Allelopathic Effects of Soil Extracts from Rhus typhina Plantations on Common Turfgrass Species in Northern China

Abstract

The allelopathic plant Rhus typhina (Rt) has a shallowly distributed root system with a high density of secretory canals, which may allow it to exhibit indirect allelopathic effects through the soil on an understory turf species in a plantation. However, how these effects occur is still not well understood. For this study, the soil from the root zone of Rt was extracted via distilled water, and extracts at different concentrations (25, 50, and 100 g L⁻¹) were used to treat six commonly planted turfgrass species, including Medicago sativa (Ms), Lolium perenne (Lp), Trifolium repens (Tr), Medicago falcata (Mf), Festuca arundinacea (Fa) and Coronilla varia (Cv), during a continuous germination–seedling culture experiment via the sand culture method. The germination, physiological, and growth indicators of the turfgrass seedlings were analyzed. The allelopathic effects of the soil extract at different concentrations on the six receivers were evaluated to provide a scientific basis for managing plantations with multilayer structures. The results indicated that, in general, the extracts of the soil from the root zone of Rt inhibited the germination and seedling growth performance characteristics of the mentioned turfgrasses; the inhibitory effects on Ms, Lp, and Mf increased with increasing concentrations of the Rt soil extracts, whereas those on Fa tended to decrease. In addition, the inhibitory effect on Tr obviously weakened at 50 g·L⁻¹ relative to that at 25 g·L⁻¹, whereas it became more obvious at 100 g·L⁻¹; however, it exhibited the opposite trend for Cv. Generally, Mf and Cv were more sensitive to Rt allelochemicals at relatively low concentrations, and these species should be avoided when planting in Rt plantations at low densities. In contrast, Lp and Tr were more sensitive than those at relatively high concentrations and should be avoided when planting in Rt plantations at high densities.

1. Introduction

Some species inhibit the germination, seedling growth, and physiological activities of other species by secreting allelochemicals to achieve competitive advantages, adversely affecting the natural regeneration and succession direction of plant communities. Recent studies have indicated that allelopathic effects result in a 25% decrease in plant performance [1], affecting agricultural, forestry, and pastoral production [2,3,4]. Thus, plant allelopathy has been widely investigated [5].

Plants can release allelochemicals via various pathways, including volatilization, precipitation leaching, residue decomposition, and root secretion [6]. Chemicals exhibit differences in their concentrations, linkages with receivers, and transformation processes in the environment, resulting in significant differences in the allelopathic effects of plant species. Most studies have extracted allelochemicals from living plants via water to treat the seeds or seedlings of the receivers. Researchers cultured seeds via filter paper, Petri dishes, and sand/soil cultures to simulate the allelopathic effects caused by precipitation leaching of living plants or their litter [7,8]. These methods have been used to study the short-term effects of allelochemicals on receivers. However, plant-derived allelochemicals are continuously released into the soil by leaching, root secretion, and litter decomposition. These allelochemicals are stored in the environment for a long time, resulting in long-term allelopathic effects [6]. Most allelochemicals are degraded by soil microorganisms or transferred into other substances. Their effects on the community and the functions of soil microorganisms can significantly alter the nutrient status and enzymatic properties of soil and change the types and concentrations of allelochemicals, thus leading to allelopathic effects that are quite different from those caused by directly released chemicals [6,9]. For example, nontoxic secondary metabolites in Juglandaceae species exhibited allelotoxicity only when they underwent hydrolyzation and oxidation in the soil [10]. Zhang et al. [11] reported that ferulic acid was degraded by Pseudomonas and Aspergillus microorganisms in the soil, significantly weakening the allelopathic inhibitory effects of this chemical. Facenda et al. [12] reported that the adsorption of umbelliferone by soil prevented its biodegradation and extended its duration of activity. In addition, several studies have indicated that allelochemicals from Centaurea stoebe, Mikania micrantha, Robinia pseudoacacia, and Juniperus virginiana disrupt soil nutrient cycling and nutrient balance, causing the collapse of soil microbial communities and limiting the growth of other plants [3]. Therefore, the lack of consideration of the role of soil can lead to erroneous results regarding allelopathic effects. Analyzing the influence of soil can improve our understanding of the mechanisms and ecological influences of the allelopathic effects of plants, especially ligneous plants. The long lives and large biomass of these plants enable them to release large amounts of allelochemicals into the soil, where they accumulate.

