Mycorrhizal Symbiosis in Forest-Grown American ginseng (Panax quinquefolius) and the Relationship Between Mycorrhizal Colonization and Root Ginsenoside Content

Filyaw, Tanner R. and Sarah C. Davis. Environmental Studies, Ohio University, OH.
tanner@ruralaction.org, tf287901@ohio.edu, daviss6@ohio.edu

(Presented at The Future of Ginseng and Forest Botanicals Symposium, July 12-14, 2017, Morgantown, WV)

Abstract

American ginseng (Panax quinquefolius L.) is a valuable medicinal plant that has been harvested from the forests of eastern North America for over 300 years, and commercially cultivated since the late 1800’s. Arbuscular Mycorrhizal Fungi (AMF) are symbiotic soil organisms that colonize plant roots, and often contribute to enhanced growth by increasing the uptake of water and nutrients. The role of AMF in the production of American ginseng has become a topic of increasing interest, but forest-based research on this subject is limited. This study quantified AMF colonization in six-year-old forest-grown ginseng roots, resolved the relationship between AMF colonization and root ginsenoside content, and identified species of AMF present in forest production sites. Roots from four production sites were measured for AMF colonization, and ginsenosides Rg1, Re, Rb1, Rc, Rb2, and Rd were quantified by High Performance Liquid Chromatography (HPLC). AMF spores were extracted from soil samples by wet-sieving, and identified morphologically. Results indicate that AMF colonization varied significantly between sites (p < 0.05), but no significant differences in ginsenoside content were resolved between sites (p = 0.104). Furthermore, ginsenoside content was determined to not be significantly influenced by AMF colonization (p = 0.0823). Significant inverse relationships between AMF colonization and Rg1 (p = 9.826e-05) were detected, and there was a positive correlation between AMF colonization and Re (p = 0.007). Due to high spore degradation, Rhizophagus intraradices (formerly Glomus intraradices) was the only species of AMF identified between production sites.

Introduction

American ginseng is a long-lived herbaceous perennial herb belonging to the Araliaceae family that is typically found growing in the deeply shaded understory of mature hardwood forests (Chandler & McGraw, 2015), ranging from southern Canada to northern Georgia, and west to states along the Mississippi River (Burkhart, 2013). Ginseng is highly valued as a medicinal plant species and has been harvested from the forests of eastern North America for over 300 years (Burkhart, 2013). Concerns about overharvesting, loss of natural habitats, and observed declines in wild populations have increased the need and demand for high-quality wild-simulated (WS) roots that are intentionally produced on private forestlands. WS roots are grown with the goal of producing a root that is wild in appearance, and is virtually indistinguishable from truly wild ginseng, thus enabling producers to capture premium prices typically paid for wild roots (Carroll & Apsley, 2013).

Symbiotic mycorrhizal fungi have the potential to improve the health, productivity, and quality of commercially produced medicinal plants. Mycorrhizal fungi are symbiotic soil organisms that form partnerships with the root systems of approximately 80% of all terrestrial plant species (Whigham, 2004). Mycorrhizae function as an extension of the plant root systems and facilitate the uptake of water and nutrients, particularly phosphorus and nitrogen, in exchange for a supply of carbohydrates (e.g. glucose) (Hodge et al., 2010). Ginseng, as well as most herbaceous plant species, form partnerships with Arbuscular Mycorrhizal Fungi (AMF), a class of endo-mycorrhizal species that penetrate and colonize the internal cortex of fibrous secondary and tertiary roots (McGonigle et al., 1999). Previously observed benefits of mycorrhizae in the production of ginseng roots include increased yields and biomass production (Li, 1995), and enhanced production of secondary metabolites (e.g. ginsenosides) that are attributed to increased medicinal potency (Zeng et al., 2013; Fournier et al., 2003).

