Preliminary correlation analysis reveals the relationship between ginsenosides in multi stem ginseng and soil properties

Compared with single stem ginseng (SSG), there is less research on the natural formation and quality of multi stem ginseng (MSG). This research measured the ginsenoside content in the roots, stems, and leaves of 4-year-old SSG, double stem ginseng (DSG), and triple stem ginseng (TSG), as well as the endogenous hormone content in the rhizomes. At the same time, the physicochemical properties and enzyme activity of the rhizosphere and non-rhizosphere soil were measured, and the differences and connections between each indicator were analyzed. The results showed significant differences in the content of ginsenosides and endogenous hormones between SSG and MSG. TSG had the highest content of ginsenosides in roots, DSG had higher content of ginsenosides in leaves, and SSG had the highest content of salicylic acid and brassinolide in rhizomes than MSG. The activities of trace elements (Ce, Nd, Pr, La, Y, Tb, Fe, Mn) and phosphorus cycling enzymes (ribonuclease, acid phosphatase, phytase) in the rhizosphere soil of TSG were significantly highest. In addition, Pr, Nd, and Ce might affect the morphological construction of TSG by influencing the accumulation of brassinolide in TSG. Finally, we found that Ce, Pr, Zn, and β-amylase significantly affect the accumulation of ginsenosides. Overall, compared to SSG, MSG undoubtedly has higher medicinal and economic value, and soil rare earth elements may play a more critical role in the formation of MSG morphology and quality.

1 Introduction

The perennial herbaceous plant ginseng (Panax ginseng C. A. Mey) in the family Araliaceae is mainly distributed in northeastern China, North Korea, South Korea, Japan, and eastern Russia, and is widely used due to its extremely high medicinal value (1). Modern pharmacological research has shown that the main pharmacological effects of ginseng are anti-tumor, anti-aging, immune enhancement, anti-cancer, and protection of the central nervous system, cardiovascular system, digestive system, and immune system (2, 3). The pharmacological effects of ginseng can be explained by its chemical composition. Ginsenosides, polysaccharides, proteins, volatile oils, peptides, amino acids, and other substances are the main chemical components in ginseng roots, all of which have different pharmacological activities. Among them, ginsenosides are the most significant component of ginseng pharmacological activity (4–6). Therefore, many scholars focus their research on ginsenosides.

Soil is the foundation for the growth of ginseng, and the quality of soil can affect or even determine the quality of ginseng. Rhizosphere soil refers to the soil surrounded and influenced by plant roots. Compared with non-rhizosphere soil (NSG), rhizosphere soil has faster nutrient cycling and material exchange rates, and can better reflect the correlation between soil and plant roots (7, 8). Relatively speaking, NSG mainly serves as a “potential nutrient” reservoir for plants, and its nutrients need to be transformed and migrated in time and space before they can be absorbed and utilized by plants. Therefore, researchers usually have limited research on NSG (9).

Ginseng has extremely strict requirements for soil quality, and the air permeability, acidity, moisture, and nutrient content of soil should be within appropriate ranges. Excessive or insufficient levels can lead to a decrease in ginseng quality and yield (10). Specifically, according to the “Code of practice on good quality culture of ginseng” (GB/T 34789-2017), the pH of soil suitable for ginseng cultivation should be between 5.5-6.5, bulk density should be between 0.9-1.0 g/cm³, soil thickness should be at least 25 cm, organic matter content should be at least 3%, and nutrient supplementation should be based on the actual nutritional needs of ginseng at different ages. For example, we previously reported that 3-year-old ginseng cultivated in farmland achieved significant levels of nitrogen, boron, and silicon consumption in the soil (11). In addition to non-biological factors in soil, the activity of enzymes in soil is also a significant factor that cannot be ignored. Soil enzymes are biologically active proteins that play indispensable roles in material metabolism and nutrient cycling processes (12). Soil enzymes respond quickly to external disturbances, reflecting not only the intensity of biochemical reactions but also the fertility status of the soil. Therefore, soil enzymes are also used as indicators to evaluate soil quality (13).

Single stem ginseng (SSG) has only one rhizome and is the most common ginseng on the market. Multi stem ginseng (MSG) has at least 2 rhizomes and is usually less common. According to reports, the multi stem phenomenon of ginseng is caused by external stimuli (chemical factors or mechanical trauma). After being stimulated by external factors, the latent buds of ginseng rhizomes begin to sprout, resulting in the production of two or more rhizomes (14). MSG mainly appears in 3-year-old and 4-year-old ginseng, and the phenomenon of double stems is the most common (15). In addition, MSG has a larger photosynthetic area and higher dry matter accumulation than SSG due to the presence of more leaves. It is worth noting that the content of ginsenosides in the roots of MSG is significantly higher than that of SSG (15), indicating that MSG has higher medicinal and economic value.

Plant hormones are small molecule compounds in plants that regulate almost all life activities such as dormancy, reproduction, and secondary metabolite synthesis (16). Correspondingly, plants can also respond to varying degrees of external environmental stimuli and promote the synthesis of secondary metabolites to resist environmental stress by regulating plant hormones such as salicylic acid and abscisic acid (17). Zou (18) reported that compared with SSG, the transcription factor BZR1 in the brassinosteroid signaling pathway of double stem ginseng (DSG) was significantly upregulated, indicating that BZR1 played a key role in the production of MSG. Similarly, Zhao et al. (19) also reported significant differential expression of significant genes 90B/724B in the biosynthesis pathway of brassinosteroids in SSG and DSG.

