Agilawood forming agent and agilawood forming method and application

Abstract

The invention relates to an agilawood forming agent and an agilawood forming method and application. It is found for the first time that a mixture of a fungus solution of appropriate wood-rotting fungi and non-wood-rotting fungi and an agilawood extract aqueous solution is adopted for inducing and treating agilawood (such as aquilaria sinensis) or quasi-aquilaria plants under appropriate conditions, agilawood without a rotting phenomenon can be obtained, and the agilawood production rate is high; meanwhile, it is found for the first time that under the appropriate condition that a fungus solution of wood-rotting fungi and non-wood-rotting fungi and an agilawood extract aqueous solution are subjected to mixed treatment, the non-wood-rotting fungi can generate volatile components with bacteriostatic activity, and the method has important significance in the aspects of preventing xylem tissue from rotting, improving stress resistance of trees and the like; therefore, the invention provides theoretical and practical scientific basis for innovation and optimization of the existing agilawood production technology, and has important significance for industrial production of agilawood.

Description

Technical Field

[0001] This invention relates to the field of biology, and in particular to an agarwood induction agent, a method for agarwood formation, and its application. Background Technology

[0002] Agarwood primarily originates from plants in the genera *Aquilaria* and *Gyrinops* of the family Thymelaeaceae. Normally, the xylem of healthy *Aquilaria* or *Gyrinops* trees is bright white and contains almost no agarwood. When these trees suffer stress from natural disasters (wind damage, lightning strikes, and insect infestation) or human disturbances (fire, cutting, drilling), secondary metabolites containing aromatic oils are produced and bonded to the xylem, forming agarwood. *Aquilaria* and *Gyrinops* trees and their surroundings harbor a large number of microorganisms, especially at the site of wounds where fungal infection occurs, resulting in browning. Over time, the discolored area deepens and expands, representing the accumulation of secondary metabolites from the agarwood formation process. However, agarwood formation is an extremely long natural process, and fungal invasion following stress is considered a significant contributing factor. Based on similar studies, existing research has identified several fungal species from *Aquilaria* or *Aquilaria* species that are conducive to inducing agarwood formation. These include *Trichoderma* sp., *Fusarium* sp., *Lasiodiplodia* sp., and *Botryodiplodia* sp. The method of fermenting fungal solutions and then injecting appropriate amounts of fungi and their mixtures into the tree using an infusion device, followed by a period of continuous stress to produce agarwood, has become an important applied technology for the artificial induction of agarwood formation.

[0003] After different fungal solutions are used to induce treatment of Aquilaria species or Aquilaria pseudoaquilaria species, the xylem tissue structure will be significantly altered, and the main components of the resulting aquilaria will also differ to some extent.

[0004] Fungi are a crucial factor in inducing agarwood formation. They act as stressors, triggering defensive responses in the tree and activating secondary metabolic pathways involved in agarwood formation. Following stress, the physiological and biochemical parameters regulating the defensive response in *Aquilaria* or *Aquilaria* species, such as *Aquilaria sinensis*, significantly increase, while parameters regulating growth decrease. Over time, the intensity of the defensive response diminishes, and growth gradually recovers. This process involves the tree consuming large amounts of non-structural carbohydrates such as starch and soluble sugars. Existing research has shown that most fungi that induce agarwood formation cause fungal diseases such as necrosis and rot, indicating that fungal agarwood induction techniques also have certain limitations. Improper fungal selection or excessive dosage can lead to poor or no agarwood formation after induction, or even excessive damage to the tree caused by pathogens, resulting in rot and death and significant economic losses. Summary of the Invention

[0005] In view of the problems existing in the prior art, and after research, in order to obtain the effects of no decay and high agarwood fragrance production rate, the technical solution proposed in this invention is as follows:

[0006] A first aspect of the present invention provides an agarwood resin-forming agent, the active ingredients of which comprise a fungal solution of a wood-rotting fungus, a fungal solution of a non-wood-rotting fungus, and an aqueous solution of agarwood extract; wherein the wood-rotting fungus is *Cladosporium cladosporioides*; the non-wood-rotting fungus is *Calvatiacyathiformis*; the aqueous solution of agarwood extract is obtained by water distillation extraction; the concentration of the fungal solution of the wood-rotting fungus is 25wt%-30wt%; the concentration of the fungal solution of the non-wood-rotting fungus is 25wt%-30wt%; the concentration of the aqueous solution of agarwood extract is 10wt%-20wt%; and the mixing volume ratio of the fungal solution of the wood-rotting fungus, the fungal solution of the non-wood-rotting fungus, and the aqueous solution of agarwood extract is 1:(2-3):(0.05-0.2).

[0007] It should be noted that the active ingredients in the agarwood resin-forming agent of the present invention can be in a non-solution form before use.

[0008] In some preferred embodiments, the volume ratio of the fungal solution of wood-rotting fungi, the fungal solution of non-wood-rotting fungi, and the aqueous solution of agarwood extract is 1:2:0.1.

[0009] In some preferred embodiments, the water distillation extraction method includes the following steps:

[0010] (1) Pretreatment: Obtain the black oil-rich area of ​​agarwood, and then crush it into agarwood powder of 50-200 mesh; soak the agarwood powder in pure water or deionized water for 20-28 hours to fully absorb water and soften it. The volume ratio of the agarwood powder to pure water or deionized water is 1:0.6-1. Then put the soaked agarwood powder into a distillation tank, add pure water or deionized water, seal it and ferment at room temperature of 20-25℃ for 7-9 days to obtain a fermentation mixture.

[0011] (2) Distillation and condensation: The fermentation mixture is added to a distillation column. During distillation, the internal temperature of the distillation column is maintained at 90-95℃. After primary condensation and secondary condensation, the secondary condensate is collected. The inlet temperature of the primary condenser is 20℃-30℃, and the inlet temperature of the secondary condenser is -5℃-0℃.

[0012] (3) Oil-water separation: The secondary condensate is separated into oil and water using an oil-water separator to obtain agarwood extract. In some preferred embodiments, the method for forming the agarwood obtained in step (1) includes the following steps:

[0013] (a) Cultivating fungal strains: Inoculate strains of Trichoderma atroviride and Fusarium solani into sterile potato dextrose agar medium and culture them at 25-30°C for 2-6 days at pH 5-6 to promote the activation of the strains.

[0014] (b) Obtaining fungal solutions: The activated strains of Trichoderma atroviride and Fusarium solani were inoculated into stirred potato dextrose medium at a stirring speed of 50-60 rpm / min and cultured at 28-30℃ and pH 5-6 for 6-10 days to obtain Trichoderma atroviride fungal solutions and Fusarium solani fungal solutions, respectively.

[0015] (c) Injection of fungal solution: Under environmental conditions of light intensity of 600-1000 Lux, temperature of 25-30℃ and humidity of 30-50%, a 1:1 volume mixture of 25wt%-30wt% Trichoderma atroviride fungal solution and 25wt%-30wt% Fusarium solani fungal solution is injected into the xylem of Aquilaria sinensis, and the environmental conditions are maintained for 12 months; the average diameter at breast height of the Aquilaria sinensis is 10-15cm, the average tree height is 5-7m, and the average tree age is 8-12 years.

