Intra- and Interspecific Competition Altered the Competitive Strategies of Alternanthera philoxeroides and Trifolium regens under Cadmium Contamination

Abstract

Heavy metal accumulation in soils has been one of the environmental and ecological issues, as it caused life and biodiversity problems. However, many invasive plants can survive in heavy metal polluted areas, but little is known about the invasiveness while under different densities either with native species or themselves. In this study, a greenhouse experiment was performed to examine how cadmium contamination with different concentrations (0, 100, and 200 mg/kg) may influence the interspecific competition between invasive plant Alternanthera philoxeroides and the landscape grass T. regens, as well as the intraspecific competition of A. philoxeroides with different densities. The results showed that stronger interspecific competition would alleviate cadmium damage to both A. philoxeroides and T. regens, but the two species adopted different allocation strategies. A. philoxeroides allocated more biomass to belowground and less to aboveground, while T. regens showed exactly the opposite allocation strategy. There was a significant density effect of intraspecific competition on A. philoxeroides. That is to say, with the increase of A. philoxeroides density, the cadmium stress on the growth of A. philoxeroides decreased. Our findings provide a theoretical basis and technical support for the effective control of A. philoxeroides invasion, as well as the restoration and reconstruction of green vegetation.

1. Introduction

Heavy metal accumulation in soils, whether caused by natural processes or human activity, severely impacts life and biodiversity [1]. The problem of soil heavy-metal pollution has been a focus of attention in China [2]. Heavy metal toxicity is one of the primary causes of environmental and ecological issues [3,4]. According to China’s soil analysis reports, the lead, cadmium, mercury, and arsenic exceed the prescribed standard [5]. Heavy metals in soils are not only difficult to remove, but also circulate and accumulate in the food chain of the whole ecosystem, threatening the stability of the ecosystem and human health [6]. Specifically, soil heavy-metal pollution not only damages soil quality and reduces crop yields, but also exacerbates global climate change and affects the sustainable development of society [7].

However, some plants are tolerant to heavy metals and may become more competitive in contaminated sites [8]. Many invasive plants have been found to readily colonize areas with high levels of heavy metal contamination [9]. Alternanthera philoxeroides is a worldwide invasive plant, which is native to South America. In the 1930s, it was introduced into Shanghai as a forage plant. Due to the high invasiveness, A. philoxeroides has caused serious damage to many ecosystems all around China. First, A. philoxeroides competed with crops for fertilizer and water, which led to the loss of crop yields [10,11]. Second, it severely blocked the rivers, obstructed water traffic and transportation, and reduced species richness, causing serious damage to the local agricultural eco-economic systems [12]. A. philoxeroides can occupy ecological niches quickly and decrease the stability and species richness of the native community. The invasion of A. philoxeroides can drastically affect biodiversity and cause yield losses in agriculture. As a consequence, much attention has been paid to studying the invasion mechanism of A. philoxeroides and taking measures to prevent the further spread of its invasion.

Trifolium repens is famous for its ornamental value, including its long green period, easy reproduction, extensive management, drought resistance, barrenness, pruning resistance, long flowering period, and low cost [13]. Therefore, it is often planted for green lawns as well as in various sports fields and parks. T. repens has a good ground coverage and greening effect in soil consolidation and slope protection, which can effectively prevent soil erosion. Its leaves are rich in nutrients, which can speed up the growth rate after grazing, so it usually forms excellent pastures [14]. It also plays a good role in regulating climate and opens up avenues for improving urban soil fertility [15]. The presence of T. repens can repair and improve the fragile soil environment and help maintain the stability of the ecosystem. However, from field investigation, we found A. philoxeroides invaded most of the T. repens lawns. Its invasion of lawns will not only increase the consumption of human, material, and financial resources in the urban landscape, but will also have an impact on soil consolidation and slope protection.

In this study, we would like to examine the invasiveness of A. philoxeroides and the competitiveness of A. philoxeroides and T. repens under cadmium stress. In 2021, we conducted a pot-experiment under different concentrations of cadmium stress to predict the invasion strategy of this alien plant. We also added a factor of planting density to quantify the response of both species to cadmium stress and planting density. We hypothesized that the relative invasive dominance of A. philoxeroides would increase with increasing cadmium concentration, while planting density may aggravate this influence as planting density could weaken the stressful effects of cadmium on invaders thereby accelerating invader invasion. Specifically, we addressed the following questions: (1) How do the performance and dominance of A. philoxeroides and T. repens vary along with different cadmium concentrations? (2) Whether the interspecific competition change with different T. repens densities under cadmium stress? (3) Will the intraspecific competition of A. philoxeroides to cadmium stress change with its different densities?

2. Material and Methods

2.1. Overview of the Experimental Site

We conducted a competition experiment from June to August 2021 in a greenhouse at China West Normal University (106°4′1″ E, 30°48′45″ N), Nanchong, China. It has a subtropical monsoon climate with an average annual temperature of 15.6 °C and annual precipitation of 1070.5 mm. The land is dominated by brown-purple clay and reddish-brown-purple clay soils with low organic matter content. The soil used for this experiment was made by mixing local soil with nutrient soil (containing a small amount of perlite, high quality grass peat, peat, and various trace elements) in a ratio of 1:4.

2.2. Experimental Design

The soil used in this experiment was from the campus of China West Normal University, and it was artificially contaminated with Cd [100 and 200 mg·kg⁻¹ supplied as CdCl₂]. Uncontaminated soil was used as a control. The metal concentrations were designed following the Guideline Values of Cd for agriculture used in China by ~33×, 100×, 200×, and 333× [16]. The weight of soil in each pot was constant in the experiment. Cadmium concentration= cadmium mass/ soil mass. Before the experiment, all soil was evenly sprayed with CdCl₂ solution containing the corresponding mass of elemental cadmium, thoroughly homogenized, and periodically mixed for 1 week.

