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Article

Effect of Plant Density on Growth and Bioactive Compounds in Salvia miltiorrhiza

by
Zhiheng Xing
1,
Guihong Bi
1,*,
Tongyin Li
1,
Qianwen Zhang
1 and
Patricia R. Knight
2
1
Department of Plant and Soil Sciences, Mississippi State University, Starkville, MS 39762, USA
2
Coastal Research and Extension Center, Mississippi State University, Poplarville, MS 39470, USA
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(6), 1157; https://doi.org/10.3390/agronomy14061157
Submission received: 29 April 2024 / Revised: 20 May 2024 / Accepted: 27 May 2024 / Published: 29 May 2024
(This article belongs to the Special Issue Medicinal and Aromatic Plants: Cultivation, Chemistry and Promising Applications)
Versions Notes

Abstract

:
Danshen (Salvia miltiorrhiza) is an herbaceous plant widely used in the pharmaceutical industry. However, the majority of medicinal plants utilized in the US are imported, posing challenges such as fluctuations in bioactive compound concentrations and insufficient supply to meet demand. Determining the optimal plant density is a key management decision for danshen production. This study aimed to investigate the effects of different plant densities on the growth and bioactive compound content of danshen cultivated in Mississippi. A field experiment was conducted to investigate the effects of different plant densities on individual plant growth, photosynthesis, and the content of bioactive components in danshen in 2020 and 2021. Six plant densities were designed: 30 × 20 cm (between row spacing × within row spacing), 30 × 30 cm, 30 × 40 cm, 45 × 20 cm, 45 × 30 cm, or 45 × 40 cm. A plant density of 45 × 40 cm resulted in danshen plants exhibiting the highest Plant Growth Index (PGI), SPAD, root number, shoot number, shoot fresh and dry weight, maximum root diameter, maximum root length, net photosynthesis, intracellular CO2 concentration, tanshinone I, and cryptotanshinone, regardless of year. Plants spaced at 45 × 30 cm had similar root fresh weight, root dry weight, and tanshinone IIA and salvianolic acid B levels compared with plants grown at the 45 × 40 cm spacing, and both were significantly higher than other densities.

1. Introduction

Salvia miltiorrhiza (danshen) is a perennial medicinal plant belonging to the Lamiaceae family. It is native to China and Japan. In its native habitat, danshen commonly grows on hillsides, meadows, and grassy areas near streams and forest edges, typically at elevations between 90 and 1200 m [1]. Because of its critical bioactive components, including salvianolic acid B, cryptotanshinon, tanshinone I, and tanshinone IIA [2], it has aroused interest from the pharmaceutical industry. Salvianolic acid B is the most abundant compound [3]. Some medicinal uses of danshen are to treat liver disease, cardiovascular disease, insomnia, dysphoria, and cancer [4]. In fact, danshen has played a crucial role in treating cardiovascular diseases and is widely used in the pharmaceutical industry [5].
Danshen refers to dried reddish-purple-colored roots and rhizomes, which are the main plant parts used in the traditional medicine. Danshen contains a variety of chemical components, which are mainly divided into two categories: one is the lipid-soluble component dominated by tanshinone diterpenes, and the other is the water-soluble component dominated by phenolic acid [4]. Compound danshen dripping pills are undergoing US Food and Drug Administration (FDA) clinical trials, and are expected to be launched in the United States [6]. They have been shown to be effective in reducing the frequency of chronic stable angina with few adverse side effects. T89 is sold as a prescription drug in China, Pakistan, Vietnam, South Korea, India, and the United Arab Emirates, where about 10 million people take it each year. It is the first compound Chinese herbal medicine in the world to have passed a multi-center Phase III clinical trial supervised by the US FDA. T89 provides important clinical scientific evidence for traditional Chinese medicine (TCM) as a potential contributor to the modern healthcare system.
Field production is the main cultivation method for most commercial crops. Tillage, irrigation, fertilization, and weed control are common practices used in field cultivation to create an optimal environment for plant growth [7]. Additionally, the strategic arrangement of plants within a given space can profoundly impact their overall health, development, and productivity [8]. Plant density affects many aspects of plant growth by affecting the environment conditions, including the intensity of sunlight, the availability of nutrients in the soil, and the efficiency of water distribution [9]. Root development plays a crucial role in danshen cultivation as it is where significant bioactive compounds accumulate, essential for medicinal purposes. Appropriate plant density can greatly increase the yield of roots [10]. Furthermore, appropriate plant density minimizes competition for vital resources, reduces the risk of diseases and pests and enhances air circulation and promotes better pollination [11]. An optimized plant density yields not only higher plant productivity but also contributes significantly to the accumulation of valuable bioactive compounds within medicinal plant parts [12]. Moreover, standardized plant density emerges as a crucial factor in guaranteeing the uniformity and consistency of these bioactive compounds in medicinal materials. In one study, danshen was planted in field conditions with plan densities of 30 × 20 cm, 30 × 30 cm, 30 × 40 cm, 45 × 20 cm, 45 × 30 cm, and 45 × 40 cm, and there were significant variations in the root yields and contents of bioactive compounds among different plant densities. The highest root yield occurred at plant densities of 45 × 30 cm and 45 × 40 cm. The highest contents of the three tanshinones occurred at plant densities of 30 × 30 cm. However, the highest content of salvianolic acid B was found at a density of 45 × 40 cm [10]. In another study, fresh and dry weights of roots, the number of roots, and the total surface area of roots were the highest when plant density was 20 cm × 25 cm [13].
Most medicinal plants, including danshen, used in the United States are imported, leading to high costs, slow delivery, and quality concerns. With market growth expected, it is crucial for US industries to access high-quality supplies. Few studies exist on danshen cultivation in the United States. The aim of this study was to examine the impact of varying plant densities on plant growth and bioactive compound contents of danshen grown in Mississippi.

