A cultivation method for promoting the growth and metabolism of acanthopanax

Abstract

The application discloses a cultivation method for promoting growth and metabolism of acanthopanax senticosus, and belongs to the technical field of cultivation. The acanthopanax senticosus seedlings are fumigated by using an alpha-pinene and / or isoprene solution. It is found that the alpha-pinene and isoprene can promote the synthesis of main active ingredients in acanthopanax senticosus leaves, are beneficial to the accumulation of phenolic acid compounds in acanthopanax senticosus leaves and roots, and can increase the contents of organic carbon, nitrogen and phosphorus elements in rhizosphere soil of acanthopanax senticosus, promote the accumulation of organic carbon and nitrogen in the soil, and promote the activity of bacteria of the phylum proteobacteria in the rhizosphere soil of acanthopanax senticosus, so as to increase the soil fertility by fixing nitrogen and the like and promote the growth of plants.

Description

Technical Field

[0001] This invention belongs to the field of cultivation technology, and in particular relates to a cultivation method for promoting the growth and metabolism of Acanthopanax senticosus. Background Technology

[0002] Acanthopanax senticosus (Rupr. & Maxim.) Harms is a deciduous shrub belonging to the genus Acanthopanax in the family Araliaceae. The entire plant has high medicinal value. Wild Acanthopanax develops new plants from the terminal buds of the underground rhizomes of the mother plant, while lateral buds develop into new rhizomes. Therefore, a single mother plant can often form a small population. However, due to long-term predatory harvesting, wild resources of Acanthopanax have been severely damaged, and reserves are decreasing year by year. Cultivation of Acanthopanax involves high production costs for cuttings and tissue culture, and seedling survival rates are low. In practice, seed propagation is the main method of propagation. However, Acanthopanax seeds have dormancy characteristics, and under untreated natural conditions in the wild, the natural regeneration capacity of Acanthopanax is weak.

[0003] Therefore, in order to rationally utilize forest resources, scientifically develop the ecological cultivation of Acanthopanax senticosus under forest cover, and effectively protect wild Acanthopanax senticosus resources and improve land use efficiency, researching new cultivation methods for Acanthopanax senticosus has become an urgent problem to be solved. Preliminary investigations have revealed differences in the growth, development, physiological metabolism, and content of medicinal active ingredients in Acanthopanax senticosus under forest cover, but its mechanism of action remains unclear. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a cultivation method to promote the growth and metabolism of Acanthopanax senticosus. Using two-year-old potted seedlings of Acanthopanax senticosus as material, this invention conducts fumigation treatment with typical plant-derived volatile organic compounds (BVOCs) (isoprene and α-pinene) to study the effects of BVOCs on growth indicators, photosynthesis, physiological indicators, content of major active ingredients, metabolic characteristics, soil physicochemical properties, and soil bacterial community structure of Acanthopanax senticosus. This preliminary analysis of the effects of BVOCs on the growth and metabolism of Acanthopanax senticosus lays a theoretical foundation for the ecological cultivation of Acanthopanax senticosus resources.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A cultivation method to promote the growth and metabolism of Acanthopanax senticosus includes the following steps: fumigating Acanthopanax senticosus seedlings with α-pinene and / or isoprene as a fumigation solution.

[0007] Furthermore, the Acanthopanax senticosus seedlings are two-year-old Acanthopanax senticosus.

[0008] Further, the concentration of the fumigation solution is 0.26 ppb. When the fumigation solution is α-pinene and isoprene, the concentration ratio of α-pinene to isoprene is 1:1 (i.e., the concentration of α-pinene is 0.13 ppb and the concentration of isoprene is 0.13 ppb); when the fumigation solution is α-pinene, the concentration of α-pinene is 0.26 ppb; when the fumigation solution is isoprene, the concentration of α-pinene is 0.26 ppb.

[0009] Furthermore, the fumigation treatment lasts for 30 days.

[0010] Furthermore, the Acanthopanax senticosus seedlings are managed routinely during the fumigation treatment.

[0011] Compared with the prior art, the present invention has the following advantages and technical effects:

[0012] This invention employs a mixed solution of α-pinene and isoprene to fumigate Acanthopanax senticosus seedlings. It was found that the combination of α-pinene and isoprene can promote the synthesis of the main active ingredients in Acanthopanax senticosus leaves, facilitate the accumulation of phenolic acid compounds in Acanthopanax senticosus leaves and roots, increase the content of organic carbon, nitrogen, and phosphorus in the rhizosphere soil, promote the accumulation of organic carbon and nitrogen in the soil, and simultaneously promote the activity of Proteobacteria in the rhizosphere soil of Acanthopanax senticosus, thereby increasing soil fertility and promoting plant growth through nitrogen fixation. Attached Figure Description

[0013] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings:

[0014] Figure 1 The photosynthetic pigment content of Acanthopanax senticosus leaves under different BVOCs treatments: (a) chlorophyll a content, (b) chlorophyll b content, (c) chlorophyll content, (d) carotenoid content.

[0015] Figure 2 The total phenol and total flavonoid content of Acanthopanax senticosus leaves under different BVOCs treatments; (a) total phenol content, (b) total flavonoid content;

[0016] Figure 3 The soluble sugar and soluble protein contents of Acanthopanax senticosus leaves under different BVOCs treatments; (a) soluble sugar content, (b) soluble protein content;

[0017] Figure 4 The antioxidant enzyme activities of Acanthopanax senticosus leaves under different BVOCs treatments: (a) SOD activity, (b) POD activity, (c) CAT activity, (d) APX activity.

[0018] Figure 5 The contents of malondialdehyde, hydrogen peroxide and superoxide anion in Acanthopanax senticosus leaves under different BVOCs treatments; (a) malondialdehyde content, (b) hydrogen peroxide content, (c) superoxide anion content;

[0019] Figure 6 The contents of the main active ingredients in Acanthopanax senticosus leaves under different BVOCs treatments: (a) Acanthopanax senticosin B content, (b) Acanthopanax senticosin E content, (c) isopyridine content, and (d) hyperoside content.

[0020] Figure 7 PCA score of primary metabolites in Acanthopanax senticosus leaves treated with different BVOCs;

[0021] Figure 8 Analysis of PLS-DA, a primary metabolite in Acanthopanax senticosus leaves treated with different BVOCs;

[0022] Figure 9 Results of differentially enriched pathways of compounds in Acanthopanax senticosus leaves treated with different BVOCs;

[0023] Figure 10 PCA score plot of primary metabolites in Acanthopanax senticosus stems treated with different BVOCs;

[0024] Figure 11 Analysis of PLS-DA, a primary metabolite of Acanthopanax senticosus stems treated with different BVOCs;

[0025] Figure 12 Results of differential compound pathway enrichment for Acanthopanax senticosus stems treated with different BVOCs;

[0026] Figure 13 PCA score plot of primary metabolites of Acanthopanax senticosus root treated with different BVOCs;

[0027] Figure 14 Analysis of PLS-DA, a primary metabolite of Acanthopanax senticosus stems treated with different BVOCs;

[0028] Figure 15 Results of differential compound pathway enrichment for Acanthopanax senticosus roots treated with different BVOCs;

[0029] Figure 16 Cluster analysis of phenolic compounds in Acanthopanax senticosus leaves;

[0030] Figure 17 Cluster analysis of phenolic compounds in Acanthopanax senticosus stems;

[0031] Figure 18 Cluster analysis of phenolic compounds in Acanthopanax senticosus root;

[0032] Figure 19Soil pH and electrical conductivity of Acanthopanax senticosus plants under different BVOCs treatments; (a) pH value, (b) electrical conductivity;

[0033] Figure 20 Soil organic carbon, total nitrogen, total phosphorus and total potassium content of Acanthopanax senticosus plants under different BVOCs treatments; (a) organic carbon content, (b) total nitrogen content, (c) total phosphorus content, (d) total potassium content;

[0034] Figure 21 The contents of available nitrogen, nitrate nitrogen, ammonium nitrogen and available phosphorus in the soil of Acanthopanax senticosus plants under different BVOCs treatments; (a) available nitrogen content, (b) nitrate nitrogen content, (c) ammonium nitrogen content, (d) available phosphorus content;

[0035] Figure 22 Dilution curves of rhizosphere soil taxonomic units (OTUs) of Acanthopanax senticosus under different BVOCs treatments;

[0036] Figure 23 Rank-abundance curves of rhizosphere soil microorganisms of Acanthopanax senticosus under different BVOCs treatments;

[0037] Figure 24 The relative abundance (phylum level) of rhizosphere soil microorganisms of Acanthopanax senticosus under different BVOCs treatments;

[0038] Figure 25 The relative abundance (genus level) of rhizosphere soil microorganisms of Acanthopanax senticosus under different BVOCs treatments;

[0039] Figure 26 PCoA analysis of rhizosphere soil microbial community of Acanthopanax senticosus under different BVOCs treatments based on UniFrac;

[0040] Figure 27 LDAse analysis of rhizosphere soil microorganisms of Acanthopanax senticosus under different BVOCs treatments. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] The technical solution of the present invention will be further illustrated by the following embodiments.

[0043] The experiments of this invention were conducted in the Key Laboratory of Forest Plant Ecology of the Ministry of Education at Northeast Forestry University (N45.75°, E126.63°).

[0044] The nutrient soil used in the following embodiments of the present invention is a conventional raw material that can meet the nutrient requirements for plant growth. It can be purchased on the market and is not considered as a factor in the present invention, nor does it affect the effect.

[0045] Example 1

[0046] I. Effects of BVOCs on the growth and physiological indicators of Acanthopanax senticosus

[0047] 1. Experimental Materials and Design

[0048] A single-factor, completely randomized pot experiment was conducted. Two-year-old Acanthopanax senticosus seedlings were transplanted into pots with a diameter of 15 cm and a height of 25 cm. The potting soil was a 1:1 mixture of nutrient soil and ordinary soil, which could meet the nutrient requirements of plant growth. In May 2023, the plants were placed in an artificial controlled culture chamber for fumigation treatment for 30 days. To simulate the emission characteristics of BVOCs in a typical forest type, four treatment groups were set up, with three biological replicates in each group and three pots in each replicate. The control groups were set as CK (no fumigation), T1 treatment group (+α-pinene 0.26 ppb), T2 treatment group (+isoprene 0.26 ppb), and T3 treatment group (+α-pinene 0.13 ppb, +isoprene 0.13 ppb). During the treatment period, routine management was carried out to ensure the supply of water and nutrients. After fumigation, fully expanded leaves with uniform growth at the top of the plant stems were selected as sample leaves to measure their growth, photosynthesis, physiological indicators, and the content of major active ingredients.

[0049] 2. Experimental Methods

[0050] (1) Growth index measurement

[0051] Plant height was measured using a ruler; base diameter was measured using a vernier caliper; and leaf fresh weight was measured using a balance. The average value of each indicator was taken for each treatment group.