On 25 May 2021, we collected a stem cutting (of approximately the same size and length) of A. philoxeroides near the experimental site and grew them in an incubator for 12 d under the same external condition before transplanting them into plastic pots (top diameter: 23.5 cm; bottom diameter: 13.2 cm; height: 14 cm) which contained 3.5 kg prepared soils. On 6 June 2021, the similarly-sized ramets were transplanted to pots for one of the following two culture types: (1) A. philoxeroides monoculture, (2) Mixed culture with A. philoxeroides and T. regens. A. philoxeroides monoculture is available in 3 planting densities: 1, 5, and 9 plants/pot. Thus, the intraspecific competition experiment included 9 treatments (3 cadmium concentration × 3 planting density). Mixed culture is available in 2 planting densities: 5 (one A. philoxeroides in the center and four T. regens evenly surrounded) and 9 (one A. philoxeroides in the center and eight T. regens evenly surrounded) plants/pot. Thus, the interspecific competition experiment included 6 treatments (3 cadmium concentration × 2 planting density). For each treatment, we used five replicates. The pots were watered in sufficient quantity every day during the experiment to keep the soil in the pots moist, cleared of weeds in a timely manner, and moved randomly once a week to avoid position effects. All plants were harvested on 26 August 2021.

2.3. Growth and Biomass Allocation

The total plants were thoroughly washed and divided into leaves, shoots, and roots; the biomass of each part was determined using the balance after oven drying at 75 ℃ for 48 h. The biomass allocation traits shoot mass ratio (SMR), root mass ratio (RMR), leaf mass ratio (LMR), root to shoot ratio (RMR), and root: shoot mass (R/S) were calculated as follows:

Total mass = leaf mass + shoot mass + root mass

SMR = shoot mass/total mass

RMR = root mass/total mass

R/S = root mass/(shoot mass + leaf mass)

2.4. Photosynthesis-Related Traits

Traits associated with photosynthetic activity, namely, specific leaf area (SLA), total leaf area, number of leaves, and leaf chlorophyll content. SLA and leaf area were calculated as follows:

SLA = leaf area/dry weight of the leaves

The total leaf area was measured by Image J after scanning by the scanner. The total chlorophyll content of leaves of both A. philoxeroides and T. regens in each pot was measured using the method of Gao [17]. The number of leaves was counted out by hand. We weighed 0.1 g of the fresh leaves of A. philoxeroides and T. regens, ground them in a mortar, then extracted chlorophyll with 95% ethanol, and then measured the absorbance with a spectrophotometer at 645 nm and 663 nm, respectively. The chlorophyll content was calculated as follows:

${\mathrm{Chl}}_{\mathrm{a}} \mathrm{content} (\mathrm{mg}·{\mathrm{g}}^{-1} \mathrm{FW})=\frac{\left(12.72\times {\mathrm{A}}_{663}-2.59\times {\mathrm{A}}_{645}\right)\times {\mathrm{V}}_{\mathrm{t}}}{\mathrm{F}\mathrm{W}\times 1000}\times n$

${\mathrm{Chl}}_{\mathrm{b}} \mathrm{content} (\mathrm{mg}·{\mathrm{g}}^{-1} \mathrm{FW})=\frac{\left(22.88\times {\mathrm{A}}_{645}-4.67\times {\mathrm{A}}_{663}\right)\times {\mathrm{V}}_{\mathrm{t}}}{\mathrm{F}\mathrm{W}\times 1000}\times n$

Chl_T content (mg·g⁻¹ FW) = Chl_a+ Chl_b

where Chl_a, Chl_b, and Chl_T denote chlorophyll a, chlorophyll b, and the total chlorophyll content, respectively; A₆₄₅ and A₆₆₃ denote the absorbance at 645 nm and 663 nm, respectively; Vt denotes the total volume of chlorophyll extract solution; FW denotes the fresh weight of the leaves (g); and n denotes the dilution times. The spectrophotometric analyses were performed by UV/Vis spectrophotometry (ONDA, mod, UV-30 Scan).

2.5. H₂O₂ Content, MAD Content, and Enzyme Activity

Hydrogen peroxide (H₂O₂) content is a very important physiological indicator of plants under stress. H₂O₂ content was determined by the colorimetric method of titanium sulfate [18]. Lipid peroxidation levels in A. philoxeroides and T. regens were measured by estimating the malondialdehyde (MDA) content. The MDA content was colorimetrically determined using the thiobarbituric acid (TBA) assay. Superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) are used to scavenge hydrogen peroxide produced in metabolism to avoid oxidative damage to cells by hydrogen peroxide accumulation, and the level of their activity is related to the resistance of plants. SOD activity was determined by photochemical reduction of nitrogen blue tetrazole (NBT), POD activity was determined by the guaiacol method, and CAT activity was determined by UV absorption. All spectrophotometric analyses were performed by UV/Vis spectrophotometry (ONDA, mod, UV-30 Scan). H₂O₂ content, MAD content, and enzyme activity were calculated as follows [17,18]:

${\mathrm{H}}_2{\mathrm{O}}_2 \mathrm{content} (\mathsf{\mu }\mathrm{mol}·{\mathrm{g}}^{-1} \mathrm{FW})=\frac{\mathrm{C} \times {\mathrm{V}}_{\mathrm{t}}}{\mathrm{FW} \times {\mathrm{V}}_{\mathrm{s}}}$

$\mathrm{MAD} \mathrm{content} (\mathrm{mmol}·{\mathrm{g}}^{-1} \mathrm{FW})=[6.452 \times (\mathrm{A}{532}_{ }-{\mathrm{A}}_{600})-0.559 \times {\mathrm{A}}_{450}] \times \frac{{\mathrm{V}}_{\mathrm{t}}}{{\mathrm{V}}_{\mathrm{s}}\times \mathrm{F}\mathrm{W}}$

$\mathrm{SOD} \mathrm{activity} (\mathrm{U}·{\mathrm{g}}^{-1} \mathrm{FW}·{\mathrm{h}}^{-1})=\frac{\left({\mathrm{A}}_0-{\mathrm{A}}_{\mathrm{s}}\right)\times {\mathrm{V}}_{\mathrm{t}}\times 60}{{\mathrm{A}}_0\times 0.5\times \mathrm{F}\mathrm{W}\times {\mathrm{V}}_{\mathrm{s}}\times \mathrm{t}}$

