Effects of Planting Density on the Growth, Taproots Yield and Quality of Glycyrrhiza uralensis

Glycyrrhiza uralensis Fisch. ex DC. is a perennial clonal plant of Leguminosae and its dried taproots and rhizomes have been used medicinally. G. uralensis is popular with consumers because the slices of its taproots can be infused for drinking and its extracts have a variety of pharmacological actions such as anti-inflammatory, antitussive, anti-bacterial, anti-viral and inhibit the proliferation of tumor cells (Qin et al., 2015; Yu et al., 2015; Deng et al., 2017; Kwon et al., 2020; Huan et al., 2021). Over the past few decades, with demands for its roots and rhizomes have been increasing significantly, G. uralensis was suffering predatory exploitation making many of its wild populations at the edge of extinction (Hayashi et al., 2009; Huang et al., 2010; Li et al., 2013). In order to ensure an adequate supply of the licorice, China has encouraged the cultivation of the medicinal plant, which has indeed alleviated the contradiction between its supply and demand to a certain extent (Alamgir, 2017). As cultivated licorice is usually harvested within two or three years and has a short growth period, its taproots and rhizomes appear to have a lower content of medicinally active components than wild licorice. Therefore, almost all of the taproots and rhizomes are processed into medicinal slices after harvesting (Zheng et al., 2020). Although the chemical compositions of the taproots and rhizomes are similar, consumers prefer the larger diameter of the taproots slices, which has a red, smooth and intact epidermis (Huang et al., 2019) and the larger the diameter of the slices, the higher the price (Long, 2017). Since there is a trade-off in resource allocation between the taproots and rhizomes, over-developed rhizomes will inevitably lead to a reduction in the yield and quality of the taproots (Wani et al., 2021).

Cultivation density is often considered a key factor affecting morphological characteristics, biomass allocation and secondary metabolite accumulation of plants. It changes the occupation of space and utilization of water and mineral nutrients of crop’s below-ground organs by intraspecific competition (Wang et al., 2016; Desouza et al., 2021), which in turn leads to variation in yield (Xu et al., 2021; Liu et al., 2021; Musavi et al., 2020) and quality of crop’s economic organs (Gunri et al., 2015; Omerand Ufuk, 2019; Zahra et al., 2021). So density plays a critical role in regulating the biomass allocation among the organs of plants and intraspecific competition is also an important factor in shaping the rhizomes’ development of clonal plants (Lima et al., 2021). Therefore, we are devoted to find the effect of cultivation density on growth, rhizomes development, yield and quality of taproots and rhizomes of G. uralensis, intending to provide a scientific basis for establishing a cultivation system that may effectively inhibit rhizomes’ development but significantly increase the yield of taproots and rhizomes of G. uralensis.

Test material

The seeds of G. uralensis used for the test were collected in October 2018 from the town of Burqin (86°28′73″E, 41°38′ 83″N), in China and were dried at room temperature. Glycyrrhizic acid (Batch No.: C11654946) and liquiritin (Batch No.: C11602211), methanol, formic acid, anhydrous ethanol, acetate, aluminum trichloride and sulfuric acid (98%) were bought from Macklin Biochemical (Shanghai Macklin Biochemical Co, Ltd) and acetonitrile was purchased from Thermo Fisher Scientific, Inc. (LCMS grade, Thermo Fisher Scientific, USA).

Experimental design

On 10 April 2019, uniformly sized seeds of G. uralensis were placed in a beaker and were soaked in sulfuric acid solution with concentration of 98% for 0.5 h, then were rinsed with running water until no sulfuric acid residue remained on the seed coat and then were soaked in distilled water for 8 hours at room temperature. The seeds with full imbibition were selected and planted in plastic buckets (75 cm in diameter and 80 cm in height) with sandy soil (sand: loam mixture ratio of 3:7) as the cultivation substrate, total nitrogen content 1.21 g·kg^-1, total phosphorus content 0.82 g·kg^-1, total potassium content 2.57 g·kg^-1, organic matter content 21.25 g·kg^-1, available phosphorus content 39.33 mg·kg^-1, available potassium 53.54 mg·kg^-1 and 45 seeds of G. uralensis were evenly sown in each bucket at a depth of 1 cm. As emerging of the fourth true leaf, excess seedlings were removed according to the experimental designas following on 10 May 2019.

Three density treatments were designed in this study:low-density (10 plants·bucket^-1, corresponding to 2.25×10⁵ plants·hm^-2), medium-density (20 plants·bucket^-1, amounting to 4.5×10⁵ plants·hm^-2), high-density (30 plants·bucket^-1, being equal to 6.75×10⁵ plants·hm^-2). Ten days after the density setting of the seeding, fertilizers were added to each bucket according to the common level of the G. uralensis field fertilization with 14.99 g·m^-2 of urea (N≥46%), 23.99 g·m^-2 of potassium calcium super phosphate (P₂O₅≥46%) and 10.49 g·m^-2 of potassium sulphate (K₂O≥50%), which were added in 5 times at 3-week intervals. Ten repetitions were set for each density treatment.