Rhus typhina (Rt), an Anacardiaceae arbor species originating in North America, is widely used in afforestation and soil and water conservation projects in barren mountains because it is highly resistant to cold, drought, and saline–alkali environments, and it is also an invasive species in some countries and regions [13]. In China, it has been widely used in urban landscapes and greening projects since the 1950s because of its unique shape and bright autumn color [14]. However, it has also been reported that Rt has an adventitious shallow root system and is favorable for tillering propagation [15]. These characteristics enable it to accumulate more allelochemicals in the surface soil layer than other ligneous plants. Thus, this species has strong allelopathic inhibitory effects on the growth of understory vegetation, especially for turfgrasses with shallow roots. However, most studies have focused on the allelopathic effects caused by water extracts (or organic solvents) from living Rt tissues [16,17,18,19]. It remains unclear whether Rt-derived allelochemicals accumulate in the soil over time and how these substances affect the growth of commonly used turfgrasses.

Hence, we conducted a follow-up study to previous studies that investigated the allelopathic effects on common turfgrasses in northern China via the same method and receivers ([20] and our accepted data are awaiting publication). In this study, the soil from the root zone of an Rt plantation in an urban green area was extracted via water. Extracts with different concentrations were used to treat the seeds and seedlings of six turf species, namely, Medicago sativa, Lolium perenne, Trifolium repens, Medicago falcata, Festuca arundinacea, and Coronilla varia. The indirect allelopathic effects (through soil media) of Rt on the germination, growth, and physiological properties of other species were investigated. These results improved the understanding of the mechanisms of the allelopathic effects of Rt under comparable conditions and provided a scientific basis for selecting suitable species to establish lawns on Rt plantations.

2. Materials and Methods

2.1. Sampling Area

The samples were collected from the campus of Yan’an University, China (109°27′20″ E, 36°37′44″ N), which is located in the loess hilly region of the Loess Plateau, with an altitude of 1092 m. The area has a warm, temperate continental monsoon climate with an average annual temperature of 10.3 °C, an average annual precipitation level of 523.7 mm, and an average annual sunshine duration of 2471.7 h. Rt was planted 20 years ago, and its density, including seedlings from root tillering regarded as independent plants in this study, was ~8 plants·m⁻². The canopy coverage of Rt was greater than 90% (Figure S1).

2.2. Soil Sampling and Extract Preparation

The allelochemicals in soil are produced mainly from the secretions of roots [21]. Because the adventitious roots of Rt are distributed mainly in the 0~20 cm layer [15], especially in the 0–15 cm layer of soil, and because of the high densities of individual Rt plants, the soil in this layer is considered the root zone soil of Rt. The soil of this layer is frequently or even continuously affected by the rain leachates and litter of Rt, and it is the main layer in which the roots of turfgrasses grow. Therefore, the soil 0–15 cm under the litter layer was used as the source of allelochemicals for the following experiments. Specifically, several 20 cm × 20 cm quadrats were established in August 2023 in the Rt garden (due to the high tree density of Rt, all quadrats were established within the crown diameter and root zone of Rt). All the visible plant litter was removed, and 15-cm-thick exposed soil was collected. The soil from the quadrats was passed through a 2-mm mesh sieve to remove the adventitious roots of Rt and other sundries and was uniformly mixed. A portion of the homogenized soil sample was used to determine the moisture content, and the remaining soil was extracted with distilled water to obtain extracts containing allelochemicals [8]. The concentrations of the extracts were set as 25, 50, and 100 g (DW)·L⁻¹ according to the precipitation levels in the growth seasons of the turfgrasses in the studied region and the thickness and bulk density of the soil in this study. In addition, the concentrations were also adjusted (1) to make the results as comparable as possible with our previous study, where we detected the direct allelopathic effects of living Rt plants in the same studied region [20] (the concentrations of Rt leaf extracts were set as 12.5, 25) and 50 g (dry weight)·L⁻¹) and, simultaneously, (2) to simulate the possible relatively high concentrations of soil allelochemicals because, relative to those directly leached by precipitation, they were dissolved in soil solution over the long term and could be concentrated in the form of enhanced evaporation during the growing season (mainly summer) of turfgrasses. Specifically, the soil samples were mixed with sterile distilled water at a concentration of 100 g/L. The mixture was shaken for 30 min at 180 r·min⁻¹, and extraction was conducted for 48 h at 4 °C. The suspension was filtered twice using trilaminar nonwoven fabrics and filter paper and diluted to 50 and 25 g·L⁻¹. The extracts were labeled as high, moderate, or low concentrations and maintained at 4 °C. Before the following treatments, the extracts were removed from the refrigerator to allow for adjustment to room temperature.

In addition, the extracts contained soluble mineral nutrients and nonallelopathic organic components from the soil, and removing these substances was difficult. Therefore, extracts from the soil that were not affected by Rt were prepared for the control test. Specifically, the soil was collected from weedy land close to the Rt plantation, which shared the same grass species and soil type. The sampling and extraction methods were the same as those used in the Rt plantation, and the 25, 50, and 100 g·L⁻¹ extracts were used as corresponding controls for the extracts of the soil from the Rt root zone, aiming to eliminate the influences of mineral nutrients and nonallelopathic organic components at every concentration as much as possible. In addition, the potential allelochemicals were qualitatively analyzed via gas chromatography–mass spectrometry, and the results are listed in Table S1.