$\mathrm{CAT} \mathrm{activity} (\mathrm{U}·{\mathrm{g}}^{-1} \mathrm{FW}·{\min }^{-1})=\frac{\mathrm{\Delta }{\mathrm{A}}_{240 }\times {\mathrm{V}}_{\mathrm{t}}}{0.1\times {\mathrm{V}}_{\mathrm{s}}\times \mathrm{t}\times \mathrm{F}\mathrm{W}}$

$\mathrm{POD} \mathrm{activity} (\mathsf{\mu }\mathrm{g}·{\mathrm{g}}^{-1} \mathrm{FW}·{\min }^{-1})=\frac{\left(X^1-X_0\right)\times {\mathrm{V}}_{\mathrm{t}}}{\mathrm{F}\mathrm{W}\times {\mathrm{V}}_{\mathrm{s}}\times \mathrm{t}}$

where C denotes the concentration of H₂O₂ in the sample found on the standard curve; V_t and V_s denote the total volume of the extract and the amount of crude enzyme solution taken for the determination, respectively; A_n denotes the absorbance at n nm; A₀ indicates the absorbance of the control tube under light; and A_s indicates the absorbance of the sample measuring tube.

2.6. Quality Control (QC)

Whole analytical procedures were monitored using strict quality control measures. Repeated tests were used for each set of experiments for monitoring interference. The standard errors (SE) were all below 25%.

2.7. Statistical Analysis

Before analysis, data of each variable were divided by the number of plants initially grown in each pot so that the data were scaled to per unit plant level. The experimental data were statistically analyzed by SPSS statistics (version 20.0; IBM Corp, Armonk, NY, USA) for each measurement and the mean and standard error were calculated. The variability of the effects of different planting densities and cadmium concentrations on the physiological and ecological characteristics of A. philoxeroides and T. repens was analyzed by two-way ANOVA. Multiple comparisons were performed by the Duncan test to examine the level of significant differences in the corresponding data (p < 0.05). Use origin to complete the drawing.

3. Results

3.1. Effects of Cadmium Concentration and Planting Density on Plant Growth

The cadmium concentration was a significant factor affecting the total biomass, biomass allocation (including SMR, RMR, LMR, and R/S), and leaf traits of plants (including chlorophyll content, SLA, and the number of leaves, excepting the total leaf area) in the process of A. philoxeroides’s invasion. Planting density only had a significant effect on chlorophyll content, the total leaf area, and the number of leaves of A. philoxeroides (p < 0.001), and the interaction between cadmium concentration and planting density on total biomass, chlorophyll content, total leaf area, and the number of leaves was significant (p < 0.005) (Table 1). The same trend was found in total biomass of A. philoxeroides and T. regens when A. philoxeroides invaded T. regens population. When the planting density of T. regens was low, the total biomass of both species showed a trend of “low promotion and high suppression” with the increase of cadmium concentration, but when the planting density of T. regens was high, both total biomass showed a trend of “first decrease then increase” with the increase of cadmium concentration (Figure 1A,B). Cadmium concentration and planting density significantly reduced the total biomass of A. philoxeroides. The total biomass of A. philoxeroides decreased progressively with increasing planting density at each cadmium concentration, and the total biomass decreased progressively with increasing cadmium concentration at each planting density (Figure 1C). The intraspecific competition of A. philoxeroides increased with increasing density, thus affecting the material accumulation.

The allocation strategy of A. philoxeroides was decreasing SMR and increasing RMR, LMR, and R/S with the addition of cadmium. RMR and R/S of A. philoxeroides significantly increased with increasing density of T. regens, while the effect on SMR was not significant under each cadmium concentration stress; RMR and R/S of A. philoxeroides showed a trend of “low promotion and high suppression” with the increase of cadmium concentration, while the leaf mass ratio showed a continuously increasing trend under each planting density (Figure 2A–C,J). SMR of T. regens showed a trend of increasing and then decreasing with the increase of cadmium concentration. The trends of changes in RMR and R/S were the same for different planting densities under low cadmium concentration (100 mg/kg), while the trends of changes in their effects were opposite for different planting densities under high cadmium concentration (200 mg/kg). It is noteworthy that the trends of RMR, SMR, LMR, and R/S with increasing planting density under each cadmium stress were exactly opposite to those presented without cadmium stress (Figure 2D–F,K). SMR of A. philoxeroides decreased and RMR and R/S increased with increasing cadmium concentration. In the absence of cadmium stress, the greater the intraspecific competition of A. philoxeroides, the greater RMR and R/S, and the smaller SMR and LMR. Overall, RMR, LMR, and R/S of A. philoxeroides increased with increasing cadmium concentration at each planting density, while SMR decreased with increasing cadmium concentration (Figure 2J–I,L).

A. philoxeroides tended to reduce leaf numbers and increase total chlorophyll content, while total leaf area and SLA showed a trend of “low promotion and high suppression” with increasing cadmium concentration when A. philoxeroides invaded the low-density T. regens population. The interspecific competition between A. philoxeroides and T. regens increased when the planting density was high, causing the number of leaves of A. philoxeroides to decrease continuously with increasing cadmium concentration. The trend of total leaf area and SLA was opposite to that of low planting density, i.e., the total leaf area and SLA showed a trend of decrease and then increase with increasing cadmium concentration. The chlorophyll content of A. philoxeroides in general continued to decline with increasing cadmium concentration. Under the same cadmium stress, the stronger the interspecific competition between A. philoxeroides and T. regens, the more A. philoxeroides tended to increase the number of leaves and decrease the total leaf area (Figure 3A–C,J–L). At low planting density, T. regens tended to reduce SLA and chlorophyll content, while leaf number and total leaf area showed a trend of “low promotion and high inhibition” with increasing cadmium concentration. The number of leaves, SLA, and chlorophyll content of T. regens tended to “first decrease then increase” with the increase of cadmium concentration when planting density was high (Figure 3D–F,M–O). When A. philoxeroides successfully invaded T. regens habitat, both cadmium concentration and density decreased leaf number and total leaf area of A. philoxeroides, and cadmium addition increased the SLA of A. philoxeroides. In the absence of cadmium stress, chlorophyll content of A. philoxeroides decreased with increasing intraspecific competition; the higher the population density, the more chlorophyll content under low cadmium concentration stress (100 mg/kg); while chlorophyll content showed a trend of first decreasing and then increasing with increasing intraspecific competition under high cadmium concentration stress (200 mg/kg) (Figure 3G–I,P–R).

