WO2018006104A1 - A biological composition stimulating aquilaria spp. to induce agarwood - Google Patents

A biological composition stimulating aquilaria spp. to induce agarwood Download PDF

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Publication number
WO2018006104A1
WO2018006104A1 PCT/VN2017/000006 VN2017000006W WO2018006104A1 WO 2018006104 A1 WO2018006104 A1 WO 2018006104A1 VN 2017000006 W VN2017000006 W VN 2017000006W WO 2018006104 A1 WO2018006104 A1 WO 2018006104A1
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agarwood
collectotrichum
biological composition
bacterial strain
induce
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PCT/VN2017/000006
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French (fr)
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Thi Thu Nguyen
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Thi Thu Nguyen
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the present invention relates to a biological composition that stimulates Aquilaria spp. to induce agarwood, in particular this biological composition helps the method to promote agarwood production to achieve a high success rate with lower cost of materials and labor, and especially the agarwood made from this biological composition has pleasant aroma like natural agarwood.
  • bioproduct used to stimulate the Aquilaria spp. to be made up of two groups, in which the first group is the magnetic disturbance of two fungal species Phialophora sp. and Fusarium sp. which are produced in the form of dry white powder; and the second group is nutrients, including starch, glucose, and potex, this is produced and stored as light brown aromatic liquid.
  • this preparation is not very effective because the success rate of the agarwood-inducing treatment is not high and the aroma of agarwood produced by this preparation does not have natural smell, because these two fungi are not endophyte fungi on Aquilaria spp. which produces natural agarwood, moreover, this preparation is produced as the fungus without bacteria.
  • An object of the invention is to find out fungal strains and endogenous bacteria that are most suitable for stimulating the Aquilaria spp. to produce a naturally aromatic scent, thereby producing a biological composition that stimulates the Aquilaria spp. to induce agarwood.
  • This inoculant achieves a higher success rate of agarwood-inducing stimulation and treatment but it is less expensive in terms of materials and labor.
  • one aspect of the invention is to provide a Biological composition that stimulates the Aquilaria spp. to induce agarwood comprising: 50% prepared by fungal strain CoUectotrichum sp. (TH1) and 50% prepared by bacterial strain Pseudomonas sp. (TH2), in which:
  • the fungal strain CoUectotrichum sp. and the bacterial strain Pseudomonas sp. have biomass of lxlO 5 to lxlO 9 CFU/g and obtained by isolation and selection, identified by biochemical method and molecular biology method of endogenous micro-organisms in Aquilaria spp. containing natural agarwood and resin crops when microorganisms are endophyte.
  • the preparation of the fungal strain CoUectotrichum sp. and the preparation of the bacterial strain Pseudomonas sp., each of preparation is prepared by steps of:
  • biomass densities from 10 to 10 tb/ml it is preferably within 48 to 96 hours to reach the density of 10 9 tb/ml;
  • the inoculant of fungal strain Collectotrichum sp. (TH1) and the inoculant of bacterial strain Pseudomonas sp. (TH2) are mixed into a paste-like biological inoculant; and packed in silver bags and stored in cool condition for good use within 3 months.
  • the fungal strain Collectotrichum sp. and the bacterial strain Pseudomonas sp. are fermented at three levels.
  • the preparation of the invention has endogenous bacterial strains, the formation of agarwood on the Aquilaria spp. is quicker at wounds (bores) and has a more rapid association between the wounds than the fungal strains.
  • Fig.1 is a scheme of a biological composition that stimulates aquilaria spp. to induce agarwood according to the preferred embodiment of the present invention.
  • Fig.l is a scheme of creating a biological composition that induces agarwood formation according to a preferred embodiment of the invention.
  • the fungal strain Collectotrichum sp. and the bacterial strain Pseudomonas sp. lxl 0 3 - lxl0 9 CFU/g, preferably lxlO 5 - lxl 0 7 CFU/g are selected as seed for production of biological compositions of the present invention, and the fungal strain Collectotrichum sp. and the bacterial strain Pseudomonas sp. to be obtained by isolation and selection from endogenous microbiologics on the Aquilaria spp.
  • Step 1 conduct fermentation of accelerated biomass in improved environments and conditions of fungal strain Collectotrichum sp. and the bacterial strain Pseudomonas sp. at three distinct breeding stages over a period of 30 to 100 hours, preferably 48 hours, to reach a density of 10 to 10 tb/ml, preferably 10 9 tb/ml of fermentation broth.
  • the fungal strain Collectotrichum sp. and the bacterial strain Pseudomonas sp. at level 1 breeds are made in vitro, at level 2 breeds are made in triangular flasks and shaken at 150 rpm, and at level 3 breeds made on fermentation tanks; and
  • Level 1 breed storage environment [0024]
  • Fungi Collectotrichum sp. Potato starch: lOg/1; Sucroza: 50g/l; MgS0 4 . 7H 2 0: 0.25g/l: KH 2 P0 4 : 5g/l; KN0 3 : lOg/1; pH ⁇ 7.0 ⁇ 0.2
  • Step 3 infect 400ml of fermentation broth obtained in step 1 into polypropylen bags which are prepared at step 2 so that the inoculant has a moisture of about 40 to 55%, preferably 45%; and 2 weeks after to obtain type 1 micro-organisms (TH1) from the fungal strain Collectotrichum sp. and type 2 micro-organisms (TH2) from the bacterial strain Pseudomonas sp..
  • TH1 type 1 micro-organisms
  • TH2 type 2 micro-organisms
  • Step 4 mix 50% TH1 with 50% TH2 to obtain a paste-like inoculant
  • Step 5 packaging the paste-like inoculant in silver packages and storing it in cool condition for good use within 3 months.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
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  • Biomedical Technology (AREA)
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  • Tropical Medicine & Parasitology (AREA)
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  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
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Abstract