2.3. Turfgrass Seed Preparation

Seeds of M. sativa ‘Mufeng’ (Ms, thousand kernel weight = 2.11 g), L. perenne ‘Mathilde’ (Lp, 4.06 g), T. repens ‘Super Haifa’ (Tr, 0.71 g), M. falcata ‘Tengri’ (Mf, 1.72 g), F. arundinacea ‘Xiashuang’ (Fa, 1.36 g), and C. varia ‘Emerald’ (Cv, 3.46 g) were purchased from Zhengdao Seed Co., Ltd. (Beijing, China). The ripe seeds were disinfected for 10 min with 0.2% NaClO solution, rinsed three times, and used for the germination experiment.

2.4. Germination and Seedling Growth Experiments

In accordance with a previous study [8], a continuous germination and seedling growth experiment was conducted via the sand culture method. The silica sand used was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and disinfected via a high-pressure steam sterilizer. Subsequently, 850 g of silica sand was placed into a three-compartment culture tray with a thickness of 2.5 cm, each of which was merged to form a culture tray with six compartments. A total of 18 merged trays were prepared, nine of which were used as the experimental group. The sand in each of the three merged trays was wetted with 400 mL of extracts from the root zone soil of Rt at high, moderate, and low concentrations (three concentrations × three replicates). In addition, the remaining merged trays were treated with the corresponding extracts of the weedy land soil using the same method. The seeds of six receiver plants were subsequently sown into each merged tray, and 50 seeds of each species were sown with given plant and row spacings in a compartment. We did not perform a control test with water because the seedlings could not grow in pure water.

The trays were weighed after sowing. They were then placed into temperature incubators with a temperature, relative humidity, and light duration of 25 °C, 75%, and 12 h·d⁻¹, respectively. The germinated seeds were counted every day with the standard that the cotyledon/leaf broke through the surface of the sand, and the dead seedlings were also counted and represented via sticks. The germination period ended when no germination was observed within three consecutive days. Beginning with the end of germination of the last type of receiver (the 17th), the seedlings were incubated for another 21 d for the following seedling growth experiment. That is, the germination–seedling growth of all receivers in any type of sand substance was the same over the same period of 38 days. During the entire experiment, the merged trays were weighed every two days, and distilled water was added according to the mass loss to maintain a consistent moisture level. Since the allelochemicals in soil typically accumulated over a long period, the soil extracts were obtained several times (the soil was kept at 4 °C before each extraction), and the new extracts were added to the trays at 10-day intervals during the experiments. A preliminary experiment indicated that the masses of distilled water or soil extracts added to each tray were similar each time. After 38 days, the seedling growth experiment ended, and the surviving seedlings were counted. Five seedlings from the same receiver were subsequently randomly selected from each tray. The shoot height, root length, and fresh aboveground and underground biomasses of these five seedlings were measured, and the average values were obtained and considered replicates. The sampling was conducted three times in trays subjected to the same treatment (concentration × extracts from two soil types), and the obtained values of the seedling growth-related indicators were considered replicates. A sufficient number of seedlings, which have the fresh biomass needed for the determination of the physiological properties according to the following method, were subsequently obtained within 3 days in every compartment for the following evaluations.

