid into the agarwood tree.

[0044] Comparative Example 2

[0045] The culture medium consists of 8 g / L glucose, 18 g / L yeast extract, 0.9 g / L ammonium nitrate, 1.5 g / L potassium nitrate, 6 g / L copper sulfate, 12 mg / L streptomycin sulfate, 18 mg / L vitamin PP and the remainder water.

[0046] The probiotic strain is Sulphur Bacillus.

[0047] The probiotic solution is prepared by mixing the probiotics and the culture medium in a mass ratio of 0.02-0.04:10.

[0048] The probiotic strain is mixed with culture medium to prepare a probiotic solution, which is then injected into the agarwood tree.

[0049] Select agarwood trees with a diameter of more than 6 cm, drill holes in the trunk more than 60 cm above the ground, with a hole diameter of 0.7 and a drilling depth of 3 cm, and inject probiotic liquid into the agarwood tree.

[0050] Comparative Example 3

[0051] The culture medium consists of 8 g / L glucose, 18 g / L yeast extract, 0.9 g / L ammonium nitrate, 1.5 g / L potassium nitrate, 6 g / L copper sulfate, 12 mg / L streptomycin sulfate, 18 mg / L vitamin PP and the remainder water.

[0052] The probiotic strain is Trichoderma viride.

[0053] The probiotic solution is prepared by mixing the probiotics and the culture medium in a mass ratio of 0.02-0.04:10.

[0054] The probiotic strain is mixed with culture medium to prepare a probiotic solution, which is then injected into the agarwood tree.

[0055] Select agarwood trees with a diameter of more than 6 cm, drill holes in the trunk more than 60 cm above the ground, with a hole diameter of 0.7 and a drilling depth of 3 cm, and inject probiotic liquid into the agarwood tree.

[0056] Comparative Example 4

[0057] The culture medium consists of 8 g / L glucose, 18 g / L yeast extract, 0.9 g / L ammonium nitrate, 1.5 g / L potassium nitrate, 6 g / L copper sulfate, 12 mg / L streptomycin sulfate, 18 mg / L vitamin PP and the remainder water.

[0058] The probiotic strains are composed of Diplodia theobromin, Sulphur Fungus and Trichoderma viride in a mass ratio of 1::1:1.

[0059] The probiotic solution is prepared by mixing the probiotics and the culture medium in a mass ratio of 0.02-0.04:10.

[0060] The probiotic strain is mixed with culture medium to prepare a probiotic solution, which is then injected into the agarwood tree.

[0061] Select agarwood trees with a diameter of more than 6 cm, drill holes in the trunk more than 60 cm above the ground, with a hole diameter of 0.7 and a drilling depth of 3 cm, and inject probiotic liquid into the agarwood tree.

[0062] Test Example 1

[0063] The probiotic solution and use method of Example 1 and Comparative Examples 1-4 were used to start the experiment in Poxin Town, Tunchang County, Hainan Province in April 2021, and 10 agarwood trees were used in each group for the experiment. The agarwood obtained in Example 1 and Comparative Examples 1-4 was used to detect the extract according to the agarwood detection standard in the 2020 edition of the Chinese Pharmacopoeia, and the phenylalanine ammonia lyase kit was used to detect the activity of phenylalanine ammonia lyase 7, 14, and 30 days after inoculation of the probiotic solution.

[0064] If the interval of induction of fragrance in the first month is less than 10 days, it will be counted as one month; if it is 11-20 days in the first month, it will be counted as 1.5 months; if it is 20-30 days in the first month, it will be counted as two months, and so on.

[0065] The samples for testing agarwood extracts are timed from the time when the agarwood forms fragrance, and the test is conducted on agarwood that has been forming fragrance for 8 months.

[0066] Table 1 Fragrant induction time

[0067] name Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Induction time (months) 1.5 10 3.5 9.5 3.5

[0068] The experimental results show that the probiotic liquid of the present invention can shorten the time it takes for agarwood to form.

[0069] The present invention uses the probiotic solution and the method of use of Example 1 to conduct an amplification test, and conducts an amplification test in Jianfengling, Ledong County, Hainan Province, Changjiang County, Hainan Province, and Tunchang County, Hainan Province in September 2021, with a sample capacity of 50 trees. The average induction time for agarwood formation is 43 days. It can be seen that the probiotic solution prepared by the present invention can stably induce agarwood formation in tropical areas and shorten the agarwood formation time.

[0070] Table 2 Extract content in agarwood

[0071] name Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Extract content 43.26 22.64 28.31 25.38 39.42

[0072] The test results show that the probiotic solution of the present invention can increase the content of extracts in agarwood.

[0073] Table 3 Activity of phenylalanine ammonia lyase (PLA)

[0074] name Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 PLA activity at 7 days (U / min) 89.4 53.6 60.7 48.4 76.1 PLA activity at 14 days (U / min) 102.6 65.7 76.9 62.9 92.7 PLA activity at 30 days (U / min) 84.1 46.2 50.4 55.8 71.3

[0075] The experimental results show that the PLA activity of the probiotic liquid of the present invention is in a rapid increase process in the tree body for 1-14 days, reflecting the synthesis rate of secondary metabolites in the tree body. By comparing the experimental results of Example 3 and Comparative Examples 1-4, it is shown that the probiotic strains of the present invention are reasonably compounded to form dominant strains in the tree body, change the fungal community composition in the tree body, further regulate the secondary metabolic pathway of the agarwood tree, promote the biological transformation process in the tree body, and achieve the purpose of promoting the formation of agarwood.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A probiotic liquid for promoting fragrance formation, characterized in that: The probiotic strains include Lasiodiplodia theobromae, Laetiporus sulphureus and Trichoderma viride.

2. The probiotic liquid for promoting fragrance formation according to claim 1, characterized in that: The probiotic strains include Lactodiplodia theobromin, Sulphur Fungus and Trichoderma viride in a mass ratio of 0.5-0.9:1-1.8:0.8-1.

3.

3. The use of a probiotic liquid for promoting fragrance formation as claimed in claim 1, characterized in that: The probiotic strain is mixed with culture medium to prepare a probiotic solution, which is then injected into the agarwood tree.

4. The use of the probiotic liquid for promoting fragrance formation as claimed in claim 3, characterized in that: The mass ratio of the probiotics to the culture medium is 0.02-0.04:

10.

5. The use of the probiotic liquid for promoting fragrance formation as claimed in claim 4, characterized in that: The culture medium comprises 7-9 g / L of glucose, 15-20 g / L of yeast extract, 0.7-0.8 g / L of ammonium nitrate, 1-2 g / L of potassium nitrate, 5-7 g / L of copper sulfate, 11-13 mg / L of streptomycin sulfate and 15-20 mg / L of vitamin PP.

6. The use of the probiotic liquid for promoting fragrance formation as claimed in claim 3, characterized in that: Select agarwood trees with a diameter of more than 6 cm, drill holes in the trunk 55-60 cm above the ground with a drilling depth of 2.5-3.2 cm, and inject probiotic liquid into the agarwood tree.

7. The use of the probiotic liquid for promoting fragrance formation as claimed in claim 6, characterized in that: The aperture is 0.5-0.8cm.