2. Materials and Methods

2.1. Plant Materials and Cultivation

Danshen seeds from Shaanxi, China, were sown in trays (54 cm × 28 cm, 128-cells) in March 2020 at a greenhouse in MSU (Mississippi State University, 33°29′ N 88°47′ W) and transferred into 0.5 L containers to further develop within the greenhouse environment. The greenhouse maintained a temperature of 25 ± 1 °C with natural light. Plants were transplanted into a field located at the R. R. Foil Plant Science Research Center at Mississippi State University in June 2020. Plants were subject to six plant densities: 30 × 20 cm (between row spacing × within row spacing), 30 × 30 cm, 30 × 40 cm, 45 × 20 cm, 45 × 30 cm, or 45 × 40 cm. The plants received slow-release fertilizer Osmocote Plus 15N-3.9P-10K (15-9-12, 8–9 months; Scotts Miracle-Grow Co., Marysville, OH, USA) at a rate of 10 g per plant. Drip irrigation lines were installed and utilized based on plant requirements. The experiment followed a randomized complete block design with four replications, with varying plant density as the experimental factor. Each replication comprised six plants (subsamples) per plant density. Each block had 6 treatment groups, and protective rows were planted between blocks, between treatment groups, and at the edges of the experimental area. The plants in the protective rows were not harvested, but were used to ensure the density of experimental plants. Harvesting took place on 2 December 2020. The experiment was repeated in 2021 with the same methods in 2020.

2.2. Morphological Characteristics and SPAD

Five plants were randomly selected and harvested from each treatment of six plant densities. Prior to harvest, measurements were taken for plant height, length 1, and length 2. Length 1 indicated the widest part of the plant, while length 2 represented the length perpendicular to length 1. To assess plant size, a Plant Growth Index (PGI) was calculated by averaging the measurements of height, length 1, and length 2. Leaf SPAD measurements were taken from three recent fully expanded leaves on each plant to assess relative chlorophyll content. Each plant’s SPAD value was derived from an average of three measurements [14]. SPAD values were measured with the SPAD-502 m (Konica Minolta, Inc., Osaka, Japan). Post-harvest, shoot fresh weight and number were evaluated. Furthermore, measurements were recorded for root fresh weight, maximum root length, root number, and maximum root diameter. Root number indicated the quantity of roots with a diameter exceeding 2 mm, while maximum root length was determined as the length of the longest root [15], The maximum root diameter was assessed at the upper middle section of the tap root [16]. Subsequently, the root and shoots dry weight was registered after plant tissues were oven-drying at 60 °C.

2.3. Photosynthesis

Photosynthesis was assessed by randomly selecting one plant from each treatment per replication between 10 am and 3 pm. A portable photosynthesis system (LI-6400 XT; LI-COR Biosciences, Lincoln, NE, USA) was utilized for the measurements. During the sampling process, the photosynthetically active radiation (PAR) was 1500 μmol.m−2 and the reference CO2 concentration inside the leaf chamber was 400 μmol.mol−1 [17]. Several parameters were measured, including the net photosynthetic rate (Pn), intercellular CO2 concentration (Ci), and leaf-to-air vapor pressure deficit (VPDL).

2.4. Extraction

Dried roots were finely pulverized and 0.5 g of each sample was extracted for 30 min with 50 mL of 75% methanol. The extraction process was assisted by ultrasound, the sealed flasks containing the mixture underwent ultrasonic at room temperature. The resultant extract was then filtered to eliminate the plant material [18,19,20].