2.5. Physiological Properties of the Seedlings

All the determinations of the physiological properties of the seedlings were conducted according to the methods of Liu et al. [22]. Briefly, 0.1 g of leaves were ground with silica sand, CaCO₃ powder, and a total of 10 mL of 95% ethanol to extract chlorophyll (Chl). The absorbancy of the extracted solution was then determined at 665 and 649 nm, and the contents of Chl a and Chl b were calculated via the method of Liu, Li, and Ding [22]. The roots (0.5 g) were soaked in a 10 mL mixture of 0.4% triphenyltetrazolium chloride solution and phosphate buffer solution for 1 h, after which the reaction was terminated with 2 mL of 1 mol·L⁻¹ H₂SO₄. The roots were then ground with silica sand 10 mL of ethyl acetate, and the absorbancy of the extracted solution was then determined at 485 nm. The root activity was calculated using the methods of Liu, Li, and Ding [22]. A total of 0.2 g of each seedling was ground with 2 mL of pH 7.8 phosphate buffer solution and centrifuged at 4 °C (12,000× g) to obtain the crude enzyme. For the determination of catalase (CAT) activity, a mixture of 0.25 mL of crude enzyme and 0.25 mL of 0.1 mol·L⁻¹ H₂O₂ solution was incubated at 30 °C for 10 min and titrated with 0.01 mol·L⁻¹ KMnO₄ solution. The activity of CAT was then calculated using the methods of Liu, Li, and Ding [22] according to the volume of the KMnO₄ solution consumed. For the determination of peroxidase (POD) activity, 30 μL of crude enzyme was added to 3 mL of phosphate buffer-guaiacol-H₂O₂ solution. The change in the absorbance of the mixture at 470 nm over 40 s was detected, and the POD activity was calculated via the method of Liu, Li, and Ding [22]. For the determination of superoxide dismutase (SOD) activity, 30 μL of the crude enzyme was added to a 3 mL mixture containing methionine, EDTA-Na2, nitroblue tetrazolium, phosphate buffer, and riboflavin solution. The mixture was then incubated under 4000 lux illumination for 20 min. The absorbancy of the mixture was determined at 560 nm, and the SOD activity was calculated via the method of Liu, Li, and Ding [22]. For each determination of enzymatic activity, corresponding control tests were performed, as described previously by Liu, Li, and Ding [22]. For the determination of malondialdehyde (MDA) content, 0.2 g of each seedling was ground with a total of 5 mL of distilled water. Five milliliters of 0.5% thiobarbituric acid solution was added to the abovementioned suspension, and the mixture was boiled and centrifuged at 8000× g. The absorbancy levels at 450, 532, and 600 nm of the supernatant were then determined, and the content of MDA was calculated via the method of Liu, Li, and Ding [22]. All mentioned chemicals and reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All mentioned titration analyses were performed with Titertte bottle-top burettes (BRANDTECH Scientific, Inc., Wertheim, Germany); colorimetric analysis was performed with an EPOCH 2 Multiscan Spectrum (BioTek Instruments Inc., Winooski, VT, USA).

2.6. Data Processing

In accordance with Ding, et al. [23], the germination rate (GR) and seedling survival rate (SR) were calculated via Equations (1) and (2). The ratio of Chl a to Chl b (Chl a/b) was calculated via Equation (3). The RIs for the germination, growth, and physiological indicators of the receivers were calculated via Equation (4).

GR (%) = number of germinated seeds/50 × 100%,

(1)

SR (%) = number of surviving seedlings on the 38th day after sowing/number of germinated seeds × 100%,

(2)

Chl a/b ratio = C_chla/C_chlb,

(3)

where C_chla and C_chlb represent the contents of Chl a and Chl b, respectively.

RI = 1 − C_i/T_i,

(4)

where T_i represents the data obtained in the treatments using the extracts of the root zone soil of Rt at a given concentration, and C_i represents the data obtained in the treatments using the extracts of soil from weedy land with the corresponding concentration. RI > 0 indicates the presence of allelopathic accelerating effects, whereas RI < 0 indicates the inhibition effect (the opposite was true for the RI for the MDA content). The absolute value of the RI represents the allelopathic intensity.

2.7. Statistical Analysis

A two-factor analysis of variance was employed to detect the influences of the concentration of extracts, receiver type, and their interactions on the indicators. As the interaction effect was significant (Table S2), a simple effect test was conducted mainly to determine the influence of the concentration. Principal component analysis (PCA) was performed on the RIs of the indicators, except for those of the antioxidant enzymatic activities and Chl a/b, because the changes in these indicators could not directly reflect the quality of germination or seedling growth. In addition, to ensure that the negative value of the RI uniformly indicated that the receiver was subjected to allelopathic stress, the RIs of the MDA content were converted to negative values when PCA was performed. As the cumulative explanatory rate of the first two principal components was less than 50% (Table S3), which could not represent the overall allelopathic effects, the integrated principal component value (F_PCA) was used to evaluate the degree of the allelopathic inhibitory effect via the method of Zhang, et al. [24], and under the above preconditions, lower values of the F_PCA indicated stronger allelopathic inhibitory effects.

All the statistical tests were performed via SPSS 23.0 software (SPSS Inc., Chicago, IL, USA), and the significance level was α = 0.05. All graphics were created via OriginPro 2021b software (Origin Lab Corp., Northampton, MA, USA).

3. Results

3.1. Allelopathic Effects of Extracts from the Root Zone Soil of Rt on the Germination of the Receivers

The results of the germination experiment (Figure 1A) indicated that extracts from the root zone soil of Rt (briefly referred to as ‘Rt soil extracts’ hereafter) significantly inhibited the germination of Lp at all concentrations, and the inhibitory effects were significantly stronger at 50 and 100 g·L⁻¹ (p < 0.05). It also significantly inhibited the germination of Tr at 25 g·L⁻¹ and that of Fa at 100 g·L⁻¹ (p > 0.05). In contrast, the Rt soil extracts significantly promoted the germination of Mf and Cv at 50 g·L⁻¹; however, the promoting effects were inhibitory effects on Cv at 100 g·L⁻¹ (p < 0.05).