Ginsenosides are chemically classified as triterpenoid saponins and are considered the major active constituents of American ginseng. More than 60 unique compounds have been isolated from the roots, shoots, leaves, flower buds, and berries, with additional novel ginsenoside compounds found to be produced through metabolic processes and biotransformation (Qi et al., 2011). Ginsenosides typically account for 3-6 % of total root mass (Robbins, 1998), with ginsenosides Rg1, Re, Rb1, Rc, Rb2, and Rd being the six most abundant by weight (Lim et al., 2005). Previous research has also shown that ginsenoside concentrations are typically correlated with root mass (Robbins, 1998; Smith et al., 1996) and plant age (Court et al. 1996), with higher concentrations in larger and older roots.

Thus far, studies examining mycorrhizal symbiosis in WS ginseng have been under-represented relative to more widely researched production methods (e.g. field cultivation). Growing conditions in these production systems are significantly different from those required to produce ginseng roots with wild characteristics (Carroll & Apsley, 2004). Key differences in habitat conditions, production practices, and harvest cycles raise questions about how ginseng-mycorrhizae interactions may differ in WS ginseng. Research sites and sources of root material for this study represent the variation in habitat conditions found within the central portion of the natural range for American ginseng, and the diversity of production practices currently used in the forest-farming community to produce “wild-simulated” ginseng.

Methods

Objectives
  • To quantify rates of mycorrhizal colonization observed in WS ginseng roots.
  • To determine if there is a statistical correlation between mycorrhizal colonization and root ginsenoside concentrations.
  • To characterize the community of mycorrhizal species present in forested ginseng production sites.
  • To determine the infectivity potential of forest soils at each production site.
Study Sites

Roots were collected from four commercial ginseng production sites located in Ohio, Pennsylvania, and Maryland. These sites are representative of two commonly used WS production systems that vary in scale, intensity of production, and cultivation/management practices (Table 1). Sites A and B are distinguished from sites C and D by four main differences: (1) the use of moderate soil tillage to prepare planting sites, (2) the removal of competitive understory vegetation prior to planting, (3) higher planting density, and (4) the application of fungicides as needed to prevent and control disease during the cropping cycle.

Table 1. Description of sampling site habitat characteristics and management practices

Root and Soil Sampling

Sixty six-year-old roots were collected using a randomized sampling design (15 roots per site). Each sampling unit was represented by a 1.5 m x 6 m grid, and roots were harvested using randomly selected coordinates. Roots were harvested between August 27, 2016 and September 10, 2016 when mycorrhizal colonization (Whitebread et al., 1996) and ginsenoside concentrations are typically at peak levels (Li et al, 1996). After harvesting, roots were gently washed, then weighed in order to resolve relationships between AMF colonization, ginsenoside concentrations, and root mass. To quantify mycorrhizal colonization the fibrous roots were removed from the rhizome, weighed, and half of the fibrous root mass was randomly selected for mycorrhizal analysis. The remaining fibrous roots were dried with the tuberous portion of the root for ginsenoside analysis. Ten soil samples were randomly collected from each sampling grid for mean infectivity assays, and AMF spore identification. Samples were collected at a depth of 10 cm where AMF spore density is greatest (Egerton-Warburton & Allen, 2000).

Mycorrhizal Analysis: Root Clearing and Staining

The fibrous roots selected for mycorrhizal analysis were cut into 2 cm segments (McGonigle et al., 1999), placed in labeled tissue cassettes and cleared (e.g. cellular contents removed) in pre-boiled 10% potassium hydroxide (KOH) for 30 minutes. Once cleared the tissue cassettes were rinsed in distilled water, and submerged in 2% hydrochloric acid (HCl) for 15 minutes prior to staining (INVAM, 2014). Tissue cassettes were then submerged in a pre-boiled solution of 0.05% direct blue histological stain (1:1:1 distilled water, glycerin, and lactic acid (v/v/v)) and soaked for 5-7 minutes (INVAM, 2014). Samples were then rinsed in distilled water to remove excess stain, and stored in distilled water until the colonization analysis was conducted (INVAM, 2014).