[0016] A second aspect of the present invention provides a method for forming agarwood, comprising the following steps:

[0017] (1) Cultivating fungal strains: Inoculate wood-rotting fungal strains and non-wood-rotting fungal strains into sterile activation medium and culture them at 25-30℃ for 2-6 days at pH 5-6 to promote the activation of the fungal strains; the wood-rotting fungus is Cladosporium cladosporioides; the non-wood-rotting fungus is Calvatia cyathiformis.

[0018] (2) Obtaining fungal solutions: The activated strains of wood-rotting fungi and non-wood-rotting fungi were inoculated into the stirring medium at a stirring speed of 50-60 rpm / min and cultured at 28-30℃ and pH 5-6 for 6-10 days to obtain fungal solutions of wood-rotting fungi and non-wood-rotting fungi, respectively.

[0019] (3) Injection of fungal solution: Under environmental conditions of light intensity of 600-1000 Lux, temperature of 25-30℃ and humidity of 30-50%, a fungal solution of 25wt%-30wt% of wood-rotting fungi, a fungal solution of 25wt%-30wt% of non-wood-rotting fungi, and an aqueous solution of 10wt%-20wt% of agarwood extract are mixed at a volume ratio of 1:(2-3):(0.05-0.2) and injected into the xylem of Aquilaria or Aquilaria species, and then the environmental conditions are maintained for at least 10 months.

[0020] In some preferred embodiments, in step (1), the fungal strain is cultured at 28-30°C for 3-4 days at pH 5-6 to promote its activation.

[0021] In some preferred embodiments, in step (1), the activation medium is potato dextrose agar medium.

[0022] In some preferred embodiments, in step (2), the culture is carried out at 28-30°C and pH 5-6 for 6-8 days.

[0023] In some preferred embodiments, in step (2), the stirring medium is potato glucose medium.

[0024] In some preferred embodiments, in step (2), the stirring medium is potato glucose medium.

[0025] In step (3) of the present invention, the injection method can be an infusion method or a pneumatic automatic injection method.

[0026] In some preferred embodiments, in step (3), the plant is a species of the genus Aquilaria. More preferably, it is Aquilaria sinensis.

[0027] In some preferred embodiments, the average diameter at breast height (DBH) of the Aquilaria genus or Aquilaria pseudoagaric tree is 10-15 cm, the average height is 5-7 m, and the average age is 8-12 years. More preferably, the average DBH of the Aquilaria genus or Aquilaria pseudoagaric tree is 12-13 cm, the average height is 5.3-6.6 m, and the average age is 10 years.

[0028] In some preferred embodiments, in step (3), the environmental conditions are maintained for 12 months.

[0029] A third aspect of the present invention provides an agarwood formed according to the method described above.

[0030] The fourth aspect of the present invention provides the application of the above-described method for forming agarwood resin in the production of agarwood.

[0031] The beneficial effects of the present invention include at least the following:

[0032] This invention is the first to discover that by using a mixture of suitable wood-rotting fungal solutions and non-wood-rotting fungal solutions with an aqueous solution of agarwood extract under appropriate conditions to induce treatment of Aquilaria species (such as Aquilaria sinensis) or Aquilaria pseudoagarwood, agarwood without decay and with a high resin production rate can be obtained. Furthermore, it is also the first to discover that under the mixed treatment of suitable wood-rotting fungal solutions and non-wood-rotting fungal solutions with an aqueous solution of agarwood extract, non-wood-rotting fungi can produce volatile components with antibacterial activity. This is of great significance for preventing the decay of xylem tissue and improving the tree's resistance to stress. Therefore, this invention provides a theoretical and practical scientific basis for innovating and optimizing existing agarwood induction technology and is of great significance for the industrial production of agarwood.

[0033] The features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0034] Figure 1 This illustrates the macroscopic structural features of the xylem tissue of *Aquilaria sinensis* treated with induction in this embodiment of the invention.

[0035] Figure 2The optical microstructure of the xylem tissue of induced-treatment Aquilaria sinensis is shown in three sections. In treatment M1, M1-a is a transverse section, M1-b is a longitudinal section, and M1-c is a tangential section; in treatment M2, M2-a is a transverse section, M2-b is a longitudinal section, and M2-c is a tangential section; in treatment M3, M3-a is a transverse section, M3-b is a longitudinal section, and M3-c is a tangential section. FS represents oily substances or fillings; XR represents wood rays; V represents vessels; IP represents phloem; and F represents wood fibers.

[0036] Figure 3 The ultrastructural characteristics of the xylem tissue of induced-treatment Aquilaria sinensis are shown. In treatment M1, M1-a: cross section, M1-b: longitudinal section, M1-c: tangential section; in treatment M2, M2-a: cross section, M2-b: longitudinal section, M2-c: tangential section; in treatment M3, M3-a: cross section, M3-b: longitudinal section, M3-c: tangential section; FS: oily substances or fillers; S: starch grains; XR: wood rays; V: vessels; IP: containing phloem; F: wood fibers.

[0037] Figure 4 The dynamic changes in starch and soluble sugar content in the xylem tissue of *Aquilaria sinensis* under induced treatment were shown. M1: *Cladosporium* solution; M2: *Lycoperdon perlatum* solution; M3: *Cladosporium* solution: *Lycoperdon perlatum* solution: *Aquilaria sinensis* extract aqueous solution = 1:2:0.1 (solution volume ratio), where the concentrations of the *Cladosporium* solution and the *Lycoperdon perlatum* solution were both 25 wt%; the concentration of the *Aquilaria sinensis* extract aqueous solution was 15 wt%; CK: distilled water treatment. Different lowercase letters represent the same treatment, and significant differences were observed between different months (P<0.05). Different uppercase letters represent significant differences between different treatments within the same month (P<0.05).

[0038] Figure 5 The activity of major antioxidant enzymes in the xylem tissue of *Aquilaria sinensis* under induced treatment was shown. M1 was a *Cladosporium* solution; M2 was a *Calvatia purpurea* solution; M3 was a *Cladosporium* solution: *Calvatia purpurea* solution: *Aquilaria sinensis* extract aqueous solution = 1:2:0.1 (volume ratio), with the *Cladosporium* solution and *Calvatia purpurea* solution both having a concentration of 25 wt% and 25 wt% respectively; the *Aquilaria sinensis* extract aqueous solution had a concentration of 15 wt%; CK was distilled water treatment. Different lowercase letters represent the same treatment, and significant differences were observed between different months (P<0.05). Different uppercase letters represent significant differences between different treatments within the same month (P<0.05).

[0039] Figure 6The activities of TPS and PKS in the xylem tissue of *Aquilaria sinensis* under induced treatment were shown. M1 was a *Cladosporium* solution; M2 was a *Calvatia purpurea* solution; M3 was a *Cladosporium* solution: *Calvatia purpurea* solution: *Aquilaria sinensis* extract aqueous solution = 1:2:0.1 (volume ratio), with the concentrations of the *Cladosporium* solution and the *Calvatia purpurea* solution both being 25 wt% and 15 wt% respectively; CK was distilled water treatment. Different lowercase letters represent the same treatment, and significant differences were observed between different months (P<0.05). Different uppercase letters represent significant differences between different treatments within the same month (P<0.05).