2.5. Analysis of Salvianolic Acid B, Tanshinone IIA, Tanshinone I, and Cryptotanshinone

HPLC (1260 Infinity II series; Agilent Technologies, Wilmington, DE, USA) assessments were conducted using a diode array detector (G1315C Diode-array Detector, Agilent Technologies) with an injection volume of 10 μL. The flow rate was set at 1 mL/min, and an oven temperature of 30 °C was maintained throughout the analysis. A C18 column (Agilent TC-C18, 4.6 mm × 250 mm, 5 μm) was used, with mobile phase A consisting of 100% acetonitrile, and mobile phase B containing 0.02% phosphoric acid.
The procedure for tanshinone IIA, tanshinone I, and cryptotanshinone was slightly adapted from the Chinese Pharmacopoeia [21]. The method for salvianolic acid B was slightly modified from Ren et al. [22]. The modified condition was oven temperature from 20 °C to 30 °C.

2.6. Statistical Analysis

Analysis of variance (ANOVA) and Tukey’s Honestly Significant Difference test were conducted. Statistical significance was set at p ≤ 0.05. All statistical assessments were performed using SAS (version 9.4, SAS Institute, Cary, NC, USA).

3. Results

3.1. PGI, Leaf SPAD Values, Shoot Number, and Root Number

There were differences in PGI, SPAD value, shoot number, and root number among danshens grown under different plant densities in both years (Table 1). As the plant density decreased (30 × 20–45 × 40 cm), PGI, SPAD value, shoot number, and root number had an ascending trend. Danshens grown at a plant density of 45 × 40 had the greatest PGI, shoot number, SPAD value, and root number compared with plants grown at any other density, regardless of year. Compared with plants grown at a density of 30 × 20 cm, plants grown at a density of 45 × 40 cm had 95.5% greater PGIs in 2020. Plants grown at a 45 × 40 cm density had the highest SPAD values in 2020 and 2021, respectively, and these values were higher than values for plants grown at any other density. Utilizing a plant density of 45 × 40 cm rather than 30 × 20 cm increased plant SPAD values by 23.4% and 28.8% in 2020 and 2021, respectively. Shoot numbers ranged from 2.6 to 6.6, and 2.4 to 6.4 in 2020 and 2021, respectively. Root numbers ranged from 21.0 to 40.4 and 19.6 to 34.2 in 2020 and 2021, respectively. Plants grown at a density of 45 × 40 had the highest root and shoot numbers compared with plants grown at any other plant density, regardless of year.

3.2. Shoot Fresh Weight, Shoot Dry Weight, Maximum Root Length, and Maximum Root Diameter

Lower plant densities had a positive effect on shoot fresh weight, shoot dry weight, maximum root length, and maximum root diameter in both years (Table 2). Among the six plant densities, plants growing in the 45 × 40 cm spacing had the highest shoot fresh weight, dry weight, maximum root length, and maximum root diameter, while danshens growing in the 30 × 20 cm plant density had the lowest shoot fresh weight, dry weight, and maximum root diameter, regardless of year. Maximum root lengths were similar for plants grown at 30 × 20 cm or 30 × 30 cm densities in 2021. Shoot fresh weight ranged from 45.5 g to 121.6 g and 45.9 g to 116.7 g in 2020 and 2021, respectively. Utilizing a plant density of 45 × 40 cm rather than 30 × 20 cm increased plant fresh weight by 62.6% and 60.7% compared with 30 × 20 cm in 2020 and 2021, respectively. Shoot dry weight exhibited the same trend among plant densities for both years. Plants grown in the 45 × 40 cm density had 156 and 142% greater shoot dry weights compared with plants that were grown in the 30 × 20 cm density for 2020 and 2021, respectively. Maximum root length in 2020 was greatest for plants grown in the 45 × 40 cm density compared with all others. Maximum root length in 2021 was greatest for plants grown at a density of 45 × 40 cm and least for plants grown at a density of 30 × 20 cm, 30 × 30 cm, or 45 × 20 cm. In 2020, maximum root diameter was greatest for plants grown at a density of 45 × 40 cm and least for plants grown at a density of 30 × 20 cm. In 2021, maximum root diameter was greatest for plants grown at a density of 45 × 40 cm and least for plants grown at a density of 30 × 20 cm or 45 × 20 cm. The maximum root diameter of the six plant densities ranged from 5.3 to 11.8 mm, and 7.6 to 13.3 mm in both years, respectively.

