Introduction

Ginseng is a perennial plant cultivated as a food and a health supplement. Four species belonging to the genus Panax are grown in Asia, North America, and Europe (Baeg and So, 2013). Ginsenosides are a class of triterpenoid saponins comprising approximately 30 unique saponins found in various parts of the plant, including the root, leaf, and berry (Chuang and Sheu, 1994). Ginsenoside is believed to be a pharmacologically active compound having antioxidative, anticarcinogenic, and antidiabetic effects in humans (Attele et al., 1999). There have been attempts to improve on the

traditional grading criterion based on root shape by grading ginseng on the basis of its ginsenoside content (Roy et al., 2003).

The effects of environmental factors on ginsenoside production of the American ginseng have been well studied (Panax quinquefolius L.). Ginsenoside content is affected by water stress (Lim et al., 2006; Lee and Mudge, 2013), light (Foumier et al., 2003; Yu et al., 2005), mineral content (Li and Mazza, 1999), and temperature (Jochum et al., 2007). It is also affected by biotic factors such as plant growth regulators, plant age, and genotype (Court et al., 1996; Lim et al., 2005; Barbara et al., 2006; Um et al., 2017).

Plants deploy secondary metabolites as defensive responses to environmental factors (Neilson et al., 2013). Ginsenosides act as a defensive compound against biotic and abiotic challenges. Ginsenoside has negative allelopathic effects on plants, pathogens, and pests (Nicol et al., 2002; Zhang et al., 2005; Yang et al., 2015). It is also involved in biological tolerance to environmental stress (Devi et al., 2012). Questions about the differential allocation of available resources for root growth and ginsenoside from both botanical and agricultural perspectives have prompted this study to characterize the relationship between ginsenoside and biomass in response to environmental factors.

There has been growing interest in developing cultural methods to control both root mass yield and ginsenoside content (Park et al., 1986; Lee et al., 2012). However, there is little information on the effects of soil quality and climate on the cultivation of P. ginseng. Soil texture varies largely among sites and influences the availability of water and nutrients (Saxton and Rawls, 2006). Cultivation site is also a critical factor determining local climate and topology. Fertilization is beneficial in crop production, and farmers apply various organic materials in the cultivation of P. ginseng (Eo and Park 2013). It is thus desirable to determine the influence of various soil nutrient conditions on ginseng culture.

It is important to investigate the influence of abiotic factors on root growth and ginsenoside content to simultaneously improve root quantity and quality. We hypothesized that fertilization, soil particle size and cultivation site are the main abiotic factors responsible for environmental stress. To verify this hypothesis, we evaluated the influence of abiotic stresses.

Materials and Methods

Fertilization Experiment

The experiment was conducted in a greenhouse located in Emsung, Chungbuk Province, Korea. Nine 2-year-old seedlings were transplanted into plastic pots (W 33 × D 24 × H 30 cm) on 21 April 2014. The treatments included an unfertilized control. Fertilization regimes were as follows: urea (46% N) at 0.1 and 0.3 g·kg^-1, Urea_0.1 and Urea_0.3, respectively; fused calcium magnesium phosphate (FMP, 17% P) at 0.2 and 0.6 g·kg^-1, FMP_0.2 and FMP_0.6, respectively; potassium chloride (60% K) at 0.2 and 0.6 g·kg^-1, KCl_0.2 and KCl_0.6, respectively. Fungicide was applied using a conventional method, and watering was performed every 2 weeks. The experiment was performed using a randomized block design with 5 replicates. Roots were harvested on 15 October 2014, and root growth parameters were measured.

Soil Texture Experiment

The experiment was performed in a greenhouse covered with a shade cloth. The soil was separated into the following 3 groups according to particle size using a sieve: < 0.5 mm, 0.5-1 mm and 1-2 mm. These treatments were compared with an unsieved control. The soil had sand, silt and clay content of 51.7%, 44.0%, and 4.3%, respectively, and was sandy loam in texture. Nine 2-year-old seedlings were transplanted into plastic pots on 21 April 2014, and roots were harvested on 15 October 2014. The experiments were conducted using a randomized block design with 4 replicates.