The Rt soil extracts also significantly affected the survival of the recipient seedlings (Figure 1B). Specifically, it significantly decreased the survival of Ms seedlings at 50 and 100 g·L⁻¹ (p < 0.05); however, the inhibitory effects were not affected by increasing the concentration in the mentioned range. Rt soil extracts also significantly decreased the survival rates of Lp seedlings at 25 g·L⁻¹ and of Mf seedlings at 100 g·L⁻¹ (p < 0.05). In contrast, it significantly increased that of the Tr seedlings at 50 g·L⁻¹ but exhibited inhibitory effects at 100 g·L⁻¹ (p < 0.05).

3.2. Allelopathic Effects of Extracts from the Root Zone Soil of Rt on the Physiological Properties of Receivers

Physiological analysis revealed that the Rt soil extract significantly increased the Chl a contents of Ms seedlings at 25 g·L⁻¹ (Figure 2A, p < 0.05). In contrast, it significantly decreased those of the Lp seedlings at 100 g·L⁻¹ and those of the Tr and Fa seedlings at 25 and g·L⁻¹, respectively (p < 0.05). It also significantly decreased the Chl a contents of Mf and Cv seedlings at all concentrations; however, its inhibitory effects weakened with increasing concentration for Mf seedlings, while it exhibited the most obvious inhibitory effects at 50 g·L⁻¹ for Cv seedlings (p < 0.05). The extracts had similar allelopathic effects on the content of Chl b (Figure 2B). In addition, the Rt soil extracts significantly increased the ratios of Chl a/b in the Ms seedlings at 50 g·L⁻¹ and significantly decreased those at 100 g·L⁻¹ (Figure 2C, p < 0.05). It significantly decreased the ratio of Chl a/b in the Tr seedlings at 25 g·L⁻¹ and significantly increased that at 100 g·L⁻¹, while it had significant inhibitory effects at 50 g·L⁻¹ (p < 0.05). The Rt soil extract significantly decreased the ratio of Chl a/b in Fa seedlings at 25 g·L⁻¹ but significantly increased it at 50 g·L⁻¹ (p < 0.05). Cv significantly increased the ratios of Chl a/b at all concentrations (p < 0.05); however, these effects first increased significantly and then decreased with increasing concentration (p < 0.05).

In general, the Rt soil extracts significantly decreased the root activities of the recipient plants (Figure 2D). It significantly decreased the activities of Ms and Lp seedlings at all concentrations (p < 0.05); however, the inhibitory effects were the most obvious at 50 g·L⁻¹ Ms, whereas they weakened with increasing concentration (p < 0.05). In addition, it significantly decreased the root activities of the Tr and Cv seedlings at 50 and 100 g·L⁻¹ (p < 0.05), and the inhibitory effects tended to increase with increasing concentration. In addition, the Rt soil extracts significantly decreased only the root activities of Mf seedlings at 100 g·L⁻¹ and significantly inhibited those of the Fa seedlings at 25 and 100 g·L⁻¹ (p < 0.05).

Rt soil extracts significantly increased the CAT activities of Ms, Lp, Fa, and Cv seedlings at 25 and 50 g·L⁻¹; however, the promoting effects were inhibited at 100 g·L⁻¹ (Figure 3A, p < 0.05). Similarly, it significantly increased that of Mf seedlings at 25 g·L⁻¹ and that of Tr seedlings at 50 g·L⁻¹, while inhibitory effects occurred at 50~100 and 100 g·L⁻¹, respectively (p < 0.05). Rt soil extracts significantly increased the POD activities of Ms and Mf seedlings at 25 g·L⁻¹ and those of Lp seedlings at 25 and 100 g·L⁻¹ (Figure 3B, p < 0.05). It also significantly increased the POD activities of the Tr and Cv seedlings at all concentrations. However, the effects on the Tr seedlings weakened with increasing concentration, whereas the effects on the Cv seedlings exhibited the opposite trend (p < 0.05). In addition, it significantly increased the POD activities of Fa seedlings at 50 g·L⁻¹; however, the promoting effects were inhibited at 100 g·L⁻¹ (p < 0.05). Rt soil extracts significantly increased the SOD activities of Ms, Tr, Mf, and Fa seedlings at 25 and 50 g·L⁻¹; however, the promoting effects tended to weaken with increasing concentration and even became inhibitory at 100 g·L⁻¹ (Figure 3C, p < 0.05). It also significantly increased the SOD activities of Lp and Cv seedlings (p < 0.05); however, its promoting effects were weakest at 50 g·L⁻¹ and even became nonsignificant for Cv seedlings (p > 0.05).