3.3. Root Fresh Weight and Dry Weight

Root fresh and dry weight followed identical patterns in 2020 and 2021 (Table 3). Plants grown at a density of 45 × 40 cm or 45 × 30 cm had greater root fresh and dry weights compared with root fresh and dry weights of plants grown at any other plant density, regardless of year. Plants grown at a density of 30 × 20 cm had the lowest root fresh and dry weights compared with all other densities, regardless of year. Plants grown at lower density (45 × 40 cm) had at least 120 or 110% greater root fresh weights compared with plants grown at the highest density (30 × 20 cm) in 2020 or 2021, respectively. Plants grown at lower plant density (45 × 40 cm) had at least 173 or 158% greater root dry weights compared with plants grown at the highest density (30 × 20 cm) in 2020 or 2021, respectively.

3.4. Photosynthetic Activities

Photosynthetic activities in danshen were affected by plant density in 2021 (Table 4). Plants grown at a density of 45 × 40 cm had the highest net photosynthesis (Pn) compared with plants grown at any other plant density. Plants grown at densities of 30 × 30 cm, 30 × 40 cm, 45 × 20 cm, or 45 × 30 cm had similar Pn, and all had greater Pn compared with plants grown at a density of 30 × 20 cm. Plants grown at the lowest density (45 × 40 cm) had the highest Pn value (1.9-fold compared with plants grown at the highest plant density). Plants grown at a density of 45 × 40 cm had the highest intracellular CO2 concentration (Ci) compared with the Ci of plants grown at any other plant density. Plants grown at a density of 45 × 30 cm or 30 × 30 cm had similar Ci levels, and both had higher Ci levels compared with plants grown at a density of 30 × 40 cm or 30 × 20 cm. Plants grown at a density of 30 × 20 cm had greater vapor pressure deficits (VPDL) compared with the VPDL of plants grown at all higher densities. The lowest VPDL values were registered in both plants grown at high density (30 × 30 cm) and in plants grown at low density (45 × 40 cm). Additionally, plants that were grown at a density of 45 × 40 cm had VPDL levels that were similar to plants grown at densities of 30 × 40 cm or 45 × 20 cm.

3.5. Tanshinone I, Tanshinone IIA, Cryptotanshinone, and Salvianolic Acid B

The content of bioactive compounds in danshen roots was affected by plant density in both years (Table 5). In both years, tanshinone I levels were greatest in plants grown at a density of 45 × 40 cm and lowest for plants grown at a density of 30 × 20 cm. Tanshinone I levels were 106 and 100% greater for plants grown at the lowest plant density (45 × 40 cm) compared with plants grown at the highest plant density (30 × 20 cm) in 2020 and 2021, respectively. In both years, tanshinone IIA levels were greatest for plants grown at densities of 45 × 40 cm or 45 × 30 cm, and lowest for plants grown at a density of 30 × 20 cm. Tanshinone IIA levels were 57 and 46% greater for plants grown at the lowest plant density (45 × 40 cm) compared with plants grown at the highest plant density (30 × 20 cm) in 2020 and 2021, respectively. In 2020, cryptotanshinone levels were greatest in plants grown at a density of 45 × 40 cm followed by plants grown at a density of 45 × 30 cm, 30 × 40 cm, and 30 × 30 cm. Plants that were grown at densities of 30 × 40 cm and 30 × 30 cm had similar crytotanshinone levels compared with plants that were grown at 45 × 20 cm. In 2021, cryptotanshinone levels were greatest in plants grown at a density of 45 × 40 cm followed by plants grown at a density of 45 × 30 cm and 30 × 40 cm. Plants that were grown at densities of 45 × 20 cm and 30 × 30 cm had similar crytotanshinone levels compared with plants that were grown at 30 × 40 cm. Plants that were grown at a density of 30 × 20 cm had the least cryptotanshinone, regardless of year. Cryptotanshinone levels were 37 and 36% greater for plants grown at the lowest plant density (45 × 40 cm) compared with plants grown at the highest plant density (30 × 20 cm) in 2020 and 2021, respectively. In both years, salvianolic acid B levels were highest in plants grown at densities of 45 × 40 cm or 45 × 30 cm and least for plants grown at a density of 30 × 20 cm. Salvianolic acid B levels were 36 and 54% greater for plants grown at the lowest plant density (45 × 40 cm) compared with plants grown at the highest plant density (30 × 20 cm) in 2020 and 2021, respectively.

4. Discussion

Plant growth, photosynthetic activities, and bioactive compound content of danshen plants differed depending on plant density (30 × 20, 30 × 30, 30 × 40, 45 × 20, 45 × 30, or 45 × 40 cm) in 2020 and 2021.