The invention relates to a biological composition that stimulates Aquilaria spp. to induce agarwood. This inoculant contains 50% prepared by the fungal strain Collectotrichum sp. (TH1) and 50% prepared by the bacterial strain Pseudomonas sp. (TH2), in which the fungal strain Collectotrichum sp. and the bacterial strain Pseudomonas sp., which have biomass of x105 to 1x109 CFU/g and obtained by isolation and selection, identified by biochemical method and molecular biology method of endogenous micro-organisms in Aquilaria spp. containing natural agarwood and resin crops when microorganisms are endogenous. With the inoculant prepared from the fungal strain Collectotrichum sp. and the bacterial strain Pseudomonas sp., the rate of incense stimulation achieves a high success rate with lower cost of materials and labor, and especially the agarwood induced from this biological composition has pleasant aroma like natural agarwood.

Description

A BIOLOGICAL COMPOSITION STIMULATING AQUILARIA SPP.
TO INDUCE AGARWOOD
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to Vietnamese Patent Application No. VN 1-2016-02427 filed July 1, 2016, the contents of which are hereby incorporated by reference in their entirety.
FIELD OF INVENTION
[0001] The present invention relates to a biological composition that stimulates Aquilaria spp. to induce agarwood, in particular this biological composition helps the method to promote agarwood production to achieve a high success rate with lower cost of materials and labor, and especially the agarwood made from this biological composition has pleasant aroma like natural agarwood.
BACKGROUND OF THE INVENTION
[0002] In the world as well as in Vietnam, there are many methods that have been studied and applied to produce agarwood, and each method is effective. However, these known methods are not really effective in the success rate of producing agarwood and agarwood made from these known methods has no natural soothing aroma.
[0003] Among the known methods, the biological composition used to Aquilaria spp. to induce agarwood is now widely applied because biological compositions for Aquilaria spp. give better scent despite its lower performance than other known methods.
[0004] For example, there is a kind of bioproduct used to stimulate the Aquilaria spp. to be made up of two groups, in which the first group is the magnetic disturbance of two fungal species Phialophora sp. and Fusarium sp. which are produced in the form of dry white powder; and the second group is nutrients, including starch, glucose, and potex, this is produced and stored as light brown aromatic liquid. However, this preparation is not very effective because the success rate of the agarwood-inducing treatment is not high and the aroma of agarwood produced by this preparation does not have natural smell, because these two fungi are not endophyte fungi on Aquilaria spp. which produces natural agarwood, moreover, this preparation is produced as the fungus without bacteria.
SUMMARY OF INVENTION
[0005] An object of the invention is to find out fungal strains and endogenous bacteria that are most suitable for stimulating the Aquilaria spp. to produce a naturally aromatic scent, thereby producing a biological composition that stimulates the Aquilaria spp. to induce agarwood. This inoculant achieves a higher success rate of agarwood-inducing stimulation and treatment but it is less expensive in terms of materials and labor.
[0006] In order to achieve the above-mentioned objects, one aspect of the invention is to provide a Biological composition that stimulates the Aquilaria spp. to induce agarwood comprising: 50% prepared by fungal strain CoUectotrichum sp. (TH1) and 50% prepared by bacterial strain Pseudomonas sp. (TH2), in which:
[0007] The fungal strain CoUectotrichum sp. and the bacterial strain Pseudomonas sp. have biomass of lxlO5 to lxlO9 CFU/g and obtained by isolation and selection, identified by biochemical method and molecular biology method of endogenous micro-organisms in Aquilaria spp. containing natural agarwood and resin crops when microorganisms are endophyte.
[0008] According to a preferred embodiment of the invention, the preparation of the fungal strain CoUectotrichum sp. and the preparation of the bacterial strain Pseudomonas sp., each of preparation is prepared by steps of:
[0009] fermenting fast the fungal strain biomass CoUectotrichum sp. and bacterial strain Pseudomonas sp. within the period from 35 to 100 hours to 1 1 1
obtain biomass densities from 10 to 10 tb/ml, it is preferably within 48 to 96 hours to reach the density of 109 tb/ml;
[0010] preparing a carrier of organic substance containing 18 to 22% of nutrient source, pH= between 6 and 7, preferably pH=6.8; putting the carrier in polypropylene bags; packaging and autoclaving at temperature between 90 and 130°C within a period of 20 to 30 minutes, preferably 121°C within 30 minutes; leaving it stay within 24 to 48 hours, preferably 48 hours; and autoclave for the second time at temperature between 90 and 130°C within a period of 5 to 20 minutes, preferably 121°C within minutes, to kill the remaining spores; and
[0011] infecting the above-mentioned fermentation into polypropylene bags containing the said carrier and inoculate for 2 weeks to obtain the inoculant of fungal strain Collectotrichum sp. (TH1) and the inoculant of bacterial strain Pseudomonas sp. (TH2) with the humidity from 40 to 55%, preferably 45%.