At present, some studies have reported differences in endogenous hormones (19), ginsenoside content, and photosynthesis (15) between SSG and DSG, but there have been no reports on the quality of triple stem ginseng. In addition, the differences in rhizosphere soil properties of ginseng with different stem numbers and the impact of rhizosphere soil properties on MSG quality formation are not yet clear. Therefore, this study measured the content of ginsenosides in the roots, stems, and leaves of 4-year-old SSG, DSG, and TSG, as well as the endogenous hormone content in the rhizomes. In addition, we also measured the physicochemical properties and enzyme activity of rhizosphere and non-rhizosphere soil of SSG, DSG, and TSG. The purpose of this study is to 1) investigate the differences in ginsenosides and endogenous hormone content of ginseng with different stem numbers; 2) clarify the differences in rhizosphere and non-rhizosphere soil properties of ginseng with different stem numbers; 3) explore the main soil factors that lead to differences in the quality of ginseng with different stem numbers. The results contributed to a deeper understanding of the application value of MSG and provided a preliminary theoretical basis for subsequent analysis of MSG rhizosphere soil microorganisms, soil metabolism, soil antibiotic resistance genes, etc.

2 Materials and methods

2.1 Experimental station description

The research area is located in Zhujiabao, Qingyishan Town, Kuandian Manchu Autonomous County, Dandong City, Liaoning Province, China (124.63’E, 40.75’N), the area belongs to the Dwa type in the Köppen climate classification, and with an altitude of 305.12 m. It belongs to the temperate continental monsoon climate, with distinct four seasons, warm winters and cool summers, and abundant sunshine as its main characteristics. The annual average temperature is 6.5°C, the annual effective accumulated temperature is 3000°C, the annual average precipitation is 1100 millimeters, mostly concentrated from June to August, the annual average humidity is 70%, and the average frost free period is 140 days per year (20).

2.2 Collection of ginseng and soil samples

With the aim of comprehensively observe the morphology of MSG, ginseng and soil samples were collected during the red fruit final period of ginseng growth (September 1, 2024). Specifically, a total of 6 sets of replicates were designed, with 6 randomly selected ginseng fields, each with an area of approximately 60 m² (2 m×30 m). 6–7 SSGs (Figure 1A), DSGs (DSG, Figure 1B), and TSGs (TSG, Figure 1C) were collected from each ginseng field using the “S” sampling method as one set of replicates. A total of 41 SSG, 43 DSG, and 37 TSG strains were collected. Took 3 ginseng roots separately for each repetition, washed the surface soil with deionized water, cut off the rhizome parts, and put them into a liquid nitrogen tank filled with liquid nitrogen and brought it back to the laboratory. Ginseng without rhizome was not used for the analysis of ginsenoside content.

Figure 1

Figure 1. Single stem ginseng (A), double stem ginseng (B), and three stem ginseng (C).

Used the root-shaking method to collect rhizosphere soil from the surface of ginseng roots, as well as NSG (21). The collection method of NSG was to use the “S” sampling method in each ginseng field to search for and collect soil within a radius of 50–100 cm without ginseng growth (0–15 cm soil layer). Mixed well and used it as a set of replicates. A total of 6 replicate samples was collected in 6 ginseng fields.

The ginseng and soil were separated and individually labelled, then placed in sterile self-sealing bags and taken back to the laboratory in a freezer with an ice pack. After washing the soil on the surface of ginseng with deionized water, the repeated roots, stems, and leaves of each group were separated and air-dried naturally. The dried stems, leaves, and roots of ginseng were ground in a grinder and passed through a 0.18 mm sieve for the analysis of ginsenoside content. Soil was divided into two parts: the first part was naturally air dried, then ground, and analyzed through corresponding sieves according to the requirements of soil property determination regulations; the other part was stored in a refrigerator at 4°C for future use.

2.3 Determination of ginsenoside content

According to the method for determining ginsenosides determined by the research group in the early stage (11), the ginsenoside content of roots, stems, and leaves of SSG, DSG, and TSG was measured. The content of ginsenosides Rg1, Re, Ro, Rb1, Rb2, Rb3, Rf, Rc, Rd, Rg3 (R-type), and Rh2 (R-type) were determined by Thermo Ultimate 3000 High-performance liquid chromatography. The chromatographic column was Elite Hypersil ODS2 (250 mm × 4.6 mm, 5 μm). The composition of the mobile phase, gradient elution program, column temperature, injection volume, flow rate, and detection wavelength of the VWD detector were presented in Supplementary Table S1.

The determination of total ginsenosides (TG) in ginseng was carried out according to the reference standard method (GB/T 18765-2015). The content of ginsenosides was measured in three replicates, and the average was taken as the final result.

2.4 Determination of endogenous hormones in ginseng

Took the rhizomes from “2.2.” in a liquid nitrogen tank, ground them into powder using liquid nitrogen, and referred to the method described in the “National Drug Standard Draft for Determination of Plant Growth Regulator Residues (China)”. The content of anti-zeaxanthin, 6-benzylaminopurine, brassinolide, jasmonic acid, abscisic acid, gibberellin, 3-indoleacetic acid, and salicylic acid were determined by Agilent 1290–6470 LC-MS/MS. The chromatographic column was Agilent ZORBAX Eclipse Plus C18 (2.1 × 100mm, 1.8 μm). The composition of the mobile phase, gradient elution program, column temperature, injection volume, flow rate, and mass spectrometry conditions were presented in Supplementary Table S1. The content of endogenous hormones was measured in three replicates, and the average was taken as the final result.

2.5 Determination of soil physicochemical properties

According to standard methods (22, 23), soil alkaline hydrolyzable nitrogen (A-N), available phosphorus (A-P), available potassium (A-K), ammonium nitrogen (NH₄⁺-N), nitrate nitrogen (NO₃^--N), available silicon (A-Si), available sulfur (A-S), available boron (A-B), complexed iron (C-Fe), bicarbonate ions (HCO₃^-), magnesium ions (Mg²⁺), calcium ions (Ca²⁺), inorganic phosphorus (IP), organic matter (OM), conductivity (EC), exchangeable hydrogen ions (E-H⁺), exchangeable aluminum ions (E-Al³⁺), exchangeable total acids (E-TA), bulk density (BD), mass moisture content (MMC), field moisture capacity (FMC), pH, cation exchange capacity (CEC), sulfate ions (SO₄^2-), and chloride ions (Cl^-) were determined. The specific measurement methods were presented in Supplementary Table S1.