Cultivation Site Experiment

The experiment was conducted at 5 sites including Eumseong, Jinan, Keumsan, Pyeongchang, and Yeoncheon. Ginseng was cultivated under artificial shade with conventional management. The sites were selected on the basis of variable latitude and climatic conditions, and information about the sites is shown in Table 1. Climate data were obtained from the Korea Meteorological Administration. A polystyrene frame (W 32.5 × D 25.0 × H 26.0 cm) was buried and filled with 20 kg of the same unsieved soil used in the soil texture experiment. Nine 2-year-old seedlings were transplanted into each frame from 24 through 28 March 2014. Because the soil in the frame was separated laterally, movement of materials could occur only vertically between the bottom and top of the filled soil. The experiment was performed using a randomized block design with 3 replicates. Roots were harvested from 30 September to 1 October in 2014.

Table 1. Location of experimental sites, mean temperature, and total precipitation during the experimental period

Soil Analysis

The soil in the pots and frame was homogenized after the ginseng roots were harvested. The nitrate concentration in the soil was determined using an auto-analyzer (AutoAnalyser 3, Bran+Luebbe, Germany). Available phosphate was analyzed using the Lancaster method and the soil organic content was determined using the Tyurin method (RDA, 2002). The exchangeable cations in the soil were extracted with 1 N NH₄OAc (pH 7.0) and measured by inductively coupled plasma analysis (Integra XMP, GBC, Australia).

Ginsenoside Analysis

Harvested roots were washed and freeze dried. Ginsenoside content was analyzed using the modified method of Kim et al. (2012). Standard ginsenosides were purchased from a chemical company (ChromaDex Inc., Santa Anna, CA, USA). Powdered 200 mg of samples were extracted with 70% MeOH. The resulting suspension was ultrasonicated for 30 min at 50°C and then centrifuged for 15 min at 13,000 rpm. The supernatant was first filtered through a Sep-Pak C18 cartridge (Waters Corp., Miford, MA, USA) and then through a 0.45 µL microfilter (Watman Mini-UniPrep Syringeless Filters, USA). An LC-1100 system (Agilent, Walbronn, Germany) was used to perform the analysis of ginsenoside. Chromatographic separations were performed on a Halo^® RP-Amide column (4.6 × 150 mm, 2.7 µm at 50°C). The mobile phase was acetonitrile:deionized water, 30:70), and the flow rate was 0.5-0.8 mL·min^-1. Ultraviolet detection was performed at 203 nm and the injection volume was 10 µL.

Data Analysis

Differences among treatments were analyzed using the Duncan’s multiple range test at p = 0.05 if a significant ANOVA result was found. Correlations between root growth parameters and ginsenoside content were calculated for each experiment. In addition, a correlation analysis between ginsenoside content and root weight was performed using combined data from the 3 separate experiments. All statistical analyses were performed using SAS 9.1 (SAS Institute, Cary, NC).

Results

Fertilization Experiment

The changes in soil chemical properties reflected the type and quantity of applied fertilizers (Table 2). Nitrate and K concentrations were increased by urea and KCl application, respectively, compared to those of the unfertilized control. Concentrations of P, Ca and Mg were increased by FMP. Root weight was greater in FMP_0.2 and KCl_0.2 than in Urea_0.3 (Fig. 1). There was no significant difference in ginsenoside content between fertilization regimes (Table 3). A correlation analysis using combined data from the 3 separate experiments showed that ginsenoside content and root weight were correlated. Root weight was positively correlated with the ginsenosides Re (r = 0.36, p = 0.003), Rf (r = 0.33, p = 0.005), Rb1 (r = 0.26, p = 0.025), Rc (r = 0.34, p = 0.004), Rb2 (r = 0.25, p = 0.032), Rg3 (r = 0.36, p = 0.002), Rd (r = 0.51, p < 0.0001) and total ginsenoside (r = 0.30, p = 0.002).

Fig. 1. Root growth of ginseng as affected by the application of inorganic fertilizers. The means of the treatments having the different letter as a suffix are significantly different at p < 0.05 according to Duncan’s multiple range test. FMP, fused calcium magnesium phosphate. The subscripted number after the fertilizer name indicates the applied rate (g·kg^-1 soil).