[0052] (2) Measurement of photosynthetic rate and gas exchange parameters

[0053] On sunny mornings from 9:00 to 11:00, leaves with good growth and uniform leaf position were selected. The net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr) of the tested plants were measured using a Li-6400 portable photosynthesis system manufactured by LI-COR Corporation, USA. The average values ​​of each index for each treatment group were taken.

[0054] (3) Chlorophyll fluorescence parameter measurement

[0055] Acanthopanax senticosus seedlings were placed in a dark environment for 15 minutes to allow the leaves to adapt to the dark. Chlorophyll fluorescence parameters, including maximum photosynthetic efficiency (Fv / Fm), actual photosynthetic efficiency (ΦPSⅡ), non-photochemical quenching coefficient (NPQ), photochemical quenching coefficient (qP), and quantum yield of PSⅡ-regulated energy dissipation (γ) were measured using a portable PAM-2500 chlorophyll fluorescence spectrometer (Shanghai Zequan Technology Co., Ltd.). NPQ Quantum production of PSII non-regulated energy dissipation (Y) NO ), and the relative electron transport rate (ETR) of PSII.

[0056] (4) Determination of photosynthetic pigment content

[0057] Weigh 0.1g of Acanthopanax senticosus leaves, accurate to 0.0001g, grind in a mortar, add 10mL (95%) ethanol, and let stand in the dark for 24h until the sample tissue turns completely white. Use a UV-Vis spectrophotometer (Shimadzu UV-2550, Japan) to measure the absorbance of the extract at 480, 649, and 665nm. Average values ​​were taken for each indicator in each treatment group. Chlorophyll a content (C Chla ), chlorophyll b content (C Chlb ), total chlorophyll content (C Chl ), carotenoid content (C Car The calculation formula is:

[0058] C Chla (mg·g -1 ·FW)=12.19×A 665 -3.45×A 649

[0059] C Chlb (mg·g -1 ·FW)=21.99×A 649 -5.32×A 665

[0060] C Chl (mg·g -1 ·FW)=C Chla +C Chlb

[0061] C Car (mg·g -1 ·FW)=(1000×A 480 -2.14×Chla-70.16×Chlb) / 220

[0062] (5) Physiological index measurement

[0063] Physiological indicators were determined according to the guidelines of "Experimental Guide to Plant Physiology" edited by Gao Junshan (2018) and "Principles and Techniques of Plant Physiological and Biochemical Experiments" edited by Li Hesheng (2000). The total phenol content in the samples was determined using the Folin-Ciocalteu method; the total flavonoid content was determined using the aluminum chloride colorimetric method; the soluble protein content was determined using the Coomassie Brilliant Blue G-250 staining method; the soluble sugar content was determined using the anthrone colorimetric method; the superoxide dismutase (SOD) activity was determined using the NBT method; the peroxidase (POD) activity was determined using the guaiacol method; the catalase (CAT) and ascorbate peroxidase (APX) activities were determined using ultraviolet spectrophotometry; the malondialdehyde content was determined using the thiobarbituric acid method; the superoxide anion content was determined using the hydroxylamine oxidation method; and the hydrogen peroxide content was determined using the potassium iodide method.

[0064] (6) Determination of medicinal component content

[0065] Experimental reagents: Chromatographic grade methanol and acetonitrile were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; chromatographic grade formic acid (Sigma-Aldrich, USA); the standards for eleutheroside B, eleutheroside E, isofraxidin, and hyperoside, all with a purity ≥98%, were purchased from Shanghai Hewu Biotechnology Co., Ltd.

[0066] Experimental instruments: LC-100 liquid chromatograph (Shanghai Wufeng Scientific Instruments Co., Ltd.), Xi-nyi-24 high-throughput grinder purchased from Ningbo Xinyi Ultrasonic Equipment Co., Ltd., KQ500-DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), HY-2 vortex mixer purchased from Shanghai Shuoguang Electronic Technology Co., Ltd., LSE-3K vacuum centrifuge (Changzhou Jintan Liangyou Instrument Co., Ltd.), benchtop high-speed centrifuge purchased from Sigma, Germany, and Millipore ultrapure water system purchased from Millipore, France.

[0067] Sample preparation: Dissolve the four standards separately in a small amount of methanol, and then dilute to 1 mg / mL for each standard. -1Obtain the standard solution (storage temperature: 4℃). Dry the Acanthopanax senticosus leaves (at a constant temperature of 60℃) to constant weight, pulverize the sample using a pulverizer and sieve it (30 mesh). Accurately weigh 0.5g of Acanthopanax senticosus leaf powder, add 10mL of extraction solution (methanol:water volume ratio = 70%:30%), sonicate for 45min (40℃, 80kHz), centrifuge for 10min (4℃, 8000rpm), and collect the supernatant. Add another 10mL of extraction solution to the filter residue, sonicate for 45min (40℃, 80kHz), centrifuge for 10min (4℃, 8000rpm), and collect the supernatant. Place the supernatant in a rotary evaporator for evaporation. After evaporation, add an equal volume of 70% methanol to all samples, seal and store for later use (-20℃).

[0068] Liquid chromatography conditions: Column (EXFORMMA, RSZG-C18, 5μm, 4.6×250mm, 5GEIC0125), flow rate 1mL·min -1 The column temperature was 30℃, the wavelength was 220nm, the injection volume was 20μL, the mobile phase A was 0.1% (formic acid: water), and the mobile phase B was acetonitrile. The elution gradient conditions are shown in Table 1.

[0069] Table 1 Mobile phase elution procedure

[0070]

[0071]

[0072] Content Calculation: After the sample is tested, the content of the target compound in the sample is calculated as follows: t=(c*v) / (m*1000)

[0073] In the above formula: t represents the sample extraction amount (μg·g) -1 ); c represents the measured concentration of the extract (mg·mL); v represents the volume of the extract (mL); m represents the weight of the sample (g).

[0074] Standard curves: The four component standards were diluted separately, and the standard solutions of different concentrations were measured under the above-described liquid chromatography conditions. The obtained concentrations (x) and peak areas (y) were subjected to linear regression to determine the linear range. The final standard curves were as follows: Acanthopanax senticosin B: y = 52.889x + 64.396 (R²). 2 =0.9996), Acanthopanax senticosin E: y = 21.642x + 151.65 (R 2 =0.9951), Isocyanide: y = 30.223x + 58.804 (R 2 =0.9995), hyperoside: y = 67.759x + 105.46 (R 2 =0.9995).

[0075] 3. Data Processing

[0076] Data analysis was performed using SPSS 22.0 and Excel 2010 software. One-way ANOVA was used to test the significance of differences between treatment groups (P<0.05). Charts were created using Excel software.

[0077] 4. Results Analysis

[0078] 4.1 Effects of BVOCs on the growth indicators of Acanthopanax senticosus

[0079] The growth indicators of Acanthopanax senticosus under different BVOCs treatments are shown in Table 2.

[0080] Table 2. Growth indicators of Acanthopanax senticosus plants under different BVOCs treatments

[0081]

[0082] Note: Lowercase letters in the same column indicate significant differences between different treatments (P<0.05).

[0083] Table 2 shows that the height of Acanthopanax senticosus plants reached its maximum in the T2 treatment group, which was 18.16±0.19 cm, an increase of 2.8% compared with the CK treatment group. There was no significant difference in the basal diameter of the plants among the treatment groups (P>0.05). The total number of leaves per plant reached its maximum in the T3 treatment group, which was 12.66±0.7 leaves, an increase of 15.7% compared with the CK treatment group. The number of branches per plant showed a decreasing trend under the T1, T2 and T3 treatments, decreasing by 12.5%, 25% and 31.2% respectively compared with the CK treatment group (P<0.05). The fresh weight of a single leaf reached its maximum in the T1 treatment group, which was 0.63±0.12 g, an increase of 1.5% compared with the CK.

[0084] 4.2 Effects of BVOCs on the photosynthetic characteristics of Acanthopanax senticosus leaves

[0085] 1) Photosynthetic rate and gas exchange parameters

[0086] The photosynthetic rate and gas exchange parameters of Acanthopanax senticosus leaves under different BVOCs treatments are shown in Table 3.

[0087] Table 3. Photosynthetic rate and gas exchange parameters of Acanthopanax senticosus leaves under different BVOC treatments.

[0088]

[0089] Note: Lowercase letters in the same column indicate significant differences between different treatments (P<0.05).

[0090] As shown in Table 3, the net photosynthetic rate Pn, stomatal conductance Gs, and intercellular carbon dioxide concentration Ci of Acanthopanax senticosus leaves were significantly increased in the T3 treatment group (P<0.05), increasing by 5.6%, 40%, and 12.4% respectively compared with the CK treatment group. The transpiration rates of the T1, T2, and T3 treatment groups decreased by 8.8%, 11.3%, and 0.3% respectively compared with the CK treatment group.

[0091] 2) Chlorophyll fluorescence parameters

[0092] The chlorophyll fluorescence parameters of Acanthopanax senticosus leaves under different BVOCs treatments are shown in Table 4.

[0093] Table 4. Chlorophyll fluorescence parameters of Acanthopanax senticosus leaves under different BVOC treatments.

[0094]

[0095] Note: Lowercase letters in the same column indicate significant differences between different treatments (P<0.05).

[0096] Table 4 shows that the maximum photosynthetic efficiency Fv / Fm of the T3 treatment group was significantly reduced (P>0.05), decreasing by 2.5% compared to the CK group. The actual photosynthetic efficiency Φ(II), photochemical quenching coefficient qP, and non-regulated energy dissipation Y were also significantly reduced. NO Electron transport rate (ETR) increased significantly in the T3 treatment group (P<0.05), increasing by 19.4%, 18.3%, 4%, and 22.3% respectively compared to CK. Non-photochemical quenching coefficient (NPQ) increased significantly in the T2 treatment group (P<0.05), increasing by 5.6% compared to CK. Modulated energy dissipation (Y)... NPQ The T1 treatment group showed a significant increase (P<0.05), with a 2.1% increase compared to the control group.

[0097] 3) Photosynthetic pigment content

[0098] The photosynthetic pigment content of Acanthopanax senticosus leaves under different BVOCs treatments, such as Figure 1 As shown. By Figure 1 It was found that BVOCs treatment significantly increased the contents of chlorophyll a, chlorophyll b, chlorophyll, and carotenoids in Acanthopanax senticosus leaves (P<0.05), and all of these contents reached their maximum in the T2 treatment group, at 1.29±0.006 mg·g⁻¹, respectively. -1 0.64±0.04mg·g -1 2.18±0.017mg·g -1 and 0.21±0.006 mg·g -1 Compared with CK, the levels were increased by 48.8%, 50%, 40.8%, and 71.4%, respectively.