To better clarify the potential nutrient supply capacity of SSG and MSG in rhizosphere and non-rhizosphere soil, this study also measured the content of total elements in the soil. The content of total boron (T-B) was determined according to GB/T 3653.1-2024, the content of total manganese (T-Mn), total titanium (T-Ti), total calcium (T-Ca), total magnesium (T-Mg), total iron (T-Fe), total aluminum (T-Al), and total silicon (T-Si) was determined according to HJ 974-2018. The content of total zinc (T-Zn) was determined according to HJ 491-2019, and the determination of total europium (T-Eu), total lanthanum (T-La), total cerium (T-Ce), total praseodymium (T-Pr), total neodymium (T-Nd), total samarium (T-Sm), total gadolinium (T-Gd), total terbium (T-Tb), total dysprosium (T-Dy), total holmium (T-Ho), total erbium (T-Er), total thulium (T-Tm), total ytterbium (T-Yb), total lutetium (T-Lu), and total yttrium (T-Y) content referred to GB/T 18115.6-2023.

2.6 Determination of soil enzyme activity

According to the standard method (24), the activity of soil dehydrogenases (DHA), amylases (AMY), α-amylases (α-AMY), β-amylases (β-AMY), invertases (Inv), cellulases (Cel), uricases (UR), polyphenol oxidase (PPO), urease (Ure), nitrate reductase (NR), arylsulfatase (ASF), acid phosphatase (ACP), acid protease (Prot), ribonuclease (NUC), hydroxylamine reductase (HR), phytase (Phy), catalase (CAT), glutaminase (GLS), L-asparaginase (ASP), and peroxidase (POD) were measured. Soil physicochemical properties and enzyme activity were measured for three replicates, and the average value was taken as the final result.

2.7 Data processing and statistical analysis

Used Microsoft Excel 2020 to organize the data, and then analyzed the data using SPSS 26.0 software. Firstly, the Shapiro-Wilk normality test was conducted. Then, one way analysis of variance (ANOVA) and Duncan’s multiple range test were performed on data that conformed to the normal distribution, while Dunnett’s T3 test was performed on data that did not conform to the normal distribution, with p < 0.05 as the criterion for significant differences. The correlation analysis based on Spearman and Pearson test was also conducted in SPSS 26.0 software, the difference was considered statistically significant when the adjusted p < 0.05 after false discovery rate (FDR) correction for the initial calculated p value. Performed random forest analysis using R (version 4.1.3) and the “RandomForest” package (versions 4.7-1.1), and visualized it in the ggplot2 package (version 3.4.0). Performed principal component analysis (PCA) using the vegan package (v2.5.4) in R (v4.1.3), and visualized it in the ggplot2 package (v3.3.3). The visualization of data and analysis results was performed in GraphPad Prism 9.5.0 and SigmaPlot 10.0.

3 Results

3.1 Appearance and ginsenoside content

According to Figures 2A–C, MSG exhibited obvious multi stem phenomenon. According to the results of the ginsenoside content in the roots, stems, and leaves (Figures 2d–f), there were significant differences in the content of ginsenosides among the three parts of SSG, DSG, and TSG.

Figure 2

Figure 2. The apparent morphology of MSG and the content of ginsenosides in different parts (n=6). (a) SSG; (b) DSG; (c) TSG. The content of ginsenosides in roots (d), stems (e), and leaves (f). Different lowercase letters above the same indicator bar chart indicate significant differences (p<0.05, Duncan’s multiple range test). The results indicate mean ± standard deviation (SD).

In the roots (Figure 2D), the content of ginsenosides Ro, Rg1, Re, Rb1, Rc, Rb2, Rb3, Rh2, Rg3, and TG in TSG were significantly higher than those in SSG and DSG (p < 0.05). The content of Ro (0.57 mg/g), Rg3 (0.58 mg/g), and TG (37.91 mg/g) in TSG differed the most from SSG and DSG, being 4.1 times (Ro), 4.0 times (Rg3), and 2.1 times (TG) of SSG, and 2.7 times (Ro), 3.3 times (Rg3), and 1.5 times (TG) of DSG, respectively.

In the stems (Figure 2E), the content of most types of ginsenosides (Rg1, Re, Rb1, Rc, Rb2, Rd, Rh2, TG) was also the highest in TSG (p < 0.05). Noticeable, the content of ginsenoside Rg3 in SSG was significantly higher than that in DSG and TSG.

In the leaves (Figure 2F), DSG showed an incredible phenomenon, as all types of ginsenosides were significantly higher in DSG than in SSG and TSG (p < 0.05). In addition, there was not a significant difference in the content of ginsenosides between SSG and TSG. Specifically, the content of ginsenosides Ro, Rg1, and Rb1 in SSG was significantly higher than that in TSG, while the content of ginsenosides Rd, Rg3, and TG in TSG was significantly higher than that in SSG. However, there was no significant difference in the content of most ginsenosides (Re, Rf, Rc, Rb2, Rb3, Rh2) between the two samples (p < 0.05).

3.2 Endogenous hormone content

The content of 8 endogenous hormones in the rhizome was determined (Table 1), and the content of anti-zeaxanthin, gibberellin, 6-benzylaminopurine, abscisic acid, and jasmonic acid were all below the minimum detection limit. Therefore, the content of salicylic acid, 3-indoleacetic acid, and brassinolide was mainly analyzed. The content of salicylic acid and brassinolide in SSG was significantly higher than that in DSG and TSG (p < 0.05), while the content of 3-indoleacetic acid showed no significant difference among the three samples (p < 0.05). In addition, the brassinolide content of DSG was significantly higher than that of TSG, and there was no significant difference in the content of salicylic acid between the two samples.