3.2. Effects of Cadmium Concentration and Planting Density on H₂O₂ Content, MAD Content, and Antioxidant Enzyme Activity

Cadmium concentration had significant effects on the H₂O₂ content, MAD content, and antioxidant enzyme activities (CAT and POD) of A. philoxeroides and T. regens in mixed culture. The planting density had a significant effect on enzyme activities of A. philoxeroides in mixed culture, while it had little effect on the enzyme activities of T. regens. The interaction of cadmium concentration and planting density only had significant effects on MAD and POD of A. philoxeroides and T. regens (Table 2). Both cadmium concentration and planting density significantly increased the content of H₂O₂ and MAD, and CAT activity of A. philoxeroides and T. regens. Specifically, the content of H₂O₂ and MAD, and CAT activity gradually increased with increasing cadmium concentration for both A. philoxeroides and T. regens at each planting density. They all gradually decreased with increasing planting density at two cadmium concentrations (Figure 4A,B,D,E,J,K). The SOD activity of A. philoxeroides showed a trend of increasing and then decreasing with the increase of cadmium concentration at low planting density, while it showed a completely opposite trend of decreasing and then increasing at high planting density (Figure 4G). Although planting density had little effect on SOD activity of T. regens under each cadmium stress, SOD activity showed a trend of decreasing and then increasing with cadmium concentration increase at two planting densities, while it increased with increasing planting density at two cadmium concentration (Figure 4H). The POD activity of A. philoxeroides decreased with increasing planting density at each cadmium concentration, while the same trend was observed for T. regens only under cadmium contamination (Figure 4M,N).

Cadmium concentration, planting density, and their interaction had significant effects on enzyme activities (except for MAD content) of A. philoxeroides in monoculture (Table 2). Similarly, both cadmium concentration and planting density significantly increased the content of H₂O₂ and MAD, and CAT activity of A. philoxeroides. Both H₂O₂ and MAD contents of A. philoxeroides decreased with increasing planting density under two cadmium contamination concentrations, while they both increased with increasing cadmium addition concentrations at different planting densities (Figure 4C,F,L). When A. philoxeroides successfully invaded uncontaminated cadmium habitats of T. regens, it gradually reduced CAT activity as its intraspecific competition increased. Under each cadmium stress, CAT activity of A. philoxeroides showed a trend of increasing and then decreasing with the increase of intraspecific competition; under each planting density, CAT activity of A. philoxeroides increased with the increase of cadmium concentration. The SOD activity gradually increased with the increase of planting density (Figure 4I). The POD activity of A. philoxeroides showed a continuously increasing trend with increasing cadmium concentration at low planting density (5 plants/pot), while its activity showed a trend of increasing and then decreasing at high planting density (9 plants/pot). And the trend of POD activity at low cadmium addition concentration was exactly opposite to that at high cadmium addition concentration. Under low cadmium stress, POD activity of A. philoxeroides gradually increased with increasing planting density, while under high cadmium stress, POD activity gradually decreased with increasing planting density.

4. Discussion

Competitive ability and heavy metal tolerance are two of the most important factors that contribute to the capacity of invasive plants to colonize polluted habitats [9,19,20]. The two species A. philoxeroides and T. regens examined in this study are both characterized by a high tolerance to heavy metals [21,22], and the competition between them represents a typical interaction between invasive and native plants in polluted sites [19]. Biomass is an important basis for the study of plant ecology and functional traits, a fundamental expression of energy accumulation [23,24], and an important parameter for measuring interspecific plant competition [25]. The total biomass of both A. philoxeroides and T. regens was promoted by low cadmium concentration (100 mg/kg) and low planting density (5 plants/pot), indicating that energy accumulation was promoted under low interspecific competition intensity and low cadmium stress. This may be since both A. philoxeroides and T. regens are tolerant of cadmium and that low interspecific competition intensity stimulated their nutrient uptake. However, their total biomass was suppressed when interspecific competition increased, probably because the nutrients they could uptake decreased with increased interspecific competition in a limited resource environment. Plant biomass decreases with increasing density, exhibiting a clear density-constrained effect to adapt to different competitive pressures and environmental conditions [26]. The total biomass of both A. philoxeroides and T. regens increased with planting density under high concentrations of cadmium stress (200 mg/kg), which may be due to the uptake of cadmium by multiple plants, thus alleviating the stressful effect of cadmium on their growth.

Allocation plasticity is a major adaptive strategy deployed by plants to counter the adverse effects of environmental stress. As for biomass allocation, A. philoxeroides tended to show a higher degree of allocation plasticity, and RMR and LMR rapidly increased with the cadmium addition, and the R/S ratio also increased. Plants develop high levels of root biomass in metalliferous soils, resulting in a higher R/S ratio, which not only enhances the capacity to store toxic metal ions but also facilitates the biosynthesis of diverse defense-related cellular biomolecules in roots [27]. Root mass allocation under mixed culture condition is indicative of an increased requirement of A. philoxeroides for nutrients or other limited resources under these circumstances [28,29]. A. philoxeroides adjusted root length, root mass distribution, and leaf mass distribution, thus increased competition for soil resources and light. Since T. regens were affected not only by cadmium concentration and interspecific competition but also by intraspecific competition, biomass allocation varied with different cadmium concentrations at different planting densities. Interspecific competition stimulated shoot biomass allocation and leaf biomass allocation, while suppressed root mass allocation of T. regens under cadmium stress. Such adaptive mechanism facilitated T. regens to occupy more space above ground and compete for more light resources. In the absence of cadmium stress, T. regens increased the biomass allocation of the belowground part and decreased the aboveground part as planting density increased, indicating that plant competition for belowground resources such as water, nutrients, and physical space increased with density, while competition for aboveground light resources decreased with density. This is consistent with the findings of Fan [30]. The high planting density of T. regens inhibited biomass allocation to roots and promoted biomass allocation to stems and leaves under cadmium stress, indicating that interspecific competition gradually dominated under the drive of cadmium stress, causing the density effect on biomass allocation to change. Overall, the R/S of A. philoxeroides was significantly greater than that of T. regens, suggesting that A. philoxeroides has more phenotypic competitive advantages.