During the experiment, each bucket was rehydrated by weighing method at 10:00 am (Beijing time) every day, maintaining the relative soil moisture at about 70%. All buckets were randomly placed in an open area on the Ministry of Education Key Laboratory of Xinjiang Phytomedicine Resource Utilization at Shihezi University campus with a distance of 100 cm between two adjacent buckets to avoid shading among the treatments and the position of each bucket was randomly changed every week to eliminate the influence of light and marginal effect.

Measurement of biomass and morphological parameters

On October 1, 2020, all the materials were harvested after the height and crown width of each plant were measured, the complete below-ground part of each plant was carefully rinsed with running water until the surface was free of floating soil. The length and the maximum diameter of the taproots were measured for each plant; the number of rhizomes (length≥1 cm), rhizome shoot (length<1 cm) and ramet were counted.

Each plant was separated into above-ground organ (stem and leaf), taproot and rhizome and the rhizomes of each treatment group were scanned by Expression 1100XL scanner (Epson company, Japan) and the scanned images were performed uniformly by using the image analysis software (WinRHIZO Pro 2013) to calculate the total length, total surface area and total volume of rhizomes of each plant. Then the above-ground organ, taproot and rhizomes were dried in an oven at 70°C to a constant weight and their biomass was measured by an analytical balance (Beijing Sartorius Instrument Systems Co. Ltd.). The biomass of below-ground organs (taproot and rhizome), the percentage of taproot biomass (taproot’s biomass/below-ground organ’s biomass) and the percentage of rhizome’s biomass (rhizome biomass/below-ground organ’s biomass) were calculated.

Measurement of the content of major active components of the taproots of G. uralensis

The contents of glycyrrhizic acid and glycyrrhizin were detected by the ultra-high performance liquid chromatography -tandem mass spectrometry (UHPLC-MS/MS) method (Agilent Technologies Inc., California, USA), the method of the standard curve, UHPLC and MS condition reference to detail indicated in a previous study (Fu et al., 2013). The ion pairs, fragmentation voltages and collision energies used for quantitative analysis were shown in Table 1. The content of total flavonoids was determined by a UV-visible spectrophotometer (UV-1900, Shimadzu Corporation, Shanghai) at 334 nm with the LI standard (CAS#551-15-5) as the control.



Data analysis

Statistical analysis of all experimental data was performed by IBM SPSS 19.0 (IBM Corp, Armonk, NY, USA) software. One-way ANOVA was used to compare the differences in plant morphological parameters, rhizome development index, taproots yield and secondary metabolite content of the licorice under the different density treatment conditions. Duncan multiple range test (α=0.05) was used to test for significant difference and the same letter means no significant difference among the two treatments, while the different means there is a significant difference between them, Origin 2019 software was used for plotting.

Effect of cultivation of density on morphological parameters of G. uralensis

Influenced by environmental factors, plant inevitably develops certain morphological, structural and physiological metabolic characteristics that are suitable for the environment. Morphological changes are the most intuitive indicators that may reflect the adaptive status of a plant to cultivation density and may reflect the energy allocation strategies of different organs (Luo and Dong, 2002). The results of the present study showed that the most vigorous growth potential of G. uralensis was observed under low-density treatment, which showed the highest values of plant height, crown width, taproots length and taproots diameter among the three treatments (Fig 1). Compared with the low-density treatment, the plant height of G. uralensis was reduced by 14.32% and 8.78%; its crown width was reduced by 15.89% and 27.18%; its taproots length was shortened by 14.19% and 16.07%; and its taproots diameter was decreased by 13.84% and 24.23% under the medium-density and high-density planting conditions, respectively. In summary, the growth and development of rhizomes of G. uralensis were significantly inhibited by the increase in planting density and their clonal spreading potential was significantly reduced. This may be due to the fact that under low-density condition, individuals have more space to grow with less intraspecific competition and the space, nutrients, water and light resources required for growth are relatively adequate. On the contrary, plant growth is poor under high-density planting condition because the available space and resources of each single plant is very limited and the intraspecific competition intensity is relatively high (Gao, 2017).