[0012] According to another embodiment of the present invention, the inoculant of fungal strain Collectotrichum sp. (TH1) and the inoculant of bacterial strain Pseudomonas sp. (TH2) are mixed into a paste-like biological inoculant; and packed in silver bags and stored in cool condition for good use within 3 months.
[0013] According to further embodiment of the present invention, the fungal strain Collectotrichum sp. and the bacterial strain Pseudomonas sp. are fermented at three levels.
EFFICENCY OF INVENTION
[0014] With the isolation and selection of fungal and bacterial strains in Aquilaria spp. with natural agarwood and resin crops when endogenous organisms exist, the most suitable fungal and bacterial strains which are able to induce agarwood and used to create an agarwood-inducing are obtained, and the agarwood produced from this inoculant has natural delicate aroma and has a higher rate of stimulating agarwood production than other conventional methods.
[0015] Furthermore, since the preparation of the invention has endogenous bacterial strains, the formation of agarwood on the Aquilaria spp. is quicker at wounds (bores) and has a more rapid association between the wounds than the fungal strains.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Fig.1 is a scheme of a biological composition that stimulates aquilaria spp. to induce agarwood according to the preferred embodiment of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0017] Hereafter, the present invention will be described in detail by way of example of preferred embodiments with reference to the accompanying drawings. It should be understood that the invention is not limited by such preferred embodiments, the invention may be modified, amended and replaced by those skilled in the art without departing from the scope of the invention.
[0018] As shown in Fig.l, which is a scheme of creating a biological composition that induces agarwood formation according to a preferred embodiment of the invention.
[0019] The fungal strain Collectotrichum sp. and the bacterial strain Pseudomonas sp. lxl 03 - lxl09 CFU/g, preferably lxlO5 - lxl 07 CFU/g are selected as seed for production of biological compositions of the present invention, and the fungal strain Collectotrichum sp. and the bacterial strain Pseudomonas sp. to be obtained by isolation and selection from endogenous microbiologics on the Aquilaria spp. with natural agarwood and resin crops for endogenous organisms such as oil bean, lavender, etc.; nextly it will be identified by biochemical methods using the GAN + B-ID System Kit by Microgen company (UK) and the 16S rDNA and 28S rRND gene sequencing molecular biology method, as well as research on BLAST SEARCH to accurately identify the species name of strain of microorganism (the gene sequence available in the gene bank of wildlife).
[0020] Followed by the fungal strain Collectotrichum sp. and the bacterial s a Pseudomonas sp. 1x10 - 1x10 CFU/g is performed on two branches as shown in Fig. 1 with the same steps, namely:
[0021] Step 1 : conduct fermentation of accelerated biomass in improved environments and conditions of fungal strain Collectotrichum sp. and the bacterial strain Pseudomonas sp. at three distinct breeding stages over a period of 30 to 100 hours, preferably 48 hours, to reach a density of 10 to 10 tb/ml, preferably 109 tb/ml of fermentation broth.
[0022] Detailed as follows:
[0023] The fungal strain Collectotrichum sp. and the bacterial strain Pseudomonas sp. at level 1 breeds are made in vitro, at level 2 breeds are made in triangular flasks and shaken at 150 rpm, and at level 3 breeds made on fermentation tanks; and
[0024] Level 1 breed storage environment:
[0025] Bacteria Pseudomonas sp.: NA (Meat extract: 1.5g/l; Yeast extract:
1.5g/l; Pepton: 5g/l; NaCl: 5g/l; Agar: 20g/l); pH ~ 6.8 ± 0.2
[0026] Fungi Collectotrichum sp.: PGA (Potato starch: lOg/1; Glucoza: 50g/l;
Aga: 20g/l); pH ~ 7.0 ± 0.2
[0027] Level 2 and level 3 breeding environment:
[0028] Pseudomonas sp. Bacteria: Meat extract: 1.5g/l; Yeast extract: 1.5g/l; Pepton: 5g/l; NaCl: 5g/l; pH ~ 6.8 ± 0.2
[0029] Fungi Collectotrichum sp.: Potato starch: lOg/1; Sucroza: 50g/l; MgS04. 7H20: 0.25g/l: KH2P04: 5g/l; KN03: lOg/1; pH ~ 7.0 ± 0.2
[0030] Step 2: prepare an organic carrier in peat soil with 15 to 25% of the nutrient source, preferably between 18 and 22%, pH= between 6 and 7, preferably pH=6,8, with an optional moisture so that the prepeation will have a moisture of 40 to 55%; then this carrier is put in polypropylene bags; packed and autoclaved for the first time at temperature between 90 to 130°C within the period of 20 to 30 minutes, preferably at 121°C within 30 minutes; stored within 24 to 48 hours, preferably within 48 hours; and autoclaved for the second time at temperature from 90 to 130°C within 5 to 20 minutes, preferably at 121°C within 15 minutes, to kill the remaining spores.
[0031] Step 3: infect 400ml of fermentation broth obtained in step 1 into polypropylen bags which are prepared at step 2 so that the inoculant has a moisture of about 40 to 55%, preferably 45%; and 2 weeks after to obtain type 1 micro-organisms (TH1) from the fungal strain Collectotrichum sp. and type 2 micro-organisms (TH2) from the bacterial strain Pseudomonas sp..
[0032] Step 4: mix 50% TH1 with 50% TH2 to obtain a paste-like inoculant; and
[0033] Step 5: packaging the paste-like inoculant in silver packages and storing it in cool condition for good use within 3 months.