Table 1

Table 1. Content of endogenous hormones in ginseng rhizomes (n=6).

3.3 Soil physicochemical properties

There were significant differences in the physicochemical properties of rhizosphere soil between SSG, DSG, and TSG (Figure 3, Supplementary Table S2). In the conventional soil physicochemical properties shown in Figure 3, the content of pH, Cl^-, A-Si, NH₄⁺-N, A-B, E-H⁺, CEC, Ca²⁺, and Mg²⁺ in SSG were significantly higher than those in other groups. The content of BD in DSG was significantly higher than those in other groups, and the content of E-TA and E-Al³⁺ in TSG were significantly higher than those in other groups. However, the content of EC, FWC, A-N, A-P, A-K, IP, SO₄^2-, and NO₃^--N in NSG were significantly higher than those in other groups (p < 0.05).

Figure 3

Figure 3. Physicochemical properties of different soil samples (n=6). Different lowercase letters above the same indicator bar chart indicate significant differences (p<0.05, Duncan’s multiple range test). The results indicate mean ± standard deviation (SD).

In the results of the total element content (Figure 3), the content of T-Zn and T-Al in DSG were significantly higher than those in other groups. The content of T-Mn, T-Fe, T-Tb, T-La, T-Pr, T-Y, T-Nd, and T-Ce in TSG were significantly higher than those in other groups, while the content of T-Ti, T-Ca, and T-Si in NSG were significantly higher than those in other groups (p < 0.05).

3.4 Soil enzyme activity

There were significant differences in rhizosphere soil enzyme activities among SSG, DSG, and TSG (Figure 4, Supplementary Table S2). SSG had significantly higher activities of AMY, α-AMY, β-AMY, Ure, Cel, PPO, GLS, and ASP than other groups. The activity of NR in DSG was significantly higher than other groups, while the activity of Inv, ACP, NUC, POD, Phy, ASF, and HR were significantly higher in TSG (p < 0.05). In addition, unlike soil physicochemical properties, there was no enzyme activity in NSG that was significantly higher than MSG and SSG.

Figure 4

Figure 4. Enzyme activity of different soil samples (n=6). Different lowercase letters above the same indicator bar chart indicate significant differences (p<0.05, Duncan’s multiple range test). The results indicate mean ± standard deviation (SD).

3.5 Ternary plot analysis

The ternary plot analysis revealed the distribution patterns of different factors in the soil and tissues of ginseng with different stem numbers. The results showed that NSG had the highest nutrient proportion in soil physicochemical properties, while DSG had the lowest nutrient proportion (Figure 5A). In terms of soil enzyme activity, the higher enzyme activity proportion was mainly concentrated in SSG and TSG (Figure 5B). In the roots (Figure 5C) and stems (Figure 5D) of ginseng, TSG was enriched with higher abundance of ginsenosides; in the leaves, DSG enriched a higher proportion of ginsenoside content (Figure 5E).

Figure 5

Figure 5. Ternary plot displaying the abundance of indicators for different soil and ginseng samples (n=6). (a) Soil physicochemical properties; (b) soil enzyme activity; (c) endogenous hormones and ginsenosides content in roots; ginsenosides content in stems (d) and leaves (e).

3.6 Principal component analysis

With the aim of clarifying the differences between soil properties and ginseng quality, principal component analysis (PCA) was conducted. The PCA results of the physicochemical properties (Figure 6A) and enzyme activity (Figure 6B) of rhizosphere and non-rhizosphere soil showed that SSG, DSG, TSG, and NSG were separated from each other, indicating significant differences between them.

Figure 6

Figure 6. Principal component analysis (PCA) of rhizosphere and non-rhizosphere soil properties, endogenous hormones, and ginsenosides from different parts (n=6). (a) Soil physicochemical properties; (b) Soil enzyme activity; (c) endogenous hormones; (d) the roots, stems, and leaves of ginseng.

The PCA results of the measured endogenous hormone content (Figure 6C) showed that SSG, DSG, and TSG were separated separately, but DSG had a higher similarity with TSG.

PCA results based on ginsenoside content were performed on the roots, stems, and leaves of ginseng (Figure 6D), showing significant separation of TSG from SSG and DSG in the roots, while SSG and DSG were not well distinguished. The differences between SSG, DSG, and TSG in the stem were minimal. The situation in leaves was completely different from that in roots, where DSG was significantly separated from SSG and TSG, while SSG and TSG were not well distinguished.

3.7 Random forest analysis

The results of PCA had confirmed significant differences among samples from different groups, but the characteristic factors that cause these differences were not yet clear. Here, we used random forest analysis to identify the characteristic factors of these differences (Figure 7). In soil physicochemical properties, T-Ce, NH₄⁺-N, T-Pr, T-Si, and T-Nd were characteristic factors of differences between different samples (Figure 7A). In soil enzyme activity, HR, POD, Inv, ASP, and Ure were characteristic factors of differences between different samples (Figure 7B).

Figure 7

Figure 7. Random forest analysis of different parts of soil and ginseng (n=6). (a) Soil physicochemical properties; (b) soil enzyme activity properties; endogenous hormones in the rhizomes (c) and ginsenosides content in the roots (d), stems (e), and leaves (f) of ginseng.

Brassinolide was a characteristic factor for the differences in endogenous hormones of ginseng among different samples (Figure 7C). Ginsenosides Rb2, TG, Rb2, Rg1, and Rb3 were characteristic factors for the quality differences of ginseng roots in different samples (Figure 7D). Ginsenosides Rd, Rg3, and Rh2 were characteristic factors for the quality differences of ginseng stems in different samples (Figure 7E), and ginsenosides Rd, Rg3, TG, Rf, and Ro were characteristic factors for the quality differences of ginseng leaves in different samples (Figure 7F).