4.1. PGI, Shoot Number, Shoot Fresh Weight, and Shoot Dry Weight

Plants growing in six plant densities varied in their PGI, shoot number, shoot fresh weight, and shoot dry weight. In this study, as the plant density decreased, the growth of the aboveground part increased, showing a similar trend to another study [15]. Increasing the plant density resulted in the development of plants with shorter, slimmer branches, and smaller leaves [23,24]; this may be due to the competition between plants for nutrients, light, air, and other factors under high plant density conditions [9,25,26]. These results support the findings of this study that danshen planted at the highest density group had a lower PGI. Limited sunlight availability results in plants dedicating less effort to developing extensive branches and larger leaves, resulting in the lowest number of shoots in the 30 × 20 cm and 45 × 20 cm plant density group in 2020 and 30 × 20 cm plant density group in 2021. This result indicates that a spacing of 20 cm still had an inhibitory effect on the shoot growth of danshen. In addition, although the densities of 45 × 20 cm and 30 × 30 cm had different distances within and between rows, their planting areas were the same (900 cm2). In other studies, it was reported that closer spacing also impedes proper air circulation between plants, leading to increased humidity and potentially promoting the occurrence of diseases [27,28]. With limited resources and space, plants might adopt a more energy-efficient strategy. Developing smaller leaves and fewer branches can reduce water loss through transpiration and overall maintenance costs [29], allowing them to thrive in a crowded environment. In present study, danshens growing at 45 × 40 cm density had the highest PGI, shoot dry weight, shoot number, shoot fresh weight, while plants grown at a density of 30 × 20 cm had the lowest PGI, shoot number, shoot fresh weight, and shoot dry weight in both years, indicating that high plant density inhibited the growth of the aboveground part of danshen.

4.2. Leaf SPAD Values and Photosynthetic Activities

High plant density limited SPAD value and various aspects of photosynthesis, while lower photosynthesis levels led to stunted plant growth [30]. Low plant density promoted SPAD value, Pn, and Ci. In this study, the highest SPAD value, Pn, and Ci were recorded for plants grown at a density of 45 × 40 cm, while the VPDL was highest in plants grown at a density of 30 × 20 cm. As plant density increases, shading from neighboring plants reduces sunlight reaching lower leaves, decreasing photosynthesis and chlorophyll production [9]. A previous study [31] reported that increasing plant density generally reduced the SPAD value and net photosynthetic rate of winter wheat, which was consistent with our study. Plants growing in high density may not have access to the necessary materials for optimal photosynthesis, resulting in reduced overall photosynthetic activity. In addition, high plant density limited air circulation within the canopy [32]. Proper air circulation is important for gas exchange, including the intake of CO2 required for photosynthesis and the release of O2 produced during photosynthesis. Reduced air circulation can lead to elevated O2 levels and a decrease in CO2 levels within the canopy, which might negatively affect photosynthesis. In this study, SPAD value, Pn, and Ci generally increased with decreasing plant density. However, VPDL decreased with decreasing plant density. A higher SPAD value indicates greater chlorophyll content, which can contribute to increased Pn [33]. Pn and Ci are closely related. Good air circulation with relatively high CO2 concentration at low plant densities allows for greater CO2 uptake, resulting in higher Ci, leading to increased photosynthetic rates (Pn). In addition, lower plant density improves air circulation, facilitating an increased uptake of CO2 from the atmosphere, boosting Ci due to enhanced photosynthesis. A similar relationship between Pn and Ci was found in Noor’s results [34]. A possible reason for the decrease in VPDL under a low plant density may be that lower plant density reduces water competition, leading to higher soil moisture levels around individual plants. This lowers transpiration rates, decreasing the VPDL [35]. Furthermore, lower plant density improves air circulation, while chlorophyll efficiently converts absorbed energy into chemical energy rather than causing excessive leaf surface heating, lowering leaf temperature, and reducing water evaporation, thus decreasing VPDL [36]. It was also reported that when VPDL increases, the stomata close, leading to a decrease in Pn [35].

4.3. Root Number, Root Fresh Weight, and Root Dry Weight

The decrease in photosynthetic products from aboveground sources can also have adverse effects on the roots, including metabolism, nutrient uptake, and stress tolerance [9,37]. In the present study, plants grown at a density of 45 × 40 cm had the highest root number, maximum root length, and maximum root diameter, and plants growing at densities of 45 × 30 and 45 × 40 cm produced the highest root fresh weight and root dry weight, which indicates growth of roots was inhibited as the plant density increased. The results of this study were similar to Sheng’s study [15], who reported that the highest root number of one-year-old danshen was found in 30 × 40 or 45 × 30 cm plant densities, and root number generally increased with decreasing plant density. Sheng reported that as plant density decreased, the maximum root diameter and maximum root length of individual danshens increased. Shao et al. [38] reported that to adapt to limited photosynthesis at high plant densities, maize plants tended to reduce the number of nodal roots and inhibit lateral root growth and avoid root-to-root competition. High plant density (90,000 plants per ha) reduced the number of nodal roots, lateral root density, and average lateral root length in field-grown maize [38]. Tegen et al. [39] reported that the highest root diameter of carrots occurred at the widest interrow spacing of 30 cm. Lower plant density has also been reported to increase average root weight and dry matter content for radishes [40].