In general, the Rt soil extracts caused significant membrane lipid peroxidation of the receiver seedlings, although their antioxidase activities increased (Figure 3D). It significantly increased the MDA contents of Ms and Fa seedlings (p < 0.05); however, these effects were weakest at 50 g·L⁻¹ and even became nonsignificant for Ms seedlings (p > 0.05). The Rt soil extracts significantly decreased the MDA contents of the Lp seedlings but did not affect those of the Cv seedlings at 25 g·L⁻¹. However, these effects increased at concentrations of 50 and 100 g·L⁻¹, although they weakened with increasing concentrations (p < 0.05). It significantly increased the MDA contents of the Tr seedlings at all concentrations, and these effects increased with increasing concentration (p < 0.05). In addition, the Rt soil extract significantly increased the MDA contents of Mf seedlings at 25 g·L⁻¹; these effects became more obvious at 50 g·L⁻¹ (p < 0.05), but they weakened to a nonsignificant degree at 50 g·L⁻¹ (p > 0.05).

3.3. Allelopathic Effects of Extracts from the Root Zone Soil of Rt on Seedling Growth of the Receivers

The results of the seedling growth experiment indicated that the Rt soil extracts significantly inhibited the shoot elongation of Ms and Tr at 25 g·L⁻¹ and of Lp at 100 g·L⁻¹ (p < 0.05, Figure 4A). It also significantly inhibited the shoot elongation of Mf at all concentrations (p < 0.05); however, the inhibitory effects were not affected by increasing the concentration (p > 0.05). For the root growth of the receivers (Figure 4B), the Rt soil extracts significantly inhibited only the root elongation of Lp and Tr at 100 g·L⁻¹ and of Mf at 50 and 100 g·L⁻¹ (p < 0.05).

With respect to biomass accumulation (Figure 4C,D), the Rt soil extracts significantly inhibited only the accumulation of root biomass in the Tr seedlings at 25 and 50 g·L⁻¹ (p < 0.05) and the Mf seedlings at 100 g·L⁻¹ (p < 0.05, Figure 4D).

3.4. Relationships Between the Growth and Physiological Indices of the Receivers

The results of Pearson’s correlation analysis (Figure 5) indicated that the Chl a content and the Chl a/b ratio were significantly positively related to the shoot height. The POD activity was significantly negatively correlated with the MDA content. The Chl a content, Chl a/b ratio, and root activity presented significant positive relationships with root length. However, the POD and SOD activities and MDA content were significantly and negatively correlated with the root length. The Chl a/b ratio was significantly positively related to shoot and root biomass accumulation, whereas the Chl b content and SOD activity were significantly negatively related to shoot biomass accumulation (p < 0.05).

3.5. Integrated Allelopathic Effects of Extracts from the Root Zone Soil of Rt on Receivers

The results of integrated principal component analysis (Figure 6) revealed that the inhibitory effects on Ms, Lp, and Mf increased with increasing concentrations of the Rt soil extracts, whereas those on Fa tended to decrease. In addition, the inhibitory effect of the Rt soil extracts on Tr obviously weakened at 50 g·L⁻¹ relative to that at 25 g·L⁻¹, whereas it became more obvious at 100 g·L⁻¹; however, the opposite trend was observed for Cv. In general, the 25 g·L⁻¹ Rt soil extract had the strongest inhibitory effects on Mf, Fa, and Cv; the 50 g·L⁻¹ Rt soil extract had the strongest inhibitory effects on Mf and Cv; and the 100 g·L⁻¹ Rt soil extract had the strongest inhibitory effects on Lp and Tr.