Measuring Mycorrhizal Colonization

The percentage of root length colonized by AMF was determined using the gridline-intersect method (Giovanetti & Mosse, 1980). Stained roots were placed in 10 cm diameter petri dish marked with a 1.3 x 1.3 centimeter grid, then examined using a dissecting microscope (Giovanetti & Mosse, 1980). The number of horizontal and vertical intersections where mycorrhizal structures were present were tallied, and the number of positive intersections was divided by the total number of intersections to determine percent colonization. Roots were quantified for Total Percent Colonization (TPC) by counting all mycorrhizal structures (e.g. internal and external hyphae, intra-radical spores, external spores, vesicles, and arbuscules), and for Percent Arbuscule Content (PAC), by solely counting intersections where arbuscules were present.

Ginsenoside Sample Preparation and Analysis

A subsample of 32 roots (8 from each site) were randomly selected for ginsenoside analysis. Ginsenoside concentrations were quantified using High Performance Liquid Chromatography (HPLC). Ginsenoside samples were prepared using a combination of methods previously described by Lim et al. (2005) and Corbit et al. (2005). Roots were dried at 35°C (95°F) in a forced-air dehydrator (Nesco Gardenmaster), then ground to powder. Extracts were prepared by combining 300 mg of root with 10 mL of 70% HPLC-grade methanol in 15 mL centrifuge tubes. Sample slurries were extracted in a water bath sonicator (Fischer Scientific, model FS20H) at 40°C for 30 minutes, centrifuged (Eppendorf Model 5810 R) for 2 minutes at 3500 rpms, and the supernatants collected. The pellet was re-extracted using the same process, and the supernatants were combined. Supernatants were roto-evaporated (IKAâ RV 10) to remove the methanol fraction, and the residues re-dissolved in 2 mL of 100% HPLC-grade methanol (Fischer Scientific, Pittsburgh PA). The 2 mL solution was lyophilized (Virtis Genesis 25 ES) to reduce to dryness, then dissolved in 500 µl of 73% acetonitrile and filtered with 0.02 µm nylon filters prior to HPLC injection (Lim et al., 2005; Corbit et al., 2005).

Extracts were analyzed against standards for ginsenosides Rg1, Re, Rb1, Rc, Rb2, and Rd (Indofine Chemical Co, Hillsborough NJ, and Sigma Aldrich, St. Louis MO), and were quantified based on peak height. Calibration curves were developed for each standard in concentrations ranging from 10 µl/ml – 2 mg/ml. Samples were analyzed with a Shimadzu Prominence HPLC system (Shimadzu Corporation, Kyoto Japan), with a LC-20AD pump, degasser, SIL-20AD HT autosampler, SPD-M20A diode array detector, and a Phenomenex C18 250 mm x 4.6 mm analytical column (5 µm pore size). The data was collected and analyzed with Shimadzu LC Solutions software. The mobile phase was a binary gradient of acetonitrile (A) and water (B) at a flow rate of 1.3 ml/min. The gradient was as follows: 0-15 min., 21% A; 16-38 min., 30% A; 39-55 min., 42% A; 56-65 min., 90% A; and 66-80 min., back to 21% A (Corbit et al., 2005).

Mean Infectivity Percentage and Species Identification

Mean Infectivity Percentage (MIP) was used to determine the infectivity potential of mycorrhizal populations at each production site (Moorman & Reeves, 1979; INVAM, 2014). Soils from each site were used to inoculate a sterile growing medium, planted with corn (Zea mays), and grown for 21 days (INVAM, 2014). Corn roots were then measured for percent colonization using the methods previously described.

AMF spores were extracted from soil samples by wet-sieving (Smith & Skipper, 1979), and identified by morphological characteristics. Mean infectivity analysis and species identification was conducted at the International Culture Collection of Vesicular Arbuscular Mycorrhizal Fungi by Dr. Joseph Morton (INVAM).