4.4. Tanshinone I, Tanshinone IIA, Cryptotanshinone, and Salvianolic Acid B

Based on the results of this study, lower plant densities significantly increased the contents of salvianolic acid B, tanshinone IIA, tanshinone I, cryptotanshinone. These results are similar to Sheng’s [15], who indicated that lower plant density (45 × 40 cm) promoted the content of salvianolic acid B of Salvia miltiorrhiza. In contrast, Chang [41] reported that a density (100 × 60 cm) greater than ones investigated in this study had the highest salvianolic acid B, and tanshinone IIA content. Similarly, in this study, plants with more space had the highest content of tanshinone I, cryptotanshinone, tanshinone IIA, and salvianolic acid B. Photosynthesis is important in influencing the synthesis of bioactive compounds within plants. Photosynthesis provides precursors and energy for bioactive compounds biosynthesis via the mevalonate and phenylpropanoid pathways. Changes in photosynthesis affect precursor availability, impacting tanshinone and salvianolic acid levels in danshen [42,43]. The efficiency of photosynthesis dictates the content of energy accessible to the plant, thus influencing both the development and the synthesis of bioactive substances. In this research, plants growing at a density of 45 × 40 cm exhibited elevated levels of salvianolic acid B, tanshinone IIA, tanshinone I, and cryptotanshinone, and also exhibiting a high Pn. However, in some cases, the relationship between plant density and the content of bioactive compounds may vary depending on factors such as plant species, growth conditions, and specific bioactive compounds [44].

5. Conclusions

In conclusion, plants grown at a density of 45 × 40 cm resulted in the highest leaf SPAD value, PGI, shoot number, photosynthetic activity, shoot fresh and dry weights, root number, maximum root diameter, maximum root length, and contents of bioactive compounds including tanshinone I and cryptotanshinone, regardless of years. Plants grown at a density of 45 × 30 cm had similar root fresh and dry weight, content of tanshinone IIA, and salvianolic acid B compared with plants grown at a density of 45 × 40 cm, and were at higher levels than plants grown at higher densities. However, reducing plant density results in a decrease in the number of plants planted in a specific area, which may reduce total yield. For future research, it is suggested that field experiments be conducted to explore the relationship between plant density, individual plant yield, and total yield.