3.8 Correlation analysis

To clarify the relationship between soil factors and ginsenoside content, we conducted a correlation analysis based on Spearman test to analyze the correlation between ginsenosides, endogenous hormones, and soil properties (Figures 8, 9, 10). We had measured many soil indicators with the main purpose of comprehensively reflecting the impact of soil abiotic factors and enzyme activity on the quality formation of MSG. According to PCA, we found significant differences in soil properties and ginseng quality between SSG and MSG; based on random forest analysis, we identified the main factors causing these differences. Therefore, we speculate that the characteristic factors of soil property differences may be the main factors leading to differences in ginseng quality, and the characteristic factors of differences in ginsenoside content are also the main manifestation of differences in ginseng quality. Therefore, we focus our attention on some characteristic factors in the results of the “3.7 Random forest analysis”. The main purpose of this analysis is to clarify certain connections between the characteristic factors of soil property differences and the characteristic factors of ginsenoside content differences. After comprehensive consideration, we mainly focus on the relationship between characteristic soil physicochemical properties (top 10) and ginsenosides (top 3) in random forest analysis.

Figure 8

Figure 8. Spearman test-based correlation analysis between soil properties and the content of ginsenosides and endogenous hormones in ginseng roots (n=6). (a) and (d): SSG; (b) and (e): DSG; (c) and (f): TSG. Only results with p < 0.05 are displayed (FDR correction), with the red line representing positive correlation and the blue line representing negative correlation.

Figure 9

Figure 9. Spearman test-based correlation analysis between soil properties and the content of ginsenosides in ginseng stems (n=6). (a) and (d): SSG; (b) and (e): DSG; (c) and (f): TSG. Only results with p < 0.05 are displayed (FDR correction), with the red line representing positive correlation and the gray line representing negative correlation.

Figure 10

Figure 10. Spearman test-based correlation analysis between soil properties and the content of ginsenosides in ginseng leaves (n=6). (a, d) SSG; (b, e) DSG; (c, f) TSG. Only results with p < 0.05 are displayed (FDR correction), with the red line representing positive correlation and the green line representing negative correlation.

Firstly, we analyzed the correlation between soil properties and the content of ginsenosides in ginseng roots and endogenous hormones in rhizomes (Figure 8, Supplementary Table S3). In SSG (Figure 8A), T-Zn, T-Pr were significantly correlated with TG, and T-Ce was significantly positively correlated with Rg1 (p < 0.05). In DSG (Figure 8B), IP, T-Zn, and Rb2 were significantly correlated, while pH was significantly positively correlated with Rg1 (p < 0.05). In TSG (Figure 8C), IP was significantly negatively correlated with Rb2, while T-Pr, T-Ce, T-Nd were significantly negatively correlated with brassinolide (p < 0.05). In the correlation analysis between enzyme activity and ginsenosides, the content of ginsenosides in SSG was significantly correlated with AMY and α-AMY (Figure 8D), the content of ginsenosides in DSG was significantly correlated with HR and β-AMY (Figure 8E), and the content of ginsenosides in TSG was significantly correlated with β-AMY, ACP, and Prot (Figure 8F).

Subsequently, we analyzed the correlation between soil properties and the content of ginsenosides in ginseng stems (Figure 9, Supplementary Table S4). In SSG (Figure 9A), NH₄⁺-N, T-Pr, IP, T-Nd, pH, T-Zn were significantly associated with characteristic ginsenosides (Rd, Rg3, Rh2). In DSG (Figure 9B), T-Si, T-La, T-Pr were significantly associated with characteristic ginsenosides (Rd, Rg3). In TSG (Figure 9c), T-Si, T-La, T-Pr, T-Zn were significantly associated with characteristic ginsenosides (Rh2, Rg3). In the correlation analysis between enzyme activity and ginsenosides, the content of ginsenosides in SSG was significantly correlated with AMY, β-AMY, ACP, and Prot (Figure 9D), the content of ginsenosides in DSG was significantly correlated with β-AMY (Figure 9E), and the content of ginsenosides in TSG was significantly correlated with ASF (Figure 9F).

Finally, we analyzed the correlation between soil properties and the content of ginsenosides in ginseng leaves (Figure 10, Supplementary Table S5). In SSG (Figure 10A), T-Ce and T-Pr were significantly associated with characteristic ginsenosides (Rd, Rg3). In DSG (Figure 10B), IP, pH, and T-Zn were significantly associated with characteristic ginsenosides (Rd, Rg3). In TSG (Figure 10C), NH₄⁺-N, T-Pr, T-Ce, T-Nd, T-Si, T-La, and T-Zn were significantly associated with characteristic ginsenosides (Rd, Rg3, TG). In the correlation analysis between enzyme activity and ginsenosides, the content of ginsenosides in SSG was significantly correlated with Ure and GLS (Figure 10D), the content of ginsenosides in DSG was significantly correlated with DHA and β-AMY (Figure 10E), and the content of ginsenosides in TSG was significantly correlated with β-AMY (Figure 10f).

4 Discussion

4.1 Ginsenoside content of ginseng with different stem numbers

Ginsenosides are the most active chemical substances in the pharmacological activity of ginseng, in this study, the content of ginsenosides was used as the evaluation standard for ginseng quality. The root is the traditional medicinal site of ginseng, and we found that the content of most ginsenosides in the roots of TSG was significantly higher (Figure 2D). The results of PCA also showed that the roots of TSG were significantly separated from SSG and DSG (Figure 6C). Although the roots of SSG and DSG were not well separated in PCA (Figure 6C), most types of ginsenosides in DSG (Ro, Rg1, Re, Rc, Rb2, Rb3, Rd, TG) were still significantly higher than those in SSG, although the difference was not significant (Figure 2D), the results are consistent with previous reports (14). Overall, the medicinal value was TSG > DSG > SSG. The aboveground part of ginseng (mainly the leaves) is the main site of photosynthesis, which helps to accumulate substances in various parts of ginseng (25). TSG has the most leaves, which may be the main reason why the roots of TSG have a higher content of ginsenosides.