Statistical Analysis

One-way analysis of variance (ANOVA) was used to test for site-based differences in root mass, fibrous root mass, AMF colonization, and ginsenoside concentrations. Two-way ANOVA was used to resolve site-level differences in the effect of AMF colonization on ginsenoside concentrations, the effect of AMF colonization on concentrations of individual ginsenosides, and the effect of root mass on total ginsenoside concentrations. Where site differences were not observed, regression analysis was used to quantify the overall relationship across all sites between AMF colonization, root mass, and ginsenoside concentrations. For ginsenoside variables that could not be rendered normally distributed, Welch’s One-way ANOVA and Spearman rank order correlations were used. Pearson product moment correlations were used to test for significant correlations between fungal colonization and total ginsenoside concentrations.

Results

Mycorrhizal Colonization

One-way analysis of variance (ANOVA) determined that Total Percent Colonization (TPC) (p = 9.835e-07) and Percent Arbuscule Content (PAC) (p = 0.0008) were significantly different between sites, with differences between sites A and C, and A and D accounting for most of the observed variation in both TPC and PAC. The highest mean TPC was recorded at Site C (66.15%), followed by Sites D (63.72%), B (53.97%), and A (48.87%), and the highest mean PAC was recorded at Site D (15.86%), followed by Sites B (13.69%), A (11.88%), and C (6.70%). The distribution of TPC and PAC across all sites is illustrated in Figures 1 and 2 respectively.

One-way ANOVA was also used to compare total root mass across production sites, and indicated that root masses (p = 5.143e-08) were significantly different across sites. The distribution total root mass is illustrated in Figure 3. Pearson product moment correlations were used to test for relationships between AMF colonization and measurements of root mass and fibrous root mass, and determined that PAC was inversely correlated with both total root mass (p = 0.021, rho = -0.2978) and fibrous root mass (p = 3.233e-05, rho = -0.5093), and this relationship was consistent across sites. The relationship between PAC and root mass are illustrated in Figure 4.  Results showed no significant correlations between TPC and either root or fiber mass.  Data for TPC, PAC, total root mass, and fibrous root mass for all sites is summarized in Table 2.

Fig 3. Mean (n=15) and distribution of root mass by site. Data was log transformed to achieve normality.

Fig 3. Mean (n=15) and distribution of root mass by site. Data was log transformed to achieve normality.

Table 2. Mean values for total percent colonization (TPC), percent arbuscule content (PAC), total root mass, and fibrous root mass (± STDEV)

Fig 4. Inverse significant relationship (p < 0.05) between arbuscule content (PAC) and root mass.
Ginsenoside Analysis

One-way ANOVA was used to compare root ginsenoside concentrations across all production sites, and there were no significant differences between sites (p = 0.104). Mean total ginsenoside concentrations were highest in roots sampled from Site B (108.73 mg/g), followed by sites C (107.51 mg/g), A (96.83 mg/g), and D (65.09 mg/g), representing 10.87%, 10.75%, 9.68%, and 6.51% of mean root mass respectively. Mean ginsenoside concentrations for each site are illustrated in Figure 5, and the percentage of root mass represented by ginsenosides is summarized in Table 3.

Fig 5. Mean (n = 8) and distribution of total root ginsenoside concentrations (mg/g) for each site.

Table 3. Mean values for total ginsenoside concentration, and percent of root mass represented by ginsenosides (± STDEV)

One-way ANOVA’s were also used to compare the levels of individual ginsenosides present in roots across sites, and determined that the amount of ginsenosides Rg1 (p = 0.002) and Re (p = 0.006) were significantly different across sites, with higher amounts of Rg1 present in roots from sites A and B, and higher amounts of Re present in roots from sites C and D.

Two-way ANOVA was used to test for an interactive effect of root mass and site on ginsenoside concentrations. The results indicated that total ginsenoside concentrations varied significantly with root mass (p = 0.031), but there was no interactive effect of root mass and site (p = 0.930). The relationship between ginsenoside concentrations and root mass are illustrated in Figure 6. Regression analyses support this finding, and suggest that approximately 12.46% of the variation in ginsenoside concentrations can be explained by root mass (p = 0.0269). When examined individually, no significant relationships between individual ginsenosides and root mass were detected.