Table 2. Soil chemical properties as affected by application of inorganic fertilizers

zFMP: fused calcium magnesium phosphate. The subscripted number after the fertilizer name indicates the applied rate (g·kg-1 soil). yThe different letters indicate significant differences by Duncan’s multiple range test at p < 0.05.

Table 3. Ginsenoside content as affected by the application of inorganic fertilizers

zFMP: fused calcium magnesium phosphate. The subscripted number after the fertilizer name indicates the applied rate (g·kg-1 soil). yThe different letters indicate significant differences by Duncan’s multiple range test at p < 0.05.

Soil Texture Experiment

Soil particle size was found to influence root growth (Fig. 2). Root weight was lower in soil with a particle size of< 0.5 mm, and roots were longer in soil with a particle size of 1-2 mm compared with the control. Ginsenoside content was affected by soil particle size (Table 4). Ginsenoside Rb1 and total ginsenoside content was lower in soil with a particle size of < 0.5 mm compared with the control.

Fig. 2. >Root growth of ginseng as affected by soil particle size. The means of the treatments having the different letter as a suffix are significantly different at p < 0.05 according to Duncan’s multiple range test.

Table 4. Ginsenoside content as affected by soil particle size

zThe different letters indicate significant differences by Duncan’s multiple range test at p < 0.05.

Cultivation Site Experiment

Cultivation site strongly affected root growth parameters (Fig. 3). Root weight was 2.8 times greater in Pyeongchang than in Eumseong. Root shape was also influenced by cultivation site. All measured ginsenoside contents differed among cultivation sites (Table 5). No significant correlation was found between root growth properties and ginsenoside content.

Fig. 3. Root growth of ginseng as affected by cultivation site. The means of the treatments having the different letter as a suffix are significantly different at p < 0.05 according to Duncan’s multiple range test.

Table 5. Ginsenoside content as affected by cultivation site

zThe different letters indicate significant differences by Duncan’s multiple range test at p < 0.05.

Discussion

Effects of Fertilization on Root Growth and Ginsenoside Content

Cultivation of P. ginseng depends on organic fertilizers, owing to a ban on inorganic fertilizers in Republic of Korea (Eo and Park, 2013). However, chemical fertilizers have been increasingly used in the cultivation of P. notoginseng (Xia et al., 2016). We used chemical fertilizers to manipulate various soil nutrient conditions created by organic amendment. Previous studies have reported conflicting results on the effects of inorganic and organic fertilizers. Studies involving inorganic fertilizers have variously reported a positive effect on both root growth and ginsenoside content (Lee and Mudge, 2013a; Xia et al., 2016), a neutral effect on both (Lee and Mudge, 2013a), and a positive effect on root growth but a negative effect on ginsenoside content (Park et al., 1986). The effectiveness of organic fertilizers also varies with type and application rate (Park et al., 2015). It is possible that at moderate levels of fertilization, the ginsenoside content of the plant is not influenced by the availability of nutrients. This hypothesis is consistent with the findings of Reelder et al. (2000), who reported that increasing planting density decreased root weight without changing the ginsenoside content.

The use of excessive NPK fertilizers in our study appeared to suppress growth while promoting ginsenoside production in the root. Root growth was suppressed by Urea₃₀₀ and KCl₆₀₀, although it was not significantly different from that of the control. FMP₂₀₀ increased root growth more than did Urea₆₀₀, but it was difficult to separate the effects of P from those of Ca and Mg. Positive effects on root yield and ginsenoside have been reported for Ca and Mg (Konsler et al., 1990; Xi et al., 2016). Excessive fertilization is a form of environmental stress. Jang et al. (2015) reported that the increase in ginsenoside level was more apparent for protopanaxatriol than for protopanaxadiol under light stress. The ginsenoside Rg1 was also found to be involved in the antioxidative defense mechanism of rats (Yu et al., 2012). This is consistent with our result that Rg1 and Rg2, which are protopanaxatriols, were negatively correlated with root weight in the fertilization experiment.