July and August are usually the harvest period for ginseng seeds, while September is usually the traditional harvest period for ginseng roots, because September is the period when the aboveground parts of ginseng wither, and the content of ginsenosides in the roots is usually the highest of the year (26). However, some scholars had reported that MSG was different from SSG. Zhang et al. (15) measured the content of ginsenosides in ginseng at different growth periods at 3 and 4 years old. The results showed that the highest content of DSG, TSG, ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1, and TG at 3-year-old and 4-year-old all occurred during the red fruit period. Due to the limited research on MSG by previous scholars, it is necessary to collect more MSG samples from different times and locations for analysis to ensure the reliability of the results.

Although leaves are not a traditional medicinal part of ginseng, the Pharmacopoeia of the People’s Republic of China has already included ginseng leaves as medicinal, so analyzing the content of active ingredients in ginseng leaves also has practical significance. According to PCA (Figure 6C), the leaves of DSG were significantly separated from SSG and TSG, and the results of ginsenoside content in the leaves also showed that the content of all types of ginsenosides in DSG was significantly higher than that in SSG and TSG (Figure 2F). One possible reason is that there is a large overlap in the leaves of TSG, which leads to the inability to maximize photosynthesis. Many leaves that can perform photosynthesis are wasted, resulting in a small difference in ginsenoside content between DSG and SSG (15). However, due to the presence of a large number of leaves, the economic value of TSG is still high in terms of quantity alone.

4.2 Endogenous hormone content of ginseng with different stem numbers

Artificial induction and natural production are the two main reasons for the formation of MSG. Artificial induction mainly involves removing it from the base when the ginseng bud grows to the size of sorghum grains (27); the excessive content of nutrients in soil and favorable ecological and climatic conditions are the natural causes of MSG production, and at this time, the endogenous hormone content of MSG will also undergo significant changes compared to SSG (19). According to local farmers, MSG is a non-human induced phenomenon in the sampling area of this study, and DSG has the highest quantity. Therefore, the exploration of soil properties and endogenous hormone content in this study is meaningful.

The results of showed that the levels of salicylic acid and brassinolide in SSG were significantly higher than those in DSG and TSG, while there was no significant difference in the content of 3-indoleacetic acid among the three samples (Table 1). However, the results of PCA showed a significant separation between MSG and SSG (Figure 6C). The results were similar to those of Zhao et al. (19), who reported that during the early period of overwintering bud formation in ginseng (early July), the 3-indoleacetic acid content in the rhizomes of 4-year-old DSG was significantly lower than that of SSG, and the difference in 3-indoleacetic acid content between DSG and SSG became larger with time. In addition, they also reported that low concentrations of 3-indoleacetic acid contributed to the production of MSG compared to high concentrations. This research found that compared to SSG, MSG had lower concentrations of salicylic acid and brassinolide. Salicylic acid has been reported to have multiple effects, including regulating plant development, promoting plant growth, ion absorption and transport, and root growth (28, 29). Brassinolide can regulate the growth of nutrient organs, participate in regulating the elongation of plant stems, regulating the growth of lateral roots and root hairs, vascular bundle differentiation, and seed germination (30, 31). Furthermore, hormones (salicylic acid, abscisic acid, 3-Indoleacetic acid, etc.) act as inducers to indirectly activate the secondary metabolism of ginseng (32), upregulate the expression of ginsenoside synthase related gene promoters, increase the accumulation of ginsenosides, and thereby enhance the pharmacological activity of ginseng (33). In addition to activating secondary metabolism, these hormones are also significantly associated with plant epigenetic traits, such as enhancing root vitality, promoting cell elongation, division, and differentiation (34). Although there is currently no clear evidence to prove the role of salicylic acid and brassinolide in inducing multi stem phenomenon in ginseng, we speculate that endogenous hormones, rare earth elements, or both jointly promote the formation of MSG, which provides a feasible direction for subsequent research.

4.3 Relationship between the physicochemical properties of rhizosphere soil and the quality of ginseng

With the aim of comprehensively analyzing the differences in rhizosphere soil physicochemical properties between MSG and SSG, we analyzed as many soil physicochemical properties as possible. There were significant differences in soil abiotic factors between SSG, MSG, and NSG (Figures 3, 6A). Through random forest analysis (Figure 7A), we found that the characteristic factors of these differences mainly came from rare earth elements (Ce, Pr, Nd, La) and trace elements (Si, Zn). In addition, NH₄⁺-N, IP, and pH were the conventional factors for these differences. The correlation analysis results showed that these characteristic factors were significantly correlated with ginsenosides (Figures 8, 9, 10).

The soil conditions are closely related to the supply of mineral nutrients, water, and gas to plants. The results of this study also indicate that there are significant differences in the physicochemical properties and enzyme activities of ginseng rhizosphere soil with different stem numbers, which may be related to the nutrient absorption and utilization of multi stem ginseng. Chen et al. (35) reported significant differences in the physicochemical properties and enzyme activity of rhizosphere soil between 3-year-old MSG and SSG, with the main feature being that MSG has significantly higher enzyme activity, which may be a necessary factor in maintaining MSG nutrient requirements. In this study, the enzyme activity ratio of DSG was significantly lower than that of other groups (Figure 5), which may be directly related to the lower nutrient supply of DSG. Therefore, the content of ginsenosides in the roots and stems of DSG was significantly lower than that of SSG and TSG, while the content of some ginsenosides in the leaves was less limited by soil nutrients due to the direct influence of photosynthesis (36).