[0040] Figure 7 The differences in agarwood aroma production rate and alcohol-soluble extract content after induced treatment were shown. M1 was a *Cladosporium* solution; M2 was a *Calvatia purpurea* solution; M3 was a *Cladosporium* solution: *Calvatia purpurea* solution: *Agarwood extract aqueous solution = 1:2:0.1 (solution volume ratio), with the *Cladosporium* solution and the *Calvatia purpurea* solution both having a concentration of 25 wt% and 25 wt% respectively; the agarwood extract aqueous solution had a concentration of 15 wt%; CK was distilled water treatment. Different lowercase letters represent the same treatment, and significant differences were observed between different months (P<0.05). Different uppercase letters represent significant differences between different treatments within the same month (P<0.05).

[0041] Figure 8 The diagram shows the ion chromatogram of agarwood components after induced treatment, where M1 is a Cladosporium solution; M2 is a purple puffball solution; M3 is a Cladosporium solution: purple puffball solution: agarwood extract aqueous solution = 1:2:0.1 (solution volume ratio), the concentration of the Cladosporium solution is 25 wt%, the concentration of the purple puffball solution is 25 wt%, the concentration of the agarwood extract aqueous solution is 15 wt%; CK: distilled water treatment.

[0042] Figure 9 This display shows partial Mantel test results between aromatic components and physiological indicators, with a heatmap showing pairwise correlations between the physiological indicators. Lines represent Mantel test results, line width represents Mantel's r-statistic, and color represents Pearson's correlation coefficient. SOD: Superoxide dismutase, POD: Peroxidase, CAT: Catalase, MDA: Multiplex drying analysis, TPS: Terpene synthase, PKS: Polyketide synthase. Detailed Implementation

[0043] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described in the following description are merely illustrative examples of specific implementations of this invention and are intended to explain the invention, but do not constitute a limitation thereof.

[0044] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein. In the description of this application, unless otherwise stated, terms such as "multiple / a variety" mean two / a kind or more.

[0045] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are illustrative rather than limiting. It should be noted that if specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. If the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased commercially.

[0046] Example 1

[0047] 1. Materials and Methods

[0048] 1.1 Soil-grown agarwood and artificial agarwood induction treatment

[0049] Ten-year-old healthy Aquilaria sinensis trees were selected as experimental materials, with an average diameter at breast height of 12.54±0.36cm and an average tree height of 6.02±0.58m.

[0050] This embodiment employs a completely randomized block design, setting up solutions of single fungi and fungal combinations, adding 200 ml of agarwood extract aqueous solution (concentration 15 wt%) to induce treatment of Aquilaria sinensis. The solutions included 25 wt% Cladosporium solution and 25 wt% Puffball solution, with a Cladosporium solution:Puffball solution:Aquilaria sinensis extract aqueous solution ratio of 1:2:0.1 (volume ratio). A control group was treated with distilled water injection. The fungal solutions used for each Aquilaria sinensis plant in the induction treatment and their amounts are shown in Table 1.

[0051] Table 1. Experiments on fungal solution-induced treatment of Aquilaria sinensis.

[0052]

[0053]

[0054] The above fungi can be obtained from the Tropical Forestry Experimental Center of the Chinese Academy of Forestry and the China Forestry Microbial Culture Collection Center.

[0055] First, strains of *Cladosporium cladosporioides* and *Calvatia cyathiformis* were inoculated separately onto autoclaved potato dextrose agar (PDA) plates and cultured at 28°C for 3-4 days at pH 5-6 to promote fungal activation. Then, the activated *Cladosporium cladosporioides* and *Calvatia cyathiformis* strains were inoculated into a 50L mechanically stirred stainless steel fermenter (Anhui Saideqirui Biotechnology Co., Ltd.) containing potato dextrose (PD) medium and cultured at 28°C at pH 5-6 for 8 days, yielding fungal solutions of both wood-rotting and non-wood-rotting fungi. To prevent clogging of the injection catheters during the experiment, the fungal solutions cultured in the fermenter were filtered through double-layered gauze and then sealed in plastic containers for later use.

[0056] Under environmental conditions of 600 Lux light intensity, 28°C temperature, and 35% humidity, a mixture of 25 wt% Cladosporium solution, 25 wt% puffball solution, and 15 wt% agarwood extract aqueous solution was prepared at a volume ratio of 1:2:0.1. Holes containing the mixture were then drilled 30-60 cm above the ground on the trunks of Aquilaria sinensis trees using a 5mm cordless electric drill (model GSR 18V-90FC, Bosch (China) Investment Co., Ltd.). The mixture was then injected into each tree using an infusion method. Five trees were randomly selected for each treatment, with three replicates, for a total of 60 trees.

[0057] The above agarwood extract was obtained by water distillation, including the following steps:

[0058] (1) Pretreatment: Obtain the black oil-rich area of ​​agarwood and then crush it into agarwood powder of 50-200 mesh; soak the agarwood powder in pure water or deionized water for 24 hours to fully absorb water and soften it. The volume ratio of the agarwood powder to pure water or deionized water is 1:1. Then, put the soaked agarwood powder into a distillation tank, add pure water or deionized water, seal it, and ferment it at room temperature of 25°C for 7 days to obtain a fermentation mixture.

[0059] (2) Distillation and condensation: The fermentation mixture is added to a distillation column. During distillation, the internal temperature of the distillation column is maintained at 90°C. After primary condensation and secondary condensation, the secondary condensate is collected. The inlet temperature of the primary condenser is 20°C, and the inlet temperature of the secondary condenser is -5°C.

[0060] (3) Oil-water separation: The secondary condensate is separated into oil and water using an oil-water separator to obtain agarwood extract. The agarwood extract aqueous solution is an aqueous solution containing more than 15 wt% agarwood extract.

[0061] The method for obtaining the agarwood rich in black resin in step (1) above includes the following steps:

[0062] (a) Cultivating fungal strains: Inoculate strains of Trichoderma atroviride and Fusarium solani into sterile potato dextrose agar medium and incubate at 25°C for 2-6 days at pH 5-6 to promote the activation of the strains.

[0063] (b) Obtaining fungal solutions: The activated strains of Trichoderma atroviride and Fusarium solani were inoculated into stirred potato dextrose medium at a stirring speed of 50-60 rpm / min and cultured at 28°C and pH 5-6 for 6 days to obtain Trichoderma atroviride fungal solutions and Fusarium solani fungal solutions, respectively.

[0064] (c) Injection of fungal solution: Under environmental conditions of light intensity of 600 Lux, temperature of 28°C and humidity of 35%, a 1:1 volume mixture of 25 wt% Trichoderma atroviride fungal solution and 25 wt% Fusarium solani fungal solution was injected into the xylem of the agarwood, and the environmental conditions were maintained for 12 months; the average diameter at breast height of the agarwood was 12-13 cm, the average tree height was 5.3-6.6 m, and the average age was 10 years.

[0065] 1.2 Observation of the structural characteristics of agarwood

[0066] 1.2.1 Macroscopic structural characteristics

[0067] In the 12th month, three agarwood trees from each treatment were felled, and a disc approximately 5.0 cm thick was taken at a position of 1.0 m for photographic observation. Based on the characteristics of the agarwood samples, such as wood color, texture, and aroma, the resin formation characteristics of each layer of xylem tissue were summarized.