Plant leaves can respond sensitively to environmental changes. Both the number of leaves and total leaf area can characterize the plant’s ability to capture light and the area of photosynthesis. The total leaf area of A. philoxeroides and T. regens showed consistent trends with increasing cadmium stress when the intensity of interspecific competition is certain, indicating that heavy metal pollution caused plants to make a consistent stress response, i.e., to affect the capture of light resources and the accumulation of organic matter. Interspecific competition reduced the area of photosynthesis of A. philoxeroides in each cadmium stress. For T. regens, interspecific competition and cadmium stress generally increased its leaf number and total leaf area, while decreased SLA (although there was no significant difference between treatments). This is well in-line with the effects of density constraints and cadmium stress on biomass allocation between A. philoxeroides and T. regens. SLA is correlated with leaf thickness [31], the thicker the leaf the more it helps to preserve water in the plant in case of water shortage, ensuring that the plant body can survive harsh and extreme environments [32]. SLA also means that the plant has more resources for photosynthesis [33]. The SLA of T. regens was generally larger than that of A. philoxeroides, but it decreased at high concentrations of individual stresses, indicating that the invasiveness of T. regens community increased. Moreover, cadmium stress and interspecific competition increased total chlorophyll content of A. philoxeroides, while inhibited total chlorophyll content of T. regens, which indicates that interspecific competition and cadmium stress stimulated the photosynthetic capacity of A. philoxeroides and inhibited the photosynthetic capacity of T. regens.

Interspecific competition increased the H₂O₂ content of A. philoxeroides and T. regens when they were not subjected to cadmium stress; their H₂O₂ content increased significantly under heavy metal stress, but interspecific competition decreased the hydrogen peroxide content. This may be because the experiment was conducted in pots with a certain amount of cadmium, and the effect of cadmium stress on each plant was alleviated when planting density was higher. Under the same cadmium stress, the H₂O₂ content of A. philoxeroides was lower than that of T. regens, and therefore A. philoxeroides had better tolerance to the heavy metal cadmium. Due to the production of reactive oxygen species--the H₂O₂, the plants underwent membrane lipid peroxidation [34], but the difference in malondialdehyde content between A. philoxeroides and T. regens was not significant, indicating that the antioxidant enzymes of T. regens responded better to interspecific competition and cadmium stress. SOD, CAT, and POD are the main enzymes that scavenge H₂O₂ [35,36,37], and their previous synergistic effects allow for the maintenance of normal growth and development of A. philoxeroides and T. regens.

Overall, A. philoxeroides increased the number of leaves, RMR, LMR, R/S, and total chlorophyll content with increasing planting density, while T. regens increased SMR and LMR, to increase competitive advantage under low concentration of cadmium stress. T. regens increased total biomass, SMR, LMR, number of leaves, total leaf area, SLA, and total chlorophyll content with increasing planting density, while A. philoxeroides increased total biomass, number of leaves, and R/S, to increase competitive advantage under high cadmium stress. That is, T. regens stimulates leaf development to capture more light resources at high cadmium contamination and planting density, resulting in a superior competitive advantage.

Both intraspecific competition and cadmium stress in A. philoxeroides significantly reduced its biomass accumulation, and its total biomass decreased with increasing density or cadmium stress. The essence of density effect is the competition for space and resources among individuals within a plant population due to increased density and the mutual interference among individuals, which leads to a decrease in plant biomass accumulation [38,39]. A. philoxeroides adapts to intraspecific competition and cadmium-stressed environments by regulating the biomass allocation of above- and below-ground parts. Density showed a significant density effect on above- and below-ground biomass of A. philoxeroides, i.e., increased root biomass allocation and decreased stem and leaf biomass allocation under low concentration and no cadmium stress. The above- and below-ground biomass allocation of A. philoxeroides showed a compensating effect under high cadmium stress, allowing it to better absorb and capture resources for its normal growth and development. Similarly, both density effect and cadmium stress reduced the photosynthetic area and leaf number of A. philoxeroides. The chlorophyll content of A. philoxeroides decreased with the increase of intraspecific competition under low concentration of cadmium stress, indicating that cadmium in the soil was absorbed by the plant under high density and alleviated the stress effect. Cadmium stress significantly increased H₂O₂ content of A. philoxeroides, and it decreased with increased intraspecific competition under cadmium contamination, which was consistent with the trend of malondialdehyde. This suggests that there is also a density effect of planting density on H₂O₂ content and malondialdehyde content, and that the stressing effect of cadmium may be alleviated by the uptake of a certain amount of cadmium by more plants in a pot, which reduced the production of reactive oxygen species and inhibited the membrane lipid peroxidation reaction. H₂O₂ content increased slightly with intraspecific competition without cadmium stress, indicating that intraspecific competition is a stress at this time. The production of hydrogen peroxide caused the production of antioxidant enzymes, but the trends of SOD, CAT, and POD were different, implying the existence of synergistic effects of each antioxidant enzyme to reduce the damage of reactive oxygen species to plants.