4. Discussion

4.1. Allelopathic Effects of Extracts from the Root Zone Soil of Rt on the Receivers

Our results indicate that in some cases, the Rt soil extracts significantly inhibit the germination of turf species, such as Lp, Tr, and Cv, which is in line with the results of several previous studies [25,26,27]. These findings can be attributed to the allelochemicals in the extracts disrupting reactive oxygen species (ROS) production and scavenging in seeds [28]; decreasing the activities of key enzymes, such as protease, amylase, lipase, and phosphorylase [29,30,31], during germination; and disrupting the balance of phytohormones [28]. For example, the coumarin present in extracts (Table S1) can lead to the accumulation of ROS in seeds and peroxidation damage [28]; benzoic acid may significantly inhibit the activity of the phosphorylase of seeds [29]; and the benzoic acid, p-hydroxybenzoic acid, and other chemicals found in our soil extracts can affect the degradation of auxin and gibberellin [31,32]. These changes might destroy the cell structure, hinder the transformation of materials and energy, and decrease the germination rate. However, the Rt soil extract treatment also significantly increases the germination rates of Mf and Cv at the given concentrations. According to previous mechanistic studies, (1) the abovementioned allelochemicals at low concentrations can be used as nutrient sources for plants, and they can also stimulate the defense system or other metabolic activities of the receivers [33,34,35,36], thus promoting the physiological activities of seeds. In addition, allelochemicals from invasive species may affect the structural and functional properties of soil microbial communities, thus preventing the loss of soil nitrogen (N) as nitrate [37] or increasing the availability of phosphorus (P) [38]. Therefore, their allelopathic effects may indirectly increase the nutrient status of the medium used for germination, thus increasing their germination rates. Rt-derived allelochemicals have been reported to increase the relative abundance of Streptomyces, a phosphate-solubilizing bacterial genus, to obtain sufficient P [38]. In addition, treatments involving allelochemicals extracted from Rt can significantly improve the availability of soil N, P, and potassium by increasing the microbial mineralization rate [39]. These might allow the root zone soil of Rt to accumulate more nutrients relative to the soil from the lawn and exhibit more accelerating effects. Of course, these are also partially seen in the form of the indirect allelopathic effects of Rt caused by the soil in their root zone in this study.

In terms of physiological properties, the Rt soil extracts significantly decrease the chlorophyll contents of the receivers in most cases. There are two potential reasons. First, allelochemicals found in Rt soil extracts, such as α-pinene and cymene (Table S1), can cause severe chloroplast injury or increase chlorophyllase activity [40,41]. In addition, the benzoic acid, p-hydroxybenzoic acid, and cymene found in the extracts can cause the downregulation of genes related to chlorophyll synthesis [7,41], reducing the uptake of nutrients and trace elements, such as N, P, and Mg [42,43]. All these effects may hinder transcription, protein expression, or the formation of porphyrin structures during chlorophyll synthesis [7,27,43]. In contrast, the Rt soil extracts also increase the chlorophyll contents of the receivers in some cases, which might also be attributed to the indirect effects of Rt-derived allelochemicals, creating nutrient-rich soil conditions relative to those of the control soils [38,39]. In addition, the Rt soil extracts also significantly decrease the root activities of the receivers. The reason is that some allelochemicals can cause root damage, including the breakdown of epidermal cells, anomalous growth, and fungal invasion [43]. We observe color changes, surface stripping, and decay in the roots of some receivers (most seedlings exhibiting these phenomena died before the physiological analysis; thus, we did not take photos), supporting the above results. In addition, allelopathic stress caused by p-hydroxybenzoic acid and phthalic acid, which were detected in our soil extracts (Table S1), might restrict the expression levels of ion channel-related genes or the activities of enzymes, such as H⁺-ATPase [25,44], thereby affecting the absorption capacities of roots. Notably, the root activities of receivers are inhibited more frequently than the content of Chl, which was also reported by Taupik, Aani, Wai, and Seng [34]. This finding might be attributed to the continuous contact between the roots and allelochemicals in this study, whereas the effects of allelochemicals on underground parts are generally weakened by the fixing and degradation of allelochemicals by the roots [45].

With respect to the antioxidant defense systems of the receivers, the Rt soil extracts generally increase the CAT, POD, and SOD activities of the receivers at low concentrations but decrease them at high concentrations, which is in line with the findings of Qu, Li, and Ma [16] and Ma, Chen, Chen, Zhang, Zhang, and He [29]. The likely reason is that an appropriate amount of allelochemicals, such as vanillin, p-hydroxybenzoic acid, and gallic acid, found in Rt soil extracts (Table S1) can activate the antioxidation system, increasing the activities of these enzymes, such as POD and SOD, to scavenge excess ROS [32,46]. However, under high allelopathic stress caused by these substances, the antioxidation system usually cannot scavenge ROS thoroughly [16,29]. Therefore, the ROS burst caused by these allelochemicals significantly decreases antioxidase activity by restricting the expression levels of related genes, hindering protein synthesis, and limiting the supply of nutrients and organic substrates [30,47]. In addition, although the Rt soil extracts stimulate antioxidoxidase activity at limited concentrations, they still cause significant membrane lipid peroxidation. The synthesis of antioxidants and the activities of other antioxidases, such as glutathione reductase, are likely inhibited by allelochemicals at low concentrations. As a result, the redox cycle of the antioxidant protection system is incomplete; thus, rapid ROS accumulation exceeds the protection threshold, leading to MDA accumulation. However, the antioxidation system collapses completely under high allelopathic stress.