Claims

1. A biological composition stimulating the Aquilaria spp. to induce agarwood, it includes: 50% prepared by fungal strain Collectotrichum sp. (TH1) and 50% prepared by bacterial strain Pseudomonas sp. (TH2), wherein:
the said fungal strain Collectotrichum sp. and the said bacterial strain Pseudomonas sp., which have biomass of lxl 05 to lxl 09 CFU/g and obtained by isolation and selection, identified by biochemical method and molecular biology method endogenous micro-organisms in Aquilaria spp. containing natural agarwood and resin crops when microorganisms are endogenous.
2. The biological composition stimulating the Aquilaria spp. to induce agarwood according to claim 1 , wherein the preparation of the fungal strain Collectotrichum sp. and the preparation of the bacterial strain Pseudomonas sp., each preparation is prepared by steps of:
fermenting fast the fungal strain Collectotrichum sp. and the bacterial strain Pseudomonas sp. within a period from 35 to 100 hours to obtain
7 1 1
fermentation broth of 10 to 10 tb/ml, preferably within a period of 48 to 96 hours to reach the density of 109 tb/ml;
preparing an organic carrier containing 18 to 22% of nutrition source, pH= between 6 and 7, preferably pH=6.8; infecting the said carrier into polypropylen bags; packaging and autoclaving for the first time at 90 to 130°C within 20 to 30 minutes, preferably 121°C within 30 minutes; then inoculating within 24 to 48 hours, preferably 48 hours; and autoclaving for the second time at a temperature of 90 to 130°C within a period of 5 to 20 minutes, preferably 121°C within 15 minutes, to kill the remaining spores; and
Infecting the above-mentioned fermentation broth into polypropylene bags containing the said carrier and storing them for 2 weeks to obtain the inoculant of the fungal strain Collectotrichum sp. (TH1) and the bacterial strain Pseudomonas sp. (TH2) with humidity of 40 to 55%, preferably 45%.
3. The biological composition stimulating the Aquilaria spp. to induce agarwood according to claim 1 or 2, wherein the inoculant of the fungal strain Collectotrichum sp. (THl) and the inoculant of bacterial strain Pseudomonas sp. (TH2) are mixed into paste-like biological composition; and the paste-like biological composition packed in silver bags and stored in cool condition for good use within 3 months.
4. The biological composition stimulating the Aquilaria spp. to induce agarwood according to claim 3, wherein the fungal strain Collectotrichum sp. and the bacterial strain Pseudomonas sp. are fermented at three levels.
PCT/VN2017/000006 2016-07-01 2017-06-29 A biological composition stimulating aquilaria spp. to induce agarwood WO2018006104A1 (en)