Abiotic factors in soil can directly or indirectly affect the accumulation of ginsenosides. For example, Xu et al. (11) reported a significant correlation between pH and the accumulation of ginsenosides Rb2, Rb3, Rc, Rd, Rg1, and Rg3 in 3-year-old ginseng, and a significant correlation between A-P and the accumulation of ginsenosides Rc, Rg1, Rg3, and Rh2 (p < 0.05). Regarding nitrogen, scholars had reported a significant negative correlation between NH₄⁺-N and the accumulation of ginsenoside Rh2 in 2-year-old, 3-year-old, and 4-year-old ginseng (21); Meanwhile, the addition of nitrogen fertilizer significantly increased the content of notoginsenoside R1 an ginsenosides (Rg1, Re, Rb1, Rg2, Rh1, Rd, and Rg3) in the main and fibrous roots of Panax notoginseng, a plant belonging to the same genus as Panax ginseng (37). Trace elements have a role in affecting the levels of secondary metabolites in plants such as carrots, onions, and potatoes (38). Although IP, T-Ce, T-Pr, T-Nd, T-La, T-Si, and T-Zn are non available nutrients in soil, belonging to the potential “nutrient pool” of plants and soil microorganisms (39), soil microorganisms can convert these non-available nutrients into available nutrients that plants can directly absorb and utilize (40, 41), so these nutrients may indirectly participate in the accumulation of ginsenosides. For example, it is generally believed that ginseng grown in forests has the best quality because forest soil has abundant mineral elements that can meet the various needs of ginseng growth. Zhu et al. (42) analyzed the correlation between the content of ginsenosides in forest cultivated ginseng and the mineral element content in rhizosphere soil. Although elements such as barium (Ba), aluminum (Al), iron (Fe), nickel (Ni), and chromium (Cr) were significantly associated with ginsenosides, comprehensive analysis showed that lead (Pb), tin (Sn), and strontium (Sr) were the main mineral elements affecting the accumulation of ginsenosides (Rb1, Rb2, Rb3, Rc). Although there is limited research on the promoting role of rare earth elements in the formation of ginseng quality, we found that Ce-induced nanoenzyme materials have been reported to significantly improve the rhizosphere microbiota of ginseng and promote sustainable production of ginseng (43). Similarly, T-Pr and T-La have been reported as characteristic factors for the differences in rhizosphere soil between SSG and MSG at the age of 3 (35), which are similar to our results on the potential role of rare earth elements in MSG quality and morphology formation.

In the correlation analysis between soil properties and ginseng chemical composition (Supplementary Table S6), we found that T-Zn, T-Pr, and IP were significantly correlated with the characteristic ginsenoside content of ginseng roots, stems, and leaves. Similarly, T-Pr and T-Nd specifically affected the accumulation of ginsenosides in MSG. Although these soil factors may have a potential promoting effect on the quality formation of MSG, their potential role in the morphological formation of MSG is still unclear. We observed a significant correlation between T-Pr, T-Nd, and T-Ce and brassinolide in TSG (Figure 8F). According to Chen et al. (35), the content of brassinolide in SSG was significantly higher than that in MSG. Our study is consistent with them, indicating that high levels of brassinolide are not conducive to the multi stem phenomenon of ginseng, which may indirectly explain why T-Pr, T-Nd, T-Ce have a negative correlation with brassinolide. Based on the role of brassinolide in regulating plant morphological changes, we speculate that these three rare earth elements may indirectly promote the formation of TSG, and we believe that future research can focus on verifying this hypothesis.

4.4 Relationship between the enzyme activity of rhizosphere soil and the quality of ginseng

Soil enzymes are bioactive substances in soil that react quickly to external disturbances and are significant indicators for evaluating soil fertility and environmental quality (13). The results showed that the activities of enzymes related to the carbon cycle (AMY, α-AMY, β-AMY, Cel) and nitrogen cycle (Ure, GLS, ASP) were significantly higher in SSG; the significantly higher enzyme activity in DSG was NR, which belonged to the nitrogen cycling enzyme; although there were significantly higher activity carbon (Inv) and nitrogen (HR) cycling enzymes in TSG, the activities of phosphorus (ACP, NUC, Phy) and sulfur (ASF) cycling enzymes were characteristic and significantly highest in TSG. Higher carbon, nitrogen, phosphorus, and sulfur cycling enzyme activity reflects the efficiency of microbial energy and nutrient acquisition, reflecting the demand of microorganisms for soil carbon substrates and nitrogen, phosphorus, and sulfur nutrients (44, 45). Specific high phosphorus and sulfur cycling enzyme activity suggest that this may be related to the formation or quality differences of TSG, which may be related to the decomposition of IP and the effectiveness of phosphorus.

The results of the correlation analysis (Figures 8–10) showed a significant correlation between β-AMY and the accumulation of ginsenosides with different stem numbers, which has been reported in previous studies. β-AMY is an essential enzyme in the formation of the basic carbon skeleton structure of ginsenosides (46). Different types of microorganisms in the soil can produce β-AMY, such as Kitasatospora sp. MK-1785 (47), Corallococcus sp. EGB (48), Bacillus subtilis, Delftia acidovorans, and Bacillus polymyxae (49). There may not be a direct or indirect link between β-AMY and the formation of MSG, as we did not find a significant association between β-AMY and characteristic endogenous hormones in the results of the correlation analysis. Anyway, our research results indicate a feasible direction for future validation experiments. In addition to β-AMY, AMY and α-AMY may also have potential effects on the accumulation of ginsenosides (Figures 8, 9, 10). It is feasible to add these enzymes from different microbial sources to the soil to explore their effects on ginseng growth.