[0068] 1.2.2 Microstructural characteristics

[0069] In the 12th month, three agarwood trees were randomly selected from each treatment, and agarwood samples (approximately 3.0 cm) were collected 5.0 cm above the infusion port.3 The collected agarwood samples were quickly immersed in 70% FAA fixative (BIOSHARP LIFE SCIENCES, Hefei Lanjieke Technology Co., Ltd.) and brought back to the laboratory for later use. The agarwood samples were prepared into regular shapes of 1.0cm × 1.0cm × 1.0cm, and thoroughly softened using a boiling method. The softened agarwood samples were then embedded in paraffin wax. Based on the cross-section, radial section, and tangential section of the wood, sections of 15.0μm thickness were prepared using a fully automated rotary microtome (Leica RM2255, Leica GmbH, Germany), with three sections cut from each side. The sections were stained with potassium iodide to detect starch grains, and Schiff's reagent was used to stain soluble reducing sugars. Finally, the sections were mounted with neutral resin. The agarwood samples were observed using an optical microscope (Olympus BX51, Olympus Corporation, Japan), and the histological structure of the prepared sections was photographed using a high-resolution digital camera (Pro600es, Pixera Corporation, USA). At the same time, the anatomical structure characteristics, starch grains, and distribution of soluble reducing sugars in the agarwood samples were recorded.

[0070] Agarwood samples were prepared into 2.0 mm thick slices and placed in the vacuum chamber of an ion sputtering instrument (JFC-1600, NEC Corporation). Gold was sputtered twice, each time for 60 seconds, at a flow rate of 30 mA / s. In the public laboratory of the South China Botanical Garden, Chinese Academy of Sciences, the agarwood samples were fixed on a special cylindrical steel column and placed in a high-resolution field emission scanning electron microscope (Regulus 8100, Hitachi, Ltd., Japan) for vacuum scanning ultra-microscopic morphology observation. The ultra-microscopic structures of parenchyma, wood rays, phloem vessels, and pores, as well as the distribution of starches, oils, or fillers, were recorded.

[0071] 1.3 Measurement of physiological indicators

[0072] At months 3, 6, 9 and 12, three Aquilaria sinensis trees were randomly selected from each treatment, and a suitable amount of Aquilaria sinensis samples were collected. The samples were wrapped in aluminum foil, labeled, and completely frozen in liquid nitrogen. They were then brought back to the laboratory and stored in an ultra-low temperature vertical freezer at -86℃ (DW-86L80, Zhejiang Jiesheng Cryogenic Equipment Co., Ltd.) for subsequent physiological index determination.

[0073] 5 mL of 80% methanol (Shanghai, China) and 1.0 mmol / L 2,6-di-tert-butyl-4-methylphenol (Zhongyin Chemical Reagent Co., Ltd.) cooling buffer were added to 1.0 g of fresh wood sample. The total sugar and soluble sugar contents were determined by anthrone colorimetry. The sample was ground and extracted, and the homogenate was incubated at 4 °C for 4 h, centrifuged at 4000 × g for 5 min, and the activities of antioxidant enzymes (including SOD, CAT, and peroxidase (POD)) and MDA content were determined by the thiobarbituric acid method. Additionally, the activity indices of TPS and PKS were sent to Shanghai Enzyme-Linked Biotechnology Co., Ltd. for determination.

[0074] 1.4 Determination of Agarwood Aroma Yield and Alcohol-Soluble Extract

[0075] 1.4.1 Agarwood Production

[0076] In the 12th month, three agarwood trees were randomly selected from each treatment. They were harvested, cut into sections, weighed, and transported back to the laboratory. First, the white wood and rotten areas of the tree were removed with a machete. Then, a resin-picking tool was used to further remove the dark-colored agarwood chips containing oil. After the agarwood chips were completely dried, they were weighed again. The agarwood yield was calculated as the ratio of the weight of dried agarwood to the weight of fresh agarwood.

[0077]

[0078] In the formula, Y is the agarwood yield, W1 is the weight of the dried agarwood sample, and W2 is the weight of the fresh agarwood sample.

[0079] 1.4.2 Alcohol-soluble extracts

[0080] In the 12th month, three agarwood trees were randomly selected from each treatment. A suitable amount of agarwood samples were extracted, wrapped in aluminum foil and labeled, and brought back to the laboratory to air dry. The agarwood samples were then ground into fine powder using a small herbal medicine grinder (YF-111B, Ruian Yongli Pharmaceutical Machinery Co., Ltd.). After grinding, each agarwood sample was passed through a 40-mesh sieve for later use.

[0081] For each treatment, accurately weigh 2.0 g of agarwood sample powder and mix it with 20.0 mL of 95% ethanol (analytical grade). Treat the mixture for 0.5 h using an Elma ultrasonic cleaner (P300H, Elma GmbH, Germany) at 60 °C and 35 kHz. Remove impurities by passing the extracted solution through a 0.45 μm filter membrane. Repeat this process three times. Allow the liquid sample to stand until it evaporates to constant weight, and finally weigh it to calculate the content of the alcohol-soluble extract.

[0082] 1.5 Determination of main agarwood components

[0083] Weigh 2.0g of powder from each agarwood sample, and obtain the alcohol-soluble extract according to the procedure in section 2.4.2. Take 10.0ml of the solution and let it evaporate, then make up to 2.0mL with ethyl acetate. Analyze the aromatic components of agarwood using GC-MS (Aglien, USA) with an HP-5MSZ column (30m×0.25mm, 5μm) at the Analysis and Testing Center of South China Agricultural University.

[0084] The chemical components were identified by searching the NIST standard mass spectrometry database (2021) using GC-MS Postrun Analysis software based on the retention index and mass spectrometry fragmentation information of the chromatographic peaks. The retention time of agaric tetraol was used as a benchmark to correct the retention times of the chromatographic peaks in the GC-MS total ion chromatogram and HPLC. Common peaks were identified and their areas were calculated using a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine.

[0085] Chromatographic conditions: First, the temperature program was used, starting at 90℃ and holding for 1 min, then increasing to 150℃ at a rate of 5℃ / min and holding for 5 min; then increasing to 260℃ at a rate of 5℃ / min and holding for 10 min. The injection port temperature was 250℃; the carrier gas was high-purity He (99.999%), with a flow rate of 0.5 mL / min, a total injection volume of 1 μL per injection, and a solvent delay of 5 min.

[0086] Mass spectrometry conditions: ion source interface temperature 250℃, ionization mode EI; electron energy 70eV, mass scan range 35~350m / z.

[0087] 1.6 Data Analysis and Charting

[0088] Experimental data were preprocessed using Excel (Microsoft Corporation, USA), and SPSS 22.0 software (IBM Corporation, USA) was used to perform t-tests and one-way ANOVA. Origin Pro 2021 software (OriginLab, USA) was used to create bar charts and perform correlation analysis. The correlations among various physiological indicators were compared, and a correlation heatmap was generated using ChiPlot (https: / / www.chiplot.online / ) (accessed October 1, 2024). The statistical significance level was set at P < 0.05.

[0089] 2 Results and Analysis

[0090] 2.1 Characteristics of Agarwood Structure

[0091] 2.1.1 Macroscopic structural characteristics

[0092] At 12 months, compared with the distilled water-treated (CK) agarwood, the fungal solution-induced treated agarwood showed obvious discoloration areas in its xylem tissue, such as... Figure 1 As shown in the diagram. After M1 induction treatment, the agarwood xylem showed a layer of dark brown and gray areas with obvious stratification, but with significant decay in the center. After M2 induction treatment, the agarwood xylem showed irregular dark brown areas with obvious stratification and some decay in the center. After M3 induction treatment, the agarwood xylem showed continuous dark brown and gray areas with obvious stratification and no decay. The CK-treated agarwood xylem was basically apricot-white (with only slight discoloration around the drilled holes), showed no decay, and had almost no special aromatic odor.