Fig 6. Positive correlation (p <0.05) between root mass (g) and ginsenoside concentrations (mg/g).

Two-way ANOVA was also used to test for interactive effects of AMF colonization and sites on ginsenosides. There was no significant interactive effect of TPC (p = 0.56706) or PAC (p = 0.4471) and there were no significant main effects of TPC (p = 0.0823) or PAC (p = 0.182) on ginsenosides, although regression analyses suggest that TPC may have a minor effect on ginsenoside concentrations (p = 0.1058), with approximately 5.4% of the variation in explained by TPC.

When examined individually, Spearman’s rank order correlation test showed that Rg1 was inversely correlated with TPC (p = 9.826e-05, rho = -0.6338) (Figure 7), while Re was positively correlated with TPC (p = 0.007, rho = 0.4691) (Figure 8).

Fig 7. Inverse relationship (p < 0.05) between total percent colonization (TPC) and concentrations of Rg1 (mg/g).
Fig 8. Positive correlation (p< 0.05) between total percent colonization (TPC) and concentrations of Re (mg/g).
AMF Species Identification and Mean Infectivity Potential

Rhizophagus intraradices (formerly Glomus intraradices) was the only species of AMF identified across all four production sites. AMF spores extracted from soil samples were heavily degraded, thus limiting the ability to identify species based on morphological characteristics. Mean infectivity analyses showed no mycorrhizal colonization in the root systems of trap plants after 21 days of growth.

Discussion

The colonization of WS ginseng roots by AMF were determined to be significantly different between production sites, with greater PAC and TPC observed in sites that are less intensively managed, suggesting that AMF may be influenced by management interventions in WS production. Measurements of PAC in WS roots (6.70%-15.86%) were lower than values previously reported by Whitebread et al. (1996) and McGonigle et al. (1999), which ranged from 23% to 57% in one to three-year-old field-cultivated roots. Hyphal colonization in WS roots (48%-66%) was substantially higher than values reported by Whitebread et al. (1996) and McGonigle et al. (1999), which ranged from 8%-33% in field-cultivated roots.

The determination of significant differences in total ginsenoside concentrations between sites is supported by findings previously reported by Lim et al. (2005) and Schlag and McIntosh (2006). Differences observed in concentrations of Rg1 and Re between production sites were previously reported by Schlag and McIntosh (2006), who suggest that chemotypic differences are likely attributed to plant genotype (e.g. seed source). This is supported by differences in seed origin between production sites utilized in this study. Among individual ginsenosides, Re was the only ginsenoside determined to be positively correlated with TPC. The correlation between Re and mycorrhizal colonization is supported by the results of Fournier et al. (2003), who also determined that Re is significantly influenced by mycorrhizal colonization. Total ginsenoside concentrations measured in WS roots during this study (6.51% – 10.87% of root mass) were consistent with previously reported values. Li and Fitzloff (2002) determined that commercially available ginseng powders and capsules contained between 5.1% and 10.9% ginsenosides by weight. Concentrations measured in WS roots were higher than those previously reported for field-cultivated (Court et al.,1996; Li et al., 1996) and young wild roots (Assinewe et al., 2003), with concentrations ranging between 3.0% and 5.0% of total root mass.

Differences in total ginsenoside concentrations were determined to not be based on differences in mycorrhizal colonization, plant genotype/chemotype (e.g. Rg1/Re ratios), or management intensity (e.g. low vs. high-intensity). Rather, ginsenoside concentrations were determined to be more significantly influenced by root mass, with the highest concentrations observed in sites B and C where the highest mean root masses were recorded. The relationship between root mass and ginsenoside concentrations is supported by results previously reported by Smith et al. (1996) and Court et al. (1996). Additionally, root mass was not shown to be significantly influenced by the extent of AMF colonization in WS roots, with results actually indicating an inverse relationship between root mass and PAC, suggesting that smaller ginseng roots may rely more heavily on fungal partnerships for nutrient acquisition and resource foraging. These results differ from those previously reported by Li (1995), which showed that inoculated one and two-year old roots had higher root masses compared to un-inoculated controls.