The relationship between soil enzymes and soil microorganisms is closely related. In previous studies (11, 21), we had reported a close relationship between certain microbial genera and ginsenosides in ginseng grown in farmland, such as the significant correlation between the relative abundance of fungal genera Coprinellus, Agaricales_uncassified, and Mortierella and the content of ginsenosides (Rb2, Rb3, Rd, Re), and the significant correlation between the relative abundance of Tomentera, Ganderma, and Exophiala and the content of ginsenosides (Rb3, Rc, Re, TG). Like soil enzymes, soil microorganisms are biological indicators for measuring soil fertility and soil health, and are known as the “second genome of plants” (50). Therefore, we believe that there must be an undeniable connection between the soil microbiome and MSG formation. As this study mainly focused on the association between soil abiotic factors and MSG, we suggest that future research still needs to focus on analyzing the relationship between the microbiome and MSG, and combining soil abiotic factors to construct a synergistic regulatory network of “soil abiotic factors-MSG-soil biotic factors” (26), so as to better understand the natural formation laws of MSG and improve the economic value of ginseng.

5 Conclusion

The results indicated that in addition to significant differences in appearance, the significant differences in ginsenoside content between roots and leaves of SSG, DSG, and TSG were also essential manifestations of their quality differences. Among them, the roots of TSG had the highest ginsenoside content, while the leaves of DSG had the highest ginsenoside content, indicating that MSG had higher medicinal value than SSG. In rhizosphere soil, TSG had significantly higher levels of trace elements (T-Ce, T-Mn, T-Fe, T-Tb, T-La, T-Pr, T-Y, T-Nd) and activities of phosphorus (ACP, NUC, Phy) and sulfur (ASF) cycling enzymes. The analysis of differences indicate that T-Ce, NH₄⁺-N, and T-Pr are characteristic factors for the differences in soil abiotic factors among SSG, MSG, and NSG, while HR, POD, and Inv are characteristic factors for the differences in soil enzyme activity among SSG, MSG, and NSG. Furthermore, we found that T-Pr, T-Nd, and T-Ce had a closer association with brassinolide in TSG, and brassinolide is related to the morphology of MSG, indicating that there may be some connection between these three, which provides a feasible direction for future research. In summary, the results indicate that T-Ce, T-Pr, T-Zn, and β-AMY are potential influencing factors in the quality formation of ginseng, while T-Nd, T-Si, and T-La may have a greater impact on the quality of MSG. Our research provides good guidance for subsequent confirmatory experiments and the cultivation of MSG.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Author contributions

ZX: Formal Analysis, Writing – original draft, Visualization. YC: Methodology, Writing – original draft, Conceptualization. YX: Writing – review & editing. KZ: Writing – review & editing. RY: Formal Analysis, Writing – review & editing. YW: Visualization, Investigation, Writing – review & editing. JS: Writing – review & editing, Formal Analysis. YS: Software, Writing – review & editing. JF: Visualization, Writing – review & editing, Investigation. QZ: Writing – review & editing, Software. CC: Funding acquisition, Project administration, Writing – review & editing. TZ: Writing – review & editing, Funding acquisition, Project administration.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This work was supported by Jilin Province Science and Technology Development Plan Project (No. 20250101032JJ), the National Natural Science Foundation of China Youth Foundation Program (No. 82204556).

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer QG declared a shared affiliation with the author(s) to the handling editor at the time of review.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fsoil.2025.1733495/full#supplementary-material

Glossary

A-N: Soil alkali hydrolyzable nitrogen

A-P: Soil available phosphorus

A-K: Soil available potassium

NH₄⁺-N: Soil ammonium nitrogen

NO₃^—N: Soil nitrate nitrogen

A-Si: Soil available silicon

A-S: Soil available sulfur

A-B: Soil available boron

C-Fe: Soil complex iron

HCO₃^-: Soil bicarbonate ion

Mg²⁺: Soil magnesium ions

Ca²⁺: Soil calcium ions

IP: Soil inorganic phosphorus

OM: Soil organic matter

EC: Soil electrical conductivity

E-H⁺: Soil exchangeable hydrogen ion

E-TA: Soil exchangeable total acid

E-Al³⁺: Soil exchangeable aluminum ion

BD: Soil bulk density

FWC: Soil field moisture capacity

MWC: Soil mass moisture content

CEC: Soil cation exchange capacity

SO₄^2-: Soil sulfate ion;Cl^-, Soil chloride ion

T-B: Soil total boron

T-Mn: Soil total manganese

T-Ti: Soil total titanium

T-Ca: Soil total calcium

T-Mg: Soil total magnesium

T-Fe: Soil total iron

T-Al: Soil total aluminum

T-Si: Soil total silicon

T-Zn: Soil total zinc

T-Eu: Soil total europium

T-La: Soil total lanthanum

T-Ce: Soil total cerium

T-Pr: Soil total praseodymium

T-Nd: Soil total neodymium

T-Sm: Soil total samarium

T-Gd: Soil total gadolinium

T-Tb: Soil total terbium

T-Dy: Soil total dysprosium

T-Ho: Soil total holmium

T-Er: Soil total erbium

T-Tm: Soil total thulium

T-Yb: Soil total ytterbium

T-Lu: Soil total lutetium

T-Y: Soil total yttrium

DHA: Soil dehydrogenase

AMY: Soil amylase

α-AMY: Soil α-amylase

β-AMY: Soil β-amylase

Inv: Soil invertase

Cel: Soil cellulose

PPO: Soil polyphenol oxidase

UR: Soil uricase

ASF: Soil arylsulfatase

Ure: Soil urease

NR: Soil nitrate reductase

POD: Soil peroxidase

ACP: Soil acid phosphatase

Prot: Soil acid protease

NUC: Soil ribonuclease

HR: Soil hydroxylamine reductase

Phy: Soil phytase

CAT: Soil catalase

GLS: Soil glutaminase

ASP: Soil L-asparaginase

TG: total ginsenosides in ginseng.

Footnotes

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