[0099] 4.3 Effects of BVOCs treatment on physiological parameters of Acanthopanax senticosus leaves

[0100] 1) Total phenol and total flavonoid content

[0101] Depend on Figure 2 It can be seen that the total phenol content of Acanthopanax senticosus leaves increased significantly after BVOCs treatment. The total phenol content of the T1, T2 and T3 treatment groups was 71.9±1.87 μg·g, respectively. -1 84.59±1.54μg·g -1 and 92.99±0.84μg·g -1 Compared with the control group (CK), the total flavonoid content increased by 8%, 21.8%, and 28.8%, respectively. The total flavonoid content was highest in the T1 treatment group, at 8.98 ± 0.27 μg·g. -1 Compared to CK, it increased by 15.7%.

[0102] 2) Content of soluble sugars and soluble proteins

[0103] Depend on Figure 3 It was found that the soluble sugar content of Acanthopanax senticosus leaves was significantly reduced after BVOCs treatment (P<0.05). Specifically, the soluble sugar content of the T1, T2, and T3 treatment groups was 0.5±0.03 mg·g, respectively. -1 0.49±0.03mg·g -1 and 0.54±0.04 mg·g -1 Compared with the control group (CK), the soluble protein content decreased by 12.2%, 14%, and 5.2%, respectively. Soluble protein content significantly increased after BVOCs treatment (P<0.05), with the soluble protein content in the T1, T2, and T3 treatment groups being 0.56±0.01 mg·g⁻¹, respectively. -1 0.43±0.01mg·g -1 and 0.52±0.02 mg·g -1 Compared with CK, these values ​​increased by 32.1%, 11.6%, and 26.9%, respectively.

[0104] 3) Antioxidant enzyme activity

[0105] Depend on Figure 4 It was found that the superoxide dismutase (SOD) activity in Acanthopanax senticosus leaves was the highest in the T1 treatment group, which was 386.92 ± 7.7 U·g. -1 ·min -1Compared with the CK treatment group, the activity of peroxidase (POD) increased by 15.1%. The activity of catalase (CAT) was the highest in the T2 treatment group, increasing by 7.5% compared with the CK treatment group. The activity of catalase (CAT) was the highest in the T1 treatment group, increasing by 19.2% compared with the CK treatment group. After BVOCs treatment, the activity of ascorbate peroxidase (APX) increased significantly (P<0.05), with the highest value in the T1 treatment group, increasing by 17.5% compared with the CK treatment group.

[0106] 4) Content of malondialdehyde, hydrogen peroxide, and superoxide anion

[0107] Depend on Figure 5 It can be seen that the malondialdehyde content in the leaves of Acanthopanax senticosus was the highest in the T1 treatment group, which was 0.047±0.0007 μmol·g. -1 FW increased by 25.5% compared to CK. The hydrogen peroxide content was highest in the T1 treatment group, with a content of 168.46 ± 3.55 μmol·g. -1 Compared with the control (CK), the superoxide anion content increased by 15.2%. The superoxide anion content increased significantly after BVOCs treatment, with the content in the T1, T2 and T3 treatment groups being 0.43±0.01 μmol·(min·gFW). -1 ,0.48±0.02μmol·(min·gFW) -1 and 0.49±0.01 μmol·(min·gFW) -1 Compared with CK, these figures increased by 9.3%, 16.6%, and 18.3%, respectively.

[0108] 4.4 Effects of BVOCs treatment on the content of active ingredients in Acanthopanax senticosus leaves

[0109] like Figure 6 As shown, the content of eleutheroside B increased significantly after BVOCs treatment (P<0.05). The contents of eleutheroside B in the T1, T2 and T3 treatment groups were 1.86 μg·g⁻¹, respectively. -1 3.22 μg·g -1 and 3.24 μg·g -1 Compared with the control group (CK), the levels of eleutheroside E increased by 55.3%, 74.2%, and 74.3%, respectively. The content of eleutheroside E significantly increased after BVOCs treatment (P<0.05). The eleutheroside E content in the T1, T2, and T3 treatment groups was 2.84 μg·g⁻¹, respectively. -1 2.29 μg·g -1 and 2.86 μg·g -1 Compared with the control group (CK), the levels increased by 54.5%, 43.6%, and 54.8%, respectively. The isozymine content reached its maximum value of 2.15 μg·g in the T2 treatment group. -1Compared with CK, it increased by 44.6%, reaching a minimum of 0.48 μg·g in the T1 treatment group. -1 Compared with the control group (CK), the content of hyperoside decreased by 59.6%. The hyperoside content reached its maximum in the T3 treatment group, increasing by 41.1% compared with the control group, and reached its minimum in the T1 treatment group, at 0.04 μg·g⁻¹. -1 Compared to CK, it decreased by 42.3%.

[0110] 5. Discussion

[0111] Compared with the control group, the malondialdehyde content in Acanthopanax senticosus leaves was significantly increased after α-pinene treatment, indicating that the degree of lipid peroxidation in Acanthopanax senticosus leaves increased after α-pinene fumigation. Furthermore, the degree of lipid peroxidation is a key factor determining cell damage. To avoid cell damage, plants produce various antioxidant enzymes, among which superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) play important roles in cell defense activities. Compared with the control group, the activities of superoxide dismutase and ascorbate peroxidase were significantly increased under α-pinene treatment, indicating that α-pinene treatment promotes the increase of antioxidant enzyme activity in Acanthopanax senticosus leaves.

[0112] The main active components of Acanthopanax senticosus leaves determined by this invention using an HPLC platform include two saponin compounds, eleutheroside B and eleutheroside E, one coumarin compound, isozyridine, and one flavonol glycoside, hyperoside. Among them, eleutheroside is a relatively abundant specific component in Acanthopanax genus plants, which has the effect of relieving anxiety, depression, insomnia and dreaminess. Isozyridine has antibacterial, anti-inflammatory and anti-tumor effects. Hyperoside has antioxidant and hypoglycemic effects. Compared with the control group, the content of eleutheroside E was significantly increased under α-pinene treatment, the content of eleutheroside B and isozyridine was significantly increased under isoprene treatment, and the content of hyperoside was significantly increased under α-pinene + isoprene treatment, indicating that BVOCs can promote the synthesis of the main active components in Acanthopanax senticosus leaves.

[0113] 6. Summary

[0114] (1) The plant height of Acanthopanax senticosus was the highest in the T1 treatment group, and the number of leaves was the highest in the T3 treatment group; the net photosynthetic efficiency, stomatal conductance, intercellular carbon dioxide concentration and transpiration rate were the highest in the T3 treatment group; the actual photosynthetic efficiency, photochemical quenching coefficient, non-regulated energy dissipation and electron transport rate of leaves were all the highest in the T3 treatment group, the non-photochemical quenching coefficient was the highest in the T2 treatment group, and the regulated energy dissipation was the highest in the T1 treatment group; compared with the control group, the contents of chlorophyll a, chlorophyll b, chlorophyll and carotenoids were all significantly increased, and all of them were the highest in the T2 treatment group.

[0115] (2) Compared with the control group, the activities of superoxide dismutase and ascorbate peroxidase were significantly increased; the contents of malondialdehyde and hydrogen peroxide were the highest in the T1 treatment group, and the contents of superoxide anion were the highest in the T3 treatment group; compared with the control group, the contents of eleutheroside B and eleutheroside E were significantly increased, the contents of isozytin were the highest in the T2 treatment group, and the contents of hyperoside were the highest in the T3 treatment group.

[0116] II. Effects of BVOCs on the metabolites of Acanthopanax senticosus

[0117] 1. Experimental reagents and instruments

[0118] (1) Experimental reagents

[0119] Gas chromatography-mass spectrometry analysis and detection: formic acid, methanol, acetonitrile (Sigma-Aldrich, USA), L-2-chloro-phenylalanine, BSTFA (containing 1% TMCS) derivatizing reagent, pyridine (Shanghai Aladdin Bio-Chem Technology Co., Ltd.), methoxyamine hydrochloride (Hunan Huateng Pharmaceutical Co., Ltd.), chloroform (Hubei Guangao Biotechnology Co., Ltd.), n-hexane (Tianjin Fuyu Fine Chemical Co., Ltd.).

[0120] HPLC-MS / MS analysis: Methanol and acetonitrile were purchased from Fisher Scientific (Pittsburgh, Pennsylvania, USA), and formic acid (mass spectrometry grade, Dikma) was used. Twenty-eight phenolic metabolite standard compounds, including L-phenylalanine, apigenin, naringenin, quercetin, luteolin, catechin, naringenin, kaempferol, glycyrrhizin, galangin, protocatechin, rutin, isoglycoside, salicylic acid, astragalin, piracetamine, isoquercitrin, p-hydroxycinnamic acid, benzoic acid, gentianic acid, abscisic acid, syringic acid, ferulic acid, cinnamic acid, caffeic acid, vanillic acid, rosmarinic acid, and sinapic acid, were analyzed by HPLC. The purity of these standard compounds was higher than 98%. Standard stock solutions were prepared using methanol as the solvent and stored at -20°C.

[0121] (2) Experimental apparatus

[0122] Gas chromatography-mass spectrometry (GC-MS) analysis and detection: 7890A-5975C GC-MS system [Agilent Technologies (China) Co., Ltd.], TGL-16 high-speed centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.), BSA224S-CW 1 / 10,000 electronic balance [Sartorius Scientific Instruments (Beijing) Co., Ltd.], SHZ-82A gas bath constant temperature shaker (Changzhou Jintan Liangyou Instrument Co., Ltd.), KQ500-DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., power 500W, ultrasonic frequency 40kHz), Xi-nyi-24 high-throughput tissue grinder (Ningbo Xinyi Ultrasonic Equipment Co., Ltd.), DK-98-ⅡA electric thermostatic water bath (Tianjin Tester Instrument Co., Ltd.), ZLS-2 vacuum centrifuge concentrator (Hunan Hexi Instrument Equipment Co., Ltd.).

[0123] Liquid chromatography-mass spectrometry (LC-MS) analysis and detection: ACQUITY UPLC Xevo G2-S Q TofMSn LC-MSn system [Waters Technology (Shanghai) Co., Ltd.]; TGL-16 high-speed centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.); BSA224S-CW 1 / 10,000 electronic balance [Sartorius Scientific Instruments (Beijing) Co., Ltd.]; SHZ-82A gas bath constant temperature shaker (Changzhou Jintan Liangyou Instrument Co., Ltd.); KQ500-DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., power 500W, ultrasonic frequency 40kHz); Xi-nyi-24 high-throughput tissue grinder (Ningbo Xinyi Ultrasonic Equipment Co., Ltd.); DK-98-ⅡA electric thermostatic water bath (Tianjin Tester Instrument Co., Ltd.); ZLS-2 vacuum centrifuge concentrator (Hunan Hexi Instrument Equipment Co., Ltd.).