[0093] The results of xylem tissue discoloration under different induction treatments showed that the M3 induction treatment had the largest discoloration area, followed by the M2 induction treatment, while the M1 treatment showed severe decay, affecting the actual discoloration area. Based on the preliminary judgment of the observation results, the M3 induction treatment is more conducive to the cultivation of agarwood.

[0094] Under an optical microscope, the anatomical structure of the xylem tissue of *Aquilaria sinensis* can be clearly seen, including the cellular structural features of tracheids, ray parenchyma, and vessels (such as...). Figure 2 (As shown). Compared with the CK treatment, the amount of oily substance filling in the fungal solution treatment was significantly increased. Specifically, in the M1 treatment, a small portion of the phloem and wood ray cells were filled with oily substance, and the color was lighter (in... Figure 2 In the diagram, M1-a: cross section, M1-b: longitudinal section, M1-c: tangential section). Treatment with M2 results in a higher filling volume of grease and a darker color than treatment with M1 (in...). Figure 2 In the diagram, M2-a is the cross section, M2-b is the longitudinal section, and M2-c is the tangential section. Treatment M3 has the highest oil content, with most of the phloem and wood ray cells completely filled with oily substances. Oily substances can also be observed in the vessels and wood fibers, exhibiting the darkest and most uniform color. Figure 2 In the diagram (M3-a: cross section, M3-b: longitudinal section, M3-c: tangential section), it is indicated that during agarwood formation, resinous substances may initially accumulate in the phloem and ray cells of the xylem tissue of *Aquilaria sinensis*. With prolonged induction treatment, the resinous substances diffuse and gradually fill the gaps between vessels and wood fibers. The analysis suggests that *Aquilaria sinensis* treated with M3 may experience a longer duration of stress from fungal fermentation broth, producing a similar anatomically based self-defense response.

[0095] 2.1.2 Ultramicroscopic structural features

[0096] Under ultra-high resolution field emission scanning electron microscopy, the distribution of inclusions such as starch grains and oily substances can be clearly seen in the anatomical structure of the xylem tissue of *Aquilaria sinensis* (e.g., ...). Figure 3 (As shown). In treatments M1 and M2, a large amount of flocculent tyloses accumulated in the phloem, ray cells, and vessels. Starch grains gradually disappeared in the ray cells, and flocculent material also began to appear in the parenchyma cells. This material entered adjacent vessels through the semi-bordered pits of the vessel-parenchyma cells, further accumulating in the vessels or parenchyma cells, but did not completely block the vessels and ray cells (corresponding to...). Figure 3 The M1-a, M1-b, M1-c, and M2-a, M2-b, and M2-c groups were included. In the M3 treatment, the area of ​​tyloses blocking the phloem, ray cells, and vessels was the largest, even completely blocked. Although the number of tyloses in the ray cells increased, and partial blockage occurred, starch grains were still observed. In the CK treatment, a small amount of starch grains were present in the phloem and ray cells; the vessel pits and wall structures were clear; and only a very small number of tyloses were observed in some vessels. Figure 3 The values ​​of CK-a, CK-b, and CK-c indicate that the formation of agarwood may be related to the decomposition, transformation, and utilization of starch grains within the xylem cells of agarwood.

[0097] 2.2 Starch content and soluble sugar content

[0098] from Figure 4 Figure A shows that fungal solution induction treatment has a significant impact on the starch content in the xylem cells of *Aquilaria sinensis*. Overall, the starch content generally decreased after fungal solution induction treatment. The decrease was smaller in the 3rd and 6th months, and larger in the 9th and 12th months, with the M3 treatment showing the largest decrease. In particular, at 12 months, the starch content of the M3 treatment decreased to 54.82 mg / g. -1 Compared to the CK treatment, the decrease was -55.11%, while the decreases for the M1 and M2 treatments were -47.21% and -28.33%, respectively.

[0099] from Figure 4 As shown in Figure B, compared with the control (CK) treatment, the soluble sugar content in the fungal solution-induced treatment initially increased and then decreased. Analysis revealed that in the early stage of induction treatment (3 months), the soluble sugar content of treatments M1 and M3 increased significantly, with increases of 34.11% and 44.63%, respectively, followed by treatment M2 (24.41%). In the later stage of induction treatment (12 months), the soluble sugar content of treatments M1, M2, and M3 decreased significantly, with decreases of -27.28%, -23.10%, and -26.07%, respectively.

[0100] The above studies indicate that, compared to the control (CK) treatment, the M3 treatment showed the greatest consumption of starch and soluble sugars, followed by the M1 and M2 treatments. This suggests that after fungal solution induction treatment, based on starch content, varying degrees of starch consumption were observed in the xylem tissue of *Aquilaria sinensis*, promoting the conversion and accumulation of soluble sugars. Overall, the M3 treatment exhibited the most efficient starch conversion rate and soluble sugar utilization.

[0101] 2.3 Physiological test indicators

[0102] 2.3.1 Main antioxidant enzyme activities

[0103] Compared with the control (CK) treatment, fungal solution induction treatment significantly increased the activity of related antioxidant enzymes and MDA content (e.g., ...) in the xylem tissue of *Aquilaria sinensis*. Figure 5 A to Figure 5 (As shown in D). After induction treatment, the SOD activity and MDA content of M1, M2, and M3 reached their maximum values ​​in the third month, being 39.50%, 48.13%, 52.44%, and 95.30%, 94.51%, 90.05% higher than those of the CK treatment, respectively. With the extension of induction time, SOD activity and MDA content showed a trend of first increasing and then decreasing. In the 12th month, only the SOD activity difference between the M2 treatment and the CK treatment was significant, while the MDA content differences between the fungal solution treatment and the CK treatment were all significant (P<0.05).

[0104] Following induction treatment, the POD activity of the M1 treatment showed a decreasing trend, while the POD and CAT activities of the M2 and M3 treatments showed a trend of first increasing and then decreasing. Specifically, the POD activity of the M1 treatment reached its maximum value in the third month, while the POD and CAT activities of the M2 and M3 treatments reached their maximum values ​​in the ninth month. The maximum POD and CAT activities of the three treatments were 35.01%, 23.94%, and 33.30% higher than those of the CK treatment, and 116.33%, 94.89%, and 92.31% higher, respectively.

[0105] In summary, as the induction time increased, a series of changes occurred in the tree's osmotic regulation. The stress and damage caused by the fungal solution to *Aquilaria sinensis* resulted in different intracellular ion states, leading to varying trends in the changes of antioxidant enzyme activity. Simultaneously, the antioxidant enzyme activity in the xylem tissue of *Aquilaria sinensis* showed significant changes, possibly because fungal infection can rapidly induce a signal transduction response in the tree.