Author Contributions

Conceptualization, G.B., P.R.K. and T.L.; methodology, G.B. and Z.X.; formal analysis, Z.X.; investigation, Z.X. and Q.Z.; writing—original draft preparation, Z.X.; writing—review and editing, G.B., T.L., P.R.K., Z.X. and Q.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the United States Department of Agriculture (USDA) National Institute of Food and Agriculture Hatch Project MIS-112050. We thank James Dao and Tom Dao for plant materials, technical guidance, and financial support. Mention of a trademark, proprietary product, or vendor, does not constitute a guarantee or warranty of the product by Mississippi State University or the USDA and does not imply its approval to the exclusion of other products or vendors that also may be suitable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Table 1. PGI, leaf SPAD value, shoot number, and root number of plants growing at six plant densities in Mississippi in 2020 and 2021.
Table 1. PGI, leaf SPAD value, shoot number, and root number of plants growing at six plant densities in Mississippi in 2020 and 2021.
Plant Density (cm)PGI 1SPADShoot
Number (per Plant)		Root Number (per Plant)
20202021202020212020202120202021
30 × 2019.9 ± 1.4 d 223.4 ± 1.8 d27.8 ± 1.4 e21.9 ± 2.2 d2.6 ± 0.9 d2.4 ± 0.5 d21 ± 2.0 d19.6 ± 1.7 e
30 × 3032.0 ± 3.1 b27.9 ± 2.3 c30.6 ± 2.8 cd24.1 ± 2.3 cd4.2 ± 0.4 c4.0 ± 0.7 c31.8 ± 2.7 c23.8 ± 1.3 cd
30 × 4033.0 ± 1.6 b31.3 ± 2.6 b31.6 ± 2.6 bc26.4 ± 2.2 bc4.4 ± 0.5 bc4.4 ± 0.5 b29.2 ± 1.9 c25.4 ± 2.2 c
45 × 2029.9 ± 2.3 c27.5 ± 2.2 c28.5 ± 1.4 de23.4 ± 2.1 d3.4 ± 1.1 cd3.8 ± 0.8 c31.8 ± 2.6 c22.6 ± 1.7 d
45 × 3035.3 ± 1.9 b33.9 ± 1.9 b31.7 ± 2.5 bc26.7 ± 1.6 bc5.4 ± 0.8 b4.8 ± 0.8 b35.6 ± 2.1 b30.0 ± 2.7 b
45 × 4038.9 ± 6.2 a36.4 ± 3 a34.3 ± 1.3 a28.2 ± 1.7 a6.6 ± 1.1 a6.4 ± 0.5 a40.4 ± 2.6 a34.2 ± 1.5 a
p-value<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001
1 Plant Growth Index (PGI). 2 Different lowercase letters within a column denote significant differences among plant densities, as determined by Tukey’s HSD test at p ≤ 0.05. Data are presented as mean ± S.D.
Table 2. Shoot fresh weight, shoot dry weight, maximum root length, and maximum root diameter of plants growing at six plant densities in Mississippi in 2020 and 2021.
Table 2. Shoot fresh weight, shoot dry weight, maximum root length, and maximum root diameter of plants growing at six plant densities in Mississippi in 2020 and 2021.
Plant Density (cm)Shoot Fresh Weight (g per Plant)Shoot Dry Weight (g per Plant)Maximum Root Length (cm)Maximum Root Diameter (cm)
20202021202020212020202120202021
30 × 2045.5 ± 3.5 e 145.9 ± 3.9 e9.8 ± 1.9 e10.1 ± 1.1 e13.9 ± 2.0 c15.8 ± 1.3 d5.3 ± 0.6 c7.6 ± 0.6 e
30 × 3071.4 ± 4.8 d71.6 ± 4.5 d13.7 ± 1.2 d14.1 ± 0.8 d26.7 ± 3.4 b21.8 ± 1.8 cd8.1 ± 0.9 b8.6 ± 0.7 cd
30 × 4090.6 ± 6.5 c84.2 ± 4.2 c20.4 ± 1.9 c17.2 ± 1.1 c25.9 ± 2.2 b25.8 ± 1.3 bc9.2 ± 1.3 b9.2 ± 0.5 c
45 × 2069.2 ± 4.2 d66.4 ± 3.9 d13.6 ± 1.1 d12.9 ± 1.1 d24.2 ± 1.9 b21.2 ± 2.2 d8.6 ± 1.0 b8.3 ± 0.7 de
45 × 3099.5 ± 3.5 b93.8 ± 5.6 b22.8 ± 1.9 b 19.3 ± 1.6 b 24.8 ± 5.1 b22.0 ± 1.6 bc9.3 ± 1.1 b10.4 ± 0.9 b
45 × 40121.6 ± 6.0 a116.7 ± 5.8 a25.1 ± 1.6 a 24.4 ± 1.1 a 33.0 ± 4.9 a38.0 ± 3.6 a11.8 ± 0.9 a13.3 ± 0.5 a
p-value<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001
1 Different lowercase letters within a column denote significant differences among plant densities, as determined by Tukey’s HSD test at p ≤ 0.05. Data are presented as mean ± S.D.
Table 3. Root fresh and dry weight of plants growing at six plant densities in Mississippi in 2020 and 2021.
Table 3. Root fresh and dry weight of plants growing at six plant densities in Mississippi in 2020 and 2021.
Plant Density (cm)Root Fresh Weight (g per Plant)Root Dry Weight (g per Plant)