[0124] 2. Experimental Methods

[0125] (1) Gas chromatography-mass spectrometry analysis and detection method

[0126] Sample preparation: Weigh 0.09 g of fresh Acanthopanax senticosus leaves, freeze-mill with liquid nitrogen, add 540 μL of cold methanol and 60 μL of internal standard, sonicate (40 kHz, 45 ℃) for 30 min, add 300 μL of chloroform and 600 μL of deionized water, sonicate (40 kHz, 45 ℃) for 30 min, centrifuge (4 ℃, 12000 r) for 10 min, collect 700 μL of supernatant, transfer to centrifuge tube, concentrate and evaporate to dryness; add 400 μL of methoxyamine hydrochloride pyridine, place in a shaking incubator (37 ℃) for oxime reaction for 90 min, add 400 μL of BSTFA derivatizing reagent and 60 μL of n-hexane for oxime reaction (70 ℃) for 60 min, cool, centrifuge (4 ℃, 12000 r) for 10 min, collect supernatant for analysis.

[0127] GC-MS conditions: Separation was performed using an Agilent 5975C gas chromatograph-mass spectrometer and a nonpolar DB-5 capillary column (250 μm × 30 m, 0.25 μm). The separated components were then analyzed by mass spectrometry. High-purity helium was used as the carrier gas at a flow rate of 1 mL / min. -1 Initial column temperature 60℃, programmed temperature increase 8℃·min. -1 60~125℃; 4℃·min -1 125~210℃; 5℃·min -1 210~270℃; 10℃·min -1 The temperature range was 270–305℃, maintained at 305℃ for 3 min. The injection port temperature was 260℃, the electron impact ion source (EI) temperature was 260℃, and the voltage was -70V. The mass scan range was 50–600 m / z, with a 5 min delay before acquisition began, and the acquisition rate was 20 spectra / s.

[0128] (2) Liquid chromatography-mass spectrometry analysis and detection method

[0129] Sample preparation: Weigh 0.6g of fresh Acanthopanax senticosus leaves, freeze-mill with liquid nitrogen, add 6mL of 70% methanol, sonicate (40kHz, 40℃) for 45min, centrifuge (4℃, 12000r) for 10min, and collect the supernatant. Add 10mL of 70% methanol to the filter residue, sonicate (40kHz, 40℃) for 45min, centrifuge (4℃, 12000r) for 15min, and collect the supernatant. Transfer the supernatant to a centrifuge tube and concentrate to dryness. Add 1mL of 70% methanol to dissolve, centrifuge (4℃, 12000r) for 10min, and collect the supernatant for analysis.

[0130] UPLC-MS conditions: Acquity UPLCBEH C18 column (2.1 mm × 50 mm, 1.7 μm) with Van Guard BEHC18 pre-column (2.1 m × 5 mm, 1.7 μm); flow rate 0.25 mL / min. -1 Column temperature: 40℃; injection volume: 5 μL; mobile phase: 0.04% formic acid-water (A), 0.04% formic acid-acetonitrile (B). ESIQTOF / MS mass spectrometry conditions: positive ion mode, scanning at m / z 50–1200, cone voltage 3 kV. Mobile phase elution program: 0–20 min, 95%–5% A; 20–22.1 min, 5%–95% A; 22.1–28 min, 95% A.

[0131] 3. Data processing methods

[0132] Raw data of characteristic peaks from GC-MS were extracted using the XCMS software package. Principal component analysis and other multivariate statistical analyses were performed using SIMCA-P (Version 13) software. Metabolic pathways were enriched using MetaboAnalyst 5.0 (https: / / www.metaboanalyst.cn / ). UPLC-MS data were analyzed using MassLynx 4.1 software, and the concentrations (μg·mL) of each phenolic compound in the extract were calculated according to the standard curve. -1 SPSS 22.0 software was used for data statistics, normalization, and analysis of variance. Origin software was used for compound cluster analysis and graphing.

[0133] 4. Results Analysis

[0134] 4.1 Effects of BVOCs treatment on primary metabolites of Acanthopanax senticosus plants

[0135] (1) Analysis of primary metabolites PCA and PLS-DA in Acanthopanax senticosus leaves

[0136] Primary metabolites of Acanthopanax senticosus leaves treated with BVOCs were determined and analyzed using GC-MS, identifying a total of 65 primary metabolites. An unsupervised principal component analysis (PCA) model was established using SIMCA-P (Version 14.1) software. The PCA score graph is shown below. Figure 7 As shown in the figure, in the first principal component (PC1), the CK treatment group is located in the positive half-axis region, while T1, T2, and T3 are located in the negative half-axis region; in the second principal component (PC2), CK and T3 are located in the positive half-axis region, while T1 and T2 are located in the positive half-axis region. This indicates that the primary metabolites of the CK, T1, T2, and T3 treatment groups can be clearly distinguished by the PCA model, and the accumulation of primary metabolites in Acanthopanax senticosus leaves under different BVOCs treatments shows significant differences.

[0137] A supervised partial least squares discriminant analysis (PLS-DA) model was built using the SIMCA-P (Version 14.1) program; the permutation test results of the PLS-DA model are as follows. Figure 8 As shown, the R value of the experimental values ​​on the left is... 2 and Q 2All values ​​were lower than the original values ​​on the right, indicating that the model did not overfit and could be used to screen for differentially expressed compounds. As shown in the PLS-DA model score plot, CK was located in the positive half-axis region of PC1, T1, T2, and T3 were located in the negative half-axis region of PC1, T2 and T3 were located in the positive half-axis region of PC2, and CK and T1 were located in the negative half-axis region of PC2. This indicates that the model can distinguish the metabolic differences of Acanthopanax senticosus leaves among the treatment groups. Differentially expressed metabolites could be screened based on the contribution score of the PLS-DA model variables (VIP>1) and the significance of one-way ANOVA (P<0.05). Finally, 39 differentially expressed metabolites among the groups were screened and classified as shown in Table 5, including 15 organic acids, 11 sugars, 5 sugar alcohols, 5 amino acids, and 3 alcohols.

[0138] Table 5. Different metabolites in Acanthopanax senticosus leaves treated with different BVOCs

[0139]

[0140] (2) Metabolic pathway enrichment analysis of differentially expressed compounds in Acanthopanax senticosus leaves

[0141] Twenty-eight differentially expressed compounds were input into the MetaboAnalyst database for metabolic pathway enrichment analysis, resulting in a bubble diagram of differential metabolic pathways in Acanthopanax senticosus leaves, as shown below. Figure 9 As shown, the larger the Impact value and -log10(P) of the metabolic pathway, the better the fit between the differential compound pathway and the metabolism. Using Impact > 0.05 and P < 0.01 as the criteria, seven metabolic pathways with high fit were selected: galactose metabolism, starch and sucrose metabolism, glyoxylate and dicarboxylic acid metabolism, citric acid (TCA cycle) metabolism, glycerol lipid metabolism, glycine, serine, and threonine metabolism, and pyruvate metabolism.

[0142] (3) Analysis of primary metabolites PCA and PLS-DA from Acanthopanax senticosus stem

[0143] Primary metabolites of Acanthopanax senticosus stems treated with BVOCs were determined and analyzed using GC-MS, identifying a total of 64 primary metabolites. An unsupervised principal component analysis (PCA) model was established using the SIMCA-P (Version 14.1) program. The PCA score plot is shown below. Figure 10 As shown, in the first principal component (PC1), the T3 treatment group is located in the positive half-axis region, while the CK, T1, and T3 treatment groups are located in the negative half-axis region; in the second principal component (PC2), T1 and T2 are located in the positive half-axis region, while CK and T3 are located in the negative half-axis region, indicating that the primary metabolites of the CK, T1, T2, and T3 treatment groups can be clearly distinguished by the PCA model, and the accumulation of primary metabolites of Acanthopanax senticosus stems under different BVOCs treatments has significant differences.

[0144] A supervised partial least squares discriminant analysis model was built using the SIMCA-P (Version 14.1) procedure. The permutation test results of the PLS-DA model are as follows: Figure 11 As shown, the R value of the experimental values ​​on the left is... 2 and Q 2 All values ​​were lower than the original values ​​on the right, indicating that the model did not overfit and could be used to screen for differentially expressed compounds. The PLS-DA model score plot is shown in the figure. CK, T1, and T3 are located in the positive half-axis region of PC1, T2 in the negative half-axis region of PC1, CK and T3 in the positive half-axis region of PC2, and T1 and T2 in the negative half-axis region of PC2. This indicates that the method can explain the metabolic differences in Acanthopanax senticosus stems among the BVOCs-treated groups. Differentially expressed metabolites can be screened based on the contribution score of the PLS-DA model variables (VIP>1) and the significance of one-way ANOVA (P<0.05). Finally, 14 differentially expressed metabolites were screened, and their classification is shown in Table 6, including 4 organic acids, 4 amino acids, 2 sugars, 3 sugar alcohols, and 1 alcohol.

[0145] Table 6. Different metabolites of Acanthopanax senticosus stems treated with different BVOCs

[0146]

[0147] (4) Enrichment analysis of metabolic pathways of differentially expressed compounds from Acanthopanax senticosus stem

[0148] Fourteen differentially expressed compounds were input into the MetaboAnalyst database for metabolic pathway enrichment analysis, resulting in a bubble diagram of differential metabolic pathways in Acanthopanax senticosus stem, as shown below. Figure 12 As shown, the larger the Impact value and -log10(P) of the metabolic pathway, the better the fit between the differential compound pathway and the metabolism. Using Impact > 0.05 and P < 0.01 as the criteria, four metabolic pathways with high fit were screened: alanine, aspartic acid, and glutamate metabolism; glycine, serine, and threonine metabolism; butyrate metabolism; and phenylalanine, tyrosine, and tryptophan biosynthesis.

[0149] (5) Analysis of primary metabolites PCA and PLS-DA from Acanthopanax senticosus root

[0150] Primary metabolites in Acanthopanax senticosus roots treated with BVOCs were determined and analyzed using GC-MS, identifying a total of 65 primary metabolites. An unsupervised principal component analysis (PCA) model was established using the SIMCA-P (Version 14.1) program. The PCA score graph is shown below. Figure 13As shown, in the first principal component (PC1), T1 and T2 are located in the positive half-axis region, while the CK and T3 treatment groups are located in the negative half-axis region; in the second principal component (PC2), CK and T1 are located in the positive half-axis region, while T2 and T3 are located in the negative half-axis region. This indicates that the primary metabolites of the CK, T1, T2, and T3 treatment groups can be clearly distinguished by the PCA model, and the accumulation of primary metabolites in Acanthopanax senticosus root under different BVOCs treatments has significant differences.

[0151] A supervised partial least squares discriminant analysis (PLS-DA) model was built using the SIMCA-P (Version 14.1) procedure. The permutation test results of the PLS-DA model are as follows: Figure 14 As shown, the R value of the experimental values ​​on the left is... 2 and Q 2 All values ​​were lower than the original values ​​on the right, indicating that the model did not overfit and could be used to screen for differentially expressed compounds. The PLS-DA model score plot showed that CK and T3 were located in the positive half-axis region of PC1, T1 and T2 were located in the negative half-axis region of PC1, CK and T1 were located in the positive half-axis region of PC2, and T2 and T3 were located in the negative half-axis region of PC2. This indicates that the method can explain the metabolic differences in Acanthopanax senticosus root among the BVOCs-treated groups. Differentially expressed metabolites could be screened based on the contribution score of the PLS-DA model variables (VIP>1) and the significance of one-way ANOVA (P<0.05). Finally, 34 differentially expressed metabolites were screened and classified as shown in Table 7, including 9 organic acids, 10 amino acids, 10 sugars, and 5 alcohols.