[0106] 2.3.2 Activities of terpene synthase TPS and polyketide synthase PKS

[0107] Compared with the control (CK) treatment, the activities of TPS and PKS in the xylem tissue of *Aquilaria sinensis* treated with fungal solution were significantly enhanced, showing a significant difference (P<0.05). Figure 6 A and Figure 6 (As shown in B). With the extension of induction time, TPS activity showed a trend of first increasing and then decreasing. At month 9, the TPS activities of M1, M2, and M3 reached their maximum values, being 39.58%, 36.10%, and 46.14% higher than the CK treatment, respectively. PKS activity, on the other hand, showed a continuous upward trend, reaching its maximum value at month 12, being 54.32%, 49.98%, and 69.31% higher than the CK treatment, respectively.

[0108] This indicates that after agarwood is subjected to stress, it initiates the synthesis pathway of secondary metabolites, rapidly increasing the activities of TPS and PKS to catalyze the synthesis of terpenes and chromones, thereby enhancing the tree's self-defense mechanisms and resistance to adverse external damage. Among these, the M3 treatment exhibits the highest TPS and PKS activity thresholds, which is more conducive to the accumulation of secondary metabolites of the main components of agarwood.

[0109] 2.4 Agarwood fragrance yield and alcohol-soluble extract content

[0110] 2.4.1 Agarwood fragrance yield

[0111] Compared with the control (CK) treatment, fungal solution induction treatment significantly increased agarwood yield and alcohol-soluble extractives content (P<0.05). Figure 7 A and Figure 7 (As shown in B). With the extension of induction time, the agarwood resin production rate showed an increasing trend. In the 12th month, the resin production rate of agarwood treated with M1, M2, and M3 reached its maximum, accounting for 27.12%, 22.35%, and 30.08% of the tree body weight, respectively. In contrast, the resin production rate of agarwood treated with CK was only 0.71%, indicating that treatment with M3 was more conducive to improving the resin production rate. The content of alcohol-soluble extracts showed a trend of first increasing and then slowing down. In the 12th month, the content of alcohol-soluble extracts of M1, M2, and M3 also reached its maximum, reaching 13.46%, 9.02%, and 16.82%, respectively. In contrast, the content of alcohol-soluble extracts obtained from treatment with CK was only 1.68%, indicating that treatment with M3 was more conducive to improving the content of alcohol-soluble extracts.

[0112] Overall, M3 (a mixed fungal solution) showed better induction effects than M1 or M2 (a single fungal solution). This may be because the mixed fungal solution caused more severe and prolonged stress damage to the Aquilaria sinensis tree. As secondary metabolites containing aquilaria components accumulated, it was more conducive to improving the aquilaria resin production rate and the content of ethanol extract.

[0113] 2.5 Main components of agarwood

[0114] At the 12th month, GC-MS analysis of agarwood samples from each induced treatment was performed, as follows: Figure 8 As shown, the total ion chromatogram (TIC) and chemical composition indicate that, between the 5th and 35th minutes, the main components of agarwood identified were terpenes, alkanes, and aromatic compounds (such as...). Figure 8 (As shown in Table 2). Between the relative retention times of 36 minutes and 60 minutes, the main components identified were chromones and alkanes.

[0115] It can be seen that after fungal solution induction treatment, the peak time and relative peak area of ​​the main aromatic components are different, indicating that different induction treatments have a certain impact on the main components and their contents of agarwood.

[0116] GC-MS analysis revealed 58 major aromatic components in the agarwood obtained from fungal solution-induced treatment, as shown in Table 2. Treatments M1, M2, and M3 identified 42, 46, and 50 aromatic components, respectively, while the CK treatment identified 28. These major aromatic components included terpenes, chromones, aromatics, and alkanes. Treatment M3 had the highest proportion of major aromatic components, accounting for 90.88% of the total relative content. Treatments M1 and M2 followed, accounting for 83.39% and 76.21%, respectively. The CK treatment had the lowest proportion, at only 33.51%.

[0117] The analysis results showed that the main aromatic components of agarwood varied depending on the induction treatment. Terpenes accounted for 42.78%, 33.14%, and 41.48% in the M1, M2, and M3 treatments, respectively, while chromones accounted for 32.48%, 30.09%, and 37.52%, respectively, which were significantly higher than those in the CK treatment (16.56% and 9.53%, respectively). However, only the aromatic compounds in the M3 treatment (5.72%) and the alkanes in the M2 treatment (10.60%) were significantly higher than those in the CK treatment.

[0118] In the M1-treated agarwood sample, the main terpene was (+)-α-longifine (C 15 H 245.72%), α-Molybdenum (C 15 H 24 3.86%), citrione (C 15 H 22 O, 3.68%), 2-methyl-5-(1-methylvinyl)cyclohexanol (C 10 H 18 O, 3.60%). M2 treatment resulted in terpenoids of 2-methyl-5-(1-methylvinyl)cyclohexanol (C 10 H 18 O, 4.86%), isoaromatene (C 15 H 24 4.40%), α-neosyringotricycloene (C 15 H 24 ,2.48%). M3 treatments were 2-methyl-5-(1-methylvinyl)cyclohexanol (C 10 H 18 O, 5.40%), α-neosyringotricycloene (C 15 H 24 3.99%), Santal alcohol (C 15 H 24 O, 3.24%), citrione (C 15 H 22 O, 3.32%). The most common chromone is 2-(2-phenylethyl)chromone (C 17 H 14 O2 (13.89%-18.69%), while the CK treatment only had 7.79%. Aromatic compounds included benzaldehyde (C7H6O, 0.21%-4.52%) and benzylacetone (C... 10 H 12 O, 0.49-1.15%. Alkanes mainly include hexadecane (C60, 0.49-1.15%). 16 H 32 ,1.32%-3.32%), palmitic acid (C 16 H 32 O2, 2.14-2.80%), 5-(2,3-dimethyltricyclo[2.2.1.02,6]hept-3-yl)pent-2-one (C 15 H 24 O, 1.01-1.79%), etc.

[0119] Table 2. Main aromatic components of agarwood after induction treatment

[0120]

[0121]

[0122]

[0123] Note: ▲: Terpene compounds, ●: Chromone compounds, ■: Aromatic compounds, ◆: Alkane compounds.

[0124] 2.6 Correlation between the main components of agarwood and physiological test indicators

[0125] After induction treatment, correlation analysis of the main physiological indicators of agarwood samples showed that starch, soluble sugar, and MDA content were highly significantly (P < 0.01) or significantly positively correlated (P < 0.05), while they were highly significantly (P < 0.01) or significantly negatively correlated (P < 0.05) with TPS, PKS activities, and agarwood aroma production rate. Agarwood aroma production rate and ethanol extract content were positively correlated with TPS and PKS activities. However, SOD, POD, and CAT activities did not show significant correlations with most other physiological indicators (P > 0.05).

[0126] Mantel's r statistical analysis of the main aromatic components and main physiological indicators of agarwood showed that terpenes, chromones, alkanes and aromatic compounds were significantly positively correlated with the content of alcohol-soluble extracts and POD activity (P < 0.05), but there was no significant correlation with other physiological indicators (P > 0.05).