5. Conclusions

The results from this study showed that both A. philoxeroides and T. regens increased their competitive advantage by changing the biomass accumulation as well as the trade-offs of each functional trait when A. philoxeroides invaded T. regens. In the presence of heavy metal cadmium stress, stronger interspecific competition would alleviate cadmium damage to both A. philoxeroides and T. regens, but their allocation strategies were different. A. philoxeroides increased its competitive ability by increasing belowground biomass allocation. T. regens increased competitive ability by increasing aboveground biomass allocation. In terms of enzyme activity, T. regens was more sensitive to cadmium stress and had stronger activity of individual antioxidant enzymes, making its membrane lipidation similar to that of A. philoxeroides. Overall, T. regens is more competitive at high cadmium stress and planting densities. Intraspecific competition and cadmium stress affected the material accumulation and nutrient uptake patterns of A. philoxeroides. Such findings provide insights into the invasion mechanism of A. philoxeroides during phytoremediation with T. regens at heavy metal cadmium-contaminated sites and some theoretical basis and technical support for the restoration and reconstruction of green vegetation.

Author Contributions

Conceptualization, Y.C.; methodology, Y.C. and Y.Z.; software, Y.C.; validation, Y.C., T.T., P.L. and C.H.; formal analysis, Y.C.; investigation, T.T., P.L. and C.H.; resources, Y.Z.; data curation, Y.C.; writing—original draft preparation, Y.C., and Q.Z.; writing—review and editing, Q.Z., X.D. and L.Z.; visualization, Y.C.; supervision, Q.Z. and Y.Z.; project administration, Y.Z.; funding acquisition, T.T. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest

The authors declare that they have no known conflicts of interests or personal relatioships that could have appeared to influence the work reported in this paper.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures and Tables

Figure 1. Total biomass of A. philoxeroides and T. regens subjected to cadmium concentration and planting density. (A–C) were the total biomass of A. philoxeroides in mixed culture, the total biomass of T. regens in mixed culture, and the total biomass of A. philoxeroides in monoculture, respectively. The data represents the means ± SE (n = 5). Lowercase letters indicate a significant difference (p < 0.05) in A. philoxeroides or T. regens present on each treatment based on post hoc multiple comparisons in Duncan with cadmium concentration and planting density as fixed factors.

Figure 2. Biomass allocation of A. philoxeroides and T. regens in monoculture and mixed culture. (A,D,G,J), (B,E,H,K), and (C,F,I,L) were the biomass allocation of A. philoxeroides in mixed culture, the biomass allocation of T. regens in mixed culture, and the biomass allocation of A. philoxeroides in monoculture, respectively. Lowercase letters indicate a significant difference (p < 0.05) in A. philoxeroides or T. regens present on each treatment based on post hoc multiple comparisons in Duncan with cadmium concentration and planting density as fixed factors.

Figure 3. Traits related to photosynthesis of A. philoxeroides and T. regens subjected to cadmium concentration and planting density. (A,D,G,J), (B,E,H,K), and (C,F,I,L) were the traits related to photosynthesis of A. philoxeroides in mixed culture, the traits related to photosynthesis of T. regens in mixed culture, and the traits related to photosynthesis of A. philoxeroides in monoculture, respectively. Lowercase letters indicate a significant difference (p < 0.05) in A. philoxeroides or T. regens present on each treatment based on post hoc multiple comparisons in Duncan with cadmium concentration and planting density as fixed factors.

Figure 4. Enzyme activity, including H2O2 content, MAD content, SOD activity, CAT activity, and POD activity, of A. philoxeroides and T. regens subjected to cadmium concentration and planting density. (A,D,G,J,M), (B,E,H,K,N), and (C,F,I,L,O) were the enzyme activity of A. philoxeroides in mixed culture, the enzyme activity of T. regens in mixed culture, and the enzyme activity of A. philoxeroides in monoculture, respectively. Lowercase letters indicate a significant difference (p < 0.05) in A. philoxeroides or T. regens present on each treatment based on post hoc multiple comparisons in Duncan with cadmium concentration and planting density as fixed factors.

Table 1

F-values and significance levels for factorial ANOVA of the effects of density and cadmium on morphological traits and biomass of A. philoxeroides and T. regens.

Variables	CadmiumConcentration		Planting Density		Interaction
	F	P	F	P	F	P
A. philoxeroides in mixed culture
Total biomass	30.123	<0.001	0.042	0.840	14.702	<0.001
SMR	10.734	<0.001	0.482	0.494	0.301	0.743
RMR	8.564	0.002	0.517	0.479	0.173	0.842
LMR	27.967	<0.001	0.080	0.780	2.414	0.111
R/S	7.501	0.003	1.255	0.274	0.388	0.683
Chlorophyll content	32.391	<0.001	192.178	<0.001	338.890	<0.001
SLA	7.501	0.003	1.255	0.274	0.388	0.683
Total leaf area	1.849	0.179	14.334	0.001	6.782	0.005
Number of leaves	204.118	<0.001	989.807	<0.001	253.300	<0.001
T. regens in mixed culture
Total biomass	1.506	0.242	0.055	0.817	4.508	0.022
SMR	0.769	0.475	1.133	0.298	1.009	0.379
RMR	1.803	0.186	1.326	0.261	4.303	0.025
LMR	1.023	0.375	0.661	0.808	1.699	0.204
R/S	2.224	0.130	0.155	0.697	4.525	0.022
Chlorophyll content	7.219	0.004	0.008	0.929	20.774	<0.001
SLA	2.224	0.130	0.155	0.697	4.525	0.022
Total leaf area	453.896	<0.001	6.791	0.015	1148.826	<0.001
Number of leaves	86.771	<0.001	43.102	<0.001	7.980	0.002
A. philoxeroides in monoculture
Total biomass	51.019	<0.001	39.612	<0.001	15.517	<0.001
SMR	27.296	<0.001	4.166	0.024	1.928	0.127
RMR	26.027	<0.001	6.583	0.004	1.748	0.161
LMR	18.900	<0.001	16.409	<0.001	3.908	0.01
R/S	17.800	<0.001	4.478	0.018	2.108	0.100
Chlorophyll content	11.951	<0.001	15.468	<0.001	34.875	<0.001
SLA	21.138	<0.001	10.731	<0.001	13.753	<0.001
Total leaf area	7.295	0.002	4476.921	<0.001	37.028	<0.001
Number of leaves	13.587	<0.001	5.304	0.030	55.681	<0.001

Table 2

F-values and significance levels for factorial ANOVA of the effects of density and cadmium on enzyme activity of A. philoxeroides and T. regens.