[0152] Table 7. Different metabolites in Acanthopanax senticosus roots treated with different BVOCs

[0153]

[0154]

[0155] (6) Metabolic pathway enrichment analysis of differential compounds in Acanthopanax senticosus root

[0156] Thirty-four differentially expressed compounds were input into the MetaboAnalyst database for metabolic pathway enrichment analysis, resulting in a bubble diagram of differential metabolic pathways in Acanthopanax senticosus root, as shown below. Figure 15 As shown, the larger the Impact value and -log10(P) of the metabolic pathway, the better the fit between the differential compound pathway and the metabolism. Using impact > 0.05 and P < 0.01 as the criteria, eight metabolic pathways with high fit were selected: glycine, serine, and threonine metabolism; galactose metabolism; starch and sucrose metabolism; glyoxylate and dicarboxylic acid metabolism; alanine, aspartic acid, and glutamic acid metabolism; arginine biosynthesis; citric acid (TCA cycle) metabolism; and glycerol lipid metabolism.

[0157] 4.2 Effects of BVOCs treatment on secondary metabolites of Acanthopanax senticosus plants

[0158] (1) Cluster analysis of phenolic substances in Acanthopanax senticosus leaves

[0159] Phenolic metabolism is one of the main metabolic pathways for secondary metabolites in plants. Phenolic compounds have a wide range of biological activities. Targeted metabolomics analysis was conducted on 28 phenolic compounds, which can be divided into three categories according to their structure: 16 flavonoids (apigenin, naringenin, quercetin, luteolin, catechin, naringenin, kaempferol, glycyrrhizin, galangin, protocatechin, rutin, isoglycoside, salicylic acid, astragalin, piperidin, isoquercitrin), 11 phenolic acids (p-hydroxycinnamic acid, benzoic acid, gentianic acid, abscisic acid, syringic acid, ferulic acid, cinnamic acid, caffeic acid, vanillic acid, rosmarinic acid, sinapic acid), and amino acids (L-phenylalanine), which are precursors for the synthesis of phenolic compounds.

[0160] After standardizing the metabolomics data of 28 target phenolic compounds obtained by UPLC-MS, cluster analysis was performed to obtain a heatmap of the distribution of phenolic compounds in Acanthopanax senticosus leaves under different BVOC treatments. Figure 16 As shown, among the 28 phenolic substances, 4 phenolic substances accumulated at a high level in the CK group, namely gentic acid, catechin, taxine, and apigenin; 5 phenolic substances accumulated at a high level in the T1 treatment group, namely protocatechin, naringin, cinnamic acid, caffeic acid, and rosmarinic acid; 4 phenolic substances accumulated at a high level in the T2 treatment group, namely syringic acid, vanillic acid, taxine, and quercetin; and 14 phenolic substances accumulated at a high level in the T3 treatment group, namely L-phenylalanine, p-hydroxycinnamic acid, benzoic acid, isoglycoside, sinapic acid, abscisic acid, ferulic acid, isoquercetin, apigenin, naringin, luteolin, kaempferol, kaempferol, and rutin.

[0161] (2) Cluster analysis of phenolic substances in Acanthopanax senticosus stem

[0162] After standardizing the metabolomics data of 28 target phenolic compounds obtained by UPLC-MS, cluster analysis was performed to obtain a heatmap of the distribution of phenolic compounds in Acanthopanax senticosus stems under different BVOC treatments. Figure 17 As shown, among the 28 phenolic substances, 4 phenolic substances accumulated at high levels in the CK group: vanillic acid, quercetin, naringenin, and piperidin; 4 phenolic substances accumulated at high levels in the T1 treatment group: catechin, kaempferol, luteolin, and astragalin; 7 phenolic substances accumulated at high levels in the T2 treatment group: L-phenylalanine, syringic acid, ferulic acid, rutin, cinnamic acid, p-hydroxycinnamic acid, and isoquercetin; and 5 phenolic substances accumulated at high levels in the T3 treatment group: gentianic acid, naringenin, caffeic acid, rosmarinic acid, and isoquercetin.

[0163] (3) Cluster analysis of phenolic substances in Acanthopanax senticosus root

[0164] After standardizing the metabolomics data of 28 target phenolic compounds obtained by UPLC-MS, cluster analysis was performed to obtain a heatmap of the distribution of phenolic compounds in Acanthopanax senticosus roots under different BVOC treatments. Figure 18 As shown, among the 28 phenolic substances, two phenolic substances accumulated at a high level in the CK group: ferulic acid and apigenin; four phenolic substances accumulated at a high level in the T1 treatment group: naringenin, p-hydroxycinnamic acid, sinapic acid, and abscisic acid; six phenolic substances accumulated at a high level in the T2 treatment group: isoquercitrin, rutin, protocatechuic acid, syringic acid, vanillic acid, and L-phenylalanine; and two phenolic substances accumulated at a high level in the T3 treatment group: caffeic acid, naringin, and rosmarinic acid.

[0165] (4) Principal component analysis of phenolic acid compounds

[0166] Principal component analysis (PCA) was performed based on metabolomics data of 11 phenolic acid compounds. Data were standardized using SPSS software (Z-score method). The KOM test and Bartlett's test of sphericity were used to test the applicability of the analysis. The KOM value was 0.791, and the Sig value was <0.001, indicating a high correlation between the variables and statistical significance suitable for PCA. Using an eigenvalue >1.0 as the criterion, two principal components were extracted, with a cumulative variance contribution rate of 78.02%, meaning the extracted components can represent most of the information. The first principal component had an eigenvalue of 5.902 and the highest variance contribution rate (53.65%), with its main determinants being syringic acid, p-hydroxycinnamic acid, abscisic acid, cinnamic acid, vanillic acid, and sinapic acid. The second principal component had an eigenvalue of 2.681 and a variance contribution rate of 24.37%, with its main determinants being rosmarinic acid and caffeic acid. The eigenvectors and loadings of the 11 phenolic acid compounds are shown in Table 8.

[0167] Table 8. Eigenvectors and loadings of principal component analysis.

[0168]

[0169] Let X1 represent syringic acid, X2 represent p-hydroxycinnamic acid, X3 represent abscisic acid, X4 represent cinnamic acid, X5 represent vanillic acid, X6 represent sinapic acid, X7 represent benzoic acid, X8 represent ferulic acid, X9 represent rosmarinic acid, and X... 10 Indicates gentianic acid, X 11 Let F1 and F2 represent the scores of the compound in the two principal components, and F represent the overall score. The principal component function expression is obtained by using the eigenvectors as weights, as follows:

[0170] F1=0.394X1+0.373X2+0.347X3+0.333X4+0.324X5+0.324X6-0.303X7-0.247X8+0.0955X9+0.201X 10 -0.247X 11

[0171] F2=0.084X1-0.195X2-0.188X3+0.277X4+0.307X5-0.203X6-0.373X7-0.036X8+0.511X9-0.406X 10 +0.373X 11

[0172] F=F1×53.653%+F2×24.37%

[0173] The principal components and comprehensive scores of phenolic acid compounds in the root, stem, and leaf treatments of Acanthopanax senticosus were calculated and are shown in Table 9. The results showed that the F1 score and comprehensive score of Acanthopanax senticosus leaves were both positive, indicating that there was a relatively high accumulation of phenolic acid compounds in the leaves. Among them, the T3 treatment group had the highest score and the greatest promoting effect on the accumulation of phenolic acid compounds. The F1 score and comprehensive score of Acanthopanax senticosus stems were both negative, indicating that there was a relatively low accumulation of phenolic acid compounds in the stems. Among them, the T1 treatment group had the lowest score and the least promoting effect on the accumulation of phenolic acid compounds. The F1 score and comprehensive score of Acanthopanax senticosus roots were both positive, indicating that there was a relatively high accumulation of phenolic acid compounds in the leaves. Among them, the T3 treatment group had the highest score and the greatest promoting effect on the accumulation of phenolic acid compounds.

[0174] Table 9 Principal Component Scores and Overall Ranking

[0175]

[0176]

[0177] 5. Discussion

[0178] Metabolism is a series of chemical reactions occurring within organisms, converting photosynthetic products or ingested nutrients into substances essential for life activities. It is an indispensable pathway for plant survival and growth, and metabolic products have a significant impact on plant growth and development. This invention comprehensively analyzes the changes in primary metabolism of Acanthopanax senticosus plants after BVOCs treatment using a GC-MS platform. Principal component analysis (PCA) and partial least squares analysis (PLS-DA) were used to identify differentially expressed compounds under different BVOCs treatments. Based on their classification, function, and metabolic pathway, the changes in primary metabolism of Acanthopanax senticosus under different BVOCs treatments were analyzed. The results showed that there were 56 significantly different metabolic compounds in Acanthopanax senticosus plants (VIP > 1, P < 0.05), indicating that the composition of primary metabolites of Acanthopanax senticosus was significantly different after BVOCs treatment. Among them, organic acids accounted for the largest proportion, including 16 organic acids, among which malic acid and limonene were the most abundant. Acids are key intermediates in the tricarboxylic acid cycle, directly participating in and accelerating its operation, promoting cellular respiration, and providing energy for cells through oxidative metabolism. Organic acids are precursors for the synthesis of secondary metabolites such as fatty acids and flavonoids, and also participate in the decomposition and metabolism of carbohydrates, lipids, and amino acids. This indicates that BVOCs treatment promotes the primary metabolism of Acanthopanax senticosus compared to the control group. In addition, organic acids secreted by plant stems and leaves participate in plant photosynthesis and respiration, and organic acids secreted by plant roots can affect the content of surrounding soil and mineral elements, suggesting that BVOCs promoting the accumulation of organic acids can enhance the adaptability of Acanthopanax senticosus to the environment.