The results of Pearson correlation analysis indicate that the growth of the seedlings is closely related to their physiological properties. Therefore, the low photosynthetic rate caused by chlorophyll loss, a lack of nutrients and water caused by decreased root activity, and the oxidative damage caused by the collapse of the antioxidation system mentioned previously inhibit the growth of the seedlings and reduce their survival rates. Notably, previous studies have indicated that the p-hydroxybenzoic acid and ferulic acid found in Rt extracts (Table S1) can also result in low phytohormone contents and activities, including those of auxin and cytokinin [31,48,49]. These effects can limit cell division and the elongation of plant organs. However, the Rt extracts do not affect biomass accumulation in the shoots or roots, indicating that the stress caused by allelochemicals might have caused compensatory radial growth of the plants; the underlying mechanism still requires further study. However, the low height and short root length indicate that the seedlings cannot compete successfully with other plants for light, water, or nutrients. The adverse effects of allelopathy might accumulate and inhibit lawn establishment.

4.2. Other Issues Should Be Addressed

This study is focused on the indirect allelopathic effects of Rt through its root zone soil. Compared with the RIs of the germination and seedling growth indicators detected by treating the receivers with extracts from living tissues of Rt at the same concentrations, we have indeed detected weaker allelopathic effects caused by the Rt soil extracts. For example, the seedlings of Mf, Ms, and Tr do not survive after being treated with Rt leaf extracts at concentrations of 25 and 50 g·L⁻¹. In contrast, these species present acceptable seedling survival rates at the same concentrations in the present study. Lp and Fo can survive both the Rt leaf and Rt soil extract treatments. The RIs for the growth indices (including the length and biomass of shoots and roots) of Lp are 0.22–0.59 and 0.50–0.63 greater in this study than in the leaf extract treatments at concentrations of 25 and 50 g·L⁻¹, respectively; in addition, the RIs for the physiological indices (including the Chl a and b contents and the root activities) of Lp are 0.26–1.08 and 0.60–0.85 greater in the present study. Similar phenomena are observed for Fo ([20] and our accepted data awaiting publication). This is similar to the findings of Bundit et al. [50], who reported that soil from a Crotalaria juncea plantation causes significantly weaker allelopathic effects than living plants, suggesting that the allelopathic inhibitory effects of Rt might be greater than expected, as most of the studies were focused on the direct effects of extracts from the living tissues of Rt. A potential reason for this is the rapid microbial degradation of some allelochemicals [51]. For example, the phenolics detected in Rt soil extracts, such as p-hydroxybenzoic acid and gallic acid, can be degraded by Pseudomonas spp. and Phanerochaete chrysosporium, and phenolic degradation-related genes can be easily found in soil microorganisms [32]. In addition, as physical, chemical, and biological transformations (such as the adsorption and desorption of soil inorganic and organic soil particles and polymerization, oxidation, reduction, decarboxylation, and amidation caused by microbial activities), the chemical composition, concentration, biotoxicity, and removal of allelochemicals may be affected by the presence of the soil medium [21]. Notably, the allelopathic potential of living Rt tissue is substantially affected by the source organ, concentration, and developmental stage characteristics of Rt plants [20]. Hence, we cannot completely assess the differences in the direct and indirect allelopathic effects in this study, and more comprehensive comparative studies are needed. In addition, we observe significant differences in the responses of different receivers to Rt soil extracts, which might be attributed to differences in their resistance to allelochemicals caused by evolution [52]. Finally, it cannot be denied that the extracts used for this study contain other chemicals, such as soluble nutrients and nonallelopathic substances, which might have resulted in unexpected effects. However, we do not find an appropriate adsorbent to remove these substances completely (or, inversely, the allelochemicals) to determine the effects of allelopathy. Therefore, we collected only the soil near the Rt plantation and used its extracts as controls for every extraction concentration. Thus, the effects of increasing contents of the mentioned nonallelopathic chemicals with increasing concentrations of extracts might be minimized, while the effects of Rt-derived allelochemicals in the Rt root zone soil and the altered soil nutrient contents caused by their allelopathic effects (which are considered sources of indirect allelopathy) can be detected. In further studies, suitable chelating agents might be used to minimize the effects of soluble nonallelopathic chemicals and explain the effects of allelochemicals more clearly.

5. Conclusions

Rt soil extracts had overall inhibitory effects on the germination and seedling growth performance of the tested turfgrasses. In general, its inhibitory effects on Ms, Lp, and Mf increased with increasing concentration, whereas the effects on Fa exhibited a decreasing trend. In addition, the inhibitory effect on Tr obviously weakened at 50 g·L⁻¹ relative to that at 25 g·L⁻¹, whereas it became more obvious at 100 g·L⁻¹; however, it exhibited the opposite trend for Cv. Generally, Mf and Cv were more sensitive to the indirect effects of Rt-derived allelochemicals with relatively low concentrations, and these species should be avoided in Rt plantations with low density; in contrast, Lp and Tr were more sensitive than those with higher concentrations, which should be avoided in Rt plantations with high densities.