Effects of planting density on the development of rhizomes of G. uralensis

Intraspecific competition is considered to be an important factor in shaping rhizome traits (Li et al., 2020). In the present study, with the increase in cultivation density, rhizome development and growth of G. uralensis were significantly suppressed and their clonal spreading potential was significantly reduced, the resource allocation pattern between taproots and rhizomes was changed and more resources were invested in the growth of the taproots, thus showing that the number of rhizomes, shoots, clonal ramets, total length, total surface area, total volume, biomass and biomass percentage of the rhizome decreased significantly with increasing of the plant density (Fig 2). Compared with the low-density treatment, these above parameters decreased by 18.44%, 9.38%, 34.29%, 24.49%, 44.89%, 51.04%, 53.36% and 19.52%, respectively under the medium-density condition and those of the high-density treatment reduced by 39.27%, 40.01%, 46.67%, 50.99%, 70.09%, 78.17%, 82.55% and 33.45%, respectively. This is similar with the results of Baoqing Dai and Yan Wang’s study on knotweed (Zoysia japonica Steud.) (Dai and Wang, 2013), which showed that the rhizome’s development and spread were significantly inhibited under high-density planting condition. This may be because the increased planting density intensified the intraspecific competition for space, light, water and mineral elements, leading to the blockage of horizontal extension of the rhizomes.



Effects of planting density on yield of the licorice’s economic organs

Cultivation density also affects the final yield of a crop (Gunaeni et al., 2021). The results of this study showed that the biomass of above-ground organs, below-ground organs and taproots of a single individual under low-density cultivation treatment were the highest among the three treatments and all the three indexes decreased with the increasing of cultivation density (Fig 3). Compared with the low-density treatment, the above-ground organs biomass decreased by 35.96% and 56.37% in the medium- and high-density treatment groups, respectively; the below-ground organs biomass declined by 42.04% and 73.77%, respectively and the taproots biomass reduced by 33.06% and 66.81%, respectively.



Although the taproots biomass of an individual was the lowest under the high-density condition, the percentage of taproots’ biomass of it was the highest, the percentage of taproots biomass in the high-density treatment group increased by 25.56% and 9.58% compared with the low and medium-density treatments, respectively, which indicated that increasing cultivation density within a certain range could effectively promote the resources allocation to taproots.

However, as planting density increases, there is often a trend towards inconsistencies in crop yield between an individual’s and a population’s. In the present study, the total yield (mean value of taproots biomass per plant × number of plants in a certain area) of taproots was the highest in the medium-density treatment in a certain area (Table 2), which increased by 34.44% and 33.87% compared with those of the low and high-density treatments, respectively. This indicated that the increase in planting density resulted in a significant decrease of taproots’ biomass of an individual, but the variation in total taproots’ yield in a certain area of farmland showed a parabolic curve pattern. This may be due to the fact that the increase in number of the taproots to some extent compensate for the decrease in single taproots’ biomass. However, when the seedling density continuously increased and reached a high level, the increase of the taproots’ number could not effectively make up the decrease in the yield of single taproot, which might be the reason why the total yield of the taproots of G. uralensis under high-density planting conditions was significantly lower than that of the medium-density treatment group.


 
Effects of planting density on quality of the licorice’s economic organs
 
The two most important factors determining the market price of medicinal material are the medicinal quality and the morphological character of the taproots and the medicinal quality plays a dominant role, which is mainly depended on the content of triterpenoids (Luo et al., 2021) and flavonoids (Hu et al., 2019) in the taproots. Content of glycyrrhetinic acid, glycyrrhizin and total flavonoids is traditionally used to characterize the quality of the licorice in the Pharmacopoeia of the People’s Republic of China (2020 edition) (Chinese Pharmacopoeia Commission 2019) and in this study, planting density had significant effects on the content of these three secondary metabolites in the taproots of G. uralensis and the content of these active components significantly decreased with the increase of cultivation density (Fig 4). Compared to the low-density treatment, the content of glycyrrhetinic acid, glycyrrhizin and total flavonoids in the medium-density treatment group decreased by 7.45%, 5.64% and 2.46%, respectively, while the content of these components in the high-density treatment group decreased by 36.43%, 37.61% and 15.86%, respectively.

The contents of glycyrrhetinic acid, glycyrrhizin and total flavonoids in the high-density treatment were reduced by about 16% to 38%, respectively, than those in the low-density treatment, while there was no difference in the contents of these three active components among low and medium-density treatments.

Exploring a reasonable planting density of G. uralensis is one of the essential ways to increase its’ taproots yield and optimize its quality. Planting with low density, G. uralensis can’t fully utilize the sunlight and the nutrients in the soil, its’ yield will be reduced; planting with high density, the competition for nutrients between individuals will increase and photosynthesis will be hindered, its’ quality will be inferior. In conclusion, G. uralensis planting with medium density not only significantly inhibits the growth and development of its rhizomes, but also ensures the maximum yield and optimal quality of the licorice population.