[0179] Secondary metabolism is the process by which plants synthesize non-essential substances for life and store secondary metabolites. It plays an important role in the growth, development, and reproduction of plants. Phenolic compounds are an important class of secondary metabolites, including simple phenols, flavonoids, phenolic polymers, and quinones. They are widely found in higher plants and can help plants form substances that maintain their growth and resist adverse environments under stress. This invention used UPLC-MS to determine 28 phenolic metabolites, clarifying the effects of different BVOCs treatments on the accumulation of phenolic metabolites in Acanthopanax senticosus plants. The results showed that, compared with the treatment groups, the α-pinene + isoprene treatment group had a higher accumulation of L-phenylalanine in Acanthopanax senticosus leaves, the isoprene treatment group had a higher accumulation of L-phenylalanine in Acanthopanax senticosus stems, and the α-pinene treatment group had a higher accumulation of L-phenylalanine in Acanthopanax senticosus roots. L-phenylalanine is a key precursor in the synthesis of secondary compounds such as lignin, coumarins, alkaloids, flavonoids, and isoflavones, playing an important role in various biosynthetic pathways. This indicates that BVOCs treatment affected the phenylpropanoid metabolic pathway in Acanthopanax senticosus plants, enhancing the plant's resistance to stress. Phenolic acids exist in plant tissues in bound or free states, affecting enzyme synthesis and function, and ultimately influencing plant growth and development. They are among the most active chemical substances in plants. This invention uses cluster analysis combined with principal component analysis to analyze the changes in phenolic acid content in Acanthopanax senticosus plants under different BVOCs treatments. Two principal components were extracted using an eigenvalue > 1.0 as the standard, with a cumulative variance contribution rate of 78.02%. Principal component 1 was mainly evaluated by the content of syringic acid and p-hydroxycinnamic acid, while principal component 2 was mainly evaluated by the content of rosmarinic acid and caffeic acid. The comprehensive score results show that the α-pinene + isoprene treatment group had the most significant accumulation of syringic acid and p-hydroxycinnamic acid in Acanthopanax senticosus leaves, and the α-pinene + isoprene treatment group had the most significant accumulation of rosmarinic acid and caffeic acid in Acanthopanax senticosus roots. This indicates that BVOCs treatment is beneficial to the accumulation of phenolic acids in Acanthopanax senticosus leaves and roots.

[0180] 6. Summary

[0181] (1) Analysis showed that Acanthopanax senticosus plants treated with BVOCs had 56 significantly different primary metabolites, among which organic acids accounted for the largest proportion, followed by sugars and amino acids. Metabolic pathway enrichment analysis showed that the main differential metabolic pathways were glycine, serine and threonine metabolism and galactose metabolism.

[0182] (2) 28 phenolic compounds were obtained by UPLC-MS analysis. Compared with the control group, the phenolic compound content in Acanthopanax senticosus leaves was the highest in the T3 treatment group, while the phenolic compound content in Acanthopanax senticosus stems and roots was the highest in the T2 treatment group. The L-phenylalanine content in Acanthopanax senticosus roots increased significantly after BVOCs treatment. Further principal component analysis of phenolic acid compounds showed that BVOCs treatment had a significant promoting effect on the accumulation of phenolic acid compounds in Acanthopanax senticosus roots and leaves.

[0183] III. Effects of BVOCs on Soil Nutrients and Bacterial Community Structure of Acanthopanax senticosus

[0184] 1. Materials and Methods

[0185] Using Acanthopanax senticosus treated in Example 1 as material, healthy plant roots were vertically dug along the base of Acanthopanax senticosus. Non-rhizosphere soil was collected by shaking and placed in a 45°C oven until it was dried to constant weight for soil physicochemical factor determination. The soil was collected into sterile self-sealing bags. Soil about 2 mm thick from the Acanthopanax senticosus roots was collected with a sterile brush as rhizosphere soil. The rhizosphere soil from 3 Acanthopanax senticosus plants was mixed in equal amounts and placed into sterile plastic-sealed bags. The bags were then quickly placed in an ultra-low temperature freezer at -80°C for storage and used for rhizosphere soil bacterial genome extraction.

[0186] 2. Experimental Methods

[0187] (1) Determination of soil physicochemical properties

[0188] Soil physicochemical parameters were determined according to the "Methods for Soil Agricultural Chemical Analysis" edited by Lu Rukun (1999). Soil pH was determined using the water immersion potential method (soil mass / water volume ratio of 1:5), and soil conductivity was determined using a DDS-307 conductivity meter (Shanghai Leici). Organic carbon was determined using the potassium dichromate external heating method; total nitrogen was determined using the semi-micro Kjeldahl method; total phosphorus was determined using the NaOH fusion-molybdenum-antimony colorimetric method; alkaline nitrogen was determined using the alkaline diffusion method; ammonium nitrogen was determined using the Nessler colorimetric method; nitrate nitrogen was determined using the phenol disulfonic acid method; and available phosphorus was determined using the sodium bicarbonate extraction-molybdenum-antimony colorimetric method.

[0189] (2) Sequencing methods

[0190] We commissioned MicroBio (Shanghai) Co., Ltd. to extract rhizosphere soil DNA using the MO-BIO PowerSoilDNAIsolation Kit and performed high-throughput sequencing on the V4-V5 region of 16S RNA.

[0191] 3. Data processing methods

[0192] Data analysis was performed using SPSS 22.0 and Excel 2010 software. One-way ANOVA was used to test the significance of differences between treatment groups (P<0.05), and charts were created using Excel software. OUT clustering was performed using USEARCH (8.1.1861) and Mothur (1.39.5), and graphs and index calculations were performed using R (3.6.0) language.

[0193] 4. Results Analysis

[0194] 4.1 Effects of BVOCs treatment on the physicochemical properties of Acanthopanax senticosus soil

[0195] (1) Soil pH and electrical conductivity of Acanthopanax senticosus plants

[0196] Depend on Figure 19 The results showed that the pH range of rhizosphere soil under different BVOCs treatments was 5.18±0.01~5.46±0.02, and the pH range of non-rhizosphere soil was 5.19±0.01~5.21±0.005, indicating that the soil was generally weakly acidic. The pH of rhizosphere soil in the CK treatment group was significantly higher than that in the VOC treatment group (P<0.05), while there was no significant difference in non-rhizosphere soil among the treatment groups (P>0.05). The electrical conductivity of non-rhizosphere soil was significantly higher than that of rhizosphere soil. The electrical conductivity of non-rhizosphere soil was the highest in the CK and T1 treatment groups, and the lowest in the T2 treatment group. Compared with the non-rhizosphere soil in the CK treatment group, the electrical conductivity of non-rhizosphere soil in the T3 treatment group decreased by 21.66%, and compared with the rhizosphere soil in the CK treatment group, the electrical conductivity of rhizosphere soil in the T2 treatment group decreased by 13.73%.

[0197] (2) Soil organic carbon, total nitrogen, total phosphorus and total potassium content of Acanthopanax senticosus plants

[0198] Depend on Figure 20 It can be seen that the organic carbon content in the rhizosphere soil was the highest in the T3 treatment group, which was 12.78±0.11 g·kg. -1 Compared with the CK treatment group, the rhizosphere soil organic carbon content increased by 16.4%, while the non-rhizosphere soil organic carbon content was the highest in the CK treatment group, at 12.4 ± 0.54 g·kg⁻¹. -1 Compared with the control group, the non-rhizosphere soil organic carbon content of treatment groups T1, T2, and T3 decreased by 15.3%, 13.3%, and 12.5%, respectively; the total nitrogen content of rhizosphere soil was highest in treatment group T3, at 7.86 ± 0.03 g·kg⁻¹. -1 Compared with the CK treatment group, the total nitrogen content in the rhizosphere soil increased by 17.4%. The total nitrogen content in the non-rhizosphere soil was the highest in the T1 treatment group, at 0.55 ± 0.04 g·kg⁻¹. -1 Compared with the CK treatment group, the total phosphorus content in the non-rhizosphere soil increased by 7.9%; the total phosphorus content in the rhizosphere soil was highest in the T3 treatment group, at 1.19 ± 0.006 g·kg⁻¹. -1Compared with the CK treatment group, the total phosphorus content in the rhizosphere soil increased by 19.3%. The total phosphorus content in the non-rhizosphere soil was the highest in the CK treatment group, at 1.23 ± 0.01 g·kg⁻¹. -1 Compared with the control group, the total phosphorus content in the non-rhizosphere soil of treatment groups T1, T2, and T3 decreased by 4.8%, 12.1%, and 7.3%, respectively; the total potassium content in the rhizosphere soil was highest in the control group, at 18.98 ± 0.46 g·kg⁻¹. -1 Compared with the CK treatment group, the total potassium content in the rhizosphere soil of the T1, T2 and T3 treatment groups decreased by 13.2%, 11.8% and 12.3%, respectively; the total potassium content in the non-rhizosphere soil was the highest in the T2 treatment group, which increased by 12.2% compared with the non-rhizosphere soil of the CK treatment group.

[0199] (3) Content of available nitrogen, nitrate nitrogen, ammonium nitrogen and available phosphorus in soil containing Acanthopanax senticosus plants

[0200] Depend on Figure 21 It was found that the content of available nitrogen in the rhizosphere soil was the highest in the T3 treatment group, which was 379.51±7.74 mg·Kg. -1 Compared with the CK treatment group, the rhizosphere soil showed a 4% increase. The alkaline nitrogen content in the non-rhizosphere soil was highest in the CK and T1 treatment groups, at 426.45±7.65 mg·Kg, respectively. -1 and 426.39±15.67 mg·Kg -1 The nitrate nitrogen content in the rhizosphere soil was highest in the control (CK) group, at 15.37 ± 0.47 mg·kg⁻¹. -1 Compared with the control group, the rhizosphere soil nitrate nitrogen content in treatment groups T1, T2, and T3 decreased by 21.6%, 10.1%, and 11.1%, respectively. The non-rhizosphere soil nitrate nitrogen content was highest in the control and T2 groups, at 32.58 ± 0.81 mg·Kg, respectively. -1 and 32.34±1.03mg·Kg -1 The contents of rhizosphere soil and ammonium nitrogen in rhizosphere soil were both highest in the T2 treatment group, at 74.32±3.64 mg·Kg, respectively. -1 and 96.52±4.48 mg·Kg -1 Compared with the control group, the rhizosphere soil ammonium nitrogen content increased by 71.3% and 19.9%, respectively; the rhizosphere soil available phosphorus content was highest in the T3 treatment group, at 4.69±0.15 mg·Kg. -1 Compared with the CK treatment group, the rhizosphere soil showed a 31.7% increase in available phosphorus. The non-rhizosphere soil had the highest available phosphorus content in the CK treatment group, at 6.4 ± 0.09 mg·Kg. -1 .

[0201] (4) Trace element content in soil of Acanthopanax senticosus plants

[0202] Table 10 shows that the mineral element content in the rhizosphere soil of Acanthopanax senticosus plants increased after BVOCs treatment compared with the CK treatment group. The potassium and calcium contents were highest in the T3 treatment group, at 2.91±0.06 g·kg⁻¹, respectively. -1 and 6.44±0.13g·Kg -1 Compared with the CK treatment group, the levels of magnesium and iron increased by 25.7% and 25.4%, respectively. The highest levels of magnesium and iron were found in the T2 treatment group, at 4.23 ± 0.09 g·kg⁻¹, respectively. -1 and 19.28±0.41g·Kg -1 Compared with the CK treatment group, the levels of manganese increased by 42.3% and 27%, respectively. The highest manganese content was found in the T3 treatment group, at 735.87 ± 15.68 mg·Kg. -1 Compared with the CK treatment group, the levels increased by 10.6%. The highest levels of zinc and copper were found in the T2 treatment group, at 66.64 ± 1.42 mg·Kg, respectively. -1 and 19.3±0.41mg·Kg -1 Compared with the CK treatment group, the levels increased by 42.5% and 36.2%, respectively. The sodium content was highest in the T1 treatment group, at 363.42 ± 7.74 mg·Kg. -1 The percentage increased by 30.4% compared to the CK treatment group.