The identification of Rhizophagus intraradices, although limited in scope, provides new insight into the AMF associates of American ginseng in WS production. No previous reports of AMF associates in wild or WS American ginseng were identified during the course of this study. Glomus intraradices (e.g. R. intraradices) was previously identified by Seok-Cho et al. (2007) in field-cultivated Korean ginseng roots, and has also been used as an inoculant in studies conducted by Fournier et al. (2003) and Li (1995). The use of more advanced identification techniques, such as DNA analysis, is recommended to help resolve the identify of fungal partners associating with American ginseng in forested production sites.

In conclusion, the results of this study may further underscore the importance of site selection and habitat quality as key factors contributing to the success of WS ginseng production. Production sites with opposing production practices and management intensity, varying levels of mycorrhizal colonization, and with differing plant genotypes and chemotypes, were observed to produce roots with the highest ginsenoside concentrations, as well as being the most productive based on root mass. Although the results of this study provide new insights regarding ginseng-mycorrhizae interactions in forest-based production systems, additional research is needed to better resolve these relationships, particularly in regards to identification of mycorrhizal associates of American ginseng, and how different AMF species may influence root development and plant phytochemistry.

References

Assinewe, V. A., Baum, B. R., Gagnon, D., & Arnason, J. T. (2003). Phytochemistry of wild populations of Panax quinquefolius L. (North American ginseng). Journal of Agricultural and Food Chemistry, 51(16), 4549-4553.

Burkhart, E. P. (2013). American ginseng (Panax quinquefolius L.) floristic associations in Pennsylvania: guidance for identifying calcium-rich forest farming sites. Agroforestry systems, 87(5), 1157-1172.

Carroll, C., & Apsley, D. (2013). Growing American Ginseng in Ohio: Site Preparation and Planting Using the Wild-Simulated Approach. Ohio State University Extension Fact Sheet F-57-13. Columbus: the Ohio State University.

Carroll, C., & Apsley, D. (2004). Growing American ginseng in Ohio: an introduction. Ohio State University Extension Fact Sheet F-56-04.

Chandler, J. L., & McGraw, J. B. (2015). Variable effects of timber harvest on the survival, growth, and reproduction of American ginseng (Panax quinquefolius L.). Forest Ecology and Management, 344, 1-9.

Corbit, R. M., Ferreira, J. F., Ebbs, S. D., & Murphy, L. L. (2005). Simplified extraction of ginsenosides from American ginseng (Panax quinquefolius L.) For high-performance liquid chromatography− ultraviolet analysis. Prevention, 4, 6.

Court, W. A., Reynolds, L. B., & Hendel, J. G. (1996). Influence of root age on the concentration of ginsenosides of American ginseng (Panax quinquefolium). Canadian Journal of Plant Science, 76(4), 853-855.

Egerton-Warburton, L. M., & Allen, E. B. (2000). Shifts in arbuscular mycorrhizal communities along an anthropogenic nitrogen deposition gradient. Ecological Applications, 10(2), 484-496.

Eo, J. K., & Eom, A. H. (2009). The effect of benomyl treatments on ginsenosides and arbuscular mycorrhizal symbiosis in roots of Panax ginseng. Journal of Ginseng Research, 33(4), 256-259.

Fournier, A. R., Khanizadeh, S., Gauthier, L., Gosselin, A., & Dorais, M. (2003). Effect of Two Glomus Species Inoculations on Survival, Photosynthetic Capacity, Growth, Morphology and Root Ginsenoside Content of Panax quinquefolius L. Journal of Ginseng Research, 27(4), 178-182.