[0127] 3 Discussion

[0128] 3.1 Effects of induction treatment on the anatomical characteristics of xylem tissue in Aquilaria sinensis

[0129] In this invention, the effects of fungal solutions on the discoloration areas of the agarwood xylem tissue varied. Observations revealed that the M3 treatment resulted in the largest discoloration area, followed by the M2 treatment, while the M1 treatment showed significant decay, affecting the actual discoloration area of ​​the agarwood. This indicates that fungal solution treatment, through transpiration pull, transports substances upwards, triggering a self-defense response in the tree and promoting the accumulation of agarwood resin. Although fungi play a crucial role in agarwood formation, most fungi that induce agarwood formation are decay fungi. These fungi infect the xylem tissue, causing extensive decay, which is considered a key step in agarwood formation. However, the inventors of this invention found that induction treatment using non-wood-decaying fungi (Mucor) significantly reduced the degree of decay in the agarwood xylem tissue, even resulting in no decay at all. This indicates that decay in the xylem tissue of agarwood can lead to agarwood formation, but this decay process is not a necessary condition for agarwood formation. Therefore, utilizing non-wood-decaying fungi to induce agarwood formation not only helps improve the agarwood yield but also has significant commercial potential.

[0130] Under an optical microscope, it was found that the oily substances treated with Cladosporium, Muscone, and their mixed solutions were mainly distributed in the phloem, ray cells, and vessels within the xylem tissue of Aquilaria sinensis. Treatment with M3 showed the highest oil content, with most of the phloem and ray cells completely filled. Oily substances were also clearly observed in the vessels and wood fibers, exhibiting the darkest color and uniform distribution. From cross-sections of Aquilaria wood samples, the oily substances initially appeared in the phloem and ray cells. Over time, the oily substances diffused, adsorbing onto the walls of parenchyma cells and vessels until they completely filled the gaps between vessels, parenchyma cells, and wood fibers, forming an insulating layer to prevent further damage from stress. Ultimately, the wood parts containing the oily substances lost their physiological activity, forming aquilaria.

[0131] Ultra-high resolution field emission scanning electron microscopy also revealed significant differences in the anatomical structure of agarwood samples from different induction treatments. In the M3 treatment, the area of ​​blockage by oily substances or fillers in the phloem, ray cells, and vessels was the largest, with only a few scattered starch grains observed. Although oily substances or fillers were also present in the phloem, ray cells, and vessels of the M1 and M2 treatments, most vessels and ray cells were not completely blocked. In the CK treatment, starch grains were also present in the phloem and ray cells, and the vessel pits and wall structures were clearly visible, with very little oily substance or filler observed. This indicates that the M3 treatment accelerated starch conversion and utilization, promoting the formation and transport rate of oily substances or fillers in the phloem, ray cells, and vessels.

[0132] 3.2 Effects of induction treatment on the physiological characteristics of xylem tissues of Aquilaria sinensis

[0133] In this invention, the starch content of each induction treatment generally showed a decreasing trend, while the soluble sugar content showed a trend of first increasing and then decreasing. Among them, the M3 treatment showed the largest changes in starch and soluble sugar content, followed by the M2 and M1 treatments, with the CK treatment showing the smallest consumption. Starch in the xylem tissue of *Aquilaria sinensis* was consumed to varying degrees, promoting the conversion and accumulation of soluble sugars, with the M3 treatment showing the most significant effect. Initially, while starch granules decreased within the phloem, wood ray cells, and parenchyma cells, the soluble sugar content showed an increasing trend. During agarwood formation, some starch in the parenchyma cells was consumed, and the soluble sugar content showed a trend of first increasing and then decreasing. There was a certain time lag between the decrease in starch content and the increase in soluble sugar to the threshold. This indicates that during agarwood formation, some soluble sugars decompose and synthesize agarwood oil substances, which is simultaneously influenced by starch content, conversion rate, and other factors.

[0134] Plants typically utilize enzymatic or non-enzymatic antioxidant defense systems to scavenge reactive oxygen species (ROS). SOD, POD, and CAT are important components of these ROS scavenging enzyme systems, while MDA content can indirectly reflect the degree of oxidative damage in plant tissues. In this invention, compared to the control (CK) treatment, the activities of SOD, POD, and CAT, as well as the MDA content, in the fungal solution-induced treatment generally showed a trend of first increasing and then decreasing. However, during the M1 induction treatment, POD activity continuously decreased, possibly due to the continuous release of superoxide anions under severe stress from the *Pterocarya stenoptera* solution, leading to a relative overconsumption of POD. The hypertonic state of cells also inhibits POD activity. Therefore, after different induction treatments, the intracellular ion levels in *Aquilaria sinensis* xylem tissues varied, resulting in different trends in antioxidant enzyme activity. In this invention, the significant change in antioxidant enzyme activity after fungal solution treatment indicates a rapid activation of signal transduction processes in the fungal-infected tree. Further systematic research is needed to further investigate the stress resistance of *Aquilaria sinensis* xylem tissues with prolonged induction time.

[0135] This invention revealed significant differences in TPS and PKS activities between the fungal solution and the CK treatment (P<0.05). With prolonged induction time, TPS activity initially increased and then decreased, while PKS activity consistently increased. The M3 treatment exhibited the highest TPS and PKS activity thresholds, indicating a greater affinity for the accumulation of secondary metabolites containing the main components of agarwood.

[0136] 3.3 Effects of induction treatment on agarwood resin yield and alcohol-soluble extract

[0137] In this invention, compared with the control (CK) treatment, the fungal solution induction treatment significantly increased the agarwood aroma production rate and the content of alcohol-soluble extracts (P<0.05). With the extension of induction time, the aroma production rate of agarwood treated with the fungal solution showed an increasing trend. At the 12th month, the aroma production rate of agarwood treated with M3 reached a maximum of 30.08%, followed by M1 and M2 treatments at 27.12% and 22.35%, respectively, which were far higher than the aroma production rate of agarwood obtained from the CK treatment (0.71%).

[0138] With the extension of induction time, the content of alcohol-soluble extracts showed a trend of first increasing and then slowing down. At the 12th month, the maximum contents of alcohol-soluble extracts in the three fungal solution treatments reached 13.46%, 9.02% and 16.82%, respectively, which were significantly higher than those in the CK treatment (P<0.05).

[0139] Overall, M3 (a mixed fungal solution) showed better induction effects than M1 or M2 (a single fungal solution), possibly because the mixed fungal solution caused more severe and prolonged stress on the tree. The continuous accumulation of secondary metabolites further enhanced the resin production rate and ethanol extract content of agarwood.

[0140] 3.4 Effects of induction treatment on the formation of major aromatic components in agarwood

[0141] This invention, using GC-MS analysis, identified 58 major aromatic compounds in the agarwood resin obtained from fungal solution-induced treatment, including terpenes (33.14%-42.78%), chromones (30.09%-37.52%), aromatic compounds (1.84%-5.72%), and alkanes (6.16%-10.60%). These compounds are unique aromatic compounds found in artificially cultivated agarwood samples and are largely consistent with the aromatic components of natural agarwood. Terpenes and chromones, in particular, are closely related to the quality of agarwood.

[0142] The present invention differs from existing technologies in its agarwood resin-forming agents, methods, and processing times, resulting in significant differences in the relative content of terpenoid compounds in the obtained agarwood. Terpenes such as (+)-α-pinene, α-lactone, citrione, 2-methyl-5-(1-methylvinyl)cyclohexanol, isocaryophyllene, α-neo-eugenol, and santalol possess active sweet, floral, and fruity aromas and are extremely important characteristic aromatic components of agarwood. 2-(2-phenylethyl)chromone and its derivatives, benzaldehyde, and benzylacetone, with woody and nutty aromas, are also among the most potent aromatic active components. In addition, aromatic components such as α-coumarene, isocaryophyllene epoxide, and β-caryophyllene also contribute to enhancing the aroma of agarwood. Agarorol, α-selinene, and β-wattene also contribute significantly to the aroma, attributing sweet fruity, floral, and herbal notes to their presence. α-Guaifenesin, longifolene, and 2-cyclohexylanisole are also major sources of the sweet aroma. This invention demonstrates that the aroma of agarwood is a balance among different volatile odor compounds, and these active components play a crucial role in effectively preventing further invasion by pathogens and reducing stress damage.