[0203] Table 10. Micronutrient content in rhizosphere soil of Acanthopanax senticosus plants under different BVOCs treatments.

[0204]

[0205] Note: Lowercase letters in the same column indicate significant differences between different treatments (P<0.05).

[0206] As shown in Table 11, the non-rhizosphere soil manganese content of Acanthopanax senticosus plants was highest in the T1 treatment group, which was 763.4 ± 16.27 mg·Kg. -1 Compared with the CK treatment group, the content of zinc increased by 2.1%. The content of other mineral elements decreased after BVOCs treatment compared with the CK treatment group, with the lowest levels of all other elements observed in the T2 treatment group. Magnesium saw the largest decrease, at 29.4% compared to the CK treatment group, while manganese saw the smallest decrease, at 14.1%. The zinc content in the T1 treatment group was 42.83 ± 0.92 mg·Kg. -1 The reduction was the largest, decreasing by 15.1% compared to the CK treatment group, with a calcium content of 6 ± 0.13 g·kg⁻¹. -1 The reduction was the smallest, at 1.4% compared to the CK treatment group; the magnesium content in the T3 treatment group was 2.86 ± 0.06 g·kg⁻¹. -1The reduction was the largest, decreasing by 21.2% compared to the CK treatment group, with potassium content at 2.47 ± 0.05 g·kg⁻¹. -1 The reduction was the smallest, decreasing by 7.8% compared to the CK treatment group.

[0207] Table 11 Trace element content in non-rhizosphere soil of Acanthopanax senticosus under different BVOCs treatments

[0208]

[0209] Note: Lowercase letters in the same column indicate significant differences between different treatments (P<0.05).

[0210] 4.2 Effects of BVOCs treatment on the rhizosphere soil bacterial community of Acanthopanax senticosus

[0211] (1) Sequencing data of rhizosphere soil microorganisms of Acanthopanax senticosus

[0212] Table 12 shows that high-throughput sequencing of rhizosphere soil from Acanthopanax senticosus yielded 277,522 initial sequences and 245,389 valid sequences after quality control filtering, representing 0.87%–0.89% of the total number of sequences. The average sequence length was 373 nt, and the number of bases in the valid data ranged from 22,472,701 to 23,205,365 nt. The proportions of guanine (G) and cytosine (C) were 55.8%–56.3%. OTU clustering of the valid sequences at a 97% similarity level yielded 3,719 operational taxonomic units (OTUs).

[0213] Table 12 Sequencing data of rhizosphere soil microorganisms of Acanthopanax senticosus under different VOC treatments.

[0214]

[0215] (2) Dilution curves of rhizosphere soil microorganisms of Acanthopanax senticosus under different VOC treatments

[0216] The number of valid sequences was randomly selected as the x-axis, and the corresponding number of OTUs was used as the y-axis to plot the dilution curves of OTU taxonomic units in the rhizosphere soil of Acanthopanax senticosus under different BVOCs treatments, as shown below. Figure 22As shown in the figure, each curve represents a sample. The total sequencing volume of the sample is around 60,000 sequences. When the number of sequences reaches 20,000, the growth of the number of OTUs gradually slows down, and the end of the curve tends to flatten. This indicates that the sequencing data volume has reached the requirement of reflecting the majority of microbial diversity information of the sample. The T2 treatment group has the largest number of OTUs under the same number of sequences, indicating that the T2 treatment group has the most species, that is, the highest species richness. The species richness of the other treatment groups from high to low is T1>CK>T3. The high-throughput sequencing of Acanthopanax senticosus rhizosphere soil yielded 277,522 initial sequences and 245,389 valid sequences after quality control filtering, representing 0.87%–0.89% of the total. The average sequence length was 373 nt, and the effective data ranged from 22,472,701 to 23,205,365 nt in base count. The proportions of guanine (G) and cytosine (C) were 55.8%–56.3%. OTU clustering of the valid sequences at a 97% similarity level yielded 3,719 operational taxonomic units (OTUs).

[0217] Table 13 Sequencing data of rhizosphere soil microorganisms of Acanthopanax senticosus under different VOC treatments.

[0218]

[0219] (3) Rank-abundance curve of rhizosphere soil microorganisms of Acanthopanax senticosus

[0220] The number of sequences corresponding to each OTU in each sample was counted, and the OUT level was used as the x-axis. The ratio of the number of sequences corresponding to that OTU to the total number of sequences was used as the y-axis. Rank-abundance curves of bacterial diversity in the rhizosphere soil of Acanthopanax senticosus under different BVOCs treatments were plotted as follows: Figure 23 As shown, when the OTU level is between 0 and 500, the relative abundance of species in each sample is relatively consistent. When the OTU level is higher than 500, the curve of the T2 treatment group is flatter than other treatment groups, and the species distribution uniformity is the highest. The curves of the CK and T1 treatment groups are similar in flatness, and the species distribution uniformity is relatively consistent. The species distribution uniformity of the T3 treatment group is the lowest. The T2 treatment group has the largest distance in the horizontal axis direction and the highest relative abundance of species. The species abundance of other treatment groups from high to low is T1>CK>T3.

[0221] (4) Composition of rhizosphere soil microbial community of Acanthopanax senticosus

[0222] The statistical results of non-repetitive sequence OTU clustering at each taxonomic level are shown in Table 14. The taxonomic range of rhizosphere soil microorganisms in the Acanthopanax senticosus treatment group is 34-42 phyla, 41-45 classes, 77-81 orders, 120-128 families, 178-220 genera, and 212-240 species. Among them, the CK and T2 treatment groups had the most microbial composition, followed by the T1 treatment group, while the T3 treatment group had the fewest microbial composition. The species ranking of each treatment group at the phylum level is: T2 = CK > T1 > T3; the species ranking of microorganisms at the genus level is: T2 > T1 > T3 > CK. From the two taxonomic levels of phylum and genus, it can be seen that the T2 treatment group has the richest microbial species, while the T3 treatment group has a lower richness of rhizosphere microbial species.

[0223] Table 14. Microbial composition of rhizosphere soil of Acanthopanax senticosus under different BVOCs treatments.

[0224]

[0225]

[0226] (5) Relative abundance of rhizosphere soil microbial community of Acanthopanax senticosus

[0227] Based on taxonomic analysis of the rhizosphere soil microbial community composition under different BVOCs treatments, species with abundance less than 1% were grouped into "others," such as... Figure 24As shown, the relative abundance of rhizosphere microorganisms in *Eleutherococcus senticosus* varied under different BVOC treatments. Proteobacteria were the dominant phylum in the rhizosphere soil, accounting for 46.3% of all soil microorganisms, with the highest relative abundance in the T2 treatment group and the lowest in the CK treatment group. Acidobacteria accounted for 21.2% of all soil microorganisms, with a significantly decreased relative abundance in the T2 treatment group (P<0.05). Gemmatimonadetes accounted for 8.8% of all soil microorganisms, with a significantly increased relative abundance in the T1 and T3 treatment groups (P<0.05). The proportions of other soil microorganisms were as follows: Chloroflexi (5.9%), Actinobacteria (4.3%), and Bacteroides (4.3%). The phyla *Isomycetes* accounted for 38.2%, *Planctomycetes* for 2.2%, *Verrucomicrobia* for 1.3%, *Elusimicrobia* for 1.2%, and *Armatimonadetes* for 0.8%. These 10 phyla accounted for 96% of the total rhizosphere microorganisms. Among them, *Proteobacteria*, *Acidobacteria*, and *Bacillus* had significantly higher relative abundances in the rhizosphere soil of *Eleutherococcus senticosus* than other microorganisms, and were the dominant microorganisms in the rhizosphere soil.

[0228] Based on taxonomic analysis of the rhizosphere soil microbial community composition under different BVOCs treatments, species with abundance less than 1% were grouped into "others," such as... Figure 25As shown, the relative abundance of rhizosphere microorganisms in Acanthopanax senticosus varied under different BVOCs treatments. Excluding unclassified genera, the most abundant genus in the rhizosphere soil of Acanthopanax senticosus was *Gemmatimonas*, accounting for 4.6% of all soil microorganisms. Its relative abundance was highest in treatment T3 and lowest in treatment T2. *Rhizomicrobium* accounted for 4.3% of all soil microorganisms, with a significantly lower relative abundance in treatment T2 (P<0.05). *Bryobacter* accounted for a significant portion of all soil microorganisms. The relative abundance of the rhizosphere microorganisms was 2.9% in the T3 treatment group, which was significantly lower (P<0.05). The proportions of other soil microorganisms were as follows: Candidatus_Solibacter 1.6%, Rhodanobacter 1.3%, Phenylobacterium 1.3%, Opitutus 1.1%, Haliangium 1.02%, and Bradyrhizobium 1.01%. These nine types of microorganisms accounted for 19.4% of the total rhizosphere microorganisms. Among them, Bacillus, Rhizopus, and Bryobacterium had significantly higher relative abundances in the rhizosphere soil of Acanthopanax senticosus than other microorganisms, and were the dominant microorganisms in the rhizosphere soil.

[0229] (6) Alpha diversity changes in the rhizosphere soil microbial community of Acanthopanax senticosus

[0230] The Alpha diversity index results of the rhizosphere soil microbial community of Acanthopanax senticosus under different BVOCs treatments are shown in Table 15. The coverage values ​​of each treatment group were all above 0.99, which can effectively obtain sample diversity information. The Sobs index, Chao index, and Ace index can reflect the community richness. The larger the values ​​of the Sobs index, Chao index, and Ace index, the more species there are. The Sobs index, Chao index, and Ace index were all the largest in the T2 treatment group. Compared with other treatment groups, the T2 treatment group had the most observed species and the highest species richness. The Shannon index can reflect community diversity. The larger the Shannon index, the higher the community diversity. The T2 treatment group had the largest Shannon index value. Compared with other treatment groups, the T2 treatment group had the most observed species and the highest community diversity.

[0231] (7) Changes in the diversity of βata microbial community in rhizosphere soil of Acanthopanax senticosus

[0232] Pairwise comparisons between treatment groups yielded Unifrac distance matrices, which were then used for principal coordinate analysis (PcoA) to reveal the similarities and differences in microbial evolution among the different treatment groups. Within the same treatment group, the closer the coordinate distances between replicate samples, the more similar the community composition within that group; conversely, the greater the coordinate distances between different treatment groups, the greater the differences in community composition between them. The PCoA analysis results are as follows: Figure 26 As shown, principal coordinate 1 (PC1) explains 41.2% of the variance, and principal coordinate 2 (PC2) explains 25.23% of the variance, with a combined contribution of 66.43%. The samples within each group are relatively concentrated, and the community composition within each group is similar. The coordinates of the CK treatment group are located on the negative axis of PC1, the coordinates of the T3 treatment group are located on the positive axis of PC2, and the coordinates of the T1 and T2 treatment groups are located in two quadrants respectively. Compared with the coordinates of the CK treatment group, the coordinates of the T1 treatment group are the farthest, indicating a larger change in the rhizosphere microbial community composition, while the coordinates of the T2 treatment group are the closest, indicating a smaller change in the rhizosphere microbial community composition.