Variables	CadmiumConcentration		Planting Density		Interaction
	F	P	F	P	F	P
A. philoxeroides in mixed culture
H2O2 content	2293.342	<0.001	60.202	<0.001	35.337	<0.001
MAD content	147.702	<0.001	27.136	<0.001	28.955	<0.001
SOD activity	35.528	<0.001	18.818	<0.001	106.077	<0.001
CAT activity	255.588	<0.001	18.811	<0.001	0.520	0.601
POD activity	121.129	<0.001	106.096	<0.001	24.361	<0.001
T. regens in mixed culture
H2O2 content	41.094	<0.001	0.241	0.628	1.524	0.238
MAD content	50.144	<0.001	0.013	0.909	15.849	<0.001
SOD activity	3.276	0.055	0.117	0.736	0.327	0.724
CAT activity	370.694	<0.001	4.100	0.054	0.405	0.672
POD activity	34.128	<0.001	5.879	0.023	19.753	<0.001
A. philoxeroides in monoculture
H2O2 content	220.223	<0.001	4.551	0.017	3.220	0.023
MAD content	1257.838	<0.001	2.494	0.097	32.358	<0.001
SOD activity	37.376	<0.001	21.781	<0.001	15.891	<0.001
CAT activity	737.560	<0.001	16.871	<0.001	21.122	<0.001
POD activity	445.598	<0.001	14.189	<0.001	90.811	<0.001

References

1. Akram, M.A.; Wahid, A.; Abrar, M.; Manan, A.; Naeem, S.; Zahid, M.A.; Gilani, M.M.; Paudyal, R.; Gong, H.Y.; Ran, J.Z. et al. Comparative study of six maize (Zea mays L.) cultivars concerning cadmium uptake, partitioning and tolerance. Appl. Ecol. Environ. Res.; 2021; 19, pp. 2305-2331. [DOI: https://dx.doi.org/10.15666/aeer/1903_23052331]

2. Adnan, M.; Xiao, B.; Xiao, P.; Zhao, P.; Li, R.; Bibi, S. Research Progress on Heavy Metals Pollution in the Soil of Smelting Sites in China. Toxics; 2022; 10, 231. [DOI: https://dx.doi.org/10.3390/toxics10050231]

3. Khan, I.; Awan, S.A.; Rizwan, M.; Ali, S.; Hassan, M.J.; Brestic, M.; Zhang, X.; Huang, L. Effects of silicon on heavy metal uptake at the soil-plant interphase: A review. Ecotoxicol. Environ. Saf.; 2021; 222, 112510. [DOI: https://dx.doi.org/10.1016/j.ecoenv.2021.112510]

4. Azam, Z.; Ayaz, A.; Younas, M.; Qureshi, Z.; Arshad, B.; Zaman, W.; Ullah, F.; Nasar, M.Q.; Bahadur, S.; Irfan, M.M. et al. Microbial synthesized cadmium oxide nanoparticles induce oxidative stress and protein leakage in bacterial cells. Microb. Pathog.; 2020; 144, 104188. [DOI: https://dx.doi.org/10.1016/j.micpath.2020.104188]

5. Liu, X. Discussion on environment protection optimization strategy of heavy metal pollution control. Shanxi Chem. Ind.; 2022; 42, pp. 354-360.

6. Sun, K.; Hua, Y.; Wang, Z. Research progress on heavy metal pollution and health risk assessment of the industrial wastewater. J. N. China Univ. Water Resour. Electr. Power (Nat. Sci. Ed.); 2022; 43, pp. 99-108.

7. Wu, Y.; Li, X.; Yu, L.; Wang, T.; Wang, J.; Liu, T. Review of soil heavy metal pollution in China: Spatial distribution, primary sources, and remediation alternatives. Resour. Conserv. Recycl.; 2022; 181, 106261. [DOI: https://dx.doi.org/10.1016/j.resconrec.2022.106261]

8. Tovar-Sánchez, E. Heavy Metal Pollution as a Biodiversity Threat. Heavy Metals; Intech Open: Rijeka, Croatia, 2018; pp. 383-399.

9. Prabakaran, K.; Li, J.; Anandkumar, A.; Leng, Z.; Zou, C.B.; Du, D. Managing environmental contamination through phytoremediation by invasive plants: A review. Ecol. Eng.; 2019; 138, pp. 28-37. [DOI: https://dx.doi.org/10.1016/j.ecoleng.2019.07.002]

10. Yu, D.; Wei, S.; Zhu, W.; Cao, A.; Zhang, C.; Song, Z. Influence of Altemanthera philoxeroides on the growth of paddy rice and its economic threshold. Acta Phytophylacica Sin.; 2008; 35, pp. 69-73.

11. Lin, J.; Qing, S.; Wu, H. Effect of Alternanthera philoxeroides, an invasive exotic weed, on plant biodiversity. Rural Eco-Environ.; 2005; 21, pp. 28-32.

12. Zhou, G.; Peng, Y.; Wang, Y.; Zhou, G.; Wang, W. Studies on the distribution, occurrence and harm of Alternanthera philoxeroides in Dongtinghu area. Weed Sci.; 2007; 3, pp. 16-18.