Giovannetti, M., & Mosse, B. (1980). An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytologist, 84(3), 489-500.

Hodge, A., Helgason, T., & Fitter, A. H. (2010). Nutritional ecology of arbuscular mycorrhizal fungi. Fungal Ecology, 3(4), 267-273.

International Culture Collection of Vesicular Arbuscular Mycorrhizal Fungi (INVAM). (2014). Staining of mycorrhizal roots. Retrieved from: http://invam.wvu.edu/methods/mycorrhizae/staining-roots.

Lim, W., Mudge, K. W., & Vermeylen, F. (2005). Effects of population, age, and cultivation methods on ginsenoside content of wild American ginseng (Panax quinquefolium). Journal of Agricultural and Food Chemistry, 53(22), 8498-8505.

Li, Thomas S. C. (1995). Effects of vesicular-arbuscular mycorrhizae on the growth of American ginseng. Journal of Ginseng Research, 19(1), 73-76.

Li, T. S. C., Mazza, G., Cottrell, A. C., & Gao, L. (1996). Ginsenosides in roots and leaves of American ginseng. Journal of Agricultural and Food Chemistry, 44(3), 717-720.

Li, W., & Fitzloff, J. F. (2002). HPLC determination of ginsenosides content in ginseng dietary supplements using ultraviolet detection. Journal of liquid chromatography & related technologies, 25(16), 2485-2500.

McGonigle, T. P., Hovius, J. P., & Peterson, R. L. (1999). Arbuscular mycorrhizae of American ginseng (Panax quinquefolius) in cultivated field plots: plant age affects the development of a colonization lag phase. Canadian Journal of Botany, 77(7), 1028-1034.

Moorman, T. and F. B. Reeves. (1979). The role of endomycorrhizae in revegetation practices in the semi-arid West: A bioassay to determine the effect of land disturbance on endomycorrhizal populations. American Journal of Botany, 66, 14-18.

Qi, L. W., Wang, C. Z., & Yuan, C. S. (2011). Ginsenosides from American ginseng: chemical and pharmacological diversity. Phytochemistry, 72(8), 689-699.

Robbins, C. S. (1998). American ginseng: the root of North America’s medicinal herb trade. TRAFFIC North America Report.

Schlag, E. M., & McIntosh, M. S. (2006). Ginsenoside content and variation among and within American ginseng (Panax quinquefolius L.) populations. Phytochemistry, 67(14), 1510-1519.

Seok-Cho, N., Kim, D., Cho, H., Shin, Y., Kim, Y., & Ohga, S. (2007). Identification of symbiotic arbuscular mycorrhizal fungi in Korean ginseng roots by 18S rDNA sequence. Journal-Faculty of Agriculture Kyushu University, 52(2), 265.

Smith, G. W., & Skipper, H. D. (1979). Comparison of methods to extract spores of vesicular-arbuscular mycorrhizal fungi. Soil Science Society of America Journal, 43(4), 722-725.

Smith, R. G., Caswell, D., Carriere, A., & Zielke, B. (1996). Variation in the ginsenoside content of American ginseng, Panax quinquefolius L., roots. Canadian Journal of Botany, 74(10), 1616-1620.

Whigham, D. F. (2004). Ecology of woodland herbs in temperate deciduous forests. Annual Review of Ecology, Evolution, and Systematics, 583-621.

Whitebread, F., Peterson, R. L., & McGonigle, T. P. (1996). Vesicular-arbuscular mycorrhizal associations of American ginseng (Panax quinquefolius) in commercial production. Canadian Journal of Botany, 74(7), 1104-1112.

Zeng, Y., Guo, L. P., Chen, B. D., Hao, Z. P., Wang, J. Y., Huang, L. Q., & Chen, M.L. (2013). Arbuscular mycorrhizal symbiosis and active ingredients of medicinal plants: current research status and prospectives. Mycorrhiza, 23(4), 253-265.