There have been contrasting opinions on the tradeoff between growth and ginsenoside production for plant defense. Production of ginsenoside is stimulated by plant defensive compounds including singlet oxygen, ethylene, jasmonate and NO (Wang et al., 2005; Xu et al., 2005; Tewari et al., 2007). Ginsenosides are synthesized from isopentenyl pyrophosphates, which are biosynthesized via the mevalonic acid and methylerythritol phosphate pathways in plants (Dubey et al., 2003; Hampel et al., 2007). These precursors do not directly compete for available nitrogen (Haukioja et al., 1998). However, Massad et al. (2012) has suggested that saponin production is limited by competition for nitrogen via photosynthesis. Fertilization with N induces plants to produce compounds with high N content, and N-limited conditions induce plants to produce terpenoids and phenolics, which contain no N (Ibrahim et al., 2013).

Effects of Soil Texture on Root Growth and Ginsenoside Content

Soil particle size affected root length, weight, and quality. We found the longest roots in soil with a particle size of 1-2 mm; this is consistent with a previous report of a positive correlation between root length and the proportion of sand in the soil (Li, 1997). However, Li (1997) also reported that roots were shorter in loamy sand than in sandy loam soils despite the higher sand content and suggested that the effect of soil particle size could be confounded by other factors. It is likely that soil with a small particle size has more moisture (Saxton and Rawls, 2006); therefore, poor drainage may decrease root weight (Lee et al., 2012). High soil moisture suppresses ginsenoside production in P. ginseng (Lee et al., 2012). Water deficit has a suppressive effect in P. notoginseng (Xia et al., 2016) but a promoting effect in P. quinquefolius (Lee and Mudge, 2013b). We found that total ginsenoside and Rb₁ were decreased in soil with a particle size of < 0.5 mm. This result is consistent with those of previous studies reporting a negative effect of high moisture on total ginsenoside (Lee et al., 2012) and on Rb1 content (Xia et al., 2016). Further study is needed to investigate the other factors such as how nutrient availability changes with soil texture.

Effects of Cultivation Site on Root Growth and Ginsenoside Content

While saponins generally make up approximately 3%-4% of P. ginseng by weight, and the precise ginsenoside composition has been found to vary among countries such as Republic of Korea, China, Japan, and Russia (Park, 1996). The ginsenoside contents of P. quinquefolius and P. notoginseng also vary regionally (Dong et al., 2003; Schlag and McIntosh, 2006). It is difficult to attribute these differences to a single factor because soil and climatic properties cannot easily be separated. We tested the effects of location using the same soil type to minimize the effect of soil parameters. The great variation in ginsenoside content in our study suggested that climatic factors including temperature and precipitation were the main influences on root growth. Root growth might be promoted by low mean temperature in Pyeongchang. However, we could not exclude the possibility that root growth was influenced by topography, which determines drainage. Nutrient transfer from below the buried soil also influenced the ginseng plant. However, this impact may have been minimal, considering the short root system of P. ginseng.

Ginsenoside production is influenced by climatic parameters such as temperature, precipitation, and light (Yu et al., 2005). High temperatures suppress root growth and increase ginsenoside content in P. quinquefolius (Jochum et al., 2007). Production of secondary metabolites is often increased with environmental stress. Neilson et al. (2013) have suggested that multifunctional biosynthesis and the optimization of primary metabolism can offset the tradeoff between resource allocation for growth and the production of secondary metabolites. If so, root yield can be promoted without the loss of production of ginsenoside for defense. Haukioja et al. (1998) has also reported that both saponin production and biomass accumulation can be increased with manipulation of environmental conditions. Our results revealed that no tradeoff occurs between root growth and ginsenoside content in response to local climate properties.

Conclusion

Our results demonstrated that moderate fertilization minimally impacted root growth and ginsenoside production. In contrast, excessive fertilization seemed to create environmental stress, leading to a tradeoff between biomass and ginsenoside production. Accordingly, blind or excessive fertilization should be prevented to control root yield and ginsenoside content. Soil particle size affected root weight and shape as well as ginsenoside production, showing that the selection of proper soil texture is important to root yield and quality. Cultivation site strongly influenced root growth and ginsenoside production, but no significant correlation was found between root weight and ginsenoside in different locations. A positive correlation between ginsenoside content and root weight suggests that these two properties can be simultaneously promoted under proper environmental conditions.