13. Wang, Y.; Rui, H.; Wang, Z. Trifolium repens L. lawn planting and maintenance management. Pract. For. Technol.; 2011; pp. 47-48. [DOI: https://dx.doi.org/10.13456/j.cnki.lykt.2011.11.018]

14. Lin, Y. Excellent Forage Grass Trifolium repens L. Technol. Briefing.; 1980; 1, 16.

15. Li, Y. Application of Trifolium repens L. in Urban Greening in Beijing. Chin. Land. Archi.; 1988; 3, pp. 43-45.

16. Duan, Q.; Lee, J.; Liu, Y.; Chen, H.; Hu, H. Distribution of heavy metal pollution in surface soil samples in China: A graphical review. Bull. Environ. Contam. Toxicol.; 2016; 97, pp. 303-309. [DOI: https://dx.doi.org/10.1007/s00128-016-1857-9]

17. Gao, J. Plant Physiology Experiment Guidance; Higher Education Press: Beijing, China, 2006; 287.

18. Shi, H. Experimental Guidance on Plant Adversity Physiology; Science Press: Beijing, China, 2016; pp. 58-60.

19. Che-Castaldo, J.P.; Inouye, D.W. Interspecific competition between a non-native metal-hyperaccumulating plant (Noccaea caerulescens, Brassicaceae) and a native congener across a soil-metal gradient. Aust. J. Bot.; 2015; 63, 141. [DOI: https://dx.doi.org/10.1071/BT15045]

20. Wang, Y.; Chen, C.; Xiong, Y.; Wang, Y.; Li, Q. Combination effects of heavy metal and inter-specific competition on the invasiveness of Alternanthera Philoxeroides. Environ. Exp. Bot.; 2021; 189, 104532. [DOI: https://dx.doi.org/10.1016/j.envexpbot.2021.104532]

21. Liu, Y.; Xie, A. Enrichment features of Trifolium pratense L. under cadmium stress. J. Henan Agric. Sci.; 2011; 40, pp. 82-84.

22. Xu, L.; Zhang, Z.; Zhang, P.; Wang, Y.; Tan, G. Study on tolerance of Altemanthera philoxeroides to heavy metals. J. Anhui Agric. Sci.; 2010; 38, pp. 6831-6832.

23. Zheng, S.; Tang, M.; Zou, J.; Mu, C. Summary of research on shrub biomass in China. J. Chengdu Univ. (Nat. Sci. Ed.); 2007; 26, pp. 189-192.

24. Xiao, Y.; Tao, Y.; Zhang, Y. Biomass allocation and leaf stoichiometric characteristics in four desert herbaceous plants during different growth periods in the gurbantünggüt desert, China. Chin. J. Plant Ecol.; 2014; 38, pp. 929-940.

25. Li, B.; Xu, B.; Chen, J. Perspectives on general trends of plant invasions with special reference to alien weed flora of Shanghai. Biodivers. Sci.; 2001; 9, pp. 446-457.

26. Zhang, S.; Gong, L.; Ge, Y.; Hong, Z.; Jiang, H.; Liu, J.; Tao, Y. Biomass allocation and allometric relationships of the invasive plant species Plantago virginica grown at different densities. Pratacultural Sci.; 2021; 38, pp. 1938-1949.

27. Emamverdian, A.; Ding, Y.; Mokhberdoran, F.; Xie, Y. Heavy metal stress and some mechanisms of plant defense response. Sci. World J.; 2015; 2015, 756120. [DOI: https://dx.doi.org/10.1155/2015/756120]

28. Audet, P.; Charest, C. Allocation plasticity and plant–metal partitioning: Meta-analytical perspectives in phytoremediation. Environ. Pollut.; 2008; 156, pp. 290-296. [DOI: https://dx.doi.org/10.1016/j.envpol.2008.02.010]

29. Broadbent, A.; Stevens, C.J.; Peltzer, D.A.; Ostle, N.J.; Orwin, K.H. Belowground competition drives invasive plant impact on native species regardless of nitrogen availability. Oecologia; 2018; 186, pp. 577-587. [DOI: https://dx.doi.org/10.1007/s00442-017-4039-5]

30. Fan, G.; Zhang, J.; Huang, Y.; Shen, X.; Yu, P.; Zhao, X. Influence of population density on morphological traits and allometric growth of Corispermum Macrocarpum. Acta Ecol. Sin.; 2018; 38, pp. 3931-3942.

31. Akram, M.A.; Zhang, Y.; Wang, X.; Shrestha, N.; Malik, K.; Khan, I.; Ma, W.; Sun, Y.; Li, F.; Ran, J. et al. Phylogenetic independence in the variations in leaf functional traits among different plant life forms in an arid environment. J. Plant Physiol.; 2022; 272, 153671. [DOI: https://dx.doi.org/10.1016/j.jplph.2022.153671]

32. Zhang, M. Study on ecological effects of two amaranth invasive plants on the functional traits of native plants. Nankai University: Tianjin, China, 2020; 54p.

33. Ruprecht, E.; Fenesi, A.; Nijs, I. Are plasticity in functional traits and constancy in performance traits linked with invasiveness? An experimental test comparing invasive and naturalized plant species. Biol. Invasions; 2014; 16, pp. 1359-1372. [DOI: https://dx.doi.org/10.1007/s10530-013-0574-0]

34. Li, G.; Zhao, P.; Zhao, C.; Zhang, H.; Zang, L. Specific activities of antioxidases and malondialdehyde contents of Valeriana jatamansi jones leaves under shading and open field cultivation. Chin. Agric. Sci. Bull.; 2021; 37, pp. 111-116.

35. Li, Z.; Li, J.; Liu, D.; Cong, R. Effects of mixed saline-alkali stress on the physiological indexes of willow seedlings. J. Northeast For. Univ.; 2021; 49, pp. 1-4.

36. Liu, D.; Liu, Y.; Yu, L.; Geng, G.; Wang, Y. Effects of manganese on the morphological, physiological and biochemical indexes of sugar beet seedlings. J. Eng. Heilongjiang Univ.; 2021; 12, pp. 90-96.

37. Sun, Y.; Yang, M.; Wen, X.; Feng, Y.; Ma, Q.; Li, Z. Effect of heavy metal pollution on physiological-biochemical indexes and safety quality of grapes. Sino-Overseas Grapevine Wine; 2021; 1, pp. 50-55.

38. An, H.; Shangguan, Z. Effects of density on biomass and allometric pattern of Robinia pseudoacacia seeding. Sci. Silvae Sin.; 2008; 44, pp. 151-155.

39. Chen, J.; Zhao, Q.; Wang, J.; Ma, J.; Feng, X.; Ma, L. A study of density effects on the biomass and anisotropic growth patterns of Catalpa seedlings. Pract. For. Technol.; 2012; 5, pp. 9-12.