[0143] 4. Conclusion

[0144] M3-induced treatment resulted in a larger discoloration area and abundant accumulation of oily substances in the xylem tissue of the agarwood, without any signs of decay. Simultaneously, the starch conversion rate and soluble sugar utilization efficiency were relatively high, and the activity of antioxidant enzymes was significantly enhanced. At the 12th month, the agarwood showed the highest resin yield (30.08%), alcohol-soluble extract content (16.82%), and total relative content of volatile components (90.88%). Among the main aromatic components, terpenes (42.78%) and chromones (37.52%) were the most abundant. This indicates that treatment with a mixed solution of Cladosporium and Muscone emulsifiable concentrate elicited a stronger and longer-lasting stress response in the agarwood, which is the main reason for the better resin yield and quality.

[0145] In existing technologies, the agarwood fragrance yield is less than 20%, and the number of main components is less than 35. This invention, using a suitable blend of wood-decaying and non-wood-decaying fungi, achieves maximum agarwood formation area and minimizes tree rot by the 12th month. The fragrance yield reaches 30.08%, and the alcohol-soluble extract content is 16.82%, producing unexpected results. The number of main components reaches over 50. In contrast, the agarwood obtained from the CK treatment has a fragrance yield of only 0.71%, an alcohol-soluble extract content of only 1.68%, and only 28 main components.

[0146] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation on the content of the present invention. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the content disclosed in the present invention and all fall within the protection scope of the present invention.

Claims

1. An agarwood inducing agent, characterized in that, The active ingredients include a fungal solution of wood-rotting fungi, a fungal solution of non-wood-rotting fungi, and an aqueous solution of linaloe extract; The wood-rotting fungi is Cladosporium cladosporioides; the non-wood-rotting fungi is Calvatia cyathiformis; the aqueous solution of linaloe extract is obtained by a water distillation extraction method; The concentration of the fungal solution of wood-rotting fungi is 25wt%-30wt%; the concentration of the fungal solution of non-wood-rotting fungi is 25wt%-30wt%; the concentration of the aqueous solution of linaloe extract is 10wt%-20wt%; The mixing volume ratio of the fungal solution of wood-rotting fungi, the fungal solution of non-wood-rotting fungi, and the aqueous solution of linaloe extract is 1:(2-3):(0.05-0.2).

2. The laggan according to claim 1, characterized in that The water distillation extraction method comprises the following steps: (1) Pretreatment: obtaining a black grease-rich area of linaloe, and then crushing it into linaloe wood powder with a particle size of 50-200 mesh; soaking the linaloe wood powder in pure water or deionized water for 20-28 hours to fully absorb water and soften, the volume ratio of the linaloe wood powder to pure water or deionized water is 1:0.6-1, then the soaked linaloe wood powder is loaded into a distillation tank, pure water or deionized water is added, and the tank is sealed for fermentation at room temperature 20-25℃ for 7-9 days to obtain a fermentation mixture; (2) Distillation and condensation: the fermentation mixture is added to a distillation column, and the temperature inside the distillation column is kept at 90-95℃ during distillation, and after primary condensation and secondary condensation, the secondary condensate is collected; the inlet temperature of the primary condenser is 20℃-30℃, and the inlet temperature of the secondary condenser is -5℃-0℃; (3) Oil-water separation: the secondary condensate is subjected to oil-water separation by an oil-water separator to obtain linaloe extract.

3. The laggan of claim 2, wherein The forming method of the linaloe obtained in step (1) comprises the following steps: (a) Culturing fungal strains: inoculating a strain of Trichoderma atroviride and a strain of Fusarium solani into sterilized potato dextrose agar medium, and culturing at 25-30℃ for 2-6 days, pH 5-6, to promote the activation of the strains; (b) Obtaining fungal solutions: inoculating the activated strain of Trichoderma atroviride and the strain of Fusarium solani into stirred potato dextrose medium, with a stirring speed of 50-60 rpm / min, at 28-30℃, pH 5-6, and culturing for 6-10 days to obtain Trichoderma atroviride fungal solution and Fusarium solani fungal solution, respectively; (c) injecting a fungus solution: injecting a mixture of 25wt%-30wt% of Trichoderma atroviride fungus solution and 25wt%-30wt% of Fusarium solani fungus solution in a volume ratio of 1:1 into the xylem of Aquilaria crassna after mixing under environmental conditions of illumination intensity of 600-1000 Lux, temperature of 25-30℃ and humidity of 30-50%, and then maintaining the environmental conditions for 12 months; the average diameter at breast height of the Aquilaria crassna is 10-15 cm, the average tree height is 5-7 m, and the average tree age is 8-12 years.

4. The laggan of claim 1, wherein The volume ratio of the fungus solution of the wood-decaying fungus, the fungus solution of the non-wood-decaying fungus and the aqueous solution of Aquilaria extract is 1:2:0.

1.

5. A method of forming an agarwood, characterized by, The method comprises the following steps: (1) culturing fungus strains: inoculating a strain of wood-decaying fungus and a strain of non-wood-decaying fungus into sterilized activation medium respectively, and culturing at 25-30℃ for 2-6 days at pH 5-6 to promote activation of the fungus strains; the wood-decaying fungus is Cladosporium cladosporioides, and the non-wood-decaying fungus is Calvatiacyathiformis; (2) obtaining fungus solutions: inoculating the activated strain of wood-decaying fungus and the activated strain of non-wood-decaying fungus into stirring culture medium respectively at a stirring speed of 50-60 rpm / min, and culturing at 28-30℃ at pH 5-6 for 6-10 days to obtain a fungus solution of wood-decaying fungus and a fungus solution of non-wood-decaying fungus respectively; (3) injecting a fungus solution: mixing 25wt%-30wt% of fungus solution of wood-decaying fungus, 25wt%-30wt% of fungus solution of non-wood-decaying fungus and 10wt%-20wt% of aqueous solution of Aquilaria extract in a volume ratio of 1:(2-3):(0.05-0.2) after mixing under environmental conditions of illumination intensity of 600-1000 Lux, temperature of 25-30℃ and humidity of 30-50%, and then injecting into the xylem of a tree body of Aquilaria or Parafusticocarya, and then maintaining the environmental conditions for at least 10 months.

6. The method of claim 5, wherein the linaloe is formed by the steps of: In step (1), culturing at 28-30℃ for 3-4 days at pH 5-6 to promote activation of the fungus strains.

7. The method of claim 5, wherein the linaloe is formed by the steps of: In step (2), culturing at 28-30℃ at pH 5-6 for 6-8 days.

8. The method of claim 5, wherein the linaloe is formed by the steps of: In step (3), the Aquilaria plant is Aquilaria crassna.

9. An agarwood, characterized in that, The Aquilaria is formed according to the method of any one of claims 5-8.

10. Use of the Aquilaria laccalizer of any one of claims 1-4 or the method of any one of claims 5-8 in producing Aquilaria.

Images