2020202120202021
30 × 2059.4 ± 7.5 d 163.9 ± 4.6 d6.6 ± 0.9 d7.4 ± 0.8 d
30 × 3097.1 ± 8.2 c98.4 ± 1.5 c11.6 ± 1.5 c12.9 ± 0.9 c
30 × 40114.2 ± 6.6 b114.5 ± 1.7 b14.9 ± 2.1 b16.8 ± 1.0 b
45 × 2097.7 ± 2 c91.8 ± 8.7 c12.4 ± 0.9 c12.3 ± 0.5 c
45 × 30124.7 ± 4.7 a126.3 ± 12.4 a17.2 ± 1.7 a18.2 ± 1.1 a
45 × 40131.3 ± 2.6 a134.1 ± 13.2 a18.0 ± 1.1 a19.1 ± 1.1 a
p-value<0.0001<0.0001<0.0001<0.0001
1 Different lowercase letters within a column denote significant differences among plant densities, as determined by Tukey’s HSD test at p ≤ 0.05. Data are presented as mean ± S.D.
Table 4. Photosynthetic activities of plants growing at six plant densities in Mississippi in 2021.
Table 4. Photosynthetic activities of plants growing at six plant densities in Mississippi in 2021.
Plant Density (cm)Pn 2 Ci 3VPDL 4
(μmol m−2 s−1)(μmol mol−1)(kPa)
30 × 209.87 ± 0.47 c 1155.79 ± 4.95 d2.64 ± 0.16 a
30 × 3016.53 ± 0.40 b221.77 ± 16.75 bc2.03 ± 0.08 d
30 × 4016.87 ± 1.63 b151.56 ± 12.18 d2.20 ± 0.01 cd
45 × 2015.97 ± 1.57 b207.61 ± 12.21 c2.24 ± 0.18 bc
45 × 3017.73 ± 1.66 b 226.48 ± 6.44 b2.43 ± 0.09 b
45 × 4019.70 ± 1.71 a288.62 ± 4.10 a2.16 ± 0.02 cd
p-value<0.0001<0.0001<0.0001
1 Different lowercase letters within a column denote significant differences among plant densities, as determined by Tukey’s HSD test at p ≤ 0.05. Data are presented as mean ± S.D. 2 Pn: Net photosynthesis. 3 Ci: Intracellular CO2 concentration. 4 VPDL: Leaf-to-air vapor pressure deficit.
Table 5. Content of tanshinone I, tanshinone IIA, cryptotanshinone, and salvianolic acid B in roots of plants growing at six plant densities in Mississippi in 2020 and 2021.
Table 5. Content of tanshinone I, tanshinone IIA, cryptotanshinone, and salvianolic acid B in roots of plants growing at six plant densities in Mississippi in 2020 and 2021.
Plant Density (cm)Tanshinone I (% w/w)Tanshinone IIA (% w/w)Cryptotanshinone (% w/w)Salvianolic Acid B (% w/w)
20202021202020212020202120202021
30 × 200.034 ± 0.003 d 10.035 ± 0.003 e0.216 ± 0.008 e0.211 ± 0.009 d0.079 ± 0.006 d0.078 ± 0.006 d2.584 ± 0.080 e2.261 ± 0.058 d
30 × 300.050 ± 0.003 c0.048 ± 0.003 d0.261 ± 0.013 c0.26 ± 0.002 c0.091 ± 0.007 bc0.088 ± 0.006 c2.918 ± 0.041 c2.896 ± 0.043 c
30 × 400.056 ± 0.003 b0.054 ± 0.002 c0.275 ± 0.005 b0.286 ± 0.005 b0.095 ± 0.004 bc0.090 ± 0.004 bc3.202 ± 0.083 b3.180 ± 0.066 b
45 × 200.045 ± 0.004 c 0.046 ± 0.002 d0.244 ± 0.09 d0.250 ± 0.015 c0.089 ± 0.008 c0.087 ± 0.004 c2.772 ± 0.053 d2.282 ± 0.074 c
45 × 300.066 ± 0.002 b0.066 ± 0.001 b0.302 ± 0.007 a0.299 ± 0.007 a0.098 ± 0.006 b0.096 ± 0.007 b3.417 ± 0.105 a3.467 ± 0.100 a
45 × 400.070 ± 0.004 a0.071 ± 0.003 a0.307 ± 0.006 a0.309 ± 0.005 a0.108 ± 0.004 a0.106 ± 0.005 a3.510 ± 0.048 a3.490 ± 0.037 a
p-value<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001
1 Different lowercase letters within a column denote significant differences among plant densities, as determined by Tukey’s HSD test at p ≤ 0.05. Data are presented as mean ± S.D.
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Xing, Z.; Bi, G.; Li, T.; Zhang, Q.; Knight, P.R. Effect of Plant Density on Growth and Bioactive Compounds in Salvia miltiorrhiza. Agronomy 2024, 14, 1157. https://doi.org/10.3390/agronomy14061157

AMA Style

Xing Z, Bi G, Li T, Zhang Q, Knight PR. Effect of Plant Density on Growth and Bioactive Compounds in Salvia miltiorrhiza. Agronomy. 2024; 14(6):1157. https://doi.org/10.3390/agronomy14061157

Chicago/Turabian Style

Xing, Zhiheng, Guihong Bi, Tongyin Li, Qianwen Zhang, and Patricia R. Knight. 2024. "Effect of Plant Density on Growth and Bioactive Compounds in Salvia miltiorrhiza" Agronomy 14, no. 6: 1157. https://doi.org/10.3390/agronomy14061157

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