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VN201602427 2016-07-01
VN1-2016-02427 2016-07-01

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106978342A (en) * 2016-01-15 2017-07-25 上海仙草堂保健食品有限公司 A kind of preparation method of the complex micro organism fungicide of efficient induction agalloch eaglewood Edgeworthia chrysantha
CN110859190A (en) * 2019-11-27 2020-03-06 中国医学科学院药用植物研究所海南分所 Incense formation promoter and method for promoting incense formation
CN111758425A (en) * 2020-07-09 2020-10-13 海南润森科技研发有限公司 Bundling and fragrance-forming method for aquilaria sinensis
CN111849265A (en) * 2020-06-18 2020-10-30 广州中医药大学(广州中医药研究院) Water-absorbing controlled-release film for agilawood formation and agilawood production method
CN115336482A (en) * 2022-07-22 2022-11-15 保亭海农农业开发有限公司 Method for promoting aquilaria sinensis agilawood formation rate

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2113794C1 (en) * 1991-05-23 1998-06-27 Корея Рисерч Инститьют Оф Кемикал Текнолоджи Method of preparing microbe pesticide with bipolar cover, microbe pesticide with biopolymeric cover, method of preparing microbe pesticide of buoyant type
CN102302041B (en) * 2011-07-01 2013-05-22 广东省微生物研究所 Method for manually inducing Aquilaria sinensis to generate agilawood
CN105420146A (en) * 2015-11-27 2016-03-23 张传金 Method achieving high-pressure strain inoculation and agilawood production on aquilaria sinensis trees

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2113794C1 (en) * 1991-05-23 1998-06-27 Корея Рисерч Инститьют Оф Кемикал Текнолоджи Method of preparing microbe pesticide with bipolar cover, microbe pesticide with biopolymeric cover, method of preparing microbe pesticide of buoyant type
CN102302041B (en) * 2011-07-01 2013-05-22 广东省微生物研究所 Method for manually inducing Aquilaria sinensis to generate agilawood
CN105420146A (en) * 2015-11-27 2016-03-23 张传金 Method achieving high-pressure strain inoculation and agilawood production on aquilaria sinensis trees

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106978342A (en) * 2016-01-15 2017-07-25 上海仙草堂保健食品有限公司 A kind of preparation method of the complex micro organism fungicide of efficient induction agalloch eaglewood Edgeworthia chrysantha
CN110859190A (en) * 2019-11-27 2020-03-06 中国医学科学院药用植物研究所海南分所 Incense formation promoter and method for promoting incense formation
CN111849265A (en) * 2020-06-18 2020-10-30 广州中医药大学(广州中医药研究院) Water-absorbing controlled-release film for agilawood formation and agilawood production method
CN111849265B (en) * 2020-06-18 2021-10-22 广州中医药大学(广州中医药研究院) Water-absorbing controlled-release film for agilawood formation and agilawood production method
CN111758425A (en) * 2020-07-09 2020-10-13 海南润森科技研发有限公司 Bundling and fragrance-forming method for aquilaria sinensis
CN115336482A (en) * 2022-07-22 2022-11-15 保亭海农农业开发有限公司 Method for promoting aquilaria sinensis agilawood formation rate
CN115336482B (en) * 2022-07-22 2023-07-21 保亭海农农业开发有限公司 Method for promoting aquilaria sinensis agilawood rate

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