[0233] (8) Analysis of LEfSe of rhizosphere soil microorganisms of Acanthopanax senticosus

[0234] Linear discriminant analysis (LDFse) can select and compare features across multiple groups. This analysis uses the Kruskal-Wallis rank-sum test to identify species with significant abundance differences between groups, combines this with the Wilcoxon rank-sum test for grouped comparisons, and employs linear discriminant analysis (LDA) to assess the influence of these differentially abundant species (LDAscore). Figure 27As shown, the data reveals differentially expressed species with an LDA score greater than the set value of 3.5, i.e., microorganisms with significant differences in abundance among different groups. The CK treatment group included 22 differentially expressed microorganisms, the T1 treatment group included 16, the T2 treatment group included 10, and the T3 treatment group included 5. This indicates significant differences in rhizosphere microorganisms under different BVOC treatments. The length of the bars in the bar chart represents the magnitude of the influence of significantly different species. One microbial group (Acidobacteriales) was significantly elevated in the rhizosphere soil of the CK treatment group (LDA > 4). Three microbial groups were significantly elevated in the rhizosphere soil of the T1 treatment group. The levels of LDA > 4 were high in the rhizosphere soil of the T2 treatment group, specifically Alphaproteobacteria at the class level, Actinobacteria and Chloroflexi at the phylum level. Two microbial groups were significantly elevated (LDA > 4) in the rhizosphere soil of the T3 treatment group, namely Proteobacteria at the phylum level and Betaproteobacteria at the class level.

[0235] 4.3 Correlation analysis between active components and relative abundance of Acanthopanax senticosus under BVOCs treatment

[0236] Table 15 shows the correlation coefficients among the contents of major active ingredients, photosynthetic pigments, alpha diversity index of bacterial communities in the rhizosphere soil of *Eleutherococcus senticosus* leaves, and the relative abundance of major bacteria at different phyla, classes, orders, families, genera, and species under different BVOCs treatments. The table shows that the content of eleutheroside B was significantly positively correlated with the relative abundance of Proteobacteria (P<0.01), the content of eleutheroside E was significantly positively correlated with the relative abundance of Proteobacteria and Bacillusaceae (P<0.05), the content of isozygoside was significantly positively correlated with the relative abundance of Proteobacteria and Rhizobium (P<0.01), and the content of hyperoside was significantly positively correlated with the alpha diversity index (P<0.05). The content of photosynthetic pigments was significantly positively correlated with the relative abundance of Proteobacteria (P<0.01).

[0237] Table 15 Correlation coefficients among the main active components, photosynthetic pigments, Alpha diversity index, and relative abundance of major bacteria in Acanthopanax senticosus under different BVOCs treatments.

[0238]

[0239] Note: * indicates a significant difference (P<0.05); ** indicates an extremely significant difference (P<0.01).

[0240] 5. Discussion

[0241] Mineral elements in soil are essential nutrients for plant growth and development, albeit in small quantities. They maintain soil homeostasis through a series of interactions with plant roots, influencing plant growth and reproduction in many ways. Nitrogen is an essential nutrient for plant growth and development and is also an important component of many compounds such as proteins, nucleic acids, enzymes, plant hormones, and alkaloids. Potassium promotes plant growth and development and significantly improves plant stress resistance. Phosphorus plays a crucial role in increasing crop yield and improving plant quality. The results of this invention show that, compared with the treatment group, the content of organic carbon, nitrogen, and phosphorus in the rhizosphere soil of Acanthopanax senticosus under BVOCs treatment was significantly increased, promoting the accumulation of organic carbon and nitrogen in the soil, thus having a beneficial effect on the growth of Acanthopanax senticosus and the reproduction of microorganisms.

[0242] The interactions of rhizosphere microorganisms influence plant growth and tolerance to biotic and abiotic stresses. Understanding their composition and interactions is crucial for improving plant productivity and ecosystem function. The results of this invention show that isoprene treatment significantly increased bacterial community richness and abundance. At the phylum level, the relative abundance of Proteobacteria increased, while the relative abundance of Acidobacteria decreased. This indicates that isoprene promoted the activity of Proteobacteria in the rhizosphere soil of *Eleutherococcus senticosus*. Proteobacteria have complex and diverse physiological and metabolic types, influencing soil ecological balance in multiple ways. They can increase soil fertility and promote plant growth by fixing nitrogen, thus isoprene can increase the abundance of certain bacterial species to promote plant growth. Monoterpenes and sesquiterpenes have multiple signal transduction functions in interspecific interactions. In forest cultivation models, the allelopathic effects of BVOCs are crucial for achieving sustainable forest resource development.

[0243] 6. Summary

[0244] (1) Compared with the control group, the contents of organic carbon, total nitrogen and total phosphorus in the rhizosphere soil of Acanthopanax senticosus increased significantly, while the contents of total potassium decreased significantly. The contents of organic carbon and total phosphorus in the non-rhizosphere soil decreased significantly, while the contents of total potassium increased significantly. The contents of alkaline nitrogen in the rhizosphere soil of Acanthopanax senticosus were the highest in the T3 treatment group.

[0245] (2) High-throughput sequencing results showed that the rhizosphere bacterial community of Acanthopanax senticosus could be classified into 48 phyla, 50 classes, 89 orders, 148 families, 292 genera, and 328 species. There were significant differences in the composition and relative abundance of the rhizosphere bacterial community of Acanthopanax senticosus among different treatment groups. Among the rhizosphere soil bacteria of Acanthopanax senticosus, Proteobacteria (46.31%), Acidobacteria (21.29%), Gemmatimonadetes (8.85%), and Chloroflexi (5.91%) were the main phylum groups, while Gemmatimonas (4.62%), Rhizomicrobium (4.37%), and Bryobacter (2.94%) were the main genus groups.

[0246] (3) Alpha diversity analysis showed that the bacterial community diversity was the highest in the T2 treatment group, while the bacterial community diversity was relatively low in the T3 treatment group; LDFse analysis showed that the Acidobacteriales were significantly different in the rhizosphere soil of the CK treatment group, Alphaproteobacteria in the T1 treatment group, Proteobacteria in the T2 treatment group, and uncultured Acidobacteriabacterium in the T3 treatment group.

[0247] IV. Conclusion

[0248] This invention measures and analyzes the photosynthetic characteristics, growth characteristics, physiological indicators, and content of major active ingredients in Acanthopanax senticosus leaves under BVOCs treatment. Combined with the metabolic characteristics of the whole plant of Acanthopanax senticosus, trace elements, rhizosphere soil physicochemical properties, and bacterial community structure, the effects of BVOCs on the growth and metabolism of Acanthopanax senticosus are preliminarily analyzed, thus laying a theoretical foundation for the ecological cultivation of Acanthopanax senticosus resources.

[0249] (1) Compared with the control group, the plant height of Acanthopanax senticosus in the isoprene treatment group was significantly increased, and the number of leaves in the α-pinene + isoprene treatment group was significantly increased; the photosynthetic rate and gas exchange parameters in the α-pinene + isoprene treatment group were significantly increased; the content of photosynthetic pigments was significantly increased after BVOCs treatment, and the content was the highest in the isoprene treatment group, indicating that isoprene can improve the photosynthetic rate of Acanthopanax senticosus; the activities of superoxide dismutase and ascorbic acid peroxidase were significantly increased; the contents of eleutheroside B and eleutheroside E were significantly increased, indicating that BVOCs treatment promoted the accumulation of the main active ingredients in Acanthopanax senticosus leaves.

[0250] (3) After BVOCs treatment, Acanthopanax senticosus plants had 56 differential primary metabolites, among which organic acids accounted for the largest proportion, followed by sugars and amino acids. Metabolic pathway enrichment analysis showed that the main differential metabolic pathways were glycine, serine and threonine metabolism and galactose metabolism. UPLC-MS analysis revealed 28 phenolic compounds. Compared with the control group, the phenolic compound content accumulated in Acanthopanax senticosus leaves in the α-pinene + isoprene treatment group was the highest, while the phenolic compound content accumulated in the stems and roots of Acanthopanax senticosus in the isoprene treatment group was the highest. This indicates that BVOCs treatment is beneficial to the accumulation of phenolic compounds in Acanthopanax senticosus plants.

[0251] (3) The content of trace elements in the non-rhizosphere soil of Acanthopanax senticosus was higher than that in the rhizosphere soil. After BVOCs treatment, the content of trace elements in the rhizosphere soil increased significantly, while the content of trace elements in the non-rhizosphere soil decreased significantly. Compared with the control group, the content of organic carbon, total nitrogen and total phosphorus in the rhizosphere soil increased significantly, while the content of total potassium decreased significantly, indicating that BVOCs treatment can promote the accumulation of organic carbon, nitrogen and phosphorus in the rhizosphere of Acanthopanax senticosus.

[0252] (4) Based on high-throughput sequencing analysis, the bacterial community structure of the rhizosphere soil of Acanthopanax senticosus was analyzed. The bacterial community composition in the rhizosphere soil of Acanthopanax senticosus included 48 phyla, 50 classes, 89 orders, 148 families, 292 genera, and 328 species. At the phylum level, Proteobacteria was the dominant species, and at the genus level, Gemmatimonas was the dominant species. The analysis results showed that there were significant differences in the composition of the rhizosphere bacterial community of Acanthopanax senticosus among different treatment groups. The relative abundance of bacteria and the Alpha diversity index of the bacterial community were higher in the isoprene treatment group. LDFse analysis results showed that Acidobacteriales were present in the rhizosphere soil of the control group, Alphaproteobacteria in the α-pinene treatment group, Proteobacteria in the isoprene treatment group, and uncultured Acidobacteria in the α-pinene + isoprene treatment group. bacterium) represents a significantly different bacterial species.

[0253] The above are merely preferred embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A cultivation method for promoting the growth and metabolism of Eleutherococcus senticosus, characterized by, The method comprises the following steps: The fumigation solution is prepared by using α-pinene and isoprene; The concentration of the fumigation solution is 0.26ppb; The concentration ratio of the α-pinene and isoprene is 1:1; The fumigation treatment time is 30 days.

2. The cultivation method of promoting growth and metabolism of Eleutherococcus senticosus according to claim 1, characterized in that, The Acanthopanax senticosus is two-year-old.

3. The cultivation method of promoting growth and metabolism of Eleutherococcus senticosus according to claim 1, characterized by, The Acanthopanax senticosus is routinely managed during the